Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses

Abstract

One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon-carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS and high resolution mass spectrometry. Modification of an IMS-capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressure trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities and in all cases the positions of carbon-carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of cis/trans double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes including human plasma.

Reaction of ionised steryl esters with ozone in the gas phase

“…The presence of the additional oxygen on the truncated acyl chain was confirmed by re-isolation of the product ions and subsequent activation by both collision-induced and ozone-induced dissociation (Supplemental Material Figure S1 & S2). This pair of ozonolysis product ions is consistent with ozone-induced dissociation performed previously across a wide range of ionized lipids incorporating mono-unsaturated fatty acid substituents and indeed, the consistency of the neutral losses observed has been used extensively for the structural characterisation of lipids in complex extracts (Batarseh et al, 2018;Brown et al, 2011;Ellis et al, 2018;Hancock et al, 2018;Poad et al, 2018aPoad et al, , 2018bVu et al, 2017). respectively.…”

Solvent composition dependent signal reduction of molecular ions generated from aromatic compounds in (+) atmospheric pressure photoionization mass spectrometry

“…Liquid chromatography coupled with mass spectrometry (LC/MS) is one of the most important analytical tools in contemporary scientific and industrial research on food, 1-3 drugs, 4,5 petroleum, [6][7][8] biomolecules, [9][10][11][12][13][14][15][16][17][18][19][20] and environmental samples. 21,22 For this reason, a considerable amount of effort has been devoted to improving the analytical sensitivity of LC/MS, [23][24][25][26] and it is well known that ionization is a critical step that can greatly influence the analytical sensitivity of LC/MS.…”

Large-scale lipid analysis with C=C location and sn-position isomer resolving power

“…Various methods have been explored for differentiating the lipid C=C location and sn-position isomers. Ozone-induced dissociation (OzID) [31][32][33] and ultraviolet photodissociation (UVPD) 28 have been used to determine both sn-positions and C=C locations in GPs. By coupling photochemical (Paternò-Bǜchi, PB) reaction with tandem MS (MS/MS), we have systematically demonstrated the qualitative and quantitative analysis of lipids with C=C specificity from complex biological samples [25][26][27]34,35 .…”