WorldWideScience

Sample records for lc-ms amt tags

  1. Simultaneous quantification of endogenous and exogenous plasma glucose by isotope dilution LC-MS/MS with indirect MRM of the derivative tag.

    Science.gov (United States)

    Yu, Lingling; Wen, Chao; Li, Xing; Fang, Shiqi; Yang, Lichuan; Wang, Tony; Hu, Kaifeng

    2018-03-01

    Quantification of endogenous and exogenous plasma glucose can help more comprehensively evaluate the glucose metabolic status. A ratio-based approach using isotope dilution liquid chromatography tandem mass spectrometry (ID LC-MS/MS) with indirect multiple reaction monitoring (MRM) of the derivative tag was developed to simultaneously quantify endo-/exogenous plasma glucose. Using diluted D-[ 13 C 6 ] glucose as tracer of exogenous glucose, 12 C 6 / 13 C 6 glucoses were first derivatized and then data were acquired in MRM mode. The metabolism of exogenous glucose can be tracked and the concentration ratio of endo/exo-genous glucose can be measured by calculating the endo-/exo-genous glucose concentrations from peak area ratio of specific daughter ions. Joint application of selective derivatization and MRM analysis not only improves the sensitivity but also minimizes the interference from the background of plasma, which warrants the accuracy and reproducibility. Good agreement between the theoretical and calculated concentration ratios was obtained with a linear correlation coefficient (R) of 0.9969 in the range of D-glucose from 0.5 to 20.0 mM, which covers the healthy and diabetic physiological scenarios. Satisfactory reproducibility was obtained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 5.16%, and relative recoveries of 85.96 to 95.92% were obtained at low, medium, and high concentration, respectively. The method was successfully applied to simultaneous determination of the endo-/exogenous glucose concentration in plasma of non-diabetic and type II diabetic cynomolgus monkeys. Graphical Abstract The scheme of the proposed ratio-based approach using isotope dilution LC-MS/MS with indirect MRM of the derivative tag for simultaneous quantification of endogenous and exogenous plasma glucose.

  2. Ferro-based derivatizing agents for LC/MS an LC/EC/MS

    NARCIS (Netherlands)

    Seiwert, Bettina

    2007-01-01

    Within this thesis, the development and application of ferrocene-based derivatizing agents for LC/MS and LC/EC/MS is presented. The advantages of derivatization by ferrocenes are the similtaneous introduction of a mass tag and an electroactive group, which make them ideally suited for LC/MS and

  3. Can LC and LC-MS ever replace immunoassays?

    Directory of Open Access Journals (Sweden)

    Timothy G. Cross

    2016-10-01

    Full Text Available Immunoassays have been the technology of choice for the analysis of biomolecules for many decades across a wide range of applications in research, diagnostics and infectious disease monitoring. There are good reasons for the wide adoption of immunoassays but even such a well established and characterised technique has limitations and as such investigators are looking at alternative technologies. One such alternative is liquid chromatography (LC and, more specifically, liquid chromatography coupled with mass spectrometry (LC-MS. This article will review both immunoassay and LC and LC-MS technologies and methodologies and discuss the advantages and limitations of both approaches. In addition, the next developments that will need to occur before there is widespread adoption of LC and LC-MS technology preferentially over immunoassays will be examined.

  4. Comparative LC-MS: A landscape of Peaks and Valleys

    NARCIS (Netherlands)

    America, A.H.P.; Cordewener, J.H.G.

    2008-01-01

    Quantitative proteomics approaches using stable isotopes are well-known and used in many labs nowadays. More recently, high resolution quantitative approaches are reported that rely on LC-MS quantitation of peptide concentrations by comparing peak intensities between multiple runs obtained by

  5. Isotope ratio monitoring LC/MS (IRM-LC/MS): new applications

    International Nuclear Information System (INIS)

    Juchelka, D; Hilkert, A.; Krummen, M.

    2005-01-01

    With the introduction of compound specific isotope analysis by isotope ratio monitoring GC/ MS (IRM-GC/MS) the immediate demand for similar applications using HPLC was created. Many compounds of biological, medical, pharmaceutical and environmental interest are not volatile or too polar. Consequently, they cannot be directly analyzed by gas chromatography. In IRM-GC/MS the carrier is helium, which does not interfere with the essential combust ion step prior to isotope ratio mass spectrometry (IRMS). In opposite the LC mobile phase has inhibited a similar direct conversion up to now. All earlier IRM-LC/MS approaches were based on the removal of the liquid phase prior to combustion risking fractionation of the isotope ratios of the eluted compounds. To avoid such restrictions we developed a new continuous flow concept for the coupling of an HPLC system to the isotope ratio MS for 13 C/ 12C isotope ratio analysis. In the Finnigan LC IsoLink, the liquid phase is not removed from the sample prior to oxidation. The sample is oxidized still in the mobile phase followed by on-line separation of the CO 2 from the liquid phase and transfer into the isotope ratio MS. Therefore, this strategy is based on water and inorganic buffers as mobile phase. The new approach opens up a whole new world in the application of gas isotope ratio mass spectrometry. The 13 C/ 12 C ratio s of organic acids, amino acids, carbohydrates and nucleotides can now be measured. These components typically within a complex matrix are separated by liquid chromatography followed by on-line determination of the isotope ratios. The draw backs of using derivatization and off-line preparation procedures can now be over come. This new technique allow s studying biochemical cycles, running tracer experiments and determining the origin of components. Applications from different scientific areas such as biogeochemistry, molecular biology, and pharmacy as well as authenticity control o f foods will be presented

  6. LC-MS-BASED METABOLOMICS OF XENOBIOTIC-INDUCED TOXICITIES

    Directory of Open Access Journals (Sweden)

    Chi Chen

    2013-01-01

    Full Text Available Xenobiotic exposure, especially high-dose or repeated exposure of xenobiotics, can elicit detrimental effects on biological systems through diverse mechanisms. Changes in metabolic systems, including formation of reactive metabolites and disruption of endogenous metabolism, are not only the common consequences of toxic xenobiotic exposure, but in many cases are the major causes behind development of xenobiotic-induced toxicities (XIT. Therefore, examining the metabolic events associated with XIT generates mechanistic insights into the initiation and progression of XIT, and provides guidance for prevention and treatment. Traditional bioanalytical platforms that target only a few suspected metabolites are capable of validating the expected outcomes of xenobiotic exposure. However, these approaches lack the capacity to define global changes and to identify unexpected events in the metabolic system. Recent developments in high-throughput metabolomics have dramatically expanded the scope and potential of metabolite analysis. Among all analytical techniques adopted for metabolomics, liquid chromatography-mass spectrometry (LC-MS has been most widely used for metabolomic investigations of XIT due to its versatility and sensitivity in metabolite analysis. In this review, technical platform of LC-MS-based metabolomics, including experimental model, sample preparation, instrumentation, and data analysis, are discussed. Applications of LC-MS-based metabolomics in exploratory and hypothesis-driven investigations of XIT are illustrated by case studies of xenobiotic metabolism and endogenous metabolism associated with xenobiotic exposure.

  7. Improved profiling of estrogen metabolites by orbitrap LC/MS

    Science.gov (United States)

    Li, Xingnan; Franke, Adrian A.

    2015-01-01

    Estrogen metabolites are important biomarkers to evaluate cancer risks and metabolic diseases. Due to their low physiological levels, a sensitive and accurate method is required, especially for the quantitation of unconjugated forms of endogenous steroids and their metabolites in humans. Here, we evaluated various derivatives of estrogens for improved analysis by orbitrap LC/MS in human serum samples. A new chemical derivatization reagent was applied modifying phenolic steroids to form 1-methylimidazole-2-sulfonyl adducts. The method significantly improves the sensitivity 2–100 fold by full scan MS and targeted selected ion monitoring MS over other derivatization methods including, dansyl, picolinoyl, and pyridine-3-sulfonyl products. PMID:25543003

  8. High-throughput fragment screening by affinity LC-MS.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  9. Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.

    Science.gov (United States)

    Kaspar, Hannelore; Dettmer, Katja; Chan, Queenie; Daniels, Scott; Nimkar, Subodh; Daviglus, Martha L; Stamler, Jeremiah; Elliott, Paul; Oefner, Peter J

    2009-07-01

    Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27+/-5.22, 21.18+/-10.94, and 18.34+/-14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39+/-5.35, 6.23+/-3.84, and 35.37+/-29.42. Both GC-MS and iTRAQ-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.

  10. Urinary Amino Acid Analysis: A Comparison of iTRAQ®-LC-MS/MS, GC-MS, and Amino Acid Analyzer

    Science.gov (United States)

    Kaspar, Hannelore; Dettmer, Katja; Chan, Queenie; Daniels, Scott; Nimkar, Subodh; Daviglus, Martha L.; Stamler, Jeremiah; Elliott, Paul; Oefner, Peter J.

    2009-01-01

    Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ® derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ®-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27±5.22, 21.18±10.94, and 18.34±14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39±5.35, 6.23±3.84, and 35.37±29.42. Both GC-MS and iTRAQ®-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines. PMID:19481989

  11. LC-MS (/MS) in clinical toxicology screening methods.

    Science.gov (United States)

    Viette, Véronique; Hochstrasser, Denis; Fathi, Marc

    2012-01-01

    Toxicological screening is the analysis of biological samples to detect and identify unknown compounds. The high selectivity and sensitivity of liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) technology provide an attractive alternative to the current methods (LC-UV, GC/MS, etc.). For these reasons, an increasing number of applications are being published. This paper is a brief overview of LC-MS(/MS) screening methods developed for clinical toxicology in recent years. Various sample treatments, chromatographic separations and detection by mass spectrometry can be combined to obtain screening methods adapted to the constraints and needs of clinical toxicology laboratories. Currently the techniques are in the hands of specialists, mainly in academic institutions. However, the evolution in technology should allow application of these techniques as a tool in toxicology laboratories, thus allowing a more widespread exploitation of their potential.

  12. LC-MS at core of university-industry link

    DEFF Research Database (Denmark)

    Linding, Rune

    2013-01-01

    , and the Orbitrap Fusion Tribrid system is designed for precisely this type of visionary research.’’ DTU is establishing the state-of-the-art laboratory to develop new experiments to dig deeper into the core machinery of the cell. The new laboratory will use four TFS Q Exactive LC-MS/MS systems, and nano-LC 1000.......’’ ‘‘We are immensely pleased to be working with this talented and motivated team of scientists,’’ said Iain Mylchreest, vice president, research and development, life science mass spectrometry, TFS. ‘‘We share with them the objective of pushing the limits of science to make the world a better place...

  13. LC-MS-based quantification method for Achyranthes root saponins.

    Science.gov (United States)

    Kawahara, Yuki; Hoshino, Tatsuro; Morimoto, Hidetaka; Shinizu, Tomofumi; Narukawa, Yuji; Fuchino, Hiroyuki; Kawahara, Nobuo; Kiuchi, Fumiyuki

    2016-01-01

    A liquid chromatography mass spectrometry (LC-MS) method was developed for simultaneous quantitative analysis of Achyranthes root saponins: chikusetsusaponins IVa (1) and V (2), achyranthosides B (3), C (4), D (5), E (6), and G (7), sulfachyranthosides B (8) and D (9), and betavulgarosides II (10) and IV (11). Satisfactory separation of the saponins was achieved with the use of a volatile ion-pair reagent (dihexyl ammonium acetate) on a phenyl-hexylated silica gel column, and the amounts of saponins extracted under three different conditions were determined. When Achyranthes root was extracted with water at room temperature, achyranthosides B (3) and D (5) were the major saponins, and smaller amounts of other saponins (4, 6-11) were present. However, the amounts of chikusetsusaponins (1 and 2) were negligible. Under the condition to make a standard decoction of a Kampo formula, the major saponins were achyranthosides B (3), C (4), and D (5), and small amounts of chikusetsusaponins IVa (1) and V (2) appeared, whereas prolonged heating largely increased the amounts of chikusetsusaponins. This method can be used for quality control of Achyranthes root.

  14. IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform.

    Science.gov (United States)

    Zhou, Ruokun; Tseng, Chiao-Li; Huan, Tao; Li, Liang

    2014-05-20

    A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS.

  15. Direct coupling of a flow-flow electromembrane extraction probe to LC-MS

    DEFF Research Database (Denmark)

    Fuchs, David; Gabel-Jensen, Charlotte; Jensen, Henrik

    2016-01-01

    the fully automated EME-LC-MS system offers a significant time saving and in addition demonstrates increased sensitivity as the analytes were automatically enriched during the extraction process. The experiment revealed 6 to 16 times higher S/N ratios of the EME-LC-MS method compared to protein...... precipitation followed by LC-MS and thus concomitantly lower LOD and LOQ. The setup integrates a complete analytical workflow of rapid extraction, enrichment, separation and detection of analytes in a fully automated manner. These attributes make the developed system a powerful alternative approach for a wide...

  16. The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments

    Directory of Open Access Journals (Sweden)

    Tradigo Giuseppe

    2007-07-01

    Full Text Available Abstract Background Isotope-coded affinity tags (ICAT is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS. The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses. Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS. Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. Results We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein

  17. Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

    Science.gov (United States)

    Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J

    2013-01-01

    In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

  18. ICPD-a new peak detection algorithm for LC/MS.

    Science.gov (United States)

    Zhang, Jianqiu; Haskins, William

    2010-12-01

    The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods.

  19. The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Shi, Tujin; Qian, Wei-Jun; Liu, Tao; Kagan, Jacob; Srivastava, Sudhir; Smith, Richard D.; Rodland, Karin D.; Camp, David G.

    2015-12-04

    Mass spectrometry-based proteomics has become an indispensable tool in biomedical research with broad applications ranging from fundamental biology, systems biology, and biomarker discovery. Recent advances in LC-MS have made it become a major technology in clinical applications, especially in cancer biomarker discovery and verification. To overcome the challenges associated with the analysis of clinical samples, such as extremely wide dynamic range of protein concentrations in biofluids and the need to perform high throughput and accurate quantification, significant efforts have been devoted to improve the overall performance of LC-MS bases clinical proteomics. In this review, we summarize the recent advances in LC-MS in the aspect of cancer biomarker discovery and quantification, and discuss its potentials, limitations, and future perspectives.

  20. LC-MS-MS identification of drug metabolites obtained by metalloporphyrin mediated oxidation

    Directory of Open Access Journals (Sweden)

    Maurin Andrea J. M.

    2003-01-01

    Full Text Available In this paper we report the application of liquid chromatography-mass spectrometry (LC-MS-MS to the identification of the products formed by oxidation of albendazole and disopyramide with metalloporphyrins in dichloroethane, using iodosylbenzene as an oxygen donor. Our results show that LC-MS-MS is a powerful tool to study the in vitro metabolism of drugs, allowing the identification of known and unknown metabolites. In addition, it was observed that the catalyst system used resulted in the formation of the same metabolites as obtained in vivo, although for disopyramide other products were also observed.

  1. A Guideline to Univariate Statistical Analysis for LC/MS-Based Untargeted Metabolomics-Derived Data

    Directory of Open Access Journals (Sweden)

    Maria Vinaixa

    2012-10-01

    Full Text Available Several metabolomic software programs provide methods for peak picking, retention time alignment and quantification of metabolite features in LC/MS-based metabolomics. Statistical analysis, however, is needed in order to discover those features significantly altered between samples. By comparing the retention time and MS/MS data of a model compound to that from the altered feature of interest in the research sample, metabolites can be then unequivocally identified. This paper reports on a comprehensive overview of a workflow for statistical analysis to rank relevant metabolite features that will be selected for further MS/MS experiments. We focus on univariate data analysis applied in parallel on all detected features. Characteristics and challenges of this analysis are discussed and illustrated using four different real LC/MS untargeted metabolomic datasets. We demonstrate the influence of considering or violating mathematical assumptions on which univariate statistical test rely, using high-dimensional LC/MS datasets. Issues in data analysis such as determination of sample size, analytical variation, assumption of normality and homocedasticity, or correction for multiple testing are discussed and illustrated in the context of our four untargeted LC/MS working examples.

  2. Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

    Science.gov (United States)

    2016-01-01

    Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing “fit-for-purpose” bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma. PMID:27034966

  3. Determination of toxins involved in ciguatera fish poisoning in the Pacific by LC/MS.

    Science.gov (United States)

    Yogi, Kentaro; Sakugawa, Satsuki; Oshiro, Naomasa; Ikehara, Tsuyoshi; Sugiyama, Kiminori; Yasumoto, Takeshi

    2014-01-01

    Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variable but species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.

  4. Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis

    NARCIS (Netherlands)

    Koster, Remco A.; Greijdanus, Ben; Alffenaar, Jan-Willem C.; Touw, Daan J.

    In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was

  5. A new high-throughput LC-MS method for the analysis of complex fructan mixtures

    DEFF Research Database (Denmark)

    Verspreet, Joran; Hansen, Anders Holmgaard; Dornez, Emmie

    2014-01-01

    In this paper, a new liquid chromatography-mass spectrometry (LC-MS) method for the analysis of complex fructan mixtures is presented. In this method, columns with a trifunctional C18 alkyl stationary phase (T3) were used and their performance compared with that of a porous graphitized carbon (PGC...

  6. Effect of trans fatty acid intake on LC-MS and NMR plasma profiles

    DEFF Research Database (Denmark)

    Gürdeniz, Gözde; Rago, Daniela; Bendsen, Nathalie Tommerup

    2013-01-01

    The consumption of high levels of industrial trans fatty acids (TFA) has been related to cardiovascular disease, diabetes and sudden cardiac death but the causal mechanisms are not well known. In this study, NMR and LC-MS untargeted metabolomics has been used as an approach to explore the impact...

  7. Quantification of free and total sialic acid excretion by LC-MS/MS

    NARCIS (Netherlands)

    van der Ham, Maria; Prinsen, Berthil H. C. M. T.; Huijmans, Jan G. M.; Abeling, Nicolaas G. G. M.; Dorland, Bert; Berger, Ruud; de Koning, Tom J.; de Sain-van der Velden, Monique G. M.

    2007-01-01

    The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic

  8. Comparison of MALDI-MSI and LC-MS for pharmacokinetic study of metformin

    Czech Academy of Sciences Publication Activity Database

    Strnad, Štěpán; Sýkora, D.; Cvačka, Josef; Maletínská, Lenka; Pirník, Z.; Majerčíková, Zuzana; Kuneš, Jaroslav; Vrkoslav, Vladimír

    2017-01-01

    Roč. 15, č. 1 (2017), s. 35 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : metformin * MALDI-MSI * LC-MS Subject RIV: CB - Analytical Chemistry, Separation

  9. Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data.

    Science.gov (United States)

    Tekwe, Carmen D; Carroll, Raymond J; Dabney, Alan R

    2012-08-01

    Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. ctekwe@stat.tamu.edu.

  10. LC-MS3 quantification of O-glycopeptides in human serum

    Czech Academy of Sciences Publication Activity Database

    Sanda, M.; Pompach, Petr; Benicky, J.; Goldman, R.

    2013-01-01

    Roč. 34, č. 16 (2013), s. 2342-2349 ISSN 0173-0835 R&D Projects: GA MŠk LH13051 Institutional support: RVO:61388971 Keywords : LC-MS3 * O-glycosylation * Quantification of glycopeptides Subject RIV: CE - Biochemistry Impact factor: 3.161, year: 2013

  11. Determination of chlormequat in pig serum and sow milk by LC-MS/MS

    DEFF Research Database (Denmark)

    Poulsen, Mette Erecius; Christensen, Hanne Bjerre; Sørensen, M.T.

    2007-01-01

    reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80...

  12. Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data

    KAUST Repository

    Tekwe, C. D.

    2012-05-24

    MOTIVATION: Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. RESULTS: Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. AVAILABILITY: The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. CONTACT: ctekwe@stat.tamu.edu.

  13. Determination of selected bisphenols, parabens and estrogens in human plasma using LC-MS/MS

    Czech Academy of Sciences Publication Activity Database

    Kolatorova Sosvorova, L.; Chlupacova, T.; Vitku, J.; Vlk, Martin; Heráček, J.; Stárka, L.; Šaman, David; Šimková, M.; Hampl, R.

    2017-01-01

    Roč. 174, NOV 1 (2017), s. 21-28 ISSN 0039-9140 Institutional support: RVO:61389030 ; RVO:61388963 Keywords : Alternative bisphenol * Bisphenol A * Bisphenol F * Endocrine disruptor * lc-ms/ms * Paraben Subject RIV: CC - Organic Chemistry OBOR OECD: Analytical chemistry; Organic chemistry (UOCHB-X) Impact factor: 4.162, year: 2016

  14. MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics.

    Science.gov (United States)

    Beisken, Stephan; Earll, Mark; Portwood, David; Seymour, Mark; Steinbeck, Christoph

    2014-04-01

    Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data.

  15. Probabilistic model for untargeted peak detection in LC-MS using Bayesian statistics

    NARCIS (Netherlands)

    Woldegebriel, M.; Vivó-Truyols, G.

    2015-01-01

    We introduce a novel Bayesian probabilistic peak detection algorithm for liquid chromatography mass spectroscopy (LC-MS). The final probabilistic result allows the user to make a final decision about which points in a 2 chromatogram are affected by a chromatographic peak and which ones are only

  16. Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation.

    Science.gov (United States)

    Chen, Lin-Zhi; Roos, David; Philip, Elsy

    2016-01-01

    Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing "fit-for-purpose" bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma.

  17. Identification of alkyl dimethylbenzylammonium surfactants in water samples by solid-phase extraction followed by ion trap LC/MS and LC/MS/MS

    Science.gov (United States)

    Ferrer, I.; Furlong, E.T.

    2001-01-01

    A novel methodology was developed for the determination of alkyl (C12, C14, and C16) dimethylbenzylammonium chloride (benzalkonium chloride or BAC, Chemical Abstract Service number: 8001-54-5) in water samples. This method is based on solid-phase extraction (SPE) using polymeric cartridges, followed by high-performance liquid chromatography/ion trap mass spectrometry (LC/MS) and tandem mass spectrometry(MS/MS) detection, equipped with an electrospray interface in positive ion mode. Chromatographic separation was achieved for three BAC homologues by using a C18 column and a gradient of acetonitrile/10 millimolar aqueous ammonium formate. Total method recoveries were higher than 71% in different water matrices. The main ions observed by LC/MS were at mass-to-charge ratios (m/z) of 304, 332, and 360, which correspond to the molecular ions of the C12, C14, and C16 alkyl BAC, respectively. The unequivocal structural identification of these compounds in water samples was performed by LC/MS/MS after isolation and subsequent fragmentation of each molecular ion. The main fragmentation observed for the three different homologues corresponded to the loss of the toluyl group in the chemical structure, which leads to the fragment ions at m/z 212, 240, and 268 and a tropylium ion, characteristic of all homologues, at m/z 91. Detection limits for the methodology developed in this work were in the low nanogram-per-liter range. Concentration levels of BAC - ranging from 1.2 to 36.6 micrograms per liter - were found in surface-water samples collected downstream from different wastewater-treatment discharges, thus indicating its input and persistence through the wastewater-treatment process.

  18. Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples

    NARCIS (Netherlands)

    Gaspari, M.; Verhoeckx, K.C.M.; Verheij, E.R.; Greef, J. van der

    2006-01-01

    LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its

  19. Electron ionization LC-MS with supersonic molecular beams--the new concept, benefits and applications.

    Science.gov (United States)

    Seemann, Boaz; Alon, Tal; Tsizin, Svetlana; Fialkov, Alexander B; Amirav, Aviv

    2015-11-01

    A new type of electron ionization LC-MS with supersonic molecular beams (EI-LC-MS with SMB) is described. This system and its operational methods are based on pneumatic spray formation of the LC liquid flow in a heated spray vaporization chamber, full sample thermal vaporization and subsequent electron ionization of vibrationally cold molecules in supersonic molecular beams. The vaporized sample compounds are transferred into a supersonic nozzle via a flow restrictor capillary. Consequently, while the pneumatic spray is formed and vaporized at above atmospheric pressure the supersonic nozzle backing pressure is about 0.15 Bar for the formation of supersonic molecular beams with vibrationally cold sample molecules without cluster formation with the solvent vapor. The sample compounds are ionized in a fly-though EI ion source as vibrationally cold molecules in the SMB, resulting in 'Cold EI' (EI of vibrationally cold molecules) mass spectra that exhibit the standard EI fragments combined with enhanced molecular ions. We evaluated the EI-LC-MS with SMB system and demonstrated its effectiveness in NIST library sample identification which is complemented with the availability of enhanced molecular ions. The EI-LC-MS with SMB system is characterized by linear response of five orders of magnitude and uniform compound independent response including for non-polar compounds. This feature improves sample quantitation that can be approximated without compound specific calibration. Cold EI, like EI, is free from ion suppression and/or enhancement effects (that plague ESI and/or APCI) which facilitate faster LC separation because full separation is not essential. The absence of ion suppression effects enables the exploration of fast flow injection MS-MS as an alternative to lengthy LC-MS analysis. These features are demonstrated in a few examples, and the analysis of the main ingredients of Cannabis on a few Cannabis flower extracts is demonstrated. Finally, the advantages of

  20. Authentication of Trappist beers by LC-MS fingerprints and multivariate data analysis.

    Science.gov (United States)

    Mattarucchi, Elia; Stocchero, Matteo; Moreno-Rojas, José Manuel; Giordano, Giuseppe; Reniero, Fabiano; Guillou, Claude

    2010-12-08

    The aim of this study was to asses the applicability of LC-MS profiling to authenticate a selected Trappist beer as part of a program on traceability funded by the European Commission. A total of 232 beers were fingerprinted and classified through multivariate data analysis. The selected beer was clearly distinguished from beers of different brands, while only 3 samples (3.5% of the test set) were wrongly classified when compared with other types of beer of the same Trappist brewery. The fingerprints were further analyzed to extract the most discriminating variables, which proved to be sufficient for classification, even using a simplified unsupervised model. This reduced fingerprint allowed us to study the influence of batch-to-batch variability on the classification model. Our results can easily be applied to different matrices and they confirmed the effectiveness of LC-MS profiling in combination with multivariate data analysis for the characterization of food products.

  1. LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine.

    Science.gov (United States)

    Clasquin, Michelle F; Melamud, Eugene; Rabinowitz, Joshua D

    2012-03-01

    MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis.

  2. Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK.

    Science.gov (United States)

    Zhang, Qian; Tomazela, Daniela; Vasicek, Lisa A; Spellman, Daniel S; Beaumont, Maribel; Shyong, BaoJen; Kenny, Jacqueline; Fauty, Scott; Fillgrove, Kerry; Harrelson, Jane; Bateman, Kevin P

    2016-04-01

    Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS. We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold. Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.

  3. Rapid Determination of Isomeric Benzoylpaeoniflorin and Benzoylalbiflorin in Rat Plasma by LC-MS/MS Method

    Directory of Open Access Journals (Sweden)

    Chuanqi Zhou

    2017-01-01

    Full Text Available Benzoylpaeoniflorin (BP is a potential therapeutic agent against oxidative stress related Alzheimer’s disease. In this study, a more rapid, selective, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS method was developed to determine BP in rat plasma distinguishing with a monoterpene isomer, benzoylalbiflorin (BA. The method showed a linear response from 1 to 1000 ng/mL (r>0.9950. The precision of the interday and intraday ranged from 2.03 to 12.48% and the accuracy values ranged from −8.00 to 10.33%. Each running of the method could be finished in 4 minutes. The LC-MS/MS method was validated for specificity, linearity, precision, accuracy, recovery, and stability and was found to be acceptable for bioanalytical application. Finally, this fully validated method was successfully applied to a pharmacokinetic study in rats following oral administration.

  4. DBS-LC-MS/MS assay for caffeine: validation and neonatal application.

    Science.gov (United States)

    Bruschettini, Matteo; Barco, Sebastiano; Romantsik, Olga; Risso, Francesco; Gennai, Iulian; Chinea, Benito; Ramenghi, Luca A; Tripodi, Gino; Cangemi, Giuliana

    2016-09-01

    DBS might be an appropriate microsampling technique for therapeutic drug monitoring of caffeine in infants. Nevertheless, its application presents several issues that still limit its use. This paper describes a validated DBS-LC-MS/MS method for caffeine. The results of the method validation showed an hematocrit dependence. In the analysis of 96 paired plasma and DBS clinical samples, caffeine levels measured in DBS were statistically significantly lower than in plasma but the observed differences were independent from hematocrit. These results clearly showed the need for extensive validation with real-life samples for DBS-based methods. DBS-LC-MS/MS can be considered to be a good alternative to traditional methods for therapeutic drug monitoring or PK studies in preterm infants.

  5. How much separation for LC-MS/MS quantitative bioanalysis of drugs and metabolites?

    Science.gov (United States)

    Tan, Aimin; Fanaras, John C

    2018-05-01

    LC-MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC-MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Specific determination of clinical and toxicological important substances in biological samples by LC-MS

    International Nuclear Information System (INIS)

    Mitulovic, G.

    2001-02-01

    This thesis of this dissertation is the specific determination of clinical and toxicological important substances in biological samples by LC-MS. Nicotine was determined in serum after application of nicotine plaster and nicotine nasal spray with HPLC-ESI-MS. Cotinine was determined direct in urine with HPLC-ESI-MS. Short time anesthetics were determined in blood and cytostatics were determined in liquor with HPLC-ESI-MS. (botek)

  7. An improved method for fast and selective separation of carotenoids by LC-MS.

    Science.gov (United States)

    Abate-Pella, Daniel; Freund, Dana M; Slovin, Janet P; Hegeman, Adrian D; Cohen, Jerry D

    2017-11-01

    Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

    Science.gov (United States)

    Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G

    2018-06-01

    Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

  9. Processing methods for differential analysis of LC/MS profile data

    Directory of Open Access Journals (Sweden)

    Orešič Matej

    2005-07-01

    Full Text Available Abstract Background Liquid chromatography coupled to mass spectrometry (LC/MS has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. Results We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. Conclusion The software is freely available under the GNU General Public License and it can be obtained from the project web page at: http://mzmine.sourceforge.net/.

  10. Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC-MS.

    Science.gov (United States)

    Brutti, Monia; Blasco, Cristina; Picó, Yolanda

    2010-01-01

    A method based on pressurized liquid extraction and LC-MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80 degrees C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC-MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995-0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC-MS/MS provides a sensitive and selective method for the determination of BUs in food.

  11. Investigation of enrofloxacin residues in broiler tissues using ELISA and LC-MS/MS.

    Science.gov (United States)

    Panzenhagen, Pedro Henrique N; Aguiar, Waldemir S; Gouvêa, Raquel; de Oliveira, Andréa M G; Barreto, Fabiano; Pereira, Virgínia L A; Aquino, Maria Helena C

    2016-01-01

    This study investigated the efficiency of an enrofloxacin ELISA test kit to detect the presence of enrofloxacin residues in broiler tissues compared with LC-MS/MS. Broiler tissues from 72 samples consisting of 60 breast muscle, six pools of livers (500 g each) and six pools of kidneys (500 g each) were obtained from six different slaughterhouses. Breast muscle from 10 carcasses and pools of livers and kidneys from approximately 200 carcasses of the same flock were collected from each slaughterhouse. ELISA and HPLC were used to identify and quantify the contamination of the samples with enrofloxacin. A total of 72% of the analysed samples contained enrofloxacin residues detected by the ELISA and 22.2% were detected by LC-MS/MS. The mean values of enrofloxacin contamination found in chicken breast by ELISA and HPLC were 8.63 and 12.25 μg kg(-1), respectively. None of the samples exceeded the maximum limit of 100 μg kg(-1) by both methods set by the European Union as well as the Brazilian Agriculture Ministry. All positive samples for enrofloxacin residues detected by LC-MS/MS were also positive by ELISA. These data confirm the efficiency of the ELISA test, and suggest its use as a screening method for enrofloxacin residues in poultry tissues due to its quick results, low price and ease of applicability.

  12. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS.

    Science.gov (United States)

    van Faassen, Martijn; Bischoff, Rainer; Kema, Ido P

    2017-08-28

    Disturbance of the circadian rhythm has been associated with disease states, such as metabolic disorders, depression and cancer. Quantification of the circadian markers such as melatonin and cortisol critically depend on reliable and reproducible analytical methods. Previously, melatonin and cortisol were primarily analyzed separately, mainly using immunoassays. Here we describe the validation and application of a high-throughput liquid chromatography in combination with mass spectrometry (LC-MS/MS) method for the combined analysis of melatonin and cortisol in plasma and saliva. The LC-MS/MS method was validated according to international validation guidelines. We used this method to analyze total plasma, free plasma (as obtained by equilibrium dialysis) and saliva melatonin and cortisol in healthy adults. Validation results for plasma and saliva melatonin and cortisol were well within the international validation criteria. We observed no difference between saliva collected by passive drooling or Salivette. Moreover, we noted a significant difference in saliva vs. free plasma melatonin. We observed on average 36% (95% CI: 4%-60%) higher salivary melatonin levels in comparison to free plasma melatonin, suggestive of local production of melatonin in the salivary glands. The novel outcome of this study is probably due to the high precision of our LC-MS/MS assay. These outcomes illustrate the added value of accurate and sensitive mass spectrometry based methods for the quantification of neuroendocrine biomarkers.

  13. Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

    Science.gov (United States)

    Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne

    2017-07-01

    The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. LC-MS n Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

    Science.gov (United States)

    Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

    2011-09-01

    Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS n . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS n fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS n experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS n methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

  15. COMPARISON OF COMMERCIAL LC MS/MS COMPATIBLE DETERGENTS WITH SODIUM DEOXYCHOLATE FOR SHOTGUN PROTEOMICS

    Directory of Open Access Journals (Sweden)

    Caleb J. Porter

    2014-12-01

    Full Text Available In this study, we compared the performance of sodium deoxycholate (SDC with several commercially available LC MS/MS compatible detergents for digestion of complex proteomic mixtures. First, the parameters affecting in-solution digestion using SDC were investigated with a full factorial experimental design. Metrics explored were trypsin ratio, digestion time, and concentration of SDC. These parameters were not found to be statistically associated with total peptide identifications in the experimental space investigated. However, in terms of digestion efficiency, digestion time was highly significant (p = 0.0095 as determined by the percent of peptides identified with missed cleavages. The optimized protocol for peptide identification and throughput was used to compare the performance of SDC with various commercially available LC MS/MS compatible surfactants namely Invitrosol, RapiGest, and PPS Silent Surfactant. The detergents were found to be similar through comparisons of the total identified peptides and the hydrophobicity of recovered peptides. We found suitable recovery across a large range of SDC concentrations determined from a bicinchoninic acid (BCA assay. In a spike down experiment, no distinct differences in total number of peptide identifications were discovered when comparing PPS (Silent Surfactant and SDC for preparation of peptide samples derived from low protein amounts (< 20 µg. Combined, these results indicate that SDC is a cost effective alternative to other commonly used LC MS/MS compatible surfactants

  16. Evaluation of Normalization Methods to Pave the Way Towards Large-Scale LC-MS-Based Metabolomics Profiling Experiments

    Science.gov (United States)

    Valkenborg, Dirk; Baggerman, Geert; Vanaerschot, Manu; Witters, Erwin; Dujardin, Jean-Claude; Burzykowski, Tomasz; Berg, Maya

    2013-01-01

    Abstract Combining liquid chromatography-mass spectrometry (LC-MS)-based metabolomics experiments that were collected over a long period of time remains problematic due to systematic variability between LC-MS measurements. Until now, most normalization methods for LC-MS data are model-driven, based on internal standards or intermediate quality control runs, where an external model is extrapolated to the dataset of interest. In the first part of this article, we evaluate several existing data-driven normalization approaches on LC-MS metabolomics experiments, which do not require the use of internal standards. According to variability measures, each normalization method performs relatively well, showing that the use of any normalization method will greatly improve data-analysis originating from multiple experimental runs. In the second part, we apply cyclic-Loess normalization to a Leishmania sample. This normalization method allows the removal of systematic variability between two measurement blocks over time and maintains the differential metabolites. In conclusion, normalization allows for pooling datasets from different measurement blocks over time and increases the statistical power of the analysis, hence paving the way to increase the scale of LC-MS metabolomics experiments. From our investigation, we recommend data-driven normalization methods over model-driven normalization methods, if only a few internal standards were used. Moreover, data-driven normalization methods are the best option to normalize datasets from untargeted LC-MS experiments. PMID:23808607

  17. Analysis of benzodiazepines and their metabolites using DBS cards and LC-MS/MS.

    Science.gov (United States)

    Lee, Heesang; Park, Yujin; Jo, Jiyeong; In, Sangwhan; Park, Yonghoon; Kim, Eunmi; Pyo, Jaesung; Choe, Sanggil

    2015-10-01

    Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC-MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50μL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC-MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30μL of whole blood was spotted on FTA DMPK card C and dried for 2h in a desiccator. A 6-mm disk was punched and vortexed for 1min in a centrifuge tube with 300μL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100μL mobile phase of LC-MS/MS and 5μL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters - LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) - were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem). Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

    Science.gov (United States)

    Beverly, Michael; Hagen, Caitlin; Slack, Olga

    2018-02-01

    The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

  19. Screening of veterinary drug residues in food by LC-MS/MS. Background and challenges.

    Science.gov (United States)

    Delatour, Thierry; Racault, Lucie; Bessaire, Thomas; Desmarchelier, Aurélien

    2018-04-01

    Regulatory agencies and government authorities have established maximum residue limits (MRL) in various food matrices of animal origin for supporting governments and food operators in the monitoring of veterinary drug residues in the food chain, and ultimately in the consumer's plate. Today, about 200 veterinary drug residues from several families, mainly with antibiotic, antiparasitic or antiinflammatory activities, are regulated in a variety of food matrices such as milk, meat or egg. This article provides a review of the regulatory framework in milk and muscle including data from Codex Alimentarius, Europe, the U.S.A., Canada and China for about 220 veterinary drugs. The article also provides a comprehensive overview of the challenge for food control, and emphasizes the pivotal role of liquid chromatography-mass spectrometry (LC-MS), either in tandem with quadrupoles (LC-MS/MS) or high resolution MS (LC-HRMS), for ensuring an adequate consumer protection combined with an affordable cost. The capability of a streamlined LC-MS/MS platform for screening 152 veterinary drug residues in a broad range of raw materials and finished products is highlighted in a production line perspective. The rationale for a suite of four methods intended to achieve appropriate performance in terms of scope and sensitivity is presented. Overall, the platform encompasses one stream for the determination of 105 compounds in a run (based on acidic QuEChERS-like), plus two streams for 23 β-lactams (alkaline QuEChERS-like) and 10 tetracyclines (low-temperature partitioning), respectively, and a dedicated stream for 14 aminoglycosides (molecularly-imprinted polymer).

  20. Direct identification of amyloids by label-free quantitative LC-MS

    DEFF Research Database (Denmark)

    Dueholm, Morten Simonsen; Danielsen, Heidi Nolsøe; Hansen, Susan Hove

    adhesive and therefore bind to pipette tips and other consumables. Pure cultures, large sample volumes and high productivity of amyloids are therefore required for successful purification. We here present a quantitative proteomics technique that allow direct identification of functional amyloid candidates......Direct identification of amyloids by label-free quantitative LC-MS H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm Amyloids are highly ordered fibrillar protein polymers used by organisms from all domains of life due to their exceptional properties. We have previously...... in complex samples based on their structural stability in the presence of increasing concentrations of formic acid....

  1. Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS

    OpenAIRE

    Joshi, Payal; Bhoir, Suvarna; Bhagwat, A. M.; Vishwanath, K.; Jadhav, R. K.

    2011-01-01

    Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itop...

  2. Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

    Science.gov (United States)

    Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

  3. Analysis of acrylamide by LC-MS/MS and GC-MS in processed Japanese foods.

    Science.gov (United States)

    Ono, H; Chuda, Y; Ohnishi-Kameyama, M; Yada, H; Ishizaka, M; Kobayashi, H; Yoshida, M

    2003-03-01

    Acrylamide concentrations in processed foods (63 samples covering 31 product types) from Japan were analysed by LC-MS/MS and GC-MS methods. The limit of detection and limit of quantification of acrylamide were 0.2 ng x ml(-1) (6 fmol) and 0.8 ng x ml(-1) (22 fmol), respectively, by LC-MS/MS, and those of 2,3-dibromopropionamide derived from acrylamide were 12 ng x ml(-1) (52 fmol) and 40 ng x ml(-1) (170 fmol), respectively, by GC-MS. Repeatability given as RSD was 1000 microg x kg(-1). The concentrations in non-whole potato-based snacks, rice crackers processed by grilling or frying, and candied sweet potatoes were lower compared with those in the potato crisps and the whole potato-based fried snacks. One of the whole potato-based fried snacks, however, showed low acrylamide concentration (instant precooked noodles and won-tons were <100 microg x kg(-1) with only one exception. Roasted barley grains for 'Mugi-cha' tea contained 200-600 microg x kg(-1) acrylamide.

  4. GeLC-MS: A Sample Preparation Method for Proteomics Analysis of Minimal Amount of Tissue.

    Science.gov (United States)

    Makridakis, Manousos; Vlahou, Antonia

    2017-10-10

    Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology. Despite the significant advances in the mass spectrometry instrumentation, tissue proteomics still faces several challenges mainly due to increased sample complexity and heterogeneity. However, the most prominent challenge is attributed to the invasive procedure of tissue sampling which restricts the availability of fresh frozen tissue to minimal amounts and limited number of samples. Application of GeLC-MS sample preparation protocol for tissue proteomics analysis can greatly facilitate making up for these difficulties. In this chapter, a step by step guide for the proteomics analysis of minute amounts of tissue samples using the GeLC-MS sample preparation protocol, as applied by our group in the analysis of multiple different types of tissues (vessels, kidney, bladder, prostate, heart) is provided.

  5. LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices.

    Science.gov (United States)

    Choi, B K; Hercules, D M; Gusev, A I

    2001-02-01

    The application of LC separation and mobile phase additives in addressing LC-MS/MS matrix signal suppression effects for the analysis of pesticides in a complex environmental matrix was investigated. It was shown that signal suppression is most significant for analytes eluting early in the LC-MS analysis. Introduction of different buffers (e.g. ammonium formate, ammonium hydroxide, formic acid) into the LC mobile phase was effective in improving signal correlation between the matrix and standard samples. The signal improvement is dependent on buffer concentration as well as LC separation of the matrix components. The application of LC separation alone was not effective in addressing suppression effects when characterizing complex matrix samples. Overloading of the LC column by matrix components was found to significantly contribute to analyte-matrix co-elution and suppression of signal. This signal suppression effect can be efficiently compensated by 2D LC (LC-LC) separation techniques. The effectiveness of buffers and LC separation in improving signal correlation between standard and matrix samples is discussed.

  6. Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS

    Science.gov (United States)

    Zeng, Xuemei; Hood, Brian L.; Zhao, Ting; Conrads, Thomas P.; Sun, Mai; Gopalakrishnan, Vanathi; Grover, Himanshu; Day, Roger S.; Weissfeld, Joel L.; Wilson, David O.; Siegfried, Jill M.; Bigbee, William L.

    2011-01-01

    Introduction Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit. Methods We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays. Results A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (pbiomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection. PMID:21304412

  7. Plasma metabolic profiling of dairy cows affected with clinical ketosis using LC/MS technology.

    Science.gov (United States)

    Li, Y; Xu, C; Xia, C; Zhang, Hy; Sun, Lw; Gao, Y

    2014-01-01

    Ketosis in dairy cattle is an important metabolic disorder. Currently, the plasma metabolic profile of ketosis as determined using liquid chromatography-mass spectrometry (LC/MS) has not been reported. To investigate plasma metabolic profiles from cows with clinical ketosis in comparison to control cows. Twenty Holstein dairy cows were divided into two groups based on clinical signs and plasma β-hydroxybutyric acid and glucose concentrations 7-21 days postpartum: clinical ketosis and control cows. Plasma metabolic profiles were analyzed using LC/MS. Data were processed using principal component analysis and orthogonal partial least-squares discriminant analysis. Compared to control cows, the levels of valine, glycine, glycocholic, tetradecenoic acid, and palmitoleic acid increased significantly in clinical ketosis. On the other hand, the levels of arginine, aminobutyric acid, leucine/isoleucine, tryptophan, creatinine, lysine, norcotinine, and undecanoic acid decreased markedly. Our results showed that the metabolic changes in cows with clinical ketosis involve complex metabolic networks and signal transduction. These results are important for future studies elucidating the pathogenesis, diagnosis, and prevention of clinical ketosis in dairy cows.

  8. Expedient data mining for nontargeted high-resolution LC-MS profiles of biological samples.

    Science.gov (United States)

    Hnatyshyn, Serhiy; Shipkova, Petia; Sanders, Mark

    2013-05-01

    The application of high-resolution LC-MS metabolomics for drug candidate toxicity screening reflects phenotypic changes of an organism caused by induced chemical interferences. Its success depends not only on the ability to translate the acquired analytical information into biological knowledge, but also on the timely delivery of the results to aid the decision making process in drug discovery and development. Recent improvements in analytical instrumentation have resulted in the ability to acquire extremely information-rich datasets. These new data collection abilities have shifted the bottleneck in the timeline of metabolomic studies to the data analysis step. This paper describes our approach to expedient data analysis of nontargeted high-resolution LC-MS profiles of biological samples. The workflow is illustrated with the example of metabolomics study of time-dependent fasting in male rats. The results from measurement of 220 endogenous metabolites in urine samples illustrate significant biochemical changes induced by fasting. The developed software enables the reporting of relative quantities of annotated components while maintaining practical turnaround times. Each component annotation in the report is validated using both calculated isotopic peaks patterns and experimentally determined retention time data on standards.

  9. Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS.

    Science.gov (United States)

    Lopez, Mayda I; Pettis, Jeffery S; Smith, I Barton; Chu, Pak-Sin

    2008-03-12

    A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.

  10. Pharmacokinetic studies of bergapten in dog plasma by using a LC-MS/MS method studies.

    Science.gov (United States)

    Gao, Y; Liu, Y Z; Zhang, X-M; Zhou, Y; Zhang, X; Dong, C-Y

    2013-07-01

    A sensitive LC-MS/MS method was developed for the determination of bergapten in dog plasma. The chromatographic separation was carried out on a Hypersil ODS column with a mobile phase consisting of methanol-water. The plasma sample was precipitated with methanol and prepare for injecting onto the LC-MS/MS system. The detection was performed on a triple quadrupole tandem mass spectrometer by MRM via electro spray ionization source. The standard curve for bergapten was linear over the concentration range of 0.5-500 ng/mL with a lower limit of quantification of 0.5 ng/mL. The inter-day and intra-day precision (R.S.D.%) for bergapten varied between 3.4 and 11.5. The corresponding inter-day and intra-day accuracy (Bias%) ranged between -3.8 and 6.9. For the pharmacokinetic analysis of serum, the mean (SD) values obtained for the bergapten were as follows: Cmax, 228.5 (14.3) ng/ml; Tmax, 4.2 (0.4) h; t1/2, 6.9 (2.3) h; AUC0-t h, 2507.2 (168.5) ng · h/mL and AUC0-∞, 3 219.2 (211.4) ng · h/mL, respectively. © Georg Thieme Verlag KG Stuttgart · New York.

  11. Larix decidua Bark as a Source of Phytoconstituents: An LC-MS Study

    Directory of Open Access Journals (Sweden)

    Valeria Baldan

    2017-11-01

    Full Text Available Larix decidua bark is a waste of the timber industry and is widely diffused in Northern Italy. This material can be considered a good source of antioxidants and phytoconstituents with possible use in cosmetic or nutraceutical products. In this study, simple extraction of larch bark was performed using mixtures of ethanol/water. Furthermore, the phytochemical composition of larch bark extract was studied using LC-MSn methods and the main constituents were identified as flavonoids, spiro-polyphenols, and procyanidins. To confirm the identification by LC-MS semi-preparative HPLC was performed in order to isolate the main constituents and verify the structures by 1H-NMR. Antioxidant properties were studied using an in vitro approach combining DPPH assay and LC-MS in order to establish different roles of the various classes of phytochemicasl of the extract. DPPH activity of some of the isolated compounds was also assessed. The overall results indicate this waste material as a good source of antioxidant compounds, mainly procyanidins, whichresulted the most active constituents in the DPPH assay.

  12. Effect of Genotype and Environment on Salvia miltiorrhiza Roots Using LC/MS-Based Metabolomics

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    2016-03-01

    Full Text Available Salvia miltiorrhiza (S. miltiorrhiza Bunge is broadly used as herbal medicine for the clinical treatments of cardiovascular and cerebrovascular diseases. Despite its commercial and medicinal values, few systematic studies on the metabolome of S. miltiorrhiza roots have been carried out so far. We systematically described the metabolic profiles of S. miltiorrhiza using high pressure liquid chromatography mass spectrometry (LC/MS in conjunction with multivariate statistical analyses, aimed at monitoring their biological variations of secondary metabolites related to three locations and four S. miltiorrhiza genotypes. A total of 40 bioactive constituents were putatively annotated in S. miltiorrhiza root samples. This study found that both the same S. miltiorrhiza genotype growing at three different locations and four S. miltiorrhiza genotypes growing at the same location had significant metabonomic differences identified by the principal component analysis (PCA approach. By using orthogonal projection to latent structure with discriminant analysis (OPLS-DA, 16 and 14 secondary metabolites can be used as potential location-specific and genotype-specific markers in S. miltiorrhiza, respectively. The specificity of LC/MS profiles offered a powerful tool to discriminate S. miltiorrhiza samples according to genotypes or locations.

  13. Determination of chlormequat in pig serum and sow milk by LC-MS/MS.

    Science.gov (United States)

    Poulsen, M E; Christensen, H B; Sørensen, M T; Leffers, H; Andersen, J H

    2007-11-01

    Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC-MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80-110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g-4.0 ng/g.

  14. Probabilistic Model for Untargeted Peak Detection in LC-MS Using Bayesian Statistics.

    Science.gov (United States)

    Woldegebriel, Michael; Vivó-Truyols, Gabriel

    2015-07-21

    We introduce a novel Bayesian probabilistic peak detection algorithm for liquid chromatography-mass spectroscopy (LC-MS). The final probabilistic result allows the user to make a final decision about which points in a chromatogram are affected by a chromatographic peak and which ones are only affected by noise. The use of probabilities contrasts with the traditional method in which a binary answer is given, relying on a threshold. By contrast, with the Bayesian peak detection presented here, the values of probability can be further propagated into other preprocessing steps, which will increase (or decrease) the importance of chromatographic regions into the final results. The present work is based on the use of the statistical overlap theory of component overlap from Davis and Giddings (Davis, J. M.; Giddings, J. Anal. Chem. 1983, 55, 418-424) as prior probability in the Bayesian formulation. The algorithm was tested on LC-MS Orbitrap data and was able to successfully distinguish chemical noise from actual peaks without any data preprocessing.

  15. Determination of Bupivacaine in Rat Myocardium by LC-MS/MS%大鼠心肌组织中布比卡因的LC-MS/MS测定

    Institute of Scientific and Technical Information of China (English)

    王贤亲; 刘乐; 陈莺; 陈丽梅; 林丹

    2009-01-01

    A LC-MS/MS method was established for the determination of bupivacaine in rat myocardium. A C18colunm was used with the mobile phase of methanol-0.1% formic acid solution (55 : 45) and lidocaine as the internal standard. Electrospray ionization (ESI) source was operated in positive ion mode and multiple-reaction monitoring (MRM) mode. The detected ions were m/z 289→140 for bupivacaine and m/z 285→86 for the internal standard. The calibration curve for bupivacaine was linear in the range of 0.01-20.0μg/g in myocardium. The recovery was 66.45%-74.47 % with the intra- and inter-day RSDs less than 7 %.%建立了液相色谱-串联质谱法测定大鼠心肌组织中的布比卡因.采用C18色谱柱,甲醇-0.1%甲酸溶液(55:45)为流动相,利多卡因为内标.采用正离子电喷雾离子源,多离子反应监测(MRM)方式,监测质核比m/z 289 →140(布比卡因)和m/z 285→86(内标).布比卡因在0.01~20μg/g浓度范围内线性关系良好,萃取回收率为66.4%~74.57%,日内和日间RSD均小于7%.

  16. Current advances and strategies towards fully automated sample preparation for regulated LC-MS/MS bioanalysis.

    Science.gov (United States)

    Zheng, Naiyu; Jiang, Hao; Zeng, Jianing

    2014-09-01

    Robotic liquid handlers (RLHs) have been widely used in automated sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. Automated sample preparation for regulated bioanalysis offers significantly higher assay efficiency, better data quality and potential bioanalytical cost-savings. For RLHs that are used for regulated bioanalysis, there are additional requirements, including 21 CFR Part 11 compliance, software validation, system qualification, calibration verification and proper maintenance. This article reviews recent advances in automated sample preparation for regulated bioanalysis in the last 5 years. Specifically, it covers the following aspects: regulated bioanalysis requirements, recent advances in automation hardware and software development, sample extraction workflow simplification, strategies towards fully automated sample extraction, and best practices in automated sample preparation for regulated bioanalysis.

  17. Rugged Large Volume Injection for Sensitive Capillary LC-MS Environmental Monitoring

    Directory of Open Access Journals (Sweden)

    Hanne Roberg-Larsen

    2017-08-01

    Full Text Available A rugged and high throughput capillary column (cLC LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL solid phase extraction (SPE for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 μm. In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD in the 0.05–12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

  18. Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

    Science.gov (United States)

    Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane

    2009-06-01

    The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

  19. Simultaneous quantitative analysis of nine vitamin D compounds in human blood using LC-MS/MS.

    Science.gov (United States)

    Abu Kassim, Nur Sofiah; Gomes, Fabio P; Shaw, Paul Nicholas; Hewavitharana, Amitha K

    2016-01-01

    It has been suggested that each member of the family of vitamin D compounds may have different function(s). Therefore, selective quantification of each compound is important in clinical research. Development and validation attempts of a simultaneous determination method of 12 vitamin D compounds in human blood using precolumn derivatization followed by LC-MS/MS is described. Internal standard calibration with 12 stable isotope labeled analogs was used to correct for matrix effects in MS detector. Nine vitamin D compounds were quantifiable in blood samples with detection limits within femtomole levels. Serum (compared with plasma) was found to be a more suitable sample type, and protein precipitation (compared with saponification) a more effective extraction method for vitamin D assay.

  20. Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS.

    Science.gov (United States)

    Joshi, Payal; Bhoir, Suvarna; Bhagwat, A M; Vishwanath, K; Jadhav, R K

    2011-05-01

    Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.

  1. LC-MS analysis of the plasma metabolome–a novel sample preparation strategy

    DEFF Research Database (Denmark)

    Skov, Kasper; Hadrup, Niels; Smedsgaard, Jørn

    2015-01-01

    Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC-MS based analysis of the plasma metabolome is a challenge...... as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis...... of plasma samples: The first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples; a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples...

  2. Short Communication: Testosterone Measured with an Automatic Immunoassay Compares Reasonbly Well to Results Obtained by LC-MS/MS

    DEFF Research Database (Denmark)

    Knudsen, Cindy Søndersø; Højskov, Carsten Schriver; Møller, Holger Jon

    2016-01-01

    Background: Previous studies have reported problems measuring testosterone with immunological assays. Here we explore an automatic second generation immunoassay compared to a LC-MS/MS method. Methods: We collected blood samples from 76 women and measured testosterone, progesterone, gender...... hormonebinding globulin (SHBG), and albumin employing Cobas e601/c501. Testosterone, androstenedione (andro), dehydroepiandrosterone sulphate (DHEAS), and 17-hydroxyprogesterone (17-OHP) concentrations were measured employing LC-MS/MS. We evaluated the difference between testosterone measured by the two methods...... and examined the potential interference from the selected steroids and bindings proteins. Results: Testosterone concentrations measured by the two methods yielded: Cobas e601 = 1.240 x (LC-MS/MS) - 0.197, r = 0.84, for testosterone concentrations between 0.22 - 4.9 nmol/L. A positive correlation was observed...

  3. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    International Nuclear Information System (INIS)

    Liu, Xinyue; St Ange, Kalib; Wang, Xiaohua; Lin, Lei; Zhang, Fuming

    2017-01-01

    on LC-MS and NMR analysis. • Monosaccharide compositional analysis relied on top-down NMR analysis. • Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS. • Differences due to parent heparin were observed using principal component analysis.

