Simultaneous determination and quantification of seven major phospholipid classes in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry and the application in diabetes nephropathy

A rapid and specific analytical method for simultaneous determination and quantification of seven major phospholipid classes in human blood was developed by normal-phase high-performance liquid chromatography tandem mass spectrometry. The optimal separation was achieved by using mobile phase hexane (A) and 2-propanol with water, formic acid and ammonia as modifiers (B) using an HPLC diol column. Isocratic elution method was used for better repeatability and no balance time. The seven major phospholipid classes in human blood that were detected including phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) phosphatidylcholine (PC), lysophosphatidylcholine (Lyso-PC), and sphingomyelin (SM). That can be separated in this condition. Every phospholipid class contains many molecular species which have similar structure. The structure of phospholipids molecular species was identified by ion-trap MSn which produced ion fragments. And the qualification was completed by TOF-MS which shows good accuracy. Through the accurate quantification of one representative phospholipids molecule in each class, a method for simultaneous estimation hundreds of molecular species in seven major classes was established. The intra-day and inter-day precision and recovery had been investigated in detail. The RSD of precision for most compound is below 8% and RE is below 10%. Recovery is almost over 80%. This method was applied to phospholipids disorder related with diabetes nephropathy successfully. The concentrations of most phospholipids for normal people are higher than that for diabetic nephropathy (DN) patients in three phases. For most of phospholipids, with the development of DN the concentration was decreasing.