%0 Generic %A D., Rathmann %A E., Rijntjes %A J., Lietzow %A J., Köhrle %D 2016 %T Supplementary Material for: Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS %U https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Quantitative_Analysis_of_Thyroid_Hormone_Metabolites_in_Cell_Culture_Samples_Using_LC-MS_MS/4276427 %R 10.6084/m9.figshare.4276427.v1 %2 https://karger.figshare.com/ndownloader/files/6971567 %2 https://karger.figshare.com/ndownloader/files/6971570 %2 https://karger.figshare.com/ndownloader/files/6971573 %2 https://karger.figshare.com/ndownloader/files/6971576 %K Thyroid hormone metabolite %K Culture media %K LC-MS/MS %K Liquid-liquid extraction %K Thyronamine %K Iodothyronine %X

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 nM (R2 0.991-0.999) for all calibrators. The lower limit of quantification was 0.078-0.234 nM. 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.

%I Karger Publishers