A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. Methods：The analytes and internal standard were isolated from 200μL samples by solid phase extraction using a ZORBAX SB-C8 column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v)containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The Agilent G6410A triple quadrupole LC/MS system was operated under a multiple reaction monitoring and single ion monitoring mode using the electrospray ionization technique in a negative mode. Results：The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients > 0.995 for all analytes. The intra-day andinter-day accuracy and precision of the assay were less than 15.0%. Conclusions：This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.
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