Elens L, Veriter S, Yombi J C, Di Fazio V, Vanbinst R, Lison D, Wallemacq P, Vandercam B, Haufroid V
Louvain Center for Toxicology and Applied Pharmacology (LTAP), Université Catholique de Louvain, Avenue E. Mounier 53.02, 1200 Brussels, Belgium.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul 1;877(20-21):1805-14. doi: 10.1016/j.jchromb.2009.04.046. Epub 2009 May 13.
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 (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 917.2 ng/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 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.
本文报道了一种液相色谱串联质谱（LC-MS/MS）方法的验证，该方法使用6种不同的同位素内标（IS）对外周血单核细胞（PBMC）中的10种抗逆转录病毒（ARV）药物进行定量分析及其临床应用。通过密度梯度离心从血液中分离出PBMC，并用含有6种IS的60%甲醇（MeOH）溶液提取药物。然后将细胞提取物注入高效液相色谱（HPLC）系统，在Symmetry Shield RP18 2.1 mm×50 mm柱上分离分析物。接着通过电喷雾正离子或负离子模式的串联质谱（MS/MS）检测不同分子，并使用多反应监测（MRM）模式记录数据。通过1/x(2)加权二次回归在0.25 - 125 ng/ml细胞提取物范围内构建校准曲线。获得的回归系数始终大于0.99，回算值始终在其标称浓度的±15%范围内。所有分析物的平均提取回收率均大于80%，该方法准确且精密，变异系数（CV）和偏差低于9.4%。不同药物的定量下限（LLOQ）在0.0125至0.2 ng/ml细胞提取物范围内。该方法已成功应用于98名接受克力芝（400/100 mg洛匹那韦/利托那韦（LPV/RTV），每日两次，n = 48）或司他夫定（600 mg，每日一次，n = 50）治疗的HIV感染患者队列，并已在2名接受阿扎那韦（500 mg，每日两次）治疗的患者中测试替拉那韦（TPV）的细胞定量。接受克力芝治疗的患者中，LPV和RTV的平均细胞相关浓度（CC）分别为1819.0和917.2 ng/ml。接受司他夫定治疗的患者平均CC为2388.11 ng/ml，而两名接受阿扎那韦治疗的患者TPV CC分别为4322.7和1078.0 ng/ml。该方法可用于分析靶组织内的ARV药物浓度。
