Quantification of 8 HIV-protease inhibitors and 2 nonnucleoside reverse transcriptase inhibitors by ultra-performance liquid chromatography with diode array detection.
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 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.


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 (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 <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.
Elens
Laure
L
Industrial Toxicology and Occupational Medicine Unit, Université Catholique de Louvain, Brussels, Belgium. laure.elens@uclouvain.be
Veriter
Sophie
S
Di Fazio
Vincent
V
Vanbinst
Roger
R
Boesmans
Daniel
D
Wallemacq
Pierre
P
Haufroid
Vincent
V
D000480:Alkynes; D000069446:Atazanavir Sulfate; D048588:Benzoxazines / Q000097:blood; D002219:Carbamates / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D003521:Cyclopropanes; D005663:Furans; D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D019469:Indinavir / Q000097:blood; D061466:Lopinavir; D019888:Nelfinavir / Q000097:blood; D019829:Nevirapine / Q000097:blood; D009842:Oligopeptides / Q000097:blood; D011725:Pyridines / Q000097:blood; D011744:Pyrimidinones / Q000097:blood; D011753:Pyrones / Q000097:blood; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000097:blood*; D019438:Ritonavir / Q000097:blood; D019258:Saquinavir / Q000097:blood; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013449:Sulfonamides / Q000097:blood
D000480:Alkynes; D048588:Benzoxazines; D002219:Carbamates; D003521:Cyclopropanes; D005663:Furans; D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011725:Pyridines; D011744:Pyrimidinones; D011753:Pyrones; D018894:Reverse Transcriptase Inhibitors; D013449:Sulfonamides; D061466:Lopinavir; D000069446:Atazanavir Sulfate; C095108:amprenavir; D019469:Indinavir; D019829:Nevirapine; D019888:Nelfinavir; C098320:efavirenz; D019258:Saquinavir; D019438:Ritonavir; C107201:tipranavir