The Lipidomics Study And Lipid Isomer Analysis In Depressed Rats Based On UHPLC-MS/MS

Lipids play an important role in organisms,but their physical and chemical properties are diverse,and lipid isomers have different physiological effects.At present,lipid analysis has the problems of narrow lipid coverage and serious co-elution,and the separation and analysis of lipid isomers is more difficult.In this study,we established a pseudotargeted lipidomics method based on three-phase liquild extraction(3PLE)and segment data-dependent acquisition(SDDA),and a qualitative and quantitative analysis method of lipid isomers based on microwave-assisted meta-chloroperoxybenzoic acid(m-CPBA)epoxidation.The changes of lipid metabolism in plasma and brain tissues of depressed rats were studied.The main contents and results are as follows:A comprehensive pseudotargeted lipidomics method based on 3PLE and SDDA was established successfully.The 3PLE method was used to extract the lipids with extensive coverage from biological matrixes.Besides,the SDDA strategy improved the detection of co-elution ions in the lipidomics analysis.A total of 554 potential lipids were detected by the developed approach in both positive and negative modes using ultra-high-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).Compared with the conventional liquid-liquid extraction approaches,including methyl tert-butyl ether(MTBE)and Bligh-Dyer(BD)methods,3PLE combined with SDDA significantly increased the lipid coverage 87.2%and 89.7%,respectively.Also,the proposed pseudotargeted lipidomics approach exhibited higher sensitivity and better repeatability than the untargeted approach.Finally,we applied the established pseudotargeted method to the plasma lipid profiling from the depressed rats and screened 61 differential variables.The results demonstrated that the pseudotargeted method based on 3PLE and SDDA broadened lipid coverage and improved the detection of co-elution ions with excellent sensitivity and precision.A qualitative and quantitative analysis method of lipid isomers based on microwave-assisted m-CPBA epoxidation was developed.This method specifically derivatizes the lipid carbon-carbon double bond(C=C),and could produce diagnostic ions after collision-induced dissociation(CID)indicating the C=C locations.The microwave-assisted epoxidation method is convenient to process and easy to be combined with commercial mass spectrometry.The yield of unsaturated lipids can reach more than 98%in only 15 minutes,and the resulting product is single,which is more conducive to quantification.The linearity r~2 of the epoxidized products was all greater than 0.99,the precision RSD was less than 15%,the recovery rate was80.5-106.5%,and the matrix effect was 86.0%-117.3%,which all met the requirements of method verification.The established microwave-assisted m-CPBA epoxidized qualitative and quantitative analysis method of lipid isomers has been used to analyze lipids in rat brain tissue and plasma.A total of 12 unsaturated lipids were successfully quantified,and 6 pairs of lipid isomers were qualitatively analyzed.