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

Simultaneous quantitative analysis of different ceramide species in cells by high-performance liquid chromatography-tandem mass spectrometry

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Agilent1290高效液相色谱仪系统，ZORBAX Eclipse XDB-C8（150．0mm×2．1mm，3．5μL）色谱柱，柱温35℃；流动相A为含lmmol／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O．3mL／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（R^2≥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。

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 （ R^2≥10. 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.