High performance liquid chromatography of platelet-activating factors

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.