Determination of endocannabinoids and cannabinoids in biological samples from patients with Parkinson`s disease by ultrahigh-performance liquid chromatography-tandem mass spectrometry

Author(s)

Camila Marchioni

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-02-01

Examining board members

Maria Eugenia Queiroz Nassur; Fabio Augusto; Cristiane Masetto de Gaitani; Jaime Eduardo Cecilio Hallak; Fernando Mauro Lanças; Eduardo Carasek da Rocha

• Advisor: Maria Eugenia Queiroz Nassur; José Alexandre de Souza Crippa

Abstract

Endocannabinoids are neurotransmitters derived from arachidonic acid; they bind to cannabinoid receptors and participate in various brain functions, such as memory, learning, and motor control. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the main endocannabinoids. Parkinson\'s disease (PD) is considered the second most common neurodegenerative pathology in the population, being characterized by reduced dopaminergic influence. Evidence shows an increase in AEA and a decrease in 2-AG levels in patients with PD and in animal models with induced parkinsonism. Cannabidiol (CBD) is a promising drug to treat PD: it is believed to have a neuroprotective effect on dopaminergic degeneration. However, the use of ?9- Tetrahydrocannabinol (?9-THC) is not indicated because it may lead to psychotropic effects and dependence. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) has been considered the benchmark analytical technique to determine endocannabinoids and cannabinoids in biological samples. Column switching allows analysis to be automated through hyphenation of the biological sample preparation stage (analytes pre-concentration and removal of most endogenous components) with the chromatographic system (chromatographic separation and detection). In this context, we have developed two online analytical methods based on UHPLC-MS/MS in the column switching mode to determine (a) AEA and 2-AG and (b) CBD and ?9-THC in biological samples from patients with PD. Endocannabinoids were determined in plasma and cerebrospinal fluid (CSF) samples. The UHPLC-MS/MS method in the column switching mode for determination of endocannabinoids with a column of restricted access material in the first dimension showed linearity between 0.100 (LIQ) and 6.00 ng mL-1 for AEA and 0.04 (LIQ) to 10.00 ng mL-1 for 2-AG in plasma samples. For CSF samples, the linear range was from 0.45 (LIQ) to 9.00 ng mL-1 for AEA and from 1.40 (LIQ) to 180 ng mL-1 for 2-AG. The accuracy values were below 14.2% and precision below 11.1% (excluding LIQ) for the two analytes. The methods had no residual effect or matrix effect. The analytes were stable after freeze/thaw cycles and in the short- and long-term test. These methods were used to analyze 70 plasma and 66 CSF samples. To determine CBD and ?9-THC in plasma samples (UHPLC-MS/MS in column switching mode), we synthesized a molecularly imprinted polymer (MIP) in the fused silica capillary (in situ). The synthesis showed good profitability because it required small mass of the template molecule (reduced CBD). The online method based on the MIP capillary in the first dimension showed linearity between 10 and 300 ng mL-1 (CBD and ?9-THC). Precision ranged from 0.2 to 10.3% for the two analytes and the accuracy was less than 4.4% (excluding LIQ). The residual effect and the matrix effect were within the parameters established by ANVISA. Finally, the method was satisfactorily applied to determine CBD and ?9-THC in plasma samples from patients on CBD therapy (AU)

• FAPESP's process: 16/13639-8 - Column-switching liquid chromatography-Tandem mass spectrometry for determination of endocannabinoids and cannabinoids in biological samples from patients with Parkinson's Disease
• Grantee: Camila Marchioni
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)