Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS.

Science.gov (United States)

Montoro, Paola; Maldini, Mariateresa; Russo, Mariateresa; Postorino, Santo; Piacente, Sonia; Pizza, Cosimo

2011-02-20

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria). Copyright © 2010 Elsevier B.V. All rights reserved.

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.

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.

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.

Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

Science.gov (United States)

Zhang, Yanhua; Dufield, Dawn; Klover, Jon; Li, Wenlin; Szekely-Klepser, Gabriella; Lepsy, Christopher; Sadagopan, Nalini

2009-02-15

An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.

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...

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.

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

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine.

Science.gov (United States)

Jeon, Byoung Wook; Yoo, Hye Hyun; Jeong, Eun Sook; Kim, Ho Jun; Jin, Changbae; Kim, Dong Hyun; Lee, Jaeick

2011-09-01

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.

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.

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

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.

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.

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.

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

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

A method for the determination of acrylamide in bakery products using ion trap LC-ESI-MS/MS.

Science.gov (United States)

Claus, Achim; Weisz, Georg M; Kammerer, Dietmar R; Carle, Reinhold; Schieber, Andreas

2005-10-01

Acrylamide levels of bakery products, e. g., bread and bread rolls, are usually below 100 microg/kg , often even below 50 microg/kg. Therefore, usual analytical methods which have an LOQ greater, not dbl equals 25 microg/kg are not sensitive enough for detailed investigations on acrylamide formation within these commodities. An improved method for trace level determination of acrylamide in bakery products was developed using ion trap LC-ESI-MS/MS. Samples were divided into crumbs and crusts to achieve an initial concentration by removing the crumbs since these are devoid of acrylamide. After sample extraction and clean-up using multimode SPE cartridges, further analyte enrichment was accomplished by solid-phase-supported liquid-liquid extraction with ethyl acetate prior to LC-MS/MS analysis. The method was evaluated using bread, bread rolls, alkali-baked bread rolls, and toast. LOQ was calculated from the confidence interval of the calibration curve and found to be 1.7 ng/mL, corresponding to 17 microg/kg of product. When crumbs and crusts were separated, an LOQ of 10.2 microg/kg of bakery product could be obtained. As demonstrated in preliminary comparative analyses, accuracy of the method met the requirements for determination of trace level acrylamide formation in bakery products. Mean recovery was 102.4% (CV 4.5%), intermediate reproducibility revealed a CV of 2.1%, and a repeatability of CV 6.0%.

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.

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

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.

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.

Measurement of methionine level with the LC-ESI-MS/MS method in schizophrenic patients.

Science.gov (United States)

Kulaksizoglu, S; Kulaksizoglu, B; Ellidag, H Y; Eren, E; Yilmaz, N; Baykal, A

2016-01-01

The purpose of this study was to evaluate plasma methionine levels by using liquid chromatography electrospray ionization-tandem mass spectroscopy (LC-ESI-MS/MS) in schizophrenic patients. A twelve-point standard graph was drawn, and the recovery rate, the intra-day and inter-day coefficients of variation (CV), the limit of detection (LOD), and the limit of quantification (LOQ) were evaluated. The y and R2 values of the standard graph equation were determined as 0.011x + 0.0179 and 0.9989, respectively, and the graph remained linear until the 200 µmol/l level. The intra-day coefficients of variation of the samples (n = 10) containing 8, 28, and 58 µmol/l methionine were determined as 2.68, 3.10, and 3.79%, respectively; while their inter-day coefficients of variation were determined as 2.98, 3.19, and 3.84%. The LOD and LOQ values were determined as 0.04 and 0.1 µmol/l, respectively, while the mean recovery rates were determined as 101.7 and 99.3%. Plasma methionine values were measured as 21.5 (19.5-24,6) µmol/l for the patient group, 17.8 (16.3-20.1) µmol/l for the control group, and the difference between the two groups was statistically significant (p = 0.03). LC-ESI-MS/MS method represents a fairly sensitive, economic, and rapid analysis that requires very little sample and is suitable for measuring methionine levels in schizophrenic patients.

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.

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.

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.

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...

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.

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.

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.

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...

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/MS

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...

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.

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.

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.

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.

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...

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

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.

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...

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.

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.

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.

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...

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.

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...

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.

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

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.

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...

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.

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.

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.

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.

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...

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.

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.

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.

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.

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.

Comparison of LC-ESI-MS and GC-MS for the Analysis of a Synthetic Tabun Sample

National Research Council Canada - National Science Library

D'Agostino, Paul

2003-01-01

Packed capillary LC-ESI-MS and capillary column GC-MS were compared for the analysis of a synthetic tabun sample as each method has advantages for the analysis of samples containing chemical warfare...

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).

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.

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.

Evaluation of various QuEChERS based methods for the analysis of herbicides and other commonly used pesticides in polished rice by LC-MS/MS.

Science.gov (United States)

Pareja, Lucía; Cesio, Verónica; Heinzen, Horacio; Fernández-Alba, Amadeo R

2011-02-15

Four different extraction and clean-up protocols based on the QuEChERS method were compared for the development of an optimized sample preparation procedure for the multiresidue analysis of 16 commonly applied herbicides in rice crops using LC-QqQ/MS. Additionally the methods were evaluated for the analysis of 26 insecticides and fungicides currently used in rice crops. The methods comprise, in general, the hydratation of the sample with water followed by the extraction with acetonitrile, phase separation with the addition of different salts and finally a clean-up step with various sorbents. Matrix effects were evaluated for the 4 studied methods using LC-QqQ/MS. Additionally LC-TOF/MS was used to compare the co-extractants obtained with the four assayed methodologies. Thirty-six pesticides presented good performance with recoveries in the range 70-120% and relative standard deviations below 20% using 7.5 g of milled polished rice and the buffered acetate QuEChERS method without clean-up at both fortification levels: 10 and 300 μg kg(-1). The other six pesticides presented low recovery rates, nevertheless all these analytes could be analyzed with at least one of the other three studied procedures. Copyright © 2010. Published by Elsevier B.V.

Identification, characterization and quantification of new impurities by LC-ESI/MS/MS and LC-UV methods in rivastigmine tartrate active pharmaceutical ingredient.

Science.gov (United States)

Thomas, Saji; Shandilya, Sanjeev; Bharati, Amber; Paul, Saroj Kumar; Agarwal, Ashutosh; Mathela, Chandra S

2012-01-05

Six impurities were detected at trace level in rivastigmine tartrate drug substance by a newly developed high performance liquid chromatography method. Three impurities were characterized rapidly and three impurities were found to be unknown. The unknown impurities were enriched and identified with a combination of semi-preparative HPLC and LC/MS/MS techniques. Proposed structures were further confirmed by characterization using NMR, FT-IR, and EA techniques of impurity standards. Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 3-[1-(dimethylamino)ethyl]phenyl N-ethyl-N-methyl carbamate N-oxide, ethyl-methyl-carbamic acid 4-(1-dimethylamino-ethyl)-phenyl ester and ethyl-methyl-carbamic acid 2-(1-dimethylamino-ethyl)-phenyl ester. A plausible mechanism for the formation of these impurities is also proposed. The method was validated according to ICH guidelines for fourteen impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. Regression analysis showed correlation coefficient value greater than 0.999 for rivastigmine tartrate and its impurities. Accuracy of the method was established based on the recovery obtained between 93.41 and 113.33% for all impurities. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

A new LC-ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

International Nuclear Information System (INIS)

Jauregui, Olga; Sierra, Adriana Y.; Carrasco, Patricia; Gratacos, Esther; Hegardt, Fausto G.; Casals, Nuria

2007-01-01

The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L -1 for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity

A new LC-ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

Energy Technology Data Exchange (ETDEWEB)

Jauregui, Olga [Scientific and Technical Services, University of Barcelona (Spain); Sierra, Adriana Y.; Carrasco, Patricia; Gratacos, Esther [Molecular and Cellular Unit, School of Health Sciences, International University of Catalonia (Spain); Hegardt, Fausto G. [Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy (Spain); Casals, Nuria [Molecular and Cellular Unit, School of Health Sciences, International University of Catalonia (Spain)], E-mail: ncasals@csc.uic.es

2007-09-05

The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L{sup -1} for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity.

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.

Establishment of a method for determination of arsenic species in seafood by LC-ICP-MS.

Science.gov (United States)

Zmozinski, Ariane V; Llorente-Mirandes, Toni; López-Sánchez, José F; da Silva, Márcia M

2015-04-15

An analytical method for determination of arsenic species (inorganic arsenic (iAs), methylarsonic acid (MA), dimethylarsinic acid (DMA), arsenobetaine (AB), trimethylarsine oxide (TMAO) and arsenocholine (AC)) in Brazilian and Spanish seafood samples is reported. This study was focused on extraction and quantification of inorganic arsenic (iAs), the most toxic form. Arsenic speciation was carried out via LC with both anionic and cationic exchange with ICP-MS detection (LC-ICP-MS). The detection limits (LODs), quantification limits (LOQs), precision and accuracy for arsenic species were established. The proposed method was evaluated using eight reference materials (RMs). Arsenobetaine was the main species found in all samples. The total and iAs concentration in 22 seafood samples and RMs ranged between 0.27-35.2 and 0.02-0.71 mg As kg(-1), respectively. Recoveries ranging from 100% to 106% for iAs, based on spikes, were achieved. The proposed method provides reliable iAs data for future risk assessment analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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.

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.

Development and validation of a method for the analysis of Ochratoxin A in roasted coffee by liquid chromatography/electrospray-mass spectrometry in Tandem (LC/ESI-MS/MS

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Raquel D. C. C. Bandeira

2012-01-01

Full Text Available A method using LC/ESI-MS/MS for the quantitative analysis of Ochratoxin A in roasted coffee was described. Linearity was demonstrated (r = 0.9175. The limits of detection and quantification were 1.0 and 3.0 ng g-1, respectively. Trueness, repeatability and intermediate precision values were 89.0-108.8%; 2.4-13.7%; 12.5-17.8%, respectively. To the best of our knowledge, this is the first report in which Ochratoxin A in roasted coffee is analysed by LC/ESI-MS/MS, contributing to the field of mycotoxin analysis, and it will be used for future production of Certified Reference Material.

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.

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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.

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

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)....

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

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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.

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

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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.

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

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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.

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

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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.

LC-MS/MS method for simultaneous determination of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin in serum of multiple myeloma patients.

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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.

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

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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.

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

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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.

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

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

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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.

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

An impulse-driven liquid-droplet deposition interface for combining LC with MALDI MS and MS/MS.

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Young, J Bryce; Li, Liang

2006-03-01

A simple and robust impulse-driven droplet deposition system was developed for off-line liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS). The system uses a solenoid operated with a pulsed voltage power supply to generate impulses that dislodge the hanging droplets from the LC outlet directly to a MALDI plate via a momentum transfer process. There is no contact between the LC outlet and the collection surface. The system is compatible with solvents of varying polarity and viscosity, and accommodates the use of hydrophobic and hydrophilic MALDI matrices. MALDI spots are produced on-line with the separation, and do not require further processing before MS analysis. It is shown that high quality MALDI spectra from 5 fmol of pyro-Glu-fibrinopeptide deposition after LC separation could be obtained using the device, indicating that there was no sample loss in the interface. To demonstrate the analytical performance of the system as a proteome analysis tool, a range of BSA digest concentrations covering about 3 orders of magnitude, from 5 fmol to 1 pmol, were analyzed by LC-MALDI quadrupole time-of-flight MS, yielding 6 and 57% amino acid sequence coverage, respectively. In addition, a complex protein mixture of an E. coli cell extract was tryptically digested and analyzed by LC-MALDI MS, resulting in the detection of a total of 409 unique peptides from 100 fractions of 15-s intervals.

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

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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.

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

Matrix Effect Evaluation and Method Validation of Azoxystrobin and Difenoconazole Residues in Red Flesh Dragon Fruit (Hylocereus polyrhizus) Matrices Using QuEChERS Sample Preparation Methods Followed by LC-MS/MS Determination.

Science.gov (United States)

Noegrohati, Sri; Hernadi, Elan; Asviastuti, Syanti

2018-03-30

Production of red flesh dragon fruit (Hylocereus polyrhizus) was hampered by Colletotrichum sp. Pre-harvest application of azoxystrobin and difenoconazole mixture is recommended, therefore, a selective and sensitive multi residues analytical method is required in monitoring and evaluating the commodity's safety. LC-MS/MS is a well-established analytical technique for qualitative and quantitative determination in complex matrices. However, this method is hurdled by co-eluted coextractives interferences. This work evaluated the pH effect of acetate buffered and citrate buffered QuEChERS sample preparation in their effectiveness of matrix effect reduction. Citrate buffered QuEChERS proved to produce clean final extract with relative matrix effect 0.4%-0.7%. Method validation of the selected sample preparation followed by LC-MS/MS for whole dragon fruit, flesh and peel matrices fortified at 0.005, 0.01, 0.1 and 1 g/g showed recoveries 75%-119%, intermediate repeatability 2%-14%. The expanded uncertainties were 7%-48%. Based on the international acceptance criteria, this method is valid.

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.

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.

Metabolism of the new psychoactive substances N,N-diallyltryptamine (DALT) and 5-methoxy-DALT and their detectability in urine by GC-MS, LC-MSn, and LC-HR-MS-MS.

Science.gov (United States)

Michely, Julian A; Helfer, Andreas G; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

2015-10-01

N,N-Diallyltryptamine (DALT) and 5-methoxy-DALT (5-MeO-DALT) are synthetic tryptamine derivatives commonly referred to as so-called new psychoactive substances (NPS). They have psychoactive effects that may be similar to those of other tryptamine derivatives. The objectives of this work were to study the metabolic fate and detectability, in urine, of DALT and 5-MeO-DALT. For metabolism studies, rat urine obtained after high-dose administration was prepared by precipitation and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HR-MS-MS). On the basis of the metabolites identified, several aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations thereof are proposed as the main metabolic pathways for both compounds. O-Demethylation of 5-MeO-DALT was also observed, in addition to extensive glucuronidation or sulfation of both compounds after phase I transformation. The cytochrome P450 (CYP) isoenzymes predominantly involved in DALT metabolism were CYP2C19, CYP2D6, and CYP3A4; those mainly involved in 5-MeO-DALT metabolism were CYP1A2, CYP2C19, CYP2D6, and CYP3A4. For detectability studies, rat urine was screened by GC-MS, LC-MS(n), and LC-HR-MS-MS after administration of low doses. LC-MS(n) and LC-HR-MS-MS were deemed suitable for monitoring consumption of both compounds. The most abundant targets were a ring hydroxy metabolite of DALT, the N,O-bis-dealkyl metabolite of 5-MeO-DALT, and their glucuronides. GC-MS enabled screening of DALT by use of its main metabolites only.

A novel chiral GC/MS method for the analysis of fluoxetine and norfluoxetine enantiomers in biological fluids

Directory of Open Access Journals (Sweden)

Janet Mifsud

2012-01-01

Full Text Available Aims: A novel robust chiral gas chromatographic/mass spectrometric (GC/MS method for the separation and measurement of fluoxetine and norfluoxetine enantiomers in urine and plasma was developed. Materials and Methods: The drug was extracted from the samples by a liquid-liquid technique, using chloroform, and the enantiomers were separated and measured on a chiral gas chromatographic column (HYDRODEX β-6TBDM®, 0.25 μm × 0.25 mm × 50 m. GC/MS instrumentation was used for the acquisition of data in the electron impact selective-ion monitoring mode. Results: The ions chosen were of a mass-to-charge ratio (m/z exactly equal to 44 units, in order to measure fluoxetine enantiomers, 134 units in order to measure norfluoxetine enantiomers, and 58 units in order to measure diphenhydramine, the internal standard. The method was found to be linear and reproducible in the 50-500 ng/mL concentration range for both urine samples and plasma samples and for both fluoxetine and norfluoxetine, with correlation coefficients ranging between 0.994 and 0.997. Conclusions: This methodology has an enormous potential for application in pharmacokinetic studies of the enantiomers of fluoxetine

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).

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.

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.

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS

DEFF Research Database (Denmark)

Dahl, Sandra Rinne; Kleiveland, Charlotte Ramstad; Kassem, Moustapha

2008-01-01

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k...

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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...

Multiresidue method for pesticide residue analysis in food of animal and plant origin based on GC or LC and MS or MS/MS.

Science.gov (United States)

Muñoz, Eva; Muñoz, Gloria; Pineda, Laura; Serrahima, Eulalia; Centrich, Francesc

2012-01-01

A multiresidue method based on GC or LC and MS or MS/MS for the determination of 204 pesticides in diverse food matrixes of animal and plant origin is described. The method can include different stages of cleanup according to the chemical characteristics of each sample. Samples were extracted using accelerated solvent extraction. Those with a high fat content or that contained chlorophyll required further purification by gel permeation chromatography and/or SPE (ENVI-Carb). The methodology developed here was fully validated; the LOQs for the 204 pesticides are presented. The LOQ values lie between 0.01 to 0.02 mg/kg. However, in some cases, mainly in baby food, they were as low as 0.003 mg/kg, thereby meeting European Union requirements on maximum residue levels for pesticides, as outlined in European regulation 396/2005 and the Commission Directive 2003/13/EC. The procedure has been accredited for a wide scope of pesticides and matrixes by the Spanish Accreditation Body (ENAC) following ISO/IEC 17025:2005, as outlined in ENAC technical note NT-19.

