Development and Validation of a Stability-indicating Hptlc Method for Analysis of Antitubercular Drugs

A simple, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method has been established and validated for analysis of isoniazid and rifampicin both as the bulk drugs and in formulations. The compounds were separated on aluminumbacked silica gel 60 F254 plates with n-hexane–2-propanol–acetone–ammonia–formic acid, 3:3.8:2.8:0.3:0.1 (v/v) as mobile phase. This system was found to give compact spots for isoniazid and rifampicin (RF values 0.59 ± 0.02 and 0.73 ± 0.04, respectively). Densitometric analysis of isoniazid and rifampicin was performed at 254 nm. Regression analysis data for the calibration plots were indicative of good linear relationships between response and concentration over the range 100–700 ng per spot. The correlation coefficients, r, were 0.994 and 0.997 for isoniazid and rifampicin respectively. The values of slope and intercept of the calibration plots were 3.755 ± 0.22 and 3099.1 ± 51.21, respectively, for isoniazid and 4.0957 ± 0.25 and 3567.6 ± 61.11, respectively, for rifampicin. The method was validated for precision, recovery, and robustness. The limits of detection and quantification were 20 ± 0.51 and 60 ± 1.05 ng, respectively, for isoniazid and 25 ± 0.63 and 75 ± 1.12 ng, respectively, for rifampicin. Isoniazid and rifampicin were subjected to acid, base, peroxide, and UV-induced degradation. In stability tests the drugs were susceptible to acid and basic hydrolysis, oxidation and photodegradation. Statistical analysis proved the method is repeatable, selective, and accurate for estimation of isoniazid and rifampicin. Because the method could effectively separate the drugs from their degradation products, it can be used as a stabilityindicating method.