DETERMINATION OF VALPROIC ACID AND ITS METABOLITES USING GAS-CHROMATOGRAPHY WITH MASS-SELECTIVE DETECTION - APPLICATION TO SERUM AND URINE SAMPLES FROM SHEEP

An improved method for the quantitative determination of valproic acid (VPA) and sixteen of its metabolites has been developed using gas chr omatography-mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single chromatographic run of 29.5 min. Ions selected for quantitativ e purposes were the characteristic [M - 57](+) ions of the tert.-butyl dimethylsilyl (tBDMS) derivatives. The method utilizes heptadeuterated VPA as well as six heptadeuterated metabolites as internal standards [i.e. 2-[H-2(7)]propyl-2-pentenoic acid (2-ene[H-2(7)]VPA), 2-[H-2(7)] propyl-4-pentenoic acid (4-ene[H-2(7)]VPA), 2-[H-2(7)]propyl-3-oxopent anoic acid (3-keto[H-2(7)]VPA), 2-[H-2(7)]propyl-4-oxopentanoic acid ( 4-keto[2H,]VPA), 2-[H-2(7)]propyl-3-hydroxypentanoic acid (3-OH[H-2(7) ]VPA), 2-[H-2(7)]propyl-5-hydroxypentanoic acid (5-OH[H-2(7)]VPA)]. Th e method demonstrates very good accuracy and precision over a large ra nge of concentrations for VPA and ail metabolites measured in both hum an and sheep biological fluids. The assay was applied to the analysis of VPA and metabolites in serum and urine samples collected from three non-pregnant ewes following intravenous bolus administration of a mix ture of VPA and [C-13(4)]VPA. Sheep were observed to produce measurabl e quantities of the majority of metabolites found in humans, with the notable exception of the di-unsaturated compounds (i.e. 2,3'-diene VPA and 2,4-diene VPA). The pharmacokinetics and metabolism of VPA and [C -13(4)]VPA appear to be equivalent in the sheep model, The elimination half-life of VPA and [C-13(4)]VPA in the ewe were estimated to be app roximately 3.5 +/- 0.4 and 3.2 +/- 0.4 h, respectively.