A Validated High-Performance Thin-Layer Chromatographic Method for the Determination of Azathioprine from Pharmaceutical Formulation

A simple, specific, and precise high-performance thin-layer chromatographic (HPTLC) method for the analysis of azathioprine, both as a bulk drug and in formulation, was developed and validated. The method employed TLC (thin-layer chromatographic) aluminum plates precoated with silica gel 60 F254 as the stationary phase. The solvent system consisted of methanol-toluene-25% ammonia (7:3:0.1 v/v). This system was found to give compact bands for azathioprine (RF 0.63 ± 0.05). Densitometric analysis of azathioprine was carried out in the absorbance mode at 285 nm. Linear regression analysis data for the calibration spots showed good relationship with regression coefficient r2 = 0.9908 in the range of 20–120 ng/band. The limits of detection and quantification were 6 and 20 ng/band, respectively. The relative standard deviation for instrumental precision, method precision, intra-assay precision, and intermediate precision was <2%. The proposed method was found to be simple, precise, accurate, and reproducible for the estimation of azathioprine in pure drugs and its formulations.