Development and validation of an isotope dilution-liquid chromatography (ID-LC–MS/MS)-based candidate reference measurement procedure for total 3,3′,5-triiodothyronine in human serum

3,3′,5-Triiodothyronine (T3) plays a crucial role in the diagnosis and evaluation of thyroid diseases. The current reference measurement procedure (RMP) has been in place for 20 years and is widely adopted by reference laboratories around the world. To enhance the performance of the RMP, we developed and validated a simplified, SI-traceable candidate RMP specifically targeting serum total T3 (TT3) using isotope dilution-liquid chromatography with tandem mass spectrometry (ID-LC–MS/MS). Specifically, T3 reference material was employed as the standard. Compared to the RMP, our method incorporates a simplified serum cleanup process involving protein precipitation and nitrogen drying. The method validation followed ISO guidelines for measurement uncertainty assessment. Our results indicated that there was no significant conversion of T4 into metabolites during the serum pretreatment. The lower limit of the measuring interval was determined to be 0.077 nmol/L, and the regression model was linear across a range of 0.016 to 13.086 nmol/L. The imprecision of intra-assays ranged from 0.1 to 1.9%, while inter-assay imprecision ranged from 0.5 to 1.2%. The bias observed when analyzing SRM971 was between − 0.34 and − 1.65%. This candidate RMP demonstrated excellent agreement with the RMPs developed by RELA activity, showing a mean bias falling within the educational mean ratio (6%). The measurement uncertainties were 0.022 nmol/L for RELA2023A and 0.120 nmol/L for RELA2023B. In conclusion, the developed candidate RMP is traceable, reliable, and easy to operate, showing promising potential for the development of serum reference materials and the standardization of routine assays.

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