Mass Spectrum Analysis Of Polar Lipids And Its Application

There are four parts in this paper. In the first part, electrospray ionization triple quadrupole electrospray tandem mass spectrometry (ESI-MS/MS) method was eatablished to analyze the molecular species of sulfatide (ST) and six types of phospholipids (PLs) by direct injection. In the second part, a combination method of high performance liquid chromatography (HPLC) and ESI-MS/MS was established to analysis the molecular species of ceramide (Cer), sphingomyelin (SM), glucosylceramide (Glucer) and Lactosylceramide (Laccer). In the third part, a simultaneous online analysis method of various polar lipids which combined the ESI-MS/MS method by direct injection with HPLC-ESI-MS/MS method was established by self-installed sample inlet system and its inlet program. In the fourth part, salmon oil was enzymatic esterificated into the soy lecithin, established simultaneous on-line analysis method of polar lipids was used to analyze and compare the differentials between soy lecithin before and after modified. As a film forming material, the modified soy lecithin was used to encapsulate collagen, forming liposome.Firstly, a ESI-MS/MS method by direct injection was established to analyze the individual species of ST and six types of PLs, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA) and phosphatidyl glycerol (PG). Chloroform/methanol method was used to extract lipids from rat brain, solid-phase extraction (SPE) column was used to separate the polar lipids. Then the polar lipids was injected into the ion source, the analysis of ST and six types PLs was achieved by relevant precursor ion scan (PIS) and neutral loss scan (NLS) mode. After proven, the extraction recoveries (82.6%-104.2%), intra-day precisions (1.44-3.87), inter-day precisions (2.74-5.87), the lowest limits of detection (0.50-0.87ng/ml) and lowest limits of quantification (1.5-2.6ng/ml) of this method could satisfied the analytical requirements of ST and PLs from complex biological samples.Secondly, HPLC-ESI-MS/MS was established to analyze the individual species of Cer, SM, Glucer and Laccer. After analyze the specific fragment ions in the collision-induced dissociation process, a comprehensive precursor ion/product ion pairs was established. Chloroform/methanol method was used to extract lipids from rat brain, solid-phase extraction (SPE) column was used to separate the polar lipids. Then the polar lipids was injected into the HPLC to separate Cer, SM, Glucer and Laccer. In relavant time windows, these four sphingolipids was analyzed by eatablished precursor ion/product ion pairs by multiple reaction monitoring (MRM) during elution. After proven, the extraction recoveries (81.5%-104.5%), intra-day precisions (1.12-2.25), inter-day precisions (2.42-3.81), the lowest limits of detection (0.03-0.08ng/ml) and lowest limits of quantification (0.10-0.25ng/ml) of this method could satisfied the analytical requirements of Cer, SM, Glucer and Laccer from complex biological samples.Subsequently, a simultaneous on-line method of polar lipids analysis was established after combine ESI-MS/MS direct infusion method with HPLC-ESI-MS/MS method. The self-installed inlet system was mainly composed of a syringe pump, a six-ways valve and a micro-tee. Firstly, the sample was inject directly into the ion source, the analysis of ST and six PLs was achieved in specific PIS and NLS mode. Then the sample was load into the sample loop in six-ways valve. Finally, a certain amount of sample was inject into the HPLC-ESI-MS/MS, the analysis of Cer, SM, Glucer and Laccer was achieved by MRM scan mode based on HPLC separation. The simultaneous on-line analysis of eleven types of polar lipids (at least300molecular species) could be accomplished within fourty minutes, which is fast and accurate. Using this method,307,308 and324molecular species of polar lipids from rat brain, duck brain and salmon brain was identified. After comparision, the difference in PLs was significant, in particular, polyene phospholipids was abundant in salmon brain, while was rarely in rat and duck brain. As for sphingolipids, the difference was mainly in content, the basic molecular species was similar between these three animal brain tissue.Finally, the salmon leftover was used to extract fish oil, polyunsaturated fatty acids was enriched using urea adduction method, then bound into the soy lecithin by enzymatic transesterification, forming new soy lecithin which was abundant with polyene phospholipids. Orthogonal experiments was designed with the content of eicosapaentenoic acid (EPA) and docosahexaenoic acid (DHA) as evaluation indicators. The optimal conditions was as follows:the ratio of soy lecithin and salmon oil was1:8; the content of phospholipase was18%; the reaction temperature was50℃; the reaction time was18h; the moisture content of the system is4%. Using the optimal conditions, the EPA and DHA content of soy lecithin was24.13%. The molecular species of soy lecithic before and after modified was analyzed using established simultaneous on-line analysis method of polar lipids, the results indicated that the major component of soy lecithin was PC, PE, PI and PS. When compared with lecithin before modified, the mainly characteristics of the soy lecithin after modified were the addition of polyene phospholipids and the rise of lysophospholipids content. Before modified soy lecithin has a lower degree of unsaturation, the main molecular species were PL34:2, PL36:4and PL36:3. After modified, the added polyene phospholipids content was27.41-37.01%. The lysophospholipids content of the soy lecithin before modified was low (3.52-4.57%), while the lysophospholipids content of the soy lecithin after modified was relatively high (26.85-36.01%). As the best antioxidant, the addition of0.02%BHT in modified soy lecithin could extend the shelf life up to390days. As a film forming material, the modified soy lecithin was used to encapsulate collagen, forming liposome. Orthogonal experiments was designed with the embedding ratio as evaluation indicators. The optimal conditions was as follows:soy lecithin concentration was3mg/ml; protein concentration was4mg/ml; the PH value of phosphate buffered was7.0; the time of ultrasound was140s. Using the optimal conditions, the embedding of the liposome was42.87%, the average particle diameter was160nm.