Development and validation of a reversed-phase HPLC method for quantification of 1’-acetoxychavicol acetate content in a nanostructured lipid carrier formulation

Abstract

1’-acetoxychavicol acetate (ACA)-loaded nanostructured lipid carriers (NLCs) were formulated for prostate cancer therapy and to determine the optimal therapeutic dose, we developed a rapid, specific, and accurate reversed-phase high-performance liquid chromatography (RPHPLC) method to quantify the ACA content in NLCs. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. Chromatographic separation of ACA from the lipid components was performed with an Agilent 1220 Infinity LC system and ultraviolet detector using an Agilent Poroshell C18 column (4.6 x 250.0 mm). The mobile phase consisted of acetonitrile and water (80:20 [v/v]) with a flow rate of 0.8 mL/min in isocratic mode. Linearity of the standard curve was assessed at an ACA concentration range of 5-200 µg/mL, and a 1/x weighted linear regression was adopted for the calibration curve. The calculated limits of detection and quantification were 0.59 µg/mL and 1.79 µg/mL, respectively. The mean percent recovery of ACA was 100.02% (relative SD, 2%), and the coefficients of variation for intraday and interday assays were within the values required by the ICH. We also demonstrated robustness of the method by altering the mobile phase ratio and flow rate. Furthermore, we proved specificity of the method for ACA by comparing chromatograms of the blank NLC and ACA-NLC. Hence, we effectively used this validated method to determine the drug-loading capacity and entrapment efficiency of the NLCs.