Conventional HPLC Method Used for Simultaneous Determination of the Seven HIV Protease Inhibitors and Nonnucleoside Reverse Transcription Inhibitor Efavirenz in Human Plasma

Abstract

We developed a simple HPLC method for the simultaneous quantitative determination of seven HIV protease inhibitors: amprenavir (APV), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and a nonnucleoside reverse transcription inhibitor, efavirenz (EFV). This method involves a rapid liquid-liquid drug extraction from plasma, the use of an isocratic elution on a reversed-phase C18 column, and an ultraviolet detection at a single wavelength (205 nm). The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. Forty-eight samples could be measured in one day since the runtime of one sample is 30 min. The assay has been validated over a concentration range of 0.05 to 12.20 microg/ml for APV, 0.09 to 12.05 microg/ml for ATV, 0.05 to 12.01 microg/ml for IDV, 0.12 to 12.36 microg/ml for LPV, 0.18 to 12.20 microg/ml for NFV, 0.12 to 12.33 microg/ml for RTV, 0.12 to 12.06 microg/ml for SQV, and 0.05 to 12.17 microg/ml for EFV. Calibration curves were linear in the described concentration ranges. The average accuracy ranged from 97.2 to 106.8%. Both the interday and intraday coefficients of variation for all drugs tested were less than 8.5%. This method provides a simple, accurate, and precise method for the therapeutic drug monitoring of the seven protease inhibitors and EFV in clinical routine use.