Stability-indicating RP-HPLC method for simultaneous determination of methoxsalen and p-aminobenzoic acid in binary combination

Abstract

An accurate, simple and specific reverse phase LC procedure is established and validated for simultaneous estimation of methoxsalen and p-aminobenzoic acid in pharmaceutical formulated products and human serum. Chromatographic separation among methoxsalen, p-aminobenzoic acid and their degradation products have been achieved in less than 5 min with Hypersil-ODS column (250 mm × 4.6 mm, 5 μm), using acetonitrile and 1.28 mM phosphate buffer as mobile phase (60:40 v/v). Flow rate of the mobile phase was set as 1.5 mL min-1 and detection of all the analytes was carried out by diode-array detector at 254 nm. The developed method was validated according to ICH guidelines by performing its linearity, accuracy, precision, specificity, robustness and LOD/LOQ values. Response was linear and proportional to the concentrations (24-48 μg mL-1) for p-aminobenzoic acid and (9-18 μg mL-1) for methoxsalen. The LOD was 2.3 ng mL-1 for p-aminobenzoic acid and 6 ng mL-1 for methoxsalen whereas LOQ was 7.8 ng mL-1 for p-aminobenzoic acid and 20.4 ng mL-1 for methoxsalen. The developed method efficiently separated the principal peaks from degradation products and therefore can be applied successfully for concurrent analysis of methoxsalen and p-aminobenzoic acid in pharmaceutical dosage form, human serum and product stability studies.