Electroanalysis of Apocynin Part 2 - Investigations on a Boron-Doped Diamond Electrode in Aqueous Buffered Solutions

At a Glance

Metadata	Details
Publication Date	2025-04-29
Journal	Materials
Authors	Agata Skorupa, Magdalena Jakubczyk, Sławomir Michałkiewicz
Institutions	Jan Kochanowski University
Analysis	Full AI Review Included

Executive Summary

This study details the development and validation of a highly sensitive voltammetric method for determining Apocynin (APO) using a Boron-Doped Diamond Electrode (BDDE) in an aqueous buffered environment.

Core Value Proposition: The method provides a simple, fast, and environmentally friendly (green chemistry aligned) alternative to complex chromatographic techniques for APO quantification in natural matrices.
Electrochemical Mechanism: The anodic oxidation of APO on BDDE is quasi-reversible and diffusion-controlled, involving a one-electron and one-proton transfer (EqC₁ mechanism) in Phosphate Buffer (pH 7.3).
Optimal Sensing Conditions: The analytical signal is the anodic peak observed at 0.605 V vs. Ag/AgCl, utilizing Differential Pulse Voltammetry (DPV) in PBS (pH 7.3) containing 5% (v/v) Ethanol.
High Sensitivity: The BDDE demonstrated superior performance compared to Glassy Carbon (GC) and Platinum (Pt) electrodes, achieving a low Limit of Detection (LOD) of 0.071 mg L^-1, comparable to high-performance liquid chromatography (HPLC) methods.
Robustness and Precision: The procedure exhibits excellent precision, with an intra-day Relative Standard Deviation (RSD) of 0.28% and high recovery rates (99.6-102.1%) in control assays.
Real-World Applicability: The method was successfully applied to determine APO content in a commercial dietary supplement (Kutki extract), confirming its selectivity against common interferents (e.g., ascorbic acid, BHA, inorganic ions).

Technical Specifications

Parameter	Value	Unit	Context
Working Electrode Material	Boron-Doped Diamond (BDDE)	3 mm diameter	Selected for resistance to fouling
Optimal Electrolyte	Phosphate Buffer Solution (PBS)	pH 7.3	Physiological pH environment
Optimal Organic Modifier	Ethanol (EtOH)	5% (v/v)	Required for APO solubility
Analytical Technique	Differential Pulse Voltammetry (DPV)	N/A	Used for high sensitivity determination
Anodic Peak Potential (E_p)	0.605	V vs. Ag/AgCl	Analytical signal
Linear Concentration Range	0.213-27.08	mg L^-1	Calibration range
Limit of Detection (LOD)	0.071	mg L^-1	Sensitivity metric (3.3S_b/a)
Limit of Quantification (LOQ)	0.213	mg L^-1	Quantification metric (3x LOD)
Intra-day Precision (RSD)	0.28	%	Repeatability (n=10)
Inter-day Precision (RSD)	1.51	%	Intermediate precision (n=5 days)
DPV Amplitude (dE)	30	mV	Optimized DPV parameter
DPV Pulse Width	60	ms	Optimized DPV parameter
CV Scan Rate Range	6.2 to 1000	mV s^-1	Used for mechanistic study
E_p vs. pH Slope	-0.0506	V pH^-1	Confirms 1 electron / 1 proton transfer
Optimal Extraction Time	6	hours	Ethanol extraction from plant material

Key Methodologies

The electrochemical analysis relies on rigorous BDDE preparation and optimized DPV parameters in a mixed aqueous/organic medium.

