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

被引:25
作者
Elens, L. [1 ]
Veriter, S. [1 ]
Yombi, J. C. [2 ]
Di Fazio, V. [3 ]
Vanbinst, R.
Lison, D. [1 ]
Wallemacq, P. [1 ,3 ]
Vandercam, B. [2 ]
Haufroid, V. [1 ,3 ]
机构
[1] Univ Catholique Louvain, Louvain Ctr Toxicol & Appl Pharmacol LTAP, B-1200 Brussels, Belgium
[2] St Luc Univ Hosp, Infect Dis Unit, Dept Internal Med, Brussels, Belgium
[3] St Luc Univ Hosp, Dept Clin Chem, Brussels, Belgium
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2009年 / 877卷 / 20-21期
关键词
Liquid chromatography tandem mass spectrometry; Anti-HIV drugs; Human peripheral blood mononuclear cells; Isotopic dilution; Quantitative determination; HIV-INFECTED PATIENTS; HUMAN-IMMUNODEFICIENCY-VIRUS; REVERSE-TRANSCRIPTASE INHIBITORS; P-GLYCOPROTEIN EXPRESSION; HUMAN PLASMA; PROTEASE INHIBITORS; ANTIRETROVIRAL DRUGS; SIMULTANEOUS QUANTIFICATION; INTRACELLULAR ACCUMULATION; ATAZANAVIR BMS-232632;
D O I
10.1016/j.jchromb.2009.04.046
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 x 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 0.25-125 ng/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 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra (R) (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin (R) (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus (R) (500 mg twice a day). The patients treated by Kaletra (R) showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin (R) showed mean CC of 2388.11 ng/ml while both patients under Aptivus (R) showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue. (C) 2009 Elsevier B.V. All rights reserved.
引用
收藏
页码:1805 / 1814
页数:10
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