Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Authors

  • Anna Somogyi ORCID
    Affiliation

    Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, H-1428 Budapest, P.O.B. 2, Hungary

  • Mária Berinkeiné Donkó
    Affiliation

    Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, H-1521 Budapest, P.O.B. 91, Hungary

  • Farkas Sarnyai
    Affiliation

    Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, H-1428 Budapest, P.O.B. 2, Hungary

  • Gergely Becskereki
    Affiliation

    Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, H-1521 Budapest, P.O.B. 91, Hungary

  • Miklós Csala ORCID
    Affiliation

    Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, H-1428 Budapest, P.O.B. 2, Hungary

  • Blanka Tóth
    Affiliation

    Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, H-1521 Budapest, P.O.B. 91, Hungary

https://doi.org/10.3311/PPch.15357

Abstract

A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well.

Keywords:

mass spectrometry, liquid chromatography, ceramides, glycerolipids, lipotoxicity

Published Online

2020-07-09

How to Cite

Somogyi, A., Berinkeiné Donkó, M., Sarnyai, F., Becskereki, G., Csala, M., Tóth, B. “Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry”, Periodica Polytechnica Chemical Engineering, 64(4), pp. 421–429, 2020. https://doi.org/10.3311/PPch.15357

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Articles