An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry.
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.
Owen
John S
JS
Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA. joowen@wfubmc.edu
Wykle
Robert L
RL
Samuel
Michael P
MP
Thomas
Michael J
MJ
D002384:Catalysis; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019963:Clinical Chemistry Tests; D020404:Glycerophospholipids / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008244:Lysophosphatidylcholines / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D009504:Neutrophils / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D013997:Time Factors
D020404:Glycerophospholipids; D008055:Lipids; D008244:Lysophosphatidylcholines; D010972:Platelet Activating Factor