A combined LC-MS/MS and LC-MS³ multi-method for the quantification of iodothyronines in human blood serum

We report here a novel approach for the extraction and analysis of thyroid hormones (TH) and their metabolites (THM) from human serum samples. Our method features a compact, 96-well micro-titre plate-based pre-analytic extraction/clean-up workflow combined with an isotope dilution LC-MS/MS-MS³ analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 10⁴, with potential to semi-automate the pre-analytics. The method was validated by the assessment of linearity, lower limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves were linear in the concentration range in sample matrix from 0.1-250 nM for T₃, rT₃, T₄ and 3-T₁AM and from 0.005-1 nM for 3,5-T₂ and 3,3'-T₂. Using a 200-μL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T₂) to 0.25 (T₄) nM and LLOD were between 0.002 (3,5-T₂) and 0.052 nM (T₄). We applied the LC-MS/MS-MS³ method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T₄, T₃ and rT₃ concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3'-T₂ concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T₂ concentrations were below the LLOQ and/or LLOD in all but a single sample (0.013 nM). This method expands the analytical spectrum to endogenous thyroid hormone metabolites such as 3,5-T₂ which exert biological actions and rT₃ which may act as surrogate markers for disturbed thyroid hormone metabolism. Graphical abstract.