High-performance liquid chromatography assay for the quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma

An accurate, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of HIV-protease inhibitors (PIs) (indinavir, IDV; amprenavir, APV; saquinavir, SQV; nelfinavir, NFV; ritonavir, RTV; and lopinavir, LPV) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, NVP; delavirdine, DLV; and efavirenz, EFV) in human blood plasma is described. The method provides excellent resolution and peak shape for nine analytes through a linear gradient (36–86%) of 25% phosphate buffer (pH 4.5), 60% acetonitrile, 15% methanol, and 0.75 ml TFA, with a gradient mobile phase flow rate (0.9–1.1 ml) over 30 min run time. The optimized solid phase extraction (SPE) extraction method using (1.0 ml, 100 mg BOND ELUT-C18 Varian) column provides a clean base line and high extraction efficiency using a 550 μl plasma sample. The method was validated over the range of 10–10,000 ng/ml for NVP, IDV, and SQV; 10–5000 ng/ml for EFV; 25–10000 ng/ml for APV; and 25–5000 ng/ml for DLV, NFV, RTV, and LPV. This method is accurate (average accuracies of three different concentrations ranged from 91 to 112%), and precise (within- and between-day precision measures ranged from 0.2 to 5.7% and 0.1 to 5.4%, respectively). This method is suitable for use in clinical pharmacokinetic studies as well as in therapeutic drug monitoring (TDM).