An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus ( = 2).

中文翻译：

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基于同位素稀释 LC-MS/MS 的候选参考方法，用于定量人全血中的环孢菌素 A、他克莫司、西罗莫司和依维莫司

提出了一种基于同位素稀释 LC-MS/MS 的候选参考测量程序，用于定量人全血中的环孢素 A、他克莫司、西罗莫司和依维莫司，用于评估和标准化用于治疗药物监测的常规测定。该测定允许使用 C4 反相柱在 9 分钟的总运行时间内对四种免疫抑制药物进行基线分离。样品制备基于用硫酸锌沉淀蛋白质，然后用​​固相萃取纯化。该参考测量程序中使用的参考材料通过 qNMR 进行表征，并计算分析物的绝对含量，以保证 SI 单位的可追溯性。使用相应的氘化和 C 标记的分析物作为内标。该方法允许测量5 ng/mL至2100 ng/mL范围内的环孢菌素A；他克莫司、西罗莫司和依维莫司的分析浓度范围为 0.25 ng/mL 至 50 ng/mL。环孢素 A 的日间测量不精确度≤3.5%，他克莫司、西罗莫司和依维莫司的日间测量不精确度≤4.4%。环孢素 A 的准确度在 101% 到 108% 之间，大环内酯类化合物的准确度在 95% 到 104% 之间。不确定性根据 GUM 进行评估。环孢素 A 的扩展测量不确定度为 ≤7.2%，他克莫司为 ≤6.8%，西罗莫司为 ≤9.0%，依维莫司为 ≤8.9% (= 2)。