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).

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.

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.

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.

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.

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

Investigation of a Quantitative Method for the Analysis of Chiral Monoterpenes in White Wine by HS-SPME-MDGC-MS of Different Wine Matrices

Directory of Open Access Journals (Sweden)

Mei Song

2015-04-01

Full Text Available A valid quantitative method for the analysis of chiral monoterpenes in white wine using head-space solid phase micro-extraction-MDGC-MS (HS-SPME-MDGC-MS with stable isotope dilution analysis was established. Fifteen compounds: (S-(−-limonene, (R-(+-limonene, (+-(2R,4S-cis-rose oxide, (−-(2S,4R-cis-rose oxide, (−-(2R,4R-trans-rose oxide, (+-(2S,4S-cis-rose oxide, furanoid (+-trans-linalool oxide, furanoid (−-cis-linalool oxide, furanoid (−-trans-linalool oxide, furanoid (+-cis-linalool oxide, (−-linalool, (+-linalool, (−-α-terpineol, (+-α-terpineol and (R-(+-β-citronellol were quantified. Two calibration curves were plotted for different wine bases, with varying residual sugar content, and three calibration curves for each wine base were investigated during a single fiber’s lifetime. This was needed as both sugar content and fiber life impacted the quantification of the chiral terpenes. The chiral monoterpene content of six Pinot Gris wines and six Riesling wines was then analyzed using the verified method. ANOVA with Tukey multiple comparisons showed significant differences for each of the detected chiral compounds in all 12 wines. PCA score plots showed a clear separation between the Riesling and Pinot Gris wines. Riesling wines had greater number of chiral terpenes in comparison to Pinot Gris wines. Beyond total terpene content it is possible that the differences in chiral terpene content may be driving the aromatic differences in white wines.

"Heart-cut" bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system.

Science.gov (United States)

Battisti, Umberto M; Citti, Cinzia; Larini, Martina; Ciccarella, Giuseppe; Stasiak, Natalia; Troisi, Luigino; Braghiroli, Daniela; Parenti, Carlo; Zoli, Michele; Cannazza, Giuseppe

2016-04-22

A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug

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.

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.

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.

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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.

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.

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.

Reduction of Solvent Effect in Reverse Phase Gradient Elution LC-ICP-MS

Energy Technology Data Exchange (ETDEWEB)

Sullivan, Patrick Allen [Iowa State Univ., Ames, IA (United States)

2005-12-17

Quantification in liquid chromatography (LC) is becoming very important as more researchers are using LC, not as an analytical tool itself, but as a sample introduction system for other analytical instruments. The ability of LC instrumentation to quickly separate a wide variety of compounds makes it ideal for analysis of complex mixtures. For elemental speciation, LC is joined with inductively coupled plasma mass spectrometry (ICP-MS) to separate and detect metal-containing, organic compounds in complex mixtures, such as biological samples. Often, the solvent gradients required to perform complex separations will cause matrix effects within the plasma. This limits the sensitivity of the ICP-MS and the quantification methods available for use in such analyses. Traditionally, isotope dilution has been the method of choice for LC-ICP-MS quantification. The use of naturally abundant isotopes of a single element in quantification corrects for most of the effects that LC solvent gradients produce within the plasma. However, not all elements of interest in speciation studies have multiple naturally occurring isotopes; and polyatomic interferences for a given isotope can develop within the plasma, depending on the solvent matrix. This is the case for reverse phase LC separations, where increasing amounts of organic solvent are required. For such separations, an alternative to isotope dilution for quantification would be is needed. To this end, a new method was developed using the Apex-Q desolvation system (ESI, Omaha, NE) to couple LC instrumentation with an ICP-MS device. The desolvation power of the system allowed greater concentrations of methanol to be introduced to the plasma prior to destabilization than with direct methanol injection into the plasma. Studies were performed, using simulated and actual linear methanol gradients, to find analyte-internal standard (AIS) pairs whose ratio remains consistent (deviations {+-} 10%) over methanol concentration ranges of 5

Metabolic profiling of Vitex agnus castus leaves, fruits and sprouts: analysis by LC/ESI/(QqQ)MS and (HR) LC/ESI/(Orbitrap)/MS n.

Science.gov (United States)

Mari, Angela; Montoro, Paola; D'Urso, Gilda; Macchia, Mario; Pizza, Cosimo; Piacente, Sonia

2015-01-01

Food supplements based on Vitex agnus castus L. (Verbenaceae) fruits, also known as chasteberry, are routinely used by women against somatic and psychic premenstrual symptoms such as depression, sadness or irritability. With the aim of highlighting the differences in the chemical profiles of cultivated fruits and different parts of wild plants (fruits, leaves and sprouts) of V. agnus castus, a method concerning with the quali-quantitative study of the derived hydroalcoholic extracts was carried out by using high-performance liquid chromatography coupled to electrospray negative ionization Orbitrap multicollisional high resolution mass spectrometry (LC/ESI/(Orbitrap)MS(n)) and high-performance liquid chromatography coupled to electrospray negative ionization triple quadrupole tandem mass spectrometry (LC/ESI/(QqQ)MS) in multiple reaction monitoring (MRM) mode. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

An analytical approach based on ESI-MS, LC-MS and PCA for the quali-quantitative analysis of cycloartane derivatives in Astragalus spp.

Science.gov (United States)

Napolitano, Assunta; Akay, Seref; Mari, Angela; Bedir, Erdal; Pizza, Cosimo; Piacente, Sonia

2013-11-01

Astragalus species are widely used as health foods and dietary supplements, as well as drugs in traditional medicine. To rapidly evaluate metabolite similarities and differences among the EtOH extracts of the roots of eight commercial Astragalus spp., an approach based on direct analyses by ESI-MS followed by PCA of ESI-MS data, was carried out. Successively, quali-quantitative analyses of cycloartane derivatives in the eight Astragalus spp. by LC-ESI-MS(n) and PCA of LC-ESI-MS data were performed. This approach allowed to promptly highlighting metabolite similarities and differences among the various Astragalus spp. PCA results from LC-ESI-MS data of Astragalus samples were in reasonable agreement with both PCA results of ESI-MS data and quantitative results. This study affords an analytical method for the quali-quantitative determination of cycloartane derivatives in herbal preparations used as health and food supplements. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox{sup ®}, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox{sup ®}, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin. - Highlights: • Low molecular weight heparins prepared from different heparin parents were analyzed. • An integrated analytical

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

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox"®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox"®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin. - Highlights: • Low molecular weight heparins prepared from different heparin parents were analyzed. • An integrated analytical approach relied

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

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.

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.

Verification of propofol sulfate as a further human propofol metabolite using LC-ESI-QQQ-MS and LC-ESI-QTOF-MS analysis.

Science.gov (United States)

Maas, Alexandra; Maier, Christoph; Michel-Lauter, Beate; Broecker, Sebastian; Madea, Burkhard; Hess, Cornelius

2017-03-01

Propofol (2,6-diisopropylphenol) is a water-insoluble, intravenous anesthetic that is widely used for the induction and maintenance of anesthesia as well as for endoscopic and pediatric sedation. After admission, propofol undergoes extensive hepatic and extrahepatic metabolism, including direct conjugation to propofol glucuronide and hydroxylation to 2,6-diisopropyl-1,4-quinol. The latter substance subsequently undergoes phase II metabolism, resulting in the formation of further metabolites (1quinolglucuronide, 4quinolglucuronide and 4quinol-sulfate). Further minor phase I propofol metabolites (2-(ω-propanol)-6-isopropylphenol and 2-(ω-propanol)-6-isopropyl-1,4-quinol)) are also described. Due to its chemical structure with the phenolic hydroxyl group, propofol is also an appropriate substrate for sulfation by sulfotransferases. The existence of propofol sulfate was investigated by liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LCESIQQQ-MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LCESI-QTOF-MS). A propofol sulfate reference standard was used for identification and method development, yielding a precursor at m/z 257 (deprotonated propofol sulfate) and product ions at m/z 177 (deprotonated propofol) and m/z 80 ([SO3]-). Propofol sulfate - a further phase II metabolite of propofol - was verified in urine samples by LC-ESI-QQQ-MS and LC-ESI-QTOF-MS. Analyses of urine samples from five volunteers collected before and after propofol-induced sedation verified the presence of propofol sulfate in urine following propofol administration, whereas ascertained concentrations of this metabolite were significantly lower compared with detected propofol glucuronide concentrations. The existence of propofol sulfate as a further phase II propofol metabolite in humans could be verified by two different detection techniques (LCESIQQQ-MS and LC-ESI-QTOFMS) on the basis of a propofol sulfate

Integrated Solid-Phase Extraction-Capillary Liquid Chromatography (speLC) Interfaced to ESI-MS/MS for Fast Characterization and Quantification of Protein and Proteomes

DEFF Research Database (Denmark)

Falkenby, Lasse Gaarde; Such-Sanmartín, Gerard; Larsen, Martin Røssel

2014-01-01

min speLC-MS/MS experiment. Analysis by selected reaction monitoring by speLC-SRM-MS/MS of distinct peptides derived from the blood proteins IGF1, IGF2, IBP2, and IBP3 demonstrated protein quantification with CV values below 10% across 96 replicates. The speLC-MS/MS system is ideally suited for fast......The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enables the utilization of fast liquid chromatographic (LC) separation techniques. We present a robust solid-phase extraction/capillary LC system (speLC) for 5-10 min separation of semicomplex peptide mixtures...

Quantification of pharmaceutical peptides in human plasma by LC-ICP-MS sulfur detection

DEFF Research Database (Denmark)

Møller, Laura Hyrup; Macherius, André; Hansen, Thomas Hesselhøj

2016-01-01

A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP-DRC-MS inst......A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP......-DRC-MS instrument and subsequent quantification by post-column isotope dilution (IDA). Plasma proteins were precipitated prior to analysis. Analytical figures of merit including linearity, precision, LOD, LOQ and accuracy were considered satisfactory for analysis of plasma samples. The selectivity of the developed...... method was demonstrated for five pharmaceutically relevant peptides: desmopressin, penetratin, substance P, PTH (1-34) and insulin. Preliminary experiments on an ICP-MS/MS system using oxygen to reduce the effect of organic solvents were also performed to compare sensitivity. The results of the study...

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.

Determination of Ten Corticosteroids in Illegal Cosmetic Products by a Simple, Rapid, and High-Performance LC-MS/MS Method

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Vita Giaccone

2017-01-01

Full Text Available The aim of our present work was the development of a rapid high-performance liquid chromatography method with electrospray ionization and tandem mass spectrometry detection (LC-ESI-MS/MS for the determination of several corticosteroids in cosmetic products. Corticosteroids are suspected to be illegally added in cosmetic preparations in order to enhance the curative effect against some skin diseases. Sample preparation step consists in a single extraction with acetonitrile followed by centrifugation and filtration. The compounds were separated by reversed-phase chromatography with water and acetonitrile (both with 0.1% formic acid gradient elution and detected by ESI-MS positive and negative ionization mode. The method was validated at the validation level of 0.1 mg kg−1. Linearity was studied in the 5–250 μg L−1 range and linear coefficients (r2 were all over 0.99. The accuracy and precision of the method were satisfactory. The LOD ranged from 0.085 to 0.109 mg kg−1 and the LOQ from 0.102 to 0.121 mg kg−1. Mean recoveries for all the analytes were within the range 91.9–99.2%. The developed method is sensitive and useful for detection, quantification, and confirmation of these corticosteroids in cosmetic preparations and can be applied in the analysis of the suspected samples under investigation.

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.

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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.

Development and validation of an LC-MS/MS method for the determination of adapalene in pharmaceutical forms for skin application

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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

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

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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

Contaminant screening of wastewater with HPLC-IM-qTOF-MS and LC+LC-IM-qTOF-MS using a CCS database.

Science.gov (United States)

Stephan, Susanne; Hippler, Joerg; Köhler, Timo; Deeb, Ahmad A; Schmidt, Torsten C; Schmitz, Oliver J

2016-09-01

Non-target analysis has become an important tool in the field of water analysis since a broad variety of pollutants from different sources are released to the water cycle. For identification of compounds in such complex samples, liquid chromatography coupled to high resolution mass spectrometry are often used. The introduction of ion mobility spectrometry provides an additional separation dimension and allows determining collision cross sections (CCS) of the analytes as a further physicochemical constant supporting the identification. A CCS database with more than 500 standard substances including drug-like compounds and pesticides was used for CCS data base search in this work. A non-target analysis of a wastewater sample was initially performed with high performance liquid chromatography (HPLC) coupled to an ion mobility-quadrupole-time of flight mass spectrometer (IM-qTOF-MS). A database search including exact mass (±5 ppm) and CCS (±1 %) delivered 22 different compounds. Furthermore, the same sample was analyzed with a two-dimensional LC method, called LC+LC, developed in our group for the coupling to IM-qTOF-MS. This four dimensional separation platform revealed 53 different compounds, identified over exact mass and CCS, in the examined wastewater sample. It is demonstrated that the CCS database can also help to distinguish between isobaric structures exemplified for cyclophosphamide and ifosfamide. Graphical Abstract Scheme of sample analysis and database screening.

Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

Science.gov (United States)

Karakawa, Sachise; Shimbo, Kazutaka; Yamada, Naoyuki; Mizukoshi, Toshimi; Miyano, Hiroshi; Mita, Masashi; Lindner, Wolfgang; Hamase, Kenji

2015-11-10

A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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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

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.

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.

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.

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.

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

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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

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.

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

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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.

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

OpenAIRE

Moaddel, Ruin; Venkata, Swarajya Lakshmi Vattem; Tanga, Mary J.; Bupp, James E.; Green, Carol E.; Iyer, Lalitha; Furimsky, Anna; Goldberg, Michael E.; Torjman, Marc C.; Wainer, Irving W.

2010-01-01

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in Complex Regional Pain Syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)-and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric ...

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.

Characterisation of the ester-substituted products of the reaction of p-t-butyl calix[4]arene and ethyl bromoacetate using LC-UV-MS and LC-DAD.

Science.gov (United States)

McMahon, Gillian; Wall, Rachel; Nolan, Kieran; Diamond, Dermot

2002-07-19

A series of derivatisation reactions between p-t-butyl calix[4]arene and ethyl bromoacetate were carried out in order to prepare 1,3 diester substituted calix[4]arene. Mass spectral data, obtained from direct injection of samples, indicated that the reactions were rich in the desired product. Since the ultra violet (UV) spectra of the desired product and possible impurities are very similar, liquid chromatography (LC) chromatographic data seemed to corroborate these results. However, when on-line LC-UV-MS was carried out and each LC peak subjected to MS analysis as it eluted, a very different picture emerged. It was found that many of these reactions actually contained high levels of the monoester product which, having less affinity for sodium in the MS, is therefore seriously underestimated in any direct injection assay. LC-diode array detection (DAD) methods were also used to help successfully identify and characterise the compounds being formed in these complex reactions. The overall results obtained in this paper allowed the optimal reaction conditions to be determined for this reaction. LC-MS analysis of the chromatographic peaks also identified the presence of two isomers of the diester substituted calix[4]arene (1,3 and 1,2 diesters). The combination of LC and UV/MS detection is required for accurate analysis of the products of such reactions.

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.

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...

Enantioselective extraction of (+)-(S)-citalopram and its main metabolites using a tailor-made stir bar chiral imprinted polymer for their LC-ESI-MS/MS quantitation in urine samples.

Science.gov (United States)

Unceta, Nora; Gómez-Caballero, Alberto; García, Deiene; Díaz, Goretti; Guerreiro, Antonio; Piletsky, Sergey; Goicolea, M Aránzazu; Barrio, Ramón J

2013-11-15

This paper reports the application of a chiral imprinted polymer (CIP)-coated stir bar for the selective extraction of (+)-(S)-citalopram (SCIT) and its main metabolites, (+)-(S)-desmethylcitalopram (SDCIT) and (+)-(S)-didesmethylcitalopram (SDDCIT), from urine samples. The developed device has been demonstrated to be capable of selectively extracting the three target analytes from urine samples without saturating the imprinted sites. A CIP-coated stir bar sorptive extraction procedure (CIP-SBSE) is proposed for the isolation of SCIT, SDCIT and SDDCIT followed by their subsequent analysis using liquid chromatography ion trap mass spectrometry (LC-ITMS). Deuterated SCIT-d6 was used as an internal standard. The method was validated using a standard procedure, which revealed that a quantification of 5 ng mL(-1) was obtained in urine samples and that the accuracy and precision were within the established values while no matrix effect was observed. Copyright © 2013 Elsevier B.V. All rights reserved.

Sewage-based epidemiology in monitoring the use of new psychoactive substances: Validation and application of an analytical method using LC-MS/MS.