  4. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xinyue, E-mail: liux22@rpi.edu [National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, 250100 (China); Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); St Ange, Kalib, E-mail: stangk2@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Wang, Xiaohua, E-mail: wangx35@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); School of Computer and Information, Hefei University of Technology, Hefei (China); Lin, Lei, E-mail: Linl5@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Zhang, Fuming, E-mail: zhangf2@rpi.edu [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); and others

    2017-04-08

    approach relied on LC-MS and NMR analysis. • Monosaccharide compositional analysis relied on top-down NMR analysis. • Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS. • Differences due to parent heparin were observed using principal component analysis.

  5. Liquid-chromatography mass spectrometry (LC-MS) of steroid hormone metabolites and its applications

    Science.gov (United States)

    Penning, Trevor M.; Lee, Seon-Hwa; Jin, Yi; Gutierrez, Alejandro; Blair, Ian A.

    2010-01-01

    Advances in liquid chromatography-mass spectrometry (LC-MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC-Electrospray Ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5α–dihydrotestosterone(DHT)-17β-glucuronide, DHT-17β-sulfate, and tibolone-17β-sulfate reduction catalyzed by human members of the aldo-keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable-isotope dilution LC-electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740 attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16α-hydroxy-E1 from 5 pg/mL to 2,000 pg/mL. Our LC-MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment. PMID:20083198

  6. Determination of Dextromethorphan in Oral Fluid by LC-MS-MS.

    Science.gov (United States)

    Amaratunga, Piyadarsha; Clothier, Morgan; Lorenz Lemberg, Bridget; Lemberg, Dave

    2016-06-01

    Dextromethorphan (DXM) is an antitussive drug found in commonly used nonprescription cold and cough medications. At low doses, DXM is a safe drug that does not produce adverse reactions. However, abuse of DXM has been reported among adolescents and young adults using the drug at higher doses. DXM is not a scheduled drug in the USA, and the primary reason for its abuse is the ease of availability. DXM is available to purchase in the form of over-the-counter cough medications, such as Robitussin(®) and Coricidin(®), or it can be purchased over the Internet in the form of a powder. In this research work, we developed an LC-MS-MS method that can quantify DXM and dextrorphan (DXO) in oral fluid in a high-throughput toxicology laboratory setting. The developed method was validated according to the Scientific Working Group for Forensic Toxicology guidelines. The linear dynamic range was 5-100 ng/mL with a lowest limit of quantitation (LLOQ) of 5.0 ng/mL for DXM and DXO. Overall, the results of the accuracy and the precision values were within the acceptance criteria for both drugs. In addition, selectivity, matrix effect and recovery were calculated for the LC-MS-MS method. Authentic samples (n = 59) were tested to evaluate the applicability of the method. Thirty samples were found to be positive for DXM and DXO and two samples were found to be positive for DXM only. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Software and Database Usage on Metabolomic Studies: Using XCMS on LC-MS Data Analysis

    Directory of Open Access Journals (Sweden)

    Mustafa Celebier

    2014-04-01

    Full Text Available Metabolome is the complete set of small-molecule metabolites to be found in a cell or a single organism. Metabolomics is the scientific study to determine and identify the chemicals in metabolome with advanced analytical techniques. Nowadays, the elucidation of the molecular mechanism of any disease with genome analysis and proteome analysis is not sufficient. Instead of these, a holistic assessment including metabolomic studies provides rational and accurate results. Metabolite levels in an organism are associated with the cellular functions. Thus, determination of the metabolite amounts identifies the phenotype of a cell or tissue related with the genetic and some other variations. Even though, the analysis of metabolites for medical diagnosis and therapy have been performed for a long time, the studies to improve the analysis methods for metabolite profiling are recently increased. The application of metabolomics includes the identification of biomarkers, enzyme-substract interactions, drug-activity studies, metabolic pathway analysis and some other studies related with the system biology. The preprocessing and computing of the data obtained from LC-MS, GC-MS, CE-MS and NMR for metabolite profiling are helpful for preventing from time consuming manual data analysis processes and possible random errors on profiling period. In addition, such preprocesses allow us to identify low amount of metabolites which are not possible to be analyzed by manual processing. Therefore, the usage of software and databases for this purpose could not be ignored. In this study, it is briefly presented the software and database used on metabolomics and it is evaluated the capability of these software on metabolite profiling. Particularly, the performance of one of the most popular software called XCMS on the evaluation of LC-MS results for metabolomics was overviewed. In the near future, metabolomics with software and database support is estimated to be a routine

  8. Information-dependent acquisition-mediated LC-MS/MS screening procedure with semiquantitative potential.

    Science.gov (United States)

    Decaestecker, Tineke N; Vande Casteele, Sofie R; Wallemacq, Pierre E; Van Peteghem, Carlos H; Defore, Dieter L; Van Bocxlaer, Jan F

    2004-11-01

    The development of a LC-MS/MS general unknown screening procedure for toxicologically relevant substances in blood samples by means of information-dependent acquisition on a Q-TOF is reported. IDA is an artificial intelligence-based product ion scan mode providing automatic "on-the-fly" MS to MS/MS switching. By performing information-dependent scanning at two different fragmentation energies, two collision-induced dissociation product ion spectra for each of the detected compounds are generated. As such, information-rich MS/MS spectra are obtained from precursor ions not known beforehand. In addition, limitation of the MS/MS acquisition time to an acceptable minimum resulted in an almost instantaneous switch back to the MS mode. As such, this approach provided MS chromatograms that still could be of use for semiquantitative purposes. Since the switching intensity threshold, unequivocally related to the background noise, proved a critical parameter, the solid-phase extraction procedure, the liquid chromatographic conditions, and the mass spectrometric parameters all were optimized to the advantage of information-dependent acquisition. Finally, the screening procedure we developed was benchmarked, on one hand, qualitatively against the results obtained from traditional GUS approaches in a number of routine toxicological laboratories (20 samples) and, on the other hand, quantitatively with respect to its potential against established LC-MS/MS methods (7 samples). The procedure performed very well from a qualitative point of view; almost all of the drugs detected by the conventional techniques were identified, as well as additional drugs that were not previously reported. The procedure proved well-suited for an initial semiquantitative assessment, as is customary in, for example, forensic toxicology before accurate intoxication levels are determined using targeted analytical analyses.

  9. 3D molecular cartography using LC-MS facilitated by Optimus and 'ili software.

    Science.gov (United States)

    Protsyuk, Ivan; Melnik, Alexey V; Nothias, Louis-Felix; Rappez, Luca; Phapale, Prasad; Aksenov, Alexander A; Bouslimani, Amina; Ryazanov, Sergey; Dorrestein, Pieter C; Alexandrov, Theodore

    2018-01-01

    Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body. Here, we provide a protocol and open-source software for 3D molecular cartography. The protocol includes step-by-step procedures for sample collection and processing, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, quality control (QC), molecular identification using MS/MS, data processing, and visualization with 3D models of the sampled environment. The LC-MS method was optimized for a broad range of small organic molecules. We enable scientists to reproduce our previously obtained results, and illustrate the broad utility of our approach with molecular maps of a rosemary plant and an ATM keypad after a PIN code was entered. To promote reproducibility, we introduce cartographical snapshots: files that describe a particular map and visualization settings, and that can be shared and loaded to reproduce the visualization. The protocol enables molecular cartography to be performed in any mass spectrometry laboratory and, in principle, for any spatially mapped data. We anticipate applications, in particular, in medicine, ecology, agriculture, biotechnology, and forensics. The protocol takes 78 h for a molecular map of 100 spots, excluding the reagent setup.

  10. Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.

    Science.gov (United States)

    Blue, Steven W; Winchell, Andrea J; Kaucher, Amy V; Lieberman, Rachel A; Gilles, Christopher T; Pyra, Maria N; Heffron, Renee; Hou, Xuanlin; Coombs, Robert W; Nanda, Kavita; Davis, Nicole L; Kourtis, Athena P; Herbeck, Joshua T; Baeten, Jared M; Lingappa, Jairam R; Erikson, David W

    2018-04-01

    The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. LC-MS metabolomic analysis of environmental stressor impacts on the metabolite diversity in Nephthea spp.

    Directory of Open Access Journals (Sweden)

    Hedi Indra Januar

    2012-01-01

    Full Text Available Context: The soft coral Nephthea spp. is a source of terpenoid class that potentially has pharmaceutical properties. However, metabolite diversity and cytotoxic activity of this species are varied among coral reefs from various sites. Aim: To analyze the water quality in Nephthea spp. environment as a possible factor causing a difference in its metabolite diversity. Settings and Design: Nephthea spp. from seven sites were taken in October 2010 at the Alor District of Marine Protected Area, Indonesia. Materials and Methods: Water quality assessment was analyzed in situ and indexed by Canadian Council of Ministry Environment-Water Quality Index (CCME-WQI method. Meanwhile, metabolite diversity was analyzed by a LC-MS metabolomic method, using C18 reversed phase and gradient water-acetonitrile system. Statistical Analysis Used: Spearman′s rho and regression analysis were applied to correlate the water quality index to ecological index (richness, diversity, and evenness from LC-MS results. Results: The water quality index had a significant positive correlation and strong linear regression determinant to the total metabolite (R 2 = 0.704, particularly to semipolar metabolite richness (R 2 = 0.809, the area of terpenoid class in the organism. Conclusion: It can be concluded that water quality may serve as a major factor that affects the amount of richness in Nephthea spp. metabolites. When the water quality is lower, as environment stresses increases, it may affect the metabolite richness within direct disrupt of metabolite biosynthesis or indirect ecological means. Terpenoids are known as a soft coral antipredator (coral fishes, the amount of which depends on the water quality.

  12. BatMass: a Java Software Platform for LC-MS Data Visualization in Proteomics and Metabolomics.

    Science.gov (United States)

    Avtonomov, Dmitry M; Raskind, Alexander; Nesvizhskii, Alexey I

    2016-08-05

    Mass spectrometry (MS) coupled to liquid chromatography (LC) is a commonly used technique in metabolomic and proteomic research. As the size and complexity of LC-MS-based experiments grow, it becomes increasingly more difficult to perform quality control of both raw data and processing results. In a practical setting, quality control steps for raw LC-MS data are often overlooked, and assessment of an experiment's success is based on some derived metrics such as "the number of identified compounds". The human brain interprets visual data much better than plain text, hence the saying "a picture is worth a thousand words". Here, we present the BatMass software package, which allows for performing quick quality control of raw LC-MS data through its fast visualization capabilities. It also serves as a testbed for developers of LC-MS data processing algorithms by providing a data access library for open mass spectrometry file formats and a means of visually mapping processing results back to the original data. We illustrate the utility of BatMass with several use cases of quality control and data exploration.

  13. LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

    Science.gov (United States)

    Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi

    2014-06-01

    Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

  14. Selectivity in the sample preparation for the analysis of drug residues in products of animal origin using LC-MS

    NARCIS (Netherlands)

    Berendsen, B.J.A.; Stolker, A.A.M.; Nielen, M.W.F.

    2013-01-01

    Sample preparation is critical in relation to analysis time, sample throughput and therefore analysis costs. Due to recent advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation, the detection of many compounds within one run became possible, and methods for the simultaneous

  15. Determination of Glyphosate Levels in Breast Milk Samples from Germany by LC-MS/MS and GC-MS/MS

    NARCIS (Netherlands)

    Steinborn, Angelika; Alder, Lutz; Michalski, Britta; Zomer, Paul; Bendig, Paul; Martinez, Sandra Aleson; Mol, Hans G.J.; Class, Thomas J.; Costa Pinheiro, Nathalie

    2016-01-01

    This study describes the validation and application of two independent analytical methods for the determination of glyphosate in breast milk. They are based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), respectively. For

  16. Comparison of 7 Published LC-MS/MS Methods for the Simultaneous Measurement of Testosterone, Androstenedione, and Dehydroepiandrosterone in Serum

    DEFF Research Database (Denmark)

    Büttler, Rahel M; Martens, Frans; Fanelli, Flaminia

    2015-01-01

    , and dehydroepiandrosterone (DHEA). METHODS: We used 7 published LC-MS/MS methods to analyze in duplicate 55 random samples from both men and women. We performed Passing-Bablok regression analysis and calculated Pearson correlation coefficients to assess the agreement of the methods investigated with the median concentration...

  17. Diet-induced perturbation of the rat liver mitochondrial acetylome studied by quantitative (iTRAQ) LC-MS/MS

    DEFF Research Database (Denmark)

    León, Ileana R.; Schwämmle, Veit; Williamson, James

    described3. Quantitation by MS/MS was achieved by using iTRAQ 8-Plex reagents (Life Technologies). LC-MS/MS analyses were performed using an Easy-nLC (Proxeon/ThermoFisher) fitted with an in-house made 17 cm C18 column that was interfaced to an LTQ-OrbiTrap XL instrument (ThermoFisher). Data analysis...

  18. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

    Science.gov (United States)

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

    2018-02-07

    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  19. Study of the performance of three LC-MS/MS platforms for analysis of perfluorinated compounds

    Energy Technology Data Exchange (ETDEWEB)

    Llorca, Marta; Farre, Marinella [IDAEA-CSIC, Department of Environmental Chemistry, Barcelona (Spain); Pico, Yolanda [Universitat de Valencia, Laboratori de Nutricio i Bromatologia, Facultat de Farmacia, Burjassot, Valencia (Spain); Barcelo, Damia [IDAEA-CSIC, Department of Environmental Chemistry, Barcelona (Spain); King Saud University, Riyadh (Saudi Arabia)

    2010-10-15

    The analytical suitabilities of three different liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems, (1) triple quadrupole (QqQ), (2) conventional 3D ion trap (IT), and (3) quadrupole-linear IT (QqLIT), to determine trace levels of perfluorinated compounds (PFCs) in fish and shellfish were compared. Sample preparation was performed using alkaline extraction and solid-phase-extraction cleanup. This evaluation was focused on both quantitative (sensitivity, precision, and accuracy) and qualitative (identification capabilities) aspects. In the three instruments, the former facet was evaluated using selected reaction monitoring (SRM), which is the standard mode for quantitative LC-MS/MS analysis. Accuracy was similar in the three systems, with recoveries always over 70 %. Precision was better for the QqLIT and QqQ systems (7-15%) than for the IT system (10-17%). The QqLIT (working in SRM mode) and QqQ systems offered a linear dynamic range of at least 3 orders of magnitude, whereas that of the IT system was 2 orders of magnitude. The QqLIT system achieved at least 20-fold higher sensitivity than the QqQ system, and this was at least tenfold higher sensitivity than for the IT system. In the IT system, identification was based on sensitive full mass range acquisition and MS{sup n} fragmentation and in the QqLIT system, it was based on the use of an information-dependent-acquisition scan function, which allows the combination of an SRM or MS full scan acting as the survey scan and an enhanced product ion scan followed by MS{sup 3} as the dependent scan in the same analysis. Three instruments were applied to monitor the content in fish and shellfish (anchovies, swordfish, tuna, mussels, and oysters) obtained from Valencia and Barcelona markets (Spain). The eight target PFCs were detected at mean concentrations in the range from 10 ng kg {sup -1} (perfluoro-7-methyloctanoic acid and perfluoro-1-decanesulfonate) to 4,200 ng kg {sup -1} (perfluoropentanoic acid

  20. Solvent optimization on Taxol extraction from Taxus baccata L., using HPLC and LC-MS

    Directory of Open Access Journals (Sweden)

    H Sadeghi-aliabadi

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Taxol, a natural antitumor agent, was first isolated from the extract of the bark of Taxus brevifolia Nutt., which is potentially a limited source for Taxol. In the search of an alternative source, optimum and cost benefit extracting solvents, various solvents with different percentage were utilized to extract Taxol from needles of Taxus baccata. "nMethods: One g of the dried needles of Taxus baccata, collected from Torkaman and Noor cities of Iran, was extracted with pure ethanol or acetone and 50% and 20% of ethanol or acetone in water. Solvents were evaporated to dryness and the residues were dissolved in 5 ml of methanol and filtered. To one ml of the filtrate was added 50 μl of cinamyl acetate as the internal standard and 20 μl of the resulting solution was subjected to the HPLC to determine the extraction efficiencies of tested solvents. Five μl of filtrate was also subjected to the LC-MS using water/acetonitrile (10/90 as mobile phase and applying positive electrospray ionization (ESI to identify the authenticity of Taxol. "nResults: Results of this study indicated that Taxol extraction efficiency was enhanced as the percentage of ethanol or acetone was increased. HPLC analysis showed that Taxol could be quantified by UV detection using standard curve. The standard curve covering the concentration ranges of 7.8 - 500 μg/ml was linear (r2= 0.9992 and CV% ranged from 0.52 to 15.36. LC-MS analysis using ESI in positive-ion mode confirmed the authenticity of Taxol (m/z 854; M+H, as well as some adduct ions such as M+Na (m/z 876, M+K (m/z 892 and M+CH3CN+H2O (m/z 913. "nConclusions: The results suggest that 100% acetone is the best solvent for the extraction of Taxol from Taxus baccata needles.

  1. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    Science.gov (United States)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  2. A Greener, Quick and Comprehensive Extraction Approach for LC-MS of Multiple Mycotoxins

    Directory of Open Access Journals (Sweden)

    Andreas Breidbach

    2017-03-01

    Full Text Available In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, forgoing sample extract clean-up, and minimizing solvent consumption. For this, 2 g of test material were suspended in 8 mL water and 16 mL ethyl acetate were added. Extraction was accelerated through sonication for 30 min and subsequent addition of 8 g sodium sulfate. After centrifugation, 500 µL supernatant were spiked with isotopologues, dried down, reconstituted in mobile phase, and measured with LC-MS. The method was validated in-house and through a collaborative study and the performance was fit-for-purpose. Repeatability relative standard deviation (RSDs between 16% at low and 4% at higher contaminations were obtained. The reproducibility RSDs were mostly between 12% and 32%. The trueness of results for T-2 toxin and Zearalenone were not different from 100%, for Deoxynivalenol and HT-2 toxin they were larger than 89%. The extraction was also adapted to a quick screening of Aflatoxin B1 in maize by flow-injection–mass spectrometry. Semi-quantitative results were obtained through standard addition and scan-based ion ratio calculations. The method proved to be a viable greener and quicker alternative to existing methods.

  3. Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

    Science.gov (United States)

    Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

    2009-11-01

    The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

  4. Multi-mycotoxin stable isotope dilution LC-MS/MS method for Fusarium toxins in beer.

    Science.gov (United States)

    Habler, Katharina; Gotthardt, Marina; Schüler, Jan; Rychlik, Michael

    2017-03-01

    A stable isotope dilution LC-MS/MS multi-mycotoxin method was developed for 12 different Fusarium toxins including modified mycotoxins in beer (deoxynivalenol-3-glucoside, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyl-deoxynivalenol, HT2-toxin, T2-toxin, enniatin B, B1, A1, A, beauvericin and zearalenone). As sample preparation and purification of beer a combined solid phase extraction for trichothecenes, enniatins, beauvericin and zearalenone was firstly developed. The validation of the new method gave satisfying results: intra-day and inter-day precision and recoveries were 1-5%, 2-8% and 72-117%, respectively. In total, 61 different organic and conventional beer samples from Germany and all over the world were analyzed by using the newly developed multi-mycotoxin method. In summary, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivaleneol and enniatin B were quantified in rather low contents in the investigated beer samples. None of the other monitored Fusarium toxins like 15-acetyldeoxynivalenol, HT2- and T2-toxin, zearalenone, enniatin B1, A1, A or beauvericin were detectable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

    Directory of Open Access Journals (Sweden)

    Rader Robert

    2007-06-01

    Full Text Available Abstract Background The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches. Results We have developed the MAss SPECTRometry Analysis System (MASPECTRAS, a platform for management and analysis of proteomics LC-MS/MS data. MASPECTRAS is based on the Proteome Experimental Data Repository (PEDRo relational database schema and follows the guidelines of the Proteomics Standards Initiative (PSI. Analysis modules include: 1 import and parsing of the results from the search engines SEQUEST, Mascot, Spectrum Mill, X! Tandem, and OMSSA; 2 peptide validation, 3 clustering of proteins based on Markov Clustering and multiple alignments; and 4 quantification using the Automated Statistical Analysis of Protein Abundance Ratios algorithm (ASAPRatio. The system provides customizable data retrieval and visualization tools, as well as export to PRoteomics IDEntifications public repository (PRIDE. MASPECTRAS is freely available at http://genome.tugraz.at/maspectras Conclusion Given the unique features and the flexibility due to the use of standard software technology, our platform represents significant advance and could be of great interest to the proteomics community.

  6. Identification of hormone esters in injection site in muscle tissues by LC/MS/MS.

    Science.gov (United States)

    Costain, R M; Fesser, A C E; McKenzie, D; Mizuno, M; MacNeil, J D

    2008-12-01

    The detection of hormone abuse for growth promotion in food animal production is a global concern. Initial testing for hormones in Canada was directed at the compounds approved for use in beef cattle, melengestrol acetate, trenbolone acetate and zeranol, and the banned compound diethylstilbestrol (DES). No hormonal growth promoters are approved for use in veal production in Canada. However, instances of use of trenbolone and clenbuterol were detected in Canada in the 1990s. During the development of a new analytical method for testosterone and progesterone, there were reports of suspicious injection sites being found in veal calves. Upon implementation of the method, analysis of investigative samples revealed significant residues of testosterone in some injection sites. To prove that the source of these residues was exogenous, a fully validated method for hormone esters was developed to confirm the presence of exogenous hormones in these injection sites. The QUECHERS model was employed in methods development and resulted in a simple, effective extraction technique that consisted of sample pre-homogenization, liquid/liquid partitioning, extract dilution, filtration and use of LC/MS/MS to provide detection selectivity. The result was an adaptable MS/MS confirmation technique that meets the needs of Canadian regulatory authorities to confirm the misuse of injectable testosterone, and potentially other hormones, in food animal production.

  7. Analysis of cocaine/crack biomarkers in meconium by LC-MS.

    Science.gov (United States)

    D'Avila, Felipe Bianchini; Ferreira, Pâmela C Lukasewicz; Salazar, Fernanda Rodrigues; Pereira, Andrea Garcia; Santos, Maíra Kerpel Dos; Pechansky, Flavio; Limberger, Renata Pereira; Fröehlich, Pedro Eduardo

    2016-02-15

    Fetal exposure to illicit drugs is a worldwide problem, since many addicted women do not stop using it during pregnancy. Cocaine consumed in powdered (snorted or injected) or smoked (crack cocaine) form are harmful for the baby and its side effects are not completely known. Meconium, the first stool of a newborn, is a precious matrix usually discarded, that may contain amounts of substances consumed in the last two trimesters of pregnancy. Analyzing this biological matrix it is possible to detect the unaltered molecule of cocaine (COC) or its metabolite benzoylecgonine (BZE) and pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC). A liquid chromatography mass spectrometry (LC-MS) method was validated for meconium samples after solvent extraction, followed by direct injection of 10μL. Linearity covered a concentration range of 15 to 500ng/mg with a lower limit of quantification (LLOQ) of 15ng/mg for all analytes. Matrix effect was evaluated and showed adequate results. Detection of illicit substances usage can be crucial for the baby, since knowing that can help provide medical care as fast as possible. The method proved to be simple and fast, and was applied to 17 real meconium samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Simultaneous quantification of seven hippocampal neurotransmitters in depression mice by LC-MS/MS.

    Science.gov (United States)

    Huang, Fei; Li, Jia; Shi, Hai-Lian; Wang, Ting-ting; Muhtar, Wahaf; Du, Min; Zhang, Bei-bei; Wu, Hui; Yang, Li; Hu, Zhi-bi; Wu, Xiao-jun

    2014-05-30

    There is no method available to simultaneously detect GABA, Glu, Epi, NE, DA, 5-HT and 5-HIAA in mouse hippocampus. A rapid and sensitive LC-MS/MS method has been developed for simultaneously measuring seven neurotransmitters in mouse hippocampus. The analytes were detected in positive mode with multiple reaction monitoring (MRM) and the procedure was completed in less than 9min. This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99, and showed good intra- and inter-day precisions (RSDneurotransmitters in a mouse depression model induced by successive methylprednisolone injections. The results indicated that this depression model was closely associated with the decreased level of Epi (p=0.002) and elevated ratio of 5-HIAA/5-HT (p=0.01), which has never been reported elsewhere. Compared with previous methods, current approach is more convenient without any pre-column derivatization of the analytes but enhances detectability with incremental neurotransmitter profile and shortens detection time. This work represents the first accurate simultaneous determination of seven neurotransmitters in the mouse depression model induced by methylprednisolone. The reliable method will benefit the research of neurological diseases with the altered neurotransmitter profile in brain. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Antioxidant properties and phenolic profile characterization by LC-MS/MS of selected Tunisian pomegranate peels.

    Science.gov (United States)

    Abid, Mouna; Yaich, Héla; Cheikhrouhou, Salma; Khemakhem, Ibtihel; Bouaziz, Mohamed; Attia, Hamadi; Ayadi, M A

    2017-08-01

    Antioxidant contents and activities of different extracts from four Tunisian pomegranate peels, locally called "Acide", "Gabsi", "Nebli" and "Tounsi", were studied. Peels samples were extracted with three solvents (water, ethanol and acetone). For each extract, the total phenol contents and antioxidant activity were evaluated. The highest values of polyphenol, tannins, flavonoids and anthocyanins were recorded in the acetone extract of Acide ecotype with 304.6 mg gallic acid equivalent/g; 292.23 mg gallic acid equivalent/g; 15.46 mg Quercetin/g and 54.51 mg cy-3-glu/100 g, respectively. The acetone extract of Acide ecotype also showed the highest free radical-scavenging and reducing power activity compared to other extracts. Besides, the phytochemical analysis by LC-MS/MS revealed a high content of ellagitannins with punicalagin and punicalagin derivatives as the major compounds that might be responsible for promising antioxidant activity of pomegranate peel extracts. Two compounds (Castalagin derivative and Galloyl-bis-HHDP-hex derivative) were detected only in "Acide" ecotype in important contents.

  10. Quantitative Determination of Ivermectin in Raw Milk Using Positive ESI LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Meenakshi Dahiya

    2010-01-01

    Full Text Available Ivermectin, a veterinary drug, is commonly used endectocide for animal husbandry. The drug is available in the form of subcutaneous or topical formulations. Its application may cause accumulation of its residues into the animal tissues, which ultimately find their way into the food products, such as milk and meat products. In order to determine the residues of ivermectin in milk, a comparatively simple, sensitive and rapid method was developed and validated using LC-MS/MS. The MRM transitions corresponding to m/z 892.71>569.6, 892.71>551.5 and 892.71>307.3 were used for the purpose of quantification and evaluation of other parameters of the method. The limit of detection of the method was found to be 0.1 μg/kg and the limit of quantitation was calculated as 0.2 μg/kg. The method was found to be linear in the range of 1.0 ng/mL to 100.0 ng/mL with correlation coefficient of 0.9992 for pure calibration curve and 0.9990 for the matrix- matched calibration curve. The recoveries of ivermectin from the spiked samples of raw milk were found between 85 to 105%.

  11. Determination of piroxicam from rat articular tissue and plasma based on LC-MS/MS.

    Science.gov (United States)

    Kim, Han Sol; Cho, Ha Ra; Ho, Myoung Jin; Kang, Myung Joo; Choi, Yong Seok

    2016-12-01

    Osteoarthritis (OA) is the most common type of arthritis. To manage OA, in general, oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) is used. Recently, the analgesic and anti-inflammatory efficacy of piroxicam (PX), a long-acting NSAID, by intra-articular (IA) administration in OA was reported, and the possibility that PX is distributed in articular tissues at a certain concentration was raised. Thus, herein, novel LC-MS/MS methods to detect PX in rat articular tissue and plasma are presented. For articular tissue, solvent extraction with acetonitrile for 12 h was employed and a protein precipitation method was used for the preparation of a plasma sample. The developed methods were validated by following the FDA guidelines, and the validated methods were successfully applied to a PK study of IA PX. The present study presents, to our knowledge, the first method of determining a drug in articular tissue. Additionally, the level of PX in articular tissue after IA PX administration was experimentally confirmed for the first time using the present methods. Therefore, the present methods provide a new direction for in vivo evaluation for IA PX formulations and contribute to the development of alternative IA PX formulations with better effects for the treatment of OA.

  12. A High-Resolution LC-MS-Based Secondary Metabolite Fingerprint Database of Marine Bacteria

    KAUST Repository

    Lu, Liang

    2014-10-09

    © 2014 Macmillan Publishers Limited. All rights reserved. Marine bacteria are the most widely distributed organisms in the ocean environment and produce a wide variety of secondary metabolites. However, traditional screening for bioactive natural compounds is greatly hindered by the lack of a systematic way of cataloguing the chemical profiles of bacterial strains found in nature. Here we present a chemical fingerprint database of marine bacteria based on their secondary metabolite profiles, acquired by high-resolution LC-MS. Till now, 1,430 bacterial strains spanning 168 known species collected from different marine environments were cultured and profiled. Using this database, we demonstrated that secondary metabolite profile similarity is approximately, but not always, correlated with taxonomical similarity. We also validated the ability of this database to find species-specific metabolites, as well as to discover known bioactive compounds from previously unknown sources. An online interface to this database, as well as the accompanying software, is provided freely for the community to use.

  13. Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

    Science.gov (United States)

    Zauber, Henrik; Schulze, Waltraud X

    2012-11-02

    The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

  14. Determination of linsidomine in human plasma by tandem LC-MS with ESI.

    Science.gov (United States)

    Sutherland, F C; de Jager, A D; Swart, K J; Hundt, H K; Scanes, T; Hundt, A F

    2000-04-01

    A sensitive method for the determination of linsidomine in plasma was developed, using high-performance liquid chromatographic (HPLC) separation with tandem mass spectrometric detection. Linsidomine was derivatised with propyl chloroformate and extracted with tert-butyl methyl ether/1,2-dichloroethane (55:45, v/v), back-extracted into HCl (0.01 M) followed by alkalinisation and back-extraction into ether; the final ether extract evaporated, reconstituted in mobile phase and then separated on a Phenomenex Luna C18 (2) 5 micron 2.1 x 150 mm column with a mobile phase consisting of methanol water formic acid (98/100%) (400:600:0.05, v/v/v) at a flow-rate of 0.4 ml min(-1). Detection was achieved by a Finnigan MAT mass spectrometer (LCQ) at unit resolution in the selected reaction monitoring (SRM) mode monitoring the transition of the protonated molecular ion m/z 257.0 to the product ion m/z 86.0. The mean recovery for linsidomine was 51% with a lower limit of quantification of 0.70 ng/ml using 1 ml plasma for extraction. This LC-MS/MS method for the determination of linsidomine in human plasma allows for better specificity and a higher sample throughput than the traditional LC-UV methods. It also demonstrates the profound effect that the composition of acidic modifiers and matrix constituents can have on the electrospray ionisation (ESI) of the analyte.

  15. Studies of alkyl porphyrin distributions in organic-rich sediments using LC-MS

    International Nuclear Information System (INIS)

    Eckardt, C.B.; Carter, J.F.; Keely, B.J.; Maxwell, J.R.; Kilpatrick, G.

    1992-01-01

    In recent years, structure elucidation of a wide variety of sedimentary tetrapyrroles has provided clear molecular evidence for the presence of primary photosynthetic communities in palaeo water columns. The reported structures indicate an origin from algal chlorophylls c for certain components, while an origin from photosynthetic bacteria is apparent from the carbon skeletons of other components. In particular, the structures of ≤C 34 porphyrin carboxylic acids in the Eocene Messel shale indicate an origin from Chloroblum bacteria. Since such bacteria are strict anaerobes, the presence of these species is evidence for anoxic conditions extending into the photic zone of Messel lake. By analogy, the presence in the more widely-occurring alkyl porphyrin distributions of components >C 33 would also suggest a Chlorobium chlorophyll origin. Hence, in this paper, the authors studied by LC-MS, the distributions of alkyl porphyrins in selected sediments and searched for the presence of such components, in order to determine photic zone anoxia in the respective palaeo environments

  16. A High-Resolution LC-MS-Based Secondary Metabolite Fingerprint Database of Marine Bacteria

    KAUST Repository

    Lu, Liang; Wang, Jijie; Xu, Ying; Wang, Kailing; Hu, Yingwei; Tian, Renmao; Yang, Bo; Lai, Qiliang; Li, Yongxin; Zhang, Weipeng; Shao, Zongze; Lam, Henry; Qian, Pei-Yuan

    2014-01-01

    © 2014 Macmillan Publishers Limited. All rights reserved. Marine bacteria are the most widely distributed organisms in the ocean environment and produce a wide variety of secondary metabolites. However, traditional screening for bioactive natural compounds is greatly hindered by the lack of a systematic way of cataloguing the chemical profiles of bacterial strains found in nature. Here we present a chemical fingerprint database of marine bacteria based on their secondary metabolite profiles, acquired by high-resolution LC-MS. Till now, 1,430 bacterial strains spanning 168 known species collected from different marine environments were cultured and profiled. Using this database, we demonstrated that secondary metabolite profile similarity is approximately, but not always, correlated with taxonomical similarity. We also validated the ability of this database to find species-specific metabolites, as well as to discover known bioactive compounds from previously unknown sources. An online interface to this database, as well as the accompanying software, is provided freely for the community to use.

  17. Multimycotoxin LC-MS/MS Analysis in Tea Beverages after Dispersive Liquid-Liquid Microextraction (DLLME).

    Science.gov (United States)

    Pallarés, Noelia; Font, Guillermina; Mañes, Jordi; Ferrer, Emilia

    2017-11-29

    The aim of the present study was to develop a multimycotoxin liquid chromatography tandem mass spectrometry (LC-MS/MS) method with a dispersive liquid-liquid microextraction procedure (DLLME) for the analysis of AFs, 3aDON, 15aDON, NIV, HT-2, T-2, ZEA, OTA, ENNs, and BEA in tea beverages and to evaluate their mycotoxin contents. The proposed method was characterized in terms of linearity, limits of detection (LODs), limits of quantification (LOQs), recoveries, repeatability (intraday precision), reproducibility (interday precision), and matrix effects to check suitability. The results show LODs in the range of 0.05-10 μg/L, LOQs in the range of 0.2-33 μg/L, and recoveries in the range of 65-127% (RSD tea, red tea, green tea, and green mint tea. The results show that, of the analyzed mycotoxins, AFB2, AFG2, 15aDON, AFG1, and ENB were detected in the samples. AFB2 (14.4-32.2 μg/L) and 15aDON (60.5-61 μg/L) presented the highest levels. Green mint tea contained the highest concentration of mycotoxins. The risk assessment study shows that the population is not much exposed to mycotoxins through the consumption of tea beverages.

  18. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

    Science.gov (United States)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

    2013-09-01

    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  19. Analyses of marketplace tacrolimus drug product quality: bioactivity, NMR and LC-MS.

    Science.gov (United States)

    Sommers, Cynthia D; Pang, Eric S; Ghasriani, Houman; Berendt, Robert T; Vilker, Vincent L; Keire, David A; Boyne, Michael T

    2013-11-01

    Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace. A combination of analytical methods, including mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and bioactivity assay were developed and validated to assess the quality of tacrolimus. These tests measured the identity, impurities and activity of tacrolimus from active pharmaceutical ingredient (API) sources and with formulated drug product from five different approved manufactures. In addition, some testing was performed on tacrolimus capsules obtained from a non US approved Indian source. The data obtained showed no discernible difference in the impurity profiles and potency between the generic and innovator tacrolimus products. Copyright © 2013. Published by Elsevier B.V.

  20. The effect of antibiotics and diet on enterolactone concentration and metabolome studied by targeted and non-targeted LC-MS metabolomics

    DEFF Research Database (Denmark)

    Bolvig, Anne Katrine; Nørskov, Natalja; Hedemann, Mette Skou

    2017-01-01

    with lower levels of ENL. Here, we investigate the link between antibiotic use and lignan metabolism in pigs using LC-MS/MS. The effect of lignan intake and antibiotic use on the gut microbial community and the pig metabolome is studied by 16S rRNA sequencing and non-targeted LC-MS. Treatment...

  1. What Is in Your Wallet? Quantitation of Drugs of Abuse on Paper Currency with a Rapid LC-MS/MS Method

    Science.gov (United States)

    Parker, Patrick D.; Beers, Brandon; Vergne, Matthew J.

    2017-01-01

    Laboratory experiments were developed to introduce students to the quantitation of drugs of abuse by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Undergraduate students were introduced to internal standard quantitation and the LC-MS/MS method optimization for cocaine. Cocaine extracted from paper currency was…

  2. Multiple testing issues in discriminating compound-related peaks and chromatograms from high frequency noise, spikes and solvent-based noise in LC-MS data sets

    NARCIS (Netherlands)

    Nyangoma, Stephen O.; van Kampen, Antoine A. H. C.; Reijmers, Theo H.; Govorukhina, Natalia I.; van der Zee, Ate G. J.; Billingham, Lucinda J.; Bischoff, Rainer; Jansen, Ritsert C.

    2007-01-01

    Liquid Chromatography - Mass Spectrometry (LC-MS) is a powerful method for sensitive detection and quantification of proteins and peptides in complex biological fluids like serum. LC-MS produces complex data sets, consisting of some hundreds of millions of data points per sample at a resolution of

  3. METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

    Science.gov (United States)

    Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensi...

  4. Development of high-performance chemical isotope labeling LC-MS for profiling the human fecal metabolome.

    Science.gov (United States)

    Xu, Wei; Chen, Deying; Wang, Nan; Zhang, Ting; Zhou, Ruokun; Huan, Tao; Lu, Yingfeng; Su, Xiaoling; Xie, Qing; Li, Liang; Li, Lanjuan

    2015-01-20

    Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively

  5. Screening and Identification of Mitragynine and 7-Hydroxymitragynine in Human Urine by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hanzhuo Fu

    2015-05-01

    Full Text Available Kratom is a tree planted in Southeast Asia, including Thailand, Malaysia, Myanmar (Burma and elsewhere in the region. A long history of usage and abuse of kratom has led to the classification of kratom as a controlled substance in its native Thailand and other Southeast Asian countries. However, kratom is not controlled in the United States, and the wide availability of kratom on the Internet and in the streets has led to its emergence as an herbal drug of misuse. With the increasing popularity of kratom, efficient protocols are needed to detect kratom use. In this study, a rapid method for the analysis of kratom compounds, mitragynine and 7-hydroxymitragynine, in human urine has been developed and validated using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS. The chromatographic system employed a 2.6-μm 100 mm × 2.1 mm phenyl-hexyl analytical column and gradient elution with a 0.4-mL/min flow rate of water and acetonitrile as mobile phases. A triple quadrupole mass spectrometer was used as the detector for data acquisition. The analyst was the quantification software. The established method demonstrated linearity of >0.99 for both analytes, and low detection limits were obtained down to 0.002581 ng/mL for mitragynine and 0.06910 ng/mL for 7-hydroxymitragynine. The validated method has been utilized for clinical analysis of urine for the purpose of mitragynine and 7-hydroxymitragynine detection.

  6. Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis.

    Science.gov (United States)

    Koster, Remco A; Greijdanus, Ben; Alffenaar, Jan-Willem C; Touw, Daan J

    2015-02-01

    In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[(2)H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[(2)H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0% and a maximum bias of -5.9%. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at -20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement.

  7. Degradation of pesticides with RSDL® (reactive skin decontamination lotion kit) lotion: LC-MS investigation.

    Science.gov (United States)

    Fentabil, Messele; Gebremedhin, Mulu; Purdon, J Garfield; Cochrane, Laura; Goldman, Virginia Streusand

    2018-09-01

    This study examined the degradation of organophosphate (OP) and carbamate pesticides using RSDL ® (Reactive Skin Decontamination Lotion Kit) lotion. Degradation occurs from a nucleophilic substitution (SN) reaction between an ingredient in the RSDL lotion, potassium 2,3-butanedione monoximate (KBDO), with susceptible sites in the pesticides. Evaluation at several molar ratios of KBDO:test articles using liquid chromatography-mass spectrometry (LC-MS) techniques was performed. The OP test articles, parathion, paraoxon, parathion-methyl, paraoxon-methyl and chlorpyrifos were effectively degraded at molar ratios of four and above in less than 6min contact time. Malathion and malaoxon were similarly converted to inactive by-products at molar ratios as low as two in less than 4min. A minimum molar ratio of nine was found to be effective against the carbamate pesticide carbofuran. In the case of aldicarb, complete destruction was achieved at a molar ratio of fifteen and a reaction time of one hour. It is important to note that these studies are based on a direct liquid phase RSDL lotion reaction with the toxic chemicals without the added physical removal decontamination efficacy component provided by the sponge component of the RSDL kit. The RSDL kit is intended to be used to remove or neutralize chemical warfare agents (CWA) and T-2 toxin from the skin. In actual use, the majority of the CWA decontamination occurs through the combined action of the sponge in both removing the chemical from the skin, and in rapidly mixing the chemicals at a high molar ratio of KBDO:CWA within the pores of the sponge to enhance rapid neutralization of the chemical. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Quantitative monitoring of corticosteroids in cosmetic products manufactured in Korea using LC-MS/MS.

    Science.gov (United States)

    Nam, Yun Sik; Kwon, Il Keun; Lee, Yeonhee; Lee, Kang-Bong

    2012-07-10

    Some cosmetic products manufactured in Korea for the treatment of eczema, seborrhea and psoriasis have been suspected to contain anti-inflammatory corticosteroids such as prednisolone, hydrocortisone, betamethasone, dexamethasone and triamcinolone acetonide without these ingredients being indicated on the label. Due to their severe side effects such as permenent skin atopy, these corticosteroids have to be monitored in cosmetic products from a forensic point of view. Many cosmetic product samples (N=65) have been collected from both local and online markets in Korea. The corticosteroid content of these samples was analyzed by LC-MS/MS with diagnostic ions (m/z). Linearity was studied with 0.1-10 μg/mL range in all corticosteroids. Good correlation coefficients (r(2)≥0.997) were found and the limits of quantification were 4.68-7.97 ng/mL for each of the corticosteroids. At three different concentrations spanning the linear dynamic ranges, mean recoveries were 97.2-113.5%and precisions (RSD) for intra-day and inter-day analysis were less than 8.9%. Also, accuracy (Bias %) was less than 11.8%. The results showed that between 0.76-0.94 μg/g levels of prednisolone were detected in four cosmetic products and triamcinolone acetonidewas detected with a concentration in the range of 11.5-272 μg/g in nine samples. This fact reveals that some manufacturers have arbitrarily added these corticosteroids in their cosmetic products without indicating them on the label. Thus, these cosmetic products have to be monitored and if proven illegal preparations removed from the market. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. Determining urea levels in exhaled breath condensate with minimal preparation steps and classic LC-MS.

    Science.gov (United States)

    Pitiranggon, Masha; Perzanowski, Matthew S; Kinney, Patrick L; Xu, Dongqun; Chillrud, Steven N; Yan, Beizhan

    2014-10-01

    Exhaled breath condensate (EBC) provides a relatively easy, non-invasive method for measuring biomarkers of inflammation and oxidative stress in the airways. However, the levels of these biomarkers in EBC are influenced, not only by their levels in lung lining fluid but also by the volume of water vapor that also condenses during EBC collection. For this reason, the use of a biomarker of dilution has been recommended. Urea has been proposed and utilized as a promising dilution biomarker due to its even distribution throughout the body and relatively low volatility. Current EBC urea analytical methods either are not sensitive enough, necessitating large volumes of EBC, or are labor intensive, requiring a derivatization step or other pretreatment. We report here a straightforward and reliable LC-MS approach that we developed that does not require derivatization or large sample volume (∼36 µL). An Acclaim mixed-mode hydrophilic interaction chromatography column was selected because it can produce good peak symmetry and efficiently separate urea from other polar and nonpolar compounds. To achieve a high recovery rate, a slow and incomplete evaporation method was used followed by a solvent-phase exchange. Among EBC samples collected from 28 children, urea levels were found to be highly variable, with a relative standard deviation of 234%, suggesting high variability in dilution of the lung lining fluid component of EBC. The limit of detection was found to be 0.036 µg/mL. Published by Oxford University Press [2013]. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. Post-acquisition data mining techniques for LC-MS/MS-acquired data in drug metabolite identification.

    Science.gov (United States)

    Dhurjad, Pooja Sukhdev; Marothu, Vamsi Krishna; Rathod, Rajeshwari

    2017-08-01

    Metabolite identification is a crucial part of the drug discovery process. LC-MS/MS-based metabolite identification has gained widespread use, but the data acquired by the LC-MS/MS instrument is complex, and thus the interpretation of data becomes troublesome. Fortunately, advancements in data mining techniques have simplified the process of data interpretation with improved mass accuracy and provide a potentially selective, sensitive, accurate and comprehensive way for metabolite identification. In this review, we have discussed the targeted (extracted ion chromatogram, mass defect filter, product ion filter, neutral loss filter and isotope pattern filter) and untargeted (control sample comparison, background subtraction and metabolomic approaches) post-acquisition data mining techniques, which facilitate the drug metabolite identification. We have also discussed the importance of integrated data mining strategy.

  11. Development of an LC-MS/MS method for the determination of pesticides and patulin in apples

    DEFF Research Database (Denmark)

    Christensen, Hanne Bjerre; Poulsen, Mette Erecius; Rasmussen, Peter Have

    2009-01-01

    A method for the simultaneous determination of 33 pesticides or degradation products together with patulin in apples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method involved homogenization of the apples, extraction with ammonium acetate-acetic acid solution...... in methanol-water by ultrasonication, filtration, and determination by LC-MS/MS. The repeatability and within-laboratory reproducibility for the three spiking levels 0.02, 0.04 and 0.2 mg kg(-1) were between 4% and 35%. In general, the repeatability and reproducibility were about 10-20%. The limits...... of quantification (LOQs) were between 0.01 and 0.14 mg kg(-1). The method was used on incurred samples from parts of the ISAFRUIT project financed by the European Commission under the 6th Framework Programme. Samples were analysed at four different stages: after harvest, after storage (controlled), after a water...

  12. LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data

    Energy Technology Data Exchange (ETDEWEB)

    Kyle, Jennifer E.; Crowell, Kevin L.; Casey, Cameron P.; Fujimoto, Grant M.; Kim, Sangtae; Dautel, Sydney E.; Smith, Richard D.; Payne, Samuel H.; Metz, Thomas O.

    2017-01-31

    We introduce an open-source software, LIQUID, for semi-automated processing and visualization of LC-MS/MS based lipidomics data. LIQUID provides users with the capability to process high throughput data and contains a customizable target library and scoring model per project needs. The graphical user interface provides visualization of multiple lines of spectral evidence for each lipid identification, allowing rapid examination of data for making confident identifications of lipid molecular species.

  13. Determination of isosteviol by LC-MS/MS and its application for evaluation of pharmacokinetics of isosteviol in rat

    Directory of Open Access Journals (Sweden)

    Bazargan M.

    2007-06-01

    Full Text Available Isosteviol has been found to have potential preventive or therapeutic effects against hypertension, ischemia reperfusion injury, diabetes and cancer, but little is known about the pharmacokinetics (PK of the compound. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS method for determination of isosteviol in rat plasma and to assess in a preliminary manner the PK of isosteviol after intravenous bolus injection.Ions of analytes were generated using electro-spray ionization and detected in the positive-ion mode in LC-MS/MS. Multiple reaction monitoring was performed, using the precursor product ion combination for isosteviol m/z 319.4→273.4. Progesterone was used as an internal standard. Nitrogen was used as the nebulising gas and unit resolution was set for Q1 and Q3. Isosteviol solution was injected through the penile vein of rats at a dose of 8 mg/kg. Blood samples were collected from a jugular vein cannula. The PK parameters were calculated using a two - compartment PK model.The LC-MS/MS assay for isosteviol in rat plasma was linear over the range of 0.5-80 μg/ml. The terminal half life of isosteviol (t 1/2 was 406 ± 31.7 min and clearance (CL was 2.9 ± 0.3 ml/min/kg. A sensitive LC-MS/MS assay for isosteviol in plasma has been successfully established and used in a preliminary PK evaluation of isosteviol in rats.

  14. Effect of Genetics, Environment, and Phenotype on the Metabolome of Maize Hybrids Using GC/MS and LC/MS.

    Science.gov (United States)

    Tang, Weijuan; Hazebroek, Jan; Zhong, Cathy; Harp, Teresa; Vlahakis, Chris; Baumhover, Brian; Asiago, Vincent

    2017-06-28

    We evaluated the variability of metabolites in various maize hybrids due to the effect of environment, genotype, phenotype as well as the interaction of the first two factors. We analyzed 480 forage and the same number of grain samples from 21 genetically diverse non-GM Pioneer brand maize hybrids, including some with drought tolerance and viral resistance phenotypes, grown at eight North American locations. As complementary platforms, both GC/MS and LC/MS were utilized to detect a wide diversity of metabolites. GC/MS revealed 166 and 137 metabolites in forage and grain samples, respectively, while LC/MS captured 1341 and 635 metabolites in forage and grain samples, respectively. Univariate and multivariate analyses were utilized to investigate the response of the maize metabolome to the environment, genotype, phenotype, and their interaction. Based on combined percentages from GC/MS and LC/MS datasets, the environment affected 36% to 84% of forage metabolites, while less than 7% were affected by genotype. The environment affected 12% to 90% of grain metabolites, whereas less than 27% were affected by genotype. Less than 10% and 11% of the metabolites were affected by phenotype in forage and grain, respectively. Unsupervised PCA and HCA analyses revealed similar trends, i.e., environmental effect was much stronger than genotype or phenotype effects. On the basis of comparisons of disease tolerant and disease susceptible hybrids, neither forage nor grain samples originating from different locations showed obvious phenotype effects. Our findings demonstrate that the combination of GC/MS and LC/MS based metabolite profiling followed by broad statistical analysis is an effective approach to identify the relative impact of environmental, genetic and phenotypic effects on the forage and grain composition of maize hybrids.

  15. FRAGMENTATION STUDIES OF D6,7-ANHIDROERITROMISIN-A BY LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY (LC-MS

    Directory of Open Access Journals (Sweden)

    Khairan Khairan

    2010-06-01

    Full Text Available Semisynthesis of D6,7-Anhydroerythromycin-A was done by biomodification technique by addition of 0.2% INH into a culture fermentation of Saccharopolyspora erythraea ATCC 11635 in medium Hutchinson. The aim of this research is to studies of fragmentation pattern from new matabolite of D6,7-Anhydroerythromycin-A by Liquid Chromatography-Mass Spectroscopy (LC-MS and the ionization of mass spectroscopy is use by ESI (Electrospray Ionization pattern. The FT-IR spectrometric analyzes showed a stretching vibration of C=C conjugated group at wave number 1602.7 cm-1. This C=C conjugated vibration indicated the existence of double bond between C6 and C7 (D6,7, this confirmed that isolate contained D6,7-Anhydroerythromycin-A (the possibility of D6,7 was positive. For complementation, a LC-MS (Liquid Chromatography-Mass Spectroscopy analyzes using ESI-MS (Electrospray Ionization-Mass Spectroscopy ionization pattern was conducted to the isolate which resulted Quassimolecular ions [M+H]+ of D7,8- and D6,7-Anhydroerythromycin-A. LC-MS spectrogram of the isolate, which gave two peaks of m/z 732.2460 and m/z 716.2522, confirmed that the m/z 732.2460 possibly was D7,8-Anhydroerythromycin-A, while the m/z 716.2502 and m/z 715.2522 possibly were D6,7-Anhydroerythromycin-A.   Keywords: isoniazid, enoyl reduction, D6,7-Anhidroeritromisin-A, fragmentation, LC-MS.

  16. Non-enzymolytic adenosine barcode-mediated dual signal amplification strategy for ultrasensitive protein detection using LC-MS/MS.

    Science.gov (United States)

    Yang, Wen; Li, Tengfei; Shu, Chang; Ji, Shunli; Wang, Lei; Wang, Yan; Li, Duo; Mtalimanja, Michael; Sun, Luning; Ding, Li

    2018-05-10

    A method is described for the determination of proteins with LC-MS/MS enabled by a small molecule (adenosine) barcode and based on a double-recognition sandwich structure. The coagulation protein thrombin was chosen as the model analyte. Magnetic nanoparticles were functionalized with aptamer29 (MNP/apt29) and used to capture thrombin from the samples. MNP/apt29 forms a sandwich with functionalized gold nanoparticles modified with (a) aptamer15 acting as thrombin-recognizing element and (b) a large number of adenosine as mass barcodes. The sandwich formed (MNP/apt29-thrombin-apt15/AuNP/adenosine) can ben magnetically separated from the sample. Mass barcodes are subsequently released from the sandwiched structure for further analysis by adding 11-mercaptoundecanoic acid. Adenosine is then detected by LC-MS/MS as it reflects the level of thrombin with impressively amplified signal. Numerous adenosines introduced into the sandwich proportional to the target concentration further amplify the signal. Under optimized conditions, the response is linearly proportional to the thrombin concentration in the range of 0.02 nM to 10 nM, with a detection limit of 9 fM. The application of this method to the determination of thrombin in spiked plasma samples gave recoveries that ranged from 92.3% to 104.7%. Graphical abstract Schematic representation of a method for the determination of thrombin with LC-MS/MS. The method is based on a double-recognition sandwiched structure. With LC-MS/MS, mass barcodes (adenosine) are detected to quantify thrombin, which amplifies the detection signal impressively.

  17. Structure Annotation and Quantification of Wheat Seed Oxidized Lipids by High-Resolution LC-MS/MS.

    Science.gov (United States)

    Riewe, David; Wiebach, Janine; Altmann, Thomas

    2017-10-01

    Lipid oxidation is a process ubiquitous in life, but the direct and comprehensive analysis of oxidized lipids has been limited by available analytical methods. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) to quantify oxidized lipids (glycerides, fatty acids, phospholipids, lysophospholipids, and galactolipids) and implemented a platform-independent high-throughput-amenable analysis pipeline for the high-confidence annotation and acyl composition analysis of oxidized lipids. Lipid contents of 90 different naturally aged wheat ( Triticum aestivum ) seed stocks were quantified in an untargeted high-resolution LC-MS experiment, resulting in 18,556 quantitative mass-to-charge ratio features. In a posthoc liquid chromatography-tandem mass spectrometry experiment, high-resolution MS/MS spectra (5 mD accuracy) were recorded for 8,957 out of 12,080 putatively monoisotopic features of the LC-MS data set. A total of 353 nonoxidized and 559 oxidized lipids with up to four additional oxygen atoms were annotated based on the accurate mass recordings (1.5 ppm tolerance) of the LC-MS data set and filtering procedures. MS/MS spectra available for 828 of these annotations were analyzed by translating experimentally known fragmentation rules of lipids into the fragmentation of oxidized lipids. This led to the identification of 259 nonoxidized and 365 oxidized lipids by both accurate mass and MS/MS spectra and to the determination of acyl compositions for 221 nonoxidized and 295 oxidized lipids. Analysis of 15-year aged wheat seeds revealed increased lipid oxidation and hydrolysis in seeds stored in ambient versus cold conditions. © 2017 The author(s). All Rights Reserved.

  18. Application of HRAM screening and LC-MS/MS confirmation of active pharmaceutical ingredient in "natural" herbal supplements.

    Science.gov (United States)

    Pascali, Jennifer P; Fais, Paolo; Vaiano, Fabio; Bertol, Elisabetta

    2018-05-01

    The growing market of herbal remedies worldwide could pose severe problems to consumers' health due to the possible presence of potentially harmful, undeclared synthetic substances or analogues of prescription drugs. The present work shows a simple but effective approach to unequivocally identify synthetic anorectic compounds in allegedly 'natural' herbal extracts, by exploiting liquid chromatography/time of flight (Q-TOF LC/MS) technology coupled to liquid chromatography/triple quadrupole (LC-MS/MS) confirmation and quantitation. The procedure was applied to five tea herbal extracts and pills sold as coadjutant for weigh loss. The method exploited liquid-liquid sample extraction (LLE) and separation in a C18 (2.1mm×150mm, 1.8μm) column. QTOF acquisitions were carried out both in scan mode and all ion MS/MS mode and results were obtained after search against ad hoc prepared library. Sibutramine, 4-hydroxyamphetamine, caffeine and theophylline were preliminary identified samples. Confirmation and quantitation of the preliminary identified compounds were obtained in LC-MS/MS after preparation of appropriated standards. Sibutramine, caffeine and theophylline were finally confirmed and quantitate. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based bioavailability determination of the major classes of phytochemicals.

