Determination of free concentration of endocannabinoids in brain tissue

The release of metabolites from their bound to free forms is the main regulatory path in living species. Therefore, the ability to determine the free concentrations of small molecules is highly critical in many biological samples. The main challenges in achieving this task are the interferences inherent to complex matrices and the ability to distinguish between the free and total concentrations. This paper presents a non-invasive microextraction method that enables the determination of endocannabinoids in brain tissue. The proposed method is based on two key principles: the availability of the free concentration of endocannabinoids for partitioning to the solid-phase microextraction (SPME) fiber; and negligible depletion enabled by the small volume of extraction phase on the fiber. These features allow the presented SPME method to provide information about the free concentration of analytes without disturbing the binding equilibrium between the analytes and the matrix. The determination of spiked samples with known concentrations enables the percentage of analyte bound to the tissue to be calculated, which can then be applied to calculate the total concentration from the determined free concentration. This manuscript focuses on the determination of the free concentration and tissue binding percentages of endocannabinoids in brain tissue. Significantly, SPME's small size and potential for non-invasive sampling enable its application in live animal subjects with minimal tissue damage.