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Journal of Lipid Research, Vol. 46, 373-382, February 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

John S. Owen¹, Robert L. Wykle, Michael P. Samuel and Michael J. Thomas

Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC

¹ To whom correspondence should be addressed. e-mail: joowen{at}wfubmc.edu

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine.

These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.

Abbreviations: d₃-16:0 PAF, 1-O-hexadecyl-2-[²H₃]acetyl-glycerophosphocholine; fMLP, N-formyl-methionyl-leucyl-phenylalanine; GPC, sn-3-glycerophosphocholine; LC-MS, high-performance liquid chromatography-mass spectrometry; LC-MS/MS, high-performance liquid chromatography-tandem mass spectrometry; lysoPC, lysophosphatidylcholine; MRM, multiple reaction monitoring; PAF, platelet-activating factor; PFPC, palmitoyl-formyl-glycerophosphocholine; PMN, polymorphonuclear neutrophil

Supplementary key words high-performance liquid chromatography tandem mass spectrometry • trace analysis • neutrophil

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