Electrode Activation and Cleaning:
- The BDDE surface was mechanically polished using a suspension of 0.01 µm alumina powder.
- The electrode was ultrasonically treated in distilled water and dried.
- Cathodic activation was performed at -2.4 V for 5 minutes in 1 mol L^-1 H₂SO₄ prior to daily measurements.
Electrolyte Preparation:
- Phosphate Buffer Solutions (PBS) were prepared by mixing 0.2 mol L^-1 solutions of disodium hydrogen phosphate (Na₂HPO₄) and potassium dihydrogen phosphate (KH₂PO₄).
- The optimal working pH was experimentally determined to be 7.3.
Analyte Solution Preparation:
- Apocynin (APO) solutions were prepared fresh daily.
- Due to low aqueous solubility, 5% (v/v) Ethanol (EtOH) was added to the PBS electrolyte, which was determined to maximize the oxidation peak current.
Voltammetric Measurement Protocol (DPV):
- Measurements were conducted using a three-electrode system (BDDE working, Ag/AgCl reference, Pt auxiliary) at a constant temperature of 25 ± 1 °C.
- Optimized DPV parameters were: amplitude (dE) 30 mV, potential step (E_s) 5 mV, and pulse width 60 ms.
- The potential range scanned was 0 to 1.3 V vs. Ag/AgCl.
Sample Extraction (Kutki Dietary Supplement):
- Plant material (1.5 g) was extracted using 10 mL of ethanol.
- Mechanical shaking was applied, and the optimal extraction time was determined to be 6 hours to ensure maximum APO recovery.

Commercial Applications

The use of BDDE in this highly sensitive and robust analytical method opens doors for commercial applications, particularly in quality control and rapid screening where traditional methods are too slow or costly.

Pharmaceutical and Nutraceutical Quality Control:
- Rapid, high-throughput determination of active phenolic compounds (like APO) and other antioxidants in raw materials, dietary supplements, and herbal extracts.
- Monitoring the stability and concentration of antioxidant ingredients in pharmaceutical formulations.
Clinical and Biomedical Diagnostics:
- Development of robust electrochemical sensors for monitoring APO and its metabolites in biological fluids (e.g., plasma, urine) due to BDDE’s resistance to biofouling and its wide potential window in aqueous media.
Environmental and Food Analysis:
- Screening for phenolic contaminants or beneficial antioxidants in complex food matrices and environmental samples.
- BDDE is ideal for these applications due to its chemical inertness and stability in harsh or mixed-solvent systems.
Green Analytical Chemistry:
- The method aligns with “green chemistry” principles, making it attractive for laboratories seeking to reduce organic solvent consumption, waste generation, and energy use compared to conventional HPLC or LC-MS/MS techniques.

View Original Abstract

In this study, the voltammetric behavior of apocynin on a boron-doped diamond electrode in a phosphate buffer (pH 7.3) has been reported for the first time. The oxidation process is quasi-reversible, diffusion-controlled, and involves one electron and one proton. The product of the electrode reaction is an unstable radical that undergoes successive chemical transformations near the working electrode. The proposed mechanism of this process can be described as EqCi and served as the basis for the development of a new voltammetric method for determining apocynin in natural samples. The analytical signal was the anodic peak on DPV curves at a potential of 0.605 V vs. Ag/AgCl. A linear response was observed in the concentration range of 0.213-27.08 mg L−1. The estimated LOD and LOQ values were 0.071 and 0.213 mg L−1, respectively. The effectiveness of the method was demonstrated both in control determinations and in the analysis of the dietary supplement. This procedure is simple, fast, sensitive, selective, and requires no complicated sample preparation, which is limited only to a simple extraction with ethanol. The low consumption of non-toxic reagents makes it environmentally friendly. To the best of our knowledge, this is the first presentation of a voltammetric procedure to determine this analyte studied in a phosphate buffer solution on a boron-doped diamond electrode. It can also be easily adapted to determine other phenolic compounds with antioxidant properties in various matrices.

Tech Support

Original Source

DOI: https://doi.org/10.3390/ma18092044

References

2018 - Novel anti-inflammatory film as a delivery system for the external medication with bioactive phytochemical “Apocynin” [Crossref]
2008 - Apocynin: Molecular aptitudes [Crossref]
2001 - Analysis of iridoid glycosides from Picrorhiza kurroa by capillary electrophoresis and high performance liquid chromatography-Mass spectrometry [Crossref]
2017 - Synthesis of apocynin dimer derivatives combining L-cysteine and alpha-lipoic acid
2019 - Plants with evidence-based therapeutic effects against neurodegenerative diseases [Crossref]
2019 - Redox- and bio-activity of apocynin (acetovanillone) in tobacco, a plant phenolic that alleviates symptoms of autoimmune diseases in animals [Crossref]
2017 - DFT study on the radical scavenging capacity of apocynin with different free radicals
2020 - Apocynin as an antidepressant agent: In vivo behavior and oxidative parameters modulation [Crossref]
2013 - Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor [Crossref]