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

• Published in: Analytical and bioanalytical chemistry 2019-08, Vol.411 (21), p.5605-5616
• Main Authors: Richards, Keith H., Monk, Ray, Renko, Kostja, Rathmann, Daniel, Rijntjes, Eddy, Köhrle, Josef
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Dilution; Feature extraction; Food Science; Hormones; Laboratory Medicine; Linearity; Mathematical analysis; Matrix methods; Metabolism; Metabolites; Monitoring/Environmental Analysis; Research Paper; Standard deviation; Thyroid; Thyroid gland; Thyroid hormones; Thyroxine; Triiodothyronine; Workflow
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-019-01941-9

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-MS 3 analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 10 4 , 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 T 3 , rT 3 , T 4 and 3-T 1 AM and from 0.005–1 nM for 3,5-T 2 and 3,3′-T 2 . Using a 200-μL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T 2 ) to 0.25 (T 4 ) nM and LLOD were between 0.002 (3,5-T 2 ) and 0.052 nM (T 4 ). We applied the LC-MS/MS-MS 3 method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T 4 , T 3 and rT 3 concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3′-T 2 concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T 2 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-T 2 which exert biological actions and rT 3 which may act as surrogate markers for disturbed thyroid hormone metabolism. Graphical abstract