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浙江大学学报（医学版）
浙江大学学报（医学版）  2015, Vol. 44 Issue (4): 429-434    DOI: 10.3785/j.issn.1008-9292.2015.07.13
原著     
应用高效液相色谱串联质谱法检测细胞内神经酰胺的含量
邱丽萍, 孙文均
浙江大学医学院公共卫生学院 浙江省生物电磁学重点实验室, 浙江 杭州 310058
Simultaneous quantitative analysis of different ceramide species in cells by high-performance liquid chromatography-tandem mass spectrometry
QIU Li-ping, SUN Wen-jun
School of Public Health, Zhejiang University School of Medicine, Bioeletromagnetics Key Laboratory of Zhejiang Province, Hangzhou 310058 China
全文: PDF(744 KB)  
摘要: 

目的:采用高效液相色谱串联质谱法建立测定细胞中不同类型神经酰胺含量的分析方法.方法:采用Agilent 1290高效液相色谱仪系统,ZORBAX Eclipse XDB-C8(150.0 mm×2.1 mm,3.5 μL)色谱柱,柱温35 ℃;流动相A为含1 mmol/L甲酸铵和0.2%甲酸的甲醇溶液,流动相B为100%甲醇;流速为0.3 mL/min,梯度洗脱;质谱检测中采用电喷雾离子源多反应监测模式.结果: C16、C17、C18、C24四种神经酰胺的色谱响应线性范围良好(R2 ≥0.9989);平均回收率分别为99.1%、99.9%、100.5%、98.2%,相对标准偏差分别为5.6%、5.1%、4.8%、5.5%(n = 9).结论:采用高效液相色谱串联质谱法建立同时测定细胞中不同类型神经酰胺的方法灵敏度高,重现性好,可以作为检测细胞中不同类型神经酰胺含量的工具.
关键词 ： 成纤维细胞,  神经酰胺类,  色谱法,  高压液相,  质谱分析法,  细胞,  培养的    
Abstract：

Objective: To establish a high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS) method for simultaneous quantitative analysis of different ceramide species in cells. Methods: The analysis was performed on an Agilent 1290 HPLC system with a ZORBAX Eclipse XDB-C8 (150.0 mm×2.1 mm, 3.5 μL) column and a temperature of 35 ℃. Methanol with 1 mmol/L ammonium formate and 0.2% formic acid was used as mobile phase A and 100% methanol was used as mobile phase B. And the ceramides were separated by gradient elution at a flow rate of 0.3 mL/min. Electrospray ionization (ESI) with multiple reaction monitoring (MRM) was used in the analysis. Results: Four ceramide species all had a good linear response in the determination ranges (R2 ≥0.9987). The average recoveries (n = 9) were 99.1%,99.9%,100.5% and 98.2% with RSDs of 5.6%, 5.1%, 4.7% and 5.5%, respectively. In addition, the levels of ceramides in FL cells were relatively stable, while the C24-ceramide had the highest level. Conclusion: The HPLC-ESI-MS method for simultaneous analysis of ceramides has high accuracy, reproducibility and linearity, which may be used for quantification of ceramide species in cells.
Key words： Fibroblasts    Ceramides    Chromatography, high pressure liquid    Mass spectrometry    Cells, cultured
收稿日期: 2015-03-06
CLC:  R331  
基金资助:

国家自然科学基金(31370831),国家重点基础研究发展计划("973"计划)(2011CB503700)
通讯作者: 孙文均(1970-),男,博士,教授,主要研究环境电磁辐射生物效应及机制;E-mail:sunwj@zju.edu.cn;http://orcid.org/0000-0002-0165-4925     E-mail: sunwj@zju.edu.cn
作者简介: 邱丽萍(1989-),女,硕士研究生,主要研究环境电磁辐射生物效应及机制;E-mail:tracyqiuok@126.com;http://orcid.org/0000-0001-6661-1849
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引用本文:

邱丽萍, 孙文均. 应用高效液相色谱串联质谱法检测细胞内神经酰胺的含量[J]. 浙江大学学报（医学版）, 2015, 44(4): 429-434.
QIU Li-ping, SUN Wen-jun. Simultaneous quantitative analysis of different ceramide species in cells by high-performance liquid chromatography-tandem mass spectrometry. Journal of ZheJiang University(Medical Science), 2015, 44(4): 429-434.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2015.07.13      或      http://www.zjujournals.com/xueshu/med/CN/Y2015/V44/I4/429

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