    Science.gov (United States)

    Stylos, Evgenios; Chatziathanasiadou, Maria V; Syriopoulou, Aggeliki; Tzakos, Andreas G

    2017-03-15

    Natural products derived from plants have served as an inexhausted source for drug discovery and drug development. They have been evolutionary amplified with drug-like properties and have already illustrated immense therapeutic potential over an array of different diseases. However, their incorporation in the drug discovery pipeline has been diminished the last two decades. This was probably due to barriers related to their inherent difficulties to be integrated in high-throughput screening assays as also their largely unexplored bioavailability. Analytical procedures have come into the spotlight, a result of the continuous development of the instrumentation's capabilities as far as detection and separation is concerned. Integral part of this technological evolution is LC-MS instrumentation and its extended use for the determination of various compounds. The fact that it provides extra sensitivity, specificity and good separation in complex samples, makes LC-MS/MS the ultimate tool in the determination of many types of chemical compounds, such as phytochemicals. Herein, we focus on the achievements of the last five years in quantitative analysis of the major classes of phytochemicals (flavonoids, alkaloids, terpenes, glycosides and saponins) in plasma, through LC-MS/MS, as also their bioavailability. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples.

    Science.gov (United States)

    Moreno-Bondi, María Cruz; Marazuela, María Dolores; Herranz, Sonia; Rodriguez, Erika

    2009-10-01

    Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS(2)). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.

  1. Sensitivity and proportionality assessment of metabolites from microdose to high dose in rats using LC-MS/MS.

    Science.gov (United States)

    Ni, Jinsong; Ouyang, Hui; Seto, Carmai; Sakuma, Takeo; Ellis, Robert; Rowe, Josh; Acheampong, Andrew; Welty, Devin; Szekely-Klepser, Gabriella

    2010-03-01

    The objective of this study was to evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to characterize metabolites in plasma and urine at microdoses in rats and to investigate proportionality of metabolite exposure from a microdose of 1.67 µg/kg to a high dose of 5000 µg/kg for atorvastatin, ofloxacin, omeprazole and tamoxifen. Only the glucuronide metabolite of ofloxacin, the hydroxylation metabolite of omeprazole and the hydration metabolite of tamoxifen were characterized in rat plasma at microdose by LC-MS/MS. The exposure of detected metabolites of omeprazole and tamoxifen appeared to increase in a nonproportional manner with increasing doses. Exposure of ortho- and para-hydroxyatorvastatin, but not atorvastatin and lactone, increased proportionally with increasing doses. LC-MS/MS has demonstrated its usefulness for detecting and characterizing the major metabolites in plasma and urine at microdosing levels in rats. The exposure of metabolites at microdose could not simply be used to predict their exposure at higher doses.

  2. ChelomEx: Isotope-assisted discovery of metal chelates in complex media using high-resolution LC-MS.

    Science.gov (United States)

    Baars, Oliver; Morel, François M M; Perlman, David H

    2014-11-18

    Chelating agents can control the speciation and reactivity of trace metals in biological, environmental, and laboratory-derived media. A large number of trace metals (including Fe, Cu, Zn, Hg, and others) show characteristic isotopic fingerprints that can be exploited for the discovery of known and unknown organic metal complexes and related chelating ligands in very complex sample matrices using high-resolution liquid chromatography mass spectrometry (LC-MS). However, there is currently no free open-source software available for this purpose. We present a novel software tool, ChelomEx, which identifies isotope pattern-matched chromatographic features associated with metal complexes along with free ligands and other related adducts in high-resolution LC-MS data. High sensitivity and exclusion of false positives are achieved by evaluation of the chromatographic coherence of the isotope pattern within chromatographic features, which we demonstrate through the analysis of bacterial culture media. A built-in graphical user interface and compound library aid in identification and efficient evaluation of results. ChelomEx is implemented in MatLab. The source code, binaries for MS Windows and MAC OS X as well as test LC-MS data are available for download at SourceForge ( http://sourceforge.net/projects/chelomex ).

  3. Improving peak detection in high-resolution LC/MS metabolomics data using preexisting knowledge and machine learning approach.

    Science.gov (United States)

    Yu, Tianwei; Jones, Dean P

    2014-10-15

    Peak detection is a key step in the preprocessing of untargeted metabolomics data generated from high-resolution liquid chromatography-mass spectrometry (LC/MS). The common practice is to use filters with predetermined parameters to select peaks in the LC/MS profile. This rigid approach can cause suboptimal performance when the choice of peak model and parameters do not suit the data characteristics. Here we present a method that learns directly from various data features of the extracted ion chromatograms (EICs) to differentiate between true peak regions from noise regions in the LC/MS profile. It utilizes the knowledge of known metabolites, as well as robust machine learning approaches. Unlike currently available methods, this new approach does not assume a parametric peak shape model and allows maximum flexibility. We demonstrate the superiority of the new approach using real data. Because matching to known metabolites entails uncertainties and cannot be considered a gold standard, we also developed a probabilistic receiver-operating characteristic (pROC) approach that can incorporate uncertainties. The new peak detection approach is implemented as part of the apLCMS package available at http://web1.sph.emory.edu/apLCMS/ CONTACT: tyu8@emory.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Simultaneous Determination of Cyclosporine A, Tacrolimus, Sirolimus, and Everolimus in Whole-Blood Samples by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Mustafa Karapirli

    2012-01-01

    Full Text Available Objectives. Cyclosporine A (CyA, tacrolimus (TRL, sirolimus (SIR, and everolimus (RAD are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. Materials and Methods. We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS in the multiple reaction monitoring (MRM detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. Results. System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. Conclusion. This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

  5. Clinical laboratory verification of thyroglobulin concentrations in the presence of autoantibodies to thyroglobulin: comparison of EIA, radioimmunoassay and LC MS/MS measurements in an Urban Hospital.

    Science.gov (United States)

    Wheeler, Sarah E; Liu, Li; Blair, Harry C; Sivak, Richard; Longo, Nancy; Tischler, Jeffery; Mulvey, Kathryn; Palmer, Octavia M Peck

    2017-12-08

    Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC-MS/MS to evaluate TgAb interference differences. We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC-MS/MS and RIA was observed at low Tg concentrations (≤ 7.55 ng/mL) in TgAb positive specimens (LC-MS/MS = 1.9 * EIA - 0.03, r = 0.68). RIA and LC-MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA - 1.4, r = 0.90). Disagreement between methods may be attributed to LC-MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC-MS/MS; implementation of LC-MS/MS by clinicians will require lower detection limits.

  6. Complete LC/MS analysis of a Tinnevelli senna pod extract and subsequent isolation and identification of two new benzophenone glucosides.

    Science.gov (United States)

    Terreaux, Christian; Wang, Qi; Ioset, Jean-Robert; Ndjoko, Karine; Grimminger, Wolf; Hostettmann, Kurt

    2002-04-01

    The hydroalcoholic extract of Tinnevelli senna is widely used as a laxative phytomedicine. In order to improve the knowledge of the chemical composition of this extract, LC/MS and LC/MS(n) studies were performed, allowing the on-line identification of most of the known constituents, i. e., flavonoids, anthraquinones and the typical dianthronic sennosides. However, the identity of four compounds could not be ascertained on-line under the given LC/MS conditions. These substances were isolated and their structures elucidated as kaempferol, the naphthalene derivative tinnevellin 8-glucoside and two new carboxylated benzophenone glucosides.

  7. Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples.

    Science.gov (United States)

    Fang, Nianbai; Yu, Shanggong; Ronis, Martin Jj; Badger, Thomas M

    2015-04-01

    High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt

  8. LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

    Science.gov (United States)

    Trettin, Arne; Jordan, Jens; Tsikas, Dimitrios

    2014-09-01

    Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Biomarker Discovery Using New Metabolomics Software for Automated Processing of High Resolution LC-MS Data

    Science.gov (United States)

    Hnatyshyn, S.; Reily, M.; Shipkova, P.; McClure, T.; Sanders, M.; Peake, D.

    2011-01-01

    Robust biomarkers of target engagement and efficacy are required in different stages of drug discovery. Liquid chromatography coupled to high resolution mass spectrometry provides sensitivity, accuracy and wide dynamic range required for identification of endogenous metabolites in biological matrices. LCMS is widely-used tool for biomarker identification and validation. Typical high resolution LCMS profiles from biological samples may contain greater than a million mass spectral peaks corresponding to several thousand endogenous metabolites. Reduction of the total number of peaks, component identification and statistical comparison across sample groups remains to be a difficult and time consuming challenge. Blood samples from four groups of rats (male vs. female, fully satiated and food deprived) were analyzed using high resolution accurate mass (HRAM) LCMS. All samples were separated using a 15 minute reversed-phase C18 LC gradient and analyzed in both positive and negative ion modes. Data was acquired using 15K resolution and 5ppm mass measurement accuracy. The entire data set was analyzed using software developed in collaboration between Bristol Meyers Squibb and Thermo Fisher Scientific to determine the metabolic effects of food deprivation on rats. Metabolomic LC-MS data files are extraordinarily complex and appropriate reduction of the number of spectral peaks via identification of related peaks and background removal is essential. A single component such as hippuric acid generates more than 20 related peaks including isotopic clusters, adducts and dimers. Plasma and urine may contain 500-1500 unique quantifiable metabolites. Noise filtering approaches including blank subtraction were used to reduce the number of irrelevant peaks. By grouping related signals such as isotopic peaks and alkali adducts, data processing was greatly simplified by reducing the total number of components by 10-fold. The software processes 48 samples in under 60minutes. Principle

  10. Determination of Scopolamine in Human Saliva Using Solid Phase Extraction and LC/MS/MS

    Science.gov (United States)

    Wang, Zuwei; Vaksman, Zalman; Boyd, Jason; Putcha, Lakshmi

    2007-01-01

    Purpose: Scopolamine is the preferred treatment for motion sickness during space flight because of its quick onset of action, short half-life and favorable side-effect profile. The dose administered depends on the mode of administration and usually ranges between 0.1 and 0.8 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids by using conventional HPLC methods. To measure scopolamine in saliva and thereby to evaluate the pharmacokinetics of scopolamine, we developed an LC/MS/MS method using off-line solid phase extraction. Method: Samples (0.5mL) were loaded onto Waters Oasis HLB co-polymer cartridges (10 mg, 1 mL) and eluted with 0.5 mL methanol without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 4 minutes. The mobile phase for separation was 90:10 (v/v) methanol: ammonium acetate (2 mM) in water, pH 5.0 +/- 0.1. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 yields 138.1 and internal standard (IS) hyoscyamine m/z = 290.2 yields 124.1. Results: The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at 1.7 and 3.2 min respectively. The linear range is 50-5000 pg/mL for scopolamine in saliva with correlation coefficients > 0.99 with a CV < 0.5 %. The intra-day and inter-day CVs are < 15 % for quality control samples with concentrations of 75, 300, 750 and 3000 pg/mL of scopolamine in human saliva. Conclusion: Solid phase extraction allows more rapid sample preparation and greater precision than liquid extraction. Furthermore, we increased the sensitivity and specificity by adjusting the LC mobile phase and using an MS

  11. The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

    Directory of Open Access Journals (Sweden)

    Saeed Alexander I

    2008-12-01

    utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. Conclusion The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.

  12. Organization of GC/MS and LC/MS metabolomics data into chemical libraries

    Directory of Open Access Journals (Sweden)

    DeHaven Corey D

    2010-10-01

    Full Text Available Abstract Background Metabolomics experiments involve generating and comparing small molecule (metabolite profiles from complex mixture samples to identify those metabolites that are modulated in altered states (e.g., disease, drug treatment, toxin exposure. One non-targeted metabolomics approach attempts to identify and interrogate all small molecules in a sample using GC or LC separation followed by MS or MSn detection. Analysis of the resulting large, multifaceted data sets to rapidly and accurately identify the metabolites is a challenging task that relies on the availability of chemical libraries of metabolite spectral signatures. A method for analyzing spectrometry data to identify and Quantify Individual Components in a Sample, (QUICS, enables generation of chemical library entries from known standards and, importantly, from unknown metabolites present in experimental samples but without a corresponding library entry. This method accounts for all ions in a sample spectrum, performs library matches, and allows review of the data to quality check library entries. The QUICS method identifies ions related to any given metabolite by correlating ion data across the complete set of experimental samples, thus revealing subtle spectral trends that may not be evident when viewing individual samples and are likely to be indicative of the presence of one or more otherwise obscured metabolites. Results LC-MS/MS or GC-MS data from 33 liver samples were analyzed simultaneously which exploited the inherent biological diversity of the samples and the largely non-covariant chemical nature of the metabolites when viewed over multiple samples. Ions were partitioned by both retention time (RT and covariance which grouped ions from a single common underlying metabolite. This approach benefitted from using mass, time and intensity data in aggregate over the entire sample set to reject outliers and noise thereby producing higher quality chemical identities. The

  13. EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)

    Science.gov (United States)

    Method MS014 describes procedures for solvent extraction of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl from water samples, followed by analysis using liquid chromatography tandem mass spectrometry (LC-MS-MS).

  14. LC-MS/MS method for the determination of clodronate in human plasma.

    Science.gov (United States)

    Hasan, Mahmoud; Schumacher, Gitta; Seekamp, Anne; Taedken, Tobias; Siegmund, Werner; Oswald, Stefan

    2014-11-01

    Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma concentrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC-MS/MS assay for the quantification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid-liquid extraction using methyl tert-butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300μl/min on a reversed-phase column (Supelco Ascentis(®), C18) temporized at 50°C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5-800ng/ml, allowing an evaluation of the plasma concentration-time profiles of clodronate for approximately 7-8 half-life (∼24h). The method was validated according to current FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6-6.9% and 0.6-8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6-8.8% and 2.2-4.5%. The recovery of the analyte was low (2-3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3h (96.0±6%) and

  15. A sensitive LC-MS/MS method for measurement of organophosphorus pesticides and their oxygen analogs in air sampling matrices.

    Science.gov (United States)

    Armstrong, Jenna L; Dills, Russell L; Yu, Jianbo; Yost, Michael G; Fenske, Richard A

    2014-01-01

    A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15-1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78-113% from XAD-2 active air sampling tubes and 71-108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74-94% after time periods ranging from 2-10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method.

  16. High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.

    Science.gov (United States)

    Jenkinson, Carl; Taylor, Angela E; Hassan-Smith, Zaki K; Adams, John S; Stewart, Paul M; Hewison, Martin; Keevil, Brian G

    2016-03-01

    Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Targeted LC-MS/MS method for quantification of Plant Lignans and Enterolignans in Biofluids from Humans and Pigs

    DEFF Research Database (Denmark)

    Nørskov, Natalja; Olsen, Anja; Tjønneland, Anne

    2015-01-01

    Lignans have gained nutritional interest due to their promising role in the prevention of lifestyle diseases. However, epidemiological studies are in need of more evidence to link the intake of lignans to this promising role. In this context, it is necessary to study large population groups...... to obtain sufficient statistical power. Therefore, there is a demand for fast, sensitive, and accurate methods for quantitation with high throughput of samples. This paper presents a validated LC-MS/MS method for the quantitation of eight plant lignans (matairesinol, hydroxymatairesinol...

  18. Non-targeted metabolomics and lipidomics LC-MS data from maternal plasma of 180 healthy pregnant women

    DEFF Research Database (Denmark)

    Luan, Hemi; Meng, Nan; Liu, Ping

    2015-01-01

    Background: Metabolomics has the potential to be a powerful and sensitive approach for investigating the low molecular weight metabolite profiles present in maternal fluids and their role in pregnancy.Findings: In this Data Note, LC-MS metabolome, lipidome and carnitine profiling data were...... collected from 180 healthy pregnant women, representing six time points spanning all three trimesters, and providing sufficient coverage to model the progression of normal pregnancy.Conclusions: As a relatively large scale, real-world dataset with robust numbers of quality control samples, the data...

  19. Analysis of the constituents in jojoba wax used as a food additive by LC/MS/MS.

    Science.gov (United States)

    Tada, Atsuko; Jin, Zhe-Long; Sugimoto, Naoki; Sato, Kyoko; Yamazaki, Takeshi; Tanamoto, Kenichi

    2005-10-01

    Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.

  20. Reported prevalence and quantitative LC-MS methods for the analysis of veterinary drug residues in honey: a review.

    Science.gov (United States)

    Venable, Ryan; Haynes, Carion; Cook, Jo Marie

    2014-04-01

    Insect pollination increases the value and productivity of three-quarters of crop species grown for food. Declining beehive health in commercial apiaries has resulted in numerous reports from government laboratories worldwide of contamination with antimicrobial chemicals in honey. This review includes pertinent discussion of legislation and events leading to increased government oversight in the commercial honey market. A detailed summary of the variety and prevalence of veterinary drug residues being found in honey as well as a selection of robust quantitative and confirmatory LC-MS methods with an emphasis on those adopted by government testing laboratories are presented.

  1. Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars

    Directory of Open Access Journals (Sweden)

    Petroczi Andrea

    2011-05-01

    Full Text Available Abstract Background Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3 and 25-hydroxyvitamin-D2 (25OHD2 collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. Methods A positive ion electrospray ionisation (ESI LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM mode for quantification. It involved i liquid-liquid extraction, ii tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter, iii Stanozolol-D3 as internal standard, and iv identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. Results The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3, and 7-α-hydroxy-4-cholesten-3-one (7αC4. The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. Conclusions To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D. The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic

  2. Accurate LC peak boundary detection for ¹⁶O/¹⁸O labeled LC-MS data.

    Science.gov (United States)

    Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang S J; Zhang, Jianqiu Michelle

    2013-01-01

    In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.

  3. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

    Science.gov (United States)

    Sinclair, John; Timms, John F

    2011-08-01

    Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. GintAMT3 – a low-affinity ammonium transporter of the arbuscular mycorrhizal Rhizophagus irregularis

    Directory of Open Access Journals (Sweden)

    Silvia eCalabrese

    2016-05-01

    Full Text Available Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules where the nutrients are released to the plant-fungal interface, i.e. to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive valuable carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P, the uptake and transfer of nitrogen (N plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices, two ammonium transporters (AMT were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium (IRM of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2. GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a Vmax of 240 nmol-1 min-1 108 cells-1, which is regulated by substrate concentration and carbon supply.

  5. Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS.

    Science.gov (United States)

    Andersen, David W; Linnet, Kristian

    2014-01-01

    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Effect of Antibiotics and Diet on Enterolactone Concentration and Metabolome Studied by Targeted and Nontargeted LC-MS Metabolomics.

    Science.gov (United States)

    Bolvig, Anne K; Nørskov, Natalja P; Hedemann, Mette S; Foldager, Leslie; McCarthy-Sinclair, Brendan; Marco, Maria L; Lærke, Helle N; Bach Knudsen, Knud E

    2017-06-02

    High plant lignan intake is associated with a number of health benefits, possibly induced by the lignan metabolite enterolactone (ENL). The gut microbiota plays a crucial role in converting dietary lignans into ENL, and epidemiological studies have shown that use of antibiotics is associated with lower levels of ENL. Here we investigate the link between antibiotic use and lignan metabolism in pigs using LC-MS/MS. The effect of lignan intake and antibiotic use on the gut microbial community and the pig metabolome is studied by 16S rRNA sequencing and nontargeted LC-MS. Treatment with antibiotics resulted in substantially lower concentrations of ENL compared with concentrations detected in untreated animals, whereas the plasma concentrations of plant lignans were unchanged. Both diet and antibiotic treatment affected the clustering of urinary metabolites and significantly altered the proportions of taxa in the gut microbiota. Diet, but not antibiotic treatment, affected the plasma lipid profile, and a lower concentration of LDL cholesterol was observed in the pigs fed a high lignan diet. This study provides solid support for the associations between ENL concentrations and use of antibiotics found in humans and indicates that the lower ENL concentration may be a consequence of the ecological changes in the microbiota.

  7. Using LC-MS to examine the fermented food products vinegar and soy sauce for the presence of gluten.

    Science.gov (United States)

    Li, Haili; Byrne, Keren; Galiamov, Renata; Mendoza-Porras, Omar; Bose, Utpal; Howitt, Crispin A; Colgrave, Michelle L

    2018-07-15

    A strict, lifelong gluten-free (GF) diet is currently the only treatment for coeliac disease (CD). Vinegar and soy sauce are fermented condiments that often include wheat and/or barley. During fermentation cereal proteins are partially degraded by enzymes to yield peptide fragments and amino acids. Whether these fermented products contain intact or degraded gluten proteins and if they are safe for people with CD remains in question. LC-MS offers the benefit of being able to detect hydrolysed gluten that might be present in commercial vinegar and soy sauce products. LC-MS revealed the presence of gluten in malt vinegar, wherein the identified peptides derived from B-, D- and γ-hordein from barley, as well as γ-gliadin, and HMW- and LMW-glutenins from wheat that are known to contain immunopathogenic epitopes. No gluten was detected in the soy sauces examined despite wheat being a labelled ingredient indicating extensive hydrolysis of gluten during soy sauce production. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. A Simple and Direct LC-MS Method for Determination of Genotoxic Impurity Hydroxylamine in Pharmaceutical compounds.

    Science.gov (United States)

    Kumar, Thangarathinam; Ramya, Mohandass; Srinivasan, Viswanathan; Xavier, N

    2017-08-01

    Hydroxylamine is a known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical processes. It is difficult to detect using conventional analytical techniques due to its physio-chemical properties like lack of chromophore, low molecular weight, absence of carbon atom and high polarity. In addition to that, analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This study describes a simple, sensitive and direct Liquid Chromatographic-Mass Spectrometric method (LC-MS) for detection of hydroxylamine in pharmaceutical compounds. The LC-MS method was detected up to 0.008 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.025 ppm of hydroxylamine with S/N ratio >10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Identification of Eupatilin from Artemisia argyi as a Selective PPARα Agonist Using Affinity Selection Ultrafiltration LC-MS

    Directory of Open Access Journals (Sweden)

    Yongsoo Choi

    2015-07-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are key nuclear receptors and therapeutic targets for the treatment of metabolic diseases through the regulation of insulin resistance, diabetes, and dyslipidemia. Although a few drugs that target PPARs have been approved, more diverse and novel PPAR ligands are necessary to improve the safety and efficacy of available drugs. To expedite the search for new natural agonists of PPARs, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry (LC-MS that is compatible with complex samples such as dietary foods or botanical extracts. The known PPARα and/or PPARγ ligands resveratrol and rosiglitazone were used as positive controls to validate the developed method. When applied to the screening of an Artemisia argyi extract, eupatilin was identified as a selective PPARα ligand. A PPAR competitive binding assay based on FRET detection also confirmed eupatilin as a selective PPARα agonist exhibiting a binding affinity of 1.18 μM (IC50. Furthermore, eupatilin activation of the transcriptional activity of PPARα was confirmed using a cell-based transactivation assay. Thus, ultrafiltration LC-MS is a suitable assay for the identification of PPAR ligands in complex matrixes such as extracts of dietary foods and botanicals.

  10. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

    Directory of Open Access Journals (Sweden)

    Caroline E. Moore

    2016-03-01

    Full Text Available Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1. A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

  11. The Fate of Sulfamethazine in Sodium-Hypochlorite-Treated Drinking Water: Monitoring by LC-MS-IT-TOF

    Directory of Open Access Journals (Sweden)

    Tyler C. Melton

    2012-01-01

    Full Text Available Pharmaceutical compounds represent a rapidly emerging class of environmental contaminants. Such compounds were recently classified by the U.S. Geological Survey, including several antibiotics. An LC-MS/MS screening method for the top five antibiotics in drinking water was developed and validated using a Shimadzu LC-MS-IT-TOF. The separation was performed using a Waters Acquity UPLC BEH C18 column with a gradient elution. Sulfamethazine was exposed to conditions intended to mimic drinking water chlorination, and samples were collected and quenched with excess sodium sulfite. Kinetics of sulfamethazine degradation was followed as well as the formation of the major chlorinated byproduct (/ 313. For the screening method, all five antibiotic peaks were baseline resolved within 5 minutes. Additionally, precision and accuracy of the screening method were less than 15%. Degradation of sulfamethazine upon exposure to drinking water chlorination occurred by first order kinetics with a half-life of 5.3×104 min (approximately 37 days with measurements starting 5 minutes after chlorination. Likewise, the formation of the major chlorinated product occurred by first order kinetics with a rate constant of 2.0×10−2. The proposed identification of the chlorinated product was 4-amino-(5-chloro-4,6-dimethyl-2-pyrimidinyl-benzenesulfonamide (C12H13N4O2SCl using MS spectra and databases searches of SciFinder and ChemSpider.

  12. LC/MS Guided Isolation of Alkaloids from Lotus Leaves by pH-Zone-Refining Counter-Current Chromatography

    Directory of Open Access Journals (Sweden)

    Rui-Lin Hu

    2011-03-01

    Full Text Available The traditional methods used in natural product separation primarily target the major components and the minor components may thus be lost during the separation procedure. Consequently, it’s necessary to develop efficient methods for the preparative separation and purification of relatively minor bioactive components. In this paper, a LC/MS method was applied to guide the separation of crude extract of lotus (Nelumbo nucifera Gaertn. leaves whereby a minor component was identified in the LC/MS analysis. Afterwards, an optimized pH-zone-refining CCC method was performed to isolate this product, identified as N-demethylarmepavine. The separation procedure was carried out with a biphasic solvent system composed of hexane-ethyl acetate-methyl alcohol-water (1:6:1:6, v/v with triethylamine (10 mM added to the upper organic phase as a retainer and hydrochloric acid (5 mM to the aqueous mobile phase eluent. Two structurally similar compounds – nuciferine and roemerine – were also obtained from the crude lotus leaves extract. In total 500 mg of crude extract furnished 7.4 mg of N-demethylarmepavine, 45.3 mg of nuciferine and 26.6 mg of roemerine with purities of 90%, 92% and 96%, respectively. Their structures were further identified by HPLC/ESI-MSn, FTICR/MS and the comparison with reference compounds.

  13. Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis.

    Science.gov (United States)

    Levin, Yishai; Jaros, Julian A J; Schwarz, Emanuel; Bahn, Sabine

    2010-01-03

    In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood. (c) 2009 Elsevier B.V. All rights reserved.

  14. Untargeted LC-MS/MS Profiling of Cell Culture Media Formulations for Evaluation of High Temperature Short Time Treatment Effects.

    Science.gov (United States)

    Floris, Patrick; McGillicuddy, Nicola; Albrecht, Simone; Morrissey, Brian; Kaisermayer, Christian; Lindeberg, Anna; Bones, Jonathan

    2017-09-19

    An untargeted LC-MS/MS platform was implemented for monitoring variations in CHO cell culture media upon exposure to high temperature short time (HTST) treatment, a commonly used viral clearance upstream strategy. Chemically defined (CD) and hydrolysate-supplemented media formulations were not visibly altered by the treatment. The absence of solute precipitation effects during media treatment and very modest shifts in pH values observed indicated sufficient compatibility of the formulations evaluated with the HTST-processing conditions. Unsupervised chemometric analysis of LC-MS/MS data, however, revealed clear separation of HTST-treated samples from untreated counterparts as observed from analysis of principal components and hierarchical clustering sample grouping. An increased presence of Maillard products in HTST-treated formulations contributed to the observed differences which included organic acids, observed particularly in chemically defined formulations, and furans, pyridines, pyrazines, and pyrrolidines which were determined in hydrolysate-supplemented formulations. The presence of Maillard products in media did not affect cell culture performance with similar growth and viability profiles observed for CHO-K1 and CHO-DP12 cells when cultured using both HTST-treated and untreated media formulations.

  15. Simultaneous Determination of Multi-Mycotoxins in Cereal Grains Collected from South Korea by LC/MS/MS

    Directory of Open Access Journals (Sweden)

    Dong-Ho Kim

    2017-03-01

    Full Text Available An improved analytical method compared with conventional ones was developed for simultaneous determination of 13 mycotoxins (deoxynivalenol, nivalenol, 3-acetylnivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, T-2, HT-2, zearalenone, and ochratoxin A in cereal grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS after a single immunoaffinity column clean-up. The method showed a good linearity, sensitivity, specificity, and accuracy in mycotoxin determination by LC/MS/MS. The levels of 13 mycotoxins in 5 types of commercial grains (brown rice, maize, millet, sorghum, and mixed cereal from South Korea were determined in a total of 507 cereal grains. Mycotoxins produced from Fusarium sp. (fumonisins, deoxynivalenol, nivalenol, and zearalenone were more frequently (more than 5% and concurrently detected in all cereal grains along with higher mean levels (4.3–161.0 ng/g in positive samples than other toxins such as aflatoxins and ochratoxin A (less than 9% and below 5.2 ng/g in positive samples from other fungal species.

  16. Discrimination of Four Marine Biofilm-Forming Bacteria by LC-MS Metabolomics and Influence of Culture Parameters.

    Science.gov (United States)

    Favre, Laurie; Ortalo-Magné, Annick; Greff, Stéphane; Pérez, Thierry; Thomas, Olivier P; Martin, Jean-Charles; Culioli, Gérald

    2017-05-05

    Most marine bacteria can form biofilms, and they are the main components of biofilms observed on marine surfaces. Biofilms constitute a widespread life strategy, as growing in such structures offers many important biological benefits. The molecular compounds expressed in biofilms and, more generally, the metabolomes of marine bacteria remain poorly studied. In this context, a nontargeted LC-MS metabolomics approach of marine biofilm-forming bacterial strains was developed. Four marine bacteria, Persicivirga (Nonlabens) mediterranea TC4 and TC7, Pseudoalteromonas lipolytica TC8, and Shewanella sp. TC11, were used as model organisms. The main objective was to search for some strain-specific bacterial metabolites and to determine how culture parameters (culture medium, growth phase, and mode of culture) may affect the cellular metabolism of each strain and thus the global interstrain metabolic discrimination. LC-MS profiling and statistical partial least-squares discriminant analyses showed that the four strains could be differentiated at the species level whatever the medium, the growth phase, or the mode of culture (planktonic vs biofilm). A MS/MS molecular network was subsequently built and allowed the identification of putative bacterial biomarkers. TC8 was discriminated by a series of ornithine lipids, while the P. mediterranea strains produced hydroxylated ornithine and glycine lipids. Among the P. mediterranea strains, TC7 extracts were distinguished by the occurrence of diamine derivatives, such as putrescine amides.

  17. Investigation on biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC-MS based metabolomics.

    Science.gov (United States)

    Xu, Jie; Hu, Feng-Lin; Wang, Wei; Wan, Xiao-Chun; Bao, Guan-Hu

    2015-11-01

    Fu brick tea (FBT) is a unique post-fermented tea product which is fermented with fungi during the manufacturing process. In this study, we investigated the biochemical compositional changes occurring during the microbial fermentation process (MFP) of FBT based on non-targeted LC-MS, which was a comprehensive and unbiased methodology. Our data analysis took a two-phase approach: (1) comparison of FBT with other tea products using PCA analysis to exhibit the characteristic effect of MFP on the formation of Fu brick tea and (2) comparison of tea samples throughout the MFP of FBT to elucidate the possible key metabolic pathways produced by the fungi. Non-targeted LC-MS analysis clearly distinguished FBT with other tea samples and highlighted some interesting metabolic pathways during the MFP including B ring fission catechin. Our study demonstrated that those fungi had a significant influence on the biochemical profiles in the FBT and consequently contributed to its unique quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. A LC-MS-MS method for determination of low doxazosin concentrations in plasma after oral administration to dogs.

    Science.gov (United States)

    Erceg, Marijana; Cindric, Mario; Pozaic Frketic, Lidija; Vertzoni, Maria; Cetina-Cizmek, Biserka; Reppas, Christos

    2010-02-01

    A rapid and sensitive reversed phase liquid chromatography- tandem mass spectrometry (LC-MS-MS) method is developed for the determination of doxazosin in canine plasma. The samples are prepared by precipitation of proteins using a mixture of methanol and acetonitrile, followed by freezing and evaporation of the organic solvent. The remaining dry residue is redissolved in mobile phase and analyzed by LC-MS-MS with positive electrospray ionization using the selected reactions monitoring mode. An XTerra MS C(18) column, a mobile phase composed of acetonitrile and 2mM ammonium acetate with gradient elution, and a flow rate of 400 microL/min are employed. The elution times for prazosin (internal standard) and doxazosin are approximately 8 and 10 min, respectively. Calibration curves are linear in the 1-20 ng/mL concentration range. Limits of detection and quantification are 0.4 ng/mL and 1.2 ng/mL, respectively. Recovery is higher than 94%. Intra- and inter-day relative standard deviations are below 7% and 8%, respectively. The method is applied for the determination of doxazosin plasma levels following a single administration of doxazosin base and doxazosin mesylate tablets (2 mg dose) to dogs in the fed state. The results indicate possible superiority of the mesylate salt on the plasma input rates of doxazosin.

  19. HR-LC-MS based analysis of two antibacterial metabolites from a marine sponge symbiont Streptomyces pharmamarensis ICN40.

    Science.gov (United States)

    Joseph, Francis-Joseph Rosemary Sharmila; Iniyan, Appadurai Muthamil; Vincent, Samuel Gnana Prakash

    2017-10-01

    On the effort to screen antibiotics against Methicillin resistant Staphylococcus aureus (MRSA), an actinomycete strain which can produce bactericidal compound was isolated from a marine sponge of Kanyakumari Coast, India. Two anti-MRSA compounds (PVI401 and PVI402) were isolated from the fermentation plates of Streptomyces pharmamarensis ICN40. TLC bioautography analysis yielded two active spots with Rf value of 0.75 (PVI401) and 0.8 (PVI402) from the crude extract. Both the compounds were characterized by HR-LC-MS analysis. LC-MS based de-replication analysis found out the compound PVI401 with an exact mass of 376.09435 Da and PVI402 with an exact mass of 273.26795 Da were found to be unidentified. Antibacterial spectrum showed significant minimal inhibitory concentration as 0.5 μg/ml of PVI401 and 2 μg/ml of PVI402 against MRSA. The whole organism zebrafish safety evaluation exhibited the compound PVI402 is safe upto 1 mg/ml 40 μg/ml of PVI401 exhibited thrombosis in cardiac chamber and this compound exhibited 44 μg/ml of LC 50 against HepG2 hepatic carcinoma cell line. Both the compounds may be identified further for its structural novelty and clinical studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Finding of pesticides in fashionable fruit juices by LC-MS/MS and GC-MS/MS.

    Science.gov (United States)

    Tran, Kevin; Eide, David; Nickols, Susan M; Cromer, Michele R; Sabaa-Srur, Armando; Smith, Robert E

    2012-10-15

    Products labelled as containing extracts from two mushrooms (cordyceps plus reishi) and the juices from açaí, goji, mangosteen, noni, pomegranate, and sea buckthorn have been analysed for 174 different pesticides, using the validated QuEChERS method for sample preparation and electrospray LC-MS/MS in the positive ion mode for analysis. Pesticides were found in 10 of the 21 samples analysed. Most pesticides found were below the tolerance levels (1-6 μg/g, depending on the pesticide), but some were not. This included boscalid, dimethomorph, iprovalicarb, pyridaben, pyrimethanil, and imazalil, for which there is no tolerance reported or zero tolerance in any fruit. However, genuine açaí that was harvested in the state of Pará and lyophilised in Rio de Janeiro had no detectable pesticides, when analysed by both LC-MS/MS and GC-MS/MS, which can detect 213 more pesticides and industrial chemicals. Likewise no pesticides were found in one sample each of cordyceps plus reishi, sea buckthorn and noni. Published by Elsevier Ltd.

  1. Detection of endocrine active substances in the aquatic environment in southern Taiwan using bioassays and LC-MS/MS.

    Science.gov (United States)

    Chen, Kuang-Yu; Chou, Pei-Hsin

    2016-06-01

    Endocrine active substances, including naturally occurring hormones and various synthetic chemicals have received much concern owing to their endocrine disrupting potencies. It is essential to monitor their environmental occurrence since these compounds may pose potential threats to biota and human health. In this study, yeast-based reporter assays were carried out to investigate the presence of (anti-)androgenic, (anti-)estrogenic, and (anti-)thyroid compounds in the aquatic environment in southern Taiwan. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was also used to measure the environmental concentrations of selected endocrine active substances for assessing potential ecological risks and characterizing contributions to the endocrine disrupting activities. Bioassay results showed that anti-androgenic (ND-7489 μg L(-1) flutamide equivalent), estrogenic (ND-347 ng L(-1) 17β-estradiol equivalent), and anti-thyroid activities were detected in the dissolved and particulate phases of river water samples, while anti-estrogenic activities (ND-10 μg L(-1) 4-hydroxytamoxifen equivalent) were less often found. LC-MS/MS analysis revealed that anti-androgenic and estrogenic contaminants, such as bisphenol A, triclosan, and estrone were frequently detected in Taiwanese rivers. In addition, their risk quotient values were often higher than 1, suggesting that they may pose an ecological risk to the aquatic biota. Further identification of unknown anti-androgenic and estrogenic contaminants in Taiwanese rivers may be necessary to protect Taiwan's aquatic environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Development of LC-MS determination method and back-propagation ANN pharmacokinetic model of corynoxeine in rat.

    Science.gov (United States)

    Ma, Jianshe; Cai, Jinzhang; Lin, Guanyang; Chen, Huilin; Wang, Xianqin; Wang, Xianchuan; Hu, Lufeng

    2014-05-15

    Corynoxeine(CX), isolated from the extract of Uncaria rhynchophylla, is a useful and prospective compound in the prevention and treatment for vascular diseases. A simple and selective liquid chromatography mass spectrometry (LC-MS) method was developed to determine the concentration of CX in rat plasma. The chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile-0.1% formic acid in water as mobile phase. Selective ion monitoring (SIM) mode was used for quantification using target ions m/z 383 for CX and m/z 237 for the carbamazepine (IS). After the LC-MS method was validated, it was applied to a back-propagation artificial neural network (BP-ANN) pharmacokinetic model study of CX in rats. The results showed that after intravenous administration of CX, it was mainly distributed in blood and eliminated quickly, t1/2 was less than 1h. The predicted concentrations generated by BP-ANN model had a high correlation coefficient (R>0.99) with experimental values. The developed BP-ANN pharmacokinetic model can be used to predict the concentration of CX in rats. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Punica granatum peel extracts: HPLC fractionation and LC MS analysis to quest compounds having activity against multidrug resistant bacteria.

    Science.gov (United States)

    Khan, Ilyas; Rahman, Hazir; Abd El-Salam, Nasser M; Tawab, Abdul; Hussain, Anwar; Khan, Taj Ali; Khan, Usman Ali; Qasim, Muhammad; Adnan, Muhammad; Azizullah, Azizullah; Murad, Waheed; Jalal, Abdullah; Muhammad, Noor; Ullah, Riaz

    2017-05-03

    Medicinal plants are rich source of traditional herbal medicine around the globe. Most of the plant's therapeutic properties are due to the presence of secondary bioactive compounds. The present study analyzed the High Pressure Liquid Chromatography (HPLC) fractions of Puncia granatum (peel) extracts (aqueous, chloroform, ethanol and hexane) against multidrug resistant bacterial pathogens (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus). All the fractions having antibacterial activity was processed for bioactive compounds identification using LC MS/MS analysis. Among total HPLC fractions (n = 30), 4 HPLC fractions of P. granatum (peel) showed potential activity against MDR pathogens. Fraction 1 (F1) and fraction 4 (F4) collected from aqueous extract showed maximum activity against P. aeruginosa. Fraction 2 (F2) of hexane showed antibacterial activity against three pathogens, while ethanol F4 exhibited antibacterial activity against A. baumannii. The active fractions were processed for LC MS/MS analysis to identify bioactive compounds. Valoneic acid dilactone (aqueous F1 and F4), Hexoside (ethanol F4) and Coumaric acid (hexane F2) were identified as bioactive compounds in HPLC fractions. Puncia granatum peel extracts HPLC fractions exhibited potential inhibitory activity against MDR bacterial human pathogens. Several bioactive compounds were identified from the HPLC fractions. Further characterization of these compounds may be helpful to conclude it as therapeutic lead molecules against MDR pathogens.

  4. Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

    Science.gov (United States)

    Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

    2016-06-01

    Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Identification of novel biomarkers for sepsis prognosis via urinary proteomic analysis using iTRAQ labeling and 2D-LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Longxiang Su

    Full Text Available Sepsis is the major cause of death for critically ill patients. Recent progress in proteomics permits a thorough characterization of the mechanisms associated with critical illness. The purpose of this study was to screen potential biomarkers for early prognostic assessment of patients with sepsis.For the discovery stage, 30 sepsis patients with different prognoses were selected. Urinary proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ coupled with LC-MS/MS. Mass spec instrument analysis were performed with Mascot software and the International Protein Index (IPI; bioinformatic analyses were used by the algorithm of set and the Gene Ontology (GO Database. For the verification stage, the study involved another 54 sepsis-hospitalized patients, with equal numbers of patients in survivor and non-survivor groups based on 28-day survival. Differentially expressed proteins were verified by Western Blot.A total of 232 unique proteins were identified. Proteins that were differentially expressed were further analyzed based on the pathophysiology of sepsis and biomathematics. For sepsis prognosis, five proteins were significantly up-regulated: selenium binding protein-1, heparan sulfate proteoglycan-2, alpha-1-B glycoprotein, haptoglobin, and lipocalin; two proteins were significantly down-regulated: lysosome-associated membrane proteins-1 and dipeptidyl peptidase-4. Based on gene ontology clustering, these proteins were associated with the biological processes of lipid homeostasis, cartilage development, iron ion transport, and certain metabolic processes. Urinary LAMP-1 was down-regulated, consistent with the Western Blot validation.This study provides the proteomic analysis of urine to identify prognostic biomarkers of sepsis. The seven identified proteins provide insight into the mechanism of sepsis. Low urinary LAMP-1 levels may be useful for early prognostic assessment of sepsis.ClinicalTrial.gov NCT01493492.

  6. Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions.

    Science.gov (United States)

    Iwatsuka, Kinya; Yasueda, Shin-ichi; Bando, Eiji; Fujii, Hiroyuki; Terada, Takashi; Okubo, Hiroya; Iwamoto, Hiroki; Kinoshita, Mitsuhiro; Kakehi, Kazuaki

    2011-10-01

    Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Comparison of methods, storage conditions, and time to analysis of serum and urine creatinine measured from microsamples by liquid chromatography mass spectrometery (LC/MS) vs. Jaffe.

    Science.gov (United States)

    Askenazi, David J; Moore, John F; Fineberg, Naomi; Koralkar, Rajesh; Clevenger, Stephanie; Sharer, Jon Daniel

    2014-09-01

    Measurement of serum creatinine (SCr) and urine creatinine (UCr) is regularly used in clinical and research settings. For small animal experiments and for studies in which sample collection is spare (i.e. neonatal cohorts), measuring SCr and UCr using tiny amounts of sample (as low as 10 mcl) would maximize exploration and minimize iatrogenic blood loss. We performed an evaluation in six healthy adults to determine differences between SCr and UCr values in different methodologies and storage environments and time. Study was conducted using 20 mcl of sample. Analyses were done using two-way repeated measures of ANOVA. Scr values showed no significant differences between LC/MS vs. Jaffe. However, the SCr using LC/MS method was lowest when measured immediately compared to other time points (F = 7.2; Psamples measured by LC/MS. UCr measurements by LC/MS vary more over time, mostly due to the sample measured after 1 year; therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results. © 2014 Wiley Periodicals, Inc.

  8. Development and Validation of a Novel LC-MS/MS Opioid Confirmation Assay: Evaluation of β-glucuronidase Enzymes and Sample Cleanup Methods.

    Science.gov (United States)

    Yang, He S; Wu, Alan H B; Lynch, Kara L

    2016-06-01

    With the rise in the use and misuse of prescription opioids, there is an increasing need for the confirmed identification of opioid analgesics in toxicology laboratories. The goals of this study were to (i) systematically evaluate the hydrolysis efficiency of four β-glucuronidase enzymes under optimized condition; (ii) evaluate compound recovery, matrix effects and precision of three protein precipitation plates and (iii) develop and validate a qualitative liquid-chromatography mass spectrometry (LC-MS/MS) assay to identify 13 opioids in urine. A recombinant β-glucuronidase exhibited the best overall hydrolysis efficiency for seven opioid glucuronide conjugates compared with β-glucuronidase from red abalone, Escherichia coli and Patella vulgata One of the protein precipitation plates tested exhibited overall better recovery of the opioids and lower ion suppression compared with the other two plates. An ESI positive mode LC-MS/MS assay for qualitative opioid analysis was developed and validated. Linearity, LOD, precision, matrix effect, recovery, carryover and interference of the method were evaluated. Sixty-two patient samples were analyzed by both a legacy GC-MS opioid method and the LC-MS/MS method, and 22 samples were analyzed by the LC-MS/MS and an LC-MS/MS reference method. The results of the comparisons showed good concordance. Overall, we described an efficient sample preparation procedure for a sensitive qualitative opioid confirmation assay in urine. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. A Modified LC/MS/MS Method with Enhanced Sensitivity for the Determination of Scopolamine in Human Plasma

    Science.gov (United States)

    Wang, Zuwei; Vaksman, Zalman; Putcha, Lakshmi

    2008-01-01

    Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC/MS

  10. LC-MS/MS determination of acrylamide in instant noodles from supermarkets in the Hebei province of China.

    Science.gov (United States)

    Yang, Li-Xin; Zhang, Gui-Xiang; Yang, Li-Xue; He, Yan

    2012-01-01

    Acrylamide (AA) concentrations in instant noodles (90 samples, covering 10 different brands) from Hebei Province of China were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The instant noodles were sampled from the southern and northern areas of Hebei Province (Shijiazhuang and Tangshan, respectively). The average content of AA for all 10 instant noodle brands was 6-145 µg/kg. The average content of AA in fried instant noodles was 4.47 times of those in non-fried ones, indicating the influence of the frying process. The average content of AA in instant noodles from Shijiazhuang was 1.64 times that of the samples from Tangshan (56 µg/kg). Eighty-four percent of the instant noodle samples in Hebei were contaminated with AA, with an average content of 80 µg/kg. These observations will be helpful for evaluating individual exposure to AA from instant noodles in China.

  11. Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Mingmei Yuan

    Full Text Available An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999, sensitivity (limit of quantitation, 0.16 mg/100 g, recovery (83.1-91.6%, precision (RSD < 5.4% and repeatability (RSD < 7.7%. To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60-150.56 mg/100 g.

  12. Skin sample preparation by collagenase digestion for diclofenac quantification using LC-MS/MS after topical application.

    Science.gov (United States)

    Nirogi, Ramakrishna; Padala, Naga Surya Prakash; Boggavarapu, Rajesh Kumar; Kalaikadhiban, Ilayaraja; Ajjala, Devender Reddy; Bhyrapuneni, Gopinadh; Muddana, Nageswara Rao

    2016-06-01

    Skin is the target site to evaluate the pharmacokinetic parameters of topical applications. Sample preparation is one of the influential steps in the bioanalysis of drugs in the skin. Evaluation of dermatopharmacokinetics at preclinical stage is challenging due to lack of proper sample preparation method. There is a need for an efficient sample preparation procedure for quantification of drugs in the skin using LC-MS/MS. The skin samples treated with collagenase followed by homogenization using a bead beater represents a best-fit method resulting in uniform homogenate for reproducible results. A new approach involving enzymatic treatment and mechanical homogenization techniques were evaluated for efficient sample preparation of skin samples in the bioanalysis.

  13. LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

    Science.gov (United States)

    Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David

    2016-12-01

    We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

  14. Antioxidant Activity of Flaxseed (Linum usitatissimum L. shell and Analysis of Its Polyphenol Contents by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hatica Han

    2018-07-01

    Full Text Available Flaxseed (Linum usitatissimum L. is important source of oil and protein for industrial, pharmaceutical, and nutritional applications. In order to estimate the effects of lyophilized aqueous extract of flaxseed shell (AEF and evaporated ethanolic extract of flaxseed shell (EEF, we studied their DPPH, ABTS, DMPD and O 2 •- scavenging effects. Total antioxidant activity by ferric thiocyanate method, Fe 3+, Cu 2+ and [Fe 3+-(TPTZ 2] 3+ reducing ability, and Fe 2+ chelating activity. Also, α-tocopherol, BHA, trolox, and BHT were used as positive controls. The results clearly AEF and EEF demonstrated effective antioxidant activity. The quantity of p-hydroxybenzoic, vanillin, p-coumaric acid, ascorbic acid, ferulic acid, and ellagic acid were investigated by LC-MS/MS. The present study will introduce a novelty for further studies on the antioxidant effects of AEF and EEF.

  15. Mycotoxin and fungicide residues in wheat grains from fungicide-treated plants measured by a validated LC-MS method.

    Science.gov (United States)

    da Luz, Suzane Rickes; Pazdiora, Paulo Cesar; Dallagnol, Leandro José; Dors, Giniani Carla; Chaves, Fábio Clasen

    2017-04-01

    Wheat (Triticum aestivum) is an annual crop, cultivated in the winter and spring and susceptible to several pathogens, especially fungi, which are managed with fungicides. It is also one of the most consumed cereals, and can be contaminated by mycotoxins and fungicides. The objective of this study was to validate an analytical method by LC-MS for simultaneous determination of mycotoxins and fungicide residues in wheat grains susceptible to fusarium head blight treated with fungicides, and to evaluate the relationship between fungicide application and mycotoxin production. All parameters of the validated analytical method were within AOAC and ANVISA limits. Deoxynivalenol was the prevalent mycotoxin in wheat grain and epoxiconazole was the fungicide residue found in the highest concentration. All fungicidal treatments induced an increase in AFB2 production when compared to the control (without application). AFB1 and deoxynivalenol, on the contrary, were reduced in all fungicide treatments compared to the control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Discovery of Food Exposure Markers in Urine and Evaluation of Dietary Compliance by Untargeted LC-MS Metabolomics

    DEFF Research Database (Denmark)

    Andersen, Maj-Britt Schmidt

    of individual foods. In this thesis, untargeted metabolomics, a relatively new method within nutrition research, has been applied to find new potential food exposure markers in urine for intake of a range of foods. In addition, it has been investigated, if it is possible to distinguish two dietary patterns...... intervention study with NND and ADD. By application of LC-MS based untargeted metabolomics, 35 markers related to intake of specific foods were found such as cabbage, citrus, beetroot, walnuts and fish. Some of the markers were found consistently in all studies and were therefore very promising new urinary......, a New Nordic Diet (NND) and an Average Danish Diet (ADD), in urine samples from a controlled intervention study. Data from three studies are included in the thesis: A cross-over meal study with nine different meals, a range of small meal studies with individual foods and a six month parallel...

  17. LC-MS/MS method development for quantitative analysis of acetaminophen uptake by the aquatic fungus Mucor hiemalis.

    Science.gov (United States)

    Esterhuizen-Londt, Maranda; Schwartz, Katrin; Balsano, Evelyn; Kühn, Sandra; Pflugmacher, Stephan

    2016-06-01

    Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Quantification of fumarate and investigation of endogenous and exogenous fumarate stability in rat plasma by LC-MS/MS.

    Science.gov (United States)

    Shi, Yao; Tse, Susanna; Rago, Brian; Yapa, Udeni; Li, Fumin; Fast, Douglas M

    2016-04-01

    Fumaric acid is a commonly used excipient in pharmaceutical products. It is not known if its presence may lead to fluctuation of endogenous fumarate levels. An LC-MS/MS method was developed and validated to quantify fumarate in support of a toxicokinetics study. Stability evaluation showed that endogenous fumarate was stable for 6 h at room temperature, while exogenously added fumaric acid was converted to malate within 1 h due to the presence of fumarase. Citric acid, a fumarase inhibitor, prevented the conversion of added fumaric acid in rat plasma. The method was validated in citric acid stabilized rat plasma using a surrogate matrix approach. A discrepancy in stability was observed between endogenous fumarate and exogenously added fumaric acid.

  19. Determination of dimethyltryptamine and β-carbolines (ayahuasca alkaloids) in plasma samples by LC-MS/MS.

    Science.gov (United States)

    Oliveira, Carolina Dizioli Rodrigues; Okai, Guilherme Gonçalves; da Costa, José Luiz; de Almeida, Rafael Menck; Oliveira-Silva, Diogo; Yonamine, Mauricio

    2012-07-01

    Ayahuasca is a psychoactive plant beverage originally used by indigenous people throughout the Amazon Basin, long before its modern use by syncretic religious groups established in Brazil, the USA and European countries. The objective of this study was to develop a method for quantification of dimethyltryptamine and β-carbolines in human plasma samples. The analytes were extracted by means of C18 cartridges and injected into LC-MS/MS, operated in positive ion mode and multiple reaction monitoring. The LOQs obtained for all analytes were below 0.5 ng/ml. By using the weighted least squares linear regression, the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.5 to 100 ng/ml; r(2)> 0.98). The method proved to be simple, rapid and useful to estimate administered doses for further pharmacological and toxicological investigations of ayahuasca exposure.

  20. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins

    DEFF Research Database (Denmark)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per

    2018-01-01

    in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining...... the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced...... accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data....

  1. On studying protein phosphorylation patterns using bottom-up LC-MS/MS: the case of human alpha-casein

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Savitski, Mikhail M; Nielsen, Michael L

    2007-01-01

    -LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, alpha(s1)-casein from human milk was selected as a model molecule representing...... moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other...... minerals to the new-born. Human alpha(s1)-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found...

  2. Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

    Science.gov (United States)

    Gao, Xinliu; Lin, Hui; Krantz, Carsten; Garnier, Arlette; Flarakos, Jimmy; Tse, Francis L S; Li, Wenkui

    2016-01-01

    Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. The validated method was robust and implemented in support of a preclinical PK/PD study.

  3. Simultaneous assay of multiple antibiotics in human plasma by LC-MS/MS: importance of optimizing formic acid concentration.

    Science.gov (United States)

    Chen, Feng; Hu, Zhe-Yi; Laizure, S Casey; Hudson, Joanna Q

    2017-03-01

    Optimal dosing of antibiotics in critically ill patients is complicated by the development of resistant organisms requiring treatment with multiple antibiotics and alterations in systemic exposure due to diseases and extracorporeal drug removal. Developing guidelines for optimal antibiotic dosing is an important therapeutic goal requiring robust analytical methods to simultaneously measure multiple antibiotics. An LC-MS/MS assay using protein precipitation for cleanup followed by a 6-min gradient separation was developed to simultaneously determine five antibiotics in human plasma. The precision and accuracy were within the 15% acceptance range. The formic acid concentration was an important determinant of signal intensity, peak shape and matrix effects. The method was designed to be simple and successfully applied to a clinical pharmacokinetic study.

  4. A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

    Science.gov (United States)

    Ji, Allena Ji; Wang, Haixing; Ziso-Qejvanaj, Enida; Zheng, Kefei; Chung, Lee Lee; Foley, Timothy; Chuang, Wei-Lien; Richards, Susan; Sung, Crystal

    2015-01-01

    Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. The new approach greatly improves assay precision and accuracy.

