Journal of Chromatography B, 877 (2009) 1805–1814 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells L. Elens a,∗ , S. Veriter a , J.C. Yombi b , V. Di Fazio c , R. Vanbinst c , D. Lison a , P. Wallemacq a,c , B. Vandercam b , V. Haufroid a,c a Louvain center for Toxicology and Applied Pharmacology (LTAP), Université catholique de Louvain, Avenue E. Mounier, 53.02, 1200 Brussels, Belgium b Department of Internal Medicine, Unit of Infectious Diseases, St-Luc University Hospital, Brussels, Belgium c Clinical Chemistry Department, St-Luc University Hospital, Brussels, Belgium article info Article history: Received 25 September 2008 Accepted 29 April 2009 Available online 13 May 2009 Keywords: Liquid chromatography tandem mass spectrometry Anti-HIV drugs Human peripheral blood mononuclear cells Isotopic dilution Quantitative determination abstract This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125ng/ml of cell extract by a 1/x 2 weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra ® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin ® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus ® (500 mg twice a day). The patients treated by Kaletra ® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin ® showed mean CC of 2388.11 ng/ml while both patients under Aptivus ® showed TPV CC of 4322.7 and 1078.0ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Analytical tools for monitoring the plasma concentrations of anti-HIV drugs are largely available in clinical settings and have already contributed to improve anti-HIV therapy through the means of therapeutic drug monitoring (TDM). Despite these advances, failures of anti-HIV therapy are still frequently encoun- tered in daily clinical practice and the reasons for these failures are not always understood. A possible explanation could be that measuring plasma drug concentration is of limited clinical value ∗ Corresponding author. Tel.: +32 2 764 53 54; fax: +32 2 764 53 38. E-mail address: laure.elens@uclouvain.be (L. Elens). because the major target of these drugs is within the infected cells and only the fraction reaching this intracellular compartment is expected to be active against HIV replication. In this respect, several studies have, however, shown a good correlation between intracel- lular and plasma drug concentrations for several protease inhibitors (PIs) [1–3] but not for NNRTIs [3,4], suggesting that plasma con- centration could constitute a valid parameter for the TDM of PIs but not, or at least less accurately, for NNRTIs. Direct measurement of intracellular drug concentrations may therefore contribute to improve and adjust antiretroviral therapy. A method to quantify the active fraction of these drugs would allow to better characterize the pharmacokinetics (PK) and the accumulation profile of these drugs in the cellular compartment and, would possibly give a better insight into understanding the reasons for some therapeutic 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.04.046