The Study Of A Novel Method Based On Derivatization Technique For High Efficient Analysis Of Phospholipids And Its Applications

Phospholipids(PLs), the main constituents of biological membranes, play important functional, structural roles and are involved in apoptosis and cell signaling. PLs have the function of promoting the lipid metabolism and transportation, ensuring vascular patency for normal liver function. Furthermore, PLs are involved in cell signaling and are associated with various diseases. By understanding the metabolism of phospholipid compounds in organisms, the roles PLs play in life activities could be comprehensive in-depth studied in combination with the physiological functions of phosphatide compounds and thus laying a significant foundation for lipidomics and lipid nutrition research. However, the profiling analysis of PLs in biological samples is a challenging task because of the presence of numerous PLspecies and their complex structures.This project focuses on the development of a novel method for high selective and sensitive qualitative and quantitative analysis of phospholipids through integrating stable isotope labeling technique with electrospray ionization tandem mass spectrometry(ESI-MS/MS), and solving the technical bottlenecks of existing MS technologies on phospholipids analysis. The major results of the paper are as follows:1. A method of profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization was developed. After acetone stable isotope derivatization, the detection sensitivity of PEs was increased about 2.1~2.5-fold with the double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis(ASID-DNLS-Shotgun ESI-MS/MS). The use of acetone(d0-acetone) and deuterium-labeled acetone(d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and hence solving the bottleneck of the quantitative analysis of PE. The ASID-DNLS-Shotgun ESI-MS/MS method was applied for the profiling and relative quantification of PE species in liver samples from rats fed with different diets(control, 20% olive oil and 20% lard oil diets). Principal component analysis(PCA)demonstrated that the PE species varied among these three groups, and the analysis result had a fixed guiding significance to trap lipid biomarkers related to chronic diseases, which were caused by high-fat diets.2. Acetone, although common accessible, can only be used for PE, PS and other amino containing types of phospholipids. Thus, a novel strategy using Hybrid solid-phase extraction followed with trimethylsilyldiazomethane stable isotope derivatization and shotgun-mass spectrometry analysis(DSPE-SIL-LC-DNLS-MS) for comprehensive profiling and accurate quantification of individual phospholipid speciesfrom biological samples was also developed. Based on the derivatization method, on the one hand, the relative quantitative analysis of PLs was achieved; And on the other hand, with the ligh labled PLs standards as internal standards, PLs absolute quantitative analysis was also realized. In addition, after methylation, the detection sensitivity of six classes of phospholipids: PC, PE, PS, PG, PA and PI was increased 1.18-, 10.21-, 14.03-, 8.60-, 104.13- and 8.44 fold, respectively. The method is simple, practical and widens the selection scope of internal standards, and also is of important significance for lipidomics and lipid nutrition research. The DSPE-SIL-LC-DNLS-MS method was applied to analysing the plasma samples. 33 PC species,25 PE species,22 PG species,15 PS species,17 PI species and 18 PA species were identified and quantified successfully, among which 30 kinds of phospholipid molecules were found out to be significant differences between hyperlipidemia group andnormal control. The study could have important implications for lipidomics research and biomarkers discover of hyperlipidemia related diseases.3. A shotgun-MS method for relative quantification of structured phospholipids was developed. In combination with single factor experiments and response surface methodology, the optimum conditions of structured phospholipids synthesis were optimized by this method. And the optimized conditions were as follows: reaction temperature 55.6℃, reaction time 25.1h, 15.3% lipase dosage, and substrate molar ratio 1:10.1. Under such conditions, the relative percentage of ARA in structured phospholipids products can reach 15.42%, and the yield of ARA structured phospholipids was 13.26%. In addition, the PC species in the structured phospholipids were identified by mass spectrometry(MS). this research laid the foundation for nutritional properties researches of structured phospholipids enriched with n-6 PUFA and development of related functional lipids products.