Journal
RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Volume 18, Issue 5, Pages 577-583Publisher
JOHN WILEY & SONS LTD
DOI: 10.1002/rcm.1373
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Funding
- NATIONAL CANCER INSTITUTE [R01CA087584] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM043825, R37GM043825] Funding Source: NIH RePORTER
- NCI NIH HHS [CA 87584] Funding Source: Medline
- NIGMS NIH HHS [GM08716, GM 43825] Funding Source: Medline
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Normal-phase high-performance liquid chromatography (NP-HPLC) coupled to atmospheric pressure ionization mass spectrometry (APCI-MS) allows quantitative analysis of endogenous ceramide and dihydroceramide species from crude lipid extracts. Qualitative information for the species comes from observation of differences in chromatographic and mass spectrometric behavior between species (Pettus et al. Rapid Commun. Mass Spectrom. 2003; 17: 1017-1026). Quantitative analysis is achieved by (1) use of a synthetic internal standard as an extraction and injection control, (2) lack of salt adduction, ion suppression, or other matrix effects in APCI mode, and (3) consistent fragmentation and ionization of external standards across the physiologically relevant concentration range found in endogenous lipid samples. Application to the analysis and quantitation of ceramide and dihydroceramide from various cell lines is demonstrated. The results from APCI-MS analysis corroborate and enhance information acquired from use of the diacylglycerol kinase assay for total ceramide measurement. This technique readily allows simultaneous quantitation of ceramide and dihydroceramide species. Copyright (C) 2004 John Wiley Sons, Ltd.
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