ID-LC-MS/MS法测定人血清雌二醇和未结合雌三醇参考系统的研究

The conventional determination methods have poor precision and unaccuracy and/or radioactivity for estradiol (E2) and unconjugated estriol (uE3). In joint committee for traceability laboratory medicine(JCTLM), the reference method of E2 is isotope dilution liquid chromatography tandem mass spectrometry(ID-LC-MS/MS) and isotope dilution gas chromatography-mass spectrometry(ID-GC-MS) method. The reference method of uE3 is ID-GC-MS method. The proposed reference methods need sample derivatization process to volatile components, therefore may bring new uncertainty. E2 has two reference materials including human serum matrix and pure substance. Unconjugated estriol has no reference material in JCTLM. E2 has only the western population reference interval. The uE3 has only 15 cases of small samples of the western population reference interval. In our country there is no research records on E2 and uE3 reference systems. Applicant had established conventional ID-LC-MS/MS method for determination uE3 to meet the performance requirements. The purpose of this study is to establish E2 and uE3 candidate reference methods based on ID-LC-MS/MS theory. In order to research a more sensitive, specific and non-derivatization candidate reference methods, we need conduct a comprehensive study on the pre-processing conditions, chromatographic conditions, mass spectrometric conditions, then make a comprehensive methodology performance evaluation. The E2 and uE3 reference materials with commutability and Chinese population reference interval will be established based on candidate reference methods. The measurement uncertainty of E2 and uE3 reference measurement results will be evaluated. At last a complete E2 and uE3 reference systems will be finished. It is to appraise performance of the conventional chemiluminescent immunoassay by reference systems. It is to help E2 and uE3 and other steroid hormones to realize standardization.

常规测定人血清雌二醇(E2)和未结合雌三醇(uE3)的方法存在精密度较差、准确度欠佳和/或放射性缺陷。国际上E2参考方法为同位素稀释气相色谱质谱(ID-GC/MS)法和同位素稀释液相色谱串联质谱(ID-LC-MS/MS)法，uE3为ID-GC/MS法，均需将样本衍生化，增加了不确定度；E2有人血清基质和纯物质两种参考物质，无uE3参考物质；E2只有西方人群的参考区间，uE3只有15例小样本的西方人群参考区间。我国没有研究E2和uE3参考系统记录。本课题在已建立uE3常规ID-LC-MS/MS法基础上，对前处理、色谱、质谱条件进行全面研究，对方法学性能全面评价，建立E2和uE3的ID-LC-MS/MS更灵敏、更特异的非衍生化候选参考方法。利用建立的候选参考方法进行E2和uE3具有互通性的参考物质以及中国人群参考区间的研究。利用参考系统评价常规化学发光系统，以期为类固醇激素标准化提供技术基础。

常规测定人血清雌二醇(E2)和未结合雌三醇(uE3)的方法存在精密度较差、准确度欠佳和/或放射性缺陷。国际上E2参考方法为同位素稀释气相色谱质谱(ID-GC/MS)法和同位素稀释液相色谱串联质谱(ID-LC-MS/MS)法，uE3为ID-GC/MS法，均需将样本衍生化，增加了不确定度；E2有人血清基质和纯物质两种参考物质，无uE3参考物质；E2只有西方人群的参考区间，uE3只有15例小样本的西方人群参考区间。我国没有研究E2和uE3参考系统记录。本课题在已建立uE3常规ID-LC-MS/MS法基础上，对前处理、色谱、质谱条件进行全面研究对方法学性能全面优化，建立了基于ID-LC-MS/MS同步检测三种雌激素的方法，并成功建立了ID-LC-MS/MS测定人血清E2和uE3候选参考方法，其测量结果具有远小于常规方法的不确定度，具有灵敏度髙、方法特异、准确性好、抗干扰等特性，与原有的参考方法可比性好。研制了E2和uE3具有互通性的参考物质。初步建立了中国人群的参考区间。形成完整的ID-LC-MS/MS测定人血清E2和uE3候选参考方法测量不确定度评定方法。得到部分质控物与校准物的互通性数据及现有E2和uE3测定常规化学发光方法的偏移结果，并提出了整改方案。初步实现了E2和uE3参考系统的建立，为该项目标准化提供了参考。