RÉSUMÉ
A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).
Sujet(s)
Acides arachidoniques/analyse , Chimie du cerveau/physiologie , Endocannabinoïdes/analyse , Glycérides/analyse , Extraction liquide-liquide/méthodes , Amides gras polyinsaturés N-alkylés/analyse , Animaux , Acides arachidoniques/isolement et purification , Chromatographie en phase liquide à haute performance/méthodes , Modèles animaux de maladie humaine , Endocannabinoïdes/isolement et purification , Glycérides/isolement et purification , Limite de détection , Modèles linéaires , Mâle , Maladie de Parkinson/métabolisme , Amides gras polyinsaturés N-alkylés/isolement et purification , Rats , Rat Wistar , Reproductibilité des résultats , Spectrométrie de masse en tandem/méthodesRÉSUMÉ
This work describes the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method that uses disposable pipette extraction (DPX-UHPLC-MS/MS) to determine the endocannabinoid anandamide (AEA) in cerebrospinal fluid samples (CSF). The DPX parameters sorption equilibrium time, sample volume, number of draw-eject cycles, washing solvent volume, and elution solvent volume were optimized by design of experiments (DOE) techniques. The simple DPX protocol proposed herein required a reduced amount of CSF sample and organic solvent. The DPX-UHPLC-MS/MS method presented linear range from 0.10â¯ngâ¯mL-1 (LLOQ) to 3.0â¯ngâ¯mL-1, inter- and intra-assay accuracy with EPR values varying from -8.2% to 9.6%, inter- and intra-assay precision with CV values ranging from 1.3% to 14.8% (except for the LLOQ), and no significant matrix effect. The innovative DPX-UHPLC-MS/MS method was successfully applied to determine AEA in CSF samples from Parkinson's disease (PD) patients and should therefore be used in clinical studies.