High Performance Liquid Chromatographic Assay for the Determination of Protease Inhibitors (PIs) and Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) in Human Plasma

Abstract

Objective: To develop and validate a high performance liquid chromatography (HPLC) method for simultaneous quantitative determination offive HIV protease inhibitors (PIs): indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), andtwonon-nucleoside reverse transcriptase inhibitors (NNRTIs):nevirapine (NVP), andefavirenz (EFV) inhumanplasma.

Methods: A sample of 200 µL of plasma and an internal standard were extracted with tert-butyl methyl ether. The compounds were separated on a reversed-phase C18 column with gradient phase of 25 mM phosphate buffer (pH 4.9) and acetonitrile. The limit of quantation, accuracy, precision, specificity, stability andrecovery were tested.

Results: The lower limit of quantitation for all drugs was 75 ng/mL. The standard curve was linear in the range of 75 ng/mL to 20,000 ng/mL. Intra-day and inter-day variability ranged from 0.1% to 2.4% and 0.3% to 4.1%, respectively. Accuracy ranged from 98.4%-102.4% for three quality controls (75, 100, and 1,000 ng/mL) for all drugs measured. The extraction recovery ranged from 98.7%-101.3%.

Conclusion: This method provides a simple, accurate, and precise method for monitoring of plasma concentrations of five PIs and two NNRTIs in the case of weak economy and out of date instrumental limitations.