Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
iC42 Clinical Research and Development, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
J Chromatogr A. 2018 Apr 27;1547:45-52. doi: 10.1016/j.chroma.2018.03.010. Epub 2018 Mar 7.
Therapeutic drug monitoring (TDM) of the immunosuppressive drug tacrolimus is essential to avoid side effects and rejection of the allograft after transplantation. In the blood circulation, tacrolimus is largely located inside erythrocytes or bound to plasma proteins and less than 0.1% is protein-unbound (free). One basic principle of clinical pharmacology is that only free drug is pharmacologically active and monitoring this portion has the potential to better reflect the drug effect than conventional measurements of total tacrolimus in whole blood. To address this, a highly sensitive and straightforward on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, validated and applied to patient plasma samples. The sample preparation included ultracentrifugation and addition of the stable isotope labeled drug analogue D2,13C-tacrolimus, followed by on-line sample extraction and measurement using a Sciex QTRAP 6500 in the multiple reaction monitoring mode. Due to very low concentrations of protein-unbound tacrolimus, it was important to develop a highly sensitive, precise and accurate assay. Here, we first report the efficient formation of tacrolimus lithium adduct ions, which greatly increased assay sensitivity. A lower limit of quantification (LLOQ) of 1 pg/mL (10 fg on column) was achieved and the assay was linear between 1 and 200 pg/mL. There was no carry-over detected. The inaccuracy ranged from -9.8 to 7.4% and the greatest imprecision was 7.5%. The matrix factor was found to be smaller than 1.1%. In summary, this method represents a suitable tool to investigate the potential clinical value of free tacrolimus monitoring in organ transplant recipients.
免疫抑制剂他克莫司的治疗药物监测对于避免移植后副作用和移植物排斥反应至关重要。在血液循环中，他克莫司主要存在于红细胞内或与血浆蛋白结合，而未与蛋白结合的部分（游离部分）不到 0.1%。临床药理学的一个基本原则是，只有游离药物具有药理活性，监测这部分药物比监测全血中总他克莫司更有潜力反映药物效果。为了解决这个问题，开发了一种高灵敏度和简单的在线液相色谱-串联质谱（LC-MS/MS）方法，并对其进行了验证和应用于患者血浆样本。样品制备包括超速离心和添加稳定同位素标记的药物类似物 D2、13C-他克莫司，然后使用 Sciex QTRAP 6500 在多重反应监测模式下在线进行样品提取和测量。由于未与蛋白结合的他克莫司浓度非常低，因此开发高灵敏度、精确和准确的测定方法非常重要。在这里，我们首次报告了他克莫司锂加合物离子的有效形成，这极大地提高了测定的灵敏度。实现了 1pg/mL（柱上 10fg）的定量下限（LLOQ），且测定在 1 至 200pg/mL 之间呈线性。未检测到拖尾。不准确度范围为-9.8%至 7.4%，最大不精密度为 7.5%。基质因子小于 1.1%。总之，该方法代表了一种适合的工具，可用于研究游离他克莫司监测在器官移植受者中的潜在临床价值。
