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An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry
John S Owen1, Robert L Wykle, Michael P Samuel
1Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA. joowen@wfubmc.edu
Journal of Lipid Research
|November 3, 2004
Summary
Researchers developed a sensitive HPLC-tandem mass spectrometry assay for platelet-activating factor (PAF). This enhanced method detects low PAF levels and identifies novel interfering lipids in human neutrophils.
Area of Science:
- Lipidomics
- Mass Spectrometry
- Biochemistry
Background:
- Platelet-activating factor (PAF) is a key lipid mediator.
- Existing LC-MS/MS assays for PAF are limited by sensitivity and interference from isobaric lipids.
Purpose of the Study:
- To develop an improved, highly sensitive LC-MS/MS assay for PAF.
- To identify and characterize novel lipids that may interfere with PAF measurements.
Main Methods:
- High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was employed.
- The assay was optimized for sensitivity and linearity in PAF detection.
- Lipid species in human neutrophils were analyzed and characterized.
Main Results:
- The improved assay detects as little as 1 pg (1.9 fmol) of PAF with linear response up to 1,000 pg.
- Three novel lipid species were detected in human neutrophils.
- One novel lipid is a new PAF molecular species; two others are formylated glycerophosphocholines.
Conclusions:
- The developed LC-MS/MS method offers significantly enhanced sensitivity for PAF detection.
- Novel PAF species and formylated lipids represent potential interferences in PAF measurements.
- These findings expand the understanding of lipid signaling and analytical challenges in lipidomics.