A simple validated RP-HPLC bioanalytical method for the quantitative determination of a novel valproic acid arylamide derivative in rat hepatic microsomes

A simple and specific bioanalytical method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet detection was developed and validated for the determination of a novel valproic acid arylamide, N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) in rat hepatic microsomes (a subcellular fraction containing phase I enzymes, especially cytochrome P450). The chromatographic separation was achieved using a reversed-phase Zorbax SB-C₁₈ column and a mobile phase of acetic acid in water (0.2% v/v) and acetonitrile (40:60v/v) with a flow rate of 0.5mL/min. The calibration curve was linear over the range of 882-7060ng/mL (r²=0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99ng/mL, respectively. The method was validated with excellent sensitivity, and intra-day accuracy and precision varied from 93.79 to 93.12%, and from 2.12 to 4.36%, respectively. The inter-day accuracy and precision ranged from 93.29 to 97.30% and from 0.68 to 3.60%, respectively. The recovery of HO-AAVPA was measured between 91.36 and 97.98%. The assay was successfully applied to the analysis of kinetic metabolism and pharmacokinetic parameters in vitro by a substrate depletion approach.