Development, Validation and Comparison of Three LC-MS/MS Methods for Determination of Endogenous Striatal Oleoyl Ethanolamine in mice

Abstract Obesity has become a severe public health problem worldwide. An endogenous fatty acid ethanolamine oleoyl ethanolamine (OEA) is reported to be capable of reducing bodyweight and food intake by increasing striatal extracellular dopamine concentration. However, association between obesity and striatal OEA level remains unknown. As such, it is necessary to develop a sensitive and reliable method to quantitate OEA concentration in striatum. Because true endogenous analytes free blank matrix is not available, surrogate analyte, surrogate matrix and background subtraction methods are often employed for the analysis of endogenous compounds. In this study, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for the determination of OEA concentration in mouse brain homogenate. Interestingly, stability results found that OEA-d4 degraded in brain homogenate under room temperature, while OEA level remarkably increased with time. Since lowering temperature could observably decelerate the endogenous transformation of OEA, sample collection and preparation were carried out under ice-bath condition. Hexane : isopropanol (9:1, v/v) was employed as an extractant for liquid-liquid extraction. After method validation, three methods were applied to quantify OEA in striatum homogenate from C57B6/L mice following normal and high fat diet feeding for 4 months. Results from three methods all showed the striatal OEA level in obesity group was significantly higher than control group and obesity-resist group, which indicated that obesity might be associated with elevated striatal OEA level.