Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed-phase liquid chromatography/multiple reaction monitoring mass spectrometry. 2011

Dipti Mangal, and Cornelius E Uboh, and Lawrence R Soma
University of Pennsylvania School of Veterinary Medicine, New Bolton Center Campus, Kennett Square, 19348, USA.

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.

UI MeSH Term Description Entries
D007553 Isotope Labeling Techniques for labeling a substance with a stable or radioactive isotope. It is not used for articles involving labeled substances unless the methods of labeling are substantively discussed. Tracers that may be labeled include chemical substances, cells, or microorganisms. Isotope Labeling, Stable,Isotope-Coded Affinity Tagging,Isotopically-Coded Affinity Tagging,Affinity Tagging, Isotope-Coded,Affinity Tagging, Isotopically-Coded,Isotope Coded Affinity Tagging,Labeling, Isotope,Labeling, Stable Isotope,Stable Isotope Labeling,Tagging, Isotope-Coded Affinity,Tagging, Isotopically-Coded Affinity
D002138 Calibration Determination, by measurement or comparison with a standard, of the correct value of each scale reading on a meter or other measuring instrument; or determination of the settings of a control device that correspond to particular values of voltage, current, frequency or other output. Calibrations
D005591 Chemical Fractionation Separation of a mixture in successive stages, each stage removing from the mixture some proportion of one of the substances, for example by differential solubility in water-solvent mixtures. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Fractionation, Chemical,Chemical Fractionations,Fractionations, Chemical
D006736 Horses Large, hoofed mammals of the family EQUIDAE. Horses are active day and night with most of the day spent seeking and consuming food. Feeding peaks occur in the early morning and late afternoon, and there are several daily periods of rest. Equus caballus,Equus przewalskii,Horse, Domestic,Domestic Horse,Domestic Horses,Horse,Horses, Domestic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013058 Mass Spectrometry An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers. Mass Spectroscopy,Spectrometry, Mass,Spectroscopy, Mass,Spectrum Analysis, Mass,Analysis, Mass Spectrum,Mass Spectrum Analysis,Analyses, Mass Spectrum,Mass Spectrum Analyses,Spectrum Analyses, Mass
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face
D015777 Eicosanoids A class of compounds named after and generally derived from C20 fatty acids (EICOSANOIC ACIDS) that includes PROSTAGLANDINS; LEUKOTRIENES; THROMBOXANES, and HYDROXYEICOSATETRAENOIC ACIDS. They have hormone-like effects mediated by specialized receptors (RECEPTORS, EICOSANOID). Eicosanoid,Icosanoid,Icosanoids
D016014 Linear Models Statistical models in which the value of a parameter for a given value of a factor is assumed to be equal to a + bx, where a and b are constants. The models predict a linear regression. Linear Regression,Log-Linear Models,Models, Linear,Linear Model,Linear Regressions,Log Linear Models,Log-Linear Model,Model, Linear,Model, Log-Linear,Models, Log-Linear,Regression, Linear,Regressions, Linear

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