Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds.
Clinical pharmacokinetic studies of antiretrovirals require accurate and precise measurement of plasma drug concentrations. Here we describe a simple, fast and sensitive HPLC-MS/MS method for determination of the commonly used protease inhibitors (PI) amprenavir, atazanavir, darunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, as well as the more recent antiretrovirals, the CCR5 antagonist maraviroc and the "second generation" NNRTI etravirine and rilpivirine. An internal standard (quinoxalone; QX) was added to plasma aliquots (100 microl) prior to protein precipitation with acetonitrile (500 microl) followed by centrifugation and addition of 0.05% formic acid (200 microl) to the supernatant. Chromatographic separation was achieved using a gradient (acetonitrile and 0.05% formic acid) mobile phase on a reverse-phase C18 column. Detection was via selective reaction monitoring (SRM) operating in positive ionization mode on a triple-quadrupole mass spectrometer. All compounds eluted within a 5 min run time. Calibration curves were validated over concentration ranges reflecting therapeutic concentrations observed in HIV-infected patients from pharmacokinetic data reported in the literature. Correlation coefficients (r2) exceeded 0.998. Inter- and intra-assay variation ranged between 1% and 10% and % recovery exceeded 90% for all analytes. The method described is being successfully applied to measure plasma antiretroviral concentrations from samples obtained from clinical pharmacokinetic studies.
Else
Laura
L
Department of Pharmacology and Therapeutics, University of Liverpool, Pharmacology Research Laboratories, Liverpool, UK. l.j.else@liv.ac.uk
Watson
Victoria
V
Tjia
John
J
Hughes
Andrew
A
Siccardi
Marco
M
Khoo
Saye
S
Back
David
D
D000704:Analysis of Variance; D019380:Anti-HIV Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003510:Cyclohexanes / Q000032:analysis; D004355:Drug Stability; D000077592:Maraviroc; D009570:Nitriles / Q000032:analysis; D011724:Pyridazines / Q000032:analysis; D011743:Pyrimidines / Q000032:analysis; D015203:Reproducibility of Results; D000068696:Rilpivirine; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014230:Triazoles / Q000032:analysis
D019380:Anti-HIV Agents; D003510:Cyclohexanes; D009570:Nitriles; D011724:Pyridazines; D011743:Pyrimidines; D014230:Triazoles; C451734:etravirine; D000068696:Rilpivirine; D000077592:Maraviroc