Development and Validation of an LC/MS/MS Method for Determination of Valproic Acid and Its Metabolite 2-Propyl-4-pentenoic Acid in Monkey Plasma

A rapid, accurate, and sensitive liquid chromatography/mass spectrometry (MS)/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid (4-ene VPA), in monkey plasma was developed and validated. The sample extraction was performed using hydrophilic-lipophilic balance cartridge. The analytes were separated on a Kinetex C18 (2.1 mm x 100 mm, 2.6 mu m) analytical column under a mobile phase consisting of 10 mM ammonium formate (pH 8.0)/methanol (20/80, v/v) and isocratic flow at 0.15 mL/min. The tandem mass spectrometer was operated ill negative electrospray ionization with selected ion monitoring conditions, 143.0, 141.0, and 121.0 for VPA, 4-ene VPA, and benzoic acid (internal standard), respectively. The linearity of calibration curve ranging from 0.1 to 20 mu g/mL was at least 0.9996 (coefficient of correlation, r) for both analytes. Intra and inter-day precisions for both analytes were lower than +/-15%, resulting from quality control (QC) samples at concentration of 0.2 (low QC), 1.6 (middle QC), and 16 (high QC) mg/mL except at the lower limit of quantification (LLOQ) (0.1 mu g/mL) level, which was less than 20%. The intra-and inter-day accuracies were within 15%. The recoveries were 84.4-90.8% for VPA and 38.2-100.6% for 4-ene VPA. Both analytes were stable throughout short-term temperature, post preparation for 24 h, and three freeze/thaw cycles, validating that this method could be applied to toxicokinetic and pharmacokinetic studies.