The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent.

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).

Analysis and quantification of DNA adducts of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in liver of rats by liquid chromatography/electrospray tandem mass spectrometry.

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to measure DNA adducts of the carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) with a microbore C-18 reversed-phase column. Quantification of the isomeric adducts N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-N(2)-MeIQx) was achieved using synthetic, isotopically labeled internal standards. The reaction of the N-acetoxy ester of 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) with calf thymus DNA (ct DNA) resulted in formation of these adducts in a ratio of 5:1 (dG-C8-MeIQx:dG-N(2)-MeIQx). The detection limit by LC/ESI-MS/MS in the selected reaction monitoring (SRM) mode ([MH(+) --> MH - 116](+)) (loss of deoxyribose) approached 500 fg (1 fmol) of adduct standard, and 1 adduct per 10(8) DNA bases using 100 microg of DNA following solid-phase extraction. The SRM analysis of rat liver DNA 24 h after an oral dose of MeIQx (10 and 0.5 mg/kg) revealed the presence of isomeric dG-MeIQx adducts at levels of 3.07 +/- 0.84 and 0.45 +/- 0.27 adducts per 10(7) bases, respectively. LC/ESI-MS/MS product ion spectra were acquired on both adducts from the elevated dose of MeIQx for unambiguous adduct identification. The contribution of dG-N(2)-MeIQx to the total adducts in vivo was significantly more important than that observed in vitro. dG-C8-MeIQx was the principal adduct formed at the 10 mg/kg dose, (dG-C8-MeIQx:dG-N(2)-MeIQx (3:2)); however, dG-N(2)-MeIQx was the major lesion detected at the 0.5 mg/kg dose (dG-C8-MeIQx:dG-N(2)-MeIQx 1:10). The striking differences between the relative amounts of dG-C8-MeIQx and dG-N(2)-MeIQx formed in vivo as a function of dose suggest that reactive esters of HONH-MeIQx other than N-acetoxy-MeIQx may be formed in vivo and react preferentially with the N(2) atom of guanine, or that dG-C8-MeIQx is removed at a significantly more rapid rate than dG-N(2)-MeIQx. The dG-N(2)-MeIQx adduct, previously thought to be a minor adduct, is likely to be an important contributor to the genotoxic damage of MeIQx.