Solid-phase extraction and reversed-phase high-performance liquid chromatographic technique for isolation and estimation of platelet activating factor in plasma.
A solid-phase extraction technique for the isolation of platelet activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)] from biological matrices was developed. Amberlite XAD-2 was effective in retaining different molecular species of AGEPC from plasma and incubation media. The recovery of the three molecular species (C14-, C16-, and C18-AGEPC) was greater than 95%. XAD-2 also removed large amounts of plasma impurities, giving a cleaner high-performance liquid chromatographic (HPLC) profile. AGEPC in plasma or incubation media was not significantly removed by passage of the sample through a column packed with ODS-silica. A reversed-phase HPLC technique for separation and estimation of different species of AGEPC was developed. Resolution of C14-, C16- and C18-AGEPC was accomplished on a Hamilton PRP-1 resin column using an aqueous acetonitrile gradient containing 1 mM methanesulphonic acid. The detection limit was of the order of 50 ng of AGEPC at 210 nm. The AGEPC purified by the technique described retained its biological activity as determined by its ability to release endogenous serotonin from rabbit platelets.
D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D013058:Mass Spectrometry; D009504:Neutrophils / Q000032:analysis; D010972:Platelet Activating Factor / Q000032:analysis* / Q000302:isolation & purification; D012701:Serotonin / Q000097:blood; D013056:Spectrophotometry, Ultraviolet
D010972:Platelet Activating Factor; D012701:Serotonin