Development of lipidomic platform and phosphatidylcholine retention time index for lipid profiling of rosuvastatin treated human plasma

Abstract

A simple and fast methodology to detect and identify multiple classes of lipid from human plasma is developed utilizing ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF) as lipidomics platform. All the conditions for the sample preparation and analytical instruments were optimized in detail to detect nine lipid classes (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), triacylglyceride (TG), phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC), lysophosphatidic acid (LysoPA), and sphingomyelin (SM)), which are the most important biologically active lipids but have different characteristics. Finally, the plasma was prepared after a liquid-liquid extraction with a mixture of chloroform/methanol (1:2v/v) including salting out by adding 0.15M of NaCl and the residue after evaporation was reconstituted with a mixture of chloroform/methanol (1:1v/v) to dissolve all lipids which have different polarity. The chromatographic conditions were set up such that mobile phase (A) comprised 10mM ammonium acetate in 40% acetonitrile and mobile phase (B) comprised 10mM ammonium acetate in acetonitrile:isopropanol=10:90(v/v) with ACQUITY BEH C18 as the stationary phase. In particular, a retention time index of PC was constructed by analyzing known standards to confirm each variant of PC without the use of any additional standards in every experiment. The lipidomic methodology and the retention time index of PC were applied to analyze the lipidomic profiling of human plasma from rosuvastatin (lipid lowering drug) treated subjects. In the developed lipidomic platform, all lipids were successfully analyzed within 16min and PCs could be confirmed with the PC retention time index. In rosuvastatin treatment, the lipid profiling was changed in all the eight lipid classes. The level of SM, TG, PI and PE decrease significantly but LysoPCs and PCs were whether decreased or increased. Those results indicated that the plasma level of overall lipids decreased by drug response, however, the changes in the lipids which are important components for biological membrane such as LysoPC and PC were more complicated, and it could be related to the side effect of rousuvastatin. In conclusion, it was found that our lipidomic methodology and the PC retention time index provided not only overall lipidomic information but also profiled specific information of drug response.