超高效液相色谱-串联质谱法测定人尿液中甲氨蝶呤浓度的不确定度评定

Uncertainty evaluation for methotrexate analysis in human urine by UHPLC-MS/MS

目的:评价超高效液相色谱-串联质谱(UHPLC-MS/MS)测定人尿液中甲氨蝶呤浓度的不确定度。方法:通过考察测定方法,分析实验过程中的不确定度来源,包括对照品称量、标准溶液配制和样品制备、回收率及基质效应、校正曲线拟合、仪器的精密性、实验温度和对照品纯度等,然后逐一评定并计算合成不确定度和扩展不确定度。结果:置信概率P为95%时,人尿液中甲氨蝶呤低浓度0.06μmol·L^(-1)和高浓度3μmol·L^(-1)质控样品的扩展不确定度分别为0.0065μmol·L^(-1)和0.20μmol·L^(-1)。结论:根据不确定度的大小及来源,改良测定方法,提高分析准确性。UHPLC-MS/MS法测定人尿液中甲氨蝶呤浓度的不确定度在低浓度时主要由曲线拟合、生物样品配制和标准液配制、回收率及基质效应引入;高浓度的不确定度主要由生物样品配制和标准液配制、回收率引入。

OBJECTIVE To evaluate the uncertainty for methotrexate analysis by ultra high performance liquid chromatograph-tandem mass spectrometry(UHPLC-MS/MS)in human urine.METHODS By examining the process of method,the potential sources of methodological uncertainty were analyzed,including weighing of reference substances,preparation of standard solution and sample,recovery rate,matrix effect,fitting of calibration curve,instrumental precision,experimental temperature and purity,etc.The uncertainty of each source component was evaluated to calculate combined uncertainty and expanded uncertainty.RESULTS With a confidence probability of 95%,the expanded uncertainty of methotrexate at low(0.06μmol·L^(-1))and high(3μmol·L^(-1))levels were 0.0065 and 0.20μmol·L^(-1) respectively.CONCLUSION The level of each uncertainty component is examined for improving the analytical method and its accuracy.The uncertainty of this UHPLC-MS/MS method is mostly caused by curve fitting,sample and solution preparation,recovery and matrix effect at a low level.At a high level,uncertainty is largely caused by preparing sample and working solutions and recovery rate.