  5. Measurement of trace levels of antibiotics in river water using on-line enrichment and triple-quadrupole LC-MS/MS.

    Science.gov (United States)

    Dinh, Quoc Tuc; Alliot, Fabrice; Moreau-Guigon, Elodie; Eurin, Joëlle; Chevreuil, Marc; Labadie, Pierre

    2011-09-15

    This study presents the development of an automated on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 23 antibiotics in environmental water samples. After optimisation of LC-MS/MS conditions, SPE parameters such as sorbent type, sample pH or sample volume were optimised. Antibiotic recoveries ranged from 64% to 98% and compared favourably with those achieved using off-line SPE. Limits of detection were in the range 0.5-13.7 ng L(-1). This on-line SPE-LC-MS/MS procedure was applied to the analysis of water samples taken in three rivers within the Seine River basin, near Paris (France). The obtained results revealed the occurrence of 12 antibiotics, including tylosin, erythromycin, tetracycline, amoxicillin, trimethoprim, sulfamethoxazole, oxolinic acid, flumequine, norfloxacin, ciprofloxacin, ofloxacin, and vancomycin (2-1435 ng L(-1)). Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

    Science.gov (United States)

    Ivleva, Vera B.; Schneck, Nicole A.; Gollapudi, Deepika; Arnold, Frank; Cooper, Jonathan W.; Lei, Q. Paula

    2018-05-01

    CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule's biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant. [Figure not available: see fulltext.

  7. Proteomic analysis of protein interactions between Eimeria maxima sporozoites and chicken jejunal epithelial cells by shotgun LC-MS/MS.

    Science.gov (United States)

    Huang, Jingwei; Liu, Tingqi; Li, Ke; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2018-04-04

    Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion.

  8. Analysis of oxcarbazepine and the 10-hydroxycarbazepine enantiomers in plasma by LC-MS/MS: application in a pharmacokinetic study.

    Science.gov (United States)

    de Jesus Antunes, Natalicia; Wichert-Ana, Lauro; Coelho, Eduardo Barbosa; Della Pasqua, Oscar; Alexandre, Veriano; Takayanagui, Osvaldo Massaiti; Tozatto, Eduardo; Lanchote, Vera Lucia

    2013-12-01

    Oxcarbazepine is a second-generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic-clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10-hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)-(+)- and R-(-)-MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert-butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD-H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC-MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S-(+)-MHD enantiomer compared to R-(-)-MHD and an AUC(0-12) S-(+)/R-(-) ratio of 5.44. © 2013 Wiley Periodicals, Inc.

  9. Supercritical CO₂assisted extraction and LC-MS identification of picroside I and picroside II from Picrorhiza kurroa.

    Science.gov (United States)

    Patil, Ajit A; Sachin, Bhusari S; Shinde, Devanand B; Wakte, Pravin S

    2013-02-01

    Picroside I and picroside II have been studied intensively because of their pharmacological actions and clinical applications. Numerous methods have been reported for extracting picroside I and picroside II from Picrorrhiza. kurroa rhizomes. This is the first report of picroside I and picroside II extraction using the supercritical carbon dioxide assisted extraction technique. To develop supercritical carbon dioxide assisted extraction and LC-MS identification of picroside I and picroside II from the Picrorrhiza kurroa Royle rhizomes. Surface response methodology based on 3³ fractional factorial design was used to extract picroside I and picroside II from P. kurroa rhizomes. The effects of various process factors, namely temperature (40-80°C), pressure (25-35 MPa) and co-solvent (methanol) concentration (0-10% v/v) on extraction yield of the two compounds were evaluated. The picroside I and picroside II contents were determined using validated LC-MS methodology. The maximum yield of picroside I (32.502 ± 1.131 mg/g) and picroside II (9.717 ± 0.382 mg/g) was obtained at the 10% v/v co-solvent concentration, 40°C temperature and 30 MPa pressure. The conventional Soxhlet assisted methanol extract of P. kurroa powder resulted in 36.743 ± 1.75 and 11.251 ± 0.54 mg/g yield of picroside I and picroside II, respectively. Variation of concentration and extraction time showed a significant effect on the picroside I and picroside II yield. Supercritical carbon dioxide assisted extraction using methanol as a co-solvent is an efficient and environmentally sustainable method for extracting picroside I and picroside II from P. kurroa rhizomes. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Immunosuppressant therapeutic drug monitoring by LC-MS/MS: workflow optimization through automated processing of whole blood samples.

    Science.gov (United States)

    Marinova, Mariela; Artusi, Carlo; Brugnolo, Laura; Antonelli, Giorgia; Zaninotto, Martina; Plebani, Mario

    2013-11-01

    Although, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. Six-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. Calibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. Our findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods. © 2013.

  11. Validation of a method for the determination of 120 pesticide residues in apples and cucumbers by LC-MS/MS.

    Science.gov (United States)

    Ramadan, Gouda; Al Jabir, Muna; Alabdulmalik, Najat; Mohammed, Ali

    2016-05-01

    Most countries have clearly defined regulations governing the use of pesticides in agricultural activity. The application of pesticides in agriculture usually leads to a residual amount of these pesticides on food products such as fruit and vegetables. The presence of pesticide residues on these foods destined for human consumption may pose food safety risks to consumers. To protect consumers, national authorities have established maximum limits for pesticide residues in foods. These limits can only be enforced if there are methods available to detect and monitor their concentrations in the applicable food products. To support the enforcement of this legislation, we have developed a multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of 120 pesticide residues in apples and cucumbers which has been validated and implemented in the routine monitoring and surveillance programme for these pesticides. In this method, apple and cucumber samples are extracted using the QuEChERS method (quick, easy, cheap, effective, rugged, and safe) and the extracts were analyzed directly by LC-MS/MS. The mean recoveries at three different concentrations of 0.01 µg/g , 0.05 µg/g, and 0.1 µg/g over the analytical range varied between 70 and 120%. The repeatability of the method expressed as %RSD was less than 20%. The limit of detection (LOD) of the method ranged between 0.0014 and 0.0110 µg/g for apples and between 0.0012 and 0.0075 µg/g for cucumbers. The limit of quantification (LOQ) of the method was 0.01 µg/g for apples and cucumbers. The method has been used for the analysis of over 600 apple and 550 cucumber samples over the past two years. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. The simultaneous detection and quantification of p-aminobenzoic acid and its phase 2 biotransformation metabolites in human urine using LC-MS/MS.

    Science.gov (United States)

    Nortje, Carla; Jansen van Rensburg, Peet; Cooke, Cecile; Erasmus, Elardus

    2015-01-01

    p-Aminobenzoic acid (PABA) can be used as a probe substance to investigate glycine conjugation, a reaction of phase 2 biotransformation. An LC-MS/MS method for simultaneous quantification of PABA and its metabolites from human urine was developed and validated. The metabolites can be quantified with acceptable precision and accuracy directly from human urine samples after ingestion of 550 mg PABA. The developed LC-MS/MS assay is to our knowledge the first method available for the simultaneous quantification of PABA and its glycine conjugation metabolites in human urine and provides important quantitative data for studies of this phase 2 biotransformation pathway.

  13. LC-MS/MS法测定饮用水中4-叔丁基苯酚的含量%Determination of 4-tert-butylphenol in drinking water by LC-MS/MS

    Institute of Scientific and Technical Information of China (English)

    丁可轩; 王雪平

    2017-01-01

    建立了液相色谱质谱法(LC-MS/MS)测定饮用水中4-叔丁基苯酚含量的方法.试样经HLB固相萃取柱富集,叔丁基醚萃取洗脱,丹酰氯衍生化后,采用BEIIC18色谱柱分离,三重四级杆质谱仪测定,外标法定量.结果表明:4-叔丁基苯酚在0.5~50.0 ng/mL范围内,具有良好的线性关系,相关系数R2为0.999 8;方法检出限为0.1 ng/mL;加标回收率为85.3%~97.4%,相对标准偏差为0.9%~5.5%.该方法检出限低,准确度高,精密度好.%A method for determination of 4-tert-butylphenol in drinking water by liquid chromatography mass spectrometry (LC-MS/MS) is established.The sample is prepared by collecting in HLB solid phase extraction column,then being extracted and eluted by tert butyl ether,and then deriving through dansyl chloride,and finally being separated by BEH C18 chromatography column.The prepared sample is determined by the triple quadrupole mass spectrometer,and quantified by external standard method.The results show that the linear correlation coefficient R2 is 0.999 8 in the 4-tertbutylphenol concentration range of 0.5-50.0 ng/mL,indicating a good linear relation.The detection limit is 0.1 ng/mL.The standard recoveries are between 85.3% and 97.4%,and the relative standard deviation is between 0.9% and 5.5%.This detection method features with low detection limit,high accuracy and good precision.

  14. Quantitative Determination of Perfluorochemicals and Fluorotelomer Alcohols in Plants from Biosolid-Amended Fields using LC/MS/MS and GC/MS

    Science.gov (United States)

    Analytical methods for determining perfluorochemicals (PFCs) and fluorotelomer alcohols (FTOHs) in plants using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) were developed, and applied to quantify a suite of analytes i...

  15. Discrimination of conventional and organic white cabbage from a long-term field trial study using untargeted LC-MS-based metabolomics

    DEFF Research Database (Denmark)

    Mie, Axel; Laursen, Kristian Holst; Åberg, K. Magnus

    2014-01-01

    The influence of organic and conventional farming practices on the content of single nutrients in plants is disputed in the scientific literature. Here, large-scale untargeted LC-MS-based metabolomics was used to compare the composition of white cabbage from organic and conventional agriculture, ...

  16. Comparison of approaches to deal with matrix effects in LC-MS/MS based determinations of mycotoxins in food and feed

    NARCIS (Netherlands)

    Fabregat-Cabello, N.; Zomer, P.; Sancho, J.V.; Roig-Navarro, A.F.; Mol, H.G.J.

    2016-01-01

    This study deals with one of the major concerns in mycotoxin determinations: The matrix effect related to LC-MS/ MS systems with electrospray ionization sources. To this end, in a first approach, the matrix effect has been evaluated in two ways: monitoring the signal of a compound (added to the

  17. Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS)

    NARCIS (Netherlands)

    Solfrizzo, M.; Girolamo, De A.; Lattanzio, V.M.T.; Visconti, A.; Stroka, J.; Alldrick, A.; Egmond, van H.P.

    2013-01-01

    Liquid chromatography coupled with single or tandem mass spectrometry (LC-MS/(MS)) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by the European Committee for Standardization and the

  18. Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE and LC-MS/MS

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Nørregaard Jensen, Ole

    2006-01-01

    was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral...

  19. APPLICATION OF LIQUID-CHROMATOGRAPHY COMBINED WITH MASS-SPECTROMETRY (LC-MS) TO ESTABLISH IDENTITY AND PURITY OF PET-RADIOPHARMACEUTICALS

    NARCIS (Netherlands)

    FRANSSEN, EJF; LUURTSEMA, G; MEDEMA, J; VISSER, GM; JERONISMUSSHALINGH, CM; BRUINS, AP; VAALBURG, W

    This article describes the application of liquid chromatography combined with mass-spectrometry (LC-MS) as a new quality control tool for PET-radiopharmaceuticals. The final step in the production of 2-[F-18]fluoro-2-deoxy-D-glucose (F-18-FDG) is a purification by HPLC. This procedure was validated

  20. Development and Validation of an LC-MS/MS Method and Comparison with a GC-MS Method to Measure Phenytoin in Human Brain Dialysate, Blood, and Saliva

    Directory of Open Access Journals (Sweden)

    Raphael Hösli

    2018-01-01

    Full Text Available Phenytoin (PHT is one of the most often used critical dose drugs, where insufficient or excessive dosing can have severe consequences such as seizures or toxicity. Thus, the monitoring and precise measuring of PHT concentrations in patients is crucial. This study develops and validates an LC-MS/MS method for the measurement of phenytoin concentrations in different body compartments (i.e., human brain dialysate, blood, and saliva and compares it with a formerly developed GC-MS method that measures PHT in the same biological matrices. The two methods are evaluated and compared based on their analytical performance, appropriateness to analyze human biological samples, including corresponding extraction and cleanup procedures, and their validation according to ISO 17025/FDA Guidance for Industry. The LC-MS/MS method showed a higher performance compared with the GC-MS method. The LC-MS/MS was more sensitive, needed a smaller sample volume (25 µL and less chemicals, was less time consuming (cleaning up, sample preparation, and analysis, and resulted in a better LOD ( 0.995 for all tested matrices (blood, saliva, and dialysate. For larger sample numbers as in pharmacokinetic/pharmacodynamic studies and for bedside as well as routine analyses, the LC-MS/MS method offers significant advantages over the GC-MS method.

  1. An LC-MS Assay with Isocratic Separation and On-Line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma

    Directory of Open Access Journals (Sweden)

    Ialongo Cristiano

    2017-04-01

    Full Text Available Background: Busulfan (Bu requires therapeutic drug monitoring (TDM in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT. To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE of samples.

  2. LC-MS/MS analysis of lipidized analogs of prolactin-releasing peptide utilizing a monolithic column and simple sample preparation

    Czech Academy of Sciences Publication Activity Database

    Zemenová, Jana; Sýkora, D.; Freislebenová, A.; Maletínská, Lenka

    2017-01-01

    Roč. 9, č. 17 (2017), s. 1319-1328 ISSN 1757-6180 R&D Projects: GA ČR(CZ) GA15-08679S Institutional support: RVO:61388963 Keywords : LC-MS * lipopeptides * monolithic column * prolactin-releasing peptide * stability Subject RIV: CE - Biochemistry OBOR OECD: Biochemical research methods Impact factor: 2.673, year: 2016

  3. A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

    DEFF Research Database (Denmark)

    Richardson, Katherine; Denny, R.; Hughes, C.

    2012-01-01

    A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data un...

  4. Multiple testing issues in discriminating compound-related peaks and chromatograms from high frequency noise, spikes and solvent-based nois in LC-MS data sets

    NARCIS (Netherlands)

    Nyangoma, S.O.; Van Kampen, A.A.; Reijmers, T.H.; Govorukhina, N.I; van der Zee, A.G.; Billingham, I.J; Bischoff, Rainer; Jansen, R.C.

    2007-01-01

    Multiple testing issues in discriminating compound-related peaks and chromatograms from high frequency noise, spikes and solvent-based noise in LC-MS data sets.Nyangoma SO, van Kampen AA, Reijmers TH, Govorukhina NI, van der Zee AG, Billingham LJ, Bischoff R, Jansen RC. University of Birmingham.

  5. Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

    Science.gov (United States)

    Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

    2004-06-29

    An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

  6. Single Laboratory Validated Method for Determination of Cylindrospermopsin and Anatoxin-a in Ambient Water by Liquid Chromatography/ Tandem Mass Spectrometry (LC/MS/MS)

    Science.gov (United States)

    This product is an LC/MS/MS single laboratory validated method for the determination of cylindrospermopsin and anatoxin-a in ambient waters. The product contains step-by-step instructions for sample preparation, analyses, preservation, sample holding time and QC protocols to ensu...

  7. Validation and implementation of liquid chromatographic-mass spectrometric (LC-MS) methods for the quantification of tenofovir prodrugs.

    Science.gov (United States)

    Hummert, Pamela; Parsons, Teresa L; Ensign, Laura M; Hoang, Thuy; Marzinke, Mark A

    2018-04-15

    The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. K 2 EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 μm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 μm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ± 7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also

  8. Enantioselective analysis of chloramphenicol residues in honey samples by chiral LC-MS/MS and results of a honey survey.

    Science.gov (United States)

    Rimkus, Gerhard G; Hoffmann, Dirk

    2017-06-01

    Chloramphenicol (CAP) is a broad-spectrum antibiotic used widely in both human and veterinary medication. Since 1994, CAP has not been authorised for use in food-producing animals in the European Union due to several adverse effects. A minimum required performance level (MRPL) of 0.3 µg kg - 1 was established in 2003. The CAP molecule contains two asymmetric centres, thus in total four para-CAP stereoisomers exist. Only the RR-CAP enantiomer is bioactive, having significant antimicrobial activity. For the first time a chiral LC-MS/MS method is reported to identify and quantify the four CAP enantiomers at residue levels in honey samples. The method was validated at two concentration levels. The decision limits (CCα) and detection capabilities (CCß) were well below 0.3 µg kg - 1 , with limits of quantification (LOQs) between 0.08 and 0.12 µg kg - 1 for all four enantiomers. The method provides a sensitive and reliable analysis of CAP enantiomers in honey, and proved its robustness during the daily routine analyses of numerous honey samples. In an internal honey survey, in total 40 honey samples from different geographical regions with identified CAP residues at or above the MRPL were reanalysed by chiral LC-MS/MS. In nine honey samples only the bioactive RR-CAP was detected as anticipated. However, in all other 31 honey samples the non-bioactive SS-CAP was also identified and quantified unambiguously. In 10 of these samples, mixtures of RR- and SS-CAP were analysed, and in 21 samples only the SS-CAP enantiomer, with concentrations up to 2.2 µg kg - 1 . Most of these samples are honeys from Ukraine and Eastern Europe. This is the first report of SS-CAP residues in food samples. The potential sources for these findings are discussed and the need of further systematic studies emphasised. It is recommended to examine in more depth the toxicological profile of the individual CAP stereoisomers.

  9. A Conversation on Data Mining Strategies in LC-MS Untargeted Metabolomics: Pre-Processing and Pre-Treatment Steps

    Directory of Open Access Journals (Sweden)

    Fidele Tugizimana

    2016-11-01

    Full Text Available Untargeted metabolomic studies generate information-rich, high-dimensional, and complex datasets that remain challenging to handle and fully exploit. Despite the remarkable progress in the development of tools and algorithms, the “exhaustive” extraction of information from these metabolomic datasets is still a non-trivial undertaking. A conversation on data mining strategies for a maximal information extraction from metabolomic data is needed. Using a liquid chromatography-mass spectrometry (LC-MS-based untargeted metabolomic dataset, this study explored the influence of collection parameters in the data pre-processing step, scaling and data transformation on the statistical models generated, and feature selection, thereafter. Data obtained in positive mode generated from a LC-MS-based untargeted metabolomic study (sorghum plants responding dynamically to infection by a fungal pathogen were used. Raw data were pre-processed with MarkerLynxTM software (Waters Corporation, Manchester, UK. Here, two parameters were varied: the intensity threshold (50–100 counts and the mass tolerance (0.005–0.01 Da. After the pre-processing, the datasets were imported into SIMCA (Umetrics, Umea, Sweden for more data cleaning and statistical modeling. In addition, different scaling (unit variance, Pareto, etc. and data transformation (log and power methods were explored. The results showed that the pre-processing parameters (or algorithms influence the output dataset with regard to the number of defined features. Furthermore, the study demonstrates that the pre-treatment of data prior to statistical modeling affects the subspace approximation outcome: e.g., the amount of variation in X-data that the model can explain and predict. The pre-processing and pre-treatment steps subsequently influence the number of statistically significant extracted/selected features (variables. Thus, as informed by the results, to maximize the value of untargeted metabolomic data

  10. Applying quantitative metabolomics based on chemical isotope labeling LC-MS for detecting potential milk adulterant in human milk.

    Science.gov (United States)

    Mung, Dorothea; Li, Liang

    2018-02-25

    There is an increasing demand for donor human milk to feed infants for various reasons including that a mother may be unable to provide sufficient amounts of milk for their child or the milk is considered unsafe for the baby. Selling and buying human milk via the Internet has gained popularity. However, there is a risk of human milk sold containing other adulterants such as animal or plant milk. Analytical tools for rapid detection of adulterants in human milk are needed. We report a quantitative metabolomics method for detecting potential milk adulterants (soy, almond, cow, goat and infant formula milk) in human milk. It is based on the use of a high-performance chemical isotope labeling (CIL) LC-MS platform to profile the metabolome of an unknown milk sample, followed by multivariate or univariate comparison of the resultant metabolomic profile with that of human milk to determine the differences. Using dansylation LC-MS to profile the amine/phenol submetabolome, we could detect an average of 4129 ± 297 (n = 9) soy metabolites, 3080 ± 470 (n = 9) almond metabolites, 4256 ± 136 (n = 18) cow metabolites, 4318 ± 198 (n = 9) goat metabolites, 4444 ± 563 (n = 9) infant formula metabolites, and 4020 ± 375 (n = 30) human metabolites. This high level of coverage allowed us to readily differentiate the six different types of samples. From the analysis of binary mixtures of human milk containing 5, 10, 25, 50 and 75% other type of milk, we demonstrated that this method could be used to detect the presence of as low as 5% adulterant in human milk. We envisage that this method could be applied to detect contaminant or adulterant in other types of food or drinks. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS.

    Science.gov (United States)

    Kikura-Hanajiri, Ruri; Kawamura, Maiko; Miyajima, Atsuko; Sunouchi, Momoko; Goda, Yukihiro

    2011-04-01

    In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid-acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out

  12. Postmortem Kanda LC-MS MS ile Yasadışı Madde Taraması 62 Postmortem Adli Olguda Uygulanması

    Directory of Open Access Journals (Sweden)

    Serap Annette Akgür

    2007-04-01

    Full Text Available Biyolojik örneklerdeki yasadışı madde taraması adli olaylarda büyük bir öneme sahiptir. Kan veya plazma, madde derişim-leri ile bu maddelerin farmakolojik etkileri arasında iyi bir korelasyonun bulunması nedeniyle genellikle tercih edilen materyallerdir. Yasadışı madde analizinde, birçok laboratuar immunoassay yöntemleri ile tarama ve Gaz Kromatografi-Kütle Spektrometresi (GC-MS ile doğrulama şeklinde bir uygulama yapmaktadır. Bununla birlikte, sıvı kromatografi tekniklerinin gelişmesiyle birlikte Sıvı Kromatografisi-Kütle Spektrometresi (LC-MS ve tandem LC-MS (LC-MS/MS birleştirilmiş tekniklerinde özellikle polar ve ısıya dayanıksız olan maddelerin nitel ve nicel analiz amaçlı olarak kullanımı zamanla artmaktadır. Bu çalışmada, LC-ESI (Electrospray İonisation-MS/MS de morfin, (±-amfetamin, (+-metamfetamin, kokain, benzolekgonin, kokaetilen ve (--ll-nor-9-karboksi-t9-THC maddeleri taranmış ve tayin edilmiştir. Sonuç olarak, postmortem kanda uygulanabilecek, türevlendirme işlemine gerek duymayan, duyarlı ve seçimli olarak, çok düşük düzeylerdeki maddelerin doğru ve kesin olarak saptanmasını sağlayacak, LC-MS/MS yöntemi geliştirilmiş ve 62 postmortem adli olguya başarıyla uygulanmıştır. Anahtar kelimeler: Yasadışı madde, tarama, LC/MS/MS, postmortem kan

  13. In Vivo Metabolism Study of Xiamenmycin A in Mouse Plasma by UPLC-QTOF-MS and LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Feng Lei

    2015-01-01

    Full Text Available Xiamenmycin A is an antifibrotic leading compound with a benzopyran skeleton that is isolated from mangrove-derived Streptomyces xiamenensis. As a promising small molecule for fibrotic diseases, less information is known about its metabolic characteristics in vivo. In this study, the time-course of xiamenmycin A in mouse plasma was investigated by relative quantification. After two types of administration of xiamenmycin A at a single dose of 10 mg/kg, the plasma concentrations were measured quantitatively by LC-MS/MS. The dynamic changes in the xiamenmycin A concentration showed rapid absorption and quick elimination in plasma post-administration. Four metabolites (M1–M4 were identified in blood by UPLC-QTOF-MS, and xiamenmycin B (M3 is the principal metabolite in vivo, as verified by comparison of the authentic standard sample. The structures of other metabolites were identified based on the characteristics of their MS and MS/MS data. The newly identified metabolites are useful for understanding the metabolism of xiamenmycin A in vivo, aiming at the development of an anti-fibrotic drug candidate for the therapeutic treatment of excessive fibrotic diseases.

  14. Orthogonal analytical methods for botanical standardization: determination of green tea catechins by qNMR and LC-MS/MS.

    Science.gov (United States)

    Napolitano, José G; Gödecke, Tanja; Lankin, David C; Jaki, Birgit U; McAlpine, James B; Chen, Shao-Nong; Pauli, Guido F

    2014-05-01

    The development of analytical methods for parallel characterization of multiple phytoconstituents is essential to advance the quality control of herbal products. While chemical standardization is commonly carried out by targeted analysis using gas or liquid chromatography-based methods, more universal approaches based on quantitative (1)H NMR (qHNMR) measurements are being used increasingly in the multi-targeted assessment of these complex mixtures. The present study describes the development of a 1D qHNMR-based method for simultaneous identification and quantification of green tea constituents. This approach utilizes computer-assisted (1)H iterative Full Spin Analysis (HiFSA) and enables rapid profiling of seven catechins in commercial green tea extracts. The qHNMR results were cross-validated against quantitative profiles obtained with an orthogonal LC-MS/MS method. The relative strengths and weaknesses of both approaches are discussed, with special emphasis on the role of identical reference standards in qualitative and quantitative analyses. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application

    Directory of Open Access Journals (Sweden)

    Zhenzhen Cai

    2013-02-01

    Full Text Available A rapid sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS method was developed for determination of timosaponin AIII (TA-III in rat plasma, using ginsenoside Re as an internal standard (IS. TA-III and the IS were detected in MRM mode with a negative ionization electrospray mass spectrometer. The calibration curves were linear over the concentration ranges from 11.14 to 1114 ng/mL and the lower limit of quantification (LLOQ was 11.14 ng/mL. Intra-day and inter-day precisions (RSD were within 10%, and accuracy ranged from 6.4% to 9.1%. The extraction recovery at three concentrations ranged from 92.3% to 95.5%. The validated method was successfully applied to monitor the concentrations of TA-III in rat plasma after intragastric administration. The best fit pharmacokinetic model to estimate the pharmacokinetic parameters was a single compartment model with weight of 1/x2 for oral administration groups of rats for TA-III.

  16. Assessment of bioavailable B vitamin content in food using in vitro digestibility assay and LC-MS SIDA.

    Science.gov (United States)

    Paalme, Toomas; Vilbaste, Allan; Kevvai, Kaspar; Nisamedtinov, Ildar; Hälvin-Tanilas, Kristel

    2017-11-01

    Standardized analytical methods, where each B vitamin is extracted from a given sample individually using separate procedures, typically ensure that the extraction conditions provide the maximum recovery of each vitamin. However, in the human gastrointestinal tract (GIT), the extraction conditions are the same for all vitamins. Here, we present an analytically feasible extraction protocol that simulates conditions in the GIT and provides a measure of the content of bioavailable vitamins using LC-MS stable isotope dilution assay. The results show that the activities of both human gastric and duodenal juices were insufficient to liberate absorbable vitamers (AV) from pure cofactors. The use of an intestinal brush border membrane (IBBM) fraction derived from the mucosal tissue of porcine small intestine ensured at least 70% AV recovery. The rate of AV liberation, however, was strongly dependent on the cofactor, e.g., in the case of NADH, it was magnitudes higher than in the case of thiamine diphosphate. For some vitamins in some food matrices, the use of the IBBM fraction assay resulted in lower values for the content of AV than conventional vitamin determination methods. Conventional methods likely overestimate the actual bioavailability of some vitamins in these cases. Graphical abstract Assessment of bioavailable B vitamin content in food.

  17. Development of a SIDA-LC-MS/MS Method for the Determination of Phomopsin A in Legumes.

    Science.gov (United States)

    Schloß, Svenja; Koch, Matthias; Rohn, Sascha; Maul, Ronald

    2015-12-09

    A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.

  18. Sample preparation prior to the LC-MS-based metabolomics/metabonomics of blood-derived samples.

    Science.gov (United States)

    Gika, Helen; Theodoridis, Georgios

    2011-07-01

    Blood represents a very important biological fluid and has been the target of continuous and extensive research for diagnostic, or health and drug monitoring reasons. Recently, metabonomics/metabolomics have emerged as a new and promising 'omics' platform that shows potential in biomarker discovery, especially in areas such as disease diagnosis, assessment of drug efficacy or toxicity. Blood is collected in various establishments in conditions that are not standardized. Next, the samples are prepared and analyzed using different methodologies or tools. When targeted analysis of key molecules (e.g., a drug or its metabolite[s]) is the aim, enforcement of certain measures or additional analyses may correct and harmonize these discrepancies. In omics fields such as those performed by holistic analytical approaches, no such rules or tools are available. As a result, comparison or correlation of results or data fusion becomes impractical. However, it becomes evident that such obstacles should be overcome in the near future to allow for large-scale studies that involve the assaying of samples from hundreds of individuals. In this case the effect of sample handling and preparation becomes very serious, in order to avoid wasting months of work from experts and expensive instrument time. The present review aims to cover the different methodologies applied to the pretreatment of blood prior to LC-MS metabolomic/metabonomic studies. The article tries to critically compare the methods and highlight issues that need to be addressed.

  19. An automated online turboflow cleanup LC/MS/MS method for the determination of 11 plasticizers in beverages and milk.

    Science.gov (United States)

    Ates, Ebru; Mittendorf, Klaus; Senyuva, Hamide

    2013-01-01

    An automated sample preparation technique involving cleanup and analytical separation in a single operation using an online coupled TurboFlow (RP-LC system) is reported. This method eliminates time-consuming sample preparation steps that can be potential sources for cross-contamination in the analysis of plasticizers. Using TurboFlow chromatography, liquid samples were injected directly into the automated system without previous extraction or cleanup. Special cleanup columns enabled specific binding of target compounds; higher MW compounds, i.e., fats and proteins, and other matrix interferences with different chemical properties were removed to waste, prior to LC/MS/MS. Systematic stepwise method development using this new technology in the food safety area is described. Selection of optimum columns and mobile phases for loading onto the cleanup column followed by transfer onto the analytical column and MS detection are critical method parameters. The method was optimized for the assay of 10 phthalates (dimethyl, diethyl, dipropyl, butyl benzyl, diisobutyl, dicyclohexyl, dihexyl, diethylhexyl, diisononyl, and diisododecyl) and one adipate (diethylhexyl) in beverages and milk.

  20. Improved Stable Isotope Dilution Assay for Dietary Folates Using LC-MS/MS and Its Application to Strawberries

    Directory of Open Access Journals (Sweden)

    Lisa Striegel

    2018-02-01

    Full Text Available Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were quantified using 13C-labeled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection, and limit of quantification and revealed to be a precise, sensitive, and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany and Australia were investigated. Total folates varied from 59 to 153 μg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16% of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.

  1. Generation of accurate peptide retention data for targeted and data independent quantitative LC-MS analysis: Chromatographic lessons in proteomics.

    Science.gov (United States)

    Krokhin, Oleg V; Spicer, Vic

    2016-12-01

    The emergence of data-independent quantitative LC-MS/MS analysis protocols further highlights the importance of high-quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP-HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP-HPLC as a "black box", while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion-pairing modifier, stationary phase and column temperature, we describe the "mysterious" effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation-a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S-values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Data processing has major impact on the outcome of quantitative label-free LC-MS analysis.

    Science.gov (United States)

    Chawade, Aakash; Sandin, Marianne; Teleman, Johan; Malmström, Johan; Levander, Fredrik

    2015-02-06

    High-throughput multiplexed protein quantification using mass spectrometry is steadily increasing in popularity, with the two major techniques being data-dependent acquisition (DDA) and targeted acquisition using selected reaction monitoring (SRM). However, both techniques involve extensive data processing, which can be performed by a multitude of different software solutions. Analysis of quantitative LC-MS/MS data is mainly performed in three major steps: processing of raw data, normalization, and statistical analysis. To evaluate the impact of data processing steps, we developed two new benchmark data sets, one each for DDA and SRM, with samples consisting of a long-range dilution series of synthetic peptides spiked in a total cell protein digest. The generated data were processed by eight different software workflows and three postprocessing steps. The results show that the choice of the raw data processing software and the postprocessing steps play an important role in the final outcome. Also, the linear dynamic range of the DDA data could be extended by an order of magnitude through feature alignment and a charge state merging algorithm proposed here. Furthermore, the benchmark data sets are made publicly available for further benchmarking and software developments.

  3. LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.

    Science.gov (United States)

    Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei

    2012-12-01

    Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Environmental contaminants of honeybee products in Uganda detected using LC-MS/MS and GC-ECD.

    Directory of Open Access Journals (Sweden)

    Deborah Ruth Amulen

    Full Text Available Pollinator services and the development of beekeeping as a poverty alleviating tool have gained considerable focus in recent years in sub-Saharan Africa. An improved understanding of the pervasive environmental extent of agro-chemical contaminants is critical to the success of beekeeping development and the production of clean hive products. This study developed and validated a multi-residue method for screening 36 pesticides in honeybees, honey and beeswax using LC-MS/MS and GC-ECD. Of the 36 screened pesticides, 20 were detected. The highest frequencies occurred in beeswax and in samples from apiaries located in the proximity of citrus and tobacco farms. Fungicides were the most prevalent chemical class. Detected insecticides included neonicotinoids, organophosphates, carbamates, organophosphorus, tetrazines and diacylhydrazines. All detected pesticide levels were below maximum residue limits (according to EU regulations and the lethal doses known for honeybees. However, future risk assessment is needed to determine the health effects on the African genotype of honeybees by these pesticide classes and combinations of these. In conclusion, our data present a significant challenge to the burgeoning organic honey sector in Uganda, but to achieve this, there is an urgent need to regulate the contact routes of pesticides into the beehive products. Interestingly, the "zero" detection rate of pesticides in the Mid-Northern zone is a significant indicator of the large potential to promote Ugandan organic honey for the export market.

  5. Mycobiota and Natural Incidence of Aflatoxins, Ochratoxin A, and Citrinin in Indian Spices Confirmed by LC-MS/MS

    Science.gov (United States)

    Jeswal, Punam; Kumar, Dhiraj

    2015-01-01

    Nine different Indian spices (red chilli, black pepper, turmeric, coriander, cumin, fennel, caraway, fenugreek, and dry ginger) commonly cultivated and highly used in India were analysed for natural occurrence of toxigenic mycoflora and aflatoxins (AFs), ochratoxin A (OTA), and citrinin (CTN) contamination. Aspergillus flavus and Aspergillus niger were the most dominant species isolated from all types of spices. Red chilli samples were highly contaminated with aflatoxins (85.4%) followed by dry ginger (77.7%). 56% Aspergillus flavus from red chilli and 45% Aspergillus ochraceus from black pepper were toxigenic and produced aflatoxins and ochratoxin A, respectively. Qualitative detection and quantitative detection of mycotoxins in spices were analyzed by ELISA and further confirmed by LC-MS/MS. Penicillium citrinum produced citrinin in red chilli, black pepper, coriander, cumin, fenugreek, and dry ginger samples. The highest amount of AFs was found in red chilli (219.6 ng/g), OTA was in black pepper (154.1 ng/g), and CTN was in dry ginger samples (85.1 ng/g). The results of this study suggest that the spices are susceptible substrate for growth of mycotoxigenic fungi and further mycotoxin production. This is the first report of natural occurrence of citrinin in black pepper and dry ginger from India. PMID:26229535

  6. Development of a LC-MS/MS Method for the Multi-Mycotoxin Determination in Composite Cereal-Based Samples

    Directory of Open Access Journals (Sweden)

    Barbara De Santis

    2017-05-01

    Full Text Available The analytical scenario for determining contaminants in the food and feed sector is constantly prompted by the progress and improvement of knowledge and expertise of researchers and by the technical innovation of the instrumentation available. Mycotoxins are agricultural contaminants of fungal origin occurring at all latitudes worldwide and being characterized by acute and chronic effects on human health and animal wellness, depending on the species sensitivity. The major mycotoxins of food concern are aflatoxin B1 and ochratoxin A, the first for its toxicity, and the second for its recurrent occurrence. However, the European legislation sets maximum limits for mycotoxins, such as aflatoxin B1, ochratoxin A, deoxynivalenol, fumonisins, and zearalenone, and indicative limits for T-2 and HT-2 toxins. Due to the actual probability that co-occurring mycotoxins are present in a food or feed product, nowadays, the availability of reliable, sensitive, and versatile multi-mycotoxin methods is assuming a relevant importance. Due to the wide range of matrices susceptible to mycotoxin contamination and the possible co-occurrence, a multi-mycotoxin and multi-matrix method was validated in liquid chromatography-tandem mass spectrometry (LC-MS/MS with the purpose to overcome specific matrix effects and analyze complex cereal-based samples within the Italian Total Diet Study project.

  7. LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.

    Science.gov (United States)

    Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H

    2017-02-05

    Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (neratinib. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Improved stable isotope dilution assay for dietary folates using LC-MS/MS and its application to strawberries

    Science.gov (United States)

    Striegel, Lisa; Chebib, Soraya; Netzel, Michael E.; Rychlik, Michael

    2018-02-01

    Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate were quantified using 13C-labelled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection and limit of quantification and revealed to be a precise, sensitive and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany were investigated. Total folates varied from 59 to 153 µg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16 % of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.

  9. Comparative Studies on the pH Dependence of DOW of Microcystin-RR and -LR Using LC-MS

    Directory of Open Access Journals (Sweden)

    Gaodao Liang

    2011-01-01

    Full Text Available Microcystins (MCs are well known worldwide as hepatotoxins produced by cyanobacteria, but little is known about the physicochemical properties of these compounds. The dependence of the n-octanol/water distribution ratio (DOW of MC-RR and -LR to pH was measured by high-performance liquid chromatography combined with mass spectrometry (LC-MS. There was a remarkable difference in such relationships between MC-RR and -LR. The log DOW of MC-LR decreased from 1.63 at pH 1.0 to -1.26 at pH 6.5, and stabilized between -1.04 and -1.56 at a pH of 6.5~12.0; log DOW of MC-RR varied between -1.24 and -0.67 at a pH of 1.00~4.00, and stabilized between -1.20 and -1.54 at a pH of 4.00~12.00. The difference of hydrophobicity in acidic condition between MC-RR and -LR is important, not only for the analytical method of both toxins, but perhaps also for understanding the difference of toxicity to animals between the two toxins.

  10. Quantitative evaluation of the matrix effect in bioanalytical methods based on LC-MS: A comparison of two approaches.

    Science.gov (United States)

    Rudzki, Piotr J; Gniazdowska, Elżbieta; Buś-Kwaśnik, Katarzyna

    2018-06-05

    Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for studying pharmacokinetics and toxicokinetics. Reliable bioanalysis requires the characterization of the matrix effect, i.e. influence of the endogenous or exogenous compounds on the analyte signal intensity. We have compared two methods for the quantitation of matrix effect. The CVs(%) of internal standard normalized matrix factors recommended by the European Medicines Agency were evaluated against internal standard normalized relative matrix effects derived from Matuszewski et al. (2003). Both methods use post-extraction spiked samples, but matrix factors require also neat solutions. We have tested both approaches using analytes of diverse chemical structures. The study did not reveal relevant differences in the results obtained with both calculation methods. After normalization with the internal standard, the CV(%) of the matrix factor was on average 0.5% higher than the corresponding relative matrix effect. The method adopted by the European Medicines Agency seems to be slightly more conservative in the analyzed datasets. Nine analytes of different structures enabled a general overview of the problem, still, further studies are encouraged to confirm our observations. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Simultaneous determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS.

    Science.gov (United States)

    Kakitani, Ayano; Inoue, Tomonori; Matsumoto, Keiko; Watanabe, Jun; Nagatomi, Yasushi; Mochizuki, Naoki

    2014-01-01

    An LC-MS/MS method was developed for the simultaneous determination of 15 water-soluble vitamins that are widely used as additives in beverages and dietary supplements. This combined method involves the following simple pre-treatment procedures: dietary supplement samples were prepared by centrifugation and filtration after an extraction step, whereas beverage samples were diluted prior to injection. Chromatographic analysis in this method utilised a multi-mode ODS column, which provided reverse-phase, anion- and cation-exchange capacities, and therefore improved the retention of highly polar analytes such as water-soluble vitamins. Additionally, the multi-mode ODS column did not require adding ion pair reagents to the mobile phase. We optimised the chromatographic separation of 15 water-soluble vitamins by adjusting the mobile phase pH and the organic solvent. We also conducted an analysis of a NIST Standard Reference Material (SRM 3280 Multi-vitamin/Multi-element tablets) using this method to verify its accuracy. In addition, the method was applied to identify the vitamins in commercial beverages and dietary supplements. By comparing results with the label values and results obtained by official methods, it was concluded that the method could be used for quality control and to compose nutrition labels for vitamin-enriched products.

  12. Statistical modelling coupled with LC-MS analysis to predict human upper intestinal absorption of phytochemical mixtures.

    Science.gov (United States)

    Selby-Pham, Sophie N B; Howell, Kate S; Dunshea, Frank R; Ludbey, Joel; Lutz, Adrian; Bennett, Louise

    2018-04-15

    A diet rich in phytochemicals confers benefits for health by reducing the risk of chronic diseases via regulation of oxidative stress and inflammation (OSI). For optimal protective bio-efficacy, the time required for phytochemicals and their metabolites to reach maximal plasma concentrations (T max ) should be synchronised with the time of increased OSI. A statistical model has been reported to predict T max of individual phytochemicals based on molecular mass and lipophilicity. We report the application of the model for predicting the absorption profile of an uncharacterised phytochemical mixture, herein referred to as the 'functional fingerprint'. First, chemical profiles of phytochemical extracts were acquired using liquid chromatography mass spectrometry (LC-MS), then the molecular features for respective components were used to predict their plasma absorption maximum, based on molecular mass and lipophilicity. This method of 'functional fingerprinting' of plant extracts represents a novel tool for understanding and optimising the health efficacy of plant extracts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity

    International Nuclear Information System (INIS)

    Dillon, Roslyn; Greig, Michael J.; Bhat, B. Ganesh

    2008-01-01

    Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids-mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1-/-) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography-mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC 50 of 247 nM. The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases

  14. Residue dynamics of pyraclostrobin in peanut and field soil by QuEChERS and LC-MS/MS.

    Science.gov (United States)

    Zhang, Fengzu; Wang, Lei; Zhou, Li; Wu, Di; Pan, Hongji; Pan, Canping

    2012-04-01

    A modified QuEChERS-LC-MS/MS (acronym of quick, easy, cheap, effective, rugged and safe-liquid chromatography tandem mass spectrometry) method for the analysis of pyraclostrobin residue in peanut and soil was developed and validated. Pyraclostrobin residue dynamics and final residues in supervised field trials at Good Agricultural Practice (GAP) conditions in peanut and soil were studied. The limits of quantitation (LOQs) for pyraclostrobin in soil, plant, shell and peanut samples were 0.00057, 0.00026, 0.003 and 0.0037 mg kg(-1), respectively. At fortification levels of 0.005, 0.05 and 0.5 mg kg(-1) in all samples, it was shown that recoveries ranged from 80.3% to 109.4% with relative standard deviations of 1.1-8.2% (n=5). The dissipation experiments showed the half-lives (T(1/2)) of pyraclostrobin in soil and plants were 13.1-16.5 days and 10.3-11.2 days, respectively. At pre-harvest intervals (PHI) of 14, 21 and 28 days, pyraclostrobin residue were 0.005-0.20 mg kg(-1) in soil, 0.006-0.27 mg kg(-1) in plants, below 0.053 mg kg(-1) in shells and not detectable in peanuts. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  15. Simultaneous determination of dihydrotestosterone and its metabolites in mouse sera by LC-MS/MS with chemical derivatization.

    Science.gov (United States)

    Gorityala, Shashank; Yang, Shuming; Montano, Monica M; Xu, Yan

    2018-07-15

    Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and β-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites. In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1 × 100 mm) using the mobile phase at a flow rate of 0.25 mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500 ng/mL to 50.0 ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Comparative Analysis of the Bufonis Venenum by Using TLC, HPLC, and LC-MS for Different Extraction Methods

    Directory of Open Access Journals (Sweden)

    Lee Hyo-Jae

    2012-12-01

    Full Text Available Objectives: Toad venom, called Chan-Su, is a traditional Oriental medicine secreted from the auricular and the skin glands of the Bufo bufo gargarizanz Cantor or B. melanosticus Schneider and has been widely used in China, Korea and other parts of Asia for the treatment of pain, heart conditions, and cancer. We examined the concentrations of the main chemical constituents within a commerciallyavailable toad venom product and compared the levels for different extraction methods. Methods: Toad venom was extracted using either cold or hot water, ethanol (EtOH, methanol (MeOH, or ethyl acetate (EtOAc, was fractionated using precipitation or reflux, and was then analyzed using thin layer chromatography (TLC, high-performance liquid chromatography (HTLC, and liquid chroma-tography - mass spectrometry (LC-MS. Individual components were identified by comparisons of the retention times, the ultraviolet spectra, and mass spectras and differences in chemical constituents for different solvents and extraction methods are presented. Results:Components with authentic standards, including serotonin and bufodienolides (cinobufagen, bufalin, cinobufalin, and resibufogenin, were detected. The water extract of toad venom contained the greatest amount of serotonin (75.7 ± 0.1 mg/g, but very small amounts of bufodienolides (3.8 ± 0.0 mg/g. In contrast, the use of MeOH or EtOH extraction solutions resulted in 5-26 times higher concentrations of bufodienolides, with only trace amounts of serotonin. The relative and the absolute concentrations of the component also varied based on the extraction method; i.e., EtOH extracts yielded the greatest total amounts of bufodienolides, and EtOAc precipitation had the lowest amounts of bufodienolides. Conclusions: Toad venom consists of serotonin and several bufodienolides, and the choice of solvent to extract chemical the constituents is important as a way to enrich the purported active components for treating different

  17. Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

    Science.gov (United States)

    Gil, Jeovanis; Cabrales, Ania; Reyes, Osvaldo; Morera, Vivian; Betancourt, Lázaro; Sánchez, Aniel; García, Gerardo; Moya, Galina; Padrón, Gabriel; Besada, Vladimir; González, Luis Javier

    2012-02-23

    Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. N-nitrosodimethylamine (NDMA) removal by reverse osmosis and UV treatment and analysis via LC-MS/MS.

    Science.gov (United States)

    Plumlee, Megan H; López-Mesas, Montserrat; Heidlberger, Andy; Ishida, Kenneth P; Reinhard, Martin

    2008-01-01

    N-nitrosodimethylamine (NDMA) is a probable human carcinogen found in ng/l concentrations in chlorinated and chloraminated water. A method was developed for the determination of ng/l levels of NDMA using liquid chromatography-tandem mass spectrometry (LC-MS/MS) preceded by sample concentration via solid-phase extraction with activated charcoal. Recoveries were greater than 90% and allowed a method reporting limit as low as 2ng/l. Using this method, the removal of NDMA was determined for the Interim Water Purification Facility (IWPF), an advanced wastewater treatment facility operated by the Orange County Water District (OCWD) in Southern California. The facility treats effluent from an activated sludge treatment plant with microfiltration (MF), reverse osmosis (RO), and an ultraviolet-hydrogen peroxide advanced oxidation process (UV-AOP). Six nitrosamines were surveyed: NDMA, N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr). Only NDMA was detected and at all treatment steps in the IWPF, with influent concentrations ranging from 20 to 59 ng/l. Removals for RO and UV ranged from 24% to 56% and 43% to 66%, respectively. Overall, 69+/-7% of the original NDMA concentration was removed from the product water across the advanced treatment process and, in combination with blending, the final concentration did not exceed the California drinking water notification level of 10 ng/l. NDMA removal data are consistent with findings reviewed for other advanced treatment facilities and laboratory studies.

  19. Plasma metabonomics study on toxicity biomarker in rats treated with Euphorbia fischeriana based on LC-MS.

    Science.gov (United States)

    Wang, Yingfeng; Man, Hongxue; Gao, Jian; Liu, Xinfeng; Ren, Xiaolei; Chen, Jianxin; Zhang, Jiayu; Gao, Kuo; Li, Zhongfeng; Zhao, Baosheng

    2016-09-01

    Lang-du (LD) has been traditionally used to treat human diseases in China. Plasma metabolic profiling was applied in this study based on LC-MS to elucidate the toxicity in rats induced by injected ethanol extract of LD. LD injection was given by intraperitoneal injection at doses of 0.1, 0.05, 0.025 and 0 g kg(-1) body weight per day to rats. The blood biochemical levels of alanine aminotransferase, direct bilirubin, creatinine, serum β2-microglobulin and low-density lipoprotein increased in LD-injected rats, and the levels of total protein and albumin decreased in these groups. The metabolic profiles of the samples were analyzed by multivariate statistics analysis, including principal component analysis, partial least squares discriminant analysis and orthogonal projection to latent structures discriminate analysis (OPLS-DA). The metabolic characters in rats injected with LD were perturbed in a dose-dependent manner. By OPLS-DA, 18 metabolites were served as the potential toxicity biomarkers. Moreover, LD treatment resulted in an increase in the p-cresol, p-cresol sulfate, lysophosphatidylethanolamine (LPE) (18:0), LPE (16:0), lysophosphatidylcholine (16:0) and 12-HETE concentrations, and a decrease in hippuric acid, cholic acid and N-acetyl-l-phenylalanine. These results suggested that chronic exposure to LD could cause a disturbance in lipids metabolism and amino acids metabolism, etc. Therefore, an analysis of the metabolic profiles can contribute to a better understanding of the adverse effects of LD. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

    Science.gov (United States)

    Mladic, Marija; de Waal, Tessa; Burggraaff, Lindsey; Slagboom, Julien; Somsen, Govert W; Niessen, Wilfried M A; Manjunatha Kini, R; Kool, Jeroen

    2017-10-01

    This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

  1. Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human plasma using LC-MS/MS.

    Science.gov (United States)

    Allard, Marie; Khoudour, Nihel; Rousseau, Benoît; Joly, Charlotte; Costentin, Charlotte; Blanchet, Benoît; Tournigand, Christophe; Hulin, Anne

    2017-08-05

    A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of regorafenib (REGO), its two metabolites regorafenib-M2 and regorafenib-M5, sorafenib (SORA), and its N-oxide metabolite in human plasma. Separation is achieved on an Hypersil Gold ® column using a gradient elution of 10mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3mL/min. After addition of two internal standards and a protein precipitation, the supernatant is diluted two-fold in a 0.1% (v/v) formic acid solution. Two selected reaction monitoring transitions are used, for each analyte, one for quantitation and the second one for confirmation. The standard curves are ranged from 50 to 5 000ng/mL for REGO and its metabolites and 80 to 5 000ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of the analytes under various conditions. The method is usually used in clinical practice in order to improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for hepatocellular carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Metabolic responses of Beauveria bassiana to hydrogen peroxide-induced oxidative stress using an LC-MS-based metabolomics approach.

    Science.gov (United States)

    Zhang, Chen; Wang, Wei; Lu, Ruili; Jin, Song; Chen, Yihui; Fan, Meizhen; Huang, Bo; Li, Zengzhi; Hu, Fenglin

    2016-06-01

    The entomopathogenic fungus, Beauveria bassiana, is commonly used as a biological agent for pest control. Environmental and biological factors expose the fungus to oxidative stress; as a result, B. bassiana has adopted a number of anti-oxidant mechanisms. In this study, we investigated metabolites of B. bassiana that are formed in response to oxidative stress from hydrogen peroxide (H2O2) by using a liquid chromatography mass spectrometry (LC-MS) approach. Partial least-squares discriminant analysis (PLS-DA) revealed differences between the control and the H2O2-treated groups. Hierarchical cluster analysis (HCA) showed 18 up-regulated metabolites and 25 down-regulated metabolites in the H2O2-treated fungus. Pathway analysis indicated that B. bassiana may be able to alleviate oxidative stress by enhancing lipid catabolism and glycometabolism, thus decreasing membrane polarity and preventing polar H2O2 or ROS from permeating into fungal cells and protecting cells against oxidative injury. Meanwhile, most of the unsaturated fatty acids that are derived from glycerophospholipids hydrolysis can convert into oxylipins through autoxidation, which can prevent the reactive oxygen of H2O2 from attacking important macromolecules of the fungus. Results showed also that H2O2 treatment can enhance mycotoxins production which implies that oxidative stress may be able to increase the virulence of the fungus. In comparison to the control group, citric acid and UDP-N-acetylglucosamine were down-regulated, which suggested that metabolic flux was occurring to the TCA cycle and enhancing carbohydrate metabolism. The findings from this study will contribute to the understanding of how the molecular mechanisms of fungus respond to environmental and biological stress factors as well as how the manipulation of such metabolisms may lead to selection of more effective fungal strains for pest control. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

    Science.gov (United States)

    Richards, S L; Cawley, A T; Cavicchioli, R; Suann, C J; Pickford, R; Raftery, M J

    2016-04-01

    Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology.

    Science.gov (United States)

    Maublanc, Julie; Dulaurent, Sylvain; Morichon, Julien; Lachâtre, Gérard; Gaulier, Jean-michel

    2015-03-01

    Despite a non-invasive sampling, hair samples are generally collected in limited amounts for an obvious esthetic reason. In order to reduce the required quantity of samples, a multianalytes method allowing simultaneous identification and quantification of 35 psychoactive drugs was developed. After incubation of 50 mg of hair in a phosphate buffer pH 5 for one night at room temperature, the substances of interest were extracted by a simple liquid-liquid extraction step, with a dichloromethane/ether mixture (70:30, v/v). After evaporation under a gentle stream of nitrogen and reconstitution in formate buffer (2 mM, pH 3)/acetonitrile (90:10, v/v), twenty microliter were injected into the LC-MS/MS system for a chromatographic run of 29 min using an Atlantis T3 column (150 × 2.1 mm, 3 μm) (Waters Corp, Milford, USA) and a gradient mixture of 2 mM, pH 3.0 ammonium formate, and 2 mM, pH 3.0 ammonium formate/acetonitrile. The data acquisition was performed in scheduled MRM mode. Intra- and inter-day precisions, estimated using the coefficient of variation and relative bias, were lower than 20 % for all concentration levels, except for two compounds. The limits of detection and quantification ranged from 0.5 to 10 pg/mg. After complete validation, this method has been successfully used in several forensic cases, three of which are reported.

  5. Studies on separation and pharmacokinetics of m-nisoldipine enantiomers in beagle dog plasma by LC/MS/MS.

    Science.gov (United States)

    Li, Min; Yuan, Lin; Li, Xiuqing; Zhi, Xuran; Sheng, Ning; Zhang, Lantong

    2013-11-01

    A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed and validated for the separation and determination of m-nisoldipine enantiomers in beagle dog plasma. Samples were pretreated by a single-step protein precipitation with acetonitrile. After m-nisoldipine racemic administration to beagle dogs, samples of m-nisoldipine enantiomers in beagle dog plasma were separated and determined on a ULTRON ES-OVM column (150 × 4.6 mm, 5 μm) at 20°C with a mobile phase consisted of methanol-acetonitrile-ammonium acetate (pH 7.0; 2mM) (15:15:70, v/v/v) at a flow rate of 0.8 mL/min. Chromatograms were monitored at 237 nm, and the API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using ElectroSpray ionization (ESI) source. The good linearity (rs=0.9958 and rr=0.9983) were found in the range 0.25-20 ng/mL. The lower limit of quantification (LLOQ) obtained was 0.25 ng/mL (n=6). All the validation data, such as accuracy, precision, intra-day and inter-day repeatability, were within the required limits. The method was successfully applied to separation and pharmacokinetics of m-nisoldipine enantiomers in beagle dog plasma. The result of statistics analysis shows that there are no significant differences between R-(-)-m-nisoldipine and S-(+)-m-nisoldipine (p>0.05). The study provides necessary evidences for the research and new drug development of m-nisoldipine enantiomers. Copyright © 2013. Published by Elsevier B.V.

