Determining carbon-carbon double bond position of unsaturated glycerophospholipids in human plasma NIST® SRM® 1950 by electron impact excitation of ions from organics-tandem mass spectrometry (EIEIO-MS/MS)

Abstract

Understanding the structural diversity and biological functions of unsaturated fatty acyl chains (FAC) esterified in complex lipids –such as glycerolipids (GL), glycerophospholipids (GP) or sphingolipids (SP)– requires precise knowledge of the degree of unsaturation, location, and geometrical isomerism of the carbon-carbon double bonds (CC). However, the complex isomeric nature of lipids, combined with the inherent limitations of conventional tandem mass spectrometry (MS/MS) in structural elucidation, remains a major challenge in accurate CC elucidation. To overcome this, advanced MS/MS strategies, such as electron impact excitation of ions from organics (EIEIO) have emerged, generating diagnostic fragment ions that enable unambiguous CC localization. In the present study, we conducted a qualitative structural analysis of the CC positions in esterified FAC of GP present in NIST® Human Plasma Standard Reference Material, SRM 1950, employing RP-UHPLC-ESI(+)-EIEIO-QTOF-MS/MS. Interpretation of ESI(+)-EIEIO-MS/MS spectra enabled CC determination in 120 unsaturated GP, revealing a predominance of ω-6 and ω-3 FAC. These results offer new insights into the FAC distribution of this reference material, enhancing the structural annotation confidence level. By integrating such detailed molecular information, EIEIO-MS/MS proves to be a powerful approach for in-depth lipid structural elucidation in complex biological matrices, thereby contributing to methodological advancements and supporting its future application in translational lipidomics.