Nonhydroxylated 1-O-acylceramides in vernix caseosa.

Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1-O-acylceramides (1-O-ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-O) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1-O-ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1-O position, 24 or 26 carbons in the N position, and sphingosine. The 1-O-ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1-O-ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1-O-ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.

Cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids in vernix caseosa.

Cholesteryl esters of ω-(O-acyl)-hydroxy FAs (Chl-ωOAHFAs) were identified for the first time in vernix caseosa and characterized using chromatography and MS. Chl-ωOAHFAs were isolated using adsorption chromatography on silica gel and magnesium hydroxide. Their general structure was established using high-resolution and tandem MS of intact lipids, and products of their transesterification and derivatizations. Individual molecular species were characterized using nonaqueous reversed-phase HPLC coupled to atmospheric pressure chemical ionization. The analytes were detected as protonated molecules, and their structures were elucidated in the negative ion mode using controlled thermal decomposition and data-dependent fragmentation. About three hundred molecular species of Chl-ωOAHFAs were identified in this way. The most abundant Chl-ωOAHFAs contained 32:1 ω-hydroxy FA (ω-HFA) and 14:0, 15:0, 16:0, 16:1, and 18:1 FAs. The double bond in the 32:1 ω-HFA was in the n-7 and n-9 positions. Chl-ωOAHFAs are estimated to account for approximately 1-2% of vernix caseosa lipids.

Combining Charge-Switch Derivatization with Ozone-Induced Dissociation for Fatty Acid Analysis.

The specific positions of carbon-carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography-mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon-carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.