Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma using liquid chromatography

Efavirenz and nevirapine are non-nucleoside reverse transcriptase inhibitors for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the simultaneous quantification of efavirenz and nevirapine in human plasma suitable for therapeutic drug monitoring is described. Sample pre-treatment consisted of protein precipitation with acetonitrile and subsequently dilution with distilled water. The drugs were separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 275 nm. The method was validated over the therapeutically relevant concentration range of 0.05–15.0 mg l−1 and 0.25–15.0 mg l−1 for efavirenz and nevirapine, respectively, using a volume of 100 μl of plasma. The calibration curves were linear over this concentration range. Carbamazepine was used as internal standard. The assay proved to be accurate (accuracies varied between −12.7 and 8.5%) and precise (intra- and inter-assay precisions were less then 5.9%). The tested batches of control human plasma and frequently co-administered drugs did not interfere with the described methodology. Efavirenz and nevirapine were stable under various relevant storage conditions. This validated assay is suited for use in pharmacokinetic studies with efavirenz and nevirapine and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz and nevirapine concentrations.