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 (40:60 v/v) with a flow rate of 0.5 mL/min. The calibration curve was linear over the range of 882-7060 ng/mL (r(2)  = 0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99 ng/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.

Objectives N‐(2‐hydroxyphenyl)‐2‐propylpentanamide (HO‐AAVPA), a derivative of valproic acid (VPA), has been proposed as a potential anticancer agent due to its improved antiproliferative effects in some cancer cell lines. Although there is evidence that VPA is metabolized by cytochrome P450 2C11 rat isoform, HO‐AAVPA CYP‐mediated metabolism has not yet been fully explored. Therefore, in this work, the biotransformation of HO‐AAVPA by CYP2C11 was investigated. Methods Kinetic parameters and spectral interaction between HO‐AAVPA and CYP were evaluated using rat liver microsomes. The participation of CYP2C11 in metabolism of HO‐AAVPA was confirmed by cimetidine (CIM) inhibition assay. Docking and molecular dynamics simulations coupled to MMGBSA methods were used in theoretical study. Key findings HO‐AAVPA is metabolized by CYP enzymes (KM = 38.94 µm), yielding a hydroxylated metabolite according to its HPLC retention time (5.4 min) and MS analysis (252.2 m/z). In addition, CIM inhibition in rat liver microsomes (Ki = 59.23 µm) confirmed that CYP2C11 is mainly involved in HO‐AAVPA metabolism. Furthermore, HO‐AAVPA interacts with CYP2C11 as a type I ligand. HO‐AAVPA is stabilized at the CYP2C11 ligand recognition site through a map of interactions similar to other typical CYP2C11 substrates. Conclusion Therefore, rat liver CYP2C11 isoform is able to metabolize HO‐AAVPA.