Identification of Ceramides in Human Cells Using Liquid Chromatography with detection by Atmospheric Pressure Chemical Ionization-Mass Spectrometry

Abstract

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-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 separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.

Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids

“…Ceramides, which are nonphosphate-containing interme- diates in the biosynthesis of sphingolipids, were analyzed by Couch et al (55) using HPLC with detection by APCI-MS. They reported the analysis of both underivatized and perbenzoylated ceramide standards and ceramides from human leukemic cells using detection by APCI-MS and UV-Vis.…”

Analysis of Sphingosine 1-Phosphate, Ceramides, and Other Bioactive Sphingolipids by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

“…Determine optimal collision energy for each subspecies in each class (such as C 12 Cer, C 16 Cer, C 18 Cer, C 20 Cer, C 22 Cer, and C 24 Cer), including the internal standards. …”

“…Other methods for the analysis of sphingolipids by mass spectrometry have been described (12,(14)(15)(16)(17)(18)(19). Besides mass spectrometry, a common method for the analysis of ceramide is to use Escherichia coli diglyceride kinase to produce a radiolabeled derivative (20), which must be done under carefully controlled conditions to be accurate (21).…”

Highly Sensitive Determination of Diverse Ceramides in Human Hair Using Reversed‐Phase High‐Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

“…While many conventional methods, such as gas chromatography-mass spectrometry (MS) [15], thinlayer chromatography (TLC) [16], diacylglycerol (DAG) kinase assay [17], liquid chromatography (LC)-evaporative light-scattering detection [18] and LC-UV detection using a derivatizing technique [19], have been used to determine CERs, Mano et al [20], Gu et al [21] and Couch et al [22] reported MS using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) to be a valuable tool in the analysis of mixed CERs in 1997. Since the methods based on ESI-MS or APCI-MS were far superior to the conventional methods in terms of sensitivity and selectivity, since after their articles, many methods using LC-MS and tandem MS (MS/MS) for the analysis of CERs have been developed.…”