Stability Indicating High‐Performance Liquid Chromatographic Method for the Assessment of Thermal Stability of Antitubercular Drug Combinations, and Characterization of Rifampicin–Isoniazid Adduct Impurity

ABSTRACT A stability indicating high‐performance liquid chromatographic method was developed for the determination of the thermal stability of antitubercular drug combinations. The method separated the analytes and degradation products using a gradient elution of a mixture of ammonium formate buffer and methanol on a C 18 column. The method demonstrated acceptable specificity, resolution, linearity, precision, and accuracy for isoniazid, pyrazinamide, and rifampicin. The method was robust against changes in %buffer composition (± 2%), flow rate (± 0.1 mL/min), and column temperature (± 2°C). The method demonstrated the thermal stability of each drug alone and their three and two‐drug combinations at 70°C for 10 days. The developed method revealed that the %degradation of isoniazid and rifampicin was relatively higher in the isoniazid–rifampicin combination than in the isoniazid–rifampicin–pyrazinamide combination. On Days 5 and 10, the respective percentages of degradation of 69.17% and 74.79% were recorded for rifampicin in the isoniazid + rifampicin combination, while the percentages of degradation of 78.99% and 80.0% were observed for isoniazid in the isoniazid + rifampicin combination. The %degradation for isoniazid and rifampicin was higher by 1.5‐2‐folds in isoniazid–rifampicin than in isoniazid + rifampicin + pyrazinamide combinations. Overall, the anti‐tuberculosis fixed‐dose combination generated 14 degradation products (Impurities 1–14), among which Impurity 12 was the most prominent. Accordingly, Impurity 12 was characterized as 3‐(isonicotinoylhydrazinomethyl) rifamycin by ultraviolet‐visible‐near infrared spectroscopy, Fourier transform infrared spectroscopy, Hydrogen‐1 nuclear magnetic resonance, and liquid chromatography‐high resolution mass spectrometry (mass‐to‐charge ratio 843.350 of M‐H). Furthermore, it was identified as a reaction product of isoniazid and rifampicin, generated at elevated temperatures. The isolated impurity was screened against Mycobacterium tuberculosis H 37 Rv and was found to be inactive with a minimum inhibitory concentration of > 250 µg/mL. Then, this method was applied to detect the isoniazid–rifampicin reaction products in marketed tablets and stability samples from an in‐house liposome formulation study.