A New Method for the Determination of Total Content of Vitamin C, Ascorbic and Dehydroascorbic Acid, in Food Products with the Voltammetric Technique with the Use of Tris(2-carboxyethyl)phosphine as a Reducing Reagent

At a Glance

Metadata	Details
Publication Date	2023-01-13
Journal	Molecules
Authors	Artur Mazurek, Marzena Włodarczyk-Stasiak
Institutions	University of Life Sciences in Lublin
Citations	13
Analysis	Full AI Review Included

Executive Summary

Core Value Proposition: Development and validation of a novel Differential Pulse Voltammetry (DPV) method using a mercury-film modified Boron-Doped Diamond Electrode (Hg-BDE) for accurate determination of total Vitamin C (Ascorbic Acid (AA) and Dehydroascorbic Acid (DHAA)) in diverse food products.
Chemical Derivatization: Tris(2-carboxyethyl)phosphine (TCEP) is utilized as the reducing agent for DHAA, enabling quantitative reduction in the standard acidic extraction environment (pH < 2) within 30 minutes.
Interference Mitigation: Interference caused by the TCEP reagent during DPV measurement is effectively eliminated by subsequent reaction with N-ethylmaleimide (NEM), requiring a 30-minute reaction time.
Electrode System: The use of a mercury film on the BDE surface minimizes matrix interference, allowing for reliable analysis across complex food matrices (juices, vegetables, powders).
Performance Metrics: The method exhibits excellent linearity (regression coefficient r = 0.9996) and low detection limits (LOD = 0.89 µg/mL).
Validation and Equivalence: Validation parameters (precision, accuracy, recovery) confirm the method provides correct results for AA and Total Vitamin C, demonstrating equivalence to the established HPLC-DAD reference method.
Limitation Noted: DHAA analysis remains imprecise due to reliance on the subtraction method (Total C minus AA_s), a limitation shared with the chromatographic reference method.

Technical Specifications

Parameter	Value	Unit	Context
Electrode Material	Boron-Doped Diamond (BDE)	3 mm diameter	Working Electrode
Electrode Modification	Mercury Film (Hg)	N/A	Applied electrochemically at -1.30 V for 180 s
Voltammetry Technique	Differential Pulse Voltammetry (DPV)	N/A	Quantitative assay method
Reducing Agent	Tris(2-carboxyethyl)phosphine (TCEP)	10 mM (effective)	Used for DHAA reduction
Interference Eliminator	N-ethylmaleimide (NEM)	40 mM (effective)	Used to eliminate TCEP interference
DHAA Reduction Time	30	min	Time required for quantitative reduction
TCEP Elimination Time	30	min	Time required for NEM reaction
Linear Regression Coefficient (r)	0.9996	N/A	Ascorbic Acid calibration curve
Limit of Detection (LOD)	0.89	µg/mL	Mean value for AA determination
Limit of Quantification (LOQ)	2.67	µg/mL	Calculated as 3 * LOD
DPV Initial Potential	-50	mV	vs. Ag/AgCl reference electrode
DPV Final Potential	200	mV	vs. Ag/AgCl reference electrode
DPV Pulse Amplitude	50	mV	N/A
Precision (CV Range)	1.61 to 8.22	%	For AA and Total Vitamin C content
Recovery Range (AA/Total C)	94.6 to 116.3	%	Conforming to AOAC requirements

Key Methodologies

Sample Preparation: Samples (food products) are weighed and extracted using a 2% metaphosphoric acid solution, homogenized (Ultra Turrax), centrifuged (12,000× g), and filtered (0.45 µm syringe filter).
Electrode Setup: A BDE (3 mm) is polished and modified with a fresh mercury film coat before each analysis to minimize matrix effects. A three-electrode system is used (Hg-BDE working, Platinum auxiliary, Ag/AgCl/KCl reference).
Ascorbic Acid (AA_s) Determination: The initial extract is analyzed directly using DPV in an acetate buffer (pH 4.6) after flushing with argon (5 min) to remove dissolved oxygen.
Total Vitamin C (Tc) Derivatization (Reduction): A portion of the extract is mixed with 100 mM TCEP solution (10 mM effective concentration) and shaken for 30 minutes to quantitatively convert DHAA to AA.
TCEP Quenching: Following reduction, 80 mM N-ethylmaleimide (NEM) solution (40 mM effective concentration) is added to the sample and shaken for 30 minutes to react with and eliminate the TCEP, preventing its interference during the subsequent DPV scan.
Total Vitamin C (Tc) Measurement: DPV analysis is performed on the quenched sample using the triple standard addition method for quantification.
DHAA Calculation: Dehydroascorbic acid content is calculated indirectly by subtracting the initial AA_s content from the measured total Vitamin C (Tc) content.

Commercial Applications

Food and Beverage Quality Control: Rapid, routine analysis of Vitamin C content in high-volume products like fruit juices (orange, grapefruit), multivitamin syrups, and processed foods, ensuring nutritional labeling accuracy.
Nutraceutical Manufacturing: Quality assurance testing for active ingredient concentration in powdered supplements and vitamin formulations, leveraging the method’s equivalence to HPLC standards.
Agricultural and Post-Harvest Monitoring: Quick assessment of Vitamin C degradation in fresh produce (e.g., kiwi, broccoli, parsley) during storage and transport, optimizing supply chain management.
Electrochemical Sensor Technology: The validated use of the Hg-BDE platform provides a robust foundation for developing field-deployable electrochemical sensors for other electrochemically active nutrients or contaminants in complex food matrices.
Analytical Laboratory Services: Offering a cost-effective and validated alternative to traditional chromatographic methods for laboratories requiring high-throughput analysis of Vitamin C without the high operational costs associated with HPLC.

View Original Abstract

The objective of the study was to develop a new method for the determination of the total content of vitamin C and dehydroascorbic acid in food, based on the technique of differential pulse voltammetry with the use of a boron-doped diamond electrode modified with mercury film. A comparison was made between the results obtained with the developed method and a proposed reference method based on high-performance liquid chromatography with spectrophotometric detection. The reduction of dehydroascorbic acid was performed with the use of tris(2-carboxyethyl)phosphine. The interference caused by the presence of tris(2-carboxyethyl)phosphine during the voltammetric determination of ascorbic acid was effectively eliminated through a reaction with N-ethylmaleimide. The conducted validation of the voltammetric method indicated that correct results of analysis of the total content of vitamin C and ascorbic acid were obtained. Analysis of the content of dehydroascorbic acid was imprecise due to the application of the differential method. The results of the analyses and the determined validation parameters of the developed method are characterised by a high degree of conformance with the results obtained with the chromatographic reference method, which indicates the equivalence of the two methods.

Tech Support

Original Source

DOI: https://doi.org/10.3390/molecules28020812

References

2005 - Biological Role of Vitamin C in Keratinocytes [Crossref]
2011 - The Genetics of Vitamin C Loss in Vertebrates [Crossref]
1994 - Healthfulness and Nutritional Quality of Fresh versus Processed Fruits and Vegetables: A Review [Crossref]
2012 - Changes of Dehydroascorbic Acid Content in Relation to Total Content of Vitamin C in Selected Fruits and Vegetables
1984 - Dehydroascorbic Acid Levels in Fresh Fruit and Vegetables in Relation to Total Vitamin C Activity [Crossref]
2000 - Non-Spectrophotometric Methods for the Determination of Vitamin C [Crossref]
2008 - HPLC Methods for Simultaneous Determination of Ascorbic and Dehydroascorbic Acids [Crossref]
2014 - Determination of Vitamin C in Foods: Current State of Method Validation [Crossref]