HPTLC-Densitometric Determination of Allopurinol and its Metabolite Oxypurinol in Human Plasma and Allopurinol in Tablet Dosage Form

Abstract

A simple, accurate and precise high-performance thin layer chromatography (HPTLC) method has been developed for the simultaneous determination of allopurinol (AP), a xanthine oxidase inhibitor and its active metabolite oxypurinol (OP) in human plasma and AP in tablet dosage form. The proposed method was developed and validated on aluminium plates pre-coated with silica gel 60G F254 as the stationary phase using methanol: chloroform: ammonia (2.0:7.9: 0.1, v/v/v) as the developing solvent system. Densitometric measurements were made at 206 nm and the retention factors (Rf) obtained were 0.38 ± 0.01 and 0.65 ± 0.01 for AP and OP respectively. System suitability testing parameters were evaluated to determine the performance of the chromatographic method. The regression plots were linear (r2 > 0.9993) in the concentration range of 100-700 ng/band for both the analytes. The limit of detection and limit of quantitation of the method were 19.56 and 59.29 ng/band for AP and 19.01 and 57.59 ng/band for OP respectively for plasma samples. For spiked plasma samples, protein precipitation with formic acid in acetonitrile afforded mean recovery of 84.67 % and 86.21 % for AP and OP, respectively. The method was validated for linearity, specificity, accuracy and precision, repeatability, stability and robustness.