🍎 Vitamin C — HPLC
In short
Determination of ascorbic acid (vitamin C) in food by high-performance liquid chromatography (HPLC) with UV detection. The test is performed according to PN-EN 14130:2004; Measurement range: 1–1000 mg/100 g. The procedure comprises 6 steps (about 1 h 25 min in total); it is used for: Food labeling control — vitamin C declaration (EU Reg. 1169/2011), Analysis of fruit and vegetable juices, Analysis of fortified foods and dietary supplements.
At a glance
- Standard: PN-EN 14130:2004
- Category: Food Chemistry
- Procedure steps: 6
- Total time of stages: 1 h 25 min
- Measurement range: 1–1000 mg/100 g
- Limit of detection: 0.5 mg/100 g
- Detection wavelength: 245 nm
Overview
Vitamin C (L-ascorbic acid) is an essential nutrient, antioxidant, and one of the most frequently declared components on food labels. Method PN-EN 14130 is the reference chromatographic method (HPLC-UV) designed for the determination of total ascorbic acid and dehydroascorbic acid (after DTT reduction) in food products.
The method involves extraction of vitamin C from the sample with metaphosphoric acid (stabilizer), reduction of the oxidized form (DHAA) with dithiothreitol (DTT), separation on a C18 column with buffered mobile phase, and UV detection at 245 nm. Measurement range: 1–1000 mg/100 g.
Method principle
The sample is homogenized with metaphosphoric acid (HPO₃) solution to prevent oxidation of vitamin C. After centrifugation, the supernatant is treated with DTT (dithiothreitol) to reduce DHAA to ascorbic acid. The solution is filtered (0.22 µm) and analyzed by HPLC on a C18 column with mobile phase: 50 mmol/L KH₂PO₄, pH 2.5. UV detection at 245 nm. Quantitative determination using a calibration curve (L-ascorbic acid, CRM).
Applications
- Food labeling control — vitamin C declaration (EU Reg. 1169/2011)
- Analysis of fruit and vegetable juices
- Analysis of fortified foods and dietary supplements
- Quality control of infant food
- Stability testing — vitamin C degradation over time
- Analysis of fresh and frozen fruits and vegetables
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 1–1000 mg/100 g |
| Limit of detection | 0.5 mg/100 g |
| Detection wavelength | 245 nm |
| Precision (CV) | 2–5% |
| Analysis time | 10–15 min/sample |
Standard
- Standard number
- PN-EN 14130:2004
- Title (PL)
- Artykuły żywnościowe — Oznaczanie witaminy C metodą HPLC
- Title (EN)
- Foodstuffs — Determination of vitamin C by HPLC
Step-by-step procedure
-
Extraction
Weigh 5–10 g sample. Add 50 mL 3% HPO₃. Homogenize 2 min. Centrifuge 10 000 g / 10 min.
-
DHAA reduction
Add 1 mL DTT (40 mg/mL) to 5 mL supernatant. Incubate 30 min in the dark at room temperature.
-
Filtration
Filter through 0.22 µm PVDF filter into HPLC vial.
-
Calibration
Prepare ascorbic acid standards: 5, 10, 25, 50, 100 mg/L in 3% HPO₃.
-
HPLC analysis
C18 column, mobile phase: 50 mM KH₂PO₄ pH 2.5 + 2% MeOH, flow 1 mL/min, detection 245 nm.
-
Calculations
Read concentration from calibration curve. Convert to mg/100 g sample. Account for dilution.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with UV/DAD detector | Agilent 1260 Infinity II, Shimadzu Nexera, Waters Alliance | 200 000–400 000 PLN |
| C18 column | Waters Atlantis T3 150×4.6 mm, 5 µm | 2 000–4 000 PLN |
| Autosampler | Integrated in HPLC system | integrated |
| Laboratory centrifuge | Eppendorf 5804R, Hettich Rotina 380R | 10 000–30 000