Supplementary Material for: Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS

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.

本研究开发了一种液液萃取-液相色谱-电喷雾电离串联质谱（LC-MS/MS）方法，用于从细胞培养基中检测碘甲状腺原氨酸类（iodothyronines）与甲状腺胺类（thyronamines, TAM）物质。甲状腺激素（thyroid hormones, TH）可通过连续脱碘代谢最终生成甲状腺原氨酸（T₀），也可经脱羧反应生成甲状腺胺类物质。本文所报道的针对DMEM/F12细胞培养基的萃取方法，可通过单次液相色谱运行精准检测9种甲状腺激素与6种甲状腺胺类物质，是一项核心实验流程。本方法采用异丙醇-叔丁基甲醚（isopropanol-TBME，体积比30:70）的液液萃取体系，从不含酚红的DMEM/F12培养基中萃取待测物与内标（internal standards, IS）。本方法以¹³C₆-甲状腺素（¹³C₆-T₄）、¹³C₆-三碘甲状腺原氨酸（¹³C₆-T₃）、¹³C₆-反式三碘甲状腺原氨酸（¹³C₆-rT₃）、¹³C₆-3,3'-二碘甲状腺原氨酸（¹³C₆-3,3′T₂）以及²H₄-甲状腺胺（²H₄-T₁AM）作为内标，在10分钟的色谱运行时长内完成甲状腺激素与甲状腺胺类的检测。针对9种甲状腺激素与6种甲状腺胺类代谢物的检测，校准曲线包含11个校准品，每个校准品均进行三次重复测定；5种内标在0.12~120 nM浓度范围内均呈现良好的线性与重现性，相关系数R²介于0.991~0.999之间。本方法的定量下限为0.078~0.234 nM。通过对精密度、准确度、过程效率、基质效应、回收率以及批内与批间稳定性的考察，验证了本方法的有效性与耐用性。针对9种甲状腺激素与6种甲状腺胺类代谢物的高、中、低浓度质量控制样本，均对上述参数进行了考察。本经验证的灵敏、无相互干扰的LC-MS/MS方法，可快速分析并精准检测不含酚红的DMEM/F12条件培养基中的甲状腺激素与甲状腺胺类物质，且易于推广应用于常用缓冲液与细胞培养基体系。