论文总字数：19981字

摘 要

体血浆中含有多种物质，本实验所要检测的雌酮、雌二醇以及雌三醇是在女性体内存在的雌激素，这三种物质都属于内源性激素，绝大部分都是由卵巢和胎盘合成并作用于机体的。本实验所检测的这三种激素在女性体内含量甚微，并且这三种激素的活性与生理作用各不相同，当属β-雌二醇的生理活性最强。本实验的最终目的是建立简单快速、灵敏度高、可靠性强并且稳定的柱前衍生化超快速液相色谱-串联质谱的实验方案来检测。取血浆样品，采取两步液液萃取方的法，因有其他内源性物质的干扰，选择丹磺酰氯进行衍生化，然后分析，选用雌二醇-d3（E2-d3）为内标。乙腈-水（含0.1%甲酸）为流动相，在0.20mL/min的流速下梯度洗脱。在ESI源正离子模式下，使用MRM模式。通过分析，E1、E2和E3及E2-d3的检测离子对分别为：m/z为504.0→171.0、m/z为506.0→171.0、m/z为522.0→171.0和m/z为509.0→171.0。实验数据表明丹磺酰氯衍生法可明显使这三种内源性激素在ESI源下的离子化效率升高，血浆中E1、E2和E3的线性范围均为0.01～5.00μg/L，LOQ均为10ng/L，三种待测物的的提取回收率≥50%，批内、批间的准确度、精密度均＜15%，可满足样品的分析要求。所以，本实验所选择的方法快速、准确、可靠性强、灵敏度高，可以用于定量分析。

关键词：内源性激素；雌激素；雌酮；雌二醇；雌三醇；LC-MS/MS；衍生化；药物分析

雌三醇

雌二醇

雌酮

Determination Estrone, Estradiol and Estriol in Human Serum by Pre-column Derivatization Ultrafast Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Human plasma contains a variety of substances, the test to be tested estrone, estradiol and estriol is present in women in the body of estrogen, these three substances are endogenous hormones, the vast majority are from the ovaries and Placenta synthesis and role in the body. The three hormones tested in this experiment in the female body content is very small, and the three kinds of hormones and physiological effects of different, when the β-estradiol is the strongest physiological activity. The ultimate goal of this experiment is to establish a simple, rapid, highly sensitive, reliable and stable pre-column derivatization of ultra-fast liquid chromatography-tandem mass spectrometry experimental program to detect. Take the plasma sample, take two-step liquid-liquid extraction method, because there are other endogenous substances interference, the choice of dansyl chloride for derivatization, and then analysis, the choice of estradiol-d3 (E2-d3) as the internal standard. Acetonitrile-water (containing 0.1% formic acid) as the mobile phase and eluted at a flow rate of 0.20 mL / min. In the ESI source positive ion mode, the MRM mode is used. The analytic ion pairs of E1, E2 and E3 and E2-d3 were m / z 504.0 → 171.0, m / z 506.0 → 171.0, m / z 522.0 → 171.0 and m / z 509.0 → 171.0 The The experimental results show that the lignin chloride derivative method can obviously increase the ionization efficiency of these three endogenous hormones under ESI source. The linear range of E1, E2 and E3 in plasma is 0.01 ~ 5.00μg / L, LOQ is 10ng / L, the recoveries of the three kinds of test materials ≥ 50%, batch, batch accuracy, precision lt;15%, to meet the sample analysis requirements. Therefore, the method chosen in this experiment is fast, accurate, reliable and sensitive, and can be used for quantitative analysis.

Keywords: endogenous hormone; estrogen; estrone; estradiol; estriol; LC-MS / MS;

derivatization; Pharmacology analysis

目录

摘要 I

Abstract II

第一章 文 献 综 述 1

1.1引言 1

1.2内源性激素 1

1.2.1皮质激素 1

1.2.2雌激素 2

1.2.3雄激素 3

1.2.4孕激素 3

1.3目前用于雌二醇、雌三醇和雌酮等内源性激素的检测方法 3

1.3.1免疫分析法 3

1.3.2高效液相色谱法 4

1.3.3气相色谱、气相色谱串联质谱法 4

1.3.4液质联用技术 4

第二章 实验材料和方法 6

2.1 实验材料 6

2.1.1实验仪器 6

2.1.2实验试剂 6

2.2实验方法 6

2.2.1质谱检测条件 6

2.2.2高效液相色谱条件 7

2.2.3溶液的制备 8

2.2.4血浆样品前处理过程 9

2.3方法学验证 9

2.3.1特异性 9

2.3.2 标准曲线和定量下限 10

2.3.3精密度和准确度 10

2.3.4 提取回收率和基质效应 10

2.3.5稳定性 11

第三章 实验结果和讨论 13

3.1方法学的建立与优化 13

3.1.1衍生化过程的优化 13

3.1.2质谱条件的优化 14

3.1.3色谱条件的优化 14

3.1.4样品前处理优化 15

3.2实验结果 15

3.2.1特异性 15

3.2.2标准曲线和定量下限 15

3.2.3精密度和准确度 16

3.2.4提取回收率与基质效应 17

3.2.5各储存条件下血浆样品的稳定性考察 18

第四章 结论 20

参考文献 21

致 谢 23

文 献 综 述

1.1引言

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