Quantification of cyclosporine A in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry using a column-switching approach
Quantification of cyclosporine A in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry using a column-switching approach
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DOI：
10.1016/j.jchromb.2007.07.001
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发表时间：
2007-09-15
影响因子：
3
通讯作者：
Rudaz, Serge
中科院分区：
文献类型：
--
作者：
Ansermot, Nicolas;Fathi, Marc;Rudaz, Serge
作为一种潜在的替代环孢素A（CsA）监测全血中，一个敏感的和选择性的方法来定量这种免疫抑制药物在人外周血单个核细胞（PBMC）的液相色谱-电喷雾电离质谱（LC-ESI-MS）。通过密度梯度离心从全血中分离PBMC。纯化后，进行细胞计数以表达每单个细胞的CsA量。然后裂解沉淀的细胞，用含有27-脱甲氧基-西罗莫司作为内标的甲醇（MeOH）提取CsA。在氮气下蒸发上清液后，将残余物在MeOH中重构，进一步用水稀释并注射到柱切换单元上。在线固相萃取使用C8柱进行，酸性水移动的相含有5%的甲醇。将分析物以反冲洗模式转移到含有65% MeOH的C18柱上，并使用MeOH梯度（65-90%）进行色谱分离。使用单四极杆分析仪进行检测，并监测钠加合物[M + Na]（+）进行定量。在5-400 ng/mL范围内对该灵敏度方法进行了充分验证。这允许测量临床样品中细胞中存在的非常小的CsA量，高达0.5 fg/PBMC。准确度（95.0-113.2%）、重复性（5.1-9.9%）和中间精密度（7.0-14.7%）均符合要求。这种方法代表了一种新的潜在的CsA治疗药物监测的工具，并可用于临床条件下，如果在前瞻性临床试验中确认的实用程序的细胞内测量。(c)2007 Elsevier B. V.保留所有权利。
As a potential alternative to cyclosporine A (CsA) monitoring in whole blood, a sensitive and selective method was developed for quantifying this immunosuppressive drug in human peripheral blood mononuclear cells (PBMCs) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). PBMCs were isolated from whole blood by density gradient centrifugation. After purification, cell counts were performed to express CsA amounts per single cell. The pelleted cells were then lysed and CsA was extracted with methanol (MeOH) containing 27-demethoxy-sirolimus as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in MeOH, further diluted with water and injected onto a column-switching unit. On-line solid-phase extraction was performed using a C8 column with an acidic aqueous mobile phase containing 5% MeOH. The analytes were transferred in the back-flush mode on a C18 column with 65% MeOH and the chromatographic separation performed with a MeOH gradient (65-90%). The detection was carried out with a single quadrupole analyzer and the sodium adducts [M + Na](+) were monitored for quantification. This sensitive method was fully validated in the range of 5-400 ng/mL. This allowed the measurement of very small CsA amounts present in cells up to 0.5 fg/PBMC in clinical samples. Trueness (95.0-113.2%), repeatability (5.1-9.9%) and intermediate precision (7.0-14.7%) were found to be satisfactory. This method represents a new potential tool for therapeutic drug monitoring of CsA and could be used in clinical conditions if the utility of intracellular measurements is confirmed in prospective clinical trials. (c) 2007 Elsevier B.V. All rights reserved.