OzNOxESI: A Novel Mass Spectrometry Ion Chemistry for Elucidating Lipid Double-Bond Regioisomerism in Complex Mixtures

Abstract

Double bond (C═C) position isomerism in unsaturated lipids can indicate abnormal lipid metabolism and pathological conditions. Novel chemical derivatization and mass spectrometry-based techniques are under continuing development to provide more accurate elucidation of lipid structure in finer structural detail. Here, we introduce a new ion chemistry for annotating lipid C═C positions, which is highly efficient for liquid chromatography-mass spectrometry-based lipidomics. This ion chemistry relies on the online derivatization of lipid C═C with ozone and nitrogen oxides upon fragmentation by tandem mass spectrometry, yielding characteristic product ions capable of unambiguously annotating C═C regioisomers. The new workflow was thoroughly evaluated with various glycerophospholipids and fatty acids and applied to human plasma lipid extract, successfully identified and quantified 270 glycerophospholipid and 36 fatty acid C═C isomers with an in-house developed software, OzNOx Companion, for automated data analysis.

Figure 1

OzNOxESI setup (A), gas-phase reaction mechanism (B), and HCD fragmentation (C). Sheath gas in the MS HESI source was modified to contain oxygen, nitrogen, and ozone. Nitric oxide is generated in situ in a HESI source. Nitrogen dioxide is then generated by the reaction of nitric oxide with ozone. Unsaturated lipids (I) react with ozone to form ozonolysis aldehydes (II) and Criegee intermediates (III). The latter two exist in a brief equilibrium with secondary ozonides (IV). Two NO₂ molecules attack the secondary ozonide (IV) forming the dominant OzNOx adduct (V). Intramolecular nucleophilic attack leads to the formation of the dominant OzNOx adduct (VI) in positive mode MS. HCD MS² of the second OzNOx adduct (VI) generates product ions (VII) analogous to the ozonolysis aldehydes (II), both of which are diagnostic of the C=C position but here with several distinct advantages by being produced and observed at the MS² level rather than MS¹.

Figure 2

LC-OzNOxESI-MS² spectra of representative PC species. (A) OzNOx HCD-MS² of internal standard d5-PC 17:0/22:4(n-6,9,12,15). For each of the 4 C=C, there is a product ion (black) of identical m/z to the corresponding OzESI-MS¹ ozonolysis aldehyde and a companion product ion (blue) corresponding to the loss of CHO. (B) OzNOx HCD-MS² spectra of PC 16:0/22:6 from human plasma. Analysis showed this species to be PC 16:0/22:6(n-3,6,9,12,15,18). As with the standard, there are a pair of product ions for each C=C in the fatty acyl.

Figure 3

LC-OzNOxESI-MS² spectra of representative (L)PE species. (A) OzNOx HCD-MS² spectra of PE 16:1_18:1 from human plasma. Analysis revealed this species to be predominately PE 16:1(n-7)_18:1(n-7). While this lipid has two C=C, their products have the same m/z. There are paired m/z for the C=C positions, detectable with and without the headgroup. (B) OzNOx HCD-MS² of LPE 18:2 from human plasma. Analysis revealed this species to be predominately LPE 18:2(n-6,9). The C=C product ions are triplets with a loss of H₂O (purple).

Figure 4

OzNOx HCD-MS² of FA 16:1 from human plasma. A trio of product ions are observed per C=C regioisomer with successive loss of the ammonium adduct and then H2O. FA 16:1 was found here to primarily be FA 16:1(n-7), but a variety of lesser regioisomers were also detected. While the proton adduct (green) is the most intense here, the ammonium adduct (red) was the species used in data processing, as it was more consistently observed over a variety of fatty acid chain lengths and degrees of unsaturation.

Figure 5

Comparing LC-OzESI-MS¹ and LC-OzNOxESI-MS² workflows using LPC 18:1 in human plasma. (A) Ozonolysis products detected by OzESI or OzNOxESI in the same run. The major OzESI-MS¹ and OzNOxESI-MS² product ions are identical for LPC, differing only in the places they are observed: OzESI products as LC-MS features and OzNOxESI product ions at the MS² level after mass selection of the OzNOx adduct. (B) OzESI EIC of LPC 18:1 and OzESI products detected in the same scan as the maximum LPC 18:1 intensity (dotted line). Poor alignment of OzESI products and multiple peaks is due to other LPC lipids with adjacent or overlapping RT. (C) Comparing LPC 18:1 C=C regioisomer quantification by OzESI and OzNOxESI. Highlighted is LPC 18:1(n–11), inflated ∼150x in OzESI vs OzNOxESI. (D) Illustrative example of the OzESI coelution challenge and explanation of OzESI’s inflated quantification of LPC 18:1(n–11) in this data set.