Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography-mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic-mass spectrometric (GC-MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human micros omal cytochrome P450 incubations. The use of the inherent soft ionization n ature of electron-capture NCI to achieve high sensitivity enabled us to con duct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M-181] fragments of pentafluorobenzyl (PFB) eaters in the NCI mode, and has the fo llowing features: (1) a micro-extraction procedure to isolate VPA metabolit es from small incubation volumes (100 mu l); (2) a second step derivatizati on with tert.-butyldimethylsilylating reagents to enhance sensitivity for h ydroxylated metabolites; (3) a short run-time (<30 min) while maintaining f ull separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and ac curacy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deutera ted derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M-1 81](-) ions while the hydroxylated metabolites gave an ion at m/z of 273, c orresponding to the [M-181](-) ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC-NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and k inetic studies of valproic acid in small volume samples typical of the in v itro cDNA-expressed micro-incubation enzymatic systems. (C) 2000 Elsevier S cience B.V. All rights reserved.