Bittersohl Heike, Herbinger Juliane, Wen Ming, Renders Lutz, Steimer Werner, Luppa Peter B
*Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; and †Department of Nephrology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
Ther Drug Monit. 2017 Jun;39(3):211-219. doi: 10.1097/FTD.0000000000000392.
Therapeutic drug monitoring (TDM) of immunosuppressants is essential to optimize patient care after organ transplantation. In blood, most immunosuppressive drugs are bound to plasma proteins or located inside blood cells. However, it is generally assumed that only protein-unbound (free) drug concentrations are pharmacologically active and could therefore better reflect the clinical outcome. Study data are still limited due to lacking rapid analytical methods. Therefore, a simple multiplex method for direct measurement of free cyclosporine A (CsA) and mycophenolic acid (MPA) has been developed.
The sample preparation included ultracentrifugation, followed by liquid-liquid extraction. Stable isotope labeled analogues of CsA and MPA were used as internal standards. The LC-MS/MS analysis was performed on a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. The validated assay was used in a study of 40 blood samples from kidney transplant patients.
The lower limits of quantification were 0.1 (CsA) and 0.5 ng/mL (MPA). Assay linearity was confirmed in the concentration ranges of 0.1-10.0 ng/mL (CsA) and 0.5-100 ng/mL (MPA). For both analytes, inaccuracy was ≤9.8% and imprecision was ≤7.8%. The extraction efficiency ranged between 91% and 96%. In the patient samples the average free CsA and MPA fractions were 5.8% (2.1%-16.8%) and 1.2% (0.5%-2.4%) respectively.
A reliable and highly sensitive LC-MS/MS method as a new suitable tool for measuring protein-unbound CsA and MPA has been developed, validated and applied in kidney transplant patient samples. Now, larger studies can be conducted to investigate the benefit of free drug monitoring in transplant recipients.
免疫抑制剂的治疗药物监测（TDM）对于优化器官移植后的患者护理至关重要。在血液中，大多数免疫抑制药物与血浆蛋白结合或存在于血细胞内。然而，一般认为只有未与蛋白结合的（游离）药物浓度具有药理活性，因此能更好地反映临床结果。由于缺乏快速分析方法，研究数据仍然有限。因此，已开发出一种直接测量游离环孢素A（CsA）和霉酚酸（MPA）的简单多重方法。
样品制备包括超速离心，然后进行液液萃取。CsA和MPA的稳定同位素标记类似物用作内标。液相色谱-串联质谱（LC-MS/MS）分析在三重四极杆质谱仪上以多反应监测模式进行。经过验证的检测方法用于对40例肾移植患者的血样进行研究。
定量下限分别为0.1（CsA）和0.5 ng/mL（MPA）。在0.1 - 10.0 ng/mL（CsA）和0.5 - 100 ng/mL（MPA）的浓度范围内确认了检测线性。对于两种分析物，误差≤9.8%，精密度≤7.8%。萃取效率在91%至96%之间。在患者样本中，游离CsA和MPA的平均比例分别为5.8%（2.1% - 16.8%）和1.2%（0.5% - 2.4%）。
已开发、验证并将一种可靠且高度灵敏的LC-MS/MS方法作为测量未与蛋白结合的CsA和MPA的新适用工具应用于肾移植患者样本。现在，可以开展更大规模的研究来探讨游离药物监测对移植受者的益处。
