Development of a liquid chromatographic method for the simultaneous analysis of six protease inhibitors

Abstract

A liquid chromatographic method for the simultaneous determination of six HIV -protease inhibitors, indinavir, saquinavir, ritonavir, amprenavir, nelfinavir and lopinavir, was developed and validated. The method was developed by systematic investigation of the various stationary phases and the effects of the pH, various organic modifiers, ionic strength and the column temperature on chromatographic parameters. Optimal separation was achieved with a PLRP-S 100 A 250 x 4.6 mm LD. column maintained at 60°C, the mobile phase consisted of tetrahydrofuran-O.1 M potassium phosphate buffer pH 5.0-0.1 M tetrabutylammonium hydrogen sulphate pH 5.0-water (35:30:10:25, v/v) at a flow rate of 1.0 ml per minute and ultraviolet detection at 254 nm. The liquid chromatography method was validated by determining the linearity of detector response, precision, limit of detection and limit of quantitation, stability and ruggedness. The method was found to be linear over the specific ranges investigated and the R values were between 0.9997 and 0.9915 for the six drugs. The limit of quantitation for the six drugs was 160 to 5120 ng, while the limit of detection was 80 to 2120 ng. The intra-day and inter-day precision was within the ranges of 0.39 to 1.79 % and 0.55 to 7.46 %, respecti vely. The method was used to evaluate the content of active pharmaceutical ingredient of some protease inhibitors in the Kenyan market. The method developed can be used in routine quantitative analysis of the HIV -protease inhibitors in commercial samples and bulk pharmaceutical raw materials.