UPLC-MS/MS法及均相酶免疫法测定人全血西罗莫司浓度

Determination of sirolimus concentration in human whole blood by UPLC-MS/MS and homogeneous enzyme immunoassay

目的建立高效液相色谱-串联质谱(UPLC-MS/MS)法监测人全血西罗莫司浓度,并将检测结果与均相酶免疫法进行比较。方法100μL全血样品加入10μL西罗莫司-D3内标、100μL 0.1 mol·L^(-1)ZnSO4溶液、300μL甲醇,涡旋离心后,上清液5μL进样。以0.1%甲酸-甲醇为有机相,0.1%甲酸-水(含10 mmol·L^(-1)乙酸铵)为水相,流速0.5 mL·min^(-1),FastCore Super C18(2.1 mm×100 mm,2.6μm)色谱柱进行梯度洗脱分离。在多反应监测(MRM)模式下采用电喷雾离子源(ESI)正离子化方式检测,西罗莫司、内标的定量离子对分别为m/z 931.6>864.6,m/z 934.6>864.6。进行方法学考察(包括特异性、定量下限、标准曲线、准确度、精密度、残留效应、基质效应、稳定性和回收率)。测定94例器官移植患者的西罗莫司浓度,与均相酶免疫法结果进行比较。结果西罗莫司标准曲线浓度范围为2.5~50 ng·mL^(-1)。西罗莫司的日内、日间准确度相对误差(RE)在-5.3%~7.5%,变异系数(CV)小于11.8%。低、中、高不同浓度的质控回收率和基质效应一致,经内标校正后回收率为100.22%~108.56%,经内标归一化后基质效应为95.45%~96.48%,CV小于7.0%。全血样本室温放置3 d,4℃放置3 d,-20℃放置20 d,反复冻融3次,处理后样本自动进样器放置20 h,室温放置24 h结果稳定。Passing-Bablok回归分析及Bland-Altman结果表明均相酶免疫法与UPLC-MS/MS两种西罗莫司测量方法存在比例偏差。结论本研究建立了一种简单、高效的UPLC-MS/MS方法检测人全血西罗莫司浓度,均相酶免疫法与UPLC-MS/MS法检测人全血西罗莫司存在比例偏差。UPLC-MS/MS方法更加准确,是免疫抑制剂治疗药物监测检测金标准。

Objective To quantify sirolimus concentration in human whole blood by UPLC-MS/MS and to evaluate the agreement with the homogeneous enzyme immunoassay.Methods Human whole blood(100μL)was spiked with 10μL sirolimus-D3(the internal standard,IS),100μL 0.1 mol·L^(-1)ZnSO4 and 300μL methanol.After the vortex and centrifugation(14000 r·min^(-1)),5μL supernatant was injected.Water(containing 10 mmol·L^(-1)ammonium acetate and 0.1%formic acid,solvent A)and methanol(containing 0.1%formic acid,solvent B)were used as the eluent,with the gradient elution at 0.5 mL·min^(-1)in FastCore Super C18 column(2.1 mm×100 mm,2.6μm).The multiple reaction monitoring(MRM)with electrospray ionization(ESI)in the positive-ion mode was used.m/z 931.6>864.6 and m/z 934.6>864.6 were used for sirolimus and the IS quantitation.Method validation,including selectivity,LLOQ,calibration curve,accuracy,precision,remaining-effect,matrix effect,stability and recovery was evaluated.Passing-Bablok regression analysis and Bland-Altman plot were used to assess the agreement between UPLC-MS/MS and homogeneous enzyme immunoassay.Results The calibration ranged 2.5~50 ng·mL^(-1)for sirolimus.The relative error of intra-and inter-day accuracy of sirolimus ranges from-5.32%to 7.46%,and the coefficient of variation was less than 11.8%.The IS normalized recovery and matrix effect were 100.22%~108.56%and 95.45%~96.48%with coefficient of variation less than 7.0%.Sirolimus was stable in the test conditions.Comparison between UPLC-MS/MS with homogeneous enzyme immunoassay showed a deviation in proportion.Conclusion UPLC-MS/MS method for the determination of sirolimus in human whole blood is rapid,easy and reliable.A proportion deviation in the two methods is observed.