Quantification of triiodothyronine and thyroxine in rat serum using liquid chromatography tandem mass spectrometry

Thyroid hormones act on almost every tissue in the body to promote catabolism in cells and are important for regulating many biological processes. Accurate quantification of endogenous thyroid hormones has become essential for clinical and non-clinical applications in the development of new drugs according to the OECD Guideline (2018). However, there are difficulties in quantitative analysis of thyroid hormones because no analyte-free biological matrices are available for analysis of endogenous substances. In this study, surrogate matrix and surrogate analyte methods were compared and validated to quantify endogenous triiodothyronine (T₃) and thyroxine (T₄) in rat serum using LC-MS/MS. Separation of analytes was performed using an Xbridge™ C18 (2.1 × 50 mm, 2.5 μm) column. In the surrogate matrix, 3,3'5-triiodo- l-thyronine-¹³C₆ (cT₃) and l-thyroxine-¹³C₆ (cT₄) were used as the internal standard (IS), and in the surrogate analyte, l-3,3'-diiodothyronine-¹³C₆ (cT₂) was used as the IS. The mobile phases consisted of 0.1 % acetic acid in purified water (A) and 0.1 % acetic acid in acetonitrile (B). Both analytical methods were suitable for selectivity, matrix effect, carryover, lower limit of quantification, linearity, accuracy, precision, recovery, stability and parallelism. The surrogate matrix method was more accurate than using the surrogate analyte method, including evaluation of parallelism at low concentrations. Additionally, the surrogate matrix is cost-effective for T₃ and T₄ analysis in biological samples because it consists only of deionized water. However, surrogate analytes difficult to evaluate parallelism by obtaining response factors for mass spectrometric signal differences between the actual and surrogate analytes. Therefore, the results of this study indicate that it is more cost-effective to use the surrogate matrix method for endogenous thyroid hormone, T₃ and T₄, analysis in biological samples.