Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization.

Abstract

RationaleLipids are a group of biomolecules known for their importance in a broad variety of biological tasks, which is attributed to the diversity of their molecular structures. The exact position of the double bond in a lipid affects its biological properties. Therefore, the determination of its position is highly important. Nevertheless, the unambiguous assignment is still a challenging task and requires the development of new approaches.MethodsA novel method for pinpointing double‐bond positions in lipids is presented based on the photochemical Paternὸ–Büchi reaction. Therefore, the reaction was performed using a post‐column derivatization by ultraviolet (UV) light irradiation (254 nm) and an additional constant flow of acetone. The acetone adduct was formed due to the reaction of acetone with the double bond resulting in an oxetane. Subsequent fragmentation experiments generated diagnostic fragments allowing the localization of double‐bond positions.ResultsIntending online hyphenation with high‐performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS), the reaction was optimized regarding parameters such as reaction time and the influence of trace oxygen as well as solvents. An additional separation of the reaction products by means of HPLC enabled further characterization of the chemical structures formed by UV irradiation. To demonstrate the applicability of the developed method, a mixture of the constitutional phospholipid isomers bis(monooleoylglycero) phosphate and dioleoyl‐phosphatidylglycerol was analyzed regarding their double‐bond positions, and lipids from an extract of the green alga Chlamydomonas reinhardtii were investigated.ConclusionsThe successful hyphenation of HPLC/MS/MS with the online reaction was demonstrated for localization of double bonds in single lipids, lipid mixtures, and complex biological samples. Matrix effects can be reduced and further structural information, for example in case of constitutional isomers, can be obtained. This opens up new avenues for pinpointing lipid double‐bond positions even in complex samples.