Development of the SALLE-UHPLC-MS/MS method to determine anandamide and 2-arachidonoylglycerol in rat brain samples submitted to a Parkinson\'s Disease animal model

Author(s)

Igor Gustavo de Carvalho Oliveira

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-07-24

Examining board members

Maria Eugenia Queiroz Nassur; Carla Beatriz Grespan Bottoli; Elaine Aparecida Del Bel Belluz Guimaraes

• Advisor: Maria Eugenia Queiroz Nassur

Abstract

The endogenous compounds anandamide (AEA) and 2-arachidonoylglycerol (2-AG), derived from the arachidonic acid, are part of a neurotransmitters class known as endocannabioids (ECs) that, together with the cellular cannabinoid receptors (CB1 and CB2) and metabolic enzymes, compose the endocannabinoid system (eCB). Studies indicate that the endogenous concentrations of these ECs suffers variations to physiological, pharmacological and pathological stimuli, making the determination of their concentrations in biological samples to be the target of studies of several pathologies, with emphasis on neurodegenerative diseases, such as Parkinson\'s Disease (PD). Experimental animal models have been developed to mimic the characteristics of PD observed in the clinic in humans, commonly, the selective acute injection of neurotoxins, such as the hydroxylated analogue of dopamine, 6-hydroxidopamine (6-OHDA) in rats, is used to create acute neurodegenerative lesions on the nigrostriatal pathways. Liquid chromatography coupled with tandem mass spectrometry has been considered the reference technique in the determination of endocannabinoids in biological samples. The sample preparation step has been required in the development of chromatographic methods, to eliminate interfering agents and to pre-concentrate the analytes, usually present at trace levels in biological samples. The salting-out assisted liquid-liquid extraction (SALLE) is an extraction technique based on the \"salting-out\" effect, which consists in reducing the mutual solubility between water and a water-miscible organic solvent by the addition of an electrolyte. In function of the salt addition, the uncharged organic analytes present in the sample undergo a reduction of solubility in the aqueous phase and are extracted in the organic phase. The developed SALLE method presented the advantage of consuming lower organic solvent volume and shorter analysis time when compared to conventional liquid-liquid extraction and solid phase extractions. In addition, the SALLE technique presented cleaner extracts due to the separation of organic/aqueous phases, when compared to protein precipitation. The SALLE-UHPLC-MS/MS method was validated, presenting linear range from 2.00 to 20.00 ng/mL for AEA and from 0.30 to 10.00 &micro;g/mL for 2-AG, values of intra-and inter-assay accuracy (CV) and precision (RSD) bellow 15%, and no significate matrix effect. The proposed method was successfully applied to determine the endogenous concentrations of AEA and 2-AG in samples of total brain, hemispheres and striatum from parkinsonian rats and healthy rats from the control group. For AEA, there was a significant difference (p<0.05) between the ipsilateral and contralateral striatums. When comparing these two groups of striatums with striatums of healthy animals, there was a large statistical difference (p <0.001). For 2-AG, a large statistical difference (p <0.001) was observed when comparing the concentrations between the ipsilateral and contralateral striatums. Likewise, a large statistical difference (p <0.001) was observed between the ipsilateral and contralateral striatum of the injured animals with the healthy animals. (AU)

• FAPESP's process: 18/06068-0 - Development of the column switching SALLE-UHPLC-MS/MS method to anandamide and 2-arachidonoylglycerol determination in brain samples of rats submitted to an animal model of Parkinsons Disease
• Grantee: Igor Gustavo de Carvalho Oliveira
• Support Opportunities: Scholarships in Brazil - Master