Quantification of 8 HIV-Protease Inhibitors and 2 Nonnucleoside Reverse Transcriptase Inhibitors by Ultra-Performance Liquid Chromatography with Diode Array Detection

Abstract

Background: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)–diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. Methods: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 μL methanol. We injected a 4-μL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 °C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. Results: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025–10 mg/L (1.875–75 mg/L for TPV) showed coefficients of determination (r2) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. Conclusions: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.