Determining Double Bond Position in Lipids Using Online Ozonolysis Coupled to Liquid Chromatography and Ion Mobility-Mass Spectrometry

Abstract

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.

Ion mobility conformational lipid atlas for high confidence lipidomics

“…Post-source decay fragment ion analysis has been utilized to assign the position and identity of fatty acid residues on the glycerol backbones of glycerophospholipids 10 , and recently an implementation of electron induced dissociation, known as electron-impact excitation of ions from organics (EIEIO), has been used to produce extensive untargeted acyl chain fragmentation and allows for determination of both sn-position and double bond location in lipid standards 11 . Additional techniques that promote carbon double bond cleavage have been developed in conjunction with MS to identify double bond position, in particular the Paternò-Büchi reaction 12 and ozonolysis 4,13–16 .…”

Chemometric Strategies for Peak Detection and Profiling from Multidimensional Chromatography

“…An example of this is the popularization of LC‐IMS‐MS that combines different separation modes allowing the analysis of highly complex samples. This is a promising tool in fields such as lipidomics in which samples may contain multiple isobaric lipid species . These new options will broaden the possibility of using these multidimensional techniques in the different omics sciences.…”

Quantification and molecular imaging of fatty acid isomers from complex biological samples by mass spectrometry

“…18,23 Another emerging strategy to locate the C]C bond position is implementation of a C]C derivatization procedure prior to CID MS/MS interrogation. C]C bond chemical derivatization methods based on the Paternò-Büchi (PB) reactions, 13,15,24,25 ozonolysis, 26,27 meta-chloroperoxybenzoic acid (m-CPBA)-epoxidation, 28,29 and plasma/electrochemical induced epoxidation, [30][31][32][33] have been utilized to induce structurally informative dissociation of FAs in CID MS/MS in recent years. Additionally, covalent adduct chemical ionization (CACI) GC MS/MS 34 and gas-phase ion/ion reactions 35 have been adopted for the characterization of isomeric FAs.…”