Quantification of total T4, T3, and reverse T3 in canine serum using UPLC-MS/MS: Analytical validation, reference intervals, and method comparison with traditional chemiluminescence immunoassays

Measuring T4 concentrations is an essential part of the diagnostic workup of dogs with suspected thyroid disease. While T3-determination provides valuable additional information in selected cases, the diagnostic benefit of rT3 still needs to be investigated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables simultaneous quantification of TT4, TT3, and TrT3 with high precision and sensitivity, offering potential improvements in canine endocrinological assessments. The aims of this study were to develop and validate a LC-MS/MS method for the measurement of TT4, TT3, and TrT3 in canine serum, to establish reference intervals in clinically healthy dogs, and to compare the LC-MS/MS values with TT4 and TT3 results evaluated by routine chemiluminescence assays (CLIA). Leftover serum samples from 105 clinically healthy dogs, with unremarkable laboratory screenings, were analyzed by LC-MS/MS and reference intervals were determined according to the ASVCP guidelines: 11.44 - 46.38 nmol/l for TT4, 0.807 - 2.152 nmol/l for TT3 and 0.165 - 0.833 nmol/l for TrT3. Further, TT4 and TT3 values were compared to CLIA measurements with a bias of 0% for TT4 and +56% for TT3. Intra- and inter-assay-precision CVs (%) were below 4.00% and 9.80% for TT4, 4.73% and 6.60% for TT3 and 5.88% and 6.50% for TrT3, respectively. This study shows that the antibody-independent LC-MS/MS is a valid method to determine total canine T4, T3 and rT3 concentrations with high accuracy and provides a sound basis for future research in this species.