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

Abstract

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-methanamine and N-(4-aminomethylphenyl)­pyridinium and subjected to reversed-phase liquid chromatography-tandem mass spectrometry. 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.

Characterization of Triacylglycerol Estolide Isomers Using High-Resolution Tandem Mass Spectrometry with Nanoelectrospray Ionization

“…OzID has previously been proven to be a useful ion activation method for the structural analysis of lipids and in particular for the resolution of lipid regioisomers [ 47 , 48 , 49 , 50 , 51 , 62 ]. This technique harnesses ozonolysis reactions of mass-selected ions within the mass spectrometer to identify the location of carbon–carbon double bonds that are either present in the native lipid structure or are formed during preceding ion activation events.…”

In-Depth Characterization of Sphingoid Bases via Radical-Directed Dissociation Tandem Mass Spectrometry

“…The C−C cleavages at the allylic positions of the C14−C15 C�C, i.e., m/z 385.3 and 331.2, are greatly enhanced relative to the other RDD channels, leading to a V-shaped pattern, which has also been reported in several other radical associated fragmentation methods 13,23,35 and chargeremote fragmentation. 36,37 This phenomenon is useful for locating the C�C along the aliphatic chain. However, RDD around C4 trans-C�C is not obvious, and thus, it cannot be determined via RDD in the negative ion mode.…”

Relative Quantification of Lipid Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions and Infrared Multiphoton Dissociation

“…Several alternative methods have recently emerged for use in imaging mass spectrometry workflows to aid in lipid isomer identification, including Paternò-Büchi reactions, − ozone-induced dissociation (OzID), − and ultraviolet photodissociation (UVPD). , On-tissue derivatization methods, including Paternò-Büchi reactions, have been used to alter the ion type generated from the tissue surface in order to gain additional structural information upon MS/MS. ,,, While Paternò-Büchi reactions have allowed for the identification of double bond positions in lipids, on-tissue derivatization methods can be time-consuming and can suffer from low reaction yields, low derivatization efficiency, and side or incomplete reactions. Ozone-induced dissociation (OzID) is an ion/molecule reaction that has been used to identify both lipid sn- positions and double bond positions in matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry experiments. − These reactions require the introduction of ozone vapor generated from an external supplemental ozone generator into the ion-trapping region of the instrument. Sequential CID/OzID allows for the identification of sn -positional isomers using diagnostic product ions and has been implemented in imaging mass spectrometry experiments with a per-pixel scan times of ∼620 ms .…”