Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS

Abstract

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 ¹³C₆-T₄, ¹³C₆-T₃, ¹³C₆-rT₃, ¹³C₆-3,3′T₂ and ²H₄-T₁AM 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> (R² 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.

In vivo Effects of Repeated Thyronamine Administration in Male C57BL/6J Mice

“…Hepatic THs and TAMs were extracted from 80 mg of mouse liver as previously described [2, 13] followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis as published [14]. A brief description of the method can be found in the online supplementary material (for all online suppl.…”

3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action

“…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)(68)(69)(70)(71)(72)(73), this consensus has not been achieved yet for most of the other TH metabolites, which occur at much lower concentrations. Analytical procedures typically work well with buffer solutions, in vitro reaction mixtures, as well as with samples of "simple" matrix composition, 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)]. Most groups reporting data employed liquid-liquid or solid-phase extraction procedures with or without previous protein precipitation to enrich and purify THM profiles from matrix components.…”

3,5-T2 alters murine genes relevant for xenobiotic, steroid, and thyroid hormone metabolism

“…Three-month-old C57BL/6N male mice weighing 20-30 g were anesthesized and primary hepatocytes were isolated by digestion of the liver with perfusion of collagenase type II, as described previously (Rathmann et al 2015).…”

“…3,5-T 2 treatment might indirectly affect the hepatic gene expression resulting from a 3,5-T 2 -induced altered balance of further locally active hepatic TH metabolites. Hepatocytes exhibit pronounced deiodination capacity (Rathmann et al 2015). It might be that TH metabolism in liver generates decarboxylated and/or deiodinated further TH metabolites that may affect gene expression even via the classical T 3 receptor.…”