Development of an organic monolithic phase with anion exchanger groups and hydrophilic coating to homocysteine and homocysteic acid determination in biological samples from patients with Alzheimer's Disease by in-tube SPME-UHPLC-MS/MS

Abstract

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that affects about 5% of the world population aged over 65 years. Physiopathologically it is characterized by the presence of neurofibrillary tangles (tau proteins) and the accumulation of senile plaques (deposits of beta-amyloid peptides), which result in synaptic loss and neuronal death observed in the brain regions responsible for cognitive functions. The diagnosis of AD has only been possible in advanced stages, thus, the identification of potential biomarkers will have significant impacts on the clinical evaluation and on the approach to disease progression, in addition to enabling the development of new therapeutic strategies. Some clinical studies have correlated the increase in concentrations of Homocysteine (Hcy) and Homocysteic Acid (HCA) with the development of AD, thus being potential biomarkers of this pathology. Hcy is a sulfur amino acid derived from methonine metabolism and has a cysteine-like structure. HCA, on the other hand, is a product of the oxidation of Hcy, although the metabolic route that gives rise to it is still unknown. Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) has been considered the reference of analytical technique for the determination of biomarkers in biological fluids. Due to the complexity of these fluids, LC-MS/MS methods require the sample preparation step for the elimination of macromolecules from the biological matrix and concentration of analytes (trace levels). The techniques for preparing biological samples have been simplified, miniaturized (less sample volume and consumption of organic solvent) and/or hyphenated with chromatographic systems (automation, high-throughput performance). In this context, we can highlight the solid phase microextraction in the capillary (in-tube SPME). The development of capillaries with innovative stationary phases for the in-tube SPME technique, such as organic monolithic phases and Restricted Access Materials (RAM) has allowed the selective pre-concentration of the analytes.The monolithic phases based on organic polymers have the following advantages: high stability over a wide pH range, easy synthesis procedure and high permeability. The RAM phases, on the other hand, combine the principles of excluding macromolecules (physical or chemical barrier) and selective sorption (hydrophobic interactions or ion exchange) of the analytes. In this project, a fused silica capillary with the organic monolithic phase with anion exchange groups and hydrophilic coating (RAM) will be developed for the in-tube SPME-UHPLC-MS/MS technique. The developed method will be validated for the determination of Hcy and HCA in biological samples from patients with AD. Therefore, in line with the state of the art of miniaturized extraction and separation techniques, this project will be of great value in the area of Neuroscience. (AU)