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

J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jun 1;878(19):1455-65. doi: 10.1016/j.jchromb.2010.03.036. Epub 2010 Apr 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Anti-HIV Agents / analysis*
  • Chromatography, High Pressure Liquid / methods*
  • Cyclohexanes / analysis
  • Drug Stability
  • Maraviroc
  • Nitriles / analysis
  • Pyridazines / analysis
  • Pyrimidines / analysis
  • Reproducibility of Results
  • Rilpivirine
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / methods*
  • Triazoles / analysis


  • Anti-HIV Agents
  • Cyclohexanes
  • Nitriles
  • Pyridazines
  • Pyrimidines
  • Triazoles
  • etravirine
  • Rilpivirine
  • Maraviroc