Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) me thod was developed for separation of phospholipid classes in human blood. T he separation was obtained using an HPLC diol column and a gradient of chlo roform 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 baselin e separation was obtained between phosphatidylglycerol. phosphatidylcholine , phosphatidylethanolamine. lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine. eluting in that order, The total run time was 30 mi n. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are s ubstances with structural similarities to the glycerophospholipids. had sim ilar retention time as phosphatidylethanolamine, but were well separated fr om the other glycerophospholipid classes. The species from each class were identified using MS2 or MS3, which forms characteristic lyse-fragments. The combination of lyse-fragment mass, molecular ion and chromatographic reten tion time was used to identify each species, including 20 species of phosph atidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlie r 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. (C) 2001 Elsevier Science B.V. All rights reserved.