Separation and identification of phosphatidylserine molecular species using reversed-phase high-performance liquid chromatography with evaporative light scattering and mass spectrometric detection.
A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C(18) and C(30) columns. The best chromatographic resolution was achieved with the C(30) column. The limit of detection for DPPS was 5 microg/ml (S/N=3) with ELS detection and 0.1 microg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.
Larsen
Asmund
A
Department of Chemistry, University of Oslo, PO Box 1033, Blindern, 0315 Oslo, Norway. aasmunl@kjemi.uio.no
Mokastet
Eva
E
Lundanes
Elsa
E
Hvattum
Erlend
E
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008027:Light; D010718:Phosphatidylserines / Q000737:chemistry / Q000302:isolation & purification*; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010718:Phosphatidylserines