A NOVEL NORMAL PHASE HPLC METHOD FOR THE QUANTIFICATION OF N-FORMYL IMPURITY IN AZACITIDINE ACTIVE PHARMACEUTICAL INGREDIENTS AND PHARMACEUTICAL DOSAGE FORMS

Abstract

Azacitidine, a drug used for the treatment of myelodysplastic syndrome is highly sensitive to water and forms N-formyl azacitidine (NFA) impurity as degradant. A novel stability-indicating isocratic normal phase liquid chromatographic method was developed for the determination of NFA impurity in Azacitidine. The method was developed using chiral pak IA (250 × 4.6 mm, 5 µm) column and the mobile phase containing n-Hexane-ethanol (50:50, v/v) was used. The eluted compounds were monitored at 242 nm. Baseline separation of Azacitidine and NFA impurity was obtained with a resolution factor of 5.1.Azacitidine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. The developed method was validated as per International Conference on Harmonization (ICH) guidelines and found to be precise, linear, accurate, and robust. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be about 0.18 and 0.60 µg/mL respectively, corresponding to 0.009 and 0.03% of the NFA impurity in Azacitidine. The calibration curve showed excellent linearity over the concentration range of 0.6 µg mL−1 (LOQ) to 6.0 µg mL−1 for NFA impurity. The percentage recovery of NFA impurity in bulk drug and in dosage forms ranged from 95.4 to 106.0%. The test solution was found to be stable in the mobile phase for 48 hr after preparation.