Laboratory of Clinical Pharmacology and Pharmacogenetics, Department of Infectious Diseases, University of Torino, Amedeo di Savoia Hospital, Corso Svizzera 164, Turin, Italy.
J Pharm Biomed Anal. 2011 Mar 25;54(4):779-88. doi: 10.1016/j.jpba.2010.10.011. Epub 2010 Nov 10.
A sensitive and accurate high performance liquid chromatography-mass spectrometric (HPLC-MS) method for the intracellular determination of 14 antiretroviral drugs in peripheral blood mononuclear cells (PBMCs) for HIV+ patients was validated. PBMCs are isolated by Ficoll density gradient centrifugation and cells count and the relative mean volume is performed with a Coulter(®) instrument. Extraction of drugs from PBMCs pellets was obtained with methanol:water (70:30, v/v), with quinoxaline added as internal standard, after a sonication step. Supernatant was dried and then dissolved in water/acetonitrile (60/40, v/v), before injection into a 2.1 mm×150 mm Atlantis(®) T3 3μ column. Chromatographic separations were performed using a gradient program with a mixture of water (0.05% formic acid), as mobile phase A and acetonitrile (0.05% formic acid), as mobile phase B. Analytes quantification was performed by electro-spray ionisation-single quadrupole mass spectrometry using the selected ion recording (SIR) detection mode. The positive ionization was used for the HIV protease inhibitors (PIs) indinavir, saquinavir, nelfinavir, nelfinavir M8 metabolite, amprenavir, darunavir, atazanavir, ritonavir, lopinavir, tipranavir, the integrase inhibitor (II) raltegravir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and etravirine, while the negative ionization is applied for efavirenz. The calibration curves were built using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.1 to 32 ng/mL (1-320 ng/mL for tipranavir) and fitted to a quadratic regression model weighted by 1/X. The mean extraction recovery for all PIs, II and NNRTIs was always above 82%. The method was precise, with a range of intra/inter-day percent standard deviation within 2.6-14.8%, and accurate with mean of percent coefficient of variation (CV%) from nominal values -7.85 to +9.7%. Each drug concentration evaluated was expressed in ng/mL and optimized using each patient medium corpuscolar volume and cell number. This analytical method is routinely used in our clinical research center for the assessment of intracellular levels of all PIs, raltegravir and NNRTIs commercially available at present.
一种灵敏、准确的高效液相色谱-质谱联用（HPLC-MS）方法，用于测定 HIV+患者外周血单个核细胞（PBMCs）中 14 种抗逆转录病毒药物的细胞内浓度。通过菲科尔密度梯度离心法分离 PBMCs，使用库尔特（®）仪器进行细胞计数和相对平均体积测定。用甲醇：水（70:30，v/v）提取 PBMCs 沉淀中的药物，并加入喹喔啉作为内标，然后进行超声处理。上清液干燥后，用乙腈（60/40，v/v）溶解，然后注入 2.1mm×150mm Atlantis（®）T3 3μ柱。采用水（0.05%甲酸）作为流动相 A 和乙腈（0.05%甲酸）作为流动相 B 的梯度程序进行色谱分离。通过电喷雾离子化-单四极杆质谱法，采用选择离子记录（SIR）检测模式进行分析物定量。HIV 蛋白酶抑制剂（PIs）茚地那韦、沙奎那韦、奈韦拉平、奈韦拉平 M8 代谢物、安普那韦、达芦那韦、阿扎那韦、利托那韦、洛匹那韦、替拉那韦、整合酶抑制剂（II）拉替拉韦和非核苷类逆转录酶抑制剂（NNRTIs）奈韦拉平和依曲韦林采用正离子化模式，而依非韦伦则采用负离子化模式。用浓度范围为 0.1-32ng/mL（替拉那韦为 1-320ng/mL）的抗逆转录病毒药物对空白 PBMCs 进行加标，建立校准曲线，并拟合二次回归模型，权重为 1/X。所有 PIs、II 和 NNRTIs 的平均提取回收率均高于 82%。该方法具有较高的精密度，日内和日间的变异系数（CV%）在 2.6-14.8%范围内，准确度高，平均相对偏差（CV%）为从名义值的-7.85%到+9.7%。用每个患者的中值细胞体积和细胞数对每种药物浓度进行优化，以 ng/mL 表示。该分析方法在我们的临床研究中心常规用于评估目前市售的所有 PIs、拉替拉韦和 NNRTIs 的细胞内水平。
