Colombo S, Beguin A, Telenti A, Biollaz J, Buclin T, Rochat B, Decosterd L A
Division de Pharmacologie clinique, Laboratoire BH 18-218, Département de Médecine, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne CHUV, Switzerland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 May 25;819(2):259-76. doi: 10.1016/j.jchromb.2005.02.010.
A sensitive and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the intracellular determination of nine antiretroviral drugs in human peripheral blood mononuclear cells (PBMCs) is proposed. PBMCs are isolated by density gradient centrifugation using Vacutainer CPT tubes and cell count is performed with a Coulter instrument. Single-step extraction of drugs from PBMCs pellets is performed with MeOH 50% (with clozapine added as internal standard, I.S.) and the supernatant is injected onto a 2.1 mm x 30 mm SymmetryShield 3.5 microm-RP18 column equipped with a 2.1 x 10 mm guard column. Chromatographic separations are performed using a gradient program with a mixture of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electro-spray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring (SRM) detection mode. The positive mode is used for the HIV protease inhibitors (PIs) indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, and the negative mode is applied for efavirenz. The calibration curves are prepared using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.5 to 100 ng/ml of cell extracts and fitted to a quadratic regression model weighted by 1/(concentration)(2). The lower limit of quantification is less than 0.5 ng/ml. The mean extraction recovery for all PIs/NNRTIs is always above 88%. The method is precise, with mean inter-day CV% within 0.6-10.2%, and accurate (range of inter-day deviation from nominal values -7.2 to +8.3%). This analytical method can be conveniently used in clinical research for the assessment of intracellular levels of all PIs/NNRTIs commercially available at present using a simple one-step cell extraction of PBMCs followed by liquid chromatography coupled with tandem triple quadripole mass detection.
本文提出了一种灵敏且准确的液相色谱 - 串联质谱法（LC-MS/MS），用于测定人外周血单核细胞（PBMCs）内的九种抗逆转录病毒药物。使用Vacutainer CPT管通过密度梯度离心法分离PBMCs，并使用库尔特仪器进行细胞计数。用50%甲醇（添加氯氮平作为内标，I.S.）从PBMCs沉淀中进行单步药物提取，将上清液注入到配备2.1×10 mm保护柱的2.1 mm×30 mm SymmetryShield 3.5微米RP18柱上。色谱分离采用梯度洗脱程序，流动相为含0.1%甲酸的2 mM乙酸铵和含0.1%甲酸的乙腈的混合物。采用电喷雾电离 - 三重四极杆质谱法，在选择反应监测（SRM）检测模式下对分析物进行定量。正离子模式用于HIV蛋白酶抑制剂（PIs）茚地那韦、安普那韦、沙奎那韦、利托那韦、奈非那韦、洛匹那韦、阿扎那韦以及非核苷类逆转录酶抑制剂（NNRTIs）奈韦拉平，负离子模式用于依法韦仑。通过在空白PBMCs中加入浓度范围为0.5至100 ng/ml细胞提取物的抗逆转录病毒药物来制备校准曲线，并拟合到以1/(浓度)(2)加权的二次回归模型。定量下限小于0.5 ng/ml。所有PIs/NNRTIs的平均提取回收率始终高于88%。该方法精密度良好，日间平均CV%在0.6 - 10.2%之间，准确度高（日间偏差与标称值的范围为 -7.2至 +8.3%）。这种分析方法可方便地用于临床研究，通过对PBMCs进行简单的单步细胞提取，随后进行液相色谱与串联三重四极杆质谱检测，来评估目前所有市售PIs/NNRTIs的细胞内水平。
