PhD Scientific Days 2021

PH_I_P: Pharmaceutical Sciences I. Posters

Validated Method for the Separation and Quantitative Determination of Gangliosides by Capillary Electrophoresis

Text of the abstract

Introduction: Gangliosides are sialic acid-containing glycolipids of the plasma membranes that are particularly enriched in the nervous system. Altered concentration and composition of gangliosides was observed in several neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, therefore their simultaneous detection is necessary in pharmacological studies.
Aims: Our aim was to optimize and validate a capillary electrophoresis (CE) method for the quantitative determination of the most abundant gangliosides in neuronal tissues.
Methods: Separation conditions were optimized and parameters of analytical performance were determined on ganglioside extracts of rat cerebral synaptosome samples. Ganglioside levels of rat cerebrum, cerebellum and spinal cord synaptosomes were measured by the validated CE method.
Results: Testing several cyclodextrins as separation buffer additives, randomly methylated alpha-cyclodextrin (RAMEA) was found suitable for the separation of neuronal gangliosides (GM1, GD1a, GD1b, GT1b, GQ1b), and the challenging separation of mono-sialylated GM3 and GM1 was also carried out. We found that RAMEA effectively forms complex with the amphiphilic gangliosides and disrupts their micellar aggregates in the aqueous separation buffer, which enables the separation of ganglioside monomers by CE. Improving resolution was observed at more alkaline pH and with increasing sodium borate concentration. The best resolution of the analytes was achieved with 20 mM RAMEA in 100 mM sodium borate buffer, pH 10.0. Within the studied range, linearity as well as intra- and interday precision and accuracy values were acceptable. In rat cerebral synaptosomes, the total tissue ganglioside concentration was approximately 5-fold higher compared to cerebellar synaptosomes and 15-fold higher compared to spinal cord synaptosomes. The predominant components were GD1a and GT1b in the studied samples.
Conclusion: Separation of the studied gangliosides was achieved using the established CE method. The method was found suitable for the quantitative analysis of neuronal gangliosides in synaptosome samples.
Funding: This study was supported by “Competitiveness and excellence cooperations” project (2018-1.3.1-VKE-2018-00030) provided by the National Research, Development and Innovation Fund.

University and Doctoral School

Semmelweis University, Doctoral School of Pharmaceutical Sciences