全血免疫抑制剂ID-LC-MS/MS候选参考方法的建立和性能评价

Establishment and performance evaluation of candidate reference measurement procedure for the determination of immunosuppressive drugs in human whole blood by ID-LC-MS/MS

目的建立同位素稀释液相色谱串联质谱(ID-LC-MS/MS)检测全血免疫抑制剂(他克莫司、西罗莫司、依维莫司和环孢素A)的候选参考方法,并评价其性能。方法采用蛋白沉淀法(PPT)对全血样本进行前处理,采用ID-LC-MS/MS定量检测他克莫司、西罗莫司、依维莫司和环孢素A。对建立的候选参考方法的分析性能(线性、定量限、基质效应、精密度和正确度等)进行评估。结果ID-LC-MS/MS检测他克莫司、西罗莫司、依维莫司和环孢素A的总检测时间为4.5 min;环孢素A的线性范围为10~500 ng/mL,定量限为10 ng/mL;他克莫司、西罗莫司和依维莫司的线性范围均为1~50 ng/mL,定量限均为1 ng/mL。4种免疫抑制剂的相对基质效应范围均≤8.64%,批内、批间变异系数(CV)均≤5%,加标回收率为98.84%~100.99%。他克莫司的扩展测量不确定度≤7.91%(k=2)、西罗莫司≤7.97%(k=2),依维莫司≤7.14%(k=2),环孢素A≤7.91%(k=2)。结论成功建立了ID-LC-MS/MS检测全血他克莫司、西罗莫司、依维莫司和环孢素A的候选参考方法,可用于相关项目的量值溯源和标准化。

Objective To establish a candidate reference measurement procedure for immunosuppressive drugs(tacrolimus,sirolimus,everolimus and cyclosporine A)in human whole blood by isotope-dilution liquid chromatography-tandem mass spectrometry(ID-LC-MS/MS),and to evaluate its analytical performance.Methods Human whole blood samples were prepared by a simple protein precipitation method,and ID-LC-MS/MS was used to determine tacrolimus,sirolimus,everolimus and cyclosporine A quantitatively.The performance,including linearity,limit of quantitation,relative matrix effect,precision and trueness,were evaluated.Results IDLC-MS/MS allowed the determination of the 4 immunosuppressive drugs within a total runtime of 4.5 min.ID-LC-MS/MS allowed the determination of cyclosporine A in the range of 10-500 ng/mL.Tacrolimus,sirolimus and everolimus were analyzed in the range of 1-50 ng/mL.The limit of quantitation was 10 ng/mL for cyclosporine A and 1 ng/mL for the others.The relative matrix effects were≤8.64%.The precisions for intra-day and inter-day coefficients of variation(CV)were≤5%.The average recovery rates were found to be 98.84%-100.99%for the 4 immunosuppressive drugs.Expanded measurement uncertainties were found to be≤7.91%for tacrolimus(k=2),≤7.97%for sirolimus(k=2),≤7.14%for everolimus(k=2)and≤7.91%for cyclosporine A(k=2).Conclusions The candidate reference measurement procedure for tacrolimus,sirolimus,everolimus and cyclosporine A in human whole blood by ID-LC-MS/MS has been established successfully,which can be used in value traceability and standardization.