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Identifier

000463795

Title

Ανάπτυξη και επικύρωση μεθόδου ανάλυσης κανναβινοειδών σε δείγματα αμφιβληστροειδούς με τη χρήση LC-MS/MS

Alternative Title

Method development and validation for the analysis of cannabinoids in retina samples using LC-MS/MS

Author

Λαποκωνσταντάκη, Εμμανουέλα Γ.

Thesis advisor

Χανιωτάκης, Νικόλαος

Reviewer

Θερμού, Κυριακή
Χατζηιωάννου, Αναστασία-Χρυσοβαλάντου

Abstract

The plant Cannabis sativa has been used for medicinal purposes for many years. Τhe endocannabinoid system (ECS) has been shown recently to be a major target for the development of CNS (central nervous system; brain and retina) disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The ECS system comprises of the endocannabinoids, cannabinoid CB1 and CB2 receptors(R) and the enzymes that synthesize and metabolize the endocannabinoids. The aim of the present master's thesis is the employment of LC-MS/MS, methodology for quantitative analysis of experimental rat retinal samples of a) the endocannabinoid 2-arachidonoylglycerol (2-AG) and the dual inhibitor, AM11920, of metabolic enzymes MAGL/ABHD6 of 2-AG, after chronic intraperitoneal administration in an excitotoxicity model and b) two synthetic analogues, AM1710 (CB2R agonist) and SR141716 (CB1R antagonist) after topical (eye drop) administration in an early stage diabetic retinopathy model. The qualitative and quantitative determination was performed on a Q Exactive™ Plus Quadrupole-Orbitrap™ Mass Spectrometer. Determination and quantification of 2-AG, AM1710 and SR141716 was performed with an Ultimate 3000 HPLC System. An Hypersil Gold separation column (100mm × 4.6mm i.d., 5μm) was used. The separation was performed at a flow rate of 0.4 mL/min by gradient elution, using H₂O-0,1%Formic Acid and ACN-0,1% Formic Acid as solvents. For the mass spectrometry, electrospray ionization in positive mode and the PRM mode for the fragmentation of the analytes were used after the optimization of the monitoring conditions for the target compound. All methods were validated for each analyte, in terms of linearity, precision, repeatability, robustness, limits of detection and quantification. The use of the developed methodology of the three cannabinoids certified for the first time that these compounds, independent of the route of administration, reached the retina and induced their neuroprotective and anti-inflammatory actions.

Language

Greek

Subject

2AG

AM1710

Electrospray

Limit of quantification

Limits of detection

Linearity

Liquid chromatography

Mass spectrometry

PRM