Validated Stability-Indicating HPLC and HPTLC Methods for the Determination of Ritonavir in Bulk Powder and in Capsules

Two sensitive and reproducible methods were described for the quantitative determination of the antireteroviral drug ritonavir in the presence of its degradation products. The first method was based on high performance liquid chromatographic (H PLC) separation of the drug from its stress degradation products with the use of a reversed phase Agilent Eclipse XDB-C18 column (5 mu m, 4.6 x 150 mm) and a mobile phase consisting of acetonitrile : 0.05 M phosphoric acid (55 : 45, v/v) at a flow rate of 1.0 mL/min. The retention time of the drug was found to be 4.82 +/- 0.002 min. Quantification was achieved with diode array detection (DAD) at 210 nm based on peak area and a linear calibration curve in the concentration range of 1-500 mu g/mL. The proposed method made use of diode array detection as a tool for peak purity and identification. The second method involved a high performance thin layer chromatographic (HPTLC) separation followed by densitometric measurement of the spots at 240 nm. The separation was carried out on Fluka TLC aluminium sheets of silica gel with fluorescent indicator (254) nm and the mobile phase was acetonitrile - water (1 : 2, v/v), adjusted to pH 5.0 using 1 M orthophosphoric acid solution. The proposed procedure gave compact spots for ritonavir (retention factor, R-f = 0.41 +/- 0.014). The linear regression equation was generated by least-squares treatment of the calibration data in the range of 0.8-12.5 mu g/spot. The reliability and analytical performance of the proposed methods, including linearity, range, precision, accuracy, detection and quantitation limits, were statistically validated. The proposed methods were applied to Norvir capsules and no chromatographic interference was observed. When ritonavir was subjected to stress conditions; according to ICH guidelines, the proposed methods could effectively separate the drug from its degradation products, and were thus considered as good stability-indicating procedures.