Development of a Catalyst-Free Ultrasound-Assisted Derivatization Method for Detection of Valproic Acid in Epilepsy Patient's Serum Using High-Performance Liquid Chromatography: A Comparison With Chemiluminescence Immunoassay

Abstract

The aim of this research was the therapeutic drug monitoring for valproic acid in epilepsy patient's serum samples by the common, sensitive, and accessible HPLC-UV method. Because of the absence of a suitable chromophore in the valproic acid structure, a facile, selective, and cost-effective pre-column derivatization was designed. This catalyst-free ultrasound-assisted derivatization assay can accomplish the derivatization very quickly only in 5.0 min and at a mild temperature of 60 degrees C. 2,4'-Dibromoacetophenone and nonanoic acid was used as derivatizing agent and internal standard, respectively. The effect of sample pH, buffer concentration, ultrasound exposure time, reaction temperature, and derivatizing agent amount were optimized. The proposed method exhibited a good linear range of 5.0-300.0 mu g/mL with acceptable correlation coefficients of 0.9981. The limit of detection was as low as 0.4 mu g/mL. Also, the limit of quantification was reported as 1.3 mu g/mL. Interday and intraday relative standard deviations (n = 10) were 1.1 and 0.3-7.0, respectively. In addition, the relative recovery ranged from 100.3 to 107.7. The measurement of valproic acid was performed in the presence of several epilepsy and non-epilepsy drugs by the developed protocol. This confirmed the specific and accurate determination of valproic acid in the patient's serum. A comparative evaluation was employed against the precise chemiluminescence immunoassay approach. The correlation coefficient between the two methods was 0.9992, which demonstrated the results were statistically the same.