  6. Determination of esculentoside A in dog plasma by LC-MS/MS method: application to pre-clinical pharmacokinetics.

    Science.gov (United States)

    Guan, Xiaoduo; Chang, Huichao; Sun, Fanlu; Chen, Xiaohui; Zhang, Wen; Fan, Guorong

    2013-01-01

    A simple and rapid high performance liquid chromatography electrospray ionization ion-trap tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitative determination of esculentoside A (EsA) in dog plasma using ginsenoside Rg1 as the internal standard (IS). After liquid-liquid extraction (LLE) with n-butanol, the analyte and IS were separated on a Diamonsil C(18) (2.1 mm × 50 mm, 3 μm) column with the mobile phase of methanol-water containing 0.1% acetic acid (70:30, v/v) at a flow rate of 0.2 ml/min. An ion trap mass spectrometer equipped with an electrospray ionization source performed in selected reaction monitoring (SRM) mode was used as the detector. The precursor-product ion transitions were m/z 849.3 [M+Na](+)→m/z 805.3 for EsA and m/z 823.3 [M+Na](+)→m/z 643.3 for IS. The total chromatographic run time was 5 min. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 5 ng/ml and had a good linearity (r(2)>0.997) over the linear range of 5-500 ng/ml. The mean extraction recovery of EsA from spiked plasma samples was over 75%. The intra- and inter-precisions were no more than 8.8% and accuracies were within the range of -4.6 to 8.7%. All the validated data were within the accepted criteria as stated in the FDA bioanalytical method validation guideline. The developed method was suitable for the quantification of EsA and successfully applied to the pharmacokinetic study of EsA after an oral administration to beagle dogs. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Helio A Martins-Júnior

    Full Text Available Golden retriever muscular dystrophy (GRMD provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD. The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR versus GRMD-gene carrier (CaGR and affected female dogs (AfCR. Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS. To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

  8. Quantitative determination of metaxalone in human plasma by LC-MS and its application in a pharmacokinetic study

    Directory of Open Access Journals (Sweden)

    Lanting Zhao

    2016-10-01

    Full Text Available A simple and rapid method using liquid chromatography–mass spectrometry (LC-MS for the determination of metaxalone in human plasma has been developed and validated. Letrozole was used as the internal standard (IS. The plasma samples were simply treated with acetonitrile which allowed the precipitation of plasma proteins. The chromatographic separation was achieved on a Sapphire C18 (2.1 mm × 150 mm, 5 µm, Newark, USA column using the mobile phase (5 mM ammonium acetate containing 0.01% formic acid: acetonitrile (45:55, v/v at a flow rate of 0.3 ml/min. The selected ion monitoring (SIM in the positive mode was used for the determination of [M + H]+ m/z 222.1 and 286.1 for metaxalone and letrozole, respectively. The standard curve obtained was linear (r2 ≥ 0.99 over the concentration range of 30.24−5040 ng/ml. Meanwhile, no interfering peaks or matrix effect was observed. The method established was simple and successfully applied to a pharmacokinetic study of metaxalone in healthy Chinese volunteers after a single oral dose administration of 800 mg metaxalone. The main pharmacokinetic parameters of metaxalone were as follow: Cmax, (1664 ± 1208 ng/ml and (2063 ± 907 ng/ml; AUC0−36, (13925 ± 6590 ng/ml h and (18620 ± 5717 ng/ml h; t1/2, (13.6 ± 7.7 h and (20.3 ± 7.7 h for the reference and test tablets, respectively. These pharmacokinetic parameters of metaxalone in healthy Chinese volunteers were reported for the first time.

  9. Multiresidue determination and potential risks of emerging pesticides in aquatic products from Northeast China by LC-MS/MS.

    Science.gov (United States)

    Fu, Lei; Lu, Xianbo; Tan, Jun; Wang, Longxing; Chen, Jiping

    2018-01-01

    A simple method for determining 33 pesticides with a wide polarity range (logK ow 0.6-4.5) in aquatic products was developed based on LC-MS/MS. The target analytes included three types of widely used pesticides: insecticides, fungicides and herbicides. Based on the optimization of ultrasonic assisted extraction and GPC clean-up procedures, the matrix effect, extraction recoveries and LOD were improved distinctively. LOQ of this method was below 0.5ng/g for all pesticides, which is superior to values in the literature, and the matrix effect was reduced effectively (-14.7% to 7.5%). The method was successfully applied to investigate the pesticide residue levels of twenty-five samples including seven common kinds of fishes from Northeast China. The results showed that all targeted pesticides were present in the fish samples; however, their levels were low, except for atrazine, linuron, ethoprophos, tetrachlorvinphos, acetochlor and fenthion. Atrazine and linuron caught our attention because the concentrations of atrazine in fish samples from Liaoning province were in the range of 0.5-8ng/g (w/w) with mean concentration of 2.3ng/g, which were far above those of other pesticides. The levels of linuron were in the range of 0.6-6ng/g (mean concentration 2.8ng/g), which were the highest among all targeted pesticides in the Inner Mongolia. This is the first systematic investigation on the characteristics and levels of these pesticides in aquatic products from northeast China. Considering their toxicity and bioaccumulation, the potential risk of atrazine and linuron from consuming aquatic products should be paid more attention. Copyright © 2017. Published by Elsevier B.V.

  10. Quantitative method for the determination of Iso-fludelone (KOS-1803) in human plasma by LC-MS/MS

    Science.gov (United States)

    Christner, Susan M.; Parise, Robert A.; Levine, Erica D.; Rizvi, Naiyer A.; Gounder, Mrinal M.; Beumer, Jan H.

    2014-01-01

    Epothilones are relatively new tubulin-poison anticancer drugs. Iso-fludelone (KOS-1803) is a synthetic third generation epothilone drug discovered at Memorial Sloan Kettering Cancer Center, and currently in Phase I clinical trials. We report an LC-MS/MS assay for the sensitive, accurate and precise quantitation of Iso-fludelone in 0.2 mL of human plasma. Validation was performed according to FDA guidance. The assay comprised of KOS-1724 as the internal standard and an MTBE liquid-liquid extraction with a water wash step. Separation was achieved with an YMC-Pack ODS-AQ column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water (70:30, v/v) at 0.3 mL/min for 4 min. Chromatographic separation was followed by electrospray, positive-mode ionization tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode. The assay was linear from 0.1– 300 ng/mL and was accurate (−9.41–7.07%) and precise (1.03–13.7%) which fulfilled FDA criteria for validation. Recovery from plasma was 73.9–79.7% and ion suppression was negligible (−22.8 to −31.3%). Plasma freeze thaw stability (99.97–105.7%), stability for 11 months at −80 °C (94.93–107.9%), and stability for 6 h at room temperature (94.75–105.5%) were all acceptable. This assay is currently being applied to quantitate Iso-fludelone in clinical samples. PMID:25168219

  11. Quantitative method for the determination of iso-fludelone (KOS-1803) in human plasma by LC-MS/MS.

    Science.gov (United States)

    Christner, Susan M; Parise, Robert A; Levine, Erica D; Rizvi, Naiyer A; Gounder, Mrinal M; Beumer, Jan H

    2014-11-01

    Epothilones are relatively new tubulin-poison anticancer drugs. Iso-fludelone (KOS-1803) is a synthetic third generation epothilone drug discovered at Memorial Sloan Kettering Cancer Center, and currently in phase I clinical trials. We report an LC-MS/MS assay for the sensitive, accurate and precise quantitation of iso-fludelone in 0.2mL of human plasma. Validation was performed according to FDA guidance. The assay comprised of KOS-1724 as the internal standard and an MTBE liquid-liquid extraction with a water wash step. Separation was achieved with an YMC-Pack ODS-AQ column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water (70:30, v/v) at 0.3mL/min for 4min. Chromatographic separation was followed by electrospray, positive-mode ionization tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode. The assay was linear from 0.1 to 300ng/mL and was accurate (-9.41 to -7.07%) and precise (1.03-13.7%) which fulfilled FDA criteria for validation. Recovery from plasma was 73.9-79.7% and ion suppression was negligible (-22.8 to -31.3%). Plasma freeze-thaw stability (99.97-105.7%), stability for 11 months at -80°C (94.93-107.9%), and stability for 6h at room temperature (94.75-105.5%) were all acceptable. This assay is currently being applied to quantitate iso-fludelone in clinical samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Vigabatrin in dried plasma spots: validation of a novel LC-MS/MS method and application to clinical practice.

    Science.gov (United States)

    Kostić, Nađa; Dotsikas, Yannis; Jović, Nebojša; Stevanović, Galina; Malenović, Anđelija; Medenica, Mirjana

    2014-07-01

    This paper presents a LC-MS/MS method for the determination of antiepileptic drug vigabatrin in dried plasma spots (DPS). Due to its zwitterionic chemical structure, a pre-column derivatization procedure was performed, aiming to yield enhanced ionization efficiency and improved chromatographic behaviour. Propyl chloroformate, in the presence of propanol, was selected as the best derivatization reagent, providing a strong signal along with reasonable run time. A relatively novel sample collection technique, DPS, was utilized, offering easy sample handling and analysis, using a sample in micro amount (∼5μL). Derivatized vigabatrin and its internal standard, 4-aminocyclohexanecarboxylic acid, were extracted by liquid-liquid extraction (LLE) and determined in positive ion mode by applying two SRM transitions per analyte. A Zorbax Eclipse XDB-C8 column (150×4.6mm, 5μm particle size) maintained at 30°C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550μL/min and total run time 4.5min. The assay exhibited excellent linearity over the concentration range of 0.500-50.0μg/mL, which is suitable for the determination of vigabatrin level after per os administration in children and youths with epilepsy, who were on vigabatrin therapy, with or without co-medication. Specificity, accuracy, precision, recovery, matrix-effect and stability were also estimated and assessed within acceptance criteria. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Quantitation of pregabalin in dried blood spots and dried plasma spots by validated LC-MS/MS methods.

    Science.gov (United States)

    Kostić, Nađa; Dotsikas, Yannis; Jović, Nebojša; Stevanović, Galina; Malenović, Anđelija; Medenica, Mirjana

    2015-05-10

    In this paper, novel LC-MS/MS methods for the determination of antiepileptic drug pregabalin in dried matrix spots (DMS) are presented. This attractive technique of sample collection in micro amount was utilized in the form of dried blood spots (DBS) and dried plasma spots (DPS). Following a pre-column derivatization procedure, using n-propyl chloroformate in the presence of n-propanol, and consecutive liquid-liquid extraction, derivatized pregabalin and its internal standard, 4-aminocyclohexanecarboxylic acid, were detected in positive ion mode by applying two SRM transitions per analyte. A YMC-Pack Octyl column (50mm×4.0mm, 3μm particle size) maintained at 30°C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550μL/min and total run time 2min. Established methods were fully validated over the concentration range of 0.200-20.0μg/mL for DBS and 0.400-40.0μg/mL for DPS, respectively, while specificity, accuracy, precision, recovery, matrix-effect, stability, dilution integrity and spot homogeneity were found within acceptance criteria. Validated methods were applied for the determination of pregabalin levels in dried blood and plasma samples obtained from patients with epilepsy, after per os administration of commercial capsules. Comparison of drug level in blood and plasma, as well as correction steps undertaken in order to overcome hematocrit issue, when analyzing DBS, are also given. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

    Science.gov (United States)

    Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

    2018-05-01

    The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

  15. Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays

    DEFF Research Database (Denmark)

    Weisser, Johan Juhl; Hansen, Cecilie Hurup; Poulsen, Rikke

    2016-01-01

    Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination...... of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis....... The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off...

  16. Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides.

    Science.gov (United States)

    Ciccimaro, Eugene; Ranasinghe, Asoka; D'Arienzo, Celia; Xu, Carrie; Onorato, Joelle; Drexler, Dieter M; Josephs, Jonathan L; Poss, Michael; Olah, Timothy

    2014-12-02

    Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

  17. Progress report on the AMT analysis

    International Nuclear Information System (INIS)

    Wood, B.

    1992-01-01

    ICF Resources Incorporated's analysis of the Alternative Minimum Tax (AMT) has examined its effect on the US oil and gas industry from several different perspectives, to estimate the effect of the three relief proposals and to better understand the source of the outcry about the AMTs ''inequities.'' This report is a brief summary of the methodology and results to date. The complexity of the accounting mechanisms that comprise the AMT and the disparity between this analytical conclusions and clauses made by the oil and gas industry (principally the IPAA) have led this analysis through several distinct phases of: Project-level analysis; firm-level analysis; and demographic analysis. These analyses are described in detail

  18. Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS.

    Science.gov (United States)

    Bisht, Rohit; Mandal, Abhirup; Rupenthal, Ilva D; Mitra, Ashim K

    2016-12-01

    This study aimed to develop and evaluate an ex vivo eye model for intravitreal drug sampling and tissue distribution of connexin43 mimetic peptide (Cx43MP) following intravitreal injection using the microdialysis technique and LC-MS/MS. An LC-MS/MS method was developed, validated, and applied for quantification of Cx43MP in ocular tissues. Microdialysis probes were calibrated for in vitro recovery studies. Bovine eyes were fixed in a customized eye holder and after intravitreal injection of Cx43MP, microdialysis probes were implanted in the vitreous body. Vitreous samples were collected at particular time intervals over 24 h. Moreover, 24 and 48 h after intravitreal injection ocular tissues were collected, processed, and analyzed for Cx43MP concentrations using LC-MS/MS. The LC-MS/MS method showed good linearity (r 2  = 0.9991). The mean percent recovery for lower (LQC), medium (MQC), and higher quality control (HQC) (0.244, 3.906, and 125 μg/mL) was found to be 83.83, 84.92, and 94.52, respectively, with accuracy ranges between 96 and 99 % and limits of detection (LOD) and quantification (LOQ) of 0.122 and 0.412 μg/mL. The in vitro recovery of the probes was found to be over 80 %. As per microdialysis sample analysis, the Cx43MP concentration was found to increase slowly in the vitreous body up to 16 h and thereafter declined. After 48 h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humor. This ex vivo model may therefore be a useful tool to investigate intravitreal kinetics and ocular disposition of therapeutic molecules after intravitreal injection.

  19. Analytical methodologies based on LC?MS/MS for monitoring selected emerging compounds in liquid and solid phases of the sewage sludge

    OpenAIRE

    Boix, C.; Ib??ez, M.; Fabregat-Safont, D.; Morales, E.; Pastor, L.; Sancho, J.V.; S?nchez-Ram?rez, J.E.; Hern?ndez, F.

    2016-01-01

    In this work, two analytical methodologies based on liquid chromatography coupled to tandem mass spectrometry (LC?MS/MS) were developed for quantification of emerging pollutants identified in sewage sludge after a previous wide-scope screening. The target list included 13 emerging contaminants (EC): thiabendazole, acesulfame, fenofibric acid, valsartan, irbesartan, salicylic acid, diclofenac, carbamazepine, 4-aminoantipyrine (4-AA), 4-acetyl aminoantipyrine (4-AAA), 4-formyl aminoantipyrine (...

  20. eMZed: an open source framework in Python for rapid and interactive development of LC/MS data analysis workflows

    OpenAIRE

    Kiefer, P; Schmitt, U; Vorholt, J A

    2013-01-01

    Summary: The Python-based, open-source eMZed framework was developed for mass spectrometry (MS) users to create tailored workflows for liquid chromatography (LC)/MS data analysis. The goal was to establish a unique framework with comprehensive basic functionalities that are easy to apply and allow for the extension and modification of the framework in a straightforward manner. eMZed supports the iterative development and prototyping of individual evaluation strategies by providing a computing...

  1. Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin.

    Science.gov (United States)

    Shibata, Kaito; Naito, Takafumi; Okamura, Jun; Hosokawa, Seiji; Mineta, Hiroyuki; Kawakami, Junichi

    2017-11-30

    Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A novel study of screening and confirmation of modafinil, adrafinil and their metabolite modafinilic acid under EI-GC-MS and ESI-LC-MS-MS ionization.

    Science.gov (United States)

    Dubey, S; Ahi, S; Reddy, I M; Kaur, T; Beotra, A; Jain, S

    2009-12-01

    Adrafinil and modafinil have received wide publicity and have become controversial in the sporting world when several athletes were discovered allegedly using these drugs as doping agents. By acknowledging the facts, the World Anti-Doping Agency (WADA) banned these drugs in sports since 2004. The present study explores the possibility of differentiating adrafinil and modafinil and their major metabolites under electron impact ionization in gas chromatograph-mass spectrometer (GC-MSD) and electrospray ionization in liquid chromatograph-mass spectrometer (LC-MS/MS) by studying the fragmentation pattern of these drugs. Adrafinil, modafinil and their major metabolite, modafinilic acid were analyzed on EI-GC-MSD and ESI-LC-MS/MS using various individual parameters on both the instruments. The analytical technique and equipment used in the analysis were an Agilent 6890N GC with 5973 mass selective detector for the GC-MSD analysis and an Agilent 1100 HPLC with API-3200 Triple quadrupole mass spectrometer for the LC-MS/MS analysis. Validation of both methods was performed using six replicates at different concentrations. The results show that adrafinil, modafinil and their major metabolite modafinilic acid could be detected as a single artifact without differentiation under EI-GC-MSD analysis. However, all drugs could be detected and differentiated under ESI-LCMS/MS analysis without any artifaction. The GC-MSD analysis gives a single artifact for both the drugs without differentiation and thus can be used as a marker for screening purposes. Further, the Multiple Reaction Monitoring (MRM) method developed under LC-MS/MS is fit for the purpose for confirmation of suspicious samples in routine sports testing and in forensic and clinical analysis.

  3. Development and Validation of a Fast Procedure To Analyze Amoxicillin in River Waters by Direct-Injection LC-MS/MS

    OpenAIRE

    Vera Homem; Arminda Alves; Lúcia Silveira Santos

    2014-01-01

    A laboratory application with a strong component in analytical chemistry was designed for undergraduate students, in order to introduce a current problem in the environmental science field, the water contamination by antibiotics. Therefore, a simple and rapid method based on direct injection and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized for the determination of amoxicillin in river water. Students learned the main optimization steps...

  4. Validated LC-MS/MS Method for the Quantification of Free and Bound Lignans in Cereal-Based Diets and Feces

    DEFF Research Database (Denmark)

    Nørskov, Natalja; Knudsen, Knud Erik Bach

    2016-01-01

    lignans (matairesinol, hydroxymatairesinol, secoisolariciresinol, lariciresinol, isolariciresinol, syringaresinol, medioresinol, and pinoresinol) and two enterolignans (enterodiol and enterolactone) in cereal-based diets/bread and feces. The method consisted of alkaline methanolic extraction combined......Despite the extensive literature describing the biological effects of phenolic compounds from cereals, little is known about their bioaccessibility in the food matrix. This paper describes a validated LC-MS/MS method for the quantification of free and total content (free + bound) of eight plant...

  5. Clustering with position-specific constraints on variance: Applying redescending M-estimators to label-free LC-MS data analysis

    Directory of Open Access Journals (Sweden)

    Mani D R

    2011-08-01

    Full Text Available Abstract Background Clustering is a widely applicable pattern recognition method for discovering groups of similar observations in data. While there are a large variety of clustering algorithms, very few of these can enforce constraints on the variation of attributes for data points included in a given cluster. In particular, a clustering algorithm that can limit variation within a cluster according to that cluster's position (centroid location can produce effective and optimal results in many important applications ranging from clustering of silicon pixels or calorimeter cells in high-energy physics to label-free liquid chromatography based mass spectrometry (LC-MS data analysis in proteomics and metabolomics. Results We present MEDEA (M-Estimator with DEterministic Annealing, an M-estimator based, new unsupervised algorithm that is designed to enforce position-specific constraints on variance during the clustering process. The utility of MEDEA is demonstrated by applying it to the problem of "peak matching"--identifying the common LC-MS peaks across multiple samples--in proteomic biomarker discovery. Using real-life datasets, we show that MEDEA not only outperforms current state-of-the-art model-based clustering methods, but also results in an implementation that is significantly more efficient, and hence applicable to much larger LC-MS data sets. Conclusions MEDEA is an effective and efficient solution to the problem of peak matching in label-free LC-MS data. The program implementing the MEDEA algorithm, including datasets, clustering results, and supplementary information is available from the author website at http://www.hephy.at/user/fru/medea/.

  6. High-throughput screening and confirmation of 22 banned veterinary drugs in feedstuffs using LC-MS/MS and high-resolution Orbitrap mass spectrometry.

    Science.gov (United States)

    Wang, Xufeng; Liu, Yanghong; Su, Yijuan; Yang, Jianwen; Bian, Kui; Wang, Zongnan; He, Li-Min

    2014-01-15

    A new analytical strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with accurate mass high-resolution Orbitrap mass spectrometry (HR-Orbitrap MS) was performed for high-throughput screening, confirmation, and quantification of 22 banned or unauthorized veterinary drugs in feedstuffs according to Bulletin 235 of the Ministry of Agriculture, China. Feed samples were extracted with acidified acetonitrile, followed by cleanup using solid-phase extraction cartridge. The extracts were first screened by LC-MS/MS in a single selected reaction monitoring mode. The suspected positive samples were subjected to a specific pretreatment for confirmation and quantification of analyte of interest with LC-MS/MS and HR-Orbitrap MS. Mean recoveries for all target analytes (except for carbofuran and chlordimeform, which were about 35 and 45%, respectively) ranged from 52.2 to 90.4%, and the relative standard deviations were screening of real samples obtained from local feed markets and confirmation of the suspected target analytes. It provides a high-throughput, sensitive, and reliable screening, identification, and quantification of banned veterinary drugs in routine monitoring programs of feedstuffs.

  7. Identification of the Chemical Constituents in Simiao Wan and Rat Plasma after Oral Administration by GC-MS and LC-MS

    Directory of Open Access Journals (Sweden)

    Yunshuang Fan

    2017-01-01

    Full Text Available Simiao Wan (SMW, an important multiherbal formula used in traditional Chinese medicine, is extensively used to treat rheumatoid arthritis. However, the knowledge of the bioactive components of SMW remains unclear. Thus, gas chromatography–mass spectrometry (GC-MS and liquid chromatography–mass spectrometry (LC-MS were used to analyze the chemical constituents of volatile and nonvolatile extracts of SMW, as well as its absorbed components in rat plasma after oral SMW administration. Identification of several compounds was enabled by comparison of retention times, MS spectra, and MS/MS spectral data with the standard substance and reference materials reported in the literature. In the volatile extracts, GC-MS identified 26 compounds in vitro, three of which observed in blood by GC-MS. In the nonvolatile extracts, LC-MS identified 49 compounds in SMW; 18 compounds containing 7 prototype compounds, 5 metabolites, and 6 unknown compounds were absorbed by blood. The proposed GC-MS and LC-MS method was appropriate not only for the rapid screening and identification of multiple components of an SMW extract but also for screening its bioactive constituents in vivo. The proposed method could be a promising tool for the quality control of other Chinese herbal medicines.

  8. Potential effect of diaper and cotton ball contamination on NMR- and LC/MS-based metabonomics studies of urine from newborn babies.

    Science.gov (United States)

    Goodpaster, Aaron M; Ramadas, Eshwar H; Kennedy, Michael A

    2011-02-01

    Nuclear magnetic resonance (NMR) and liquid chromatography/mass spectrometry (LC/MS) based metabonomics screening of urine has great potential for discovery of biomarkers for diseases that afflict newborn and preterm infants. However, urine collection from newborn infants presents a potential confounding problem due to the possibility that contaminants might leach from materials used for urine collection and influence statistical analysis of metabonomics data. In this manuscript, we have analyzed diaper and cotton ball contamination using synthetic urine to assess its potential to influence the outcome of NMR- and LC/MS-based metabonomics studies of human infant urine. Eight diaper brands were examined using the "diaper plus cotton ball" technique. Data were analyzed using conventional principal components analysis, as well as a statistical significance algorithm developed for, and applied to, NMR data. Results showed most diaper brands had distinct contaminant profiles that could potentially influence NMR- and LC/MS-based metabonomics studies. On the basis of this study, it is recommended that diaper and cotton ball brands be characterized using metabonomics methodologies prior to initiating a metabonomics study to ensure that contaminant profiles are minimal or manageable and that the same diaper and cotton ball brands be used throughout a study to minimize variation.

  9. Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup.

    Science.gov (United States)

    Cheng, Keding; Sloan, Angela; McCorrister, Stuart; Peterson, Lorea; Chui, Huixia; Drebot, Mike; Nadon, Celine; Knox, J David; Wang, Gehua

    2014-12-01

    The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

    Directory of Open Access Journals (Sweden)

    Laëtitia Théron

    2016-10-01

    Full Text Available Mass spectrometry imaging (MSI is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

  11. Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS system with timed and highly selective reaction monitoring.

    Science.gov (United States)

    Zhao, Zhiyong; Liu, Na; Yang, Lingchen; Deng, Yifeng; Wang, Jianhua; Song, Suquan; Lin, Shanhai; Wu, Aibo; Zhou, Zhenlei; Hou, Jiafa

    2015-09-01

    Mycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC-MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. Graphical abstract Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS.

  12. Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific.

    Science.gov (United States)

    Yogi, Kentaro; Oshiro, Naomasa; Inafuku, Yasuo; Hirama, Masahiro; Yasumoto, Takeshi

    2011-12-01

    Toxin profiles of representative ciguatera species caught at different locations of Japan were investigated in fish flesh by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Identification and quantification of 16 toxins were facilitated by the use of 14 reference toxins prepared by either synthesis or isolation from natural sources and the previous LC-MS data thereof. Sodium adduct ions [M + Na](+) were used as parent and product ions. Distinct regional differences were unveiled: ciguatoxin-1B type toxins were found in snappers and groupers from Okinawa, ciguatoxin-3C type toxins were found in a spotted knifejaw, Oplegnathus punctatus, from Miyazaki located 730 km north of Okinawa, and both types of toxins were found in a red snapper, Lutjanus bohar, from Minamitorishima (Marcus) Island. Twelve toxins were identified in a dinoflagellate, Gambierdiscus toxicus, collected as the primary toxin source in French Polynesia. Occurrence of M-seco-toxins in fish and oxidized toxins in the dinoflagellate was confirmed for the first time. The present LC-MS/MS method is rapid, specific, and accurate. It not only outperforms the currently employed mouse bioassays but also enables the study of the toxin dynamics during the food chain transmission.

  13. Improvement of Nicotinic Acid and Nicotinamide Analysis in Meats and Meat Products by HPLC and LC-MS/MS with Solid-Phase Extraction.

    Science.gov (United States)

    Hiki, Asako; Yamajima, Yukiko; Uematsu, Yoko

    2016-01-01

    A method for nicotinic acid (NA) and nicotinamide (NAA) analysis in meats was developed. NA and NAA were extracted from meats or meat products with metaphosphate aqueous solution. The extract was cleaned up with an Oasis MCX cartridge. The cartridge was washed with 2% acetic acid (v/v) and acetic acid-methanol solution. NA and NAA were eluted with ammonia-methanol solution. NA and NAA in the eluate were chromatographed on a Scherzo SM-C18 (3.0×150 mm, 3.0 μm) column with 20 mmol/L ammonium acetate containing 0.1% acetic acid-acetonitrile (97 : 3) as a mobile phase and were monitored at 261 nm. Quantification was performed by LC and LC-MS/MS. Calibration curves showed high linearity (correlation coefficient>0.998) between 1-25 μg/mL for LC and LC-MS/MS. Recoveries were 84-108% (CV≦5.8%) by HPLC and 79-105% (CV≦9.0%) by LC-MS/MS. The limit of quantitation for NA was 0.005-0.01 g/kg and that for NAA was 0.01-0.02 g/kg.

  14. Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA.

    Science.gov (United States)

    Li, Jie; Leung, Elvis M K; Choi, Martin M F; Chan, Wan

    2013-05-01

    Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10(9) nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

  15. Bioavailability of Oral Hydrocortisone Corrected for Binding Proteins and Measured by LC-MS/MS Using Serum Cortisol and Salivary Cortisone.

    Science.gov (United States)

    Johnson, T N; Whitaker, M J; Keevil, B; Ross, R J

    2018-01-01

    The assessment absolute bioavailability of oral hydrocortisone is complicated by its saturable binding to cortisol binding globulin (CBG). Previous assessment of bioavailability used a cortisol radioimmunoassay which has cross reactivity with other steroids. Salivary cortisone is a measure of free cortisol and LC-MS/MS is the gold standard method for measuring steroids. We here report the absolute bioavailability of hydrocortisone calculated using serum cortisol and salivary cortisone measured by LC-MS/MS. 14 healthy male dexamethasone suppressed volunteers were administered 20 mg hydrocortisone either intravenously or orally by tablet. Samples of serum and saliva were taken and measured for cortisol and cortisone by LC-MS/MS. Serum cortisol was corrected for saturable binding using published data and pharmacokinetic parameters derived using the program WinNonlin. The mean (95% CI) bioavailability of oral hydrocortisone calculated from serum cortisol, unbound serum cortisol and salivary cortisone was 1.00 (0.89-1.14); 0.88 (0.75-1.05); and 0.93 (0.83-1.05), respectively. The data confirm that, after oral administration, hydrocortisone is completely absorbed. The data derived from serum cortisol corrected for protein binding, and that from salivary cortisone, are similar supporting the concept that salivary cortisone reflects serum free cortisol levels and that salivary cortisone can be used as a non-invasive method for measuring the pharmacokinetics of hydrocortisone.

  16. Label-free LC-MS analysis of HER2+ breast cancer cell line response to HER2 inhibitor treatment.

    Science.gov (United States)

    Di Luca, Alessio; Henry, Michael; Meleady, Paula; O'Connor, Robert

    2015-08-04

    Human epidermal growth-factor receptor (HER)-2 is overexpressed in 25 % of breast-cancers and is associated with an aggressive form of the disease with significantly shortened disease free and overall survival. In recent years, the use of HER2-targeted therapies, monoclonal-antibodies and small molecule tyrosine-kinase inhibitors has significantly improved the clinical outcome for HER2-positive breast-cancer patients. However, only a fraction of HER2-amplified patients will respond to therapy and the use of these treatments is often limited by tumour drug insensitivity or resistance and drug toxicities. Currently there is no way to identify likely responders or rational combinations with the potential to improve HER2-focussed treatment outcome. In order to further understand the molecular mechanisms of treatment-response with HER2-inhibitors, we used a highly-optimised and reproducible quantitative label-free LC-MS strategy to characterize the proteomes of HER2-overexpressing breast-cancer cell-lines (SKBR3, BT474 and HCC1954) in response to drug-treatment with HER2-inhibitors (lapatinib, neratinib or afatinib). Following 12 ours treatment with different HER2-inhibitors in the BT474 cell-line; compared to the untreated cells, 16 proteins changed significantly in abundance following lapatinib treatment (1 μM), 21 proteins changed significantly following neratinib treatment (150 nM) and 38 proteins changed significantly following afatinib treatment (150 nM). Whereas following 24 hours treatment with neratinib (200 nM) 46 proteins changed significantly in abundance in the HCC1954 cell-line and 23 proteins in the SKBR3 cell-line compared to the untreated cells. Analysing the data we found that, proteins like trifunctional-enzyme subunit-alpha, mitochondrial; heterogeneous nuclear ribonucleoprotein-R and lamina-associated polypeptide 2, isoform alpha were up-regulated whereas heat shock cognate 71 kDa protein was down-regulated in 3 or more comparisons. This proteomic

  17. Simultaneous determination of riboflavin and pyridoxine by UHPLC/LC-MS in UK commercial infant meal food products.

    Science.gov (United States)

    Zand, Nazanin; Chowdhry, Babur Z; Pullen, Frank S; Snowden, Martin J; Tetteh, John

    2012-12-15

    An assay for the simultaneous quantitative determination of riboflavin and pyridoxine in eight different complementary infant meal products has been developed in order to (1) estimate the daily intake of these vitamins from commercial infant food consumption, and (2) ascertain their nutritional suitability relative to dietary guidelines for the 6-9 months age group. The method involves mild hydrolysis of the foods, an extraction of the supernatant by centrifugation followed by quantitative determination using ultra-high performance liquid chromatography. Separation of the two water soluble vitamins is achieved within one minute and the resultant sample is also LC-MS compatible. Despite wide individual differences between brands (p=6.5e-12), no significant differences were observed in the level of pyridoxine between the meat and vegetable-based varieties (p=0.7) per 100g of commercial infant food. Riboflavin was not detected in any of the samples where the detection limit was below 0.07 μg/mL. In terms of the Reference Nutrient Intake (RNI) of pyridoxine for 6-9 months old infants, the complementary infant meal products analysed herein provided less than 15% of the RNI values with mean (SD) values of 12.87 (± 4.46)% and 13.88 (± 4.97)% for the meat- and vegetable-based recipes, respectively. The estimated total daily intake of riboflavin and pyridoxine from the consumption of commercial complementary food was found to be satisfactory and in accordance with the Dietary Reference Values (DRVs). The intake of both riboflavin and pyridoxine was estimated to be mainly derived from the consumption of formula milk which could be a cause of concern if the quality of an infant's milk diet is compromised by an inadequate or lack of supplemented milk intake. The results of this study suggest that the selected commercial complementary infant foods in the UK market may not contain the minimum levels of riboflavin and pyridoxine required for the labelling declaration of the

  18. Incidence of pharmaceuticals in soils, sediments and waters of Pego-Oliva Marsh by LC-MS/MS.

    Science.gov (United States)

    Vazquez-Roig, P.; Andreu, V.; Blasco, C.; Picó, Y.

    2012-04-01

    The presence of pharmaceutical residues in the environmental compartments is a growing problem that could have unexpected consequences. In recent years, the number of pharmaceuticals detected in the environment had increased spectacularly, reaching a broad number of the most consumed drugs and including virtually all the existing therapeutic classes. These compounds come mainly from human excretions, waste effluents of manufacturing processes and animal farms. In Spain, obsolete sewage treatment plants, and even the absence of those, are the main problem to be solved. Some pharmaceuticals have shown toxicity to bacteria, algae and invertebrates. Besides that reproductive problems in fishes have been observed in "in vitro" studies. By the other hand, synergistic effects of exposure to mixtures of drugs or toxic effects due to accumulation would be expected. A method developed in our laboratory was utilized to monitor the occurrence of 16 relevant pharmaceuticals in the Pego-Oliva Marsh Natural Reserve (Valencian Community, Spain). A total 46 samples of soils (at two different depths), 15 sediments and 34 waters were collected in June 2009. Solid samples were concentrated by pressurized liquid extraction (ASE® 200) using water at 90°C as extracting solvent and three cycles of extraction of 7 minutes. The aqueous extract obtained was passed through two cartridges connected in series: to an Isolute® SAX cartridge (strong anion exchange) on the top and an Oasis® HLB cartridge below. Extraction was carried out with 6mL of methanol. Quantification was performed by a Quattro Micro LC-MS/MS with an ESI interface working in both positive and negative mode. Two transitions were utilized for each compound to obtain an unequivocal confirmation, with the exception of ibuprofen which only gave one transition with adequate sensitivity. All water samples appeared contaminated with at least with two compounds. Ibuprofen and codeine were the compounds more frequently detected in

  19. LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2.

    Science.gov (United States)

    Kathiresan, Meena; English, Ann M

    2017-02-01

    We recently reported that cytochrome c peroxidase (Ccp1) functions as a H 2 O 2 sensor protein when H 2 O 2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (Cyc II ), determines whether Ccp1 acts as a H 2 O 2 sensor or peroxidase. For H 2 O 2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H 2 O 2 in the absence of Cyc II to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H 2 O 2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H 2 O 2 shuts down heterolytic cleavage of H 2 O 2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H 2 O 2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O 2 reactivity.

  20. Assessment of the effects of As(III) treatment on cyanobacteria lipidomic profiles by LC-MS and MCR-ALS.

    Science.gov (United States)

    Marques, Aline S; Bedia, Carmen; Lima, Kássio M G; Tauler, Romà

    2016-08-01

    Cyanobacteria are a group of photosynthetic, nitrogen-fixing bacteria present in a wide variety of habitats such as freshwater, marine, and terrestrial ecosystems. In this work, the effects of As(III), a major toxic environmental pollutant, on the lipidomic profiles of two cyanobacteria species (Anabaena and Planktothrix agardhii) were assessed by means of a recently proposed method based on the concept of regions of interest (ROI) in liquid chromatography mass spectroscopy (LC-MS) together with multivariate curve resolution alternating least squares (MCR-ALS). Cyanobacteria were exposed to two concentrations of As(III) for a week, and lipid extracts were analyzed by ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry in full scan mode. The data obtained were compressed by means of the ROI strategy, and the resulting LC-MS data sets were analyzed by the MCR-ALS method. Comparison of profile peak areas resolved by MCR-ALS in control and exposed samples allowed the discrimination of lipids whose concentrations were changed due to As(III) treatment. The tentative identification of these lipids revealed an important reduction of the levels of some galactolipids such as monogalactosyldiacylglycerol, the pigment chlorophyll a and its degradation product, pheophytin a, as well as carotene compounds such as 3-hydroxycarotene and carotene-3,3'-dione, all of these compounds being essential in the photosynthetic process. These results suggested that As(III) induced important changes in the composition of lipids of cyanobacteria, which were able to compromise their energy production processes. Graphical abstract Steps of the proposed LC-MS + MCR-ALS procedure.

  1. ChromAlign: A two-step algorithmic procedure for time alignment of three-dimensional LC-MS chromatographic surfaces.

    Science.gov (United States)

    Sadygov, Rovshan G; Maroto, Fernando Martin; Hühmer, Andreas F R

    2006-12-15

    We present an algorithmic approach to align three-dimensional chromatographic surfaces of LC-MS data of complex mixture samples. The approach consists of two steps. In the first step, we prealign chromatographic profiles: two-dimensional projections of chromatographic surfaces. This is accomplished by correlation analysis using fast Fourier transforms. In this step, a temporal offset that maximizes the overlap and dot product between two chromatographic profiles is determined. In the second step, the algorithm generates correlation matrix elements between full mass scans of the reference and sample chromatographic surfaces. The temporal offset from the first step indicates a range of the mass scans that are possibly correlated, then the correlation matrix is calculated only for these mass scans. The correlation matrix carries information on highly correlated scans, but it does not itself determine the scan or time alignment. Alignment is determined as a path in the correlation matrix that maximizes the sum of the correlation matrix elements. The computational complexity of the optimal path generation problem is reduced by the use of dynamic programming. The program produces time-aligned surfaces. The use of the temporal offset from the first step in the second step reduces the computation time for generating the correlation matrix and speeds up the process. The algorithm has been implemented in a program, ChromAlign, developed in C++ language for the .NET2 environment in WINDOWS XP. In this work, we demonstrate the applications of ChromAlign to alignment of LC-MS surfaces of several datasets: a mixture of known proteins, samples from digests of surface proteins of T-cells, and samples prepared from digests of cerebrospinal fluid. ChromAlign accurately aligns the LC-MS surfaces we studied. In these examples, we discuss various aspects of the alignment by ChromAlign, such as constant time axis shifts and warping of chromatographic surfaces.

  2. Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses.

    Science.gov (United States)

    Preis, Anna; Mugisha, Lawrence; Hauser, Barbara; Weltring, Anja; Deschner, Tobias

    2011-12-01

    The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC-MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC-MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC-MS is a valuable tool for the validation of immunoassays. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs.

    Science.gov (United States)

    Paoloni, Angela; Solfrizzo, Michele; Bibi, Rita; Pecorelli, Ivan

    2018-05-04

    The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L -1 for OTA and from 0.60 to 17.85 µg L -1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg -1 and 10 µg kg -1 . LODs were 0.27 and 0.26 µg kg -1 while LOQs were fixed at 1.0 and 1.2 µg kg -1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSD r ) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

  4. Examination of segmental average mass spectra from liquid chromatography-tandem mass spectrometric (LC-MS/MS) data enables screening of multiple types of protein modifications.

    Science.gov (United States)

    Liu, Nai-Yu; Lee, Hsiao-Hui; Chang, Zee-Fen; Tsay, Yeou-Guang

    2015-09-10

    It has been observed that a modified peptide and its non-modified counterpart, when analyzed with reverse phase liquid chromatography, usually share a very similar elution property [1-3]. Inasmuch as this property is common to many different types of protein modifications, we propose an informatics-based approach, featuring the generation of segmental average mass spectra ((sa)MS), that is capable of locating different types of modified peptides in two-dimensional liquid chromatography-mass spectrometric (LC-MS) data collected for regular protease digests from proteins in gels or solutions. To enable the localization of these peptides in the LC-MS map, we have implemented a set of computer programs, or the (sa)MS package, that perform the needed functions, including generating a complete set of segmental average mass spectra, compiling the peptide inventory from the Sequest/TurboSequest results, searching modified peptide candidates and annotating a tandem mass spectrum for final verification. Using ROCK2 as an example, our programs were applied to identify multiple types of modified peptides, such as phosphorylated and hexosylated ones, which particularly include those peptides that could have been ignored due to their peculiar fragmentation patterns and consequent low search scores. Hence, we demonstrate that, when complemented with peptide search algorithms, our approach and the entailed computer programs can add the sequence information needed for bolstering the confidence of data interpretation by the present analytical platforms and facilitate the mining of protein modification information out of complicated LC-MS/MS data. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Validation of an semi-automated multi component method using protein precipitation LC-MS-MS for the analysis of whole blood samples

    DEFF Research Database (Denmark)

    Slots, Tina

    BACKGROUND: Solid phase extraction (SPE) are one of many multi-component methods, but can be very time-consuming and labour-intensive. Protein precipitation is, on the other hand, a much simpler and faster sample pre-treatment than SPE, and protein precipitation also has the ability to cover a wi......-mortem whole blood sample preparation for toxicological analysis; from the primary sample tube to a 96-deepwell plate ready for injection on the liquid chromatography mass spectrometry (LC-MS/MS)....

  6. Part 3: Solid phase extraction of Russian VX and its chemical attribution signatures in food matrices and their detection by GC-MS and LC-MS.

    Science.gov (United States)

    Williams, Audrey M; Vu, Alexander K; Mayer, Brian P; Hok, Saphon; Valdez, Carlos A; Alcaraz, Armando

    2018-08-15

    Chemical attribution signatures indicative of O-isobutyl S-(2-diethylaminoethyl) methylphosphonothioate (Russian VX) synthetic routes were investigated in spiked food samples. Attribution signatures were identified using a multifaceted approach: Russian VX was synthesized using six synthetic routes and the chemical attribution signatures identified by GC-MS and LC-MS. Three synthetic routes were then down selected and spiked into complex matrices: bottled water, baby food, milk, liquid eggs, and hot dogs. Sampling and extraction methodologies were developed for these materials and used to isolate the attribution signatures and Russian VX from each matrix. Recoveries greater than 60% were achieved for most signatures in all matrices; some signatures provided recoveries greater than 100%, indicating some degradation during sample preparation. A chemometric model was then developed and validated with the concatenated data from GC-MS and LC-MS analyses of the signatures; the classification results of the model were > 75% for all samples. This work is part three of a three-part series in this issue of the United States-Sweden collaborative efforts towards the understanding of the chemical attribution signatures of Russian VX in crude materials and in food matrices. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    Science.gov (United States)

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  8. Simultaneous LC-MS/MS determination of phenylbutyrate, phenylacetate benzoate and their corresponding metabolites phenylacetylglutamine and hippurate in blood and urine.

    Science.gov (United States)

    Laryea, Maurice D; Herebian, Diran; Meissner, Thomas; Mayatepek, Ertan

    2010-12-01

    Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 μmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.

  9. A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

    Directory of Open Access Journals (Sweden)

    Elisabeth J. Faassen

    2016-02-01

    Full Text Available Exposure to β-N-methylamino-l-alanine (BMAA might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS analysis, or directly followed by LC-MS/MS analysis for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80% and precise (mean relative standard deviation 10%, except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds showed higher variation (relative standard deviation 21%–32%, implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%. Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

  10. Analysis of membrane proteome by data-dependent LC-MS/MS combined with data-independent LC-MSE technique

    Directory of Open Access Journals (Sweden)

    Joseph Kwon

    2010-03-01

    Full Text Available Proteomics work resembles the search for a needle in a haystack. The identification of protein biomarker requires the removal of the false protein data from the whole protein mixture. For high quality proteomic data, even a strict filtration step using the false discovery rate (FDR is insufficient for obtaining perfect protein information from the biological samples. In this study, the cyanobacterial whole membrane fraction was applied to the data-dependent analysis (DDA mode of LC-MS/MS, which was used along with the data-independent LC-MSE technique in order to evaluate the membrane proteomic data. Furthermore, the identified MSE-information (MSE-i data based on the peptide mass and the retention time were validated by the other database search, i.e., the probability-based MASCOT and de novo search engine PEAKS. In this present study, 208 cyanobacterial proteins with FDR of 5% were identified using the data-independent nano-UPLC/MSE acquisition with the Protein Lynx Global Server (PLGS, and 56 of these proteins were the predicted membrane proteins. When a total of 208 MSE-i proteomic data were applied to the DDA mode of LC-MS/MS, the number of identified membrane proteins was 26 and 33 from MASCOT and PEAKS with a FDR of 5%, respectively. The number of totally overlapped membrane proteins was 25. Therefore, the data-independent LC-MSE identified more proteins with a high confidence.

  11. Rapid and reliable QuEChERS-based LC-MS/MS method for determination of acrylamide in potato chips and roasted coffee

    Science.gov (United States)

    Stefanović, S.; Đorđevic, V.; Jelušić, V.

    2017-09-01

    The aim of this paper is to verify the performance characteristics and fitness for purpose of rapid and simple QuEChERS-based LC-MS/MS method for determination of acrylamide in potato chips and coffee. LC-MS/MS is by far the most suitable analytical technique for acrylamide measurements given its inherent sensitivity and selectivity, as well as capability of analyzing underivatized molecule. Acrylamide in roasted coffee and potato chips wasextracted with water:acetonitrile mixture using NaCl and MgSO4. Cleanup was carried out with MgSO4 and PSA. Obtained results were satisfactory. Recoveries were in the range of 85-112%, interlaboratory reproducibility (Cv) was 5.8-7.6% and linearity (R2) was in the range of 0.995-0.999. LoQ was 35 μg kg-1 for coffee and 20 μg kg-1 for potato chips. Performance characteristic of the method are compliant with criteria for analytical methods validation. Presented method for quantitative determination of acrylamide in roasted coffee and potato chips is fit for purposes of self-control in food industry as well as regulatory controls carried out by the governmental agencies.

  12. Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Fumio; Morino, Keiko; Miyashita, Masahiro; Miyagawa, Hisashi [Kyoto Univ. (Japan). Department of Agriculture

    2003-05-01

    The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d{sub 5}-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW){sup -1}h{sup -1}, respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW){sup -1}h{sup -1}, respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds. (author)

  13. eMZed: an open source framework in Python for rapid and interactive development of LC/MS data analysis workflows.

    Science.gov (United States)

    Kiefer, Patrick; Schmitt, Uwe; Vorholt, Julia A

    2013-04-01

    The Python-based, open-source eMZed framework was developed for mass spectrometry (MS) users to create tailored workflows for liquid chromatography (LC)/MS data analysis. The goal was to establish a unique framework with comprehensive basic functionalities that are easy to apply and allow for the extension and modification of the framework in a straightforward manner. eMZed supports the iterative development and prototyping of individual evaluation strategies by providing a computing environment and tools for inspecting and modifying underlying LC/MS data. The framework specifically addresses non-expert programmers, as it requires only basic knowledge of Python and relies largely on existing successful open-source software, e.g. OpenMS. The framework eMZed and its documentation are freely available at http://emzed.biol.ethz.ch/. eMZed is published under the GPL 3.0 license, and an online discussion group is available at https://groups.google.com/group/emzed-users. Supplementary data are available at Bioinformatics online.

  14. Development of an LC/MS/MS method in order to determine arctigenin in rat plasma: its application to a pharmacokinetic study.

    Science.gov (United States)

    Zou, Quanfei; Gu, Yuan; Lu, Rong; Zhang, Tiejun; Zhao, Guang-Rong; Liu, Changxiao; Si, Duanyun

    2013-09-01

    In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2-500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and -80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half-life and area under the concentration-time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Nuclear magnetic resonance and LC/MS characterization of native and new mass-labeled fluorinated telomer alcohols, acids and unsaturated acids

    Energy Technology Data Exchange (ETDEWEB)

    Arsenault, G.; Chittim, B.; McAlees, A.; Yeo, B. [Wellington Laboratories Inc., Guelph, ON (Canada); Ellis, D.; Mabury, S.; Stock, N. [Toronto Univ., ON (Canada); Halldorson, T.; Tomy, G. [Dept. of Fisheries and Oceans, Winnipeg, MB (Canada); McCrindle, R. [Guelph Univ., ON (Canada)

    2004-09-15

    A variety of fluorinated compounds are used in a multitude of consumer products because of their ability to repel water and oil, resistance to heat, and chemical inertness. Recently, scientists and regulators have begun raising concerns about the potential health and environmental impact of perfluorinated compounds. Exposure to perfluoroalkyl acids, such as Perfluorooctanoic acid (PFOA), has been identified as a potential human health concern. A study has shown that telomer alcohols such as 2-perfluorooctylethanol can be metabolized by living organisms or biodegrade under environmental conditions to sequentially give the saturated fluorinated telomer acid (2- perfluorooctylethanoic acid), then the unsaturated telomer acid (2H-Perfluorooct-2-enoic acid), and eventually PFOA. Additional experimental work is necessary to determine the extent, if any, to which telomer product degradation may be a source of PFOA. The analysis for fluorinated compounds in environmental samples is performed, primarily, using LC/MS techniques. These analyses have been hindered by the lack of any commercially available mass-labeled fluorinated compounds for use as surrogates and thus may be restricting the amount of research conducted in this area. We have now synthesized the mass-labeled perfluoroalkyl telomer alcohols and the corresponding acids and unsaturated acids. We report in this study their 1H-, 2H-, 19F- and 13C-NMR characterizations along with GC/MS and LC/MS data and evaluation of their use as surrogate standards.

  16. ESI-LC-MS based-metabolomics data of mangosteen (Garcinia mangostana Linn. fruit pericarp, aril and seed at different ripening stages

    Directory of Open Access Journals (Sweden)

    Siti Farah Mamat

    2018-04-01

    Full Text Available Fruit ripening is a complex phenomenon involving a series of biochemical, physiological and organoleptic changes. Ripening process in mangosteen (Garcinia mangostana Linn. is unique of which the fruit will only ripen properly if harvested during its middle stage (emergence of purple/pink colour but not earlier (green stage. The knowledge on the molecular mechanism and regulation behind this phenomenon is still limited. Hence, electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS based metabolomics analysis was applied to determine the metabolome of mangosteen ripening. Specifically, mangosteen pericarp, aril and seed were collected at four different ripening stages (stage 0: green, stage 2: yellowish with pink patches, stage 4: brownish red and stage 6: dark purple and subjected to metabolite profiling analysis. The data provided in this article have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gks1004. PubMed PMID: 23109552 with the identifier MTBLS595. The complete dataset can be accessed here https://www.ebi.ac.uk/metabolights/MTBLS595. Keywords: Ripening, Garcinia mangostana Linn., Metabolomics, ESI-LC-MS

  17. Determination and Identification of a Specific Marker Compound for Discriminating Shrub Chaste Tree Fruit from Agnus Castus Fruit Based on LC/MS Metabolic Analysis.

    Science.gov (United States)

    Yahagi, Tadahiro; Masada, Sayaka; Oshima, Naohiro; Suzuki, Ryuta; Matsufuji, Hiroshi; Takahashi, Yutaka; Watanabe, Masato; Yahara, Shoji; Iida, Osamu; Kawahara, Nobuo; Maruyama, Takuro; Goda, Yukihiro; Hakamatsuka, Takashi

    2016-01-01

    Shrub Chaste Tree Fruit (SCTF) is defined as the fruits of Vitex rotundifolia L. f. and V. trifolia L. and has been used as a component of some traditional Japanese medicines (Kampo formulations). Agnus Castus Fruit (ACF) is defined as the dried ripe fruits of V. agnus-castus L.; it is used in traditional European medicines, but is becoming popular in Japan as both an over-the-counter drug and as an ingredient in health foods for treating premenstrual syndrome (PMS). To ensure the efficacy and safety of both SCTF and ACF products, it is important to precisely authenticate their botanical origins and to clearly distinguish between SCTF and ACF. Therefore, we tried to identify SCTF-specific marker compounds based on LC/MS metabolic analysis. The multivariate analysis of LC/MS data from SCTF and ACF samples furnished candidate marker compounds of SCTF. An SCTF-specific marker was isolated from SCTF crude drugs and identified as 3-O-trans-feruloyl tormentic acid on the basis of spectroscopic data from NMR and MS. Since avoiding contamination from closely related species is a significant requirement for pharmaceuticals of natural origin, this information will be valuable for the quality control of both SCTF and ACF products from the viewpoint of regulatory science.

  18. Compatibility study of a parenteral microdose polyethylene glycol formulation in medical devices and identification of degradation impurity by 2D-LC/MS.

    Science.gov (United States)

    Dai, Lulu; Yeh, Geoffrey K; Ran, Yingqing; Yehl, Peter; Zhang, Kelly

    2017-04-15

    Polyethylene glycol (PEG) based formulation and polyvinylchloride (PVC) tubing are frequently used for drug delivery and administration. The compatibility of a parenteral drug microdose formulation in intravenous infusion (IV) devices was studied to support the clinical determination of absolute bioavailability by the microdosing method. The investigational microdose formulation containing PEG was found prone to significant loss of potency within hours of storage in the PVC IV tubing due to degradation. Degradation occurred only when both PEG and PVC tubing were present. The degradation product could not be detected by LC/MS due to the significant interference from the high concentration of PEG (4%) matrix and the extremely low level of drug (0.6ppm). To obtain structural information of the degradation impurity and understand the cause of the degradation, a simple heart-cutting 2D-LC/MS approach was utilized to effectively separate the impurity from the complex PEG oligomers and overcome the matrix interference, enabling mass spectrometric analysis of the impurity. An oxidation- dominated mechanism was proposed in which the combination of PEG auto-oxidation and dehydrochlorination of the PVC tubing yielded an oxidative environment that enhanced radical propagation and accelerated degradation of the investigational parent drug. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Ultra-sensitive LC-MS/MS method for the quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma for a microdose clinical trial.

    Science.gov (United States)

    van Nuland, M; Hillebrand, M J X; Rosing, H; Burgers, J A; Schellens, J H M; Beijnen, J H

    2018-03-20

    In microdose clinical trials a maximum of 100 μg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC-MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC-MS/MS method for quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5-500 pg/mL for gemcitabine and 250-50,000 pg/mL for dFdU were linear, with a correlation coefficient (r 2 ) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

    Science.gov (United States)

    Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

    2016-12-01

    The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

  1. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  2. Development of a LC-MS/MS Method for the Simultaneous Detection of Tricarboxylic Acid Cycle Intermediates in a Range of Biological Matrices

    Directory of Open Access Journals (Sweden)

    Omar Al Kadhi

    2017-01-01

    Full Text Available It is now well-established that perturbations in the tricarboxylic acid (TCA cycle play an important role in the metabolic transformation occurring in cancer including that of the prostate. A method for simultaneous qualitative and quantitative analysis of TCA cycle intermediates in body fluids, tissues, and cultured cell lines of human origin was developed using a common C18 reversed-phase column by LC-MS/MS technique. This LC-MS/MS method for profiling TCA cycle intermediates offers significant advantages including simple and fast preparation of a wide range of human biological samples. The analytical method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. The limits of detection were below 60 nM for most of the TCA intermediates with the exception of lactic and fumaric acids. The calibration curves of all TCA analytes showed linearity with correlation coefficients r2>0.9998. Recoveries were >95% for all TCA analytes. This method was established taking into consideration problems and limitations of existing techniques. We envisage that its application to different biological matrices will facilitate deeper understanding of the metabolic changes in the TCA cycle from in vitro, ex vivo, and in vivo studies.

  3. Rapid extraction combined with LC-tandem mass spectrometry (CREM-LC/MS/MS) for the determination of ciguatoxins in ciguateric fish flesh.

    Science.gov (United States)

    Lewis, Richard J; Yang, Aijun; Jones, Alun

    2009-07-01

    Ciguatera is a significant food borne disease caused by potent polyether toxins known as ciguatoxins, which accumulate in the flesh of ciguateric fish at risk levels above 0.1 ppb. The management of ciguatera has been hindered by the lack of analytical methods to detect and quantify clinically relevant levels of ciguatoxin in easily prepared crude extracts of fish. Here we report a ciguatoxin rapid extraction method (CREM) that allows the rapid preparation of fish flesh extracts for the detection and quantification of ciguatoxin by gradient reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS). CREM-LC/MS/MS delivers a linear response to P-CTX-1 spiked into fish prior to extraction. A similar response was obtained for P-CTX-1 spiked after extraction, indicating >95% extraction efficiency was achieved overall and 85% at the limit of quantification (0.1 ppb). Using this approach, levels >or=0.1 ppb P-CTX-1 could be detected and quantified from an extract of 2g fish flesh, making it suitable as a confirmatory assay for suspect ciguateric carnivorous fish in the Pacific Ocean. The approach is designed to simplify the extraction and analysis of multiple samples per day.

  4. Validation approach for a fast and simple targeted screening method for 75 antibiotics in meat and aquaculture products using LC-MS/MS.

