Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray...

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS2 or MS3, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1-5 ng of injected substance.