色谱

色谱 ›› 2017, Vol. 35 ›› Issue (12): 1251-1256.DOI: 10.3724/SP.J.1123.2017.09020

• 研究论文 • 上一篇    下一篇

亲水相互作用色谱-四极杆/静电场轨道阱高分辨质谱法测定肝组织中羟脯氨酸

刘伟1,2, 戚胜兰4, 徐莹1,2, 肖准1,2,3, 付亚东1,2,3, 陈佳美1,2, 杨涛5, 刘平1,2,3   

  1. 1. 上海中医药大学附属曙光医院肝病研究所, 上海 201203;
    2. 上海中医药大学肝肾疾病病证教育部重点 实验室, 上海 201203;
    3. 上海中医药大学上海高校中医内科学E-研究院, 上海 201203;
    4. 上海中医药大学中药研究所, 上海 201203;
    5. 上海中医药大学附属曙光医院心血管研究所, 上海 201203
  • 收稿日期:2017-09-09 出版日期:2017-12-08 发布日期:2017-12-15
  • 通讯作者: 刘平,Tel:(021)20256526,E-mail:liuliver@vip.sina.com;杨涛,Tel:(021)20256778,E-mail:yangtao8579@163.com
  • 基金资助:

    国家自然科学基金重点项目（81530101）；上海市青-科技英才扬帆计划资助项目（17YF1419800）；中国博士后科学基金资助项目（2016M601643）.

Determination of hydroxyproline in liver tissue by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry

LIU Wei1,2, QI Shenglan4, XU Ying1,2, XIAO Zhun1,2,3, FU Yadong1,2,3, CHEN Jiamei1,2, YANG Tao5, LIU Ping1,2,3   

  1. 1. Institute of Liver Disease, Shuguang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
    2. Key Laboratory of Liver and Kidney Disease of the Ministry of Education, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
    3. E-Institute of Shanghai Municipal Education Commission, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
    4. Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
    5. Institute of Cardiovascular Disease, Shuguang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
  • Received:2017-09-09 Online:2017-12-08 Published:2017-12-15
  • Supported by:

    Key Project of National Natural Science Foundation of China (No. 81530101); Shanghai Sailing Program (No. 17YF1419800); Project Funded by China Postdoctoral Science Foundation (No. 2016M601643).

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被引次数

2

摘要：

采用亲水相互作用色谱-四极杆/静电场轨道阱高分辨质谱系统，建立了肝组织中胶原蛋白水解物羟脯氨酸（Hyp）的快速定量检测方法。将正常及四氯化碳肝纤维化模型小鼠的肝组织样品酸水解，经过滤、稀释后，采用Hypersil GOLD HILIC色谱柱（100 mm×2.1 mm，3 μm）分离，以水-乙腈（28：72，v/v）为流动相进行等度洗脱，最后用配有电喷雾离子源的四极杆/静电场轨道阱高分辨质谱在正离子模式下进行检测。结果表明，Hyp在0.78~100.00 μg/L范围内呈良好的线性关系，相关系数（R2）为0.9983，方法的定量限为0.78 μg/L，样品加标回收率为97.4%~100.9%，相对标准偏差为1.4%~2.0%（n=6）。此外，该方法与传统的氯胺T法进行比较，发现两种方法的检测结果相关性良好，Pearson相关系数为0.927；较氯胺T法，该法具有操作简便、准确度高的优点。该方法可用于肝组织中Hyp的快速定量分析。

关键词: 肝纤维化, 肝组织, 羟脯氨酸, 亲水相互作用色谱, 四极杆/静电场轨道阱高分辨质谱

Abstract:

A method for the determination of hydroxyproline (Hyp) in liver tissue of mice by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HILIC-HRMS) was developed. The liver tissue samples of normal mice and liver fibrosis mice induced by carbon tetrachloride were hydrolyzed by concentrated hydrochloric acid. After filtrated and diluted by solution, the diluent was separated on an Hypersil GOLD HILIC column (100 mm×2.1 mm, 3 μm). Water-acetonitrile (28:72, v/v)were used as the mobile phases with isocratic elution. Finally, the target analytes were detected in positive model by HRMS equipped with an electrospray ionization source. The linear range of hydroxyproline was from 0.78 to 100.00 μg/L with the correlation coefficient (R2) of 0.9983. The limit of quantification was 0.78 μg/L. By detecting the spiked samples, the recoveries were in the range of 97.4%-100.9% with the relative standard deviations (RSDs) between 1.4% and 2.0%. In addition, comparison of the measurement results by this method and the chloramine T method was proceeded. It was found that the linear correlation between the two methods was very good, and the Pearson correlation coefficient was 0.927. And this method had simpler operation procedure and higher accuracy than chloramine T method. This method can be used for the quick determination of hydroxyproline in liver tissue samples.

Key words: hydrophilic interaction chromatography (HILIC), hydroxyproline (Hyp), liver fibrosis, liver tissue, quadrupole/orbitrap high resolution mass spectrometry (HRMS)

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