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Journal of ZheJiang University(Medical Science)  2015, Vol. 44 Issue (4): 429-434    DOI: 10.3785/j.issn.1008-9292.2015.07.13
    
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
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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 wordsFibroblasts      Ceramides      Chromatography, high pressure liquid      Mass spectrometry      Cells, cultured     
Received: 06 March 2015     
CLC:  R331  
Cite this article:

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.

URL:

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


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

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 
[[1]]   KOLESNICK R N, GONI F M, ALONSO A. Compartmentalization of ceramide signaling: physical foundations and biological effects[J]. J Cell Physiol,2000,184(3):285-300.
[[2]]   VAN BLITTERSWIJK W J,VAN DER LUIT A H, VELDMAN R J, et al. Ceramide: second messenger or modulator of membrane structure and dynamics?[J]. Biochem J, 2003,369(Pt 2):199-211.
[[3]]   GEILEN C C, WIEDER T, ORFANOS C E. Ceramide signalling: regulatory role in cell proliferation, differentiation and apoptosis in human epidermis[J]. Arch Dermatol Res, 1997,289(10):559-566.
[[4]]   OBEID L M, LINARDIC C M, KAROLAK L A, et al. Programmed cell death induced by ceramide[J]. Science, 1993,259(5102):1769-1771.
[[5]]   VENABLE M E, LEE J Y, SMYTH M J, et al. Role of ceramide in cellular senescence[J]. J Biol Chem, 1995,270(51):30701-30708.
[[6]]   SENKAL C E, PONNUSAMY S, ROSSI M J, et al. Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas[J]. Mol Cancer Ther, 2007,6(2):712-722.
[[7]]   SENKAL C E, PONNUSAMY S, BIELAWSKI J, et al. Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways[J]. FASEB J, 2010,24(1):296-308.
[[8]]   SASSA T, SUTO S, OKAYASU Y, et al. A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells[J]. Biochim Biophys Acta, 2012,1821(7):1031-1037.
[[9]]   LIEBISCH G, DROBNIK W, REIL M, et al. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)[J]. J Lipid Res, 1999,40(8):1539-1546.
[[10]]   HAYNES T A, DUERKSEN-HUGHES P J, FILIPPOVA M, et al. C18 ceramide analysis in mammalian cells employing reversed-phase high-performance liquid chromatography tandem mass spectrometry[J]. Anal Biochem, 2008,378(1):80-86.
[[11]]   KASUMOV T, HUANG H, CHUNG Y M, et al. Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry[J]. Anal Biochem, 2010,401(1):154-161.
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