Vitamin C Detection in Food Practical Analysis Methods and Case Studies
Submit Your InquiryWhat is Vitamin C?
Vitamin C, chemically known as L-ascorbic acid, is a water-soluble vitamin essential for human health. Its molecular formula is C₆H₈O₆, with a molecular weight of 176.12 g/mol. Structurally, it resembles glucose, featuring a six-carbon skeleton with two adjacent enolic hydroxyl groups at positions 2 and 3, which confer its acidic properties (pKa ≈ 4.2). Unlike most mammals, humans cannot synthesize vitamin C endogenously due to the absence of the enzyme L-gulono-γ-lactone oxidase, necessitating dietary intake from sources like citrus fruits, leafy greens, and supplements.
Vitamin C plays critical roles in biological processes, including collagen synthesis, antioxidant defense, iron absorption, and immune modulation. Deficiency leads to scurvy, characterized by bleeding gums, joint pain, and impaired wound healing.
Properties of Vitamin C
Physical Properties
Appearance: White crystalline powder or needle-like crystals, odorless with a sour taste.
Solubility: Highly soluble in water (33 g/100 mL at 20°C), slightly soluble in ethanol, and insoluble in nonpolar solvents like ether or chloroform.
Thermal Stability: Melts at 190–192°C and decomposes at 553°C. Dry forms are stable, but aqueous solutions degrade rapidly under heat, light, or alkaline conditions.
Chemical Properties
Redox Activity: Vitamin C is a potent reducing agent, readily donating electrons to neutralize free radicals (e.g., reactive oxygen species) and regenerate other antioxidants like α-tocopherol. Its oxidation produces dehydroascorbic acid (reversible) and further degrades into 2,3-diketogulonic acid (irreversible), losing biological activity.
Acidity: The enolic hydroxyl groups dissociate protons, forming ascorbate anions. This acidity enables participation in enzymatic hydroxylation reactions (e.g., collagen formation) and metal ion chelation.
Light and pH Sensitivity: Degrades under UV light or in alkaline environments, necessitating storage in dark, acidic conditions (pH < 7) to preserve stability.
Detection Principles of Vitamin C
Vitamin C quantification relies on its redox properties and UV absorption characteristics. Common analytical methods include:
High-Performance Liquid Chromatography (HPLC)
Principle: Separation using a C18 reverse-phase column with a mobile phase of methanol and phosphate buffer (pH 2.5–2.8). Detection is performed via UV absorbance at 242–254 nm, where ascorbic acid exhibits strong absorption.
Sample Preparation: To prevent oxidation, samples are extracted with 2% metaphosphoric acid, which stabilizes vitamin C by precipitating proteins and inhibiting enzymatic degradation. Filtration and centrifugation remove interferents.
Advantages: High sensitivity (detection limit ~0.04 μg/mL), specificity, and suitability for complex matrices (e.g., foods, cosmetics, pharmaceuticals).
Spectrophotometric Methods
2,6-Dichlorophenolindophenol (DCPIP) Titration: Vitamin C reduces blue DCPIP to a colorless form. The endpoint is determined by colorimetry, proportional to ascorbic acid concentration.
Limitations: Susceptible to interference from reducing sugars, pigments, and other antioxidants, requiring careful sample cleanup.
Electrochemical Detection
Amperometric Sensors: Utilize vitamin C's electroactive properties. Oxidation at a working electrode generates a current proportional to concentration. Used in portable devices for rapid screening
Sample Preparation Methods for Vitamin C Analysis in Food
Various sample preparation techniques are employed depending on the food matrix, including homogenization, acidification, extraction, centrifugation, filtration, and the use of antioxidants.
Factors Influencing Sample Preparation
Several factors must be considered when preparing food samples for vitamin C analysis:
- Matrix Complexity: Different foods contain varying levels of proteins, fats, and sugars that can interfere with analysis.
- Oxidation Sensitivity: Vitamin C is highly susceptible to oxidation when exposed to air, light, or elevated temperatures.
