Development and validation of a new HPLC method for valproic acid determination in human plasma and its application to a therapeutic drug monitoring study

Abstract

The aim of this study was to develop a new, simple and reliable high performance liquid chromatography (HPLC) method for analysis of valproic acid (VPA) in human plasma and apply to it to a therapeutic drug monitoring study. Also, the relationship between plasma-VPA concentrations and the amount of VPA used by patients was aimed to be evaluated. Plasma samples (0.25 mL) were precipitated with the same volume of acetonitrile and after centrifugation, aliquots were applied to a C18 column (250 mm x 4.6 mm). Mobile phase was prepared with phosphate buffer and acetonitrile (47.5:52.5, v/v). The flow-rate was 1.2 mL/min. Accuracy was between -2.9 and 3.2% and precision was ≤6.6%. Method was specific and sensitive with a detection limit of 2.2 µg/mL and the average recovery was 94.3%. Calibration curve was linear (r2>0.9968) from 10 to 150 µg/mL. Plasma-VPA levels of the epileptic patient population (n=33) treated with VPA between 0.5 and 1.5 g/day were also determined. Patient plasma-VPA concentrations ranged from 2.9 to 166.4 µg/g/mL (56.3±38.8). High RSD% (68.8%) was observed in dose-rated plasma-VPA results. Moreover, VPA plasma levels were found to be outside the recommended treatment range in 30.3% of the patients examined. The procedure described was found to be relatively simple, precise, and applicable for routine therapeutic drug monitoring (TDM) especially in neurology clinics or in toxicology reference laboratories. The high standard deviation (SD) observed in the dose depended plasma-VPA values of the volunteers proved the importance of TDM during the use of this drug. The results showed that for rational drug use, it is important to identify individual polymorphisms in the CYP2C9, CYP2A6 and CYP2B6 subtypes responsible for VPA metabolism, and to rearrange drug doses taking these enzyme activities into account.