    Science.gov (United States)

    Dubreil, Estelle; Gautier, Sophie; Fourmond, Marie-Pierre; Bessiral, Mélaine; Gaugain, Murielle; Verdon, Eric; Pessel, Dominique

    2017-04-01

    An approach is described to validate a fast and simple targeted screening method for antibiotic analysis in meat and aquaculture products by LC-MS/MS. The strategy of validation was applied for a panel of 75 antibiotics belonging to different families, i.e., penicillins, cephalosporins, sulfonamides, macrolides, quinolones and phenicols. The samples were extracted once with acetonitrile, concentrated by evaporation and injected into the LC-MS/MS system. The approach chosen for the validation was based on the Community Reference Laboratory (CRL) guidelines for the validation of screening qualitative methods. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest, generally the maximum residue limit (MRL). A robustness study was also performed to test the influence of different factors. The validation showed that the method is valid to detect and identify 73 antibiotics of the 75 antibiotics studied in meat and aquaculture products at the validation levels.

  5. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.

    Science.gov (United States)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per; Holmbjerg, Anne Fich; Grimstrup, Marie; Mørtz, Ejvind; Kofoed, Thomas; Højrup, Peter

    2018-07-01

    Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins.

    Science.gov (United States)

    Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

    2014-01-01

    N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

  7. Chemometrically assisted development and validation of LC-MS/MS method for the analysis of potential genotoxic impurities in meropenem active pharmaceutical ingredient.

    Science.gov (United States)

    Grigori, Katerina; Loukas, Yannis L; Malenović, Anđelija; Samara, Vicky; Kalaskani, Anastasia; Dimovasili, Efi; Kalovidouri, Magda; Dotsikas, Yannis

    2017-10-25

    A sensitive Liquid Chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb range, a high concentration of meropenem API (30mg/mL) had to be injected. Therefore, efficient determination of meropenem from its impurities became a critical aim of this study, in order to divert meropenem to waste, via a switching valve. ‎ After the selection of the important factors affecting analytes' elution, a Box-Behnken design was utilized to set the plan of experiments conducted with UV detector. As responses, the separation factor s between the last eluting impurity and meropenem, as well as meropenem retention factor k were used. Grid point search methodology was implemented aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5v/v. All impurities and internal standard omeprazole were eluted before 7.5min and at 8.0min the eluents were directed to waste. The protocol was transferred to LC-MS/MS and validated according to ICH guidelines. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Multiresidue method for detection of pesticides in beef meat using liquid chromatography coupled to mass spectrometry detection (LC-MS) after QuEChERS extraction.

    Science.gov (United States)

    Oliveira, Fabiano Aurélio da Silva; Pereira, Elba Nathália Corrêa; Gobbi, Jennifer Mattedi; Soto-Blanco, Benito; Melo, Marília Martins

    2018-01-01

    Beef meat is an important food that can be contaminated by pesticides. This study aimed to optimize a multiresidue method for identification and quantification of pesticides in beef meat by liquid chromatography coupled to mass spectrometry detection (LC-MS). The extraction and clean-up procedures were adapted from the QuECHERS method. From the 188 analytes tested, the method was validated as qualitative method for 19 compounds and as quantitative method for 152 compounds. The results were satisfactory, yielding coefficients of variation of less than 20% and recoveries ranging from 70% to 120% and expanded uncertainty of less than 50%. The quantification limit was typically 10 µg kg -1 (but 25 µg kg -1 for 12 of the compounds) and the detection limit was 5.0 µg kg -1 . Thirty-two real samples of commercialized beef meat were analyzed without any residual pesticide being found. Thus, the results showed that the multiresidue method for detecting 171 pesticides, using adapted QuECHERS for extraction and LC-MS for detection, is suitable for analyzing beef meat.

  9. A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

    Science.gov (United States)

    Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro

    2018-02-01

    Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Validation of a LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study

    Science.gov (United States)

    Rower, Joseph E.; Bushman, Lane R.; Hammond, Kyle P.; Kadam, Rajendra S.; Aquilante, Christina L.

    2011-01-01

    Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate, and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6% and 10.7%, and inter-assay precision ranging from 4.4% to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6% and 108.7% of the expected value, and inter-assay concentrations within 89.4 to 104.0% of the expected value. The linear concentration range was between 0.5 and 50 μg/mL with a lower limit of quantitation of 0.5 μg/mL when 125 μL of plasma were extracted. This LC/MS method yielded a quick, simple, and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. PMID:21077249

  11. Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Rower, Joseph E; Bushman, Lane R; Hammond, Kyle P; Kadam, Rajendra S; Aquilante, Christina L

    2010-12-01

    Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6 and 10.7%, and inter-assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6-108.7% of the expected value, and inter-assay concentrations within 89.4-104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.

  12. Distinctive and Complementary MS2 Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow

    Science.gov (United States)

    Kuo, Chu-Wei; Guu, Shih-Yun; Khoo, Kay-Hooi

    2018-04-01

    High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies. Metal oxide has been applied to enrich phosphopeptides and sialylated glycopeptides, but its use to capture sulfated glycopeptides has not been investigated. Likewise, various complementary MS2 fragmentation modes have yet to be tested against sialylated and non-sialylated sulfoglycopeptides due to limited appropriate sample availability. In this study, we have investigated the feasibility of sequencing tryptic sulfated N-glycopeptide and its MS2 fragmentation characteristics by first optimizing the enrichment methods to allow efficient LC-MS detection and MS2 analysis by a combination of CID, HCD, ETD, and EThcD on hybrid and tribrid Orbitrap instruments. Characteristic sulfated glyco-oxonium ions and direct loss of sulfite from precursors were detected as evidences of sulfate modification. It is anticipated that the technical advances demonstrated in this study would allow a feasible extension of our sulfoglycomic analysis to sulfoglycoproteomics. [Figure not available: see fulltext.

  13. Simultaneous determination of seven alkaloids from Rhizoma Corydalis Decumbentis in rabbit aqueous humor by LC-MS/MS: Application to ocular pharmacokinetic studies.

    Science.gov (United States)

    Mao, Zhengsheng; Wang, Xin; Liu, Yangdan; Huang, Yuting; Liu, Youping; Di, Xin

    2017-07-01

    This study aims to establish a fast and sensitive LC-MS/MS method for simultaneous determination of seven alkaloids from Rhizoma Corydalis Decumbentis in rabbit aqueous humor. Aqueous humor samples were processed by protein precipitation and then separated on a Thermo Syncronis C 18 column (50mm×2.1mm, 5μm) with a mobile phase using acetonitrile-0.05% formic acid (28:72, v/v). Detection of the analytes and the internal standard (coptisine) were performed in positive electrospray ionization with selected reaction monitoring. The method showed good linearity (r>0.9931) for all the seven alkaloids. This fully validated method was applied to the studies of aqueous humor pharmacokinetics of seven alkaloids from Rhizoma Corydalis Decumbentis and the effects of borneol on corneal penetration of these alkaloids into aqueous humor. This is the first work that presents a reliable LC-MS/MS method for simultaneous determination of seven alkaloids in rabbit aqueous humor and its application of ocular pharmacokinetics of seven alkaloids from Rhizoma Corydalis Decumbentis. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Multivariate DoE Optimization of Asymmetric Flow Field Flow Fractionation Coupled to Quantitative LC-MS/MS for Analysis of Lipoprotein Subclasses

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Kuklenyik

    2015-02-01

    Full Text Available In this report we demonstrate a practical multivariate design of experiment (DoE approach for asymmetric flow field-flow fractionation (AF4 method optimization using separation of lipoprotein subclasses as an example. First, with the aid of commercially available software, we built a full factorial screening design where the theoretical outcomes were calculated by applying established formulas that govern AF4 channel performance for a 5–35 nm particle size range of interest for lipid particles. Second, using the desirable ranges of instrumental parameters established from theoretical optimization, we performed fractional factorial DoE for AF4 separation of pure albumin and ferritin with UV detection to narrow the range of instrumental parameters and allow optimum size resolution while minimizing losses from membrane immobilization. Third, the optimal range of conditions were tested using response surface DoE for sub-fractionation of high and low density lipoproteins (HDL and LDL in human serum, where the recovery of the analytes were monitored by fraction collection and isotope-dilution LC-MS/MS analysis of each individual fraction for cholesterol and apolipoproteins (ApoA-1 and ApoB-100. Our results show that DoE is an effective tool in combining AF4 theoretical knowledge and experimental data in finding the most optimal set of AF4 instrumental parameters for quantitative coupling with LC-MS/MS measurements.

  15. LC-MS/MS method for simultaneous determination of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin in serum of multiple myeloma patients.

    Science.gov (United States)

    Shu, Chang; Zeng, Tianmei; Gao, Shouhong; Xia, Tianyi; Huang, Lifeng; Zhang, Feng; Chen, Wansheng

    2016-08-15

    Multiple myeloma (MM), a malignant neoplastic serum-cell disorder, has been a serious threat to human health. The determination of 6 commonly used drug concentrations, including thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin, in MM patients was of great clinical interest. Herein, we reported a method for the rapid and simultaneous measurement of the above therapeutics by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method with solid phase extraction. Analysis was performed on a Waters XBridge(®) BEH C18 column (2.5μm, 2.1 mm×50mm), with formic acid aqueous solution and acetonitrile as the mobile phase at flow rate 0.3mL/min. All analytes showed good correlation coefficients (r>0.996), and LLOQ of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin were 4, 2, 2, 2, 2 and 2ng/mL, respectively. The inter- and intra-day precisions and stability were expressed as variation coefficients within 15% and relative error less than 15%. Dilution effect, carryover and incurred sample reanalysis were investigated according to the 2015 edition Chinese Pharmacopoeia guidelines, as US FDA (2013, revision 1) required. The LC-MS/MS based assay described in this article may improve future clinical studies evaluating common therapeutics for MM treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Differential Proteomic Analysis of Syncytiotrophoblast Extracellular Vesicles from Early-Onset Severe Preeclampsia, using 8-Plex iTRAQ Labeling Coupled with 2D Nano LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hongmei Li

    2015-06-01

    Full Text Available Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM may lead to preeclampsia (PE. We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8 related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE.

  17. Vigna subterranea ammonium transporter gene (VsAMT1: Some bioinformatics insights

    Directory of Open Access Journals (Sweden)

    Adewole T. Adetunji

    2015-12-01

    Full Text Available Ammonium transporters (AMTs play a role in the uptake of ammonium, the form in which nitrogen is preferentially absorbed by plants. Vigna subterranea (VsAMT1 and Solanum tuberosum (StAMT1 AMT1s were characterized using molecular biology and bioinformatics methods. AMT1-specific primers were designed and used to amplify the AMT1 internal regions. Nucleotide sequencing, alignment and phylogenetic analysis assigned VsAMT1 and StAMT1 to the AMT1 family. The deduced amino acid sequences showed that VsAMT1 is 92% and 89% similar to Phaseolus vulgaris PvAMT1.1 and Glycine max AMT1 respectively, while StAMT1 is 92% similar to Solanum lycopersicum LeAMT1.1, and correspond to the 5th–10th trans-membrane domains. Residues VsAMT1 D23 and StAMT1 D15 are predicted to be essential for ammonium transport, while mutations of VsAMT1 W1A-L and S87A and StAMT1 S76A may further enhance ammonium transport. In addition to nitrogen uptake from the roots, VsAMT1 may also contribute to interactions with rhizobia.

  18. Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Bhagwat, Aditya M; Cotton, Richard J; Dib, Shaima S; Kumar, Pankaj; Hayat, Shahina; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2017-01-06

    Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC

  19. Effects of sample injection amount and time-of-flight mass spectrometric detection dynamic range on metabolome analysis by high-performance chemical isotope labeling LC-MS.

    Science.gov (United States)

    Zhou, Ruokun; Li, Liang

    2015-04-06

    The effect of sample injection amount on metabolome analysis in a chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) platform was investigated. The performance of time-of-flight (TOF) mass spectrometers with and without a high-dynamic-range (HD) detection system was compared in the analysis of (12)C2/(13)C2-dansyl labeled human urine samples. An average of 1635 ± 21 (n = 3) peak pairs or putative metabolites was detected using the HD-TOF-MS, compared to 1429 ± 37 peak pairs from a conventional or non-HD TOF-MS. In both instruments, signal saturation was observed. However, in the HD-TOF-MS, signal saturation was mainly caused by the ionization process, while in the non-HD TOF-MS, it was caused by the detection process. To extend the MS detection range in the non-HD TOF-MS, an automated switching from using (12)C to (13)C-natural abundance peaks for peak ratio calculation when the (12)C peaks are saturated has been implemented in IsoMS, a software tool for processing CIL LC-MS data. This work illustrates that injecting an optimal sample amount is important to maximize the metabolome coverage while avoiding the sample carryover problem often associated with over-injection. A TOF mass spectrometer with an enhanced detection dynamic range can also significantly increase the number of peak pairs detected. In chemical isotope labeling (CIL) LC-MS, relative metabolite quantification is done by measuring the peak ratio of a (13)C2-/(12)C2-labeled peak pair for a given metabolite present in two comparative samples. The dynamic range of peak ratio measurement does not need to be very large, as only subtle changes of metabolite concentrations are encountered in most metabolomic studies where relative metabolome quantification of different groups of samples is performed. However, the absolute concentrations of different metabolites can be very different, requiring a technique to provide a wide detection dynamic range to allow the detection of as

  20. Detection and differentiation of 22kDa and 20kDa Growth Hormone proteoforms in human plasma by LC-MS/MS

    DEFF Research Database (Denmark)

    Sanmartín, Gerard Such; Bache, N.; Bosch, J.

    2015-01-01

    Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20kDa GH proteoforms that demonstrate the administration of the synthetic hormone....... Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report...... a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH...

  1. Parabens in urine, serum and seminal plasma from healthy Danish men determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Jørgensen, Niels; Andersson, Anna-Maria

    2011-01-01

    Parabens are used as anti-microbial preservatives in a range of consumer products, especially in cosmetics. In vitro and animal studies have shown weak estrogenic and other endocrine disrupting effects of parabens, including reduced testosterone levels in exposed male rats. The knowledge of paraben...... exposure, distribution and excretion in humans is limited. In this study we determined the concentration of five parabens; methyl-, ethyl-, n-propyl-, n-butyl- and benzylparaben in urine, serum and seminal plasma samples from 60 healthy Danish men. To conduct the study a sensitive and specific method using...... LC-MS/MS for simultaneous determination of the five parabens was developed for all three different matrices. Highest concentrations of the parabens were found in urine, wherein methyl-, ethyl-, n-propyl- and n-butyl parabens were measurable in 98%, 80%, 98% and 83% of the men, respectively. Benzyl...

  2. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

    Directory of Open Access Journals (Sweden)

    Zulezwan A. Malik

    2013-12-01

    Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

  3. A comparative study on the skin penetration of pure tryptanthrin and tryptanthrin in Isatis tinctoria extract by dermal microdialysis coupled with isotope dilution ESI-LC-MS.

    Science.gov (United States)

    Oberthür, Christine; Heinemann, Christian; Elsner, Peter; Benfeldt, Eva; Hamburger, Matthias

    2003-05-01

    The indolo[2,1- b]quinazoline alkaloid tryptanthrin has recently been identified as a pharmacologically active compound in Isatis tinctoria, with potent dual inhibitory activity on prostaglandin and leukotriene synthesis. To investigate the skin penetration of tryptanthrin from solutions of pure compound and Isatis extracts, we developed and validated a cutaneous microdialysis model using ex vivo pig foreleg. Microdialysis was performed by placing linear probes in the dermis of the skin in situ, and tryptanthrin concentrations in the dialysates were determined by isotope dilution electrospray ionization LC-MS in the selected ion mode. Measurable concentrations of tryptanthrin were detected 30 min after application. A dose-dependent increase in tryptanthrin concentrations in the dialysate was observed for the Isatis extracts, but not for pure tryptanthrin. Microscopic analysis showed that the pure compound crystallized from the solution but remained in an amorphous state in the extracts.

  4. Development and validation of methodology SPE-LC-MS/MS for pharmaceuticals and illicit drug determination in the waters of Guarapiranga dam - Sao Paulo/SP, Brazil

    OpenAIRE

    SHIHOMATSU, HELENA M.

    2015-01-01

    Este estudo apresenta o desenvolvimento da metodologia de extração em fase sólida e separação em cromatográfica líquida acoplada a espectrometria de massas em sequencia, SPE-LC-MS/MS, para a determinação de 21 (vinte e um) fármacos pertencentes a diferentes classes terapeuticas, 1 (uma) droga de abuso e seu principal metabólito, em amostras de água superficial. A separação cromatográfica foi otimizada estudando o desempenho de fases estacionárias e fases móvies. A quantificação dos compostos ...

  5. LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea.

    Science.gov (United States)

    Krock, Bernd; Tillmann, Urban; John, Uwe; Cembella, Allan

    2008-11-01

    Phycotoxins produced by various species of toxigenic microalgae occurring in the plankton are a global threat to the security of seafood resources and the health of humans and coastal marine ecosystems. This has necessitated the development and application of advanced methods in liquid chromatography coupled to mass spectrometry (LC-MS) for monitoring of these compounds, particularly in plankton and shellfish. Most such chemical analyses are conducted in land-based laboratories on stored samples, and thus much information on the near real-time biogeographical distribution and dynamics of phycotoxins in the plankton is unavailable. To resolve this problem, we conducted ship-board analysis of a broad spectrum of phycotoxins collected directly from the water column on an oceanographic cruise along the North Sea coast of Scotland, Norway, and Denmark. We equipped the ship with a triple-quadrupole linear ion-trap hybrid LC-MS-MS system for detection and quantitative analysis of toxins, such as domoic acid, gymnodimine, spirolides, dinophysistoxins, okadaic acid, pectenotoxins, yessotoxins, and azaspiracids (AZAs). We focused particular attention on the detection of AZAs, a group of potent nitrogenous polyether toxins, because the culprit species associated with the occurrence of these toxins in shellfish has been controversial. Marine toxins were analyzed directly from size-fractionated plankton net tows (20 microm mesh size) and Niskin bottle samples from discrete depths, after rapid methanolic extraction but without any further clean-up. Almost all expected phycotoxins were detected in North Sea plankton samples, with domoic acid and 20-methylspirolide G being most abundant. Although AZA was the least abundant of these toxins, the high sensitivity of the LC-MS-MS enabled detailed quantification, indicating that the highest amounts of AZA-1 were present in the southern Skagerrak in the 3-20 microm size-fraction. The direct on-board toxin measurements enabled isolation

  6. Validation Study on a Rapid Method for Simultaneous Determination of Pesticide Residues in Vegetables and Fruits by LC-MS/MS.

    Science.gov (United States)

    Sato, Tamaki; Miyamoto, Iori; Uemura, Masako; Nakatani, Tadashi; Kakutani, Naoya; Yamano, Tetsuo

    2016-01-01

    A validation study was carried out on a rapid method for the simultaneous determination of pesticide residues in vegetables and fruits by LC-MS/MS. Preparation of the test solution was performed by a solid-phase extraction technique with QuEChERS (STQ method). Pesticide residues were extracted with acetonitrile using a homogenizer, followed by salting-out and dehydration at the same time. The acetonitrile layer was purified with C18 and PSA mini-columns. The method was assessed for 130 pesticide residues in 14 kinds of vegetables and fruits at the concentration level of 0.01 μg/g according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. As a result 75 to 120 pesticide residues were determined satisfactorily in the tested samples. Thus, this method could be useful for a rapid and simultaneous determination of multi-class pesticide residues in various vegetables and fruits.

  7. DI/LC-MS/MS-Based Metabolic Profiling for Identification of Early Predictive Serum Biomarkers of Metritis in Transition Dairy Cows.

    Science.gov (United States)

    Zhang, Guanshi; Deng, Qilan; Mandal, Rupasri; Wishart, David S; Ametaj, Burim N

    2017-09-27

    The objectives of this study were to evaluate alterations of metabolites in the blood of dairy cows before, during, and after diagnosis of metritis and identify predictive serum metabolite biomarkers for metritis. DI/LC-MS/MS was used to analyze serum samples collected from both healthy and metritic cows during -8, -4, disease diagnosis, +4, and +8 wks relative to parturition. Results indicated that cows with metritis experienced altered concentrations of serum amino acids, glycerophospholipids, sphingolipids, acylcarnitines, and biogenic amines during the entire experimental period. Moreover, two sets of predictive biomarker models and one set of diagnostic biomarker models for metritis were developed, and all of them showed high sensitivity and specificity (e.g., high AUC values by the ROC curve evaluation), which indicate that serum metabolites identified have pretty accurate predictive, diagnostic, and prognostic abilities for metritis in transition dairy cows.

  8. LC-MS/MS analysis of visceral and subcutaneous adipose tissue proteomes in young goats with focus on innate immunity and inflammation related proteins

    DEFF Research Database (Denmark)

    Restelli, Laura; Codrea, Marius Cosmin; Savoini, Giovanni

    2014-01-01

    and visceral adipose tissues of goat, focusing on proteins involved in immune and inflammatory response. A 2-D LC-MS/MS approach followed by cluster analysis shows a clear distinction between subcutaneous and visceral fat tissue proteomes, and qualitative RT-PCR based analysis of 30 potential adipokines...... further confirmed the individual expression patterns of 26 of these, including 7 whose mRNA expression was observed for the first time in adipose tissues. This study provides a first description of adipose tissue proteomes in goat, and presents observations on novel proteins related to metabolic...... inflammation, detoxification and coagulation pathways, as well as regulation of body fat mobilization in dairy animals. These findings are of particular interest in farm animals where health and production traits are important for animal welfare and for economic gains. (C) 2014 Elsevier B.V. All rights...

  9. Comparison of HPLC-RI, LC/MS-MS and enzymatic assays for the analysis of residual lactose in lactose-free milk.

    Science.gov (United States)

    Trani, A; Gambacorta, G; Loizzo, P; Cassone, A; Fasciano, C; Zambrini, A V; Faccia, M

    2017-10-15

    Lactose intolerance is the decreased ability to digest lactose, and the population involved is rapidly increasing all over the world. Different procedures have been reported in the literature to quantify lactose in dairy products, but the official method of analysis is based on enzymatic assay. In this paper, the effectiveness of two enzymatic kits in detecting residual lactose in lactose-free milk was investigated, and a comparison with two alternative chromatographic methods was done. The investigation used several samples of UHT milk containing different levels of lactose, and the results highlighted the inadequacy of the enzymatic assays and of the HPLC-RI method to analyse lactose-free milk. An LC-MS/MS method using the formate adduct was developed, and it allowed quantitation of lactose and lactulose in all samples at a high level of precision and repeatability. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

    Science.gov (United States)

    Geib, Timon; Sleno, Lekha; Hall, Rabea A.; Stokes, Caroline S.; Volmer, Dietrich A.

    2016-08-01

    We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease.

  11. Screening of 23 β-lactams in foodstuffs by LC-MS/MS using an alkaline QuEChERS-like extraction.

    Science.gov (United States)

    Bessaire, Thomas; Mujahid, Claudia; Beck, Andrea; Tarres, Adrienne; Savoy, Marie-Claude; Woo, Pei-Mun; Mottier, Pascal; Desmarchelier, Aurélien

    2018-04-01

    A fast and robust high performance LC-MS/MS screening method was developed for the analysis of β-lactam antibiotics in foods of animal origin: eggs, raw milk, processed dairy ingredients, infant formula, and meat- and fish-based products including baby foods. QuEChERS extraction with some adaptations enabled 23 drugs to be simultaneously monitored. Screening target concentrations were set at levels adequate to ensure compliance with current European, Chinese, US and Canadian regulations. The method was fully validated according to the European Community Reference Laboratories Residues Guidelines using 93 food samples of different composition. False-negative and false-positive rates were below 5% for all analytes. The method is adequate for use in high-routine laboratories. A 1-year study was additionally conducted to assess the stability of the 23 analytes in the working standard solution.

  12. Determination of pesticides in coconut (Cocos nucifera Linn.) water and pulp using modified QuEChERS and LC-MS/MS.

    Science.gov (United States)

    Ferreira, Jordana Alves; Ferreira, Joana Maria Santos; Talamini, Viviane; Facco, Janice de Fátima; Rizzetti, Tiele Medianeira; Prestes, Osmar Damian; Adaime, Martha Bohrer; Zanella, Renato; Bottoli, Carla Beatriz Grespan

    2016-12-15

    The use of pesticides is directly linked to improvements in productivity and to the preservation of coconut palms. However pesticide analysis is necessary to determine whether pesticide residues in the food products containing coconut are within the maximum residue limits (MRLs), ensuring the quality of these products. This work aimed to develop a method for multiresidue determination of ten pesticides in coconut water and pulp using QuEChERS and LC-MS/MS. The method was effective in terms of selectivity, linearity, matrix effect, accuracy and precision, providing LOD of 3μgkg(-1), LOQ of 10μgkg(-1) and recoveries between 70 and 120% with RSD lower than 20%. The developed method was applied to 36 samples in which residues of carbendazim, carbofuran, cyproconazole and thiabendazole were found below the LOQ in coconut water and pulp. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Solid-phase extraction based on hydrophilic interaction liquid chromatography with acetone as eluent for eliminating matrix effects in the analysis of biological fluids by LC-MS.

    Science.gov (United States)

    Van Damme, T; Lachová, M; Lynen, F; Szucs, R; Sandra, P

    2014-01-01

    Analysis of drugs and metabolites in biological matrices such as blood or plasma by LC-MS is routinely challenged by the presence of large quantities of competing molecules for ionization in soft ionization sources, such as proteins and phospholipids. While the former can easily be removed by protein precipitation, pre-analytical extraction of the latter is necessary because they show very high retention in reversed-phase LC resulting in long analysis times or in ion suppression effects when not eluted before the next runs. A novel HILIC-based SPE approach, making use of silica cartridges and of acetone as organic solvent, is introduced as a potent alternative to current commercial methods for phospholipid removal. The methodology was developed and tested for a broad polarity range of pharmaceutical solutes (log P from 0 to 6.6) and broad applicability can therefore be envisaged.

  14. Determination of 105 antibiotic, anti-inflammatory, antiparasitic agents and tranquilizers by LC-MS/MS based on an acidic QuEChERS-like extraction.

    Science.gov (United States)

    Desmarchelier, Aurélien; Fan, Kaïli; Minh Tien, Mai; Savoy, Marie-Claude; Tarres, Adrienne; Fuger, Denis; Goyon, Alexandre; Bessaire, Thomas; Mottier, Pascal

    2018-04-01

    A procedure for screening 105 veterinary drugs in foods by liquid chromatography tandem mass-spectrometry (LC-MS/MS) is presented. Its scope encompasses raw materials of animal origin (milk, meat, fish, egg and fat) but also related processed ingredients and finished products commonly used and manufactured by food business operators. Due to the complexity of the matrices considered and to efficiently deal with losses during extraction and matrix effects during MS source ionisation, each sample was analysed twice, that is 'unspiked' and 'spiked at the screening target concentration' using a QuEChERS-like extraction. The entire procedure was validated according to the European Community Reference Laboratories Residues Guidelines. False-negative and false-positive rates were below 5% for all veterinary drugs whatever the food matrix. Effectiveness of the procedure was further demonstrated through participation to five proficiency tests and its ruggedness demonstrated in quality control operations by a second laboratory.

  15. Direct analysis of [6,6-(2)H2]glucose and [U-(13)C6]glucose dry blood spot enrichments by LC-MS/MS.

    Science.gov (United States)

    Coelho, Margarida; Mendes, Vera M; Lima, Inês S; Martins, Fátima O; Fernandes, Ana B; Macedo, M Paula; Jones, John G; Manadas, Bruno

    2016-06-01

    A liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-(2)H2]glucose and [U-(13)C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-(13)C6]glucose and an oral glucose load enriched with [6,6-(2)H2]glucose. The sensitivity is sufficient for analysis of the equivalent to blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Real Time Extraction Kinetics of Electro Membrane Extraction Verified by Comparing Drug Metabolism Profiles Obtained from a Flow-Flow Electro Membrane Extraction-Mass Spectrometry System with LC-MS

    DEFF Research Database (Denmark)

    Fuchs, David; Jensen, Henrik; Pedersen-Bjergaard, Stig

    2015-01-01

    A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable...... alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics...... offering a significant time saving as compared to conventional LC-MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time...

  17. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  18. The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics

    International Nuclear Information System (INIS)

    Elmsjö, Albert; Haglöf, Jakob; Engskog, Mikael K.R.; Nestor, Marika; Arvidsson, Torbjörn; Pettersson, Curt

    2017-01-01

    Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics. By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting. For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation. - Highlights: • The co-feature ratio (CFR) is introduced. • CFR measures chromatographic and signal selectivity of a feature. • CFR can be used for evaluating experimental procedures in metabolomics. • CFR can aid in locating features with poor selectivity.

  19. Simultaneous determination of LSD and 2-oxo-3-hydroxy LSD in hair and urine by LC-MS/MS and its application to forensic cases.

    Science.gov (United States)

    Jang, Moonhee; Kim, Jihyun; Han, Inhoi; Yang, Wonkyung

    2015-11-10

    Lysergic acid diethylamide (LSD) is administered in low dosages, which makes its detection in biological matrices a major challenge in forensic toxicology. In this study, two sensitive and reliable methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were established and validated for the simultaneous determination of LSD and its metabolite, 2-oxo-3-hydroxy-LSD (O-H-LSD), in hair and urine. Target analytes in hair were extracted using methanol at 38°C for 15h and analyzed by LC-MS/MS. For urine sample preparation, liquid-liquid extraction was performed. Limits of detection (LODs) in hair were 0.25pg/mg for LSD and 0.5pg/mg for O-H-LSD. In urine, LODs were 0.01 and 0.025ng/ml for LSD and O-H-LSD, respectively. Method validation results showed good linearity and acceptable precision and accuracy. The developed methods were applied to authentic specimens from two legal cases of LSD ingestion, and allowed identification and quantification of LSD and O-H-LSD in the specimens. In the two cases, LSD concentrations in hair were 1.27 and 0.95pg/mg; O-H-LSD was detected in one case, but its concentration was below the limit of quantification. In urine samples collected from the two suspects 8 and 3h after ingestion, LSD concentrations were 0.48 and 2.70ng/ml, respectively, while O-H-LSD concentrations were 4.19 and 25.2ng/ml, respectively. These methods can be used for documenting LSD intake in clinical and forensic settings. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

    Science.gov (United States)

    Rybakowska, I M; Slominska, E M; Romaszko, P; Olkowicz, M; Kaletha, K; Smolenski, R T

    2015-06-01

    AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

  1. Preservation of urine free catecholamines and their free O-methylated metabolites with citric acid as an alternative to hydrochloric acid for LC-MS/MS-based analyses.

    Science.gov (United States)

    Peitzsch, Mirko; Pelzel, Daniela; Lattke, Peter; Siegert, Gabriele; Eisenhofer, Graeme

    2016-01-01

    Measurements of urinary fractionated metadrenalines provide a useful screening test to diagnose phaeochromocytoma. Stability of these compounds and their parent catecholamines during and after urine collection is crucial to ensure accuracy of the measurements. Stabilisation with hydrochloric acid (HCl) can promote deconjugation of sulphate-conjugated metadrenalines, indicating a need for alternative preservatives. Urine samples with an intrinsically acidic or alkaline pH (5.5-6.9 or 7.1-8.7, respectively) were used to assess stability of free catecholamines and their free O-methylated metabolites over 7 days of room temperature storage. Stabilisation with HCl was compared with ethylenediaminetetraacetic acid/metabisulphite and monobasic citric acid. Catecholamines and metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free catecholamines and their O-methylated metabolites were stable in acidic urine samples over 7 days of room temperature storage, independent of the presence or absence of any stabilisation method. In contrast, free catecholamines, but not the free O-methylated metabolites, showed rapid degradation within 24 h and continuing degradation over 7 days in urine samples with an alkaline pH. Adjustment of alkaline urine samples to a pH of 3-5 with HCl or 4.8-5.4 with citric acid completely blocked degradation of catecholamines. Ethylenediaminetetraacetic acid/metabisulphite, although reducing the extent of degradation of catecholamines in alkaline urine, was largely ineffectual as a stabiliser. Citric acid is equally effective as HCl for stabilisation of urinary free catecholamines and minimises hazards associated with use of strong inorganic acids while avoiding deconjugation of sulphate-conjugated metabolites during simultaneous LC-MS/MS measurements of free catecholamines and their free O-methylated metabolites.

  2. Quantitative analysis of polyethylene glycol (PEG) and PEGylated proteins in animal tissues by LC-MS/MS coupled with in-source CID.

    Science.gov (United States)

    Gong, Jiachang; Gu, Xiaomei; Achanzar, William E; Chadwick, Kristina D; Gan, Jinping; Brock, Barry J; Kishnani, Narendra S; Humphreys, W Griff; Iyer, Ramaswamy A

    2014-08-05

    The covalent conjugation of polyethylene glycol (PEG, typical MW > 10k) to therapeutic peptides and proteins is a well-established approach to improve their pharmacokinetic properties and diminish the potential for immunogenicity. Even though PEG is generally considered biologically inert and safe in animals and humans, the slow clearance of large PEGs raises concerns about potential adverse effects resulting from PEG accumulation in tissues following chronic administration, particularly in the central nervous system. The key information relevant to the issue is the disposition and fate of the PEG moiety after repeated dosing with PEGylated proteins. Here, we report a novel quantitative method utilizing LC-MS/MS coupled with in-source CID that is highly selective and sensitive to PEG-related materials. Both (40K)PEG and a tool PEGylated protein (ATI-1072) underwent dissociation in the ionization source of mass spectrometer to generate a series of PEG-specific ions, which were subjected to further dissociation through conventional CID. To demonstrate the potential application of the method to assess PEG biodistribution following PEGylated protein administration, a single dose study of ATI-1072 was conducted in rats. Plasma and various tissues were collected, and the concentrations of both (40K)PEG and ATI-1072 were determined using the LC-MS/MS method. The presence of (40k)PEG in plasma and tissue homogenates suggests the degradation of PEGylated proteins after dose administration to rats, given that free PEG was absent in the dosing solution. The method enables further studies for a thorough characterization of disposition and fate of PEGylated proteins.

  3. Development and validation of a simple, rapid and sensitive LC-MS/MS method for the measurement of urinary neurotransmitters and their metabolites.

    Science.gov (United States)

    Yan, Jingya; Kuzhiumparambil, Unnikrishnan; Bandodkar, Sushil; Solowij, Nadia; Fu, Shanlin

    2017-12-01

    Neurotransmitters play crucial roles in physiological functions and their imbalances have demonstrated association in the pathology of several diseases. The measurement of neurotransmitters possesses a great potential as a significant clinical tool. This study presents the development and validation of an LC-MS/MS method for simultaneous quantification of multi-class neurotransmitters associated with dopamine, tryptophan and glutamate-γ-aminobutyric acid pathways. A total of ten neurotransmitters and their metabolites (dopamine, epinephrine, metanephrine, tryptophan, serotonin, kynurenic acid, kynurenine, anthranilic acid, GABA, glutamic acid) were determined based on a simple and rapid 'dilute and shoot' method using minimal urine volume. The chromatographic separation was achieved using a Poroshell 120 Bonus-RP LC Column in combination with a gradient elution within an 8.5-min time frame. The method exhibited good sensitivity as the limits of quantification ranged between 0.025 and 0.075 μg/mL with acceptable matrix effects ( 0.98). The accuracy and precision for all analytes were within tolerances, at neurotransmitter concentrations in urine of healthy donors. Furthermore, the undertaken stability experiments indicated that acidified urine specimens allowed the analytes to be stable for prolonged durations in comparison to those untreated. The study also reveals the performance of the method is unaffected by the absence of expensive deuterated reference standards under the experimental conditions employed which further simplifies the analytical procedures and provides a significant cost saving for running the assay. Graphical abstract The quantification of multi-class neurotransitters associated with the dopamine, tryptophan and GABA-glutamate pathways using a simple 'dilute and shoot' LC-MS/MS method.

  4. A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Bonaventure Gustavo

    2009-11-01

    Full Text Available Abstract Background Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices. Results A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA and small polar molecules (e.g., jasmonic acid (JA, salicylic acid (SA containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods. Conclusion The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the

  5. LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean

    Directory of Open Access Journals (Sweden)

    Bernd Krock

    2017-12-01

    Full Text Available A liquid chromatography-tandem mass spectrometry (LC-MS/MS method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5, one amphidinol (AM-18, and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA, which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

  6. UNE-EN ISO/IEC 17025:2005-accredited method for the determination of pesticide residues in fruit and vegetable samples by LC-MS/MS.

    Science.gov (United States)

    Camino-Sánchez, F J; Zafra-Gómez, A; Oliver-Rodríguez, B; Ballesteros, O; Navalón, A; Crovetto, G; Vílchez, J L

    2010-11-01

    A rapid, simple and sensitive multi-residue method was developed and validated for the simultaneous quantification and confirmation of 69 pesticides in fruit and vegetables using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted following the quick, easy, cheap, effective, rugged and safe method known as QuEChERS. Mass spectrometric conditions were individually optimised for each analyte in order to achieve maximum sensitivity in multiple reaction monitoring (MRM) mode. Using the developed chromatographic conditions, 69 pesticides can be separated in less than 17 min. Two selected reaction monitoring (SRM) assays were used for each pesticide to obtain simultaneous quantification and identification in one run. With this method in SRM mode, more than 150 pesticides can be analysed and quantified, but their confirmation is not possible in all cases according to the European regulations on pesticide residues. Nine common representative matrices (zucchini, melon, cucumber, watermelon, tomato, garlic, eggplant, lettuce and pepper) were selected to investigate the effect of different matrices on recovery and precision. Mean recoveries ranged from 70% to 120%, with relative standard deviations (RSDs) lower than 20% for all the pesticides. The proposed method was applied to the analysis of more than 2000 vegetable samples from the extensive greenhouse cultivation in the province of Almeria, Spain, during one year. The methodology combines the advantages of both QuEChERS and LC-MS/MS producing a very rapid, sensitive, accurate and reliable procedure that can be applied in routine analytical laboratories. The method was validated and accredited according to UNE-EN-ISO/IEC 17025:2005 international standard (accreditation number 278/LE1027).

  7. Exploring the Metabolism of (+-[18F]Flubatine In Vitro and In Vivo: LC-MS/MS Aided Identification of Radiometabolites in a Clinical PET Study †

    Directory of Open Access Journals (Sweden)

    Friedrich-Alexander Ludwig

    2018-02-01

    Full Text Available Both (+-[18F]flubatine and its enantiomer (−-[18F]flubatine are radioligands for the neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs by positron emission tomography (PET. In a clinical study in patients with early Alzheimer’s disease, (+-[18F]flubatine ((+-[18F]1 was examined regarding its metabolic fate, in particular by identification of degradation products detected in plasma and urine. The investigations included an in vivo study of (+-flubatine ((+-1 in pigs and structural elucidation of formed metabolites by LC-MS/MS. Incubations of (+-1 and (+-[18F]1 with human liver microsomes were performed to generate in vitro metabolites, as well as radiometabolites, which enabled an assignment of their structures by comparison of LC-MS/MS and radio-HPLC data. Plasma and urine samples taken after administration of (+-[18F]1 in humans were examined by radio-HPLC and, on the basis of results obtained in vitro and in vivo, formed radiometabolites were identified. In pigs, (+-1 was monohydroxylated at different sites of the azabicyclic ring system of the molecule. Additionally, one intermediate metabolite underwent glucuronidation, as also demonstrated in vitro. In humans, a fraction of 95.9 ± 1.9% (n = 10 of unchanged tracer remained in plasma, 30 min after injection. However, despite the low metabolic degradation, both radiometabolites formed in humans could be characterized as (i a product of C-hydroxylation at the azabicyclic ring system, and (ii a glucuronide conjugate of the precedingly-formed N8-hydroxylated (+-[18F]1.

  8. Untargeted metabolomics studies employing NMR and LC-MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis.

    Science.gov (United States)

    Hamerly, Timothy; Tripet, Brian P; Tigges, Michelle; Giannone, Richard J; Wurch, Louie; Hettich, Robert L; Podar, Mircea; Copié, Valerie; Bothner, Brian

    2015-08-01

    Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans , including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans 's consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans . Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites.

  9. Microdose study of a P-glycoprotein substrate, fexofenadine, using a non-radioisotope-labelled drug and LC/MS/MS.

    Science.gov (United States)

    Yamazaki, A; Kumagai, Y; Yamane, N; Tozuka, Z; Sugiyama, Y; Fujita, T; Yokota, S; Maeda, M

    2010-04-01

    Fexofenadine is a P-glycoprotein substrate of low bioavailability. It is primarily excreted into faeces as a parent drug via biliary excretion. The predictability from microdose data for the drug absorbed via transporters such as P-glycoprotein is not known. Therefore, this study assessed the predictability of therapeutic-dose pharmacokinetics of fexofenadine from microdosing data using non-radioisotope-labelled drug and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). In a single dose, randomized, two-way crossover study, eight subjects received a microdose (100 microg) or a therapeutic dose (60 mg) of fexofenadine. Blood samples were collected until 12 h after dosing, and assayed using LC/MS/MS. Plasma concentration-time curves of fexofenadine between microdose and therapeutic dose were similar. The mean +/- SD of C(max) normalized to 60 mg dose after microdose and therapeutic dose were 379 +/- 147 and 275 +/- 145 ng/mL respectively. The mean AUC(last) normalized to 60 mg dose after microdose and therapeutic dose were 1914 +/- 738 and 1431 +/- 432 ng/h/mL respectively. The mean dose-adjusted C(max) and AUC(last) after microdose were higher compared with those after therapeutic dose. Individual plots of C(max) and AUC(last) normalized to 60 mg dose, were similar for microdose and therapeutic dose. None of the pharmacokinetic parameters were statistically different using anova. Overall, the microdose pharmacokinetics profile was similar to, and hence predictive of, that of the therapeutic dose. For the P-glycoprotein substrate fexofenadine, the predictability of therapeutic-dose pharmacokinetics from microdose data was good. A microdose study using a non-radioisotope-labelled drug and LC/MS/MS is convenient, and has the potential to aid the early selection of drug candidates.

  10. Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

    Science.gov (United States)

    Newman, Carla F; Havelund, Rasmus; Passarelli, Melissa K; Marshall, Peter S; Francis, Ian; West, Andy; Alexander, Morgan R; Gilmore, Ian S; Dollery, Colin T

    2017-11-21

    ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

  11. Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula - Comparative LC-MS Analysis, Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials.

    Science.gov (United States)

    Milutinović, Violeta; Niketić, Marjan; Ušjak, Ljuboš; Nikolić, Dejan; Krunić, Aleksej; Zidorn, Christian; Petrović, Silvana

    2018-01-01

    Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce. Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts. Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC-MS. Multivariate statistical data analysis included non-metric multidimensional scaling (nMDS), unweighted pair-group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests. Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition. Comparative LC-MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non-hybrid species and groups from the same location. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley

  12. Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples.

    Science.gov (United States)

    Dupuy, Aude; Le Faouder, Pauline; Vigor, Claire; Oger, Camille; Galano, Jean-Marie; Dray, Cédric; Lee, Jetty Chung-Yung; Valet, Philippe; Gladine, Cécile; Durand, Thierry; Bertrand-Michel, Justine

    2016-05-19

    Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain. Copyright © 2016 Elsevier B.V. All rights

  13. Rapid and Sensitive LC-MS/MS Method for the Determination of Metoprolol in Beagle Dog Plasma with a Simple Protein Precipitation Treatment and Its Pharmacokinetic Applications

    Directory of Open Access Journals (Sweden)

    Aihua Hong

    2012-03-01

    Full Text Available : A rapid LC-MS/MS method with good accuracy and sensitivity was developed and validated for the pharmacokinetics study of metoprolol (MP in beagle dogs. The plasma samples were simply precipitated by methanol and then analyzed by LC-MS/MS. An Ultimate XB-C18 column (150 × 2.1 mm ID, 5 μm was used for separation, with methanol-water containing 0.2% formic acid (65:35, v/v as the mobile phase at a flow rate of 0.2 mL/min. Monitoring ions of MP and internal standard (hydroxypioglitazone were m/z 268.1/115.6 and m/z 373.1/150.2, respectively. The linear range was 3.03–416.35 ng/mL with an average correlation coefficient of 0.9996, and the limit of quantification was 3.03 ng/mL. The intra- and inter-day precision was less than 15%. At low, middle and high concentrations, the recovery, the matrix effect and the accuracy was in the range of 76.06%–95.25%, 93.67%–104.19% and 95.20%–99.96% respectively. The method was applied for the pharmacokinetics study of MP tartrate tablets (50 mg. The AUC0-t, Tmax and Cmax were respectively 919.88 ± 195.67 μg/L·h, 0.96 ± 0.33 h, 349.12 ± 78.04 ng/mL.

  14. The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Elmsjö, Albert, E-mail: Albert.Elmsjo@farmkemi.uu.se [Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala University (Sweden); Haglöf, Jakob; Engskog, Mikael K.R. [Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala University (Sweden); Nestor, Marika [Department of Immunology, Genetics and Pathology, Uppsala University (Sweden); Arvidsson, Torbjörn [Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala University (Sweden); Medical Product Agency, Uppsala (Sweden); Pettersson, Curt [Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala University (Sweden)

    2017-03-01

    Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics. By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting. For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation. - Highlights: • The co-feature ratio (CFR) is introduced. • CFR measures chromatographic and signal selectivity of a feature. • CFR can be used for evaluating experimental procedures in metabolomics. • CFR can aid in locating features with poor selectivity.

  15. Analysis of U-47700, a Novel Synthetic Opioid, in Human Urine by LC-MS-MS and LC-QToF.

    Science.gov (United States)

    Fleming, Steven W; Cooley, Justin C; Johnson, Leonard; Frazee, C Clinton; Domanski, Kristina; Kleinschmidt, Kurt; Garg, Uttam

    2017-04-01

    The illicit drug market has rapidly evolved from synthetic cannabinoids to cathinone derivatives and now a new emerging threat of synthetic opioids. These compounds were mostly developed by pharmaceutical companies during drug discovery. The new psychoactive substances are not routinely covered in drug screening and may go undetected. Recently fentanyl analogous, AH-7921, MT-45 and now U-47700 have been encountered in clinical and forensic casework. U-47700 is gaining popularity on drug user forms as a legal alternative to heroin. It is a µ-receptor agonist that is part of the trans-1-2-diamine opioid analgesic drug class developed by The Upjohn Company in an attempt to develop a non-addicting analgesic. A LC-MS-MS method was developed and validated to detect and quantify U-47700. Additional analysis was conducted with an LC-QToF to identify the presence of the parent drug and metabolites. A total of four cases have been evaluated by the LC-MS-MS methodology which has an analytical range of 1-1,250 ng/mL and limit of detection of 1 ng/mL. The concentration of U-47700 in urine specimens ranged from below the limit of quantification to 224 ng/mL. The ToF analysis detected the presence of suspected phase I demethylated metabolites that may assist future analysis of this compound. The prevalence of designer opioids in casework highlights the importance of analysis for new psychoactive substances. Traditional opiates/opioids were not detected in the presented cases, but the available case histories revealed an opioid toxidrome. These findings suggest that U-47700 drug may cause significant morbidity and mortality within the United States as an emerging drug threat. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. A fit-for-purpose LC-MS/MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood.

    Science.gov (United States)

    Kim, Hyeryun; Kosinski, Penelope; Kung, Charles; Dang, Lenny; Chen, Yue; Yang, Hua; Chen, Yuan-Shek; Kramer, Jordyn; Liu, Guowen

    2017-09-01

    Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K 2 EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000μg/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within ±10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K 2 EDTA blood for at least 8h at 4°C, 96days when stored at -70°C and after three freeze/thaw cycles. The assay has been successfully applied to K 2 EDTA human whole blood samples to support clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. LC-MS/MS method for the simultaneous determination of PA-824, moxifloxacin and pyrazinamide in rat plasma and its application to pharmacokinetic study.

    Science.gov (United States)

    Wang, Libin; Xu, Yue; Liang, Li; Diao, Chunyan; Liu, Xueying; Zhang, Jianchun; Zhang, Shengyong

    2014-08-01

    A simple, sensitive and rapid LC-MS/MS method has been developed and validated for simultaneous determination of PA-824, moxifloxacin, and pyrazinamide in rat plasma using metronidazole as internal standard. Sample preparation involved a simple one-step protein precipitation with methanol, followed by centrifugation and evaporation of the organic solvent. The residue was redissolved in mobile phase and analyzed by LC-MS/MS. An Inertsil(®) ODS3 C18 column (150mm×4.6mm, 5μm), a mobile phase composed of methanol-0.03% TEA (triethylamine) in water (85:15, v/v), and a flow rate of 0.5mL/min were employed, and the total run time was 6.0min. The mass spectrometer was run in positive ion ESI-APCI combined mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. The LLOQ was 1.0μg/mL for pyrazinamide and 0.1μg/mL for PA-824 and moxifloxacin. The recoveries obtained for PA-824, moxifloxacin and pyrazinamide were ≥85%. Intra-day and inter-day coefficients of variation were less than 10%. The method had been successfully applied to a pharmacokinetic study of fixed dose administration of PA-824, moxifloxacin, pyrazinamide and their combination in SD rat. Significant differences of Tmax, Cmax, AUC(0-t) and CLz/F were observed between the single and combined groups after equal dose of PA-824 and moxifloxacin administration, which revealed the possibility of drug-drug interaction (DDI) between the PaMZ combination. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Validation of a LC-MS/MS method for quantifying urinary nicotine, six nicotine metabolites and the minor tobacco alkaloids--anatabine and anabasine--in smokers' urine.

    Directory of Open Access Journals (Sweden)

    James E McGuffey

    Full Text Available Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling--i.e., metabolite ratios. In addition, the minor tobacco alkaloids--anabasine and anatabine--can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1'-N-oxide, anatabine, and anabasine are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76-99%, specificity, accuracy (0-10% bias and reproducibility (2-9% C.V. make this method ideal for large high through-put studies.

  19. LC-MS/MS analytical procedure to quantify tris(nonylphenyl)phosphite, as a source of the endocrine disruptors 4-nonylphenols, in food packaging materials.

    Science.gov (United States)

    Mottier, Pascal; Frank, Nancy; Dubois, Mathieu; Tarres, Adrienne; Bessaire, Thomas; Romero, Roman; Delatour, Thierry

    2014-01-01

    Tris(nonylphenyl)phosphite, an antioxidant used in polyethylene resins for food applications, is problematic since it is a source of the endocrine-disrupting chemicals 4-nonylphenols (4NP) upon migration into packaged foods. As a response to concerns surrounding the presence of 4NP-based compounds in packaging materials, some resin producers and additive suppliers have decided to eliminate TNPP from formulations. This paper describes an analytical procedure to verify the "TNPP-free" statement in multilayer laminates used for bag-in-box packaging. The method involves extraction of TNPP from laminates with organic solvents followed by detection/quantification by LC-MS/MS using the atmospheric pressure chemical ionisation (APCI) mode. A further acidic treatment of the latter extract allows the release of 4NP from potentially extracted TNPP. 4NP is then analysed by LC-MS/MS using electrospray ionisation (ESI) mode. This two-step analytical procedure ensures not only TNPP quantification in laminates, but also allows the flagging of other possible sources of 4NP in such packaging materials, typically as non-intentionally added substances (NIAS). The limits of quantification were 0.50 and 0.48 µg dm⁻² for TNPP and 4NP in laminates, respectively, with recoveries ranging between 87% and 114%. Usage of such analytical methodologies in quality control operations has pointed to a lack of traceability at the packaging supplier level and cross-contamination of extrusion equipment at the converter level, when TNPP-containing laminates are processed on the same machine beforehand.

  20. Simultaneous determination of lercanidipine, benazepril and benazeprilat in plasma by LC-MS/MS and its application to a toxicokinetics study.

    Science.gov (United States)

    Chen, Keguang; Zhang, Jing; Liu, Sha; Zhang, Dujuan; Teng, Yanni; Wei, Chunmin; Wang, Benjie; Liu, Xiaoyan; Yuan, Guiyan; Zhang, Rui; Zhao, Wenjing; Guo, Ruichen

    2012-06-15

    We aim to develop a rapid, simple, sensitive and specific LC-MS/MS method for the simultaneous quantification of lercanidipine, benazepril and benazeprilat in plasma. It is performed on the Agilent 6410 LC-MS/MS under the multiple-reaction monitoring (MRM) mode with electrospray ionization. Gliclazide was used as the internal standard (IS). Analytes and IS were extracted from plasma by solid-phase extraction. The reconstituted samples were chromatographed on a Diamond C₁₈(150 mm × 4.6 mm, 5 μm) column. The mobile phase was composed of 0.1% acetic acid-acetonitrile (50:50, v/v), with gradient flow rates: 0.6 mL/min (0-4.55 min); 4.55-4.65 min, 1 mL/min; 1 mL/min (4.65-9.5 min); 9.5-9.6 min, 0.6 mL/min; 0.6 mL/min (9.6-10 min). Method validation demonstrated that the method was of satisfactory specificity, sensitivity, precision and accuracy in linear ranges of 1-2000 ng/mL for lercanidipine, 1-2000 ng/mL for benazepril and 1-1600 ng/mL for benazeprilat, respectively. The precision (RSD%) was better than 15, and the lower limit of quantitation was identifiable and reproducible at 1 ng/mL for the three analytes. The plasma samples were stable after being stored for more than 60 days and after two freeze-thaw cycles (-20 to -25 °C). It is demonstrated that this method was successfully applied to samples from a toxicokinetics study of a compound of lercanidipine and benazepril in beagle dogs. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

    Science.gov (United States)

    Nilsson, Jonas; Noborn, Fredrik; Gomez Toledo, Alejandro; Nasir, Waqas; Sihlbom, Carina; Larson, Göran

    2017-02-01

    Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl- O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides.

  2. Comparison of the anti-inflammatory active constituents and hepatotoxic pyrrolizidine alkaloids in two Senecio plants and their preparations by LC-UV and LC-MS.

    Science.gov (United States)

    Chen, Pinghong; Wang, Yi; Chen, Lulin; Jiang, Wei; Niu, Yan; Shao, Qing; Gao, Lu; Zhao, Quancheng; Yan, Licheng; Wang, Shufang

    2015-11-10

    Two Senecio plants, Senecio cannabifolius Less. and its variety S. cannabifolius Less. var. integrifolius (Kiodz.) Kidam., were both used as the raw material of Feining granule, a traditional Chinese medicine product for treating respiratory diseases. In this study, the chemical profiles of these two plants were investigated and compared by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). A total number of 83 constituents, including 55 organic acids, 11 flavonoids, 4 alkaloids, 3 terpenes and 10 other types of compounds, were characterized. The results indicated that the levels of most flavonoids were higher in S. cannabifolius than in S. cannabifolius var. integrifolius, however, the levels of hepatotoxic pyrrolizidine alkaloids (PAs) were higher in S. cannabifolius var. integrifolius than in S. cannabifolius. Fifteen constituents were evaluated on lipopolysaccharides (LPS) induced RAW 264.7 cells, and eleven of them showed inhibition effect against nitric oxide (NO) production. Finally, the levels of ten major constituents (including seven anti-inflammatory active ones) and two PAs in Feining granule from two Senecio plants were determined and compared by the LC-UV and LC-MS methods, respectively. It was found that one organic acid (homogentisic acid) and two PAs (seneciphylline and senecionine) had higher contents in the preparation of S. cannabifolius var. integrifolius than in that of S. cannabifolius, however, the situations were inverse for the levels of four organic acids and flavonoids (chlorogenic acid, hyperoside, isoquercitrin, and isochlorogenic acid B). Based on the above results, S. cannabifolius might be a better raw material for Feining granule than S. cannabifolius var. integrifolius, because it contained more anti-inflammatory constituents and less hepatotoxic PAs than the latter. However, more pharmacological evaluations should be carried out to support the selection. The results in this study were helpful

  3. Quantitation of 25-hydroxyvitamin D in dried blood spots by 2D LC-MS/MS without derivatization and correlation with serum in adult and pediatric studies.

