The core aim of present work was to develop a simple, precise, rapid and reproducible isocratic reverse phase high performance liquid chromatographic (RP-HPLC) method for the estimation of Azathioprine in pure and in tablet dosage form. An isocratic RP-HPLC was performed by utilizing Welchrom C18 column, (250 mm × 4.6 mm i.d., particle size 5 μm) maintained at ambient temperature and mobile phase composed of a mixture of acetonitrile: water (50:50 v/v) with apparent pH of 3.3 adjusted with o-phosphoric acid. The flow rate was adjusted 1.0 mL/min and UV detection was performed at 276 nm. The developed method was statistically validated for its linearity, precision, accuracy, specificity, Robustness and ruggedness. The retention time of Azathioprine peak was found at 3.080 minutes. The developed method was linear in the range of 1-5 μg/mL with correlation coefficient of 0.999. The method was found to be specific and accurate with the overall mean % recovery of 99.74%. The % RSD of intra and inter-day precision was found to be 0.833 and 0.877 respectively. The developed method was highly sensitive with LOD of 0.0480 μg/mL and LOQ of 0.1456 μg/mL. Assay content of Azathioprine was determined and the mean % found for Azathioprine was in good agreement with the label claim. The proposed method was found to be simple, highly sensitive, precise, accurate and rapid and can be employed for quantification of Azathioprine in bulk and tablet dosage form.
