An isocratic HPLC method for the quantitation of eicosanoids in human platelets

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Abstract
We describe here a modified protocol for the simultaneous quantification of specific eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B2 (TXB2), arachidonic acid (AA), 12‐R‐hydroxyeicosatetraenoic acid (12‐R‐HETE), 12‐S‐hydroxyheptadecatrienoic acid (12‐S‐HHTrE) and the internal standard prostaglandin B1 (PGB1) were extracted from human platelets by liquid–liquid extraction using ethyl acetate. This was followed by derivatization and fluorescent 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). TXB2 and AA plasma calibration curves were linear between 6.25 and 125 ng mL−1 (r2 > 0.997), whereas for 12‐R‐HETE and 12‐S‐HHTrE the curves were linear between 5.0 and 40 ng mL−1 (r2 > 0.998). All calibration curve standards had 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.

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