Buchwald Armin, Winkler Karl, Epting Thomas
Division of Clinical Chemistry, Department of Medicine, University Medical Center Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany.
BMC Clin Pharmacol. 2012 Jan 11;12:2. doi: 10.1186/1472-6904-12-2.
Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards.
Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes.
Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards.
The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.
对器官移植患者的免疫抑制药物进行治疗药物监测对于预防因剂量不足导致的中毒或移植排斥至关重要。常用的免疫分析方法已逐渐被质谱法所取代，因为这种物理方法具有更高的灵敏度和特异性。然而，转换时应谨慎考虑，因为这是一个具有挑战性的过程，需要进行全面验证。从经济角度来看，将霉酚酸纳入分析是合理的，因为这样无需额外测量。然而，迄今为止，针对包括霉酚酸在内的免疫抑制剂测量的验证方案非常少。为了充分补偿基质效应，建议使用稳定同位素标记的内标。在此，作者描述了一种基于氘代内标的单一方法，适用于定量环孢素A、他克莫司、西罗莫司、依维莫司和霉酚酸。
用硫酸锌沉淀血浆蛋白，然后沿流通方向进行在线固相萃取。采用C18 - 苯基 - 己基柱进行色谱分离。使用稳定同位素标记的内标进行后续质谱分析。3.5分钟后可得结果。
低定量限（准确度：104 - 118%）和线性范围为：环孢素A为2 - 1250 ng/ml；他克莫司为0.5 - 42. ng/ml；西罗莫司为0.6 - 49.2 ng/ml；依维莫司为0.5 - 40.8 ng/ml；霉酚酸为0.01 - 7.5 μg/ml。批内精密度显示变异系数（CV）为0.9 - 14.7%，准确度为89 - 138%。批间精密度的CV为2.5 - 12.5%，准确度为90 - 113%。回收率在76.6%至84%之间。氘代内标很好地补偿了基质效应。
作者提出了一种快速、经济且稳健的方法，用于对包括霉酚酸在内的五种免疫抑制剂进行常规治疗药物监测。
