An LC/MS/MS method for the quantitation of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a bioconversion product of temozolomide, in rat and dog plasma.

A sensitive and selective HPLC/electrospray ionization tandem mass spectrometric (LC/ESI/MS/MS) method for the quantitative determination of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a pharmacologically active hydrolysis product of temozolomide, was developed and validated over a linear range from 10 to 400 ng ml(-1) in dog plasma and from 10 to 500 ng ml(-1) in rat plasma. This HPLC method utilized small plasma volumes (70 microl), rapid sample processing, and isocratic elusion conditions to achieve sensitive and selective MS/MS detection. Samples were processed and analyzed one at a time every 4.5 min in order to compensate for the inherent instability of MTIC. Both MTIC and the internal standard DTIC [5-(3,3'-N,N'-dimethyltriazen-1-yl)-imidazole-4-carboxamide] were quantitated in the positive ion, selected reaction monitoring (SRM) mode. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in the plasma from both species. Inter-assay accuracy and precision of all calibration standards and quality control (QC) samples were within +/- 11 and 12%, respectively, with the exception of the LLOQ in rat plasma (17%). The validated method was used to determine the time dependent plasma concentration of MTIC in rats and dogs following a single oral dose of temozolomide. The standard curve and the quality control data indicate that the method performed acceptably throughout the sample analysis period.

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid.

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.

In vitro metabolism of 10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzo[b][1,8]naphthyridin-5(7H)- one, a topical antipsoriatic agent. Use of precision-cut rat, dog, monkey and human liver slices, and chemical synthesis of metabolites.

The metabolism of SCH 40120, which is the clinically effective antipsoriatic drug 10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzol[b][1,8]naphthyrid in-5(7H)-one, was determined in vitro. Rat, dog, cynomolgus monkey, and human liver slices hydroxylated the aliphatic, cyclohexenyl ring of the drug and conjugated the resulting carbinol. The identified metabolites comprised the corresponding 6-, 7-, and 9-carbinols, the glucuronide of the 6-carbinol, and the 6-ketone derived from the parent drug. Although the three carbinols appeared in the liver isolates of all species studied, the relative amounts of these metabolites varied across species. With a high, non-physiological ratio of substrate to liver, the 6-carbinol and its glucuronide were the major metabolites in human and monkey, whereas the 6-ketone was a minor metabolite in dog. Containing a stereogenic axis and center, the 6-carbinol existed as diastereomeric atropisomers. Its structure was established by 13C and 1H NMR spectroscopy, mass spectrometry, and comparison to an authentic sample.