“自上而下”法评定液质联用技术检测人血浆中EVT201浓度的不确定度

“Top-down” approach to evaluate the measurement uncertainty of EVT201 in human plasma by UHPLC-MS/MS

目的评定UHPLC-MS/MS法测定人血浆中EVT201浓度的不确定度。方法本研究用'自上而下'法对EVT201生物分析过程中的不确定度进行评估。以实际样本分析过程中随行质控作为室内质控、以加标质控替代有证标准物质,估算室内测量复现性和偏移2个分量引入的不确定度,对影响这2个分量的因素进行分析和量化,并对相对标准不确定度结果进行合成及扩展。结果对所有分量合成后,4个质控水平的相对标准不确定度分别为9. 6%,7. 9%,5. 3%和4. 1%,均小于指导原则对于准确度的要求。对标准不确定度进行扩展后,质控结果可表示为(0. 600±0. 115),(8. 000±1. 260),(80. 000±8. 500)和(160. 000±13. 000) ng·mL-1(k=2),待测样本的扩展不确定度则由附近质控的相对标准不确定度计算获得。结论本文所建立的'自上而下'法是一种快速、简便地评定生物样本分析过程中不确定度的方法,可以从不确定度角度对生物分析过程进行快速评估。

Objective To evaluate the measurement uncertainty of the quantification of EVT201 in human plasma with UHPLC-MS/MS method using'top-down'approach.Methods With the'Top-down'approach,the internal quality control data and spiking quality control samples were used to estimate the uncertainties associated to imprecision and bias.Factors affecting the two components were analyzed and quantified.Then,the combined uncertainty was obtained by combining the relative standard uncertainties together.And the expanded uncertainty was also calculated.Results The relative standard measurement uncertainties at four quality control levels were 9.6%,7.9%,5.3%and 4.1%,respectively.The results meet with the accuracy requirement of bioanalytical method validation guidance.The final results of the quality control samples could be expressed with the expanded measurement uncertainty as follo-wing:(0.600±0.115),(8.000±1.260),(80.000±8.500)and(160.000±13.000)ng·mL-1(k=2).The relative measurement uncertainty of a real sample was the same with that of the control samples close to it.Conclusion The'top-down'approach provided a practical tool for the evaluation of measurement uncertainty in bioanalysis process.It could be used to estimate the measurement uncertainty of laboratory developed tests in bioanalysis laboratory.