Z-Project: Analytical procedures for the determination of the thyroid hormone status in biological samples collected in the framework of the priority programme SPP 1629

甲状腺激素(TH)及其代谢物(THM)，如甲状腺原氨酸()、甲状腺醋酸(TA)和几种结合形式在血液中以游离和蛋白质结合的形式循环。在第一个SPP资助期内，与SPP 1629合作伙伴合作，对人血清中3，5-T2和3-T1AM的两种免疫分析(IA)进行了优化、验证并应用于数据采集。一种基于液-液萃取(LLE)的液质联用(LC-MS/MS)检测细胞培养上清液中THM谱的方法已经得到充分验证，并使用一种改进的固相萃取(SPE)预分析方法进一步用于细胞和组织提取物的检测。LC-MS/MS方法的优化和验证，包括人和小鼠血清的分析前样品工作仍在进行中。由于各种基质干扰在回收率和过程效率方面造成的一些实验限制，导致了MS中的离子抑制。放射免疫分析(RIA)用于分析人血清中的rt3以及小鼠血清中的T4和t3已成功地与SPP合作伙伴一起用于数据生成。在第二个资助期内，将实现以下目标：(1)应用免疫分析确定儿童和成人血清中3T1AM的特定年龄和性别参考范围(S)，(2)确定儿童和成人血清中不同发育阶段、不同性别、不同年龄和不同疾病的THM图谱，用于(个性化)诊断和监测治疗干预措施，(3)一旦达到当局要求的质量控制参数，即完成对人和小鼠血清的THM LC-MS/MS分析的验证，(4)优化从含血清基质中的分析前提取方法。Z项目作为一个中心平台，将为SPP合作伙伴提供经过验证的IA和MS方法，用于临床和实验样本中THM图谱的分析任务和定量分析。

Thyroid hormones (TH) and their metabolites (THM) such as thyronamines (TAM), thyroacetic acids (TA) and several conjugated forms circulate in the blood in free and protein-bound form. During the first SPP funding period two immunoassays (IA) for 3,5-T2 and 3-T1AM in human serum have been optimized, validated and applied for data acquisition in cooperation with SPP 1629 partners. A liquid-liquid extraction (LLE)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of THM profiles in cell culture supernatants has been fully validated and further advanced for cell and tissue extracts using a modified pre-analytical approach involving solid phase extraction (SPE). Optimization and validation of LC-MS/MS methods including pre-analytical sample workup for human and mouse serum is still ongoing. Several experimental limitations in recovery and process efficiency caused by various matrix interferences lead to ion suppression in MS. Radioimmunoassays (RIA) for analysis of rT3 in human serum as well as T4 and T3 in mouse serum have been successfully applied for data generation together with SPP partners. During the second funding period the following aims will be pursued: (1) application of IA to define age and sex-specific reference range(s) for 3T1AM in human serum of children and adults, (2 characterization of development-, gender-, age-, and disease-specific THM profiles in human serum for (personalized) diagnostics and monitoring of therapeutic interventions, (3) completion of validation of THM LC-MS/MS analytics for human and mouse serum once quality control parameters requested by authorities have been accomplished, (4) optimization of pre-analytic TAM extraction from serum-containing matrices. The Z-project as a central platform will provide validated IA and MS-based methods to SPP partners for analytical tasks and quantitative analysis of THM profiles in clinical and experimental samples.