A NOVEL HPLC-PDA METHOD FOR QUANTITATIVE DETERMINATION OF RELATED IMPURITIES IN AZATHIOPRINE ACTIVE PHARMACEUTICAL INGREDIENT
An innovative and cost-effective method has been devised using high-performance liquid chromatographywithPDA detection for the assessment of Azathioprine and its related impurities (1-methyl-4-nitro-1H-imidazol-5-amineand 7H-purine-6-thiol). Validation was performed in accordance with ICH guidelines, confirming that the methodislinear, accurate, precise, and robust. The established method is suitable for routine quality control analysis of Azathioprine in pharmaceutical formulations (SDG-3). The chromatographic separations were conducted ona(150×4.6 mm), 3.5 µm Waters Symmetry C18 column, utilizing a mobile phase of 0.1% orthophosphoric acid buffer and Acetonitrile in a 30:70 ratio at a flow rate of 1.0 mL/min. Azathioprine and its two impurities were eluted inlessthan 10 minutes. The column oven temperature was maintained at 30° C, the sample temperature was kept at 20° C, and a detector wavelength of 235 nm was used. The method was validated successfully by establishing SystemSuitability, Specificity, Linearity, Accuracy, Limit of Detection, and Limit of Quantification. Specificitywasvalidated by assessing the separation of impurities and confirming the absence of interference fromthe placebo. Linearity was established by analysing a series of standard solutions at various concentration levels, withthecorrelation coefficient determined to be greater than 0.999. Accuracy was determined through recovery studiesconducted at three concentration levels, with the percentage recovery remaining within the acceptable range of 80- 120%. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the signal-to-noiseratio method.
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