    Science.gov (United States)

    Jensen, Berit P; Saraf, Rajneeta; Ma, Jing; Berry, Sarah; Grant, Cameron C; Camargo, Carlos A; Sies, Christiaan W

    2018-06-01

    Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d 6 -25OHD 3 in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD 3 and 11-91 nmol/L 25OHD 2 with a limit of detection of 3 nmol/L. Intra- and inter-day imprecision was <13% CV and bias <12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of ≤13% for 25OHD 3. No 25OHD 2 was detected. DBS calculated serum 25OHD 3 concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays

    OpenAIRE

    Saleh, Lanja; Mueller, Daniel; von Eckardstein, Arnold

    2015-01-01

    BACKGROUND: We evaluated the analytical and clinical performance of the new Lumipulse® G 25-OH vitamin D assay from Fujirebio, and compared it to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and three other commercial automated assays. METHODS: Total 25 hydroxy vitamin D (25(OH)D) levels were measured in 100 selected serum samples from our routine analysis with Fujirebio 25(OH)D assay. The results were compared with those obtained with LC-MS/MS and three other automat...

  5. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) in toxicological analysis. Studies on the detection of clobenzorex and its metabolites within a systematic toxicological analysis procedure by GC-MS and by immunoassay and studies on the detection of alpha- and beta-amanitin in urine by atmospheric pressure ionization electrospray LC-MS.

    Science.gov (United States)

    Maurer, H H; Kraemer, T; Ledvinka, O; Schmitt, C J; Weber, A A

    1997-02-07

    GC-MS is the method of choice for toxicological analysis of toxicants volatile in GC while non-volatile and/or thermally labile toxicants need LC-MS for their determination. Studies are presented on the toxicological detection of the amphetamine-like anorectic clobenzorex in urine by GC-MS after acid hydrolysis, extraction and acetylation and by fluorescence polarization immunoassay (FPIA, TDx (meth)amphetamine II). After ingestion of 60 mg of clobenzorex, the parent compound and/or its metabolites could be detected by GC-MS for up to 84 h or by FPIA for up to 60 h. Since clobenzorex shows no cross-reactivity with the used immunoassay, the N-dealkylated metabolite amphetamine is responsible for the positive TDx results. The intake of clobenzorex instead of amphetamine can be differentiated by GC-MS detection of hydroxyclobenzorex which is detectable for at least as long as amphetamine. In addition, the described GC-MS procedure allows the simultaneous detection of most of the toxicologically relevant drugs. Furthermore, studies are described on the atmospheric pressure ionization electrospray LC-MS detection of alpha- and beta-amanitin, toxic peptides of amanita mushrooms, in urine after solid-phase extraction on RP-18 columns. Using the single ion monitoring mode with the ions m/z 919 and 920 the amanitins could be detected down to 10 ng/ml of urine which allows us to diagnose intoxications with amanita mushrooms.

  6. Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Meera Jay [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    The purpose of this research was to develop sensitive LC-MS methods for enantiomeric separation and detection, and then apply these methods for determination of enantiomeric composition and for the study of pharmacokinetic and pharmacodynamic properties of a chiral nutraceutical. Our first study, evaluated the use of reverse phase and polar organic mode for chiral LC-API/MS method development. Reverse phase methods containing high water were found to decrease ionization efficiency in electrospray, while polar organic methods offered good compatibility and low limits of detection with ESI. The use of lower flow rates dramatically increased the sensitivity by an order of magnitude. Additionally, for rapid chiral screening, the coupled Chirobiotic column afforded great applicability for LC-MS method development. Our second study, continued with chiral LC-MS method development in this case for the normal phase mode. Ethoxynonafluorobutane, a fluorocarbon with low flammability and no flashpoint, was used as a substitute solvent for hexane/heptane mobile phases for LC-APCI/MS. Comparable chromatographic resolutions and selectivities were found using ENFB substituted mobile phase systems, although, peak efficiencies were significantly diminished. Limits of detection were either comparable or better for ENFB-MS over heptane-PDA detection. The miscibility of ENFB with a variety of commonly used organic modifiers provided for flexibility in method development. For APCI, lower flow rates did not increase sensitivity as significantly as was previously found for ESI-MS detection. The chiral analysis of native amino acids was evaluated using both APCI and ESI sources. For free amino acids and small peptides, APCI was found to have better sensitivities over ESI at high flow rates. For larger peptides, however, sensitivity was greatly improved with the use of electrospray. Additionally, sensitivity was enhanced with the use of non-volatile additives, This optimized method was then

  7. A Collaborative Study: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

    Science.gov (United States)

    Zhang, Kai; Schaab, Matthew R; Southwood, Gavin; Tor, Elizabeth R; Aston, Linda S; Song, Wenlu; Eitzer, Brian; Majumdar, Sanghamitra; Lapainis, Theodore; Mai, Huy; Tran, Kevin; El-Demerdash, Aref; Vega, Victor; Cai, Yanxuan; Wong, Jon W; Krynitsky, Alexandra J; Begley, Timothy H

    2017-08-23

    A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B 1 , B 2 , G 1 , and G 2 ; deoxynivalenol; fumonisins B 1 , B 2 , and B 3 ; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 13 C uniformly labeled mycotoxins ( 13 C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. Using their available LC-MS/MS platform, each laboratory developed in-house instrumental conditions for analysis. The majority of recoveries ranged from 80 to 120% with relative standard derivations (RSDs) <20%. Greater than 90% of the average recoveries of the participating laboratories were in the range of 90-110%, with repeatability RSD r (within laboratory) < 10% and reproducibility RSD R (among laboratory) < 15%. All Z scores of the results of certified reference materials were between -2 and 2. Using 13 C-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of multiple mycotoxins in a simple LC-MS/MS procedure.

  8. Simultaneous quantification of vitamin D3, 25-hydroxyvitamin D-3 and 24,25-dihydroxyvitamin D3 in human serum by LC-MS/MS

    DEFF Research Database (Denmark)

    Burild, Anders; Frandsen, Henrik Lauritz; Jakobsen, Jette

    2014-01-01

    Introduction. Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D. Method. An LC-MS/MS method was developed...

  9. HPLC and LC-MS/MS methods for determination of sodium benzoate and potassium sorbate in food and beverages: performances of local accredited laboratories via proficiency tests in Turkey.

    Science.gov (United States)

    Gören, Ahmet C; Bilsel, Gökhan; Şimşek, Adnan; Bilsel, Mine; Akçadağ, Fatma; Topal, Kevser; Ozgen, Hasan

    2015-05-15

    High Performance Liquid Chromatography LC-UV and LC-MS/MS methods were developed and validated for quantitative analyses of sodium benzoate and potassium sorbate in foods and beverages. HPLC-UV and LC-MS/MS methods were compared for quantitative analyses of sodium benzoate and potassium sorbate in a representative ketchup sample. Optimisation of the methods enabled the chromatographic separation of the analytes in less than 4 min. A correlation coefficient of 0.999 was achieved over the measured calibration range for both compounds and methods (HPLC and LC-MS/MS). The uncertainty values of sodium benzoate and potassium sorbate were found as 0.199 and 0.150 mg/L by HPLC and 0.072 and 0.044 mg/L by LC-MS/MS, respectively. Proficiency testing performance of Turkish accredited laboratories between the years 2005 and 2013 was evaluated and reported herein. The aim of the proficiency testing scheme was to evaluate the performance of the laboratories, analysing benzoate and sorbate in tomato ketchup. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays.

    Science.gov (United States)

    Saleh, Lanja; Mueller, Daniel; von Eckardstein, Arnold

    2016-04-01

    We evaluated the analytical and clinical performance of the new Lumipulse® G 25-OH vitamin D assay from Fujirebio, and compared it to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and three other commercial automated assays. Total 25 hydroxy vitamin D (25(OH)D) levels were measured in 100 selected serum samples from our routine analysis with Fujirebio 25(OH)D assay. The results were compared with those obtained with LC-MS/MS and three other automated 25(OH)D assays (Abbott, Beckman, and Roche). The accuracy of each assay tested was evaluated against a Labquality reference serum panel for 25(OH)D (Ref!25OHD; University of Ghent). Intra- and inter-day imprecision of the Fujirebio 25(OH)D assay was Lumipulse G 25-OH vitamin D assay from Fujirebio demonstrated a good correlation with LC-MS/MS and some immunoassays. The performance of the assay is well-suited for routine 25(OH)D measurement in clinical serum samples. A correction for the observed negative bias vs. LC-MS/MS could be considered.

  11. Development of a universal metabolome-standard method for long-term LC-MS metabolome profiling and its application for bladder cancer urine-metabolite-biomarker discovery.

    Science.gov (United States)

    Peng, Jun; Chen, Yi-Ting; Chen, Chien-Lun; Li, Liang

    2014-07-01

    Large-scale metabolomics study requires a quantitative method to generate metabolome data over an extended period with high technical reproducibility. We report a universal metabolome-standard (UMS) method, in conjunction with chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS), to provide long-term analytical reproducibility and facilitate metabolome comparison among different data sets. In this method, UMS of a specific type of sample labeled by an isotope reagent is prepared a priori. The UMS is spiked into any individual samples labeled by another form of the isotope reagent in a metabolomics study. The resultant mixture is analyzed by LC-MS to provide relative quantification of the individual sample metabolome to UMS. UMS is independent of a study undertaking as well as the time of analysis and useful for profiling the same type of samples in multiple studies. In this work, the UMS method was developed and applied for a urine metabolomics study of bladder cancer. UMS of human urine was prepared by (13)C2-dansyl labeling of a pooled sample from 20 healthy individuals. This method was first used to profile the discovery samples to generate a list of putative biomarkers potentially useful for bladder cancer detection and then used to analyze the verification samples about one year later. Within the discovery sample set, three-month technical reproducibility was examined using a quality control sample and found a mean CV of 13.9% and median CV of 9.4% for all the quantified metabolites. Statistical analysis of the urine metabolome data showed a clear separation between the bladder cancer group and the control group from the discovery samples, which was confirmed by the verification samples. Receiver operating characteristic (ROC) test showed that the area under the curve (AUC) was 0.956 in the discovery data set and 0.935 in the verification data set. These results demonstrated the utility of the UMS method for long-term metabolomics and

  12. Matrix removal in state of the art sample preparation methods for serum by charged aerosol detection and metabolomics-based LC-MS.

    Science.gov (United States)

    Schimek, Denise; Francesconi, Kevin A; Mautner, Anton; Libiseller, Gunnar; Raml, Reingard; Magnes, Christoph

    2016-04-07

    Investigations into sample preparation procedures usually focus on analyte recovery with no information provided about the fate of other components of the sample (matrix). For many analyses, however, and particularly those using liquid chromatography-mass spectrometry (LC-MS), quantitative measurements are greatly influenced by sample matrix. Using the example of the drug amitriptyline and three of its metabolites in serum, we performed a comprehensive investigation of nine commonly used sample clean-up procedures in terms of their suitability for preparing serum samples. We were monitoring the undesired matrix compounds using a combination of charged aerosol detection (CAD), LC-CAD, and a metabolomics-based LC-MS/MS approach. In this way, we compared analyte recovery of protein precipitation-, liquid-liquid-, solid-phase- and hybrid solid-phase extraction methods. Although all methods provided acceptable recoveries, the highest recovery was obtained by protein precipitation with acetonitrile/formic acid (amitriptyline 113%, nortriptyline 92%, 10-hydroxyamitriptyline 89%, and amitriptyline N-oxide 96%). The quantification of matrix removal by LC-CAD showed that the solid phase extraction method (SPE) provided the lowest remaining matrix load (48-123 μg mL(-1)), which is a 10-40 fold better matrix clean-up than the precipitation- or hybrid solid phase extraction methods. The metabolomics profiles of eleven compound classes, comprising 70 matrix compounds showed the trends of compound class removal for each sample preparation strategy. The collective data set of analyte recovery, matrix removal and matrix compound profile was used to assess the effectiveness of each sample preparation method. The best performance in matrix clean-up and practical handling of small sample volumes was showed by the SPE techniques, particularly HLB SPE. CAD proved to be an effective tool for revealing the considerable differences between the sample preparation methods. This detector can

  13. The flexibility of a generic LC-MS/MS method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G and related constructs in animal studies.

    Science.gov (United States)

    Lanshoeft, Christian; Wolf, Thierry; Walles, Markus; Barteau, Samuel; Picard, Franck; Kretz, Olivier; Cianférani, Sarah; Heudi, Olivier

    2016-11-30

    An increasing demand of new analytical methods is associated with the growing number of biotherapeutic programs being prosecuted in the pharmaceutical industry. Whilst immunoassay has been the standard method for decades, a great interest in assays based on liquid chromatography tandem mass spectrometry (LC-MS/MS) is evolving. In this present work, the development of a generic method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G (hIgG) in rat serum is reported. The method is based on four generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), VVSVLTVLHQDWLNGK (VVS) and FNWYVDGVEVHNAK (FNW) originating from different parts of the fraction crystallizable (Fc) region of a reference hIgG1 (hIgG1A). A tryptic pellet digestion of rat serum spiked with hIgG1A and a stable isotope labeled protein (hIgG1B) used as internal standard (ISTD) was applied prior LC-MS/MS analysis. The upper limit of quantification was at 1000μg/mL. The lower limit of quantitation was for GPS, TTP and VVS at 1.00μg/mL whereas for FNW at 5.00μg/mL. Accuracy and precision data met acceptance over three days. The presented method was further successfully applied to the quantitative analysis of other hIgG1s (hIgG1C and hIgG1D) and hIgG4-based therapeutic proteins on spiked quality control (QC) samples in monkey and rat serum using calibration standards (Cs) prepared with hIgG1A in rat serum. In order to extend the applicability of our generic approach, a bispecific-bivalent hIgG1 (bb-hIgG1) and two lysine conjugated antibody-drug conjugates (ADC1 and ADC2) were incorporated as well. The observed values on spiked QC samples in monkey serum were satisfactory with GPS for the determination of bb-hIgG1 whereas the FNW and TTP peptides were suitable for the ADCs. Moreover, comparable mean concentration-time profiles were obtained from monkeys previously dosed intravenously with ADC2 measured against Cs samples prepared either with hIgG1A in rat serum

  14. Enhancement of sensitivity in the determination of organic trace compounds in complex matrices with liquid chromatography-mass spectrometry (LC-MS)

    International Nuclear Information System (INIS)

    Mascher, D.G.

    2002-05-01

    The PhD-thesis deals with 'enhancement of sensitivity in the determination of organic trace compounds in complex matrices with liquid chromatography-mass spectrometry (LC-MS)'. Almost the most important factor is the enhancement of the ionization yield with Atmospheric Pressure Chemical Ionization (APCI) or Electrospray Ionization (ESI) in LC-MS. Ionization yields of different compounds can vary by a factor of 10000. Three ways to solve this problem of little ionization yield were tried: 1) Modification of the mobile phase in HPLC 2) Chemical modification of the analytes 3) A new type of ionization called Atmospheric Pressure Photo Ionization (APPI). ad 1) By using specific additives to mobile phases ion suppression that might derive from an ion pair reagent that was necessary for chromatography could be omitted. General remarks cannot be done. ad 2) Chemical modification or so called derivatization is well known for UV- and fluorescence-detection for a long period of time. As substances containing nitrogene (e.g. primary, secondary or tertiary amines) often have good ionization yields, poor or relatively poor ionizable substances like carboxylic acids, sugars and partially phenolic steroids were used as analytes for derivatization reactions. By using Dansyl as Dansylchlorid or Dansylhydrazine a basic derivatization agent could be found that ionizes very well. A 200 times more sensitive determination of estrogenes is possible after derivatization with Dansylchlorid. Using a tandem-mass-spectrometer a lower limit of quantification of 2 pg/mL plasma could be reached by using 1 mL of plasma. For ketones like carvon and campher an enhancement by a factor of 500 and 4000 could be reached by using Dansylhydrazine as the derivatization agent. For fatty acids DMEQ as derivatization agent enhanced the sensitivity by a factor of 20 to 100. ad 3) APPI as a new ionization mode showed really good results for specific molecules. Relatively unpolar substances as diphenylsulfide

  15. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    Science.gov (United States)

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    earlier transcriptome profiling work and show LC-MS is a viable means of profiling the abundance of almost all major metabolic enzymes of skeletal muscle in a highly parallel manner. Moreover, our approach is relatively more time efficient than techniques relying on orthogonal separations, and we demonstrate LC-MS profiling of the HCR/LCR selection model was able to highlight biomarkers that also exhibit differences in trained and untrained human muscle.

  16. Comparison of two-concentration with multi-concentration linear regressions: Retrospective data analysis of multiple regulated LC-MS bioanalytical projects.

    Science.gov (United States)

    Musuku, Adrien; Tan, Aimin; Awaiye, Kayode; Trabelsi, Fethi

    2013-09-01

    Linear calibration is usually performed using eight to ten calibration concentration levels in regulated LC-MS bioanalysis because a minimum of six are specified in regulatory guidelines. However, we have previously reported that two-concentration linear calibration is as reliable as or even better than using multiple concentrations. The purpose of this research is to compare two-concentration with multiple-concentration linear calibration through retrospective data analysis of multiple bioanalytical projects that were conducted in an independent regulated bioanalytical laboratory. A total of 12 bioanalytical projects were randomly selected: two validations and two studies for each of the three most commonly used types of sample extraction methods (protein precipitation, liquid-liquid extraction, solid-phase extraction). When the existing data were retrospectively linearly regressed using only the lowest and the highest concentration levels, no extra batch failure/QC rejection was observed and the differences in accuracy and precision between the original multi-concentration regression and the new two-concentration linear regression are negligible. Specifically, the differences in overall mean apparent bias (square root of mean individual bias squares) are within the ranges of -0.3% to 0.7% and 0.1-0.7% for the validations and studies, respectively. The differences in mean QC concentrations are within the ranges of -0.6% to 1.8% and -0.8% to 2.5% for the validations and studies, respectively. The differences in %CV are within the ranges of -0.7% to 0.9% and -0.3% to 0.6% for the validations and studies, respectively. The average differences in study sample concentrations are within the range of -0.8% to 2.3%. With two-concentration linear regression, an average of 13% of time and cost could have been saved for each batch together with 53% of saving in the lead-in for each project (the preparation of working standard solutions, spiking, and aliquoting). Furthermore

  17. Monitoring ovarian cycle activity via progestagens in urine and feces of female mountain gorillas: A comparison of EIA and LC-MS measurements.

    Science.gov (United States)

    Habumuremyi, Sosthene; Robbins, Martha M; Fawcett, Katie A; Deschner, Tobias

    2014-02-01

    Understanding the reproductive biology of endangered mountain gorillas (Gorilla beringei beringei) is essential for optimizing conservation strategies, determining any demographic impact of socioecological changes, and providing information for comparative studies of primates. Non-invasive techniques have been used to assess the reproductive function of many primates and the importance of validating the measurements of hormones metabolites is widely recognized because they may vary even within closely related species. To determine if it is possible to non-invasively monitor ovarian activity in wild mountain gorillas, we used enzyme immunoassays (EIA) to quantify both urinary and fecal excretion of immunoreactive pregnanediol-3-glucuronide (iPdG), defined as all metabolites detected by a pregnanediol-3-glucuronide immunoassay (PdG EIA). Simultaneously, we performed the liquid chromatography mass spectrometry (LC-MS) to quantify the excretion of pregnanediol in urine and feces. Samples were analyzed over nine cycles of five females from the habituated gorillas monitored by Karisoke Research Center, Rwanda. As an additional indicator for ovulation timing, estrone conjugates (E1C) were measured in a subset of urine samples. The concentrations of iPdG and pregnanediol measured in the same samples were significantly correlated. Urinary concentrations of iPdG and pregnanediol fluctuated over the menstrual cycle but did not reveal any cyclic pattern, whereas a typical preovulatory urinary E1C surge and postovulatory increases of fecal iPdG and pregnanediol were detected. The luteal peaks of iPdG and pregnanediol levels in feces were on average 2.8 and 7.6 times higher, respectively, than averaged levels in the corresponding follicular phase. The relative number of days with observed matings was higher within the presumed fertile window than in the preceding period. Overall, the results indicate that fecal analysis of iPdG and pregnanediol is suitable for detecting

  18. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    International Nuclear Information System (INIS)

    Koivunen, Marja E.; Dettmer, Katja; Vermeulen, Roel; Bakke, Berit; Gee, Shirley J.; Hammock, Bruce D.

    2006-01-01

    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL -1 ), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL -1 ) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R 2 values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R 2 = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine

  19. Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

    Science.gov (United States)

    Fernández-Fernández, Mario; Rodríguez-González, Pablo; Añón Álvarez, M Elena; Rodríguez, Felix; Menéndez, Francisco V Álvarez; García Alonso, J Ignacio

    2015-04-07

    This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors

  20. Mass Spectrometry (LC-MS-MS) as a Tool in the Maillard Reaction Optimisation and Characterisation of New 6-methoxy-tetrahydro-β-carboline Derivatives

    International Nuclear Information System (INIS)

    Goh, T.B.; Mordi, M.N.; Mansor, S.M.

    2015-01-01

    Four new 6-methoxy-tetrahydro-β-carboline derivatives (1-6- methoxy-1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole,2-6-methoxy-1- (4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, 3-6-methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole and 4-2-methoxy-4-(6-methoxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl) phenol) were prepared via the Maillard reaction using 5-methoxytryptamine and various aldehydes in water. The synthesis reaction conditions were optimised in catalyst loading, temperature and time using LC-MS for optimum yields. Surface response methodology and contour plot was selected as an approach for optimisation. The optimum yield could be achieved below 50 degree Celsius within 5 h at 7 mole % catalyst loading at yields > 70 %. The β-carboline compounds produced were characterised using electrospray ionization mass spectrometry (ESI-MS) and electrospray tandem mass (ESI-MS/MS). The mass fragmentation patterns of this group of heterocyclic tetrahydro-β-carboline compounds are described herein. (author)

  1. Determination of propineb and its metabolites propylenethiourea and propylenediamine in banana and soil using gas chromatography with flame photometric detection and LC-MS/MS analysis.

    Science.gov (United States)

    Song, Shiming; Wei, Jie; Chen, Zhaojie; Lei, Yuhao; Zhang, Yan; Deng, Cheng; Tan, Huihua; Li, Xuesheng

    2018-03-04

    A sensitive and specific method for the determination of propineb and its metabolites, propylenethiourea (PTU) and propylenediamine (PDA), using gas chromatography with flame photometric detection (GC-FPD) and LC-MS/MS was developed and validated. Propineb and its metabolite residue dynamics in supervised field trials under Good Agricultural Practice (GAP) conditions in banana and soil were studied. Recovery of propineb (as CS 2 ), PDA and PTU ranged from 75.3 to 115.4% with RSD (n = 5) of 1.3-11.1%. The limit of quantification (LOQ) of CS 2 , PDA and PTU ranged from 0.005 to 0.01 mg kg -1 , and the limit of detection (LOD) ranged from 0.0015 to 0.0033 mg kg -1 . Dissipation experiments showed that the half-life of propineb in banana and soil ranged from 4.4 to 13.3 days. PTU was found in banana with a half-life of 31.5-69.3 days, while levels of PDA were less than 0.01 mg kg -1 in banana and soil. It has been suggested that PTU is the major metabolite of propineb in banana. The method was demonstrated to be reliable and sensitive for the routine monitoring of propineb and its metabolites in banana and soil. It also serves as a reference for the detection and monitoring of dithiocarbamates (DTCs) residues and the evaluation of their metabolic pathway.

  2. Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS

    Science.gov (United States)

    Bilbao, Aivett; Zhang, Ying; Varesio, Emmanuel; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard

    2016-01-01

    Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which in turn is detrimental for accurate quantification. The “non-outlier fragment ion” (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high priority fragment ions these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative dataset (i.e. the SWATH Gold Standard), indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest dataset, NOFI properly assigns low priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80 against 0.76 for the Spectronaut interference detection algorithm. PMID:26412574

  3. Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Giuseppe Dionisio

    2018-01-01

    Full Text Available Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1 seed coats; (2 aleurone/subaleurone; (3 embryo; and (4 endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation, and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE followed by polyvinylidene fluoride (PVDF membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn2+-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS. Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed.

  4. Optimization and Comparison of ESI and APCI LC-MS/MS Methods: A Case Study of Irgarol 1051, Diuron, and their Degradation Products in Environmental Samples

    Science.gov (United States)

    Maragou, Niki C.; Thomaidis, Nikolaos S.; Koupparis, Michael A.

    2011-10-01

    A systematic and detailed optimization strategy for the development of atmospheric pressure ionization (API) LC-MS/MS methods for the determination of Irgarol 1051, Diuron, and their degradation products (M1, DCPMU, DCPU, and DCA) in water, sediment, and mussel is described. Experimental design was applied for the optimization of the ion sources parameters. Comparison of ESI and APCI was performed in positive- and negative-ion mode, and the effect of the mobile phase on ionization was studied for both techniques. Special attention was drawn to the ionization of DCA, which presents particular difficulty in API techniques. Satisfactory ionization of this small molecule is achieved only with ESI positive-ion mode using acetonitrile in the mobile phase; the instrumental detection limit is 0.11 ng/mL. Signal suppression was qualitatively estimated by using purified and non-purified samples. The sample preparation for sediments and mussels is direct and simple, comprising only solvent extraction. Mean recoveries ranged from 71% to 110%, and the corresponding (%) RSDs ranged between 4.1 and 14%. The method limits of detection ranged between 0.6 and 3.5 ng/g for sediment and mussel and from 1.3 to 1.8 ng/L for sea water. The method was applied to sea water, marine sediment, and mussels, which were obtained from marinas in Attiki, Greece. Ion ratio confirmation was used for the identification of the compounds.

  5. Analytical methodologies based on LC-MS/MS for monitoring selected emerging compounds in liquid and solid phases of the sewage sludge.

    Science.gov (United States)

    Boix, C; Ibáñez, M; Fabregat-Safont, D; Morales, E; Pastor, L; Sancho, J V; Sánchez-Ramírez, J E; Hernández, F

    2016-01-01

    In this work, two analytical methodologies based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed for quantification of emerging pollutants identified in sewage sludge after a previous wide-scope screening. The target list included 13 emerging contaminants (EC): thiabendazole, acesulfame, fenofibric acid, valsartan, irbesartan, salicylic acid, diclofenac, carbamazepine, 4-aminoantipyrine (4-AA), 4-acetyl aminoantipyrine (4-AAA), 4-formyl aminoantipyrine (4-FAA), venlafaxine and benzoylecgonine. The aqueous and solid phases of the sewage sludge were analyzed making use of Solid-Phase Extraction (SPE) and UltraSonic Extraction (USE) for sample treatment, respectively. The methods were validated at three concentration levels: 0.2, 2 and 20 μg L(-1) for the aqueous phase, and 50, 500 and 2000 μg kg(-1) for the solid phase of the sludge. In general, the method was satisfactorily validated, showing good recoveries (70-120%) and precision (RSD metabolites of dipyrone had not been studied before in sewage sludge.

  6. Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC-MS analysis.

    Science.gov (United States)

    Oppermann, Sebastian; Oppermann, Christina; Böhm, Miriam; Kühl, Toni; Imhof, Diana; Kragl, Udo

    2018-04-25

    Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K 2 HPO 4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC-MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

  7. Cocaine and crack cocaine abuse by pregnant or lactating mothers and analysis of its biomarkers in meconium and breast milk by LC-MS-A review.

    Science.gov (United States)

    D'Avila, Felipe Bianchini; Limberger, Renata Pereira; Fröehlich, Pedro Eduardo

    2016-09-01

    Abusive use of drugs is a public health problem worldwide. The use of these substances by pregnant or lactating women can have many serious side effects in newborns. Among the commonest causes of addiction in drug users is cocaine in powdered form, inhaled, intravenously injected or smoked form (crack). Fast screening and a confirmation test using high specificity and sensitivity instruments such as LC-MS or GC/MS, can provide data to qualify and quantify chemical substances present in biological samples such as breast milk or meconium. Cocaine and/or crack can be detected through biomarkers or the unchanged molecule, enabling the form of cocaine use to be distinguished through the analytes. These methods must be carefully developed and validated according to internationally recognized guidelines. Thus, the study of biological matrices in which it can be detected through the development of simple and quick analytical methods can help prevent intoxication and diagnose the symptoms of dependency such as seizures, especially in babies, providing appropriate medical care. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  8. Eco-friendly LC-MS/MS method for analysis of multi-class micropollutants in tap, fountain, and well water from northern Portugal.

    Science.gov (United States)

    Barbosa, Marta O; Ribeiro, Ana R; Pereira, Manuel F R; Silva, Adrián M T

    2016-11-01

    Organic micropollutants present in drinking water (DW) may cause adverse effects for public health, and so reliable analytical methods are required to detect these pollutants at trace levels in DW. This work describes the first green analytical methodology for multi-class determination of 21 pollutants in DW: seven pesticides, an industrial compound, 12 pharmaceuticals, and a metabolite (some included in Directive 2013/39/EU or Decision 2015/495/EU). A solid-phase extraction procedure followed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (offline SPE-UHPLC-MS/MS) method was optimized using eco-friendly solvents, achieving detection limits below 0.20 ng L -1 . The validated analytical method was successfully applied to DW samples from different sources (tap, fountain, and well waters) from different locations in the north of Portugal, as well as before and after bench-scale UV and ozonation experiments in spiked tap water samples. Thirteen compounds were detected, many of them not regulated yet, in the following order of frequency: diclofenac > norfluoxetine > atrazine > simazine > warfarin > metoprolol > alachlor > chlorfenvinphos > trimethoprim > clarithromycin ≈ carbamazepine ≈ PFOS > citalopram. Hazard quotients were also estimated for the quantified substances and suggested no adverse effects to humans. Graphical Abstract Occurrence and removal of multi-class micropollutants in drinking water, analyzed by an eco-friendly LC-MS/MS method.

  9. A QC approach to the determination of day-to-day reproducibility and robustness of LC-MS methods for global metabolite profiling in metabonomics/metabolomics.

    Science.gov (United States)

    Gika, Helen G; Theodoridis, Georgios A; Earll, Mark; Wilson, Ian D

    2012-09-01

    An approach to the determination of day-to-day analytical robustness of LC-MS-based methods for global metabolic profiling using a pooled QC sample is presented for the evaluation of metabonomic/metabolomic data. A set of 60 urine samples were repeatedly analyzed on five different days and the day-to-day reproducibility of the data obtained was determined. Multivariate statistical analysis was performed with the aim of evaluating variability and selected peaks were assessed and validated in terms of retention time stability, mass accuracy and intensity. The methodology enables the repeatability/reproducibility of extended analytical runs in large-scale studies to be determined, allowing the elimination of analytical (as opposed to biological) variability, in order to discover true patterns and correlations within the data. The day-to-day variability of the data revealed by this process suggested that, for this particular system, 3 days continuous operation was possible without the need for maintenance and cleaning. Variation was generally based on signal intensity changes over the 7-day period of the study, and was mainly a result of source contamination.

  10. A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development, validation and application in the CUMS rat model.

    Science.gov (United States)

    Zhai, Xue-jia; Chen, Fen; Zhu, Chao-ran; Lu, Yong-ning

    2015-11-01

    Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC-MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25-12,800 ng mL(-1) with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Clean-up and matrix effect in LC-MS/MS analysis of food of plant origin for high polar herbicides.

    Science.gov (United States)

    Kaczyński, Piotr

    2017-09-01

    This study reports an innovative and sensitive procedure for analysis of difficult high polar herbicides (HPH) in diverse foods of plant origin. The QuPPe (Quick Polar Pesticides) method followed by determination by LC-MS/MS was modified. Chromatographic conditions, extraction, clean-up, and matrix effect were studied. Several liquid chromatography stationary and mobile phases were evaluated, and it was found that hydrophilic interaction chromatography (HILIC) gives good retention and sensitivity. An acidified methanol-water mixture was used as an effective extraction solvent of eleven HPH. Dispersive solid-phase clean-up sorbents (C18, GCB, Florisil, chitosan and graphene) were evaluated. The efficiency of the method was examined using data on recovery, precision and matrix effects. High extraction yields were achieved, and recoveries were within the 64-97% range with relative standard deviations <20% for all HPH in all commodities. Low matrix effects were observed when graphene was used during clean-up of onion extract and when chitosan was used for wheat, potato and pea extract. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Hydrolysis is the dominating in vivo metabolism pathway for arctigenin: identification of novel metabolites of arctigenin by LC/MS/MS after oral administration in rats.

    Science.gov (United States)

    Gao, Qiong; Zhang, Yufeng; Wo, Siukwan; Zuo, Zhong

    2013-04-01

    The phenylpropanoid dibenzylbutyrolactone lignan arctigenin, a key component found in Arctium lappa, or burdock, has been reported with a variety of therapeutic effects including anticancer, anti-inflammation, and antivirus effects. Using LC/MS/MS, three novel metabolites of arctigenin, namely, arctigenic acid, arctigenin-4-O'-glucuronide, and 4-O-demethylarctigenin were identified after oral administration of arctigenin in rats for the first time. Another potential metabolite of arctigenin, arctigenin-4'-O-sulfate, was identified in vitro but not in vivo. Structure of arctigenic acid, the major metabolite of arctigenin, was confirmed by 13C-NMR and 1H-NMR. Rapid hydrolysis in plasma was identified as the major metabolic pathway of arctigenin after its oral administration, with Vmax, Km, and Clint in rat plasma determined to be 2.21 ± 0.12 nmol/min/mg, 89.12 ± 9.44 µM, and 24.74 µL/min/mg, respectively. Paraoxonase 1 was further confirmed to be the enzyme responsible for arctigenin hydrolysis, with Vmax, Km, and Clint determined to be 55.39 ± 1.49 nmol/min/mg, 300.3 ± 10.86 µM, and 184.45 µL/min/mg, respectively. Georg Thieme Verlag KG Stuttgart · New York.

  13. [Rapid screening and quality evaluation for the harmful substance 5-hydroxymethyl furfural in commercially available traditional Chinese medicine injection using LC-MS/MS method].

    Science.gov (United States)

    Zang, Qing-ce; He, Jing-jing; Bai, Jin-fa; Zheng, Ya-jie; Zhang, Rui-ping; Li, Tie-gang; Wang, Zhong-hua; He, Jiu-ming; Abliz, Zeper

    2013-11-01

    To screen the harmful substance 5-hydroxymethyl furfural content in commercially available traditional Chinese medicine injection which are commonly used, and to preliminarily evaluate the quality of these injections, 5-hydroxymethyl furfural was taken as an index. The contents of 5-hydroxymethyl furfural in 56 samples which consist of 23 kinds of traditional Chinese medicine injections and glucose injection were determined using LC-MS/MS, and 5-hydroxymethyl furfural was detected in 52 of these samples. The minimal content was 0.0038 microg x L(-1) and the maximum content was 1420 microg x mL(-1). The contents of 5-hydroxymethyl furfural were significantly different in traditional Chinese medicine injection which came from different kinds, manufacturers or batches. The results showed the quality difference of commercially available traditional Chinese medicine injection is significant taking 5-hydroxymethyl furfural content as assessment index. More attention should be paid to the safety of 5-hydroxymethyl furfural in traditional Chinese medicine injection, and unified limitation standard should be set to improve medication safety of traditional Chinese medicine injection.

  14. Survey on acrylamide in roasted coffee and barley and in potato crisps sold in Italy by a LC-MS/MS method.

    Science.gov (United States)

    Bertuzzi, Terenzio; Rastelli, Silvia; Mulazzi, Annalisa; Pietri, Amedeo

    2017-12-01

    A survey on the occurrence of acrylamide (AA) in roasted coffee, barley, and potato crisps was carried out using an intra-lab validated liquid chromatography (LC)-MS (mass spectrometry)/MS method. Over the years 2015-2016, 66 samples of coffee, 22 of roasted barley, and 22 of potato crisps were collected from retail outlets in Italy. AA was detected in almost all samples. In roasted coffee, the level exceeded 450 µg kg -1 , the limit recommended by the European Commission (EC), in 36.4% of the samples. In roasted barley, mean contamination was slightly lower than in coffee and no sample exceeded the EC limit of 2000 µg kg -1 . The AA contamination in potato crisps was remarkable. A percentage of 36.4 (n = 8) showed a value higher than the EC limit of 1000 µg kg -1 . Considering the average consumption of coffee and potato crisps by Italian people, AA exposure is significant and should be decreased.

  15. Matrix-calibrated LC-MS/MS quantitation and sensory evaluation of oak Ellagitannins and their transformation products in red wines.

    Science.gov (United States)

    Stark, Timo; Wollmann, Nadine; Wenker, Kerstin; Lösch, Sofie; Glabasnia, Arne; Hofmann, Thomas

    2010-05-26

    Aimed at investigating the concentrations and taste contribution of the oak-derived ellagitannins castalagin and vescalagin as well as their transformation products acutissimin A/B, epiacutissimin A/B, and beta-1-O-ethylvescalagin in red wine, a highly sensitive and accurate quantification method was developed on the basis of LC-MS/MS-MRM analysis with matrix calibration. Method validation showed good recovery rates ranging from 102.4 +/- 5.9% (vescalagin) to 113.7 +/- 15.2% (epiacutissimin A). In oak-matured wines, castalagin was found as the predominant ellagitannin, followed by beta-1-O-ethylvescalagin, whereas the flavano-C-ellagitannins (epi)acutissimin A/B were present in significantly lower amounts. In contrast to the high threshold concentration levels (600-1000 micromol/L) and the puckering astringent orosensation induced by flavan-3-ols, all of the ellagitannin derivatives were found to induce a smooth and velvety astringent oral sensation at rather low threshold concentrations ranging from 0.9 to 2.8 micromol/L. Dose/activity considerations demonstrated that, among all the ellagitannins investigated, castalagin exclusively exceeded its threshold concentration in various oak-matured wine samples.

  16. Evaluation of a multiresidue method for measuring fourteen chemical groups of pesticides in water by use of LC-MS-MS.

    Science.gov (United States)

    Carvalho, J J; Jerónimo, P C A; Gonçalves, C; Alpendurada, M F

    2008-11-01

    European Council Directive 98/83/EC on the quality of water intended for human consumption brought a new challenge for water-quality control routine laboratories, mainly on pesticides analysis. Under the guidelines of ISO/IEC 17025:2005, a multiresidue method was developed, validated, implemented in routine, and studied with real samples during a one-year period. The proposed method enables routine laboratories to handle a large number of samples, since 28 pesticides of 14 different chemical groups can be quantitated in a single procedure. The method comprises a solid-phase extraction step and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS-MS). The accuracy was established on the basis of participation in interlaboratory proficiency tests, with encouraging results (majority |z-score| <2), and the precision was consistently analysed over one year. The limits of quantitation (below 0.050 microg L(-1)) are in agreement with the enforced threshold value for pesticides of 0.10 microg L(-1). Overall method performance is suitable for routine use according to accreditation rules, taking into account the data collected over one year.

  17. Crystal violet: Study of the photo-fading of an early synthetic dye in aqueous solution and on paper with HPLC-PDA, LC-MS and FORS

    International Nuclear Information System (INIS)

    Confortin, Daria; Brustolon, Marina; Franco, Lorenzo; Neevel, Han; Bommel, Maarten R van; Kettelarij, Albert J; Williams, Rene M

    2010-01-01

    The photo-fading of crystal violet (CV), one of the earliest synthetic dyes and an ink component, is examined both in solution and on paper. Aqueous solutions of CV were exposed to UV light (365nm) and samples were taken at constant time intervals and analysed with a High Performance Liquid Chromatography-Photo Diode Array (HPLC-PDA) and Liquid Chromatography-Mass Spectroscopy (LC-MS). Demethylation products were positively identified. Also, deamination probably occurred. The oxidation at the central carbon likely generates Michler's ketone (MK) or its derivatives, but still needs confirmation. To study CV on paper, Whatman paper was immersed in CV and exposed to UV light. Before and after different irradiation periods, reflectance spectra were recorded with Fibre Optic Reflectance Spectrophotometry (FORS). A decrease in CV concentration and a change in aggregation type for CV molecules upon irradiation was observed. Colorimetric L*a*b* values before and during irradiation were also measured. Also, CV was extracted from paper before and after different irradiation periods and analysed with HPLC-PDA. Photo-fading of CV on paper produced the same products as in solution, at least within the first 100 hours of irradiation. Finally, a photo-fading of CV in the presence of MK on Whatman paper was performed. It was demonstrated that MK both accelerates CV degradation and is consumed during the reaction. The degradation pathway identified in this work is suitable for explaining the photo/fading of other dyes belonging to the triarylmethane group.

  18. Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

    Science.gov (United States)

    Ferrer, I.; Furlong, E.T.

    2002-01-01

    Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed by ASE, were 85% for C12BAC and 79% for C14-BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 ??g/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.

  19. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components.

    Science.gov (United States)

    Scheltema, Richard A; Mann, Matthias

    2012-06-01

    With the advent of high-throughput mass spectrometry (MS)-based proteomics, the magnitude and complexity of the performed experiments has increased dramatically. Likewise, investments in chromatographic and MS instrumentation are a large proportion of the budget of proteomics laboratories. Guarding measurement quality and maximizing uptime of the LC-MS/MS systems therefore requires constant care despite automated workflows. We describe a real-time surveillance system, called SprayQc, that continuously monitors the status of the peripheral equipment to ensure that operational parameters are within an acceptable range. SprayQc is composed of multiple plug-in software components that use computer vision to analyze electrospray conditions, monitor the chromatographic device for stable backpressure, interact with a column oven to control pressure by temperature, and ensure that the mass spectrometer is still acquiring data. Action is taken when a failure condition has been detected, such as stopping the column oven and the LC flow, as well as automatically notifying the appropriate operator. Additionally, all defined metrics can be recorded synchronized on retention time with the MS acquisition file, allowing for later inspection and providing valuable information for optimization. SprayQc has been extensively tested in our laboratory, supports third-party plug-in development, and is freely available for download from http://sourceforge.org/projects/sprayqc .

  20. Chemical and Physical Methods to Analyze a Multicomponent Traditional Chinese Herbal Prescription Using LC-MS/MS, Electron Microscope, and Congo Red Staining

    Directory of Open Access Journals (Sweden)

    Chia-Ming Lu

    2013-01-01

    Full Text Available This study develops several chemical and physical methods to evaluate the quality of a traditional Chinese formulation, Jia-Wei-Xiao-Yao-San. Liquid chromatography-tandem mass spectrometry (LC-MS/MS coupled with electrospray ionization was used to measure the herbal biomarkers of saikosaponin A, saikosaponin D, ferulic acid, and paeoniflorin from this herbal formula. A scanning electron microscope (SEM and light microscopy photographs with Congo red staining were used to identify the cellulose fibers if raw herbal powder had been added to the herbal pharmaceutical product. Moreover, water solubility and crude fiber content examination were used to inspect for potential herbal additives to the herbal pharmaceutical products. The results demonstrate that the contents of the herbal ingredients of saikosaponin A, saikosaponin D, ferulic acid, and paeoniflorin were around 0.351 ± 0.017, 0.136 ± 0.010, 0.140 ± 0.005, and 2.281 ± 0.406 mg/g, respectively, for this herbal pharmaceutical product. The physical examination data demonstrate that the raw herbal powder had rough, irregular, lumpy, filamentous, and elongated shapes, as well as strong Congo red staining. In addition, water solubility and crude fiber content were not consistent in the herbal pharmaceutical products.

  1. Anti-Trypanosoma, anti-Leishmania and cytotoxic activities of natural products from Psidium brownianum Mart. ex DC. and Psidium guajava var. Pomifera analysed by LC-MS.

    Science.gov (United States)

    de Souza, Celestina Elba Sobral; da Silva, Ana Raquel Pereira; Gomez, Maria Celeste Vega; Rolóm, Míriam; Coronel, Cathia; da Costa, José Galberto Martins; Sousa, Amanda K; Rolim, Larissa A; de Souza, Francisco Hugo Sobral; Coutinho, Henrique Douglas Melo

    2017-12-01

    Neglected diseases are those that are prevalent in developing countries, even with a rich biodiversity. These diseases still persist because of the lack of scientific studies, government negligence or failures of the public health system. This study aims to identify the composition of extracts and fractions from Psidium brownianum and Psidium guajava through LC-MS, to evaluate its in vitro anti-parasitic and cytotoxic activity against Trypanosoma cruzi, Leishmania brasiliensis and L. infantum epismastigote and promastigote forms, as well as mammalian cells. The results showed the presence of chemical constituents in the two Psidium species as quercetin, myricetin and gallic acid derivatives. The P. brownianum extract and fractions showed low toxicity at all tested concentrations and all samples were effective at the concentration of 1000μg/mL against the parasites, with the extract being the most efficient against the L. infantum promastigote form. The ethanolic extract, and the flavonoid and tannic fractions, from P. guajava showed low toxicity for the fibroblasts. All samples showed effectiveness at the highest concentration tested and the extract was more effective against the promastigote forms tested. The results showed that the species Psidium brownianum and Psidium guajava demonstrated an anti-parasitic activity against the T. cruzi, L. brasiliensis and L. infantum parasite cell lines indicating these species as an alternative therapy given their efficacy in the in vitro assays performed, opening the possibility for new biological studies to further this knowledge through in vivo assays. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Development and validation of an improved method for the quantitation of sertraline in human plasma using LC-MS-MS and its application to bioequivalence studies.

    Science.gov (United States)

    Zhang, Mengliang; Gao, Feng; Cui, Xiangyong; Zhang, Yunhui; Sun, Yantong; Gu, Jingkai

    2011-02-01

    A rapid and sensitive LC-MS-MS method for the quantitation of sertraline in human plasma was developed and validated. Sertraline and the internal standard, telmisartan, were cleaned up by protein precipitation from 100 μL of plasma sample, and analyzed on a TC-C18 column (5 μm, 150 × 4.6 mm i.d.) using 70% acetonitrile and 30% 10 mM ammonium acetate (0.1% formic acid) as mobile phase. The method was demonstrated to be linear from 0.1 ng/mL to 50 ng/mL with the lower limit of quantitation of 0.1 ng/mL. Intra- and inter-day precision were below 4.40% and 3.55%. Recoveries of sertraline at low, medium, and high levels were 88.0 ± 2.3%, 88.2 ± 1.9%, and 90.0 ± 2.0%, respectively. The method was successfully applied to a bioequivalence study of sertraline after a single oral administration of 50 mg sertraline hydrochloride tablets.

  3. Development of a Multi-class Steroid Hormone Screening Method using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

    Science.gov (United States)

    Boggs, Ashley S. P.; Bowden, John A.; Galligan, Thomas M.; Guillette, Louis J.; Kucklick, John R.

    2016-01-01

    Monitoring complex endocrine pathways is often limited by indirect measurement or measurement of a single hormone class per analysis. There is a burgeoning need to develop specific direct-detection methods capable of providing simultaneous measurement of biologically relevant concentrations of multiple classes of hormones (estrogens, androgens, progestogens, and corticosteroids). The objectives of this study were to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for multi-class steroid hormone detection using biologically relevant concentrations, then test limits of detection (LOD) in a high-background matrix by spiking charcoal-stripped fetal bovine serum (FBS) extract. Accuracy was tested with National Institute of Standards and Technology Standard Reference Materials (SRMs) with certified concentrations of cortisol, testosterone, and progesterone. 11-Deoxycorticosterone, 11-deoxycortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, adrenosterone, androstenedione, cortisol, corticosterone, dehydroepiandrosterone, dihydrotestosterone, estradiol, estriol, estrone, equilin, pregnenolone, progesterone, and testosterone were also measured using isotopic dilution. Dansyl chloride (DC) derivatization was investigated maintaining the same method to improve and expedite estrogen analysis. Biologically relevant LODs were determined for 15 hormones. DC derivatization improved estrogen response two- to eight-fold, and improved chromatographic separation. All measurements had an accuracy ≤ 14 % difference from certified values (not accounting for uncertainty) and relative standard deviation ≤ 14 %. This method chromatographically separated and quantified biologically relevant concentrations of four hormone classes using highly specific fragmentation patterns and measured certified values of hormones that were previously split into three separate chromatographic methods. PMID:27039201

  4. High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis.

    Science.gov (United States)

    Halladay, Jason S; Delarosa, Erlie Marie; Tran, Daniel; Wang, Leslie; Wong, Susan; Khojasteh, S Cyrus

    2011-08-01

    Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.

  5. Development of a quantitative multi-compound method for the detection of 14 nitrogen-rich adulterants by LC-MS/MS in food materials.

    Science.gov (United States)

    Frank, Nancy; Bessaire, Thomas; Tarres, Adrienne; Goyon, Alexandre; Delatour, Thierry

    2017-11-01

    The increasing number of food frauds using exogenous nitrogen-rich adulterants to artificially raise the protein content for economically motivated adulteration has demonstrated the need for a robust analytical methodology. This method should be applicable for quality control in operations covering a wide range of analyte concentrations to be able to analyse high levels as usually found in adulteration, as well as low levels due to contamination. The paper describes a LC-MS/MS method covering 14 nitrogen-rich adulterants using a simple and fast sample preparation based on dilution and clean-up by dispersive SPE. Quantification is carried out by isotopic dilution reaching LOQs of 0.05-0.20 mg/kg in a broad range of food matrices (infant formula, liquid milk, dairy ingredient, high protein meal, cereal, infant cereal, and meat/fish powder). Validation of seven commodity groups was performed according to SANCO 12571/2013, giving satisfactory results demonstrating the method's fitness for purpose at the validated range at contamination level. Method ruggedness was further assessed by transferring the developed method into another laboratory devoted to routine testing for quality control. Next to the method description, emphasis is placed on challenges and problems appearing during method development as well as validation. They are discussed in detail and solutions are provided.

  6. Simultaneous determination of multicomponent of acetylkitasamycin and kitasamycin by LC-MS/MS in swine plasma and its application in a pharmacokinetic study.

    Science.gov (United States)

    Pan, Yuanhu; Zhang, Heying; Xi, Chenglong; Huang, Lingli; Xie, Shuyu; Chen, Dongmei; Tao, Yanfei; Liu, Zhenli; Yuan, Zonghui

    2018-05-02

    A simple and reliable LC-MS/MS method was established for simultaneous determination of twelve components from acetylkitasamycin and kitasamycin in swine plasma. The analytes were separated by a Shim-pack VP-ODS column with a 25 min gradient elution using 5 mmol/L ammonium acetate and acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. Identification and quantification were accomplished by electrospray ionization (ESI) in positive mode using multiple reaction monitoring (MRM). The LOQ S of acetylkitasamycin A 1 A 3 , A 13 and kitasamycin A 3 , A 13 were 3 μg/L, and that of the other 8 components were 5 μg/L. The mean recoveries of kitasamycin and acetylkitasamycin ranged from 85.3 to 103.5 %. The developed method was successfully applied to a pharmacokinetic study in swine after intravenous (IV) and oral (PO) administration of acetylkitasamycin. The result showed that the plasma concentrations of acetylkitsamycin components were much higher than that of kitasamycin in swine after IV and PO, in which acetylkitsamycin A 4 A 5 was the highest component at each time point. This article is protected by copyright. All rights reserved.

  7. Development and validation of an LC-MS/MS method for the determination of mesalazine in beagle dog plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Qin, Juan; Di, Xin; Wang, Xin; Liu, Youping

    2015-02-01

    A simple, specific and sensitive LC-MS/MS method was developed and validated for the determination of mesalazine in beagle dog plasma. The plasma samples were prepared by protein precipitation, then the separation of the analyte was achieved on a Waters Spherisorb C6 column (150 × 4.6 mm, 5 µm) with a mobile phase consisting of 0.2% formic acid in water-methanol (20:80, v/v). The flow rate was set at 1.0 mL/min with a split ratio of 3:2. Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 154 → m/z 108 for mesalazine and m/z 285 → m/z 193 for diazepam (internal standard). The linear calibration curve of mesalazine was obtained over the concentration range 50-30,000 ng/mL. The matrix effect of mesalazine was within ±9.8%. The intra- and inter-day precisions were dogs after rectal administration of mesalazine suppository. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Prescription and illicit psychoactive drugs in oral fluid--LC-MS/MS method development and analysis of samples from Brazilian drivers.

    Science.gov (United States)

    Zancanaro, Ivomar; Limberger, Renata Pereira; Bohel, Paula O; dos Santos, Maíra Kerpel; De Boni, Raquel B; Pechansky, Flavio; Caldas, Eloisa Dutra

    2012-11-30

    This study is part of a larger project designed to investigate the prevalence of psychoactive drug (PAD) use among Brazilian drivers. In this paper we describe the development and validation of an analytical method to analyze 32 prescription and illicit PADs (amphetamines, benzodiazepines, cocaine, cannabis, opioids, ketamine and m-CPP) and metabolites in oral fluid samples collected with a Quantisal™ device. Samples were extracted with ethyl acetate:hexane and analyzed by LC-MS/MS. Instrumental LOD ranged from 0.26 to 0.65 ng/mL. Mean procedural recoveries at 1.3 ng/mL (LLOQ) ranged from 50% to 120% for 24 compounds. Recoveries were concentration independent, with the exception of femproporex, heroin and ecgonine methyl-ester (EME) for which the recovery decreased significantly at higher levels (13 and 52 ng/mL). RSD was drugs, cocaine/metabolites were the analytes most detected in the samples (129; 5.8%), followed by amphetamines/metabolite (69; 3.1%), benzodiazepines (28; 1.2%), cannabinoids (23; 1.1%) and opioids (8; 0.4%). Detection of at least two PADs from different classes accounted for 9.3% of the 236 positive samples. Cocaine was found at higher levels in the samples (up to 1165 ng/mL). Preventive measures aimed at reducing the use of PADs by drivers in Brazil will certainly contribute to decrease the country's highway death rates. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. Paralytic toxin profile of the marine dinoflagellate Gymnodinium catenatum Graham from the Mexican Pacific as revealed by LC-MS/MS.

    Science.gov (United States)

    Bustillos-Guzmán, José J; Band-Schmidt, Christine J; Durán-Riveroll, Lorena M; Hernández-Sandoval, Francisco E; López-Cortés, David J; Núñez-Vázquez, Erick J; Cembella, Allan; Krock, Bernd

    2015-01-01

    The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

  10. Development and characterization of a novel plug and play liquid chromatography-mass spectrometry (LC-MS) source that automates connections between the capillary trap, column, and emitter.

    Science.gov (United States)

    Bereman, Michael S; Hsieh, Edward J; Corso, Thomas N; Van Pelt, Colleen K; Maccoss, Michael J

    2013-06-01

    We report the development and characterization of a novel, vendor-neutral ultra-high pressure-compatible (~10,000 p.s.i.) LC-MS source. This device is the first to make automated connections with user-packed capillary traps, columns, and capillary emitters. The source uses plastic rectangular inserts (referred to here as cartridges) where individual components (i.e. trap, column, or emitter) can be exchanged independent of one another in a plug and play manner. Automated robotic connections are made between the three cartridges using linear translation powered by stepper motors to axially compress each cartridge by applying a well controlled constant compression force to each commercial LC fitting. The user has the versatility to tailor the separation (e.g. the length of the column, type of stationary phase, and mode of separation) to the experimental design of interest in a cost-effective manner. The source is described in detail, and several experiments are performed to evaluate the robustness of both the system and the exchange of the individual trap and emitter cartridges. The standard deviation in the retention time of four targeted peptides from a standard digest interlaced with a soluble Caenorhabditis elegans lysate ranged between 3.1 and 5.3 s over 3 days of analyses. Exchange of the emitter cartridge was found to have an insignificant effect on the abundance of various peptides. In addition, the trap cartridge can be replaced with minimal effects on retention time (<20 s).

  11. LC/MS/MS analysis of α-tocopherol and coenzyme Q10 content in lyophilized royal jelly, beebread and drone homogenate.

