一种快速简单的超高效液相色谱串联四极杆质谱方法检测激素的葡糖醛酸化代谢产物

doi:10.5246/jcps.2016.04.033

中国药学(英文版) ›› 2016, Vol. 25 ›› Issue (4): 291-301.DOI: 10.5246/jcps.2016.04.033

一种快速简单的超高效液相色谱串联四极杆质谱方法检测激素的葡糖醛酸化代谢产物

戎怿, 黄碧云, 黄珺珺, 朱柳, 刘夏雯^*

广州医科大学药物研发中心, 广东广州 511436

收稿日期:2016-01-04 修回日期:2016-02-18 出版日期:2016-04-21 发布日期:2016-03-08
通讯作者: Tel.: 020-37104150, E-mail: melody_12@163.com
基金资助:
Science and Technology Plan Project of Guangzhou Municipal College (Grant No. 1201430376) and National Natural Science Foundation of China (Grant No. 81503131).

A rapid and simple ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of glucuronic acid-conjugated steroid metabolites

Yi Rong, Biyun Huang, Junjun Huang, Liu Zhu, Xiawen Liu^*

Pharmaceutical Research and Development Center, Guangzhou Medical University, Guangzhou 511436, China

Received:2016-01-04 Revised:2016-02-18 Online:2016-04-21 Published:2016-03-08
Contact: Tel.: 020-37104150, E-mail: melody_12@163.com
Supported by:
Science and Technology Plan Project of Guangzhou Municipal College (Grant No. 1201430376) and National Natural Science Foundation of China (Grant No. 81503131).

摘要/Abstract

摘要：

本研究应用超高效液相色谱串联四极杆质谱(UPLC-MS/MS)在12分钟内有效地检测了10种激素和激素葡糖醛酸代谢产物。激素睾酮(T)、二氢睾酮(DHT)、雄甾酮(ADT)、本胆烷醇酮(ETIO)、雌二醇(E₂)以及它们的葡糖醛酸化产物能够在Eclipse Plus C₁₈色谱柱(2.1 mm × 50 mm, RRHD 1.8 µm)上很好地分离。流动相为甲醇和含1 mM甲酸铵水溶液(60:40, v/v), 流速为0.25 mL/min。液相流出液使用电喷雾离子源(ESI)在正负模式下进行检测。使用中性丢失(丢失分子量为正模式下的176、194、211和229 Da)和母离子扫描(母离子分子量m/z为正模式下的141、159、177和负模式下的75、85、133)的方法鉴定激素的葡萄醛酸化产物。质谱多反应监测模式(MRM)在正模式下检测睾酮、二氢睾酮、雄甾酮、本胆烷醇酮和雌二醇的检测离子分别为m/z 289.3→97.1、291.3→105、291.3→199.2、273.2→145.4和255.2→159.1; 在负模式下睾酮的葡糖醛酸产物(T-G)的检测离子为m/z 463.3→85, 二氢睾酮、雄甾酮、本胆烷醇酮的葡糖醛酸产物(DHT-G、ADT-G、ETIO-G)的检测离子为m/z 465.3→75, 雌二醇的葡糖醛酸产物(E₂-G)的检测离子为m/z 447.3→271。此外,本分析方法还用于检测人肝微粒体孵育体系中激素的葡糖醛酸化产物, 这可能为深入研究激素在人体内外的代谢过程奠定基础。

关键词: 激素的葡糖醛酸化产物, 超高效液相色谱串联质谱法, 人肝微粒体

Abstract:

In the present study, we effectively detected 10 steroids and glucuronic acid-conjugated steroid metabolites in 12 min by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Steroids testosterone (T), 5α-dihydrotestosterone (DHT), androsterone (ADT), etiocholanolone (ETIO), estradiol (E₂) and their glucuronide conjugates were well-separated on an Eclipse Plus C₁₈ column (2.1 mm×50 mm, RRHD 1.8 µm). The mobile phase consisted of a mixture of methanol and ultrapure water (containing 1 mM ammonium formate) at a ratio of 60:40 (v/v), and the flow rate was set at 0.25 mL/min. The LC eluate was detected by electrospray ionization (ESI) source in both positive and negative ion modes. Neutral loss (NL of 176, 194, 211 and 229 Da in positive mode) and precursor ion (PI of m/z 141, 159 and 177 in positive mode and 75, 85 and 133 in negative mode) methods were applied for the detection of steroid glucuronides. The multiple reaction monitoring (MRM) transitions were m/z 289.3→97.1, 291.3→105, 291.3→199.2, 273.2→145.4 and 255.2→159.1 for T, DHT, ADT, ETIO and E₂in positive mode, respectively; as well as m/z 463.3→85 for T glucuronide (T-G), m/z 465.3→75 for DHT glucuronide (DHT-G), ADT glucuronide (ADT-G), ETIO glucuronide (ETIO-G) and m/z 447.3→271 for E₂ glucuronide (E₂-G) in negative mode. In addition, the analytical method was also applied for the detection of steroid glucuronides in pooled human liver microsomes (HLM), which might serve as a basis for further investigation of steroid metabolism in vivo and in vitro.

Key words: Steroid glucuronides, Ultraperformance liquid chromatography coupled to tandem mass spectrometry, Human liver microsome

中图分类号:

R917

Supporting:

戎怿, 黄碧云, 黄珺珺, 朱柳, 刘夏雯. 一种快速简单的超高效液相色谱串联四极杆质谱方法检测激素的葡糖醛酸化代谢产物[J]. 中国药学(英文版), 2016, 25(4): 291-301.

Yi Rong, Biyun Huang, Junjun Huang, Liu Zhu, Xiawen Liu. A rapid and simple ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of glucuronic acid-conjugated steroid metabolites[J]. Journal of Chinese Pharmaceutical Sciences, 2016, 25(4): 291-301.

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一种快速简单的超高效液相色谱串联四极杆质谱方法检测激素的葡糖醛酸化代谢产物

A rapid and simple ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of glucuronic acid-conjugated steroid metabolites

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