LAUSR

Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) represent two of the most important endocannabinoids (ECs) investigated in neurobiology as therapeutic targets for several mental disorders. However, the determination of these ECs in biological matrices remains a challenging task because of the low concentrations, low stability and high protein-bound (LogP ∼ 6). This work describes innovative analytical methods based on biocompatible SPME (Bio-SPME), SPME-UHPLC-MS/MS and Bio-SPME-Nano-ESI-MS/MS, to determine AEA and 2-AG in human plasma samples. The direct coupling of Bio-SPME with nano-ESI-MS/MS can be considered an alternative tool for faster analysis. Different Bio-SPME fibers based on silica and polymeric coating (i.e. C18, C30, and HLB) were evaluated. Different desorption solvents based on combinations of methanol, acetonitrile, and isopropanol were also evaluated for efficient elution with minimum carry-over. Given the high protein binding analytes and the fact that SPME extracts the free-concentration of the analytes, the plasma samples were modified with additives such as guanidine hydrochloride (Gu-HCl), trifluoroacetic acid, and acetonitrile. This study was carried out by experimental design to achieve complete protein denaturation and the release of target analytes. The maximum extraction efficiency was obtained under the following conditions: HLB coated fibers (10 mm length, 20 μm coating thickness), matrix modified (300 μL of plasma) with 50 μL of Gu-HCL 1 mol L-1, 75 μL of ACN and 75 μL of water, and desorption with methanol/isopropanol solution (50:50, v/v). Both methods were validated based on current international guidelines and can be applied for monitoring of concentrations of endocannabinoids in plasma samples. SPME-UHPLC-MS/MS method presented lower LOQ values than SPME-nanoESI-MS/MS. The additional separation (chromatographic column) favored the detectability of LC-MS/MS method. However, the SPME-nano-ESI-MS/MS decrease the total analysis time, due to significant reductions in desorption and detection times.