Quantitative analysis of thyroid hormone metabolites in cell culture samples using LC-MS/MS
Open accessAbstract
A liquid-liquid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) method to determine iodothyronines and thyronamines (TAM) from cell culture media was developed. Thyroid hormones (TH) are metabolized by sequential deiodination to eventually yield thyronine (T₀), but can also be decarboxylated, resulting in TAM. The method presented here for extraction of DMEM/F12 cell culture media is a fundamental procedure for a precise determination of 9 TH and 6 TAM from a single LC run. Analytes and internal standards (IS) were extracted from DMEM/F12 (w/o phenol red) by liquid-liquid extraction using isopropanol-TBME (30:70 v/v). Measurement of TH and TAM was performed during a 10-min run time using 13 C 6-T 4, 13 C 6-T 3, 13 C 6-rT 3, 13 C 6-3,3′T 2 and 2 H 4-T 1 AM as IS. Calibration curves covered 11 calibrators measured as triplicates each for the analysis of the 9 TH and 6 TAM metabolites, and the 5 IS were linear and reproducible in the range of 0.12-120 n<smlcap>M</smlcap> (R 2 0.991-0.999) for all calibrators. The lower limit of quantification was 0.078-0.234 n<smlcap>M</smlcap>. Method validation and robustness were demonstrated by the analysis of precision, accuracy, process efficiency, matrix effects and recoveries, as well as intra- and interassay stability. These parameters were investigated for high, middle and low concentrations of quality controls of all 9 TH and 6 TAM metabolites. This validated, sensitive and interaction-free LC-MS/MS method allows rapid analysis and accurate determination of TH and TAM from DMEM/F12 (w/o phenol red) conditioned media and seems to be easily transferable and applied to commonly used buffers and cell culture media.
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Reported concentrations of most other TH metabolites in human or experimental animal serum, plasma, and tissue show wide variations and marked insufficiencies in method validation, standardization, and quality assessment.
“while reported concentrations in human or experimental animal serum, plasma, and tissue show wide variations and marked insufficiencies in terms of method validation, standardization, and quality assessment parameters [see (23, 24, 74)].”
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 [74]
The Introduction (page unavailable) mentions measuring thyroid hormone metabolites in human serum, plasma, and animal tissues, but does not assess variation in reported concentrations. The Method Validation and Discussion sections (pages unavailable) describe validation of this paper’s cell-culture assay, not deficiencies in validation, standardization, or quality assessment of reported serum, plasma, or tissue measurements. Searches for those comparative claims found no supporting passage in our copy. (LLM, conf=0.76)
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Cited by
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Groeneweg, S., van Geest, F. S., Peeters, R. P., Heuer, H., & Visser, W. E. (2020) · Endocrine Reviews · doi:10.1210/endrev/bnz008 · Cited by 216 · 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-citation
- Authors
- Rathmann, D., Rijntjes, E., Lietzow, J., & Köhrle, J.
- Affiliations
- Charité – Universitätsmedizin Berlin (DE)
- Venue
- European Thyroid Journal
- Publisher
- Bioscientifica
- Published
- 2015-05-28
- Volume
- 4
- Issue
- 1_Suppl
- Pages
- 51-58
- ISSN
- 2235-0640 (ISSN-L)
- 2235-0802
- PMC
- PMC4640291
- OpenAlex
- W332128574
- Cited by
- 34
- References
- 0
- Citations by year
- 2026: 2 · 2024: 3 · 2023: 2 · 2022: 4 · 2020: 2 · 2019: 3 · 2018: 3 · 2017: 8 · 2016: 6 · 2015: 1
- FWCI
- 5.22
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- European Thyroid Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Deutsche Forschungsgemeinschaft
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Disorders and Treatments
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical chemistry methods development
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Keywords
- thyroid hormone metabolites, iodothyronines, thyronamines, deiodination, decarboxylation, cell culture media, DMEM/F12, liquid-liquid extraction, LC-MS/MS method development, method validation, internal standard, conditioned medium
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