Science.gov (United States)

Kinyua, Juliet; Covaci, Adrian; Maho, Walid; McCall, Ann-Kathrin; Neels, Hugo; van Nuijs, Alexander L N

2015-09-01

Sewage-based epidemiology (SBE) employs the analysis of sewage to detect and quantify drug use within a community. While SBE has been applied repeatedly for the estimation of classical illicit drugs, only few studies investigated new psychoactive substances (NPS). These compounds mimic effects of illicit drugs by introducing slight modifications to chemical structures of controlled illicit drugs. We describe the optimization, validation, and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine (MPA), 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA). Sample preparation was performed using solid-phase extraction (SPE) with Oasis MCX cartridges. The LC separation was done with a HILIC (150 x 3 mm, 5 µm) column which ensured good resolution of the analytes with a total run time of 19 min. The lower limit of quantification (LLOQ) was between 0.5 and 5 ng/L for all compounds. The method was validated by evaluating the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recoveries and matrix effects. The method was applied on sewage samples collected from sewage treatment plants in Belgium and Switzerland in which all investigated compounds were detected, except MPA and PMA. Furthermore, a consistent presence of MXE has been observed in most of the sewage samples at levels higher than LLOQ. Copyright © 2015 John Wiley & Sons, Ltd.

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.

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

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...

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.

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...

[Identification of Methamphetamine Abuse and Selegiline Use： Chiral Analysis of Methamphetamine and Amphetamine in Urine].

Science.gov (United States)

Xiang, P; Bu, J; Qiao, Z; Zhuo, X Y; Wu, H J; Shen, M

2017-12-01

To study the content variation of selegiline and its metabolites in urine, and based on actual cases, to explore the feasibility for the identification of methamphetamine abuse and selegiline use by chiral analysis. The urine samples were tested by chiral separation and LC-MS/MS method using CHIROBIOTIC™ V2 chiral liquid chromatography column. The chiral analysis of methamphetamine and amphetamine were performed on the urine samples from volunteers of selegiline use and drug addicts whom suspected taking selegiline. After 5 mg oral administration, the positive test time of selegiline in urine was less than 7 h. The mass concentrations of R（-）-methamphetamine and R（-）-amphetamine in urine peaked at 7 h which were 0.86 μg/mL and 0.18 μg/mL and couldn't be detected after 80 h and 168 h, respectively. The sources of methamphetamine and amphetamine in the urine from the drug addicts whom suspected taking selegiline were analysed successfully by present method. The chiral analysis of methamphetamine and amphetamine, and the determination of selegiline's metabolites can be used to distinguish methamphetamine abuse from selegiline use. Copyright© by the Editorial Department of Journal of Forensic Medicine

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

Science.gov (United States)

Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

2017-12-06

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

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

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.

Relative quantification of enantiomers of chiral amines by high-throughput LC–ESI-MS/MS using isotopic variants of light and heavy L-pyroglutamic acids as the derivatization reagents

International Nuclear Information System (INIS)

Mochizuki, Toshiki; Taniguchi, Sayuri; Tsutsui, Haruhito; Min, Jun Zhe; Inoue, Koichi; Todoroki, Kenichiro; Toyo’oka, Toshimasa

2013-01-01

Highlights: ► Development of chiral labeling reagent for a pair of amine enantiomers. ► High-throughput analysis of diastereomers by UPLC–ESI-MS/MS. ► Highly efficient separation and detection of the enantiomers. ► Differential analysis of enantiomer ratio in different sample groups using light and heavy labeling reagents. -- Abstract: L-Pyroglutamic acid (L-PGA) was evaluated as a chiral labeling reagent for the enantioseparation of chiral amines in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. Several amines and amino acid methyl esters were used as typical representatives of the chiral amines. Both enantiomers of the chiral amines were easily labeled with L-PGAs at room temperature for 60 min in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxy-1H-benzotriazole as the activation reagents. The resulting diastereomers were completely separated by reversed-phase chromatography using the small particle (1.7 μm) ODS column (Rs = 1.6–6.8). A highly sensitive detection at a low-fmol level (1–4 fmol) was also obtained from the multiple reaction monitoring (MRM) chromatograms. Therefore, a high-throughput determination was achieved by the present UPLC–ESI-MS/MS method. An isotope labeling strategy using light and heavy L-PGAs for the differential analysis of chiral amines in different sample groups was also proposed in this paper. As a model study, the differential analysis of the R and S ratio of 1-phenylethylamine (PEA) was performed according to the proposed procedure using light and heavy reagents, i.e., L-PGA and L-PGA-d 5 . The R/S ratio of PEA, spiked at the different concentrations in rat plasma, was almost similar to the theoretical values. Consequently, the proposed strategy using light and heavy chiral labeling reagents seems to be applicable for the differential analysis of chiral amine enantiomers in different sample groups, such as healthy persons and

A high-throughput urinalysis of abused drugs based on a SPE-LC-MS/MS method coupled with an in-house developed post-analysis data treatment system.

Science.gov (United States)

Cheng, Wing-Chi; Yau, Tsan-Sang; Wong, Ming-Kei; Chan, Lai-Ping; Mok, Vincent King-Kuen

2006-10-16

A rapid urinalysis system based on SPE-LC-MS/MS with an in-house post-analysis data management system has been developed for the simultaneous identification and semi-quantitation of opiates (morphine, codeine), methadone, amphetamines (amphetamine, methylamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)), 11-benzodiazepines or their metabolites and ketamine. The urine samples are subjected to automated solid phase extraction prior to analysis by LC-MS (Finnigan Surveyor LC connected to a Finnigan LCQ Advantage) fitted with an Alltech Rocket Platinum EPS C-18 column. With a single point calibration at the cut-off concentration for each analyte, simultaneous identification and semi-quantitation for the above mentioned drugs can be achieved in a 10 min run per urine sample. A computer macro-program package was developed to automatically retrieve appropriate data from the analytical data files, compare results with preset values (such as cut-off concentrations, MS matching scores) of each drug being analyzed and generate user-defined Excel reports to indicate all positive and negative results in batch-wise manner for ease of checking. The final analytical results are automatically copied into an Access database for report generation purposes. Through the use of automation in sample preparation, simultaneous identification and semi-quantitation by LC-MS/MS and a tailored made post-analysis data management system, this new urinalysis system significantly improves the quality of results, reduces the post-data treatment time, error due to data transfer and is suitable for high-throughput laboratory in batch-wise operation.

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.

Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana).

Science.gov (United States)

Hurtado-Fernández, Elena; Carrasco-Pancorbo, Alegría; Fernández-Gutiérrez, Alberto

2011-03-23

A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.

ESI-MSn and LC-ESI-MS studies to characterize forced degradation products of bosentan and a validated stability-indicating LC-UV method.

Science.gov (United States)

Bansal, Gulshan; Singh, Ranjit; Saini, Balraj; Bansal, Yogita

2013-01-01

The present study reports the characterization of forced degradation products of bosentan and a validated stability-indicating HPLC method for the stability testing of bosentan tablets. The forced degradation was carried out under the conditions of hydrolysis, oxidation, dry heat and photolysis. The drug was found unstable in acid, alkali and oxidative media whereas stable to the hydrolysis in water, to dry heat and to photolysis. In total, six degradation products were formed in all conditions which were resolved in a single run on a C-18 column with gradient elution using ammonium acetate buffer (pH 4.5, 5.0mM), methanol and acetonitrile. Structures of all the degradation products were characterized through +ESI-MS(n) and LC-ESI-MS spectral data of bosentan as well as LC-ESI-MS spectral data of the products. The products II-VI were characterized as 6-amino-[2,2']bipyrimidinyl-4,5-diol, 6-amino-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-ol, 2-[6-amino-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-yloxy]-ethanol, 4-tert-butyl-N-[6-(1-methoxyethoxy)-5-(2-methoxyphenoxy)-[2,2']-bipyrimidinyl-4-yl]-benzenesulfonamide and 4-tert-butyl-N-[6-hydroxy-5-(2-methoxyphenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamide, respectively. The peak of the product I was found to be due to two secondary degradation products which co-eluted and were characterized as β-hydroxyethyl p-tert-butylphenylsulfonate (Ia) and 2-[2-(2-hydroxyethoxy)-phenoxy]-ethanol (Ib). These products were formed due to hydrolysis of sulfonamide and alkylaryl ether and the diaryl ether linkages as well as dehydration of the primary alcohol group. The most probable degradation mechanisms were proposed. The HPLC method was found to be stability-indicating, linear (2-100 μg ml(-1)), accurate, precise, sensitive, specific, rugged and robust for quantitation of the drug. The method was applied to the stability testing of the commercially available bosentan tablets successfully. Copyright © 2012 Elsevier B.V. All

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.

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.

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

Development and Validation of a LC/MS/MS Method for the Determination of Duloxetine in Human Plasma and Its Application to Pharmacokinetic Study

Directory of Open Access Journals (Sweden)

D. Chandrapal Reddy

2012-01-01

Full Text Available A selective, high sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS method has been developed and validated for the chromatographic separation and quantitation of duloxetine in human EDTA plasma using fluoxetine (IS as an internal standard. Analyte and IS were extracted from human plasma by liquid-liquid extraction using MTBE-n Hexane (80:20.The eluted samples were chromatographed on X-terra RP8 (50 mmx4.6 mm, 5 μm particle size column by using mixture of 30 mM ammonium formate (pH-5.0±0.05 and acetonitrile as an isocratic mobile phase at a flow rate of 0.40 mL/min and analyzed by mass spectrometer in the multiple reaction monitoring (MRM using the respective m/z 298.08→154.0 for duloxetine and 310.02→148.07 for IS. The linearity of the response/ concentration curve was established in human plasma over the concentration range 0.100-100.017 ng/mL. The lower detection limit (LOD,S/N>3 was 0.04 ng/mL and the lower limit of quantization (LOQ,S/N>10 was 0.100 ng/mL. This LC-MS/MS method was validated with Intra-batch and Inter-batch precision of 5.21-7.02. The Intra-batch and Inter-batch accuracy was 97.14-103.50 respectively. Recovery of duloxetine in human plasma is 80.31% and ISTD recovery is 81.09%. The main pharmacokinetic parameters were Tmax (hr = (7.25±1.581, Cmax (ng/mL (44.594±18.599, AUC0→t, = (984.702±526.502 and AUC0→∞, (1027.147±572.790 respectively.

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...

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.

Analytical Method Details (MS): SE29_MS1 [Metabolonote[Archive

Lifescience Database Archive (English)

Full Text Available SE29_MS1 LC-FT-ICR-MS ESI positive method 1 Agilent1100 HPLC (Agilent), LTQ-FT (The... (100mg) are solved in 300uL 80% methanol solution. 20uL sample is injected into HPLC after 0.2um membrane filter treatment. HPLC

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.

Development and validation of a rapid, selective, and sensitive LC-MS/MS method for simultaneous determination of D- and L-amino acids in human serum: application to the study of hepatocellular carcinoma.

Science.gov (United States)

Han, Minlu; Xie, Mengyu; Han, Jun; Yuan, Daoyi; Yang, Tian; Xie, Ying

2018-04-01

A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of D- and L-amino acids in human serum. Under the optimum conditions, except for DL-proline, L-glutamine, and D-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for L-amino acids (including glycine) and D-amino acids were 5-56.25 μM and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of D- and L-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of L-arginine, L-isoleucine, L-aspartate, L-tryptophan, L-alanine, L-methionine, L-serine, glycine, L-valine, L-leucine, L-phenylalanine, L-threonine, D-isoleucine, D-alanine, D-glutamate, D-glutamine, D-methionine, and D-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P hepatocellular carcinoma research. Graphical abstract Simultaneous determination of D- and L-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram.

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.

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.

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

Science.gov (United States)

Everest-Dass, Arun V.; Abrahams, Jodie L.; Kolarich, Daniel; Packer, Nicolle H.; Campbell, Matthew P.

2013-06-01

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

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

[Simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma by LC-HESI/MS/MS method].

Science.gov (United States)

Pan, Hua-Ling; Lin, Li-Shan; Ding, Jue-Fang; Chen, Xiao-Yan; Zhong, Da-Fang

2014-01-01

The study aims to develop a rapid, sensitive and specified method of liquid chromatography with heated electrospray ionization tandem mass spectrometry (LC-HESI/MS/MS) for simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma using amlodipine-d4 and ubenimex as internal standards (ISs). Selected reaction monitoring (SRM) with heated electrospray ionization (HESI) was used in the positive mode for mass spectrometric detection. Analytes and ISs were extracted from plasma by simple protein precipitation. The reconstituted samples were chromatographed on a C18 (100 mm x 4.6 mm, 5 microm) column with mixture of methanol-acetonitrile-5 mmol.L- ammonium acetate-formic acid (30 : 30 : 40 : 0.1) as mobile phase at a flow rate of 0.6 mL.min-1. The standard curves were demonstrated to be linear in the range of 0.02 to 6.00 ng.mL-1 for amlodipine, 0.2 to 1,500 ng.mL-1 for benazepril and benazeprilat with r2>0.99 for each analyte. The lower limit of quantitation was identifiable and reproducible at 0.02, 0.2 and 0.2 ng mL-1 for amlodipine, benazepril and benazeprilat, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limit across all concentrations. The plasma samples were stable after four freeze-thaw cycles and being stored for 93 days at -20 degrees C. The method was applied to a pharmacokinetic study of a fixed-dose combination of amlodipine and benazepril on Chinese healthy volunteers.

Fast and simple online sample preparation coupled with capillary LC-MS/MS for determination of prostaglandins in cell culture supernatants

DEFF Research Database (Denmark)

Rinne, Sandra; Ramstad Kleiveland, Charlotte; Kassem, Moustapha

2007-01-01

An online 2-D strong cation exchange (SCX)-RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG) A(1), PGD(2), PGE(1), PGE(2), PGF(2a), 6-keto-(6k)PGF(1a), and 15-Delta(12,14)-deoxy-PGJ(2) (15dPGJ(2...

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.

High-sensitive LC-MS/MS method for the simultaneous determination of mirodenafil and its major metabolite, SK-3541, in human plasma: application to microdose clinical trials of mirodenafil.

Science.gov (United States)

Cho, Doo-Yeoun; Bae, Soo Hyeon; Shon, Ji-Hong; Bae, Soo Kyung

2013-03-01

A high-sensitivity LC/MS/MS method was developed and validated for the simultaneous determination of mirodenafil and its major metabolite, SK-3541, in human plasma. Mirodenafil, SK-3541, and udenafil as an internal standard were extracted from plasma samples with methyl tert-butyl ether. Chromatographic separation was performed on a Luna phenyl-hexyl column (100 × 2.0 mm) with an isocratic mobile phase consisting of 5 mM ammonium formate and ACN (23:77, v/v) at a flow rate of 0.35 mL/min. Detection and quantification were performed using a mass spectrometer in selected reaction monitoring mode with positive ESI at m/z 532.3 → 296.1 for mirodenafil, m/z 488.1 → 296.1 for SK-3541, and m/z 517.3 → 283.2 for udenafil. The calibration curves were linear over a concentration range of 2-500 pg/mL using 0.5 mL plasma for the microdose of mirodenafil (100 μg). Analytical method validation of the clinical dose (100 mg), with a calibration curve range of 2-500 ng/mL using 0.025-mL plasma, was also conducted. The other LC-MS/MS conditions were similar to those used for the microdosing. Each method was applied successfully to pharmacokinetic studies after a microdose or clinical dose of mirodenafil to six healthy Korean male volunteers. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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) (

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...

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.

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.

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.

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.

Development and validation of an enantioselective SFC-MS/MS method for simultaneous separation and quantification of oxcarbazepine and its chiral metabolites in beagle dog plasma.

Science.gov (United States)

Yang, Zhichao; Xu, Xueyu; Sun, Lingxia; Zhao, Xue; Wang, Hao; Fawcett, John Paul; Yang, Yan; Gu, Jingkai

2016-05-01

A rapid and sensitive assay based on supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) has been developed and validated for the determination of oxcarbazepine (OXC) and its chiral metabolite licarbazine (Lic) in beagle dog plasma using carbamazepine as internal standard. Chiral analysis in a run time of only 3 min was performed on an ACQUITY UPC(2) ™ Trefoil™ CEL2 column (3.0 × 150 mm, 2.5 μm) at 50 °C by isocratic elution with a mobile phase of supercritical carbon dioxide (purity ≥ 99.99%) and methanol (60:40, v/v) at a flow rate of 2.3 mL/min. The assay was linear over the concentration ranges 5-1000 ng/mL for OXC and 0.5-100 ng/mL for the enantiomers of Lic with corresponding lower limits of quantitation of 5 ng/mL and 0.5 ng/mL. Intra- and inter-day precisions were in the range 0.78-14.14% with accuracies in the range -10.80% to 0.42%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 16 mg/kg OXC as Trileptal(@) tablets to beagle dogs. Copyright © 2016. Published by Elsevier B.V.

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.

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

Energy Technology Data Exchange (ETDEWEB)

Senyuva, Hamide Z. [Ankara Test and Analysis Laboratory, Scientific and Technological Research Council of Turkey, Ankara 06330 (Turkey)], E-mail: hamide.senyuva@tubitak.gov.tr; Gilbert, John [Central Science Laboratory, Sand Hutton, York YO41 1LZ (United Kingdom); Oztuerkoglu, Sebnem [Ankara Test and Analysis Laboratory, Scientific and Technological Research Council of Turkey, Ankara 06330 (Turkey)

2008-06-09

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.

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.

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.

A new simple and rapid LC-ESI-MS/MS method for quantification of plasma oxysterols as dimethylaminobutyrate esters. Its successful use for the diagnosis of Niemann-Pick type C disease.

