Development of biocompatible phases for solid-phase microextraction and applications of its in-tube and in vivo variants in the determination of biomarkers of neurodegenerative diseases by LC-MS/MS

Author(s)

Igor Gustavo de Carvalho Oliveira

• 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: 2025-05-09

Examining board members

Maria Eugenia Queiroz Nassur; Marco Aurelio Zezzi Arruda; Emanuel Carrilho; Eduardo Carasek da Rocha

• Advisor: Maria Eugenia Queiroz Nassur

Abstract

Liquid chromatography coupled with mass spectrometry (LC-MS) has been the reference analytical technique for determining biomarkers of neurodegenerative diseases, such as Parkinson\'s disease (PD) and Alzheimer\'s disease (AD), in biological samples. However, due to the complexity of these samples, a sample preparation step is required. In this context, solid-phase microextraction (SPME) and its variants, in-tube SPME and in vivo SPME, stand out as non-exhaustive sample preparation techniques that integrate sampling, extraction, and preconcentration into a single step. In these techniques, a small amount of sorbent is immobilized on a solid support (SPME device), which is then exposed to the sample. The extraction is based on the partition equilibrium of analytes between the sample and the sorbent. In this thesis, different biocompatible phases were developed for use in SPME combined with GC or LC, with a focus on determining biomarkers of neurodegenerative diseases in biological samples. Chapter 1 presents the theoretical foundations of SPME, as well as the definitions of biomarkers. Chapter 2 describes the development of novel biocompatible SPME devices coated with carbon nanotubes (CNTs) combined with polybenzimidazole (PBI). These SPME devices exhibited high mechanical, chemical, and thermal stability in LC-MS and/or GC-MS analyses. When analyzing benzene, toluene, ethylbenzene, and o-xylene, the innovative CNT-PBI coating outperformed carboxen-polydimethylsiloxane (CAR-PDMS) and CAR-PBI coatings in extraction efficiency. For non-polar drugs of abuse analyzed by LC-MS, the CNT-PBI coating demonstrated more efficient extractions compared to the conventional polyacrylonitrile (CNT-PAN) binder, while also achieving performance comparable to the C18-PAN coating. In untargeted analyses of water samples using CNT-PBI- and CNT-PAN-coated blades, the binders (PBI and PAN) significantly influenced the extraction capabilities of the materials. Chapter 3 presents the development of a capillary containing a novel anion-exchange restricted-access organic monolithic phase, used as an extraction device for in-tube SPME to determine homocysteine (Hcy) and homocysteic acid (HCA) - potential biomarkers of AD - in cerebrospinal fluid (CSF) samples. The developed phase enabled selective extraction of the analytes while simultaneously excluding approximately 90% of macromolecules (proteins) from the matrix. Optimization of in-tube SPME parameters enhanced extraction efficiency. The proposed method exhibited a linear range of 8 - 250 ng mL-1 for Hcy and 5 - 150 ng mL-1 for HCA, with appropriate precision and accuracy values and no significant matrix effect. This method was applied in a clinical study analyzing CSF samples from AD patients, individuals with mild cognitive impairment (MCI), and control group volunteers. A significant increase in Hcy concentrations was observed in the AD (p = 0.0487) and MCI (p = 0.0022) groups compared to the control group, demonstrating the potential of this analyte as an AD biomarker. In Chapter 4, an in vivo SPME method using biocompatible fibers coated with hydrophilic-lipophilic balance (HLB-PAN, 5 mm, 12 µm) particles was developed for the determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain. Extraction was performed under negligible depletion conditions, using a reduced extraction phase volume to enable the quantitative determination of both free and total analyte concentrations. The equilibrium time was set at 15 minutes for both analytes. For free concentration determination, the distribution coefficients (Kfs) were calculated, with values of 10,500 and 7,300 for AEA and 2-AG, respectively. For total concentration determination, the analyte binding percentage to the matrix (MB%) was determined, reaching 99% for both analytes. The in vivo SPME method was successfully applied to determine AEA concentrations in the striatum region of a PD animal model, revealing a significant increase in AEA levels in parkinsonian animals compared to control animals, with free concentrations of 0.57 ng mL-1 and 0.45 ng mL-1, respectively. (AU)

• FAPESP's process: 20/06526-8 - 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
• Grantee: Igor Gustavo de Carvalho Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate