The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 4 - Improved detection of reactive drug metabolites with bromine-containing glutathione analog using mass defect and isotope pattern matching
Lekha Sleno; Andre LeBlanc; Tze Chieh Shiao; Rene Roy
UQAM, Montreal, Canada
Introduction: Reactive metabolite detection represents a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them in vitro with glutathione followed by LC-MS analysis. GSH adducts can then be screened by neutral loss and precursor ion scanning, or mass spectral filtering using the mass defect of the supposed adduct. Certain methods have used analogs of GSH with fluorescent tags, quaternary amines or stable-isotope labels in order to increase sensitivity or decrease false positive rates. This presentation will describe the use of a brominated analog of GSH for determining reactive metabolites by LC-MS.
Methods: Rat microsomal incubations were performed to characterize reactive metabolites in vitro using glutathione and the 2-bromo-carbobenzyloxy (CBZ) derivative of GSH. Acetaminophen, clozapine, diclofenac, fipexide and estradiol were incubated under oxidative conditions, with and without trapping agents. Liquid chromatography was performed on a 1200 series Agilent rapid resolution LC system using a Thermo BetaBasic-18 150 x 2.1 mm column filled with 5 µm particles with 0.1% formic acid/acetonitrile gradient. Adducts were screened by LC-MS on an Agilent 6210 time of flight (ESI-TOF). MassHunter Mass Profiler software (version B.02, Agilent Technologies) was employed for comparing incubations and filtering the data based on isotope patterns and mass defects of the formed adducts.
Results: The 2-Br-CBZ derivative of GSH was synthesized by reacting oxidized glutathione (GSSG) with N-(2-bromo-benzyloxycarbonyloxy)-succinimide under alkaline conditions, followed by reduction with DTT, and extraction with ethyl acetate. The purity of the final product was verified by LC-MS and NMR. Following in vitro microsomal incubations with GSH and Br-CBZ-GSH, all adducts were screened using LC-MS with accurate mass measurements on an ESI-TOF instrument, followed by mass spectral filtering and statistical comparison of control vs. samples using Mass Profiler software. This software allowed the specific detection of Br-CBZ-GSH adducts using the isotope pattern of Br (with additional isotope contributions from the parent drug, if necessary) as well as the removal of all “molecular features” not consistent with the mass defect calculated for the studied adducts. The data were compared for the unlabeled and bromine-containing trapping agents.
Conclusions: We have combined mass defect and isotope filtering for screening reactive metabolites by accurate mass LC-MS with a novel bromine-containing GSH analog. Due to its increased hydrophobicity, this new analog increased the chromatographic retention of GSH adducts resulting in higher sensitivity, since they elute at higher organic content. The preparation of this analog was also very simple and inexpensive; in comparison to previously reported analogs (or stable-isotope labeled compounds) used for this type of work.