Science.gov (United States)

Boenzi, Sara; Deodato, Federica; Taurisano, Roberta; Martinelli, Diego; Verrigni, Daniela; Carrozzo, Rosalba; Bertini, Enrico; Pastore, Anna; Dionisi-Vici, Carlo; Johnson, David W

2014-11-01

Two oxysterols, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been recently proposed as diagnostic markers of Niemann-Pick type C (NP-C) disease, representing a potential alternative diagnostic tool to the more invasive and time consuming filipin test in cultured fibroblasts. Usually, the oxysterols are detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using atmospheric pressure chemical ionization (APCI) or electro-spray-ionization (ESI) sources, after a variety of derivatization procedures to enhance sensitivity. We developed a sensitive LC-MS/MS method to quantify the oxysterols in plasma as dimethylaminobutyrate ester, suitable for ESI analysis. This method, with an easy liquid-phase extraction and a short derivatization procedure, has been validated to demonstrate specificity, linearity, recovery, lowest limit of quantification, accuracy and precision. The assay was linear over a concentration range of 0.5-200ng/mL for C-triol and 1.0-200ng/mL for 7-KC. Intra-day and inter-day coefficients of variation (CV%) were <15% for both metabolites. Receiver operating characteristic analysis estimates that the area under curve was 0.998 for C-triol, and 0.972 for 7-KC, implying a significant discriminatory power for the method in this patient population of both oxysterols. In summary, our method provides a simple, rapid and non-invasive diagnostic tool for the biochemical diagnosis of NP-C disease. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Chiral Analysis of Methamphetamine in Oral Fluid Samples: A Method to Distinguish Licit from Illicit Drug Use.

Science.gov (United States)

Borg, Damon; Kolb, Elizabeth; Lantigua, Cindy; Stripp, Richard

2018-01-01

Methamphetamine (MAMP) is a popular illicit drug abused for its central nervous system stimulating effects. MAMP is also used therapeutically in the treatment of overeating disorders, narcolepsy, attention deficit disorder, in over-the-counter (OTC) products to ease nasal congestion. MAMP exists in two enantiomeric forms, dextrorotary (d-MAMP) or levorotary (l-MAMP). The compounds are similar in chemical structure, simply differing in the orientation of functional groups around the asymmetric carbon. In part because of the availability of l-MAMP in OTC nasal inhalers, forensic guidelines require a sample to contain greater than 20% d-MAMP to consider illicit drug use when interpreting results. Standard analytical methods readily detect MAMP in biological specimens but cannot resolve the enantiomeric composition of the sample. Specialized analytical techniques based on chiral separation of the enantiomers are required to differentiate d-MAMP from l-MAMP. Our laboratory sought to develop and validate a method for the analysis of oral fluid specimens for d/l-MAMP using a chiral derivatizing agent and traditional reverse phase liquid chromatography tandem mass spectrometry (LC-MS-MS). MAMP was extracted from dilute oral fluid samples using Strata-XC solid phase extraction (SPE) cartridges and derivatized with Marfey's reagent. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 columns. Linearity, accuracy and precision, recovery, matrix effects and specificity of the method were all within acceptable criteria. Intraday accuracy ranged from 93.3 to 103.4% and precision 0.1 to 1.6%. Interday accuracy ranged from 90.0 to 103.4% and precision 3.8 to 11.6%. Finally, having previously tested positive for MAMP using non-chiral analysis, 256 de-identified authentic oral fluid samples were analyzed using this validated method. 98% of all samples tested positive for d-MAMP at greater than 20%.

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.

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.

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

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.

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

Chiral recognition and determination of enantiomeric excess by mass spectrometry: A review

International Nuclear Information System (INIS)

Yu, Xiangying; Yao, Zhong-Ping

2017-01-01

Chiral analysis is of great importance to fundamental and applied research in chemical, biological and pharmaceutical sciences. Due to the superiority of mass spectrometry (MS) over other analytical methods in terms of speed, specificity and sensitivity, chiral analysis by MS has attracted much interest in recent years. Chiral analysis by MS typically involves introduction of a chiral selector to form diastereomers with analyte enantiomers, and comparison of the behaviors of diastereomers in MS. Chiral differentiation can be achieved by comparing the relative abundances of diastereomers, the thermodynamic or kinetic constants of ion-molecule reactions of diastereomers in the gas phase, the dissociation of diastereomers in MS/MS, or the mobility of diastereomers in ion mobility mass spectrometry. In this review, chiral recognition and determination of enantiomeric excess by these chiral MS methods were summarized, and the prospects of chiral analysis by MS were discussed. - Highlights: • Both chiral recognition and determination of enantiomeric excess by mass spectrometry are systematically reviewed. • Classification is based on the behavioral differences of diastereomers formed between chiral analytes and chiral selectors. • Development of ion mobility mass spectrometry for chiral differentiation is covered. • Various methods are highlighted and compared.

Chiral recognition and determination of enantiomeric excess by mass spectrometry: A review

Energy Technology Data Exchange (ETDEWEB)

Yu, Xiangying [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of Hong Kong Polytechnic University, Shenzhen 518057 (China); Yao, Zhong-Ping, E-mail: zhongping.yao@polyu.edu.hk [State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of Hong Kong Polytechnic University, Shenzhen 518057 (China); Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules (Yanbian University), Ministry of Education, Yanji 133002, Jilin (China); State Key Laboratory of Chirosciences, Food Safety and Technology Research Centre and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region (China)

2017-05-22

Chiral analysis is of great importance to fundamental and applied research in chemical, biological and pharmaceutical sciences. Due to the superiority of mass spectrometry (MS) over other analytical methods in terms of speed, specificity and sensitivity, chiral analysis by MS has attracted much interest in recent years. Chiral analysis by MS typically involves introduction of a chiral selector to form diastereomers with analyte enantiomers, and comparison of the behaviors of diastereomers in MS. Chiral differentiation can be achieved by comparing the relative abundances of diastereomers, the thermodynamic or kinetic constants of ion-molecule reactions of diastereomers in the gas phase, the dissociation of diastereomers in MS/MS, or the mobility of diastereomers in ion mobility mass spectrometry. In this review, chiral recognition and determination of enantiomeric excess by these chiral MS methods were summarized, and the prospects of chiral analysis by MS were discussed. - Highlights: • Both chiral recognition and determination of enantiomeric excess by mass spectrometry are systematically reviewed. • Classification is based on the behavioral differences of diastereomers formed between chiral analytes and chiral selectors. • Development of ion mobility mass spectrometry for chiral differentiation is covered. • Various methods are highlighted and compared.

Characterization and quantitation of yohimbine and its analogs in botanicals and dietary supplements using LC/QTOF-MS and LC/QQQ-MS for determination of the presence of bark extract and yohimbine adulteration.

Science.gov (United States)

Lucas, Derick; Neal-Kababick, James; Zweigenbaum, Jerry

2015-01-01

The compound yohimbine HCl has been restricted in Australia and categorized as a scheduled prescription drug in other parts of the world, including the United States where it is monographed as a drug in the U. S. Pharmacopeia. However, the bark of the yohimbe plant and its extract is considered a botanical that can be used as a dietary supplement in some parts of the world. For these reasons, methods to characterize the indole alkaloids of the bark and quantify yohimbine and its analogs are presented using accurate mass LC/quadrupole time-of-flight (QTOF)-MS and triple quadrupole LC/MS, respectively. Samples were extracted with a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method to characterize and quantify the indole alkaloids. With the LC/QTOF-MS in auto MS/MS mode the indole alkaloids were identified, and the isomeric response of each could be used to determine whether the actual bark or extract was in samples of dietary supplements and not adulteration with yohimbine HCl. Analogs were identified and include yohimbic acid, methyl yohimbine, and hydroxyl yohimbine. Many isomers of each were also detected, but identified only by the number of chromatographic peaks. Quantification of yohimbine and ajmalicine spiked extracts showed recoveries of 99 to 103% with RSD of 3.6% or lower and LODs of less than 100 ppt. Calibration of the two standards gave r(2) = 0.9999 in a range from 0.1 to 100 ppb. Dietary supplements quantified for these two compounds showed a range from not detected to 3x the amounts found in the bark.

Proanthocyanidin screening by LC-ESI-MS of Portuguese red wines made with teinturier grapes.

Science.gov (United States)

Teixeira, Natércia; Azevedo, Joana; Mateus, Nuno; de Freitas, Victor

2016-01-01

Proanthocyanidins (PAs) are one of the most important polyphenolic compounds in wine. Among PAs, prodelphinidin (PD) dimers and trimers have not been widely detected in wines due to the lack of available commercial standards and the difficulty to detect and isolate them from natural sources. LC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry) with the right chromatographic conditions has proven to be a powerful tool for PAs detection and identification in complex samples. This technique has been applied to an exhaustive study of PA composition of two Portuguese red wines made with teinturier grapes, especially for the identification of PD dimers and trimers. Tandem mass spectrometry (MS/MS) with ion trap provided additional information about the structures of these compounds through the fragmentation patterns of the pseudomolecular ions. A LC-ESI-MS method was optimized and 41 different compounds were found. Among them are included 8 PD dimers and 13 PD trimers. Copyright © 2015 Elsevier Ltd. All rights reserved.

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/MS

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed- and chiral-phase LC–MS

Czech Academy of Sciences Publication Activity Database

Řezanka, Tomáš; Kolouchová, I.; Čejková, A.; Cajthaml, Tomáš; Sigler, Karel

2013-01-01

Roč. 36, č. 20 (2013), s. 3310-3320 ISSN 1615-9306 R&D Projects: GA ČR(CZ) GAP503/11/0215 Institutional support: RVO:61388971 Keywords : Atmospheric pressure chemical ionization mass spectrometry * Chiral LC * Reversed phase LC Subject RIV: EE - Microbiology, Virology Impact factor: 2.594, year: 2013

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.

Separation and identification of selenotrisulfides in epithelial cell homogenates by LC-ICP-MS and LC-ESI-MS after incubation with selenite

DEFF Research Database (Denmark)

Gabel-Jensen, Charlotte; Gammelgaard, Bente; Bendahl, Lars

2006-01-01

. The presence of Cys-Se-SG and GS-Se-SG was verified by LC-ESI-MS. Selenotrisulfides were synthesized by reaction of L-cysteine and L-glutathione with sodium selenite. The reaction mixture contained three main products: selenodicysteine (Cys-Se-Cys), selenocysteine glutathione (Cys...

Effective application of freezing lipid precipitation and SCX-SPE for determination of pyrrolizidine alkaloids in high lipid foodstuffs by LC-ESI-MS/MS.

Science.gov (United States)

Yoon, Soo Hwan; Kim, Min-Sun; Kim, Sang Hoon; Park, Hyun Mee; Pyo, Heesoo; Lee, Yong Moon; Lee, Kyung-Tae; Hong, Jongki

2015-06-15

Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with serious hepatic disease in humans and animals. In this study, rapid and sensitive analytical method was developed for the determination of 9 toxic PAs in popularly high lipid foodstuffs by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). PAs in lipid foodstuff were effectively purified by freezing lipid precipitation (FLP) and strong cation exchange (SCX)-solid-phase extraction (SPE). Especially, FLP could easily remove the large amounts of triacylglycerols in the lipid sample extract and effectively combine with SPE cleanup. During the FLP procedure, over 77% of the lipids in the foodstuff extracts were rapidly eliminated without any significant loss of the PAs with over 81% recovery. The elimination efficiency of lipids by FLP was tested with LC-atmospheric chemical ionization (APCI)-MS. For further purification, SCX-SPE cartridge could successfully purify PAs from the remaining interfering substances by the variation pH with 5% NH4OH in methanol. For precise quantification and confirmation of PAs in complicate sample matrices, appropriate transition ions in LC-MS/MS-multiple-ion reaction monitoring (MRM) mode were selected on the basis of MS/MS fragmentation pathways of PAs. The established analytical method was validated in terms of the linearity, limits of detection (LOD), and quantification (LOQ), precision, and accuracy. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation<11.06%). Overall limits of detection and quantitation of PAs were approximately 0.06-0.60ng/mL at a signal-to-noise ratio (S/N) of 3 and were about 0.20-1.99ng/mL at a S/N of 10 for all foodstuffs. The established method was successfully applied for the monitoring of toxic PAs in several types of high lipid foodstuffs such as soybeans, seed oil, milk, and margarine. Copyright �

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

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.

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.

Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

Science.gov (United States)

Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T; Mozayani, Ashraf

2013-01-01

Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

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

Identification of Eight Synthetic Cannabinoids, Including 5F-AKB48 in Seized Herbal Products Using DART-TOF-MS and LC-QTOF-MS as Nontargeted Screening Methods.

Science.gov (United States)

Moore, Katherine N; Garvin, Demetra; Thomas, Brian F; Grabenauer, Megan

2017-09-01

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana-like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time-of-flight mass spectrometry (DART-TOF-MS) in combination with liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH-018, JWH-019, AM2201, JWH-122, 5F-AKB48, AKB48-N-(4-pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART-TOF-MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids. © 2017 American Academy of Forensic Sciences.

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

Science.gov (United States)

Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

2012-01-01

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

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...

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.

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.

Evaluation of peptide adsorption-controlled liquid chromatography-tandem mass spectrometric (PAC-LC-MS/MS) method for simple and simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides in dog cerebrospinal fluid.

Science.gov (United States)

Goda, Ryoya; Kobayashi, Nobuhiro

2012-05-01

To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography-tandem mass spectrometry (PAC-LC-MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water-acetic acid-methanol (2:6:1, v/v/v) was loaded on PAC-LC-MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H(5+)] and b(5+) ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC-MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze-thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC-MS/MS method enables reproducible Aβ quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

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.

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

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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.

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

A General LC-MS/MS Method for Monitoring Potential β-Lactam Contamination in Drugs and Drug-Manufacturing Surfaces.

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Qiu, Chen; Zhu, Hongbin; Ruzicka, Connie; Keire, David; Ye, Hongping

2018-05-15

Penicillins and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight β-lactams. From various drug products, the recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of β-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.

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

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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%.

Analysis of pesticide residues in strawberries and soils by GC-MS/MS, LC-MS/MS and two-dimensional GC-time-of-flight MS comparing organic and integrated pest management farming.

Science.gov (United States)

Fernandes, Virgínia C; Lehotay, Steven J; Geis-Asteggiante, Lucía; Kwon, Hyeyoung; Mol, Hans G J; van der Kamp, Henk; Mateus, Nuno; Domingues, Valentina F; Delerue-Matos, Cristina

2014-01-01

This study analysed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming with integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. We applied three different methods for analysis: (1) 27 pesticides were targeted using LC-MS/MS; (2) 143 were targeted using low pressure GC-tandem mass spectrometry (LP-GC-MS/MS); and (3) more than 600 pesticides were screened in a targeted and untargeted approach using comprehensive, two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS). Comparison was made of the analyses using the different methods for the shared samples. The results were similar, thereby providing satisfactory confirmation of both similarly positive and negative findings. No pesticides were found in the organic-farmed samples. In samples from integrated pest management practices, nine pesticides were determined and confirmed to be present, ranging from 2 µg kg(-1) for fluazifop-p-butyl to 50 µg kg(-1) for fenpropathrin. Concentrations of residues in strawberries were less than European maximum residue limits.

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

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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.

Development and validation of a highly sensitive LC-ESI-MS/MS method for estimation of IIIM-MCD-211, a novel nitrofuranyl methyl piperazine derivative with potential activity against tuberculosis: Application to drug development.

Science.gov (United States)

Magotra, Asmita; Sharma, Anjna; Gupta, Ajai Prakash; Wazir, Priya; Sharma, Shweta; Singh, Parvinder Pal; Tikoo, Manoj Kumar; Vishwakarma, Ram A; Singh, Gurdarshan; Nandi, Utpal

2017-08-15

In the present study, a simple, sensitive, specific and rapid liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was developed and validated according to the Food and Drug Administration (FDA) guidelines for estimation of IIIM-MCD-211 (a potent oral candidate with promising action against tuberculosis) in mice plasma using carbamazepine as internal standard (IS). Bioanalytical method consisted of one step protein precipitation for sample preparation followed by quantitation in LC-MS/MS using positive electrospray ionization technique (ESI) operating in multiple reaction monitoring (MRM) mode. Elution was achieved in gradient mode on High Resolution Chromolith RP-18e column with mobile phase comprised of acetonitrile and 0.1% (v/v) formic acid in water at the flow rate of 0.4mL/min. Precursor to product ion transitions (m/z 344.5/218.4 and m/z 237.3/194.2) were used to measure analyte and IS, respectively. All validation parameters were well within the limit of acceptance criteria. The method was successfully applied to assess the pharmacokinetics of the candidate in mice following oral (10mg/kg) and intravenous (IV; 2.5mg/kg) administration. It was also effectively used to quantitate metabolic stability of the compound in mouse liver microsomes (MLM) and human liver microsomes (HLM) followed by its in-vitro-in-vivo extrapolation. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Multi-residue analysis method for analysis of pharmaceuticals using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS) in water sample

Science.gov (United States)

Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali

2013-11-01

In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.

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.

