Validation of a high-performance liquid chromatography method for the assay of and determination of related organic impurities in nevirapine drug substance.

Nevirapine (Viramune), a dipyridiodiazepinone, is a potent and highly specific nonnucleoside inhibitor of HIV-1 reverse transcriptase. This paper describes the validation of a specific, sensitive, and stability-indicating high-performance liquid chromatography method for the assay and determination of related organic impurities in nevirapine drug substance. This method uses a Supelcosil LC-ABZ column, a mobile phase of 20:80 (v/v) acetonitrile-25mM NH4H2PO4 (pH 5.0), and ultraviolet detection at a wavelength of 220 nm. This method was validated for specificity, linearity, accuracy, repeatability, detection limit, quantitation limit, stability of analyte solutions, robustness, and intermediate precision. Nevirapine is completely separated from all impurities. The method is shown to be linear with coefficients of determination r2 greater than 0.999. Average accuracy is 100.4% with a relative standard deviation of 0.7% for the assay. Accuracy ranges from 100.1 to 102.6% for related organic impurities. Repeatability is good, with relative standard deviations not more than 1.4%. The detection limit and the quantitation limit are determined to be 0.001 and 0.003%, respectively. Relative response factors of known organic impurities are determined, permitting the use of nevirapine at the 0.1% level as an external standard for the quantitation of these impurities. Analyte solutions are shown to be stable for at least 2 days at ambient temperature. The method is validated as robust, and intermediate precision is high. A system suitability test is developed and validated, and requirements are set.