Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin‐enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal‐phase high‐performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M+H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in‐source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long‐chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI‐MS/MS via collision‐induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB–FA combinations(s) using APCI+‐MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting ∽90% of the total ion current in ESI) were composed of at least 25 different LCB–FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB–FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented. © 1998 John Wiley & Sons, Ltd.