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 of five HIV protease inhibitors (PIs) indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV), and two non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine (NVP), and efavirenz (EFV) in human plasma. 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 and recovery 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and precise method for monitoring of plasma concentrations of five PIs and two NNRTIs in the case of weak economy and out of date instrumental limitations.