A high throughput and selective method for the estimation of valproic acid an antiepileptic drug in human plasma by tandem LC-MS/MS

A high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of valproic acid, an antiepileptic drug, in human plasma is described. It is a rapid and sensitive isocratic reversed-phase liquid chromatography-tandem mass spectrometric method equipped with turbo ion spray (TIS) source, operating in the negative ion and pseudo selective reaction monitoring (SRM) acquisition mode to quantify valproic acid. The extraction of valproic acid and hydrochlorothiazide (IS) from the plasma involved sample treatment with phosphoric acid followed by solid-phase extraction using Waters hydrophilic-lipophilic balance (HLB) cartridge giving extracts free from endogenous interferences. Sample preparation by this method yielded very good and consistent mean recoveries of 99.73 and 74.47% for valproic acid and IS, respectively. The method was linear over the dynamic range of 2.0-200.0 μg/ml (covering entire therapeutic range) with a correlation coefficient r ≥ 0.9989. The coefficient of variance (CV, %) was 7.03% at 2.0 μg/ml (LLOQ). This method was fully validated for its accuracy, precision, recovery and matrix effect especially because the pattern of elution of all the analytes may appear as flow injection type. The analyte stability was examined under conditions mimicking the sample storage, handling and analysis procedures. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 500 mg formulations.