Identification of Carbon-Carbon Double Bond Stereochemistry in Unsaturated Fatty Acids by Charge-Remote Fragmentation of Fixed- Derivatives

Separation and identification of fatty acid (FA) isomers in biological samples represents a challenging problem for lipid chemists. Notably, FA regio-and stereo-isomers differing in the location or (cis/trans) geometry of carbon-carbon double bonds are often incompletely separated and ambiguously assigned in conventional chromatography-mass spectrometry analyses. To address this challenge, FAs have been derivatized with the charge-switch derivatization reagents N-methyl-pyridinium-3-meth-anamine and N-(4-aminomethylphenyl)pyridinium and subjected to reversed-phase liquid chromatography-tandem mass spectrom-etry. Charge-remote fragmentation of the fixed-charge derivatives leads to characteristic product ions arising from dissociation at allylic positions that enable assignment of position(s) of unsaturation, while a newly discovered dihydrogen neutral loss was found to be dominant for double bonds with cis- stereochemistry. The structure of the [M -2]+ product ions was probed by gas-phase ozonolysis revealing the presence of two new carbon-carbon bonds on either side of the initial position of unsaturation consistent with an electrocyclic mechanism of 1,4-dihydrogen elimination. Charge-remote fragmentation pathways diagnostic of double bond position and stereochemistry were found to be generalized for FAs of different carbon-chain lengths, double bond positions, and degrees of unsaturation and were effective in the unequivocal assignment of the FA structure in complex mixtures of FA isomers, including bovine milk powder.

Automated summary

Separation and identification of fatty acid isomers in biological samples is a challenging task. Researchers have addressed this challenge by derivatizing fatty acids and using mass spectrometry techniques, successfully determining the structure of fatty acids in complex samples.