LC/ESI-MS method applied to characterization of flavonoids glycosides in B. forficata subsp. pruinosa

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Lidiane da Silveira Farias

2014-06-01

Full Text Available Bauhinia forficata is used in folk medicine for its hypoglycemiant effect. In the south of Brazil, the subspecies pruinosa is found in a region with the characteristic flora, pampa biome. This species has been consumed by the local population as a tea for diabetes treatment. We studied the chemical composition of hydroethanolic extracts using LC/ESI-MS. The leaf extracts were prepared by percolation with 50% (v/v ethanol. The chromatographic analyses were performed using a reverse-phase system, gradient elution with acetonitrile:phosphoric acid 0.05%, and ESI-MS in the positive ion mode. The chemical profile of the flavonoids was suggested to involve four quercetin and kaempferol glycosides.

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.

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.

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

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.

Determination of etoricoxib in human plasma using automated on-line solid-phase extraction coupled with LC-APCI/MS/MS

Directory of Open Access Journals (Sweden)

Sérgio Luiz Dalmora

2008-01-01

Full Text Available A liquid chromatography-tandem mass spectrometry method with atmospheric pressure chemical ionization (LC-APCI/MS/MS was validated for the determination of etoricoxib in human plasma using antipyrin as internal standard, followed by on-line solid-phase extraction. The method was performed on a Luna C18 column and the mobile phase consisted of acetonitrile:water (95:5, v/v/ammonium acetate (pH 4.0; 10 mM, run at a flow rate of 0.6 mL/min. The method was linear in the range of 1-5000 ng/mL (r²>0.99. The lower limit of quantitation was 1 ng/mL. The recoveries were within 93.72-96.18%. Moreover, method validation demonstrated acceptable results for the precision, accuracy and stability studies.

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

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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.

A LC-MS/MS method for the determination of BADGE-related and BFDGE-related compounds in canned fish food samples based on the formation of [M+NH(4)](+) aducts.

Science.gov (United States)

Míguez, J; Herrero, C; Quintás, I; Rodríguez, C; Gigosos, P G; Mariz, O C

2012-12-01

A new and simple liquid chromatography tandem mass-spectrometry method for the determination of different bisphenol A (BPA) derivatives such as bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their reaction products with water and hydrochloric acid in different fish food products was developed. The extraction procedure and the chromatographic conditions were optimised for complex food matrices such as fish products. Food samples were homogenised and extracted with a 1:1 solution of acetonitrile-hexane, the solvent was eliminated in a N(2) stream and the extract was reconstituted with 0.5mL of a 0.01M solution of ammonium formate. The sample solution obtained was directly measured by LC-MS/MS without any further purification under the developed conditions. The use of a mobile phase composed by ammonium formate-methanol in a binary gradient mode produced [M+NH(4)](+) aducts for the different BADGEs and BFDGEs. These aduct's fragmentations were employed for the LC-MS/MS quantification of BPA derivatives in canned fish samples. The results of the validation were appropriate: the method was linear for BADGE and its hydrolysed derivatives up to 1000μgkg(-1), for the remaining compounds linearity achieved up to 100μgkg(-1). Quantification limits were in the range 2-10μgkg(-1). RSD (intra and inter-day) was 6-12% and the recovery was comprised between 89% and 109%. Under the optimised conditions, the chromatographic separation was performed in 8min per sample. The method was applied to the determination of BADGE, BFDGE and their reaction products in different samples of canned fish from Spanish origin. Migration results obtained were in compliance with the EU regulations. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Development and application of a quantitative method based on LC-QqQ MS/MS for determination of steviol glycosides in Stevia leaves.

Science.gov (United States)

Molina-Calle, M; Sánchez de Medina, V; Delgado de la Torre, M P; Priego-Capote, F; Luque de Castro, M D

2016-07-01

Stevia is a currently well-known plant thanks to the presence of steviol glycosides, which are considered as sweeteners obtained from a natural source. In this research, a method based on LC-MS/MS by using a triple quadrupole detector was developed for quantitation of 8 steviol glycosides in extracts from Stevia leaves. The ionization and fragmentation parameters for selected reaction monitoring were optimized. Detection and quantitation limits ranging from 0.1 to 0.5ng/mL and from 0.5 to 1ng/mL, respectively, were achieved: the lowest attained so far. The steviol glycosides were quantified in extracts from leaves of seven varieties of Stevia cultivated in laboratory, greenhouse and field. Plants cultivated in field presented higher concentration of steviol glycosides than those cultivated in greenhouse. Thus, the way of cultivation clearly influences the concentration of these compounds. The inclusion of branches together with leaves as raw material was also evaluated, showing that this inclusion modifies, either positively or negatively, the concentration of steviol glycosides. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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.

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.

Authenticity of raspberry flavor in food products using SPME-chiral-GC-MS

DEFF Research Database (Denmark)

Hansen, Anne-Mette Sølvbjerg; Frandsen, Henrik Lauritz; Fromberg, Arvid

2015-01-01

A fast and simple method for authenticating raspberry flavors from food products was developed. The two enantiomers of the compound (E)-α-ionone from raspberry flavor were separated on a chiral gas chromatographic column. Based on the ratio of these two enantiomers the naturalness of a raspberry...... flavor can be evaluated due to the fact that a natural flavor will consist almost exclusively of the R enantiomer, while a chemical synthesis of the same compound will result in a racemic mixture. 27 food products containing raspberry flavors where investigated using SPME-chiral-GC-MS. We found raspberry...... distribution of the R and S isomer. Two products were labelled to contain natural raspberry flavors but were found to contain almost equal amounts of both enantiomers indicating a presence of synthetic raspberry flavors only. Additionally, two products labelled to contain both raspberry juice and flavor showed...

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

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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/.

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

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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.

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

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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.

An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study.

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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, �

Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

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Kayacelebi, Arslan Arinc; Schauerte, Celina; Kling, Katharina; Herbers, Jan; Beckmann, Bibiana; Engeli, Stefan; Jordan, Jens; Zoerner, Alexander A; Tsikas, Dimitrios

2017-03-15

2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d 8 -2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d 8 -AA; range 0-1μM) as the MAGL product. Unlabelled AA (d 0 -AA, 1μM) serves as the internal standard. d 8 -AA and d 0 -AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H] - →[M-H - CO 2 ] - , i.e., m/z 311→m/z 267 for d 8 -AA and m/z 303→m/z 259 for d 0 -AA. Prior to GC-MS analysis d 8 -AA and d 0 -AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB] - , i.e., m/z 311 for d 8 -AA and m/z 303 for d 0 -AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d 8 -AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r 2 =0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination

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

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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.

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

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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.

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

Photolysis of sulfamethoxypyridazine in various aqueous media: Aerobic biodegradation and identification of photoproducts by LC-UV–MS/MS

Energy Technology Data Exchange (ETDEWEB)

Khaleel, Nareman D.H., E-mail: drndahshan@yahoo.com [Sustainable Chemistry and Material Resources, Institute of Sustainable and Environmental Chemistry, Leuphana University Lüneburg, C13, DE-21335 Lüneburg (Germany); Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522 (Egypt); Mahmoud, Waleed M.M. [Sustainable Chemistry and Material Resources, Institute of Sustainable and Environmental Chemistry, Leuphana University Lüneburg, C13, DE-21335 Lüneburg (Germany); Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522 (Egypt); Hadad, Ghada M.; Abdel-Salam, Randa A. [Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522 (Egypt); Kümmerer, Klaus, E-mail: Klaus.Kuemmerer@uni.leuphana.de [Sustainable Chemistry and Material Resources, Institute of Sustainable and Environmental Chemistry, Leuphana University Lüneburg, C13, DE-21335 Lüneburg (Germany)

2013-01-15

Highlights: ► Sulfonamides are one of the most extensively used antibiotics in human and veterinary medicine. ► Sulfamethoxypyridazine (SMP) underwent photodegradation in three different media. ► SMP was not readily biodegradable. ► SMP and some of its degradation products were identified by LC-UV–MS/MS. -- Abstract: Sulfonamides are one of the most frequently used antibiotics worldwide. Therefore, mitigation processes such as abiotic or biotic degradation are of interest. Photodegradation and biodegradation are the potentially significant removal mechanisms for pharmaceuticals in aquatic environments. The photolysis of sulfamethoxypyridazine (SMP) using a medium pressure Hg-lamp was evaluated in three different media: Millipore water pH 6.1 (MW), effluent from sewage treatment plant pH 7.6 (STP), and buffered demineralized water pH 7.4 (BDW). Identification of transformation products (TPs) was performed by LC-UV–MS/MS. The biodegradation of SMP using two tests from the OECD series was studied: Closed Bottle test (OECD 301 D), and Manometric Respirometry test (OECD 301 F). In biodegradation tests, it was found that SMP was not readily biodegradable so it may pose a risk to the environment. The results showed that SMP was removed completely within 128 min of irradiation in the three media, and the degradation rate was different for each investigated type of water. However, dissolved organic carbon (DOC) was not removed in BDW and only little DOC removal was observed in MW and STP, thus indicating the formation of TPs. Analysis by LC-UV–MS/MS revealed new TPs formed. The hydroxylation of SMP represents the main photodegradation pathway.

Photolysis of sulfamethoxypyridazine in various aqueous media: Aerobic biodegradation and identification of photoproducts by LC-UV–MS/MS

International Nuclear Information System (INIS)

Khaleel, Nareman D.H.; Mahmoud, Waleed M.M.; Hadad, Ghada M.; Abdel-Salam, Randa A.; Kümmerer, Klaus

2013-01-01

Highlights: ► Sulfonamides are one of the most extensively used antibiotics in human and veterinary medicine. ► Sulfamethoxypyridazine (SMP) underwent photodegradation in three different media. ► SMP was not readily biodegradable. ► SMP and some of its degradation products were identified by LC-UV–MS/MS. -- Abstract: Sulfonamides are one of the most frequently used antibiotics worldwide. Therefore, mitigation processes such as abiotic or biotic degradation are of interest. Photodegradation and biodegradation are the potentially significant removal mechanisms for pharmaceuticals in aquatic environments. The photolysis of sulfamethoxypyridazine (SMP) using a medium pressure Hg-lamp was evaluated in three different media: Millipore water pH 6.1 (MW), effluent from sewage treatment plant pH 7.6 (STP), and buffered demineralized water pH 7.4 (BDW). Identification of transformation products (TPs) was performed by LC-UV–MS/MS. The biodegradation of SMP using two tests from the OECD series was studied: Closed Bottle test (OECD 301 D), and Manometric Respirometry test (OECD 301 F). In biodegradation tests, it was found that SMP was not readily biodegradable so it may pose a risk to the environment. The results showed that SMP was removed completely within 128 min of irradiation in the three media, and the degradation rate was different for each investigated type of water. However, dissolved organic carbon (DOC) was not removed in BDW and only little DOC removal was observed in MW and STP, thus indicating the formation of TPs. Analysis by LC-UV–MS/MS revealed new TPs formed. The hydroxylation of SMP represents the main photodegradation pathway

Development and validation of a LC-MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers.

Science.gov (United States)

Tan, Zhirong; Ouyang, Dongsheng; Chen, Yao; Zhou, Gan; Cao, Shan; Wang, Yicheng; Peng, Xiujuan; Zhou, Honghao

2010-08-01

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid-liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C(18) (5 microm, 150 mm x 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H](+) ions, m/z 373.9-->m/z184.0 for clebopride, m/z 359.9-->m/z71.5 for itopride. The method was validated over the concentration range of 69.530-4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from -8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride. Copyright 2010. Published by Elsevier B.V.

Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis).

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Zhang, Gong; Huang, Guangwei; Xiao, Lu; Mitchell, Alyson E

2011-11-23

A sensitive and reliable LC-(ESI)MS/MS method was developed and validated for the simultaneous analysis of five common advanced glycation endproducts (AGEs) after enzymatic digestion in raw and roasted almonds. AGEs included carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), pyralline (Pyr), argpyrimidine (Arg-p), and pentosidine (Pento-s). This method allows accurate quantitation of free and AGE-protein adducts of target AGEs. Results indicate that CML and CEL are found in both raw and roasted almonds. Pyr was identified for the first time in roasted almonds and accounted for 64.4% of free plus bound measured AGEs. Arg-p and Pento-s were below the limit of detection in all almond samples tested. Free AGEs accounted for 1.3-26.8% of free plus bound measured AGEs, indicating that protein-bound forms predominate. The roasting process significantly increased CML, CEL, and Pyr formation, but no significant correlation was observed between these AGEs and roasting temperature.

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.

LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea.

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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

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

Phytochemical Analysis of Pfaffia glomerata Inflorescences by LC-ESI-MS/MS

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Daniele F. Felipe

2014-09-01

Full Text Available Pfaffia glomerata contains high levels of β-ecdysone, which has shown a range of beneficial pharmacological effects. The present study demonstrated that inflorescences of P. glomerata contain other important bioactive compounds in addition to β-ecdysone. The identification of compounds from inflorescences using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS was performed for the first time. The eight compounds identified were β-ecdysone, flavonoid glycosides such as quercetin-3-O-glucoside, kaempferol-3-O-glucoside and kaempferol-3-O-(6-p-coumaroyl-glucoside, oleanane-type triterpenoid saponins such as ginsenoside Ro and chikusetsusaponin IV, in addition to oleanonic acid and gluconic acid. This study provided information on the phytochemicals contained in P. glomerata inflorescences revealing the potential application of this plant part as raw material for the phytotherapeutic and cosmetic industries.

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.

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

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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.

Chiral analysis of bambuterol, its intermediate and active drug in human plasma by liquid chromatography-tandem mass spectrometry: Application to a pharmacokinetic study.

Science.gov (United States)

Zhou, Ting; Liu, Shan; Zhao, Ting; Zeng, Jing; He, Mingzhi; Xu, Beining; Qu, Shanshan; Xu, Ling; Tan, Wen

2015-08-01

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous chiral analysis of an antiasthma drug bambuterol, its key intermediate monocarbamate bambuterol and its active drug terbutaline in human plasma. All samples were extracted with ethyl acetate and separated on an Astec Chirobiotic T column under isocratic elution with a mobile phase consisting of methanol and water with the addition of 20mm ammonium acetate and 0.005% (v/v) formic acid at 0.6mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method has high sensitivity with the lower limit of quantifications of 25.00pg/mL for bambuterol enantiomers, and 50.00pg/mL for monocarbamate bambuterol and terbutaline enantiomers, respectively. The calibration curves for bambuterol enantiomers were linear in the range of 25.00-2500pg/mL, and for monocarbamate bambuterol and terbutaline enantiomers were linear in the range of 50.00-5000pg/mL. The intra- and inter-day precisions were <12.4%. All the analytes were separated in 18.0min. For the first time, the validated method was successfully applied to an enantioselective pharmacokinetic study of rac-bambuterol in 8 healthy volunteers. According to the results, this chiral LC-MS/MS assay provides a suitable and robust method for the enantioselectivity and interaction study of the prodrug bambuterol, the key intermediate monocarbamate bambuterol and its active drug terbutaline in human. Copyright © 2015 Elsevier B.V. All rights reserved.

A simple LC-MS/MS method facilitated by salting-out assisted liquid-liquid extraction to simultaneously determine trans-resveratrol and its glucuronide and sulfate conjugates in rat plasma and its application to pharmacokinetic assay.

Science.gov (United States)

Qiu, Zhixia; Yu, Jiaojiao; Dai, Yu; Yang, Yue; Lu, Xiaoyu; Xu, Jiaqiu; Qin, Zhiying; Huang, Fang; Li, Ning

2017-11-01

A simple LC-MS/MS method facilitated by salting-out assisted liquid-liquid extraction (SALLE) was applied to simultaneously investigate the pharmacokinetics of trans-resveratrol (Res) and its major glucuronide and sulfate conjugates in rat plasma. Acetonitrile-methanol (80:20, v/v) and ammonium acetate (10 mol L -1 ) were used as extractant and salting-out reagent to locate the target analytes in the supernatant after the aqueous and organic phase stratification, then the analytes were determined via gradient elution by LC-MS/MS in negative mode in a single run. The analytical method was validated with good selectivity, acceptable accuracy (>85%) and low variation of precision (extraction efficiency of target glucuronide and sulfate conjugates (>80%). The method was successfully applied to determine Res and its four conjugated metabolites in rat after Res administration (intragastric, 50 mg kg -1 ; intravenous, 10 mg kg -1 ). The systemic exposures to Res conjugates were much higher than those to Res (AUC 0-t , i.v., 7.43 μm h; p.o., 8.31 μm h); Res-3-O-β-d-glucuronide was the major metabolite (AUC 0-t , i.v., 66.1 μm h; p.o., 333.4 μm h). The bioavailability of Res was estimated to be ~22.4%. The reproducible SALLE method simplified the sample preparation, drastically improved the accuracy of the concomitant assay and gave full consideration of extraction recovery to each target analyte in bio-samples. Copyright © 2017 John Wiley & Sons, Ltd.

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.

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.

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.

A Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (LC-Q-TOF MS) Study for Analyzing 35 Corticosteroid Compounds: Elucidation of MS/MS Fragmentation Pathways

International Nuclear Information System (INIS)

Noh, Eunyoung; Yoon, Chang-Yong; Lee, Ji Hyun; Baek, Sun-Young; Do, Jung-Ah; Lee, Jung-min; Oh, Han Bin

2016-01-01

Corticosteroids have been often found to be added to a dietary supplement for the purpose of illegally improving the effect of their products. Thus, it is imperative to develop or improve a method that enables one to rapidly and reliably analyze corticosteroids in health or dietary supplements, for the safety management purpose. In the present study, results from liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) experiments for the selected 35 corticosteroid compounds are presented, which can be useful for the qualitative screening of corticosteroids in health or dietary supplements. Specifically, retention times, accurate mass data of the protonated steroids, m/z values of major fragment ions are given for the 35 corticosteroids. Further, fragmentation pathways for the selected steroids are also suggested. Based on the suggested fragmentation pathways, it was shown that an unknown steroid compound can be readily identified using the knowledge of a group of unique and specific common skeletal fragments. The high selectivity and sensitivity of the LC-Q-TOF-MS/MS results combined with the knowledge of the fragmentation pathways can offer a new opportunity for rapid and accurate screening of corticosteroids, thus preventing health-related incidents involving adulterated products and clamping down on illegally circulated health products.

A Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (LC-Q-TOF MS) Study for Analyzing 35 Corticosteroid Compounds: Elucidation of MS/MS Fragmentation Pathways

Energy Technology Data Exchange (ETDEWEB)

Noh, Eunyoung; Yoon, Chang-Yong; Lee, Ji Hyun; Baek, Sun-Young; Do, Jung-Ah [Ministry of Food and Drug Safety, Cheongju (Korea, Republic of); Lee, Jung-min; Oh, Han Bin [Sogang University, Seoul (Korea, Republic of)

2016-07-15

Corticosteroids have been often found to be added to a dietary supplement for the purpose of illegally improving the effect of their products. Thus, it is imperative to develop or improve a method that enables one to rapidly and reliably analyze corticosteroids in health or dietary supplements, for the safety management purpose. In the present study, results from liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) experiments for the selected 35 corticosteroid compounds are presented, which can be useful for the qualitative screening of corticosteroids in health or dietary supplements. Specifically, retention times, accurate mass data of the protonated steroids, m/z values of major fragment ions are given for the 35 corticosteroids. Further, fragmentation pathways for the selected steroids are also suggested. Based on the suggested fragmentation pathways, it was shown that an unknown steroid compound can be readily identified using the knowledge of a group of unique and specific common skeletal fragments. The high selectivity and sensitivity of the LC-Q-TOF-MS/MS results combined with the knowledge of the fragmentation pathways can offer a new opportunity for rapid and accurate screening of corticosteroids, thus preventing health-related incidents involving adulterated products and clamping down on illegally circulated health products.

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

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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.

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.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean

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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.

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.

Study on the determination and chiral inversion of R-salbutamol in human plasma and urine by liquid chromatography-tandem mass spectrometry.

Science.gov (United States)

Zhou, Ting; Zeng, Jing; Liu, Shan; Zhao, Ting; Wu, Jie; Lai, Wenshi; He, Mingzhi; Xu, Beining; Qu, Shanshan; Xu, Ling; Tan, Wen

2015-10-01

The chiral inversion has been a concerned issue during the research and development of a chiral drug. In this study, a sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for determination of salbutamol enantiomers in human plasma and urine. The chiral inversion mechanism of R-salbutamol was fully investigated for the first time by studying the effects of physicochemical factors, including pH, temperature and time. A fitted model to predict the chiral inversion ratio of R-salbutamol was proposed using a Box-Behnken design. All the samples were separated on an Astec Chirobiotic T column and detected by a tandem mass spectrometer in multiple reaction monitoring mode. Lower limit of quantification of 0.100ng/mL was achieved under the optimized conditions. The method was fully validated and successfully applied to the clinical pharmacokinetic study of R-salbutamol in healthy volunteers. Chiral inversion of R-salbutamol to S-salbutamol has been detected in urine samples. The results indicated that pH and temperature were two dominant factors that caused the chiral inversion of R-salbutamol, which should be taken into consideration during the analysis of chiral drugs. The chiral inversion of R-salbutamol determined in this study was confirmed resulted from the gastric acid in stomach rather than caused by the analysis conditions. Moreover, the calculated results of the fitted model matched very well with the enantioselective pharmacokinetic study of R-salbutamol, and the individual difference of the chiral inversion ratio of R-salbutamol was related to the individual gastric environment. On the basis of the results, this study provides important and concrete information not only for the chiral analysis but also for the metabolism research of chiral drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS).

Science.gov (United States)

Kokina, Aija; Pugajeva, Iveta; Bartkevics, Vadims

2016-01-01

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS(3)) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol-water mixture and clean-up by immunoaffinity columns and detection using the MS(3) scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r(2) > 0.998), excellent precision (RSD coffee beans and espresso beverages was 0.010 and 0.003 µg kg(-1), respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018-1.80 µg kg(-1) and 0.020-0.440 µg l(-1), respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg(-1)).

Multivariate DoE Optimization of Asymmetric Flow Field Flow Fractionation Coupled to Quantitative LC-MS/MS for Analysis of Lipoprotein Subclasses

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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.

A surrogate analyte-based LC-MS/MS method for the determination of γ-hydroxybutyrate (GHB) in human urine and variation of endogenous urinary concentrations of GHB.

Science.gov (United States)

Kang, Soyoung; Oh, Seung Min; Chung, Kyu Hyuck; Lee, Sooyeun

2014-09-01

γ-Hydroxybutyrate (GHB) is a drug of abuse with a strong anesthetic effect; however, proving its ingestion through the quantification of GHB in biological specimens is not straightforward due to the endogenous presence of GHB in human blood, urine, saliva, etc. In the present study, a surrogate analyte approach was applied to accurate quantitative determination of GHB in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to overcome this issue. For this, (2)H6-GHB and (13)C2-dl-3-hydroxybutyrate were used as a surrogate standard and as an internal standard, respectively, and parallelism between the surrogate analyte approach and standard addition was investigated at the initial step. The validation results proved the method to be selective, accurate, and precise, with acceptable linearity within calibration ranges (0.1-1μg/ml). The limit of detection and the limit of quantification of (2)H6-GHB were 0.05 and 0.1μg/ml, respectively. No significant variations were observed among urine matrices from different sources. The stability of (2)H6-GHB was satisfactory under sample storage and in-process conditions. However, in vitro production of endogenous GHB was observed when the urine sample was kept under the in-process condition for 4h and under the storage conditions of 4 and -20°C. In order to facilitate the practical interpretation of urinary GHB, endogenous GHB was accurately measured in urine samples from 79 healthy volunteers using the surrogate analyte-based LC-MS/MS method developed in the present study. The unadjusted and creatinine-adjusted GHB concentrations in 74 urine samples with quantitative results ranged from 0.09 to 1.8μg/ml and from 4.5 to 530μg/mmol creatinine, respectively. No significant correlation was observed between the unadjusted and creatinine-adjusted GHB concentrations. The urinary endogenous GHB concentrations were affected by gender and age while they were not significantly influenced by habitual

Analytical Method Details (MS): SE53_MS03 [Metabolonote[Archive

Lifescience Database Archive (English)

Full Text Available SE53_MS03 CE-TOF/MS Agilent CE capillary electrophoresis system,Agilent G3250AA LC/...s were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germ

Enantioselective analysis of citalopram and demethylcitalopram in human whole blood by chiral LC-MS/MS and application in forensic cases

DEFF Research Database (Denmark)

Johansen, Sys Stybe

2017-01-01

OH. The analytes were separated on a 250 x 4.6 mm Chirobiotic V, 5 µm column by isocratic elution with methanol:ammonia:acetic acid (1000:1:1) using an ultra-high-pressure liquid chromatography (UHPLC) system. Quantification was performed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring...... in the positive mode. The total chromatographic run time was 20 min. The limit of detection (LOD) and quantification (LOQ) were 0.001 and 0.005 mg/kg of all four enantiomers, respectively. Linear behaviour was obtained for all four enantiomers from LOQ to 0.50 mg/kg blood with absolute recoveries from 71 to 80......%. The method showed an acceptable precision and accuracy as the obtained coefficient of variation, and bias values were ≤ 16% for all enantiomers. After the validation of the method, a correlation with the racemic method was assessed and found to be acceptable. Then, the method was successfully applied...

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

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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.

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

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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.

LC-IMS-MS Feature Finder: detecting multidimensional liquid chromatography, ion mobility and mass spectrometry features in complex datasets.

Science.gov (United States)

Crowell, Kevin L; Slysz, Gordon W; Baker, Erin S; LaMarche, Brian L; Monroe, Matthew E; Ibrahim, Yehia M; Payne, Samuel H; Anderson, Gordon A; Smith, Richard D

2013-11-01

The addition of ion mobility spectrometry to liquid chromatography-mass spectrometry experiments requires new, or updated, software tools to facilitate data processing. We introduce a command line software application LC-IMS-MS Feature Finder that searches for molecular ion signatures in multidimensional liquid chromatography-ion mobility spectrometry-mass spectrometry (LC-IMS-MS) data by clustering deisotoped peaks with similar monoisotopic mass, charge state, LC elution time and ion mobility drift time values. The software application includes an algorithm for detecting and quantifying co-eluting chemical species, including species that exist in multiple conformations that may have been separated in the IMS dimension. LC-IMS-MS Feature Finder is available as a command-line tool for download at http://omics.pnl.gov/software/LC-IMS-MS_Feature_Finder.php. The Microsoft.NET Framework 4.0 is required to run the software. All other dependencies are included with the software package. Usage of this software is limited to non-profit research to use (see README). rds@pnnl.gov. Supplementary data are available at Bioinformatics online.

Accuracy of the Precision® point-of-care ketone test examined by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in the same fingerstick sample.

Science.gov (United States)

Janssen, Marcel J W; Hendrickx, Ben H E; Habets-van der Poel, Carin D; van den Bergh, Joop P W; Haagen, Anton A M; Bakker, Jaap A

2010-12-01

The Precision(®) (Abbott Diabetes Care) point-of-care biosensor test strips are widely used by patients with diabetes and clinical laboratories for measurement of plasma β-hydroxybutyrate (β-HB) concentrations in capillary blood samples obtained by fingerstick. In the literature, this procedure has been validated only against the enzymatic determination of β-HB in venous plasma, i.e., the method to which the Precision(®) has been calibrated. In this study, the Precision(®) Xceed was compared to a methodologically different and superior procedure: determination of β-HB by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in capillary blood spots. Blood spots were obtained from the same fingerstick sample from out of which Precision(®) measurements were performed. Linearity was tested by adding varying amounts of standard to an EDTA venous whole blood matrix. The Precision(®) was in good agreement with LC-MS/MS within the measuring range of 0.0-6.0 mmol/L (Passing and Bablok regression: slope=1.20 and no significant intercept, R=0.97, n=59). Surprisingly, the Precision(®) showed non-linearity and full saturation at concentrations above 6.0 mmol/L, which were confirmed by a standard addition experiment. Results obtained at the saturation level varied between 3.0 and 6.5 mmol/L. The Precision(®) β-HB test strips demonstrate good comparison with LC-MS/MS. Inter-individual variation around the saturation level, however, is large. Therefore, we advise reporting readings above 3.0 as >3.0 mmol/L. The test is valid for use in the clinically relevant range of 0.0-3.0 mmol/L.

LC clean-up and GC/MS analysis of polycyclic aromatic hydrocarbons in river sediment

International Nuclear Information System (INIS)

Nondek, L.; Kuzilek, M.; Krupicka, S.

1993-01-01

An LC clean-up procedure based upon a complexation between polycyclic aromatic hydrocarbons (PAHs) and silica with chemically bonded 2,4-dinitroaniline has been combined with GC/MS. The LC pre-separation makes it possible to obtain a relatively clean fraction of PAHs free from alkanes, alkylbenzenes and naphthalenes, PCBs, chlorinated pesticides and many other interfering compounds. This fraction has been analyzed using capillary GC and mass selective detector (MSD). Substantial improvement of the MS spectra of PAHs with three or more fused benzene rings is achieved. (orig.)

Identification of adulterants in a Chinese herbal medicine by LC-HRMS and LC-MS-SPE/NMR and comparative in vivo study with standards in a hypertensive rat model.

Science.gov (United States)

Kesting, Julie Regitze; Huang, Jingqi; Sørensen, Dan

2010-02-05

Based on anecdotal evidence of anti-hypertensive effect of Gold Nine Soft Capsules, an in vivo study of this complex Chinese "herbal-based" medicine was initiated. Dosage of the content of Gold Nine capsules in spontaneous hypertensive rats showed a remarkably good effect. This led to further investigation of the components of the preparation and eventual identification of three known anti-hypertensive drugs; amlodipine, indapamide and valsartan, which were not declared on the label. Compounds were rapidly identified using LC-HRMS and LC-MS-SPE/NMR, quantified by HPLC, and the in vivo activity of a combination of commercially purchased standards was shown to be equivalent to that of the capsule content. Adulteration of herbal remedies and dietary supplements with synthetic drugs is an increasing problem that may lead to serious adverse effects. LC-MS-SPE/NMR as a method for the rapid identification of such adulterants is highlighted in this case study.

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.

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.

Analysis of banned veterinary drugs and herbicide residues in shellfish by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and gas chromatography-tandem mass spectrometry (GC/MS/MS)

International Nuclear Information System (INIS)

Chang, Geng-Ruei; Chen, Hui-Shan; Lin, Feng-Yi

2016-01-01

Seafood safety is a crucial public health concern for consumers. In this study, we applied a validated method to analyze the residue of banned veterinary drugs in shellfish, namely chloramphenicol, malachite green, leucomalachite green, and nitrofuran metabolites; additionally, the QuEChERS method was employed to detect 76 herbicides by LC/MS/MS and GC/MS/MS. In total, 42 shellfish samples, which included hard clams, freshwater clams, and oysters, were collected from aquafarms and production areas in Taiwan during 2012. Our results revealed 3.8 ng/g of chloramphenicol in one hard clam, 19.9–32.1 ng/g of ametryn in two hard clams, 16.1–60.1 ng/g of pendimethalin in four hard clams, and 17.0 ng/g of mefenacet in one oyster, indicating that 19.1% of the samples contained residues from banned veterinary drugs and pesticides. These data can be used to monitor the residue of veterinary drugs and pesticides in aquatic organisms and as a reference for food safety. - Highlights: • A certified method was employed for analyzing residues of banned veterinary drugs and herbicides in shellfish samples. • The trace levels of chloramphenicol, ametryn, pendimethalin were detected in hard clam samples. • For ensuring food safety, continual monitoring of aquatic products is necessary.

Identification and characterization by LC-UV-MS/MS of melanotan II skin-tanning products sold illegally on the Internet.

Science.gov (United States)

Breindahl, Torben; Evans-Brown, Michael; Hindersson, Peter; McVeigh, Jim; Bellis, Mark; Stensballe, Allan; Kimergård, Andreas

2015-02-01

New methods were developed and validated to determine the identity, contents, and purity of samples of melanotan II, a synthetic melanocortin receptor agonist, sold in vials as injectable skin-tanning products that were purchased from three online shops. Methods were based on liquid chromatography with ultra-violet detection (LC-UV) at wavelength 218 nm, and tandem mass spectrometric detection (MS/MS) after collision-induced fragmentation of the double charged [M+2H](2+) precursor ion (m/z 513). Identification of melanotan II was verified by correct chromatographic retention time, and relative abundance ratios of five qualifying fragment ions. LC-UV was used to quantify melanotan II as well as impurities. Method validation was performed with reference to guidelines for assessing active substances in authorized medicinal products to reach acceptable accuracy and precision. Vials from two shops contained unknown impurities ranging from 4.1 to 5.9%; impurities from one shop were below the quantification limit. The total amount of melanotan II in vials ranged between 4.32 and 8.84 mg, although each shop claimed that vials contained 10 mg melanotan II. A broad range of drugs used for enhancement purposes can be obtained from the illicit market. However, users of these drugs may be exposed to a range of potential harms, as shown in this study, given that these products are manufactured, distributed and supplied from an illicit market. Copyright © 2014 John Wiley & Sons, Ltd.

Intoxicación letal con aldicarb: análisis de sangre post mortem mediante LC-ESI-MS/MS

Directory of Open Access Journals (Sweden)

Diana Jazmín Mariño Gaviria

2015-07-01

Full Text Available El Aldicarb es un plaguicida carbamato de alta toxicidad asociado a intoxicaciones agudas fatales en Colombia.  El presente estudio describe un método analítico para la determinación cuantitativa de aldicarb en sangre postmortem.  Básicamente el método consiste en una precipitación de proteínas de la sangre para luego ser analizada por LC-ESI-MS/MS utilizando el aldicarb-d3 como estándar interno. El método desarrollado en este trabajo fue aplicado a siete casos de intoxicación letal presuntamente con este plaguicida. El aldicarb se encontró en sangre en niveles desde 0,10 y 2,5 µg/mL. De los resultados obtenidos no sólo se expone la utilidad de emplear LC-ESI-MS/MS en el análisis de aldicarb en sangre para su aplicación en toxicología forense con el propósito de contribuir a la búsqueda de la causa de muerte, sino que además evidencia el empleo de este plaguicida en actos suicidas.

Enantioselective determination of 3-n-butylphthalide (NBP) in human plasma by liquid chromatography on a teicoplanin-based chiral column coupled with tandem mass spectrometry.

