Liu Chuanxin, Sheng Xue, Wang Yuming, Yin Jia, Huang Wei, Fan Yunshuang, Li Yubo, Zhang Yanjun
Tianjin State Key Laboratory of Modern Chinese Medicine, School of Traditional Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine 312 Anshan West Road Tianjin 300193 China
State Key Laboratory of Separation Membranes and Membrane Processes, Tianjin Polytechnic University Tianjin 300387 China.
RSC Adv. 2018 May 30;8(35):19713-19723. doi: 10.1039/c8ra01372a. eCollection 2018 May 25.
Steroid hormones are crucial substances that mediate a wide range of vital physiological functions. Because of the important biological significance of steroids, this paper presents a new targeted metabolic method based on adding stable isotope tags to hydroxyl containing and carbonyl containing steroid hormones with two pairs of synthesized derivatization reagents: deuterium 4-(dimethylamino)-benzoic acid (D-DMBA), and D-Girard P (D-GP) using of ultra performance liquid chromatography-multiple reaction monitoring (UPLC-MRM). Firstly, an Oasis PRiME hydrophilic-lipophilic balance (HLB) 96-well solid phase extraction plate was used to pretreat a number of biological samples simultaneously. Secondly, hydroxyl and carbonyl steroids were labeled using two pairs of synthetic reagents, namely DMBA and D-DMBA, and GP and D-GP, respectively. Thirdly, the mixed products were detected using UPLC-MRM and the mass spectroscopy conditions were optimized. Methodology development showed that the sensitivity was enhanced 1 to >500-fold. Finally, the new method was applied to analysis of urine samples of healthy males, females and rats. The results revealed that the method can be sensitive and reliable for simultaneous quantification of steroid hormones containing hydroxyl and carbonyl groups in 12 min in a single run. This method provided a powerful tool for studying the metabolic mechanism of steroids and contributed to the development of targeted metabolomics.
类固醇激素是介导多种重要生理功能的关键物质。由于类固醇具有重要的生物学意义，本文提出了一种新的靶向代谢方法，该方法基于使用两对合成衍生试剂：氘代4-(二甲氨基)苯甲酸（D-DMBA）和D-吉拉德P（D-GP），对含羟基和含羰基的类固醇激素添加稳定同位素标签，并结合超高效液相色谱-多反应监测（UPLC-MRM）技术。首先，使用Oasis PRiME亲水亲脂平衡（HLB）96孔固相萃取板同时对多个生物样品进行预处理。其次，分别使用两对合成试剂，即DMBA和D-DMBA以及GP和D-GP对羟基和羰基类固醇进行标记。第三，使用UPLC-MRM对混合产物进行检测，并优化质谱条件。方法学开发表明，灵敏度提高了1至>500倍。最后，将该新方法应用于健康男性、女性和大鼠的尿液样本分析。结果表明，该方法能够在单次运行12分钟内灵敏且可靠地同时定量含羟基和羰基的类固醇激素。该方法为研究类固醇的代谢机制提供了有力工具，有助于靶向代谢组学的发展。
