A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum
Abstract
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 sam…
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Reported serum concentrations for 3,3′-T2 covered a very narrow concentration range between 36 and 68 pM.
“Reported serum concentrations covered a very narrow concentration range between 36 and 68 pM for 3,3′-T2 (see Table 1) (23–29, 32–34).”
From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [25]
The 3,5-T2 antibody efficiently extracted 3,5-T2 from serum but did not bind and extract spiked 3-T1AM or other endogenous THM.
“Evaluation of extracts from mAb beads by LC-MS analysis (25) revealed that the 3,5-T2 antibody efficiently extracted 3,5-T2 from serum as expected but did not bind and extract spiked 3-T1AM or other endogenous THM.”
From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [25]
Several laboratories have developed procedures to accurately determine total T4 and total T3 in human plasma or serum and largely agree on concentration ranges, but this consensus has not been achieved for most other TH metabolites.
“While meanwhile several laboratories developed procedures to accurately determine total T4 and total T3 in human plasma or serum and largely agree on concentrations ranges (25, 67–73), this consensus has not been achieved yet for most of the other TH metabolites, which occur at much lower concentrations.”
From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [25]
Our own LC-MS results indicated that most human sera analyzed for 3,5-T2 have lower than 5 pM 3,5-T2 concentrations, the lower limit of quantification.
“while our own results (25) indicated that most human sera analyzed for 3,5-T2 have lower than 5 pM 3,5-T2 concentrations, which is the lower limit of quantification in our method.”
From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [25]
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Van Uytfanghe, K., Ehrenkranz, J., Halsall, D., Hoff, K., Loh, T. P., Spencer, C. A., Köhrle, J., & ATA Thyroid Function Tests Writing Group (2023) · Thyroid® · doi:10.1089/thy.2023.0169 · Cited by 100 · see citing works
Self-citationClinical and laboratory aspects of 3,3′,5′-triiodothyronine (reverse T3)
Halsall, D. J., & Oddy, S. (2021) · Annals of Clinical Biochemistry: International Journal of Laboratory Medicine · doi:10.1177/0004563220969150 · Cited by 28 · see citing works
Köhrle, J., Lehmphul, I., Pietzner, M., Renko, K., Rijntjes, E., Richards, K., Anselmo, J., Danielsen, M., & Jonklaas, J. (2020) · Frontiers in Endocrinology · doi:10.3389/fendo.2019.00787 · Cited by 23 · see citing works
Self-citationBinding Characteristics of Thyroid Hormone Distributor Proteins to Thyroid Hormone Metabolites
Jongejan, R. M. S., Meima, M. E., Visser, W. E., Korevaar, T. I. M., van den Berg, S. A. A., Peeters, R. P., & de Rijke, Y. B. (2022) · Thyroid® · doi:10.1089/thy.2021.0588 · Cited by 20 · see citing works
Köhrle, J., & Richards, K. H. (2020) · Experimental and Clinical Endocrinology & Diabetes · doi:10.1055/a-1175-4610 · Cited by 7 · see citing works
Self-citationDiab, N., Desale, S., Danielsen, M., Köhrle, J., Shara, N., & Jonklaas, J. (2022) · Metabolites · doi:10.3390/metabo12060476 · Cited by 1 · see citing works
Self-citation
- Authors
- Richards, K. H., Monk, R., Renko, K., Rathmann, D., Rijntjes, E., & Köhrle, J.
- Affiliations
- Charité – Universitätsmedizin Berlin (DE)
- Menarini Group (Germany) (DE)
- Corresponding author
- Köhrle, Josef
- Venue
- Analytical and Bioanalytical Chemistry
- Published
- 2019-06-15
- Volume
- 411
- Issue
- 21
- Pages
- 5605-5616
- ISSN
- 1618-2642 (ISSN-L)
- 1618-2650
- OpenAlex
- W2950995063
- Cited by
- 27
- References
- 0
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 2 · 2023: 1 · 2022: 7 · 2021: 3 · 2020: 9 · 2019: 1
- FWCI
- 2.64
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical and Bioanalytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Deutsche Forschungsgemeinschaft (KO922-17/1-2)
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Disorders and Treatments
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical chemistry methods development
- Keywords
- iodothyronines, thyroid hormones, thyroid hormone metabolites, human serum, isotope dilution, reverse T3, triiodothyronine, thyroxine, thyroid hormone metabolism
- MeSH terms
- Calibration, Chromatography, Liquid, Humans, Reference Standards, Thyronines, Reproducibility of Results, Tandem Mass Spectrometry, Limit of Detection
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