Science.gov (United States)

Diao, Xingxing; Ma, Zhiyu; Lei, Peng; Zhong, Dafang; Zhang, Yifan; Chen, Xiaoyan

2013-11-15

A novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine the exposure of 3-n-butylphthalide (NBP) enantiomers in human plasma. The NBP enantiomers were extracted from human plasma using methyl tert-butyl ether. The baseline separation of R-(+)-NBP and S-(-)-NBP was achieved within 11.0min using a teicoplanin-based Astec Chirobiotic T column (250mm×4.6mm i.d., 5μm) under isocratic conditions at a flow rate of 0.6mL/min. The selection of the chiral stationary phase and the effect of the mobile phase composition on the resolution of the enantiomers were discussed. The selectivity, linearity, precision, accuracy, matrix effect, recovery, and stability were evaluated under optimized conditions. The LC-MS/MS method using 200μL of human plasma was linear over the concentration range of 5.00-400ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for both enantiomers was 5.00ng/mL. The intra- and inter-assay precision values of the replicated quality control samples were within 8.0% for each enantiomer. The mean accuracy values for the quality control samples were within ±6.1% of the nominal values for R-(+)-NBP and S-(-)-NBP. No chiral inversion was observed during sample storage, preparation, and analysis. The method proved suitable for enantioselective pharmacokinetic studies of NBP after an oral administration of a therapeutic dose of racemic NBP. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Asymmetric polymerisation in liquid crystals and resultant electro-chiroptical effect: Structure organising polymerisation and chiral charge carrier ''chiralion''

International Nuclear Information System (INIS)

Goto, Hiromasa

2014-01-01

Electrochemical synthesis in liquid crystal (LC) affords conducting polymers having LC molecular order and electro-activity. The polymerisation method can be referred to as structure organising polymerisation (SOP). The optical textures of the polymers thus prepared appear very similar to that of the LC electrolyte solution used for the polymerisation. Especially, polymers prepared in cholesteric LC (chiral LC) having structural chirality show doping-dedoping (redox) driven change in chiroptical activity (controllable circular dichroism and optical rotation), as e lectro-chiroptical effect . The polymer films exhibit interference colour and electrochemically driven refractive index modulations. The chiroptical activity of the polymer prepared in cholesteric LC comes from axial chirality of the helical structure

Determination of vandetanib in human plasma and cerebrospinal fluid by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).

Science.gov (United States)

Bai, Feng; Johnson, Jennifer; Wang, Fan; Yang, Lei; Broniscer, Alberto; Stewart, Clinton F

2011-09-01

A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [(13)C,d(3)]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid-liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μm, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate=50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC-MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25-50 ng/mL (R(2) ≥ 0.990) for the CSF curve and from 1.0 to 3000 ng/mL (R(2) ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤ 8.8% and ≤ 5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF=0.98, %CV ≤ 4.97, n=6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib. Copyright © 2011 Elsevier B.V. All rights reserved.

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)

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.

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.

Analysis of skin derived peptides from the Cuyaba Dwarf Frog Physalaemus nattereri by off-line LC MALDI MS/MS

DEFF Research Database (Denmark)

Castro, Mariana S; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner

2017-01-01

We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has...... for future studies of peptides secreted from frogs....

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.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT -MS/MS.

Science.gov (United States)

Bajpai, Vikas; Singh, Awantika; Chandra, Preeti; Negi, M P S; Kumar, Nikhil; Kumar, Brijesh

2016-01-01

The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers. Copyright © 2015 John Wiley & Sons, Ltd.

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.

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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 .

ANALYSIS OF SULFONATES IN AQUEOUS SAMPLES BY ION-PAIR LC/ESI-MS/MS WITH IN-SOURCE CID FOR ADDUCT PEAK ELIMINATION

Energy Technology Data Exchange (ETDEWEB)

OUYANG,S.; VAIRAVAMURTHY,M.A.

1999-06-13

Determination of low-molecular-weight organic sulfonates (e.g. taurine and cysteic acid) in aqueous solutions is important in many applications of biological, environmental and pharmaceutical sciences. These compounds are difficult to be determined by commonly used reversed-phase liquid chromatographic separation combined with UV-Visible detection because of their high solubility and the lack chromophoric moieties. Here the authors report a method combining ion-pair liquid chromatography and electrospray ionization tandem mass spectrometry (IPLC/ESI-MS/MS)for determining sulfonates. The ability of low-molecular-weight sulfonates to form ion-pairs with quaternary ammonium cations in aqueous solutions allowed LC separation with a C{sub 18} column. Detection of the sulfonates was accomplished with ESI-MS that lends a universal mode of mass detection for polar, water soluble compounds. An in-source collision induced dissociation (CID) was applied to eliminate the adduct peaks in mass spectra. Characteristic marker ions showed in the second stage mass spectra lent a method for identifying sulfonates.

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...

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.

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.

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.

Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Science.gov (United States)

Roberts, Michael S; Turner, David C; Owens, Thandranese S; Ramachandran, Abhijit; Wetmore, Cynthia; Throm, Stacy L; Stewart, Clinton F

2013-06-15

A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50μl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3μm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

NARCIS (Netherlands)

Kretschmer, A.; Giera, M.A.; Wijtmans, M.; de Vries, L.; Lingeman, H.; Irth, H.; Niessen, W.M.A.

2011-01-01

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The

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.

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.

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.

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...

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...

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

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.

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.

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

Full Text Available Abstract Background Mass spectrometry (MS based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007 described a modified spectral counting technique, Absolute Protein Expression (APEX, which improves on basic spectral counting methods by including a correction factor for each protein (called Oi value that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (Oi. This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a

Application of LC-ESI-MS-MS for detection of synthetic adulterants in herbal remedies.

Science.gov (United States)

Bogusz, Maciej J; Hassan, Huda; Al-Enazi, Eid; Ibrahim, Zuhour; Al-Tufail, Mohammed

2006-05-03

Adulteration of allegedly "natural herbal medicines" with undeclared synthetic drugs is a common and dangerous phenomenon of alternative medicine. The purpose of the study was to develop a procedure for detection of most common synthetic adulterants in herbal remedies, using high-pressure liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS-MS). Eighty drugs belonging to various pharmacological classes were included in the study. For most drugs two transitions were monitored, using protonated or deprotonated molecules as precursor ions. The drugs were isolated from herbal remedies using simple methanol extraction. Chromatographic separation was done in gradient of acetonitrile-10 mM ammonium formate buffer (pH 3.0). Drugs tested were grouped in suites, comprising analgesic drugs, antibiotics, antidiabetic drugs, antiepileptic drugs, aphrodisiacs, hormones and anabolic drugs, psychotropic drugs, and weight reducing compounds. These suites were used according to the declared benefits of examined preparations. Limits of detection ranged from 5 pg to 1 ng per injected sample. Drug-free herbal remedy spiked with eight various pharmaceuticals occurring in adulterated herbal preparations was used for internal proficiency testing. The recoveries of spiked drugs ranged from 63 to 100%. The procedure was applied in everyday casework. Several undeclared drugs were identified in "herbal" remedies, like e.g. sildenafil, tadalafil, testosterone, or glibenclamide. Pharmacological properties of detected drugs always corresponded with the claims of the "natural" remedies. The method presents a valuable extension of standard GC-MS screening used for this purpose.

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.

LC-MS/MS methods for sulfadimethoxine and ormetoprim analysis in feed and fish fillet and a leaching study for feed after alternative procedures for the incorporation of drugs.

Science.gov (United States)

Fais, Ana Paula; Franco, Rodolfo Scarpino Barboza; da Silva, Agnaldo Fernando Baldo; de Freitas, Osvaldo; Paschoal, Jonas Augusto Rizzato

2017-04-01

This paper describes the method development for sulfadimethoxine (SDM) and ormetoprim (OMP) quantitation in fish feed and fish fillet employing LC-MS/MS. In order to assess the reliability of the analytical method, valuation was undertaken as recommended by guidelines proposed by the Brazilian Ministry of Agriculture. The calibration curve for the quantification of both drugs in feed showed adequate linearity (r > 0.99), precision (CV  0.99), precision (CV < 16%) and trueness around 100%, with CCα < 100.2 ng g - 1 and CCβ < 100.4 ng g - 1 . With a goal of avoiding the risk of drug leaching from feed into the aquatic environment during fish medication via the oral route, different procedures for drug incorporation into feed were evaluated. Coating feed pellets with ethyl cellulose polymer containing the drug showed promising results. In this case, medicated feed released drugs to water at a level below 6% when the medicated feed stayed in the water for up to 15 min.

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

Development and validation of an LC-MS/MS method to quantify lysergic acid diethylamide (LSD), iso-LSD, 2-oxo-3-hydroxy-LSD, and nor-LSD and identify novel metabolites in plasma samples in a controlled clinical trial

OpenAIRE

Dolder, Patrick C.; Liechti, Matthias E.; Rentsch, Katharina M.

2018-01-01

Lysergic acid diethylamide (LSD) is a widely used recreational drug. The aim of this study was to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of LSD, iso-LSD, 2-oxo-3-hydroxy LSD (O-H-LSD), and nor-LSD in plasma samples from 24 healthy subjects after controlled administration of 100 μg LSD in a clinical trial. In addition, metabolites that have been recently described in in vitro studies, including lysergic acid monoethylamide...

Development and validation of a rapid turboflow LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD in serum and urine samples of emergency toxicological cases.

Science.gov (United States)

Dolder, Patrick C; Liechti, Matthias E; Rentsch, Katharina M

2015-02-01

Lysergic acid diethylamide (LSD) is a widely used recreational drug. The aim of the present study is to develop a quantitative turboflow LC-MS/MS method that can be used for rapid quantification of LSD and its main metabolite 2-oxo-3-hydroxy LSD (O-H-LSD) in serum and urine in emergency toxicological cases without time-consuming extraction steps. The method was developed on an ion-trap LC-MS/MS instrument coupled to a turbulent-flow extraction system. The validation data showed no significant matrix effects and no ion suppression has been observed in serum and urine. Mean intraday accuracy and precision for LSD were 101 and 6.84%, in urine samples and 97.40 and 5.89% in serum, respectively. For O-H-LSD, the respective values were 97.50 and 4.99% in urine and 107 and 4.70% in serum. Mean interday accuracy and precision for LSD were 100 and 8.26% in urine and 101 and 6.56% in serum, respectively. For O-H-LSD, the respective values were 101 and 8.11% in urine and 99.8 and 8.35% in serum, respectively. The lower limit of quantification for LSD was determined to be 0.1 ng/ml. LSD concentrations in serum were expected to be up to 8 ng/ml. 2-Oxo-3-hydroxy LSD concentrations in urine up to 250 ng/ml. The new method was accurate and precise in the range of expected serum and urine concentrations in patients with a suspected LSD intoxication. Until now, the method has been applied in five cases with suspected LSD intoxication where the intake of the drug has been verified four times with LSD concentrations in serum in the range of 1.80-14.70 ng/ml and once with a LSD concentration of 1.25 ng/ml in urine. In serum of two patients, the O-H-LSD concentration was determined to be 0.99 and 0.45 ng/ml. In the urine of a third patient, the O-H-LSD concentration was 9.70 ng/ml.

Development of a rapid and sensitive LC-ESI/MS/MS assay for the quantification of propofol using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity.

Science.gov (United States)

Beaudry, Francis; Guénette, Sarah Annie; Winterborn, Andrew; Marier, Jean-Francois; Vachon, Pascal

2005-09-15

A rapid, selective and sensitive method was developed for the determination of propofol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by liquid chromatography ionspray tandem mass spectrometry (LC-ESI/MS/MS). The separation was achieved using a 100 mm x 2 mm C8 analytical column combined with an isocratic mobile phase composed of 80:20 acetonitrile: 0.5% formic acid in water. Signal intensity of the propofol-dansyl chloride derivative was increased up to 200-fold as compared to the underivatized propofol in positive electrospray mode. An analytical range of 20-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. A pharmacokinetic study was performed in rats and the novel analytical method was used as a routine analysis to provide enhanced measurements of plasma and blood concentrations of propofol. Blood and plasma pharmacokinetic results show that a very important fraction of propofol distributes into red blood cells. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of propofol. Routine analysis with the novel method provided accurate results and enhanced the detection levels of plasma and blood concentrations of propofol to better characterize the in vivo biodisposition of propofol.

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

Pistachio oil (Pistacia vera L. cv. Uzun): Characterization of key odorants in a representative aromatic extract by GC-MS-olfactometry and phenolic profile by LC-ESI-MS/MS.

Science.gov (United States)

Sonmezdag, Ahmet Salih; Kelebek, Hasim; Selli, Serkan

2018-02-01

Volatile, aroma-active, and phenolic compounds of pistachio oil obtained from cv. Uzun were investigated in the current study. To obtain a representative aromatic extract, three of the most widely used extraction methods were compared using a representative test; the solvent-assisted flavour extraction (SAFE) aromatic extract from pistachio oil was found to be the most representative. A total of 50 aroma compounds were determined in pistachio oil and it was found that terpenes, aldehydes, and alcohols were the most abundant volatile compounds. Applying GC-MS-olfactometry and aroma extract dilution analysis (AEDA) resulted in a total of 14 aroma-active areas being detected in the extract of pistachio oil. In the phenolic fraction obtained by the LC-ESI-MS/MS method, a total of 12 phenolic compounds was found in the pistachio oil, of which seven compounds were reported for the first time. Eriodictyol-7-O-glucoside and protocatechuic acid were the most dominant phenolic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

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 ).

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.

A novel screening method for 64 new psychoactive substances and 5 amphetamines in blood by LC-MS/MS and application to real cases.

Science.gov (United States)

Vaiano, Fabio; Busardò, Francesco P; Palumbo, Diego; Kyriakou, Chrystalla; Fioravanti, Alessia; Catalani, Valeria; Mari, Francesco; Bertol, Elisabetta

2016-09-10

Identification and quantification of new psychoactive substances (NPS), both in biological and non-biological samples, represent a hard challenge for forensic toxicologists. NPS are increasingly emerging on illegal drug market. Many cases of co-consumption of NPS and other substances have also been reported. Hence, the development of analytical methods aiming at the detection of a broad-spectrum of compounds (NPS and "traditional" drugs) could be helpful. In this paper, a fully validated screening method in blood for the simultaneous detection of 69 substances, including 64 NPS (28 synthetic cannabinoids, 19 synthetic cathinones, 5 phenethylamines, 3 indanes, 2 piperazines, 2 tryptamines, 2 phencyclidine, methoxetamine, ketamine and its metabolite) and 5 amphetamines (amphetamine, methamphetamine, MDMA, MDA, 3,4-methylenedioxy-N-ethylamphetamine - MDEA-) by a dynamic multiple reaction monitoring analysis through liquid chromatography - tandem mass spectrometry (LC-MS/MS) is described. This method is very fast, easy to perform and cheap as it only requires the deproteinization of 200μL of blood sample with acetonitrile. The chromatographic separation is achieved with a C18 column. The analysis is very sensitive, with limits of quantification ranging from 0.1 to 0.5ng/mL. The method is linear from 1 to 100ng/mL and the coefficient of determination (R(2)) was always above 0.9900. Precision and accuracy were acceptable at any quality control level and recovery efficiency range was 72-110%. Matrix effects did not negatively affect the analytical sensitivity. This method was successfully applied to three real cases, allowing identification and quantification of: mephedrone and methamphetamine (post-mortem); ketamine, MDMA and MDA (post-mortem); AB-FUBINACA (ante-mortem). Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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.

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.

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

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.

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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.

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.

Analytical Method Details (MS): SE52_MS01 [Metabolonote[Archive

Lifescience Database Archive (English)

Full Text Available stem and Waters Q-Tof Premier UPLC-QTOF-MS ESI Negative The eluates (3 μl) were subsequently subjected to metabolome...DS column (LC-ESI-Q-Tof/MS, HPLC: Waters Acquity UPLC system; MS: Waters Q-Tof Premier). The metabolome...d (Matsuda et al., 2009c, 2010a). Briefly, the metabolome data were obtained in the negative ion mode (m/z 1... the complex nature of the metabolome data. To construct MS2T libraries, the extracts of five ecotypes were

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

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.

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

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.

Monitoring of drug intake during pregnancy by questionnaires and LC-MS/MS drug urine screening: evaluation of both monitoring methods.

Science.gov (United States)

Hoeke, Henrike; Roeder, Stefan; Bertsche, Thilo; Lehmann, Irina; Borte, Michael; von Bergen, Martin; Wissenbach, Dirk K

2015-08-01

Various studies pointed towards a relationship between chronic diseases such as asthma and allergy and environmental risk factors, which are one aspect of the so-called Exposome. These environmental risk factors include also the intake of drugs. One critical step in human development is the prenatal period, in which exposures might have critical impact on the child's health outcome. Thereby, the health effects of drugs taken during gestation are discussed controversially with regard to newborns' disease risk. Due to this, the drug intake of pregnant women in the third trimester was monitored by questionnaire, in addition to biomonitoring using a local birth cohort study, allowing correlations of drug exposure with disease risk. Therefore, 622 urine samples were analyzed by an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening and the results were compared to self-administered questionnaires. In total, 48% (n = 296) reported an intake of pharmaceuticals, with analgesics as the most frequent reported drug class in addition to dietary supplements. 182 times compounds were detected by urine screening, with analgesics (42%; n = 66) as the predominantly drug class. A comparison of reported and detected drug intake was performed for three different time spans between completion of the questionnaires and urine sampling. Even if the level of accordance was low in general, similar percentages (~25%, ~19%, and ~ 20%) were found for all groups. This study illustrates that a comprehensive evaluation of drug intake is neither achieved by questionnaires nor by biomonitoring alone. Instead, a combination of both monitoring methods, providing complementary information, should be considered. Copyright © 2014 John Wiley & Sons, Ltd.

