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.

A validated LC-MS/MS method for cellular thyroid hormone metabolism: Uptake and turnover of mono-iodinated thyroid hormone metabolites by PCCL3 thyrocytes.

Tyrosine and phenolic ring de-iodination of thyroid hormones (TH) is crucial for regulating their physiological activity. Furthermore, reactions such as de-carboxylation to thyronamines (TAM) and de-amination to thyroacetic acids (TAc) produce TH metabolites (THM) with distinct biological properties. This needs to be considered when studying effects of TH and THM. The accurate and precise quantitative analysis of TH and THM in cell culture supernatants and cell lysates are key procedures required for studying the in vitro metabolism of TH. We report here the development of a liquid-liquid extraction/isotope dilution-liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the quantification of 9 thyronines (TN) and 6 TAM in human hepatocellular carcinoma Hep G2 cell lysate extracts. In addition, we adapted the method to quantify TH, TAM and TAc, in cell lysates of FBS-depleted rat thyroid epithelium PCCL3 cells. The methods for both cell lines were validated by rigorous assessment of linearity, 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 covering 11 concentrations (based on 400 µl of lysate) were linear in the range 0.016-50 nM and 0.010-50 nM for Hep G2 and PCCL3 cells respectively. The lower limits of quantification were in the range 0.031 to 1 nM. We applied the PCCL3 version of the LC-MS/MS method to the analysis of lysed cell extracts from PCCL3 cells that had been incubated with 3-iodo-L-thyronine (T1), 3-iodothyronamine (3-T1AM) and 3-iodothyroacetic acid (3-T1Ac). Over the course of 30 minutes incubation 3-T1AM was de-iodinated to 4-[4-(2-aminoethylphenoxy)]phenol (thyronamine, T0AM) and de-aminated to 3-T1Ac respectively, whilst T1 underwent de-iodination to T0. This data indicates avid metabolism of these mono-iodinated compounds and the utility of LC-MS/MS to quantify such cellular metabolism.