Determination of valproic acid in human plasma by HPLC with fluorescence detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of valproic acid in human plasma. The method is based on pre-column derivatization using a new fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The internal standard for the assay procedure was cyclohexanecarboxylic acid. The optimum monitoring conditions and the stability of the derivatives were investigated. The derivatization reaction proceeds in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30degreesC with a reaction time of 30 min. The resulting derivatives were separated under isocratic conditions (acetonitrile-water, 60:40, v/v) on a LiChrospher RP-18 column (125 x 4.0 mm, i.d. 5 mum) and were detected fluorimetrically at a wavelength of 435 nm with an excitation of 345 nm. All chromatographic experiments were carried out at a flow rate 1.0 mL/min at ambient temperature. The method was fully validated and applied for the determination of valproic acid over the concentration range of 6.0-150.0 mg/mL. The detection limit for valproic acid added to plasma sample was 0.13 mug/mL plasma (3 pg on column). The recovery of valproic acid averaged 100.4%. The accuracy of the assay ranged from -0.5% to 1.3%. The method was shown to be highly reproducible and it seems to be adequate for routine therapeutic drug monitoring.