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

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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.

Development of a fast isocratic LC-MS/MS method for the high-throughput analysis of pyrrolizidine alkaloids in Australian honey.

Science.gov (United States)

Griffin, Caroline T; Mitrovic, Simon M; Danaher, Martin; Furey, Ambrose

2015-01-01

Honey samples originating from Australia were purchased and analysed for targeted pyrrolizidine alkaloids (PAs) using a new and rapid isocratic LC-MS/MS method. This isocratic method was developed from, and is comparable with, a gradient elution method and resulted in no loss of sensitivity or reduction in chromatographic peak shape. Isocratic elution allows for significantly shorter run times (6 min), eliminates the requirement for column equilibration periods and, thus, has the advantage of facilitating a high-throughput analysis which is particularly important for regulatory testing laboratories. In excess of two hundred injections are possible, with this new isocratic methodology, within a 24-h period which is more than 50% improvement on all previously published methodologies. Good linear calibrations were obtained for all 10 PAs and four PA N-oxides (PANOs) in spiked honey samples (3.57-357.14 µg l(-1); R(2) ≥ 0.9987). Acceptable inter-day repeatability was achieved for the target analytes in honey with % RSD values (n = 4) less than 7.4%. Limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked PAs and PANOs samples; giving an average LOD of 1.6 µg kg(-1) and LOQ of 5.4 µg kg(-1). This method was successfully applied to Australian and New Zealand honey samples sourced from supermarkets in Australia. Analysis showed that 41 of the 59 honey samples were contaminated by PAs with the mean total sum of PAs being 153 µg kg(-1). Echimidine and lycopsamine were predominant and found in 76% and 88%, respectively, of the positive samples. The average daily exposure, based on the results presented in this study, were 0.051 µg kg(-1) bw day(-1) for adults and 0.204 µg kg(-1) bw day(-1) for children. These results are a cause for concern when compared with the proposed European Food Safety Authority (EFSA), Committee on Toxicity (COT) and Bundesinstitut für Risikobewertung (BfR - Federal Institute of Risk Assessment Germany) maximum

Analytical Method Details (MS): SE59_MS01 [Metabolonote[Archive

Lifescience Database Archive (English)

Full Text Available argeted metabolomic analysis using LC-QTOF-MS and the metabolome data processing of alignment, de-isotoping, noise cutting, and normalization was conducted as previously described. ...

Sensitive Determination of Onco-metabolites of D- and L-2-hydroxyglutarate Enantiomers by Chiral Derivatization Combined with Liquid Chromatography/Mass Spectrometry Analysis

Science.gov (United States)

Cheng, Qing-Yun; Xiong, Jun; Huang, Wei; Ma, Qin; Ci, Weimin; Feng, Yu-Qi; Yuan, Bi-Feng

2015-01-01

2-hydroxyglutarate (2HG) is a potent competitor of α-ketoglutarate (α-KG) and can inhibit multiple α-KG dependent dioxygenases that function on the epigenetic modifications. The accumulation of 2HG contributes to elevated risk of malignant tumors. 2HG carries an asymmetric carbon atom in its carbon backbone and differentiation between D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) is crucially important for accurate diagnosis of 2HG related diseases. Here we developed a strategy by chiral derivatization combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive determination of D-2HG and L-2HG enantiomers. N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was used to derivatize 2HG. The formed diastereomers by TSPC labeling can efficiently improve the chromatographic separation of D-2HG and L-2HG. And derivatization by TSPC could also markedly increase the detection sensitivities by 291 and 346 folds for D-2HG and L-2HG, respectively. Using the developed method, we measured the contents of D-2HG and L-2HG in clear cell renal cell carcinoma (ccRCC) tissues. We observed 12.9 and 29.8 folds increase of D-2HG and L-2HG, respectively, in human ccRCC tissues compared to adjacent normal tissues. The developed chiral derivatization combined with LC-ESI-MS/MS analysis offers sensitive determination of D-2HG and L-2HG enantiomers, which benefits the precise diagnosis of 2HG related metabolic diseases. PMID:26458332

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.

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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.

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.

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.

AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

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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.

LC-MS/MS methods for albendazole analysis in feed and its metabolite residues in fish fillet and a leaching study in feed after an alternative procedure for drug incorporation.

Science.gov (United States)

Busatto, Zenaís; da Silva, Agnaldo Fernando Baldo; de Freitas, Osvaldo; Paschoal, Jonas Augusto Rizzato

2017-04-01

This paper describes the development of analytical methods for the quantification of albendazole (ABZ) in fish feed and ABZ and its main known metabolites (albendazole sulfoxide, albendazole sulfone and albendazole aminosulfone) in fish fillet employing LC-MS/MS. In order to assess the reliability of the analytical methods, evaluation was undertaken as recommended by related guides proposed by the Brazilian Ministry of Agriculture for analytical method validation. The calibration curve for ABZ quantification in feed showed adequate linearity (r > 0.99), precision (CV  0.99) for all analytes, precision (CV < 13%) and trueness around 100%, with CCα < 122 ng g - 1 and CCβ < 145 ng g - 1 . Besides, by aiming to avoid the risk of ABZ leaching from feed into the aquatic environment during fish medication via the oral route, a promising procedure for drug incorporation in the feed involving coating feed pellets with ethyl cellulose polymer containing ABZ was also evaluated. The medicated feed had good homogeneity (CV < 3%) and a lower release of ABZ (< 0.2%) from feed to water when the medicated feed stayed in the water for up to 15 min.

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.

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.

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.

A comparative tissue-specific metabolite analysis and determination of protodioscin content in Asparagus species used in traditional Chinese medicine and Ayurveda by use of laser microdissection, UHPLC-QTOF/MS and LC-MS/MS.

Science.gov (United States)

Jaiswal, Yogini; Liang, Zhitao; Ho, Alan; Chen, Hubiao; Zhao, Zhongzhen

2014-01-01

Asparagus is esteemed in Traditional Chinese Medicine and Ayurveda, and it is commercially one of the most important drugs in the global herbal market. Comparative metabolite profiling of different species would help in determining the similarities and ascertain their validity for being used as substitutes for each other. Laser microdissection (LMD) facilitates identification of metabolites in specific tissues, and thus it can aid in exploration of metabolic pathways in target tissues. To compare tissue-specific metabolites and protodioscin content of Asparagus cochinchinensis (Lour.) Merr. and Asparagus racemosus Willd. used in China and India. Metabolite analysis of laser-dissected tissues was carried out using UHPLC-QTOF/MS and LC-MS/MS. The protodioscin contents were determined and the method was validated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. Metabolite analysis reveals that the velamen tissue, among other tissues such as cortex, vascular bundles and pith, contained maximum components, specifically those belonging to the steroidal saponin class. Although the metabolite profiles were similar, the content of protodioscin was found to be higher in Chinese than Indian species. The study provided a suitable methodology for metabolite profiling and protodioscin content determination of Asparagus by use of LMD, UHPLC-QTOF/MS and LC-MS/MS. The similarities in metabolite profiles indicate that Asparagus species from India and China can serve as substitute for each other in various therapeutic and pharmaceutical applications. Copyright © 2014 John Wiley & Sons, Ltd.

Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies.

Science.gov (United States)

Chhonker, Yashpal S; Sleightholm, Richard L; Li, Jing; Oupický, David; Murry, Daryl J

2018-01-01

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50×4.6mm, 3μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. Copyright © 2017 Elsevier B.V. All rights reserved.

The validation & verification of an LC/MS method for the determination of total docosahexaenoic acid concentrations in canine blood serum.

Science.gov (United States)

Dillon, Gerald Patrick; Keegan, Jason D; Wallace, Geoff; Yiannikouris, Alexandros; Moran, Colm Anthony

2018-06-01

Docosahexaenoic acid (DHA), is an omega 3 fatty acid (n-3 FA) that has been shown to play a role in canine growth and physiological integrity and improvements in skin and coat condition. However, potential adverse effects of n-3 FA specifically, impaired cellular immunity has been observed in dogs fed diets with elevated levels of n-3 FA. As such, a safe upper limit (SUL) for total n-3 FAs (DHA and EPA) in dogs has been established. Considering this SUL, sensitive methods detecting DHA in blood serum as a biomarker when conducting n-3 FA supplementation trials involving dogs are required. In this study, an LC-ESI-MS/MS method of DHA detection in dog serum was validated and verified. Recovery of DHA was optimized and parallelism tests were conducted with spiked samples demonstrating that the serum matrix did not interfere with quantitation. The stability of DHA in serum was also investigated, with -80 °C considered suitable when storing samples for up to six months. The method was linear over a calibration range of 1-500 μg/mL and precision and accuracy were found to meet the requirements for validation. This method was verified in an alternative laboratory using a different analytical system and operator, with the results meeting the criteria for verification. Copyright © 2018. Published by Elsevier Inc.

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...

APPLICATION OF DNPH DERIVATIZATION WITH LC/MS TO THE IDENTIFICATION OF POLAR CARBONYL DRINKING WATER DISINFECTION BY-PRODUCTS IN DRINKING WATER

Science.gov (United States)

A qualitative method using 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by analysis with liquid chromatography (LC)/negative ion-electrospray mass spectrometry (MS) was developed for analyzing and identifying highly polar aldehydes and ketones in ozonated drinking wa...

Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.

Science.gov (United States)

Li, Lingyun; Zhang, Fuming; Zaia, Joseph; Linhardt, Robert J

2012-10-16

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

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

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.

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.

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...

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

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.

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.

Authenticity of raspberry flavor in food products using SPME-chiral-GC-MS.

Science.gov (United States)

Hansen, Anne-Mette S; Frandsen, Henrik L; Fromberg, Arvid

2016-05-01

A fast and simple method for authenticating raspberry flavors from food products was developed. The two enantiomers of the compound (E)-α-ionone from raspberry flavor were separated on a chiral gas chromatographic column. Based on the ratio of these two enantiomers, the naturalness of a raspberry flavor can be evaluated due to the fact that a natural flavor will consist almost exclusively of the R enantiomer, while a chemical synthesis of the same compound will result in a racemic mixture. Twenty-seven food products containing raspberry flavors where investigated using SPME-chiral-GC-MS. We found raspberry jam, dried raspberries, and sodas declared to contain natural aroma all contained almost only R-(E)-α-ionone supporting the content of natural raspberry aroma. Six out of eight sweets tested did not indicate a content of natural aroma on the labeling which was in agreement with the almost equal distribution of the R and S isomer. Two products were labeled to contain natural raspberry flavors but were found to contain almost equal amounts of both enantiomers indicating a presence of synthetic raspberry flavors only. Additionally, two products that were labeled to contain both raspberry juice and flavor showed equal amounts of both enantiomers, indicating the presence of synthetic flavor.

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.

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 (...

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.

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.

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.

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.

Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

Energy Technology Data Exchange (ETDEWEB)

Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; Moore, Ronald J.; Lim, Sujung; Orphan, Victoria J.; Paša-Tolić, Ljiljana; Qian, Wei-Jun; Smith, Richard D.; Kelly, Ryan T.

2018-04-01

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.

Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-IT-TOF MS and study of their negative-ion fragmentation regularities.

Science.gov (United States)

Yu, Xu; Wang, Fan; Li, Jiani; Shan, Weiguang; Zhu, Bingqi; Wang, Jian

2017-06-05

Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS)with positive and negative modes of electrospray ionization method for further improvement of official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the negative ion mode were studied in detail, and new negative-ion fragmentation regularities were discovered. Chromatographic separation was performed on a Kromasil C18 column (250mm×4.6mm, 5μm). The mobile phase consisted of (A) ammonium formate aqueous solution (10mM)-methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10mM)-methanol (47:53, v/v). In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full scan LC-MS in both positive and negative ion modes was firstly executed to obtain the m/z value of the molecules. Then LC-MS 2 and LC-MS 3 were carried out on target compounds to obtain as much structural information as possible. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of these impurities. Structures of thirteen unknown degradation products in flomoxef sodium were deduced based on the high resolution MS n data with both positive and negative modes. The forming mechanisms of degradation products in flomoxef sodium were also studied. Copyright © 2017. Published by Elsevier B.V.

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.

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%.

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.

Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS

DEFF Research Database (Denmark)

Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte

2016-01-01

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were...

LC-ESI-MS/MS determination of 4-methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs.

Science.gov (United States)

Chandrasekhar, Devaraj V; Suresh, Ponnayyan Sulochana; Dittakavi, Sreekanth; Hiremath, Rakesh A; Bhamidipati, Ravi Kanth; Richter, Wolfgang; Srinivas, Nuggehally R; Mullangi, Ramesh

2018-02-01

A simple, specific, sensitive and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of 4-methylpyrazole in dog plasma using N-methylnicotinamide-d 4 as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4-methylpyrazole and the IS was performed on a monolithic (Chromolith RP 18e ) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4-methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96-4955 ng/mL. The intra- and inter-day accuracy and precision were in the ranges 1.81-12.9 and 3.80-11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC-ESI-MS/MS and flow-injection-ESI-MS/MS.

Science.gov (United States)

John, Harald; Eddleston, Michael; Clutton, R Eddie; Worek, Franz; Thiermann, Horst

2010-05-15

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC-ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD(intra-day) 1-8%; RSD(inter-day) 5-14%), accurate (intra-day: 95-107%; inter-day: 90-115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24-0.49 microg/ml plasma and 0.78-1.56 microg/ml urine) and lower limits of detection (0.12-0.24 microg/ml plasma and 0.39-0.78 microg/ml urine) were equivalent to quite low absolute on-column amounts (1.1-2.1 pg for plasma and 2.0-3.9 pg for urine). The calibration range (0.24-250 microg/ml plasma and 0.78-200 microg/ml urine) was subdivided into two linear ranges (r(2)>or=0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the

High-Throughput LC-MS/MS Method for Direct Quantification of Glucuronidated, Sulfated and Free Enterolactone in Human Plasma

DEFF Research Database (Denmark)

Nørskov, Natalja; Kyrø, Cecilie; Olsen, Anja

2016-01-01

Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized that ente......Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized......−MS/MS and a fluoroimmunoassay; however, most of these methods measure the total concentration of enterolactone, without any specification of its conjugation pattern. Here for the first time we present a high-throughput LC−MS/MS method to quantify enterolactone in its intact form as glucuronide, sulfate, and free enterolactone....... The method has shown good accuracy and precision at low concentration and very high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM, and free enterolactone at 86 pM. The short run time of 2.6 min combined with simple sample clean up and high sensitivity make...

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...

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.

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...

Solid phase microextraction headspace sampling of chemical warfare agent contaminated samples : method development for GC-MS analysis

Energy Technology Data Exchange (ETDEWEB)

Jackson Lepage, C.R.; Hancock, J.R. [Defence Research and Development Canada, Medicine Hat, AB (Canada); Wyatt, H.D.M. [Regina Univ., SK (Canada)

2004-07-01

Defence R and D Canada-Suffield (DRDC-Suffield) is responsible for analyzing samples that are suspected to contain chemical warfare agents, either collected by the Canadian Forces or by first-responders in the event of a terrorist attack in Canada. The analytical techniques used to identify the composition of the samples include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy. GC-MS and LC-MS generally require solvent extraction and reconcentration, thereby increasing sample handling. The authors examined analytical techniques which reduce or eliminate sample manipulation. In particular, this paper presented a screening method based on solid phase microextraction (SPME) headspace sampling and GC-MS analysis for chemical warfare agents such as mustard, sarin, soman, and cyclohexyl methylphosphonofluoridate in contaminated soil samples. SPME is a method which uses small adsorbent polymer coated silica fibers that trap vaporous or liquid analytes for GC or LC analysis. Collection efficiency can be increased by adjusting sampling time and temperature. This method was tested on two real-world samples, one from excavated chemical munitions and the second from a caustic decontamination mixture. 7 refs., 2 tabs., 3 figs.

Characterization of foot-and-mouth disease virus's viral peptides with LC-ESI-MS

International Nuclear Information System (INIS)

Pzdemir, Z.O.; Bulut, E.K.; Mustafeva, Z.; Karahan, M.

2010-01-01

Peptides and proteins play a central role in numerous biological and physiological processes in living organisms. Viral capsid peptides are part of the viruses' outer shell of genetic materials. Viruses are recognized by immune system via capsid peptides. Depending on this property of capsid peptides, prototypes synthetic peptide-based vaccine can be developed. In this work, we synthesized three different viral peptide sequences of foot-and-mouth disease virus with microwave enhanced solid phase synthesis method. These peptides were characterized by using liquid chromatography electro spray interface mass spectrometry (LC-ESI-MS) with electro spray ionization. We briefly describe the essential facts for peptide characterization. (author)