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High‐throughput screening for glutathione conjugates using stable‐isotope labeling and negative electrospray ionization precursor‐ion mass spectrometry
Author(s) -
Liao Shengkai,
Ewing Nigel P.,
Boucher Brian,
Materne Olivier,
Brummel Christopher L.
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6135
Subject(s) - chemistry , glutathione , electrospray ionization , metabolite , mass spectrometry , ion suppression in liquid chromatography–mass spectrometry , adduct , electrospray , chromatography , isotope , tandem mass spectrometry , biochemistry , organic chemistry , enzyme , physics , quantum mechanics
RATIONALE It has been proposed that the increase in the instances of idiosyncratic adverse drug reactions (IADRs) and black box warnings may be attributed to the occurrence of reactive metabolites. Consequently, a high‐throughput screen for reactive metabolites formed from liver microsome extracts with added glutathione (GSH) was developed for use in the early stages of drug discovery. METHODS To enhance sensitivity and specificity, as well as accelerate data processing, a mixture of a stable‐isotope probe consisting of natural GSH (light GSH) and stable‐isotope‐labeled [ 15 N, 13 C 2 ] GSH (heavy GSH) at a ratio of 1:1 was used. Any metabolite that reacted with the GSH results in the formation of light and heavy GSH conjugates with a 3 Da difference. Employing a precursor‐ion scan using negative ion electrospray ionization (ESI) corresponding to the expected fragments, signals with the appropriate ratio in the precursor ion scan are then further examined. RESULTS The new method greatly simplifies data collection by assuming molecules containing GSH will fragment to form specific ions. As such, this approach accelerates data processing (and collection) at the risk of missing compounds that do not fragment as expected. The assay was validated with 33 diverse drugs known to form GSH conjugates, 5 drugs known to not form GSH adducts and over 100 samples containing components of the normal in vitro matrix. In all cases data collected matched the expected result. CONCLUSIONS The observed sensitivity, specificity, and fast data processing make this assay an excellent fit for high‐throughput screening of reactive metabolites in the early stages of drug discovery. This method is not intended to eliminate compounds or terminate their development. Instead, it is to bring forward molecules with one less liability and thus a greater probability of ultimate success. Copyright © 2012 John Wiley & Sons, Ltd.