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Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

ABSTRACT: Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes.

SUBMITTER: Huang K

PROVIDER: S-EPMC4487545 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

Huang Ke K Huang Lingyi L van Breemen Richard B RB

Analytical chemistry 20150325 7

Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS ...[more]

PMID: 25774910

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Project description:BackgroundPlant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation.ResultsThis study established an optimized and advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6-60 and 0.3-30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5-93.4%, with intra- and inter-day precisions of 5.5-9.1 and 4.2-10.2%, respectively.ConclusionsThe assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.

Dataset Information

Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

Publications

Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

Similar Datasets

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