IDENTIFICATION OF CERAMIDES IN HUMAN-CELLS USING LIQUID-CHROMATOGRAPHY WITH DETECTION BY ATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION MASS-SPECTROMETRY

Ceramides are intermediates in the biosynthesis of membrane sphingolip ids, These biomolecules are also important as second messengers in sig nal transduction pathways controlling cell growth. We have developed t wo reversed-phase high pressure liquid chromatography (RPHPLC) techniq ues for identification and quantification of ceramides from mammalian cells, One method was based on atmospheric pressure chemical ionizatio n-mass spectrometry (APCI-MS) detection of ceramides and had the advan tage of requiring minimal sample preparation, yielding significant str uctural information, and affording high sensitivity, The second method relied on perbenzoylation of the ceramides and detection at 230 mn, T he predominant ceramides detected in the human leukemic HL-60 cell wer e N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignocer oyl)-sphingosine. When selected ion monitoring was used with RPHPLC/AP CI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N -(nervonyl)-sphingosine were observed in an extract from 40 000 HL-60 cells, Perbenzoylation with benzoyl chloride permitted RPHPLC separati on and 230 nm UV absorbance detection of the trisbenzoyl derivatives o f sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, an d N-(lignoceroyl)-sphingosine in the HL-60 cells, These results demons trate the utility of utilizing two different methods coupled with APCI -MS for the quantification and identification of ceramides in biologic al samples. (C) 1997 by John Wiley & Sons, Ltd.