HPLC with Electrochemical Detection Systems for Quantitative Analysis of Functional Components in Foods
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2021-07-05 |
| Journal | BUNSEKI KAGAKU |
| Authors | A. Kotani, Yuji Miyaguchi, Naoto Miyashita, Fumiyo Kusu, Kiyoko Takamura |
| Institutions | Tokyo University of Pharmacy and Life Sciences |
| Citations | 1 |
| Analysis | Full AI Review Included |
Executive Summary
Section titled âExecutive SummaryâThis research details the development and application of specialized High-Performance Liquid Chromatography with Electrochemical Detection (HPLC-ECD) systems for quantifying functional components in complex food matrices.
- High Sensitivity: Achieved ultra-high sensitivity for catechin analysis in tea, demonstrating a 40,000-fold improvement in detection limits compared to conventional HPLC-UV methods.
- Advanced Electrode Technology: Successfully implemented Boron-Doped Diamond (BDD) electrodes in non-aqueous media (acetonitrile) for the highly sensitive and selective detection of cholesterol in meat, yielding a Signal-to-Background (S/B) ratio improvement of approximately 10 times over glassy carbon electrodes.
- Complex Matrix Handling: Developed a Column-Switching HPLC-ECD (2LC-ECD) system for isoflavones (glycosides and aglycones) in soymilk, ensuring stable electrochemical detection under optimized isocratic conditions.
- Derivatization-Free Acid Sensing: Introduced a novel electrochemical method utilizing quinone mediators (e.g., VK3, DBBQ) to detect electro-inactive acidic compounds (organic acids, short-chain fatty acids, free fatty acids) by monitoring the positive shift in the quinone reduction potential.
- Broad Applicability: The developed HPLC-ECD methods were successfully applied to quantify components across diverse samples, including tea, soymilk, meat, wine, fermented foods (cheese, kusaya), and plant oils.
- Simplified Sample Prep: A core advantage across all methods is the simplified sample preparation, often requiring only dilution or simple extraction/centrifugation, enhancing the systemâs practical utility and versatility.
Technical Specifications
Section titled âTechnical Specificationsâ| Parameter | Value | Unit | Context |
|---|---|---|---|
| Catechin LOD (S/N=3) | 5 | fmol | HPLC-ECD using Glassy Carbon (GC) electrode |
| Catechin Sensitivity Gain | ~40,000 | times | Compared to standard HPLC-UV |
| Cholesterol Working Electrode | BDD | - | Used for high anodic potential detection |
| Cholesterol Applied Potential | +2.2 | V vs. Ag/AgCl | BDD electrode in acetonitrile mobile phase |
| Cholesterol LOD (S/N=3) | 8 | nmol L-1 | BDD HPLC-ECD system |
| Cholesterol S/B Improvement | ~10 | times | BDD vs. GC electrode |
| Organic Acid Applied Potential | -0.7 | V vs. SCE | Quinone reduction method (VK3 mediator) |
| Short-Chain Fatty Acid RSD | <3.4 | % | Analysis of fermented foods (n=5) |
| Free Fatty Acid Recovery | 91-109 | % | Analysis of various plant oils (n=5) |
| Isoflavone Separation Method | 2LC-ECD | - | Column switching (isocratic separation) |
| BDD Mobile Phase (Cholesterol) | 10 | mmol L-1 | LiClO4 in Acetonitrile |
| Organic Acid Column Type | RSpak KC-811 | - | Ion exclusion chromatography (300 x 8.0 mm i.d.) |
Key Methodologies
Section titled âKey MethodologiesâThe following methodologies were developed or optimized using HPLC-ECD for specific functional components:
-
Catechin Quantification (Tea Beverages):
- Electrode: Glassy Carbon (GC) working electrode.
- Detection Mode: Amperometric oxidation of the catechol moiety (B-ring).
- Applied Potential: +0.6 V vs. Ag/AgCl.
- Selectivity: High selectivity achieved by setting the potential below the oxidation potential of methylxanthines (caffeine).
-
Cholesterol Quantification (Meat Homogenates):
- Electrode: Boron-Doped Diamond (BDD) working electrode.
- Solvent System: Non-aqueous mobile phase (Acetonitrile containing 10 mmol L-1 LiClO4).
- Applied Potential: High anodic potential (+2.2 V vs. Ag/AgCl) to oxidize the 3-hydroxy group of cholesterol.
- Sample Preparation: Simple saponification followed by hexane extraction, minimizing matrix interference.
-
Isoflavone Quantification (Soymilk) using 2LC-ECD:
- System: Column-switching system (2LC-ECD) with one pre-column (C0) and two analytical columns (C1, C2).
- Separation Strategy: Hydrophilic components (glycosides) eluted first using MP1 (low methanol), followed by switching the valve to elute hydrophobic components (aglycones) using MP2 (high methanol).
- Detection: Two separate amperometric detectors (D1, D2) operating under stable isocratic conditions to maintain baseline stability and high sensitivity.
-
Detection of Electro-Inactive Acidic Compounds (Organic/Fatty Acids):
- Principle: Voltammetric acid sensing based on the reduction of a quinone mediator (e.g., Vitamin K3, DBBQ).
- Setup: Post-column addition of the quinone solution (QS) to the column eluent.
- Detection Mode: Monitoring the reduction current at a negative potential (e.g., -0.7 V vs. SCE).
- Mechanism: Acidic protons (HA) enhance the electrochemical reduction of the quinone (Q), causing a positive shift in the reduction potential (pre-wave), the height of which is proportional to the acid concentration.
Commercial Applications
Section titled âCommercial ApplicationsâThe developed HPLC-ECD systems provide robust analytical solutions for quality control and nutritional profiling in the food and beverage industry.
| Application Area | Target Analytes | Specific Products/Context | Analytical Value Proposition |
|---|---|---|---|
| Beverage Quality Control | Catechins, Polyphenols | Green tea, Oolong tea, Functional beverages | High-sensitivity quantification of antioxidants; selective measurement over caffeine. |
| Fermentation Monitoring | Organic Acids (Lactic, Tartaric, Acetic) | Wine, Yogurt, Vinegar, Fruit Juices | Tracking fermentation kinetics and ensuring product consistency and flavor profile. |
| Lipid Quality Assessment | Free Fatty Acids (FFAs) | Plant oils (Olive, Corn, Rapeseed), Cooking oils | Rapid determination of oil degradation (rancidity index) without complex derivatization steps. |
| Nutritional Supplement Analysis | Polyunsaturated Fatty Acids (EPA, DHA) | Fish oil capsules, Omega supplements | Accurate quantification of essential fatty acids in complex oil matrices. |
| Functional Food Profiling | Isoflavones (Glycones/Aglycones) | Soymilk, Tofu, Soy-based products | Efficient separation and quantification of key phytoestrogens influencing health claims. |
| Meat and Dairy Analysis | Cholesterol | Beef, Pork, Lamb, Dairy products | Accurate determination of cholesterol content for nutritional labeling and quality control using BDD-ECD. |
| Specialty Food Characterization | Short-Chain Fatty Acids (SCFAs) | Fermented foods (Cheese, Kusaya) | Profiling volatile acids critical for flavor, aroma, and preservation characteristics. |
View Original Abstract
In the present review, various high-performance liquid chromatography with electrochemical detection (HPLC-ECD) systems are proposed for the quantitative analysis of functional components in foods. Determinations of catehines in tea were performed by an HPLC-ECD system using a glassy carbon working electrode. The distribution analysis of rutin, catechin, epicatechin, and epicatechin gallatein in buckwheat seed was achieved by HPLC-ECD with high sensitivity. Column-switching HPLC-ECD, which consists of one pre-column, two separation columns, and two amperometric detectors, called 2LC-ECD, was developed for determining isoflavones in soymilk. The determination of cholesterol, which was difficult to detect electrochemically in aqueous media, was performed by an HPLC-ECD system using a boron-doped diamond (BDD) working electrode and acetonitrile media; the present method was applied to analyze meat samples, such as pork loin, ground beef, and shoulder of mutton. Moreover, voltammetric acid sensing based on the reduction of quinone is provided for determining electro-inactive acidic compounds, such as fatty acids. The HPLC-ECD systems by means of the present sensing method were applied to determine organic acids in wine and yogurt, short-chain fatty acids (SCFAs) in fermented foods, free fatty acids (FFAs) in plant oils, and polyunsaturated fatty acids (PUFAs) in fish oil. The HPLC-ECD system for determining organic acids was applied to monitor a changing in organic acid concentrations in grape and milk during fermentation with wine yeast and yogurt, respectively. In conclusion, HPLC-ECD systems are shown as a powerful analytical strategy for the quantitative analysis of functional components in foods using simple sample preparations.