Abstract: 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-d₃,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.

Key words: Digitoxin, Isotope dilution ultra high performance liquid chromatography tandem mass spectrometry, Candidate reference method, Methodological assessment