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Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed‐phase liquid chromatography/multiple reaction monitoring mass spectrometry
Author(s) -
Mangal Dipti,
Uboh Cornelius E.,
Soma Lawrence R.
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4893
Subject(s) - chemistry , chromatography , isotope dilution , mass spectrometry , selected reaction monitoring , liquid chromatography–mass spectrometry , reversed phase chromatography , tandem mass spectrometry , high performance liquid chromatography
Abstract Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid‐liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl‐ tert ‐butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avoid artifact formation of eicosanoids, deferoxamine was added to plasma to chelate residual transition metal ions. The calibration curve showed excellent linearity within 0.1 to 10 ng/mL. Slopes of the calibration curves generated by adding known quantities of eicosanoids in plasma were higher than those prepared in methanol/mobile phase A. Addition of deferoxamine decreased the slope of calibration curves generated using plasma. Limit of detection (LOD) was 1–10 pg on‐column for 25 different eicosanoids. Inter‐day accuracy was 86–111%, whereas intra‐day accuracy was from 88–110%, and precision did not exceed 15% for all quality control (QC) samples. To evaluate the formation of eicosanoids, AA was exogenously added or endogenous AA was released from esterified lipids by calcium ionophore (CI) A23187 treatment of equine whole blood. Pre‐treatment of equine whole blood with dexamethasone (DEX) significantly inhibited AA or CI A23187‐ mediated formation of eicosanoids. The validated method is now employed in studies undertaken to better understand the mechanism of action and pharmacokinetics/pharmacodynamics of eicosanoids after administration of glucocorticoids to horses. This method is reliably reproducible. Copyright © 2011 John Wiley & Sons, Ltd.