Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism.

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R²>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Noninvasive assessment of the role of cyclooxygenases in cardiovascular health: a detailed HPLC/MS/MS method.

A robust method for the routine quantitation of a selected group of urinary eicosanoid metabolites of interest to cardiovascular research in human and mouse is described and discussed. Included are the addition of stable isotope-labeled internal standards, solid phase extraction, and quantitation by liquid chromatography/tandem mass spectrometry using selected reaction monitoring (SRM) techniques.

Enantiomeric separation of hydroxy and hydroperoxy eicosanoids by chiral column chromatography.

We describe high-performance liquid chromatography (HPLC) methods for the enantiomeric resolution of hydroxy and hydroperoxy fatty acids/eicosanoids using a Chiralpak AD or AD-RH chiral stationary phase. These columns achieve baseline resolution of all six positional/conjugated isomers of the hydroxy as well as of the hydroperoxy derivatives of arachidonic acid in chromatographic runs of less than 20 min. Hydro(pero)xy derivatives of linoleic and linolenic acids can be resolved with similar efficiencies. The individual hydroperoxy isomers are best resolved using the reversed-phase Chiralpak AD-RH column. For the synthesis of milligram quantities of enantiomerically pure hydro(pero)xy arachidonic acids, a simple scheme is presented starting with the autoxidation of the fatty acid methyl ester in the presence of 10% alpha-tocopherol followed by chromatographic purification of the positional isomers using a combination of reversed- and straight-phase HPLC columns. Mild alkaline hydrolysis of the methyl ester derivatives affords the free acids suitable for biological testing. The Chiralpak AD column appears to be efficient for the chiral resolution of prostaglandins and isoprostanes although a comprehensive evaluation is yet to be reported. For chiral analysis of endogenous hydroxy eicosanoids the availability of novel microflow Chiralpak capillary columns (0.3 mm i.d.) will be of great advantage, because sample sizes of a few nanograms can be analyzed using simple UV detection.