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

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2009-01, Vol.55 (1), p.170-174
• Main Authors: Elens, Laure, Veriter, Sophie, Di Fazio, Vincent, Vanbinst, Roger, Boesmans, Daniel, Wallemacq, Pierre, Haufroid, Vincent
• Format: Article
• Language: English
• Subjects: Ammonium; Analytical, structural and metabolic biochemistry; Aqueous solutions; Atazanavir Sulfate; Benzoxazines - blood; Biological and medical sciences; Blood banks; Carbamates - blood; Chromatography; Chromatography, High Pressure Liquid; Drugs; Fundamental and applied biological sciences. Psychology; HIV; HIV Protease Inhibitors - blood; Human immunodeficiency virus; Humans; Indinavir - blood; Investigative techniques, diagnostic techniques (general aspects); Liquid chromatography; Lopinavir; Medical sciences; Methanol; Methods; Nelfinavir - blood; Nevirapine - blood; Oligopeptides - blood; Plasma; Proteinase inhibitors; Pyridines - blood; Pyrimidinones - blood; Pyrones - blood; Reproducibility of Results; Reverse Transcriptase Inhibitors - blood; Ritonavir; Ritonavir - blood; Saquinavir - blood; Sensitivity and Specificity; Solid Phase Extraction; Sulfonamides - blood; Wavelengths
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2008.108647

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. 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 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees 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. 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 (r(2)) 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