Determination of platelet-activating factor and alkyl-ether phospholipids by gas chromatography-mass spectrometry via direct derivatization.
A mass spectrometric method has been developed for the quantitative analysis of platelet-activating factor (PAF) and lyso-platelet-activating factor (lyso-PAF) based on electron-capture gas chromatography-mass spectrometry using a stable-isotope dilution technique. The cleavage and derivatization was accomplished in a single step by direct reaction of phospholipid with pentafluorobenzoyl chloride at 150 degrees C. Spectroscopic and chromatographic data indicated that PAF and lyso-PAF were converted into derivatives containing a pentafluorobenzoyl group in place of the original phosphocholine group with 95 and 51% yield, respectively. Additionally, in the lyso-PAF derivative, the free hydroxyl group was found to be replaced by chlorine. Phosphatidylcholines containing an arachidonoyl group can be derivatized with a solution of PFBCl/chloroform at 120 degrees C for 18 h, producing 90% derivative. Analysis by GC/MS and LC/MS allowed the detection of 1 or 250 pg derivative, respectively, injected onto the column with S/N greater than 3. Newly available analogues of high isotopic purity containing either three or four deuterium atoms located in the 1-O-hexadecyl chain were used as internal standards. The developed GC/MS assay was used to quantitate PAF and lyso-PAF in rabbit leukocytes before and after stimulation with calcium ionophore. The levels of PAF in unstimulated cells were in the order of 2.27 pmol/10(6) cells and increased about 17-fold during 10-min stimulation with 2 microM ionophore A23187. The lyso-PAF levels in resting cells were in the order of 3.76 pmol/10(6) cells and increased 1.7-fold during stimulation. This assay exhibited satisfactory sensitivity, reproducibility, and accuracy.
Balazy
M
M
Department of Ophthalmology, Louisiana State University Medical Center School of Medicine, New Orleans.
Braquet
P
P
Bazan
N G
NG
D000818:Animals; D001565:Benzoates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000032:analysis; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000032:analysis*; D011817:Rabbits; D005464:Fluorobenzenes; D005466:Fluorocarbons
D001565:Benzoates; D010713:Phosphatidylcholines; D010972:Platelet Activating Factor; C029271:O-deacetyl platelet activating factor; C014737:pentafluorobenzoyl chloride; D005464:Fluorobenzenes; D005466:Fluorocarbons