Development of spectrometric methods (UHPLC-MS/MS and MS/MS) for the determination of β-amyloid peptides in cerebrospinal fluid samples from patients with Alzheimer\'s disease

Author(s)

Caroline Fernandes Grecco

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-09-01

Examining board members

Maria Eugenia Queiroz Nassur; Eduardo Costa de Figueiredo; Anderson Rodrigo Moraes de Oliveira; Jonas Augusto Rizzato Paschoal

• Advisor: Maria Eugenia Queiroz Nassur

Abstract

Alzheimer\'s Disease (AD) is a progressive neurodegenerative dementia that affects about 50 million people around the world. AD is pathologically characterized by the presence of senile plaques and neurofibrillary tangles in the brain, caused by the extracellular accumulation of β-amyloid peptides (Aβ) and tau protein, respectively. The conclusive diagnosis of AD has been eminently clinical and, in these cases, neurodegeneration has already taken root in the patient\'s brain. Therefore, efforts have been made to find a molecular biomarker for identifying patients at risk of developing AD, as well as for early diagnosis and monitoring of the progression of neurodegenerations. In this project, the methods employing in-tube solid phase microextraction (in-tube SPME) associated with ultra-efficiency liquid chromatography coupled with sequential mass spectrometry (UHPLC-MS/MS) and Disposable Pipette Extraction (DPX) associated with MS/MS were developed and validated for the determination of Aβ peptides in cerebrospinal fluid (CSF) samples from patients with AD. During the development of the in-tube SPME UHPLC-MS/MS method, different chromatographic columns and mobile phase compositions were evaluated. The Acquity UPLC Peptide BEH C18 reverse phase analytical column (150 mm × 2.1 mm and 1.7 µm) with the mobile phase consisting of (A) water + 0.3% ammonium hydroxide and (B) acetonitrile and temperature of 35 °C presented adequate chromatographic resolution, symmetrical peaks and shorter analysis time. An organic monolithic phase with sulfonic groups was synthesized inside the fused silica capillary (6 cm × 530 µm i.D.) as an extractor phase for the in-tube SPME technique and characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The optimized conditions for monolithic phase synthesis were: 1-propanol (0.65 mL) and water (0.52 mL) as porogenic solvents; 3-sulfopropyl methacrylate potassium salt (SPM) as functional monomer (0.3306 g); ethylene glycol dimethacrylate (EDMA) as crosslinking agent (0.2 ml); 2,2\'-azobisisobutyronitrile (AIBN) as radical initiator (0.016 g); and polymerization time (24 hours) and temperature (60°C). The in-tube SPME variables (pH and sample volume, wash conditions, and elution conditions) were evaluated and optimized. The in-tube SPME UHPLC-MS/MS method presented adequate linearity with concentrations ranging from LOQ - 10 ng mL-1 with coefficients of determination (R2 ) greater than 0.99. Accuracy (EPR) and precision (CV) values ranged from -11.7 to 14.3% and 1.0 to 18.9%, respectively. The in-tube SPME UHPLC-MS/MS method was applied to determine Aβ peptides in six CSF samples from patients with Alzheimer\'s disease. The DPX MS/MS method used DPX tips (1 mL) containing 60 mg of Oasis MCX® commercial phase, connected to a polypropylene syringe. The optimization of the DPX variables (sample pH, sorption equilibrium time, sample volume/number of cycles and elution solution/number of cycles) favored the sensitivity and selectivity of the method and the use of small volumes of biological sample and of organic solvents. 5 µL of the final evaporated extract and reconstituted in 50 µL of mobile phase were injected into the MS/MS system with the mobile phase consisting of (A) water containing 0.3% ammonium hydroxide and (B) acetonitrile, with a flow rate of 0. 05 ml min-1 ; and direct infusion of a methanol solution containing 10% concentrated ammonium hydroxide to enhance detectability. The DPX-MS/MS method showed adequate linearity with concentrations ranging from LOQ - 1.5 ng mL-1 with coefficients of determination (R2 ) greater than 0.99. Accuracy (EPR) and precision (CV) values ranged from -13.6 to 13.2 and 0.3 to 12.7%, respectively. Therefore, according to the analytical validation parameters evaluated, the DPX MS/MS method can be used to determine Aβ peptides in CSF samples from patients with AD. (AU)

• FAPESP's process: 19/04386-7 - Development of an organic monolithic phase for the determination of b-amyloid peptides AB40 and AB42 in plasma samples from patients with Alzheimer's Disease by UHPLC-MS/MS in column switching mode
• Grantee: Caroline Fernandes Grecco
• Support Opportunities: Scholarships in Brazil - Doctorate