DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING RP-HPLC ASSAY METHOD FOR AZACITIDINE AND ITS BULK DRUG
Keywords:
Azacitidine, Impurities, Method Development, YMC ODS AQ-5, RP-HPLCAbstract
Objective: A novel gradient reverse phase High Performance Liquid Chromatographic (HPLC) method was developed for the quantification of Azacitidine impurities and degradation products in the Azacitidine tablets.
Methods: The effective separation was achieved on an YMC ODS AQ-5, (250 x 4.6 mm), 5 µm column using a gradient mode by the mobile phase A: 3.1g of ammonium acetate dissolved in 1000 mL of water filtered through 0.45μm filter paper and mobile phase B: mixture of buffer – A, methanol and acetonitrile In the ratio of 500:300:200 v/v. The flow rate of the mobile phase was 1.0 mL/minute and the total elution time, including the column equilibration was approximately 60 minutes. The UV detection was carried at the wavelength 242 nm and experiments were conducted at 35 0 C.
Results: The retention times of Azacitidine and its impurities are 15.3, 3.9, 4.9, 25.0 and 33 respectively. Azacitidine tablets were subjected to the stress conditions of oxidation, acid, base, hydrolytic, thermal and light degradation. The assay method was found to be linear in the range of 400 μg·mL–1 to with 1000 μg·mL–1 correlation coefficient is 0.998 and the linearity of the impurities was established from LOQ to 150%. Recoveries of assay and impurities were found between 99.0% and 103.6%.
Conclusion: The developed method was validated in terms of system suitability, specificity, linearity range, precision, accuracy, limits of detection and quantification for the impurities following the ICH guidelines. Therefore, the proposed method is suitable for the simultaneous determination of Azacitidine and its four related impurities.
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