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

Abstract

Triacylglycerol estolides (TG-EST) are biologically active lipids extensively studied for their anti-inflammatory and anti-diabetic properties. In this work, eight standards of TG-EST were synthesized and systematically investigated by nanoelectrospray tandem mass spectrometry. Mass spectra of synthetic TG-EST were studied with the purpose of enabling the unambiguous identification of these lipids in biological samples. TG-EST glycerol sn-regioisomers and isomers with the fatty acid ester of hydroxy fatty acid (FAHFA) subunit branched in the ω-, α-, or 10-position were used. Ammonium, lithium, and sodium adducts of TG-EST formed by nanoelectrospray ionization were subjected to collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD). Product ion spectra allowed for identification of fatty acid (FA) and FAHFA subunits originally linked to the glycerol backbone and distinguished the α-branching site of the FAHFA from other estolide-branching isomers. The ω- and 10-branching sites were determined by combining CID with ozone-induced dissociation (OzID). Lithium adducts provided the most informative product ions, enabling characterization of FA, hydroxy fatty acid (HFA), and FAHFA subunits. Glycerol sn-regioisomers were distinguished based on the relative abundance of product ions and unambiguously identified using CID/OzID of lithium and sodium adducts.

From Oxidized Fatty Acids to Dimeric Species: In Vivo Relevance, Generation and Methods of Analysis

“…The question of whether FAHFAs (due to their structural similarity to FFAs) can be incorporated into other lipid classes like diacylglycerols (DAG) has been recently addressed [59]. The resulting FAHFA-containing TAGs (FAHFA-TAGs; with a somewhat uncommon residue in the sn2 position) are an important storage pool for branched-chain FAHFAs [59,60]. There is one remarkable issue regarding this lipid class: FAHFA-TAG concentrations were about 100 times higher than those of free FAHFAs.…”

“…There is also a computer-generated library with MS/MS spectra for more than 1000 FAHFA species available [74]. Using high-resolution tandem MS with nanoelectrospray ionization, FAHFA-TAGs were recently investigated in detail (i.e., the position of the FAHFAs and the positions of modification were successfully elucidated) [60].…”