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An isocratic HPLC method for the quantitation of eicosanoids in human platelets
Author(s) -
Moraes Leonardo A.,
Giner Rosa M.,
PaulClark Mark J.,
Perretti Mauro,
Perrett David
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.349
Subject(s) - chemistry , chromatography , arachidonic acid , hydroxyeicosatetraenoic acid , calibration curve , derivatization , high performance liquid chromatography , thromboxane , quantitative analysis (chemistry) , thromboxane b2 , prostaglandin , platelet , reversed phase chromatography , standard curve , enzyme , detection limit , biochemistry , medicine
We describe here a modied protocol for the simultaneous quantication of specic eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B 2 (TXB 2 ), arachidonic acid (AA), 12‐ R ‐hydroxyeicosatetraenoic acid (12‐ R ‐HETE), 12‐ S ‐hydroxyheptadecatrienoic acid (12‐ S ‐HHTrE) and the internal standard prostaglandin B 1 (PGB 1 ) were extracted from human platelets by liquid–liquid extraction using ethyl acetate. This was followed by derivatization and uorescent detection prior to analysis by reversed phase liquid chromatography. The highperformance liquid chromatographic method consisted of ODS reversed‐phase column (3 µm) and a mobile phase of acetonitrile–water (85:15). TXB 2 and AA plasma calibration curves were linear between 6.25 and 125 ng mL −1 ( r 2 > 0.997), whereas for 12‐ R ‐HETE and 12‐ S ‐HHTrE the curves were linear between 5.0 and 40 ng mL −1 ( r 2 > 0.998). All calibration curve standards had <15% CV (coefcient of variation) and between‐run precision, and the percentage relative deviation for replicate ( n = 6) quality controls was less than 5.5%. The method was adapted to allow the screening of drugs that may affect either one or both of the lipoxygenase and cyclo‐oxygenase pathways. Copyright © 2004 John Wiley & Sons, Ltd.