洋地黄毒苷候选参考方法的建立及评价

Establishment and evaluation for the determination of serum digitoxin as a candidate reference method

目的建立一种基于同位素稀释超高效液相色谱-串联质谱(ID-UPLC-MS/MS)的血清洋地黄毒苷检测候选参考方法,并对此方法进行性能评价。方法在血清中加入内标物质洋地黄毒苷-21,23,23-d_(3),采用0.5 mol/L硫酸锌溶液进行蛋白沉淀,采用叔丁基甲基醚对样本进行液液萃取,上清液采用氮气吹干,流动相复溶后采用ID-UPLC-MS/MS检测。采用五点包括法计算血清洋地黄毒苷水平。参考美国临床实验室标准化协会(CLSI)C62-A文件和CLSI EP15-A3文件对建立的ID-UPLC-MS/MS方法进行性能评价(基质效应、特异性、携带污染、精密度、准确度),同时评估其检测洋地黄毒苷的测量不确定度。结果建立的ID-UPLCMS/MS方法检测血清洋地黄毒苷的线性范围为2.8~94.9 nmol/L,批内变异系数(CV)为0.44~1.98%、总CV为0.41%~1.06%;加标回收率为100.03%~100.48%;检测限[信噪比(R_(SN))=3]及定量限(R_(SN)=10)分别为0.032和0.095 nmol/L;检测2020 RELA-A、2020 RELA-B样本,相对偏移分别为0.62%、0.29%,其中2020 RELA-A样本的不确定度为0.35 nmol/L。结论建立了检测血清洋地黄毒苷的ID-UPLC-MS/MS方法。该方法灵敏度高,重复性好,准确度高,有望作为人血清洋地黄毒苷测定的候选参考方法。

Objective To establish a candidate reference method for the determination of serum digitoxin based on isotope dilution ultra high performance liquid chromatography tandem mass spectrometry(ID-UPLC-MS/MS),and to evaluate the performance of this method.Methods The human serum was added with digitoxin-21,23,23-d_(3),the protein was precipitated in 0.5 mol/L zinc sulfate solution.Then the samples were extracted with tert-butyl methyl ether,the upper layer was dried with nitrogen flow.The residual was reconstituted with mobile phase and analyzed by ID-UPLC-MS/MS.Serum digitoxin concentration was calculated by 5-point bracketing method.The ID-UPLC-MS/MS(matrix effect,specificity,carrying contamination,precision,accuracy)was validated according to the Clinical and Laboratory Standards Institute(CLSI)C62-A and CLSI EP15-A3.The uncertainty of the measurement of digitoxin by ID-UPLC-MS/MS.Results The ID-UPLC-MS/MS for the determination of digitoxin was validated over a concentration range of 2.8-94.9 nmol/L.The intra-batch coefficient of variation(CV)was 0.44%-1.98%,and the total CV was 0.41%-1.06%.The recoveries were 100.03%-100.48%.The limits of detection[signal-to-noise ratio(R_(SN))=3]and quantitation(R_(SN)=10)were 0.032 and 0.095 nmol/L,respectively.According to RELA 2020,RELA-A and RELA-B 2020 relative biases of 0.62%and 0.29%were determined to be the uncertainty of 0.35 nmol/L.Conclusions The ID-UPLC-MS/MS established successfully for serum digitoxin has good precision,repeatability and accuracy,which can be used as a candidate reference method.