    Science.gov (United States)

    Hryniewicka, Marta; Karpinska, Agnieszka; Kijewska, Marta; Turkowicz, Monika Joanna; Karpinska, Joanna

    2016-11-01

    This study shows the results of application liquid chromatography-tandem mass spectrometry (LC/MS/MS) for assay of the content of α-tocopherol and coenzyme Q 10 in bee products of animal origin, i.e. royal jelly, beebread and drone homogenate. The biological matrix was removed using extraction with n-hexane. It was found that drone homogenate is a rich source of coenzyme Q 10 . It contains only 8 ± 1 µg/g of α-tocopherol and 20 ± 2 µg/g of coenzyme Q 10 . The contents of assayed compounds in royal jelly were 16 ± 3 and 8 ± 0.2 µg/g of α-tocopherol and coenzyme Q 10 , respectively. Beebread appeared to be the richest of α-tocopherol. Its level was 80 ± 30 µg/g, while the level of coenzyme Q 10 was only 11.5 ± 0.3 µg/g. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Development and validation of an LC-MS/MS method for the determination of adapalene in pharmaceutical forms for skin application

    Directory of Open Access Journals (Sweden)

    Dobričić Vladimir

    2016-01-01

    Full Text Available Development and validation of a liquid chromatography - tandem mass spectrometry (LC-MS/MS method for the determination of adapalene in pharmaceutical forms for skin application were presented. The MS/MS analysis of adapalene was performed by use of three mobile phases, consisted of acetonitrile and (a 0.1 % formic acid, (b 0.1 % trifluoroacetic acid and (c 20 mM ammonium acetate. The strongest signals of parent ion and dominant product ion were obtained in negative mode by use of the mobile phase (c. Validation of this method was performed according to the ICH guidelines. Small variations of selected chromatographic parameters (concentration of ammonium acetate, mobile phase composition, column temperature and flow rate did not affect qualitative and quantitative system responses significantly, which proved method’s robustness. The method is specific for the determination of adapalene. Linearity was proved in the concentration range 6.7 - 700.0 ng mL-1 (r = 0.9990, with limits of detection and quantification 2.0 ng mL-1 and 6.7 ng mL-1, respectively. Accuracy was confirmed by calculated recoveries (98.4 % - 101.5 %. Precision was tested at three levels: injection repeatability, analysis repeatability and intermediate precision. Calculated relative standard deviations were less than 1, 2 and 3 %, respectively. [Projekat Ministartsva nauke Republike Srbije, br. OI172041 i br. TR34031

  13. LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.; Smolders, Arne; Brewer, Heather M.; Camp, David G.; Smith, Richard D.; Braeckman, Bart P.

    2014-02-20

    The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediary metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.

  14. Importance of MS selectivity and chromatographic separation in LC-MS/MS-based methods when investigating pharmaceutical metabolites in water. Dipyrone as a case of study.

    Science.gov (United States)

    Ibáñez, M; Gracia-Lor, E; Sancho, J V; Hernández, F

    2012-08-01

    Pharmaceuticals are emerging contaminants of increasing concern because of their presence in the aquatic environment and potential to reach drinking-water sources. After human and/or veterinary consumption, pharmaceuticals can be excreted in unchanged form, as the parent compound, and/or as free or conjugated metabolites. Determination of most pharmaceuticals and metabolites in the environment is commonly made by liquid chromatography (LC) coupled to mass spectrometry (MS). LC coupled to tandem MS is the technique of choice nowadays in this field. The acquisition of two selected reaction monitoring (SRM) transitions together with the retention time is the most widely accepted criterion for a safe quantification and confirmation assay. However, scarce attention is normally paid to the selectivity of the selected transitions as well as to the chromatographic separation. In this work, the importance of full spectrum acquisition high-resolution MS data using a hybrid quadrupole time-of-flight analyser and/or a suitable chromatographic separation (to reduce the possibility of co-eluting interferences) is highlighted when investigating pharmaceutical metabolites that share common fragment ions. For this purpose, the analytical challenge associated to the determination of metabolites of the widely used analgesic dipyrone (also known as metamizol) in urban wastewater is discussed. Examples are given on the possibilities of reporting false positives of dypirone metabolites by LC-MS/MS under SRM mode due to a wrong assignment of identity of the compounds detected. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Determination of urinary aromatic amines in smokers and nonsmokers using a MIPs-SPE coupled with LC-MS/MS method.

    Science.gov (United States)

    Yu, Jingjing; Wang, Sheng; Zhao, Ge; Wang, Bing; Ding, Li; Zhang, Xiaobing; Xie, Jianping; Xie, Fuwei

    2014-05-01

    Urinary aromatic amines (AAs) could be used as biomarkers for human exposure to AAs in cigarette smoke. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of urinary AAs (i.e. 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP)) in smokers and nonsmokers. A molecularly imprinted polymers (MIPs) solid phase extraction (SPE) cartridge was applied to purify urine samples and no derivatization reaction was involved. Each analytes used respective stable isotope internal standards, which could well compensate matrix effect. Lower limit of detections (LODs) for four AAs were obtained and in the range of 1.5-5ngL(-1). Recovery ranged from 87.7±4.5% to 111.3±6.4% and precision were less than 9.9%. The method was applied to analyze urine samples of 40 smokers and 10 nonsmokers. The 24h urinary excretion amounts of total AAs were higher for smokers compared with nonsmokers. What's more, 1-NA, 3-ABP and 4-ABP excretion amounts showed significant differences (p<0.05) between smokers and nonsmokers. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. LC-NMR, NMR, and LC-MS identification and LC-DAD quantification of flavonoids and ellagic acid derivatives in Drosera peltata.

    Science.gov (United States)

    Braunberger, Christina; Zehl, Martin; Conrad, Jürgen; Fischer, Sonja; Adhami, Hamid-Reza; Beifuss, Uwe; Krenn, Liselotte

    2013-08-01

    The herb of Drosera peltata, commonly named the shield sundew, is used as an antitussive in phytotherapy, although the plants' composition has not been determined in detail so far. Hence, in this study, we present a validated, sensitive, reliable, and cheap narrow-bore LC-DAD method for the simultaneous quantification of flavonoids and ellagic acid derivatives in this herbal drug. In addition, the structures of 13 compounds have been elucidated by LC-MS, LC-NMR, and offline NMR experiments after isolation: herbacetin-3-O-glucoside (1), gossypitrin (2), ellagic acid (3), quercetin-7-O-glucoside (4), isoquercitrin (5), kaempferol-3-O-(6″-O-galloyl)-glucoside (6), herbacetin-7-O-glucoside (7), astragalin (8), gossypetin (9), herbacetin (10), quercetin (11), 3,3'-di-O-methyl ellagic acid (12), and kaempferol (13). Compounds 1, 2, 4, 5, 6, 7, and 10 have been identified in D. peltata for the first time, and compounds 1, 4, 6, 7, and 10 have not been detected in any Drosera species before. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC-DAD, LC-NMR, NMR, and LC-MS.

    Science.gov (United States)

    Zehl, Martin; Braunberger, Christina; Conrad, Jürgen; Crnogorac, Marija; Krasteva, Stanimira; Vogler, Bernhard; Beifuss, Uwe; Krenn, Liselotte

    2011-06-01

    Droserae herba is a drug commonly used for treatment of convulsive or whooping cough since the seventeenth century. Because of the contribution of flavonoids and ellagic acid derivatives to the therapeutic activity of Droserae herba, an LC-DAD method has been developed for quantification of these analytes in four Drosera species used in medicine (Drosera anglica, D. intermedia, D. madagascariensis, and D. rotundifolia). During elaboration of the method 13 compounds, including three substances not previously described for Drosera species, were detected and unambiguously identified by means of extensive LC-MS and LC-NMR experiments and by off-line heteronuclear 2D NMR after targeted isolation. The most prominent component of D. rotundifolia and D. anglica, 2″-O-galloylhyperoside, with myricetin-3-O-β-glucopyranoside and kaempferol-3-O-(2″-O-galloyl)-β-galactopyranoside, were identified for the very first time in this genus. The LC-DAD method for quantification was thoroughly validated, and enables, for the first time, separation and precise analysis of these analytes in Droserae herba. Simple sample preparation and use of a narrow-bore column guarantee low cost and simplicity of the suggested system, which is excellently suited to quality control of the drug or herbal medicinal products containing this drug.

  18. IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers.

    Science.gov (United States)

    Frantzi, Maria; Zoidakis, Jerome; Papadopoulos, Theofilos; Zürbig, Petra; Katafigiotis, Ioannis; Stravodimos, Konstantinos; Lazaris, Andreas; Giannopoulou, Ioanna; Ploumidis, Achilles; Mischak, Harald; Mullen, William; Vlahou, Antonia

    2013-09-06

    Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

  19. Development of a LC-MS/MS method for the simultaneous screening of seven water-soluble vitamins in processing semi-coarse wheat flour products.

    Science.gov (United States)

    Nurit, Eric; Lyan, Bernard; Piquet, Agnès; Branlard, Gérard; Pujos-Guillot, Estelle

    2015-05-01

    Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 μg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 μg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products.

  20. A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples.

    Science.gov (United States)

    Ediage, Emmanuel Njumbe; Di Mavungu, José Diana; Monbaliu, Sofie; Van Peteghem, Carlos; De Saeger, Sarah

    2011-05-25

    This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (cake samples while fumonisin B(1) (4-21 μg/kg), aflatoxin B(2) (flour samples. Fumonisin B(1) (13-836 μg/kg), fumonisin B(2) (5-221 μg/kg), fumonisin B(3) (

  1. A new, direct analytical method using LC-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils.

    Science.gov (United States)

    Yamazaki, K; Ogiso, M; Isagawa, S; Urushiyama, T; Ukena, T; Kibune, N

    2013-01-01

    A new, direct analytical method for the determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) was developed. The targeted 3-MCPD esters included five types of monoester and 25 [corrected] types of diester. Samples (oils and fats) were dissolved in a mixture of tert-butyl methyl ether and ethyl acetate (4:1), purified using two solid-phase extraction (SPE) cartridges (C(18) and silica), then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Five monoesters and five diesters with the same fatty acid group could be separated and quantified. Pairs of 3-MCPD diesters carrying the same two different fatty acid groups, but at reversed positions (sn-1 and sn-2), could not be separated and so were expressed as a sum of both compounds. The limits of quantification (LOQs) were estimated to be between 0.02 to 0.08 mg kg(-1), depending on the types of 3-MCPD ester. Repeatability expressed as relative standard deviation (RSD(r)%) varied from 5.5% to 25.5%. The new method was shown to be applicable to various commercial edible oils and showed levels of 3-MCPD esters varying from 0.58 to 25.35 mg kg(-1). The levels of mono- and diesters ranged from 0.10 to 0.69 mg kg(-1) and from 0.06 to 16 mg kg(-1), respectively.

  2. LC-MS/MS determination of antiretroviral drugs in influents and effluents from wastewater treatment plants in KwaZulu-Natal, South Africa.

    Science.gov (United States)

    Abafe, Ovokeroye A; Späth, Jana; Fick, Jerker; Jansson, Stina; Buckley, Chris; Stark, Annegret; Pietruschka, Bjoern; Martincigh, Bice S

    2018-06-01

    South Africa has the largest occurrence of the human immune deficiency virus (HIV) in the world but has also implemented the largest antiretroviral (ARV) treatment programme. It was therefore of interest to determine the presence and concentrations of commonly used antiretroviral drugs (ARVDs) and, also, to determine the capabilities of wastewater treatment plants (WWTPs) for removing ARVDs. To this end, a surrogate standard based LC-MS/MS method was optimized and applied for the detection of thirteen ARVDs used in the treatment and management of HIV/acquired immune deficiency syndrome (HIV/AIDS) in two major and one modular WWTP in the eThekwini Municipality in KwaZulu-Natal, South Africa. The method was validated and the detection limits fell within the range of 2-20 ng L -1 . The analytical recoveries for the ARVDs were mainly greater than 50% with acceptable relative standard deviations. The concentration values ranged from effluent) in a decentralized wastewater treatment facility (DEWATS); effluent) in Northern WWTP and 61-34000 ng L -1 (influent), effluent) in Phoenix WWTP. Whilst abacavir, lamivudine and zidovudine were almost completely removed from the effluents, atazanavir, efavirenz, lopinavir and nevirapine persisted in the effluents from all three WWTPs. To estimate the ecotoxicological risks associated with the discharge of ARVDs, a countrywide survey focussing on the occurrence of ARVDs in WWTPs, surface and fresh water bodies, and aquatic organisms, is necessary. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Simultaneous determination of sibutramine and its active metabolites in human plasma by LC-MS/MS and its application to a pharmacokinetic study.

    Science.gov (United States)

    Bae, Jung-Woo; Choi, Chang-Ik; Jang, Choon-Gon; Lee, Seok-Yong

    2011-11-01

    A simple and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed and validated for the determination of sibutramine and its N-desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t-butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse-phase Luna C18 column with a mobile phase of acetonitrile-10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI-MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05-20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra- and inter-day validation for sibutramine, M1 and M2 were acceptable. This LC-MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.

  4. LC-MS/MS profiling and neuroprotective effects of Mentat® against transient global ischemia and reperfusion-induced brain injury in rats.

    Science.gov (United States)

    Viswanatha, Gollapalle Lakshminarayanashastry; Kumar, Lakkavalli Mohan Sharath; Rafiq, Mohamed; Kavya, Kethaganahalli Jayaramaiah; Thippeswamy, Agadi Hiremath; Yuvaraj, Huvvinamadu Chandrashekarappa; Azeemuddin, Mohammed; Anturlikar, Suryakanth Dattatreya; Patki, Pralhad Sadashiv; Babu, Uddagiri Venkanna; Ramakrishnan, Shyam

    2015-01-01

    The aim of this study was to evaluate the possible beneficial effects of Mentat against transient global ischemia and reperfusion-induced brain injury in rats. The neuroprotective effects of Mentat were evaluated against transient global ischemia and reperfusion (I/R)-induced brain injury in rats. Various neurobehavioral and biochemical parameters were assessed, followed by morphologic and histopathologic evaluation of brain tissue to conclude the protective effect of Mentat. Additionally, in vitro antioxidant assays were performed to explore the antioxidant capacity of Mentat and detailed liquid chromatography-mass spectrometry (LC-MS/MS) profiling was carried out to identify the active phytoconstituents responsible for the protective effects of Mentat. Sixty minutes of transient global ischemia followed by 24 h reperfusion (I/R) caused significant alterations in the cognitive and neurologic functions in the ischemia control group (P cerebral infarct area (P protective effects. These findings suggest that Mentat is a neuroprotective agent that may be a useful adjunct in the management of ischemic stroke and its rehabilitation especially with respect to associated memory impairment and other related neurologic conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy.

    Science.gov (United States)

    Wilson, Matthew P; Footitt, Emma J; Papandreou, Apostolos; Uudelepp, Mari-Liis; Pressler, Ronit; Stevenson, Danielle C; Gabriel, Camila; McSweeney, Mel; Baggot, Matthew; Burke, Derek; Stödberg, Tommy; Riney, Kate; Schiff, Manuel; Heales, Simon J R; Mills, Kevin A; Gissen, Paul; Clayton, Peter T; Mills, Philippa B

    2017-09-05

    We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B 6 , affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B 6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.

  6. Preparation of open tubular capillary columns by in situ ring-opening polymerization and their applications in cLC-MS/MS analysis of tryptic digest.

    Science.gov (United States)

    Wang, Hongwei; Yao, Yating; Li, Ya; Ma, Shujuan; Peng, Xiaojun; Ou, Junjie; Ye, Mingliang

    2017-08-01

    An open tubular (OT) column (25 μm i.d.) was prepared by in situ ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxanes (POSS-epoxy) with 4-aminophenyl disulfide (APDS) in a binary porogenic system of ethanol/H 2 O. It was found that porogenic composition played an important role in the formation of OT stationary phases. The ratio of ethanol/H 2 O at 6/1 (v/v) would lead to the fabrication of hybrid monoliths, while the ratio of ethanol/H 2 O at 13/1 (v/v) would result in the synthesis of OT phases. In addition, the effects of precursor content and reaction duration on the thickness of OT stationary phases were investigated. Either lower precursor content or shorter reaction duration would produce thinner layer of OT column. The repeatability of OT columns was evaluated through relative standard deviation (RSD%) with benzene as the analyte. The run-to-run, column-to-column and batch-to-batch repeatabilities were 1.7%, 4.8% and 5.6%, respectively, exhibiting satisfactory repeatability of the OT column. Then tryptic digest of mouse liver proteins was used to evaluate the performance of the resulting OT columns (25 μm i.d. × 2.5 m in length) by cLC-MS/MS analysis, demonstrating their potential in proteome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The influence of effective microorganisms (EM) and yeast on the degradation of strobilurins and carboxamides in leafy vegetables monitored by LC-MS/MS and health risk assessment.

    Science.gov (United States)

    Wołejko, Elżbieta; Łozowicka, Bożena; Kaczyński, Piotr; Jankowska, Magdalena; Piekut, Jolanta

    2016-01-01

    The aim of this study was to determine the behaviour of strobilurin and carbocyamides commonly used in chemical protection of lettuce depending on carefully selected effective microorganisms (EM) and yeast (Y). Additionally, the assessment of the chronic health risk during a 2-week experiment was performed. The statistical method for correlation of physico-chemical parameters and time of degradation for pesticides was applied. In this study, the concentration of azoxystrobin, boscalid, pyraclostrobin and iprodione using liquid chromatography-mass spectrometry (LC-MS/MS) in the matrix of lettuce plants was performed, and there was no case of concentration above maximum residues levels. Before harvest, four fungicides and their mixture with EM (1 % and 10 %) and/or yeast 5 % were applied. In our work, the mixtures of 1%EM + Y and 10%EM + Y both stimulated and inhibited the degradation of the tested active substances. Adding 10%EM to the test substances strongly inhibited the degradation of iprodione, and its concentration decreased by 30 %, and in the case of other test substances, the degradation was approximately 60 %. Moreover, the addition of yeast stimulated the distribution of pyraclostrobin and boscalid in lettuce leaves. The risk assessment for the pesticides ranged from 0.4 to 64.8 % on day 1, but after 14 days, it ranged from 0.0 to 20.9 % for children and adults, respectively. It indicated no risk of adverse effects following exposure to individual pesticides and their mixtures with EM and yeast.

  8. Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

    2015-11-06

    Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

  9. Qualitative analysis of MDR-reversing Anastasia Black (Russian black sweet pepper, Capsicum annuum, Solanaceae) extracts and fractions by HPLC and LC-MS-MS methods.

    Science.gov (United States)

    Schelz, Zsuzsanna; Molnár, Joseph; Fogliano, Vincenzo; Ferracane, Rosalia; Pernice, Rita; Shirataki, Yoshiaki; Motohashi, Noboru

    2006-01-01

    In earlier experiments, the MDR (multidrug resistance)-reversal activities of Anastasia Black (Russian black sweet pepper) extracts had been analysed. Recently, the most effective MDR reversing extracts and fractions have been separated by HPLC (high-performance liquid chromatography, for carotenoids) and LC-MS-MS (HPLC combined with mass spectrometry, for phenolic compounds) methods. As a result of the analytical studies, the following flavonoids had been identified: feruloyl glucopyranoside, quercetin rhamnopyranoside glucopyranoside, luteolin glucopyranoside arabinopyranoside, apigenin glucopyranoside arabinopyranoside, quercetin rhamnopyranoside, luteolin arabinopyranoside diglucopy-ranoside, hesperidine and luteolin glucuronide. According to the literature, the aglycones of these phenolic compounds exhibit MDR-reversal activity in vitro, and the connection between the phenolic content of Anastasia Black and MDR-reversal action was therefore studied by different analytical methods. The results of this study revealed that the identified flavonoids of Anastasia Black may be only partially responsible for the modulation of the MDR of mouse lymphoma cells. Other lipophilic compounds, most probably carotenoids, present in Russian black sweet pepper may act as inhibitors of MDR reversal.

  10. DBS-platform for biomonitoring and toxicokinetics of toxicants: proof of concept using LC-MS/MS analysis of fipronil and its metabolites in blood

    Science.gov (United States)

    Raju, Kanumuri Siva Rama; Taneja, Isha; Rashid, Mamunur; Sonkar, Ashish Kumar; Wahajuddin, Muhammad; Singh, Sheelendra Pratap

    2016-03-01

    A simple, sensitive and high throughput LC-MS/MS method was developed and validated for quantification of fipronil, fipronil sulfone and fipronil desulfinyl in rat and human dried blood spots (DBS). DBS samples were prepared by spiking 10 μl blood on DMPK-C cards followed by drying at room temperature. The whole blood spots were then punched from the card and extracted using acetonitrile. The total chromatographic run time of the method was only 2 min. The lower limit of quantification of the method was 0.1 ng/ml for all the analytes. The method was successfully applied to determine fipronil desulfinyl in DBS samples obtained from its toxicokinetic study in rats following intravenous dose (1 mg/kg). In conclusion, the proposed DBS methodology has significant potential in toxicokinetics and biomonitoring studies of environmental toxicants. This microvolume DBS technique will be an ideal tool for biomonitoring studies, particularly in paediatric population. Small volume requirements, minimally invasive blood sampling method, easier storage and shipping procedure make DBS a suitable technique for such studies. Further, DBS technique contributes towards the principles of 3Rs resulting in significant reduction in the number of rodents used and refinement in sample collection for toxicokinetic studies.

  11. Billfish Tagging

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The SWFSC's constituent-based Billfish Tagging Program began in 1963 and since that time has provided conventional spaghetti type tags and tagging supplies to...

  12. Tag questions Tag questions

    Directory of Open Access Journals (Sweden)

    David Brazil

    2008-04-01

    Full Text Available The so-called 'tag' structures of English have received a lot of attention in language teaching programmes, attention that is not hard to justify when one considers the problems and anxiety they can occasion for many foreign learners. Most teachers one speaks to seem fairly willing to agree, however, that traditional treatments of the topic leave much to be desired. It happens, also, that, when considered collectively, the tags and some related phenomena have a special heoretical interest. For they constitute a field in which it seems essential to bring together insights that derive from the study of several aspects of linguistic organisation, aspects which in some recent work have been held to need distinctive kinds of descriptive category to handle. Traditional treatments have found it necessary to recognise different syntactic types (e.g. 'same polarity' and 'reversed polarity' tags and ifferent intonational treatments ("falling'and 'rising' tag; while the way the communicative significance of the various permutations is described normally requires reference to the expectations they signal regarding the immediately following behaviour of the other party (in the common phrase, 'What kind of answer they expect'. This last consideration places the matter squarely in the arena of recent work on the analysis of interactive discourse. The so-called 'tag' structures of English have received a lot of attention in language teaching programmes, attention that is not hard to justify when one considers the problems and anxiety they can occasion for many foreign learners. Most teachers one speaks to seem fairly willing to agree, however, that traditional treatments of the topic leave much to be desired. It happens, also, that, when considered collectively, the tags and some related phenomena have a special heoretical interest. For they constitute a field in which it seems essential to bring together insights that derive from the study of several aspects

  13. LC-MS based Metabolomics

    DEFF Research Database (Denmark)

    Magdenoska, Olivera

    . The analytical tools applied for analysis of intracellular metabolites should be capable to cope with the large number of metabolites to be analyzed and the complex matrix in the samples. Therefore the combination of separation and detection techniques is commonly applied for analysis of intracellular........ In the studies conducted during this Ph.D. the developed method was used to understand how the genetic manipulations in various organisms, influence the levels of their intracellular metabolites. The method development was divided into three steps: i) optimization of the MS detection, ii) establishment...... of the MS detection aimed to determine multiple reaction monitoring (MRM) transitions of the analytes and to increase the sensitivity by testing different ion-source parameters and collision energies. This resulted in optimized detection of more than 50 intracellular metabolites. During the optimization...

  14. An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Wang, Qian; Xiao, Bing-Xin; Pan, Rui-Le; Liu, Xin-Min; Liao, Yong-Hong; Feng, Li; Cao, Fang-Rui; Chang, Qi

    2015-07-01

    Radix Polygala has a long history of use as a sedative in traditional Chinese medicine and its major ingredients are saponins, which are recognized effective in memory improvement but highly toxic to gastricintestinal mucosa. Polygala saponin hydrolysates (PSH), an alkaline hydrolysis product and also the intestinal metabolites of the saponins, exhibited stronger effects in improving memory of mice and had less toxicity than its original saponins. The present study aims to develop a sensitive LC-MS/MS method for simultaneously determining PSH three major active components, 3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), in rat plasma and apply the method to a pharmacokinetic study. The acidic plasma (100μl) was treated by liquid-liquid extraction with ethyl acetate and reconstituted sample was analyzed on a C18 column eluted with acetonitrile-water (50:50) containing 0.2% formic acid at 0.4ml/min. The mass detection in negative electrospray ionization was used. The ion pairs for multiple reaction monitoring were set at m/z 237.0/103.0, 177.0/116.6 and 679.5/425.3 for TMCA, PMCA and TF, respectively. Their pharmacokinetic profiles were studied in rats after intravenous and oral dose of PSH at 20 and 100mg/kg, respectively. The calibration curves had good linearity (r(2)>0.99) for TMCA, PMCA and TF within the tested concentration ranges. The limits of detection and quantification were 1, 10, 0.5ng/ml and 10.0, 20.0, 1.0ng/ml, respectively. The intra-day and inter-day precisions were less than 18.9% and accuracies between 93.2% and 113.3%, and the extraction recovery ranged from 91.2% to 112.1% for all analytes. The pharmacokinetic study showed that TMCA, PMCA and TF could be rapidly absorbed into the circulation and reached their peak concentrations at about 9.1, 9.0 and 24.0min, respectively. TF had a lower oral bioavailability (2.0%) than TMCA (90.1%) and PMCA (96.5%), but it remained in the body much longer (t1/2,

  15. A semi-automated LC-MS/MS method for the determination of LCI699, a steroid 11β-hydroxylase inhibitor, in human plasma.

    Science.gov (United States)

    Li, Wenkui; Luo, Suyi; Rebello, Sam; Flarakos, Jimmy; Tse, Francis L S

    2014-06-01

    A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500-50.0 ng/mL and 1.00-1,000 ng/mL using 0.0500 mL and 0.100mL, respectively, of human plasma. LCI699 and the internal standard, [M+6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC-MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6mm, 3 μm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500-50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were -13.0 to 2.0% bias and 3.4-19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -11.3 to 8.0% and -7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00-1,000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0-15.0% bias and 7.4-9.2% CV

  16. Validation of an LC-MS/MS method for analysis of anti-diabetic drugs in botanical dietary supplements labeled for blood sugar management.

    Science.gov (United States)

    Ma, Jun; Pawar, Rahul S; Grundel, Erich

    2018-03-01

    We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantitate 14 anti-diabetic, 2 anti-obesity, and 3 cholesterol-lowering drugs in botanical dietary supplements marketed for blood sugar management. Many botanical dietary supplements which carry label statements related to blood sugar management are available over the Internet. Potential adulteration of such dietary supplements with anti-diabetic and other prescription drugs, some of which have been removed from the market due to adverse events, is of concern. No significant matrix effects were observed and mean recoveries of all 19 analytes from a single product matrix were 88 to 113% at spiking concentrations from 500 to 2000 μg/g. Mean recoveries of metformin, phenformin, and sibutramine from matrices prepared from multiple product composites ranged from 93 to 115% at a spiking concentration of 100 μg/g. The relative standard deviations (RSD) (%) of intra-day analyses ranged from 0.2 to 13 for all recovery studies. Eighty dietary supplements obtained in the USA and carrying label statements related to blood sugar management were analyzed using this method and none were found to be adulterated with the above 19 drugs. Two products obtained outside of the USA and known to be adulterated were also analyzed by this method and found to contain phenformin, glibenclamide, and sibutramine. This method provided satisfactory selectivity, linearity, accuracy, precision, and sensitivity for rapid determination of 19 drugs and has broad applicability for the analysis of dietary supplements for possible adulteration with these compounds. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  17. Simultaneous quantification of lenalidomide, ibrutinib and its active metabolite PCI-45227 in rat plasma by LC-MS/MS: application to a pharmacokinetic study.

    Science.gov (United States)

    Veeraraghavan, Sridhar; Viswanadha, Srikant; Thappali, Satheeshmanikandan; Govindarajulu, Babu; Vakkalanka, Swaroopkumar; Rangasamy, Manivannan

    2015-03-25

    Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the development of an analytical method for determination of both drugs in plasma with precision. A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of lenalidomide, ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide, ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 μl rat plasma was carried out by liquid-liquid extraction with ethyl acetate:dichloromethane (90:10) ratio. Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm × 4.6 mm, 5 μm) column under gradient conditions with acetonitrile:0.1% formic acid buffer as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.72-183.20 ng/ml for ibrutinib, 0.76-194.33 ng/ml for PCI-45227 and 1.87-479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227, lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze-thaw and long term stability was evaluated for all the analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Determination and Pharmacokinetic Study of Gentiopicroside, Geniposide, Baicalin, and Swertiamarin in Chinese Herbal Formulae after Oral Administration in Rats by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Chia-Ming Lu

    2014-12-01

    Full Text Available A sensitive and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS method was developed for the simultaneous determination of gentiopicroside, geniposide, baicalin, and swertiamarin in rat plasma. To avoid the stress caused by restraint or anesthesia, a freely moving rat model was used to investigate the pharmacokinetics of herbal medicine after the administration of a traditional Chinese herbal prescription of Long-Dan-Xie-Gan-Tang (10 g/kg, p.o.. Analytes were separated by a C18 column with a gradient system of methanol–water containing 1 mM ammonium acetate with 0.1% formic acid. The linear ranges were 10–500 ng/mL for gentiopicroside, geniposide, and baicalin, and 5–250 ng/mL for swertiamarin in biological samples. The intra- and inter-day precision (relative standard deviation ranged from 0.9% to 11.4% and 0.3% to 14.4%, respectively. The accuracy (relative error was from −6.3% to 10.1% at all quality control levels. The analytical system provided adequate matrix effect and recovery with good precision and accuracy. The pharmacokinetic data demonstrated that the area under concentration-time curve (AUC values of gentiopicroside, geniposide, baicalin, and swertiamarin were 1417 ± 83.8, 302 ± 25.8, 753 ± 86.2, and 2.5 ± 0.1 min µg/mL. The pharmacokinetic profiles provide constructive information for the dosage regimen of herbal medicine and also contribute to elucidate the absorption mechanism in herbal applications and pharmacological experiments.

  19. Detection of carbapenemase activity in Enterobacteriaceae using LC-MS/MS in comparison with the neo-rapid CARB kit using direct visual assessment and colorimetry.

    Science.gov (United States)

    Huber, Charlotte A; Sidjabat, Hanna E; Zowawi, Hosam M; Kvaskoff, David; Reed, Sarah; McNamara, John F; McCarthy, Kate L; Harris, Patrick; Toh, Benjamin; Wailan, Alexander M; Paterson, David L

    2016-12-01

    It has been described that the sensitivity of the Carba NP test may be low in the case of OXA-48-like carbapenamases and mass spectrometry based methods as well as a colorimetry based method have been described as alternatives. We evaluated 84 Enterobacteriaceae isolates including 31 OXA-48-like producing isolates and 13 isolates that produced either an imipenemase (IMP; n=8), New Delhi metallo-β-lactamase (NDM; n=3), or Klebsiella pneumoniae carbapenemase (KPC; n=2), as well as 40 carbapenemase negative Enterobacteriaceae isolates. We used the Neo-Rapid CARB kit, assessing the results with the unaided eye and compared it with a colorimetric approach. Furthermore, we incubated the isolates in growth media with meropenem and measured the remaining meropenem after one and 2h of incubation, respectively, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Whilst all carbapenemase producing isolates with the exception of the OXA-244 producer tested positive for both the Neo-rapid CARB test using the unaided eye or colorimetry, and the 13 isolates producing either IMP, NDM or KPC hydrolysed the meropenem in the media almost completely after 2h of incubation, the 31 OXA-48-like producing isolates exhibited very variable hydrolytic activity when incubated in growth media with meropenem. In our study, the Neo-Rapid CARB test yielded a sensitivity of 98% for both the traditional and the colorimetric approach with a specificity of 95% and 100% respectively. Our results indicate that the Neo-Rapid CARB test may have use for the detection of OXA-48 type carbapenemases and that it may be particularly important to ensure bacterial lysis for the detection of these weaker hydrolysers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Development of SPME-LC-MS method for screening of eight beta-blockers and bronchodilators in plasma and urine samples.

    Science.gov (United States)

    Goryński, Krzysztof; Kiedrowicz, Alicja; Bojko, Barbara

    2016-08-05

    The current work describes the development and validation of a simple, efficient, and fast method using solid phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) for the concomitant measurement of eight beta-blockers and bronchodilators in plasma and urine. The presented assay enables quantitative determination of acebutolol, atenolol, fenoterol, nadolol, pindolol, procaterol, sotalol, and timolol. In this work, samples were prepared on a high-throughput platform using the 96-well plate format of the thin film solid phase microextraction (TFME) system, and a biocompatible extraction phase made of hydrophilic-lipophilic balance particles. Analytes were separated on a pentafluorophenyl column (100mm×2.1mm, 3μm) by gradient elution using an UPLC Nexera coupled with an LCMS-8060 mass spectrometer. The mobile phase consisted of water-acetonitrile (0.1% formic acid) at a flow rate of 0.4mLmin(-1). The linearity of the method was checked within therapeutic blood-plasma concentrations, and shown to adequately reflect typically expected concentrations of future study samples. Post-extraction addition experiments showed that the matrix effect ranged in plasma from 98% for procaterol to 115% for nadolol, and in urine, from 85% for nadolol and pindolol to 119% for atenolol. The method was successfully validated using Food and Drug Administration (FDA) guidelines, and met all acceptance criteria for bioanalytical assays at five concentration levels for all selected drugs. The final protocol can be successfully applied for monitoring concentrations of the selected drugs in both plasma and urine matrices obtained from patients or athletes. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. LC-MS/MS methods for the determination of edaravone and/or taurine in rat plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Tang, Dao-quan; Bian, Ting-ting; Zheng, Xiao-xiao; Li, Ying; Wu, Xiao-wen; Li, Yin-jie; Du, Qian; Jiang, Shui-shi

    2014-09-01

    Three liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3-methyl-1-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid-methanol, isocratic 0.1% formic acid-methanol (90:10) and 0.02% formic acid-methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H](+) 175.1 → 133.0 and [M + H](+) 189.2 → 147.0 for edaravone and its IS, m/z [M - H](-) 124.1 → 80.0 and [M - H](-) 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co-administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration-time curve, mean residence time, half-life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to study the excretion and metabolic interaction of edaravone and taurine in rats.

    Science.gov (United States)

    Tang, Dao-quan; Zheng, Xiao-xiao; Li, Yin-jie; Bian, Ting-ting; Yu, Yan-yan; Du, Qian; Yang, Dong-zhi; Jiang, Shui-shi

    2014-11-01

    In this study, two independent and complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the determination of edaravone or taurine in rat urine, feces and bile after intravenous administration, using 3-methyl-l-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Edaravone was separated on an Agilent Eclipse Plus C18 column (100×2.1 mm, 3.5 μm) using methanol and water (containing 5 mM ammonium formate and 0.02% formic acid) as mobile phase, while taurine was performed on a Waters Atlantis HILIC Silica column (150×2.1 mm, 3 μm) using acetonitrile and water (containing 5mM ammonium formate and 0.2% formic acid) as mobile phase. The mass analysis was performed in a Triple Quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative ionization mode. The optimized mass transition ion pairs (m/z) for quantification were 173.1→92.2 and 187.2→106.0 for edaravone and its IS, 124.1→80.0 and 172.0→80.0 for taurine and its IS, respectively. The validated methods have been successfully applied to the excretion and metabolism interaction study of edaravone and taurine in rats after independent intravenous administration and co-administration with a single dose. The results demonstrated that there were no significant alternations on the metabolism and cumulative excretion rate of edaravone and taurine, implying that the proposed combination therapy was pharmacologically viable. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Determination of Tinopal CBS-X in rice papers and rice noodles using HPLC with fluorescence detection and LC-MS/MS.

    Science.gov (United States)

    Ko, Kyung Yuk; Lee, Chae A; Choi, Jae Chon; Kim, Meehye

    2014-01-01

    To date there have been no reports of methods to determine Tinopal CBS-X. We developed a rapid and simple method to determine the Tinopal CBS-X content in rice noodles and rice papers using HPLC equipped with fluorescence detection. Heating the rice noodles and rice papers to 80°C after adding 75% methanol solution induced the release of Tinopal CBS-X from processed rice products. Tinopal CBS-X was separated using an isocratic mobile phase comprising 50% acetonitrile/water containing 0.4% tetrabutyl ammonium hydrogen sulphate at pH 8.0. The samples suspected to be positive by HPLC analysis were then confirmed by LC-MS/MS analysis. This study also investigated the Tinopal CBS-X content of three rice noodle products and two rice papers. The limits of quantification for rice papers and rice noodles were 1.58 and 1.51 µg kg(-1), respectively, and their correlation curves showed good linearity with r(2) ≥ 0.9997 and ≥ 0.9998, respectively. Moreover, rice papers had recoveries of 70.3-83.3% with precision ranging from 5.0% to 7.9%, whereas rice noodles had slightly lower recoveries of 63.4-78.7% and precisions of 8.5-11.5%. Only one rice noodle product contained Tinopal CBS-X, at around 2.1 mg kg(-1), whereas it was not detected in four other samples. Consequently, Tinopal CBS-X from rice noodles and rice papers can be successfully detected using the developed pre-treatment and ion-pairing HPLC system coupled with fluorescence detection.

  4. Investigation of the profile and kinetics of degradation of rivaroxaban using HPLC, TLC-densitometry and LC/MS/MS: Application to pre-formulation studies

    Directory of Open Access Journals (Sweden)

    Mohamed A. Abdallah

    2015-06-01

    Full Text Available Rivaroxaban (RIVA, an amide group-containing oral anticoagulant was subjected to stress conditions commonly required for the registration of pharmaceuticals: base and acid-catalyzed hydrolysis (0.1 M, 60 °C, 3–6 h, oxidation (10% H2O2, 24 h, photodegradation (300–800 nm, 24 h and thermal decomposition (50 °C, 6 h. Two major degradation products were separated and identified using TLC and LC/MS/MS, respectively. An orthogonal stability-indicating testing protocol (RP-HPLC and NP-TLC-densitometry was developed and validated according to ICH guidelines. Both assays enabled the determination of RIVA in the presence of its degradation products as well as the kinetics of degradation. Determination was carried out over a concentration range of (5.00–50.00 μg/mL and (0.40–12.00 μg/band with an accuracy of (100.81% ± 1.03 and (100.29% ± 1.08 for HPLC and TLC-densitometry, respectively. Results indicated that RIVA was stable towards oxidation, photodegradation and thermal decomposition but extremely sensitive to hydrolysis. Two major degradation products were detected in the case of base-catalyzed hydrolysis while only one degradation product was detected upon acid-catalyzed hydrolysis. This could be attributed to the presence of amide groups in RIVA structure of different stability profiles. The kinetics of hydrolysis was investigated in more detail and the reaction was found to follow the pseudo first order kinetics, as confirmed by the results of both HPLC and TLC-densitometric assays. The applicability of the assay for the determination of RIVA content and dissolution pattern of the innovator product as well as three generic formulations was demonstrated.

  5. SIMPATIQCO: a server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on Orbitrap instruments.

    Science.gov (United States)

    Pichler, Peter; Mazanek, Michael; Dusberger, Frederico; Weilnböck, Lisa; Huber, Christian G; Stingl, Christoph; Luider, Theo M; Straube, Werner L; Köcher, Thomas; Mechtler, Karl

    2012-11-02

    While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC-MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge.

  6. Fast and simultaneous monitoring of organic pollutants in a drinking water treatment plant by a multi-analyte biosensor followed by LC-MS validation.

    Science.gov (United States)

    Rodriguez-Mozaz, Sara; de Alda, Maria J López; Barceló, Damià

    2006-04-15

    This work describes the application of an optical biosensor (RIver ANALyser, RIANA) to the simultaneous analysis of three relevant environmental organic pollutants, namely, the pesticides atrazine and isoproturon and the estrogen estrone, in real water samples. This biosensor is based on an indirect inhibition immunoassay which takes place at a chemically modified optical transducer chip. The spatially resolved modification of the transducer surface allows the simultaneous determination of selected target analytes by means of "total internal reflection fluorescence" (TIRF). The performance of the immunosensor method developed was evaluated against a well accepted traditional method based on solid-phase extraction followed by liquid chromatography-mass spectrometry (LC-MS). The chromatographic method was superior in terms of linearity, sensitivity and accuracy, and the biosensor method in terms of repeatability, speed, cost and automation. The application of both methods in parallel to determine the occurrence and removal of atrazine, isoproturon and estrone throughout the treatment process (sand filtration, ozonation, activated carbon filtration and chlorination) in a waterworks showed an overestimation of results in the case of the biosensor, which was partially attributed to matrix and cross-reactivity effects, in spite of the addition of ovalbumin to the sample to minimize matrix interferences. Based on the comparative performance of both techniques, the biosensor emerges as a suitable tool for fast, simple and automated screening of water pollutants without sample pretreatment. To the author's knowledge, this is the first description of the application of the biosensor RIANA in the multi-analyte configuration to the regular monitoring of pollutants in a waterworks.

  7. Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis.

    Science.gov (United States)

    Zhuo, Yue; Wu, Jian-Lin; Yan, Xiaojing; Guo, Ming-Quan; Liu, Ning; Zhou, Hua; Liu, Liang; Li, Na

    2017-12-19

    Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.

  8. LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

    Science.gov (United States)

    Dong, Xue; Zhou, Shiyue; Mechref, Yehia

    2016-06-01

    Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Simultaneous determination of vitamins A and D3 in dairy products by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

    Science.gov (United States)

    Barakat, I. S. A.; Hammouri, M. K.; Habib, I.

    2015-10-01

    A potential method for simultaneous determination of vitamin A and vitamin D3 (25- hydroxyvitamin D3) in fresh milk samples is addressed. The method is based on combination of high performance liquid chromatography and mass spectrometry during the course of analysis. The method applied for determination of vitamins A and D3 on eighteen (18) different fresh milk samples using liquid chromatography along with tandem -mass spectrometry. The work describes the suitability of the proposed method for the simultaneous determination of both vitamins using LC-MS/MS as a specific and quantitative technique. The vitamins of milk were separated by C18 Thermo gold column(100mm × 4.6mm × 5 μm) with a flow rate of 1ml/min (using an isocratic mobile phase). The method was validated using duplicate analyses, relative recovery experiment, and comparative analysis with control samples. Liquid- liquid extraction was employed as a pre-concentration step with n-hexane - dichloromethane mixture (90%:10%) as an extraction solvent. The molecular ions (m/z) appeared near 286 and 385nm and for the base peaks were appeared near 255 and 355nm for vitamins A and D3. Good correlation coefficients were obtained, 0.9999 for vitamin D3 and 0.9994 for vitamin A. The limit of detection and the limit of quantification were found to be 0.09ng/ml and 0.54ng/ml for vitamin D3 and 0.32ng/ml and 1.8ng/ml and for vitamin A. The proposed method showed excellent recoveries, about 98% for both vitamins A and D3.

  10. Simultaneous determination of multi-mycotoxins in palm kernel cake (PKC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Yibadatihan, S; Jinap, S; Mahyudin, N A

    2014-01-01

    Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.

  11. A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

    Science.gov (United States)

    Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

    2013-01-01

    A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

  12. A validated LC-MS/MS method for the determination of senkyunolide I in dog plasma and its application to a pharmacokinetic and bioavailability studies.

    Science.gov (United States)

    Li, Jin-Qi; Wang, Jia-Feng; Li, Jie; Zhang, Shu-Han; He, Dan; Tong, Rong-Sheng; She, Shu-Ya

    2018-05-01

    Senkyunolide I is one of the major bioactive components in the herbal medicine Ligusticum chuanxiong. The aim of this study was to develop and validate a fast, simple and sensitive LC-MS/MS method for the determination of senkyunolide I in dog plasma. The plasma samples were processed with acetonitrile and separated on a Waters Acquity UPLC BEH C 18 column (50 × 2.1 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid aqueous and acetonitrile was delivered at a flow rate of 0.3 mL min -1 . The detection was achieved in the positive selected reaction monitoring mode with precursor-to-product transitions at m/z 225.1 → 161.1 for senkyunolide I and at m/z 349.1 → 305.1 for an internal standard. The assay was linear over the tested concentration range, from 0.5 ng mL -1 to 1000 ng mL -1 , with a correlation coefficient >0.9992. The mean extraction recovery from dog plasma was within the range of 85.78-93.25%, while the matrix effect of the analyte was within the range of 98.23-108.89%. The intra- and inter-day precisions (RSD) were dogs. The results demonstrated that (a) senkyunolide I showed short elimination half-life (dog, (b) its oral bioavailability was >40% and (c) senkyunolide I showed dose-independent pharmacokinetic profiles in dog plasma over the dose range of 1-50 mg kg -1 . Copyright © 2018 John Wiley & Sons, Ltd.

  13. A rapid MCM-41 dispersive micro-solid phase extraction coupled with LC/MS/MS for quantification of ketoconazole and voriconazole in biological fluids.

    Science.gov (United States)

    Yahaya, Noorfatimah; Sanagi, Mohd Marsin; Abd Aziz, Noorizan; Wan Ibrahim, Wan Aini; Nur, Hadi; Loh, Saw Hong; Kamaruzaman, Sazlinda

    2017-02-01

    A rapid dispersive micro-solid phase extraction (D-μ-SPE) combined with LC/MS/MS method was developed and validated for the determination of ketoconazole and voriconazole in human urine and plasma samples. Synthesized mesoporous silica MCM-41 was used as sorbent in d-μ-SPE of the azole compounds from biological fluids. Important D-μ-SPE parameters, namely type desorption solvent, extraction time, sample pH, salt addition, desorption time, amount of sorbent and sample volume were optimized. Liquid chromatographic separations were carried out on a Zorbax SB-C 18 column (2.1 × 100 mm, 3.5 μm), using a mobile phase of acetonitrile-0.05% formic acid in 5 mm ammonium acetate buffer (70:30, v/v). A triple quadrupole mass spectrometer with positive ionization mode was used for the determination of target analytes. Under the optimized conditions, the calibration curves showed good linearity in the range of 0.1-10,000 μg/L with satisfactory limit of detection (≤0.06 μg/L) and limit of quantitation (≤0.3 μg/L). The proposed method also showed acceptable intra- and inter-day precisions for ketoconazole and voriconazole from urine and human plasma with RSD ≤16.5% and good relative recoveries in the range 84.3-114.8%. The MCM-41-D-μ-SPE method proved to be rapid and simple and requires a small volume of organic solvent (200 μL); thus it is advantageous for routine drug analysis. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Determination of pharmaceuticals in sewage sludge by pressurized liquid extraction (PLE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Radjenović, J; Jelić, A; Petrović, M; Barceló, D

    2009-03-01

    In this study, we aimed at optimizing a sensitive and reliable method for a simultaneous determination of 31 pharmaceuticals belonging to predominant therapeutic classes identified in different types of sewage sludge proceeding from conventional and advanced wastewater treatment. Freeze-dried sewage sludge was extracted by pressurized liquid extraction technique using accelerated solvent extractor Dionex 300. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid phase extraction was introduced in the method as a clean-up step. The entire method was validated for linearity, precision, accuracy, and method detection limits (MDLs). The method turned out to be specific, sensitive, and reliable for the analysis of sludge of different composition, type, and retention time in the process. The developed sample preparation protocol and previously published method for LC-MS/MS analysis (Gros et al., Talanta 70:678-690, 2006) were successfully applied to monitor the target pharmaceuticals in different types of sewage sludge, i.e., primary sludge, secondary sludge, treated sludge, and sludge proceeding from pilot-scale membrane bioreactors (MBRs) operating in parallel to the conventional activated sludge treatment. Among the investigated pharmaceuticals, 20 were detected in the sludge proceeding from full-scale installations, whereas the MBR sludge concentrations were below MDLs for several compounds. The highest concentrations were recorded for treated and primary sludge. For example, the mean concentration of ibuprofen in the digested sludge was 299.3 +/- 70.9 ng g(-1) dw, whereas in the primary sludge, it was enriched up to 741.1 ng g(-1) dw. Other pharmaceuticals detected at relatively high concentrations were diclofenac, erythromycin, glibenclamide, ketoprofen, ofloxacin, azithromycin (up to 380.7, 164.2, 190.7, 336.3, 454.7, 299.6 ng g(-1) dw in the primary sludge, respectively), gemfibrozil, loratidine, and fluoxetine (up

  15. Measurement of tissue azithromycin levels in self-collected vaginal swabs post treatment using liquid chromatography and tandem mass spectrometry (LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Lenka A Vodstrcil

    Full Text Available Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure. One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment.Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11 or over 9 days (N = 10. Azithromycin concentrations were normalised to an internal standard (leucine enkephalin, and the bulk lipid species phosphatidylcholine [PC(34:1], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1, and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens.Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg. In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg, and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg on day 9.Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting that poor antibiotic absorption is unlikely to be an explanation for treatment failure.

  16. Proteomic analysis of Pinus radiata needles: 2-DE map and protein identification by LC/MS/MS and substitution-tolerant database searching.

    Science.gov (United States)

    Valledor, Luis; Castillejo, Maria A; Lenz, Christof; Rodríguez, Roberto; Cañal, Maria J; Jorrín, Jesús

    2008-07-01

    Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

  17. LC-MS/MS Determination and Pharmacokinetic Study of Pedunculoside in Rat Plasma after Oral Administration of Pedunculoside and Ilex rotunda Extract

    Directory of Open Access Journals (Sweden)

    Waiou Zhao

    2015-05-01

    Full Text Available Ilex rotunda is widely used to treat many disorders as a traditional Chinese medicine (TCM containing 4%–5% pedunculoside (PDC. A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS was developed and validated to determine PDC in rat plasma by using 3β,19α-dihydroxyurs-12-en-28-oic acid 28-β-D-glucopyranosyl ester (DEOG as an internal standard. The analytes were extracted by protein precipitation and eluted on a C18 chromatography column using a mobile phase of methanol–H2O (70:30, v/v delivered at a flow rate of 0.6 mL/min. Detection was performed using positive ion electrospray ionization in multiple reaction monitoring modes. The assay was linear over the concentration range of 0.60 ng/mL to 200 ng/mL, with a quantification limit of 0.60 ng/mL. Intra-day and inter-day precisions (%RSD ranged from 2.12 to 9.51 for PDC, whereas the accuracy was within −7.83%~9.40%. The validated method was successfully applied to the pharmacokinetic study of PDC in rat plasma after oral administration of pure PDC and Ilex rotunda extract (IRE. Pharmacokinetic parameters of PDC in IRE, such as Cmax, AUC0–t, AUC0–∞, t1/2z, and CLz/F, statistically differed from those of the pure monomer (p < 0.01. However, Tmax and MRT showed no significant differences between the two groups. Results suggested that other coexisting components in IRE may decrease the absorption of PDC. Compound-compound interactions between PDC and other herbal extract components can alter the pharmacokinetic behavior of PDC. The study will be helpful in providing references for understanding the action mechanism and clinical application of Ilex rotunda.

  18. Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum

    Directory of Open Access Journals (Sweden)

    Maryam Khaksari

    2017-04-01

    Full Text Available Two separate liquid chromatography (LC-mass spectrometry (MS methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B1, B2, B3 (nicotinamide, B5, B6 (pyridoxine, B7, B9 and B12 while the fat-soluble vitamin method detected vitamins A, D3, 25(OHD3, E and K1. These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD and limits of quantification (LOQ are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

  19. Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum.

    Science.gov (United States)

    Khaksari, Maryam; Mazzoleni, Lynn R; Ruan, Chunhai; Kennedy, Robert T; Minerick, Adrienne R

    2017-04-01

    Two separate liquid chromatography (LC)-mass spectrometry (MS) methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B 1 , B 2 , B 3 (nicotinamide), B 5 , B 6 (pyridoxine), B 7 , B 9 and B 12 while the fat-soluble vitamin method detected vitamins A, D 3 , 25(OH)D 3, E and K 1 . These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD) and limits of quantification (LOQ) are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

  20. Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows

    Science.gov (United States)

    Gokce, Emine; Shuford, Christopher M.; Franck, William L.; Dean, Ralph A.; Muddiman, David C.

    2011-12-01

    Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with

  1. Simultaneous determination of neonicotinoid insecticides and insect growth regulators residues in honey using LC-MS/MS with anion exchanger-disposable pipette extraction.

    Science.gov (United States)

    Song, Shiming; Zhang, Cuifang; Chen, Zhaojie; He, Fengmei; Wei, Jie; Tan, Huihua; Li, Xuesheng

    2018-07-06

    In this study, we developed an anion exchanger-disposable pipette extraction (DPX) method to detect the residual concentrations of eight neonicotinoid insecticides (dinotefuran, acetamiprid, clothianidin, thiacloprid, imidachloprid, imidaclothiz, nitenpyram, and thiamethoxam) and eight insect growth regulators (IGRs; triflumuron, cyromazine, buprofezin, methoxyfenozide, tebufenozide, chromafenozide, fenoxycarb, and RH 5849) in Chinese honey samples collected from different floral sources and different geographical regions using liquid chromatography tandem mass spectrometry (LC-MS/MS). QAE Sephadex A-25 was used as the anion exchanger in the DPX column for the purification and cleanup of honey samples. Analytes were eluted with a mixture of acetonitrile and 0.1 M HCl, and the elution was subjected to LC analysis. This method was thoroughly validated for its reproducibility, linearity, trueness, and recovery. Satisfactory recovery of pesticides was obtained ranging from 72% to 111% with intraday RSDs (n = 5) of 1%-10%. High linearity (R 2  ≥ 0.9987) was observed for all 16 pesticides. Limits of detection and quantification for all 16 compounds ranged from 0.3 to 3 μg/kg and from 1 to 10 μg/kg, respectively. Pesticide residues (9-113 μg/kg) were found in Chinese honey samples. The anion exchanger-DPX method was effective for removing sugars and retaining target analytes. Moreover, this method was highly reliable and sensitive for detecting neonicotinoids and IGRs in different floral sources of honey and will be applicable to matrixes with high sugar content. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Food Safety is an Important Public Health Issue: Chloramphenicol Residues Determination by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) in honey.

    Science.gov (United States)

    Krivohlavek, Adela; Žuntar, Irena; Ivešić, Martina; Andačić, Ivana Mandić; Šikić, Sandra

    2014-12-01

    Honey is used for nutritional, medicinal and industrial purposes and antibiotic residues may harm its quality and constitute a danger to human health. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in European Union (EU) because of its many serious side effects (e.g. aplastic anaemia, grey syndrome, severe bone marrow depression and hypersensitivity). The aim of this study was to facilitate analyses of the quality and safety of Croatian honey distributed to whole European Union market; an assessment that has not previously been made. CAP in honey was qualifying and quantifying by validated liquid chromatography tandem mass spectrometry with negative electrospray ionisation method (LC-MS/MS). The target antibiotic was separated on chromatographic column Zorbax SB C18 (150 mm x 2.1 mm, 3.5 μm) with a gradient elution using acetonitrile - 0.1% formic acid mobile phase at a flow rate of 0.3 mL/min, with column temperature 35°C for CAP and 5D-CAP as internal standard. Homogenised honey samples were diluted with acetate buffer solution and extracted on Oasis Hydrophilic-Lipophilic-Balanced (HLB) sorbents. The method was used to analyse 280 domestic honey samples collected throughout Croatia between 2005.-2013. Recoveries of the method for real (acacia, chestnut, linden and flower) honey samples were 102% with RSD 8.4%. The value CCα and CCβ were 0.09 and 0.12 μg/kg, respectively. Results showed only three subsequent positive detections (1.1%) of CAP in honey. Analysed honey samples from Croatia showed good quality and safety what is the one of the main objective in consumer health policy in EU.

  3. Development of an HPLC fluorescence method for determination of boldine in plasma, bile and urine of rats and identification of its major metabolites by LC-MS/MS.

    Science.gov (United States)

    Hroch, Miloš; Mičuda, Stanislav; Cermanová, Jolana; Chládek, Jaroslav; Tomšík, Pavel

    2013-10-01

    Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid-liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core-shell column (Kinetex PFP, 150×3mm, 2.6μm) in gradient elution mode with solvent system consisting of an acetonitrile-ammonium formate buffer (5mM, pH=