| Metadata | Details |
|---|
| Publication Date | 2020-07-24 |
| Journal | International Journal of Molecular Sciences |
| Authors | Gulyaim Sagandykova, Justyna WalczakâSkierska, Fernanda Monedeiro, PaweĆ Pomastowski, BogusĆaw Buszewski |
| Institutions | Nicolaus Copernicus University |
| Citations | 7 |
| Analysis | Full AI Review Included |
- Novel Methodology: A new off-line Electrochemistry (EC) coupled with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (EC-MALDI-TOF-MS) system was developed to simulate and identify Phase I oxidative metabolites of low molecular weight compounds (LMWC).
- BDD Electrode Use: Boron-Doped Diamond (BDD) electrodes were successfully employed as the working electrode for the robust electrochemical conversion of saccharides (glucose, fructose, galactose) and cyclitols (pinitol, inositols).
- Metabolite Identification: The process generated numerous candidate metabolites, primarily through reactions mimicking Phase I metabolism, such as hydrogenation, dehydrogenation, and hydroxylation. Cyclitols showed a greater variety of EC products than saccharides.
- Structural Complexity: EC-treated analytes exhibited increased propensity for recombination, dimerization, and adduct formation (with sodium or matrix compounds), resulting in more complex MS spectra compared to the original compounds.
- Data Correlation: Exploratory data analysis (HCA, Network Analysis) confirmed that EC processing increased the similarity (coincidence of fragments) between the MS spectra of the different analytes, indicating interconversion into common structural motifs.
- Analytical Advantage: The DHB matrix in positive ionization mode provided the highest number of discriminating ions, while the HCCA matrix showed greater correlation between original and treated analytes.
| Parameter | Value | Unit | Context |
|---|
| Working Electrode Material | Boron-Doped Diamond (BDD) | N/A | Used for electrochemical conversion |
| EC Potential Range | 0 to 3000 | mV | Applied potential for BDD electrode |
| EC Flow Rate | 10 | ”L/min | Infusion rate during electrochemical conversion |
| EC Oven Temperature | 37 | °C | Temperature maintained during EC conversion |
| Analyte Concentration (Final) | 10 | ”g/mL | Concentration in 10 mM ammonium acetate solution |
| MALDI Laser Wavelength | 355 | nm | Nd:YAG laser (Smart beam IITM) |
| MALDI Laser Frequency | 2 | kHz | Laser operation frequency |
| MALDI m/z Range (Reflective) | 60-1600 | m/z | Positive and negative ionization modes |
| MALDI Fragmentation Mode | LIFT | N/A | Used for fragment spectra (50-1000 m/z range) |
| MALDI Matrix Concentration | 10 | mg/mL | DHB (2,5-dihydroxybenzoic acid) and HCCA (α-cyano-4-hydroxycinnamic acid) |
| Matrix Solvent Composition | 30% ACN, 70% H2O, 0.1% TFA | N/A | Standard solution for matrix preparation |
| Statistical Significance (p) | < 0.05 | N/A | Criterion for Mann-Whitney U test |
- Working Solution Preparation: Cyclitol and saccharide standards were prepared at 10 ”g/mL concentration in a neutral pH solution (10 mM ammonium acetate with 2 mL acetonitrile).
- Electrochemical Conversion (EC): The solutions were subjected to oxidation using the ROXYTMEC system. A BDD working electrode and a HyREF (Pd/H2) reference electrode were used, operating at 10 ”L/min flow rate and 37 °C.
- Fraction Collection: Two fractions were collected manually after EC conversion (Fraction 1: first 10 min; Fraction 2: next 7 min). Control samples were prepared identically but without EC treatment.
- Sample Processing: Collected fractions were evaporated to dryness (Centri Vap DNA concentrator), frozen, and subsequently re-dissolved in 50 ”L of methanol-water (1:1) mixture.
- MALDI Sample Preparation: 1 ”L of the processed sample was mixed with 1 ”L of MALDI matrix solution (DHB or HCCA, 10 mg/mL) and spotted onto an MTP AnchorChip 384 plate in triplicate.
- Mass Spectrometry Analysis: Spectra were acquired using MALDI-TOF-MS in both positive and negative reflective ionization modes (60-1600 m/z). Cesium triiodide-cluster (CsI3) was used for mass calibration.
- Data Analysis: Exploratory data analysis (Network Analysis, Hierarchical Cluster Analysis, Weighted Scatter Plots) was performed in R environment to visualize correlations and identify statistically significant changes in ion intensity after EC treatment.
- Drug Metabolism and Pharmacological Design: The methodology provides a rapid, non-enzymatic simulation of Phase I oxidative metabolism, crucial for identifying potential drug metabolites early in the development pipeline.
- Bioactive Compound Screening: Applicable for assessing the biological activity and toxicity of natural compounds (like cyclitols and sugars) by characterizing their transformation products, which may possess different bioactivities than the parent molecule.
- Advanced Analytical Chemistry: The use of BDD electrodes in EC systems offers a robust platform for simulating high-potential redox reactions, valuable for generating and studying reactive intermediates in complex organic mixtures.
- Metabolomics Research: Provides a high-throughput tool for identifying candidate LMWC metabolites, complementing traditional LC-MS approaches, particularly where chromatographic separation is challenging.
- Food Science and Nutrition: Used for characterizing the fragmentation and transformation pathways of carbohydrates and related compounds (sugars, inositols) in food extracts, aiding in quality control and nutritional science.
View Original Abstract
A combination of electrochemistry (EC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (off-line EC-MALDI-TOF-MS) was applied for determination of the studied biologically active compounds (D-glucose, D-fructose, D-galactose, D-pinitol, L-chiro-inositol, and myo-inositol) and their possible electrochemical metabolites. In this work, boron-doped diamond electrode (BDD) was used as a working electrode. MALDI-TOF-MS experiments were carried out (both in positive and negative ion modes and using two matrices) to identify the structures of electrochemical products. This was one of the first applications of the EC system for the generation of electrochemical products produced from saccharides and cyclitols. Moreover, exploratory data analysis approaches (correlation networks, hierarchical cluster analysis, weighted plots) were used in order to present differences/similarities between the obtained spectra, regarding the class of analyzed compounds, ionization modes, and used matrices. This work presents the investigation and comparison of fragmentation patterns of sugars, cyclitols, and their respective products generated through the electrochemistry (EC) process.
- 2019 - Simultaneous Determination of Cyclitols and Sugars Following a Comprehensive Investigation of 40 Plants [Crossref]
- 2017 - Sugars and health: A review of current evidence and future policy [Crossref]
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