- Enzymatic Activity: Ascorbate oxidase and peroxidase enzymes can degrade vitamin C, especially in fresh fruits and vegetables.
- pH Sensitivity: Maintaining an acidic environment prevents vitamin C degradation during sample handling.
Sample Homogenization and Size Reduction
Efficient homogenization is essential for accurate vitamin C analysis, particularly for solid or semi-solid samples. Common techniques include:
- Blending and Grinding: High-speed blenders or mortar and pestles ensure sample uniformity, especially for fruits, vegetables, and tablets.
- Cryogenic Grinding: Liquid nitrogen can be used to freeze samples before grinding, minimizing enzymatic degradation.
- Ultrasonication: Effective for breaking down fibrous materials, releasing vitamin C into the extraction solvent.
Prevention of Oxidation During Sample Preparation
Vitamin C is highly susceptible to oxidation during sample preparation. The following strategies are commonly used to prevent its degradation:
Acidification:
Maintaining a low pH (below 4) protects vitamin C from oxidative degradation. Suitable acids include:
- Metaphosphoric Acid (HPO₃): Provides both acidification and chelation, effectively preserving vitamin C.
- Oxalic Acid: Prevents oxidation and forms a stable complex with metal ions.
- Perchloric Acid (HClO₄): Commonly used for simultaneous deproteinization and antioxidant protection.
Antioxidant Addition:
Adding antioxidants during sample preparation can further protect vitamin C. Examples include:
- Ethylenediaminetetraacetic Acid (EDTA): Chelates metal ions that catalyze oxidation.
- Sodium Metabisulfite (Na₂S₂O₅): Acts as a reducing agent to prevent oxidation.
Use of Inert Atmosphere:
Processing samples under nitrogen or argon gas minimizes exposure to oxygen, reducing oxidative degradation.
Extraction Techniques for Vitamin C
Extraction is a critical step to isolate vitamin C from complex food matrices. The choice of extraction method depends on the sample type and intended analytical technique.
Liquid-Liquid Extraction (LLE)
- Suitable for aqueous food samples such as fruit juices, milk, and beverages.
- Acidified water or buffer solutions are commonly used as extraction solvents.
- Requires careful pH control to maintain vitamin C stability.
Solid-Liquid Extraction (SLE)
- Applied for solid food samples like vegetables, fruits, and vitamin supplements.
- Samples are often homogenized in acidic solutions (e.g., metaphosphoric acid).
- Ultrasonication can enhance the release of vitamin C from plant cell walls.
Solid-Phase Extraction (SPE)
- Used to concentrate and purify vitamin C from complex matrices.
- Effective for reducing interference from pigments, fats, and proteins.
- Often applied as a pre-treatment before chromatographic analysis.
Enzymatic Hydrolysis
For samples containing bound or oxidized vitamin C derivatives, enzymes like ascorbate oxidase can be used to convert these forms into detectable vitamin C.
Removal of Interferences
To ensure accurate quantification, interferences from proteins, lipids, and other biomolecules should be removed. Techniques include:
Protein Precipitation:
- Trichloroacetic acid (TCA) or perchloric acid is commonly used to precipitate proteins.
- Centrifugation at high speeds separates the clear supernatant containing vitamin C.
Fat Removal:
- For lipid-rich samples, organic solvents like hexane or chloroform can be used for defatting before extraction.
Filtration and Centrifugation:
- Samples are filtered through 0.45 µm or 0.22 µm membrane filters.
- Centrifugation (10,000–15,000 rpm for 10–20 minutes) further clarifies the sample.
Determination Methods of Vitamin C in Food and Pharmaceuticals
| Sample Type | Pre-treatment Method | Detection Method & Conditions | Linear Range | Detection Limit | Recovery (%) | RSD (%) |
|---|---|---|---|---|---|---|
| Beverages, vegetables, vitamin C tablets | Dissolve and dilute a certain amount of the sample, then measure | Spectrophotometry: Reducing property of vitamin C decolorizes methylene blue; Reagents: p-Aminobenzenesulfonic acid (sensitizer); pH = 2 buffer (HCl-KCl); Detection wavelength: 660 nm | 0.4~40 mg/L | 0.4 mg/L | N.A. | N.A. |
| Vitamin C tablets, effervescent tablets, multivitamin tablets | Ground and extracted with hydrochloric acid solution, then filtered and diluted | Spectrophotometry: Vitamin C reduces Cu(II) to Cu(I) forming a copper complex (pH = 9.5); Detection wavelength: 600 nm | 0.8~6 mmol | 0.26 mmol | 97~101 | 1.00~5.00 |
| Food, pharmaceuticals, biological samples | Dissolved or extracted with 0.2 mol/L oxalic acid solution, centrifuged with 5% EDTA, then diluted | Spectrophotometry: Vitamin C forms a blue compound with methyl violet in an alkaline solution; Detection wavelength: 600 nm | 0.1~1.0 μg/L | 0.1 μg/mL | 98.5~99.3 | 1.9 |
| Vitamin C tablets | Ground and extracted with water | Fluorescence quenching: Vitamin C forms a complex with methylene blue, reducing fluorescence intensity; Emission wavelength: 682 nm; Excitation wavelength: 664 nm | 3.0 × 10⁻⁷ ~ 6.0 × 10⁻⁶ mol/L | 2.5 × 10⁻⁷ mol/L | N.A. | N.A. |
| Aspirin | Ground and extracted with water | Electrochemical method (cyclic voltammetry): Graphite electrode (PEG); Molecularly imprinted film of polypyrrole (PPy); Anodic peak voltage: +0.15 V; Cathodic peak voltage: +0.00 V | 0.25~7.0 mmol/L | 7.4 × 10⁻⁵ mol/L | 93~97.1 | <1.62 |
| Fruit juice | Dissolved in 50 mmol/L phosphate buffer (PBS, pH = 6.5) | Electrochemical biosensor: GCE working electrode; Platinum wire impedance electrode; Saturated calomel electrode; Response voltage: 0.3 V; Frequency: 10 kHz~0.1 Hz | 4.0 × 10⁻⁵ ~ 3 × 10⁻³ mol/L | 13 μmol/L | N.A. | 0.43~0.91 |
| Vitamin tablets | Ground and extracted with water | Capillary electrophoresis–electrochemical detection: Carbon disk electrode; Injection: 20 KV for 10 s; Optimum working potential: 1.2 V | 1.0 × 10⁻⁵ ~ 1.0 × 10⁻² mol/L | 1.0 × 10⁻⁶ mol/L | 96 | <3.0 |
| Fruits and vegetables | Mashed, extracted with trichloroacetic acid, reacted with DL-cysteine | Capillary electrophoresis–electrochemical detection: Phosphate buffer (pH = 6.90); Injection conditions: 14 KV/(42~44) μA | N.A. | N.A. | N.A. | N.A. |
| Soft drinks | Diluted | Chemiluminescence (indirect): Vitamin C quenches Fe-chlorophyll-peroxide chemiluminescence system; Emission wavelength: 400~600 nm; Photomultiplier tube voltage: 700 V | 4.0 × 10⁻¹² ~ 4.0 × 10⁻⁴ mol/L | 4.0 × 10⁻¹² mol/L | N.A. | 3.8 |
| Hematinics, lemon juice, shampoo, etc. | Dissolved in water, filtered, and diluted | Flow Injection Analysis (FI): Vitamin C reduces Ti(III) forming fluorescent TiCl3²⁻; Excitation wavelength: 227 nm; Emission wavelength: 419 nm | 1 × 10⁻⁶ ~ 5 × 10⁻⁵ mol/L | 8 × 10⁻⁷ mol/L | 91~117 | 2.1 |
| Tablets, Food, Fruits or Beverages | Ground, homogenized, or diluted | AAS (Indirect Method): Vitamin C reduces excess Fe³⁺ to Fe²⁺, detected using flame atomic absorption spectrometry (FAAS) after ion exchange resin column adsorption and elution with HNO₃; Flow rate: 7 mL/min; Current: 6.0 mA; Wavelength: 248.3 nm | 0.1~50 mg/L | 0.06 mg/L | 96~106 | 1.6 |
| Juice, Tea Beverages, Cola | Direct determination, filtration and dilution if necessary | HPLC-UVD: Column: ODS or Mediterranea Sea 18; Mobile phase: 0.1% formic acid aqueous solution; Detection wavelength: 254 nm | 0.2~400 mg/L | 0.01 mg/L | N.A. | <2.0 |
| Rosehip | Liquid nitrogen freezing or low-temperature drying, extraction with 5% metaphosphoric acid solution, centrifugation | HPLC-UVD: Column: RP C18; Mobile phase: 0.5% NaH2PO4-acetonitrile = (93:7), pH = 2.25; Detection wavelength: 254 nm | 0.5~200 mg/L | 0.05 mg/L | 97~102 | 2.42~6.24 |
Case Study of Vitamin C Detection in Foods
Case Study 1: Determination of Vitamin C in Commercial Orange Juice by HPLC-UV[1]
Overview:
In this example, vitamin C (ascorbic acid) was quantified in commercially available orange juice using a rapid reverse-phase HPLC-UV method. The juice samples were first acidified with a 2% metaphosphoric acid solution to stabilize vitamin C and precipitate interfering proteins. After centrifugation and filtration (using a 0.45 µm filter), the clear extract was injected into an HPLC system equipped with a C18 column. Detection was performed at 254 nm, where ascorbic acid shows strong absorbance. Calibration curves constructed from standard solutions yielded a linear range that covered typical vitamin C concentrations in juice, and recovery studies confirmed the method's accuracy (recoveries between 94% and 101%).
Key Points:
- Sample Preparation: Acid extraction with metaphosphoric acid, centrifugation, and filtration.
- Chromatographic Conditions: Reversed-phase C18 column; UV detection at 254 nm; analysis time less than 4 minutes.
- Validation: Excellent linearity and recovery rates demonstrated that the method is both rapid and reliable for routine quality control.
Chromatograph of Standards
Chromatogram of Spiked Vitamin-C.10ul Spot of 1 mg/mL
Chromatogram of Standard Vitamin-C.10ul Spot of 1mg/mL
Case Study 2: Rapid Determination of Vitamin C in Soft Drinks Using an Electrochemical Biosensor[2]
Overview:
This case demonstrates the use of an electrochemical biosensor based on a screen-printed electrode for the determination of vitamin C in soft drink samples. The sensor operates on the principle of ascorbic acid oxidation at the electrode surface, which generates a current proportional to its concentration. Minimal sample preparation is required; soft drink samples are diluted with a supporting electrolyte and filtered to remove particulates. Cyclic or square wave voltammetry is then performed, and the oxidation peak is recorded. Calibration with standard vitamin C solutions yields a sensitive linear response with detection limits in the sub-µmol/L range. The method has been shown to provide rapid on-site analysis, making it ideal for quality control in beverage manufacturing.
Key Points:
- Sample Preparation: Simple dilution and filtration; no extensive pretreatment needed.
- Electrochemical Measurement: Oxidation of vitamin C is measured by voltammetry at a screen-printed electrode.
- Performance: High sensitivity, rapid response, and low detection limit make the method suitable for routine analysis in complex matrices like soft drinks.
References
- Shafqat Ullah, Arshad Hussain, Javid Ali, Khaliq Urrehman, & Asad Ullah. (2012). A simple and rapid HPLC method for analysis of Vitamin-C in local packed juices of Pakistan. Middle East Journal of Scientific Research, 12(8), 1085–1091. doi: 10.5829/idosi.mejsr.2012.12.8.6675
- Škugor Rončević, I., Skroza, D., Vrca, I., Kondža, A. M., & Vladislavić, N. (2022). Development and Optimization of Electrochemical Method for Determination of Vitamin C. Chemosensors, 10(7), 283. doi: 10.3390/chemosensors10070283
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