Bahmany Soma, de Wit Lucia E A, Hesselink Dennis A, van Gelder Teun, Shuker Nauras M, Baan Carla, van der Nagel Bart C H, Koch Birgit C P, de Winter Brenda C M
Department of Hospital Pharmacy, Erasmus MC, University Medical Center, Rotterdam, the Netherlands.
Department of Internal Medicine, Rotterdam Transplant Group, Erasmus MC, University Medical Center Rotterdam, the Netherlands.
Biomed Chromatogr. 2019 Jan;33(1):e4416. doi: 10.1002/bmc.4416. Epub 2018 Nov 14.
After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC-MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS-TDM . To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing C H -tacrolimus as internal standard, 40 μL MagSiMUS-TDM Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol-based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC-MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r = 0.997) over the range 5.0-1250 pg/1 × 10 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.
实体器官移植后，给予他克莫司以预防排斥反应。采用治疗药物监测来达到全血中他克莫司的目标浓度。由于他克莫司的作用部位是淋巴细胞，且他克莫司约80%与红细胞结合，淋巴细胞内的他克莫司浓度可能更具相关性。为此，我们旨在开发、改进和验证一种超高效液相色谱-串联质谱法（UPLC-MS/MS）来测定分离的外周血单个核细胞（PBMC）中的他克莫司浓度。使用Ficoll分离技术分离PBMC，随后用红细胞裂解液进行洗涤步骤。将含有100万个PBMC的50 μL细胞悬液与MagSiMUS-TDM联用。向每个样品中加入30 μL裂解缓冲液、20 μL含有C H -他克莫司作为内标的重构缓冲液、40 μL MagSiMUS-TDM I型颗粒混合物和175 μL用于甲醇基蛋白沉淀的有机沉淀试剂VI。取10 μL上清液注入UPLC-MS/MS系统。该方法经过验证，具有高灵敏度和特异性。该方法在5.0-1250 pg/1×10 PBMCs范围内呈线性（r = 0.997）。不准确度<5%，精密度<15%。Ficoll分离后的洗涤步骤可在室温或冰上进行，温度对结果无影响。开发并成功验证了一种分析PBMC中他克莫司浓度的方法。将进行进一步研究以探讨PBMC中浓度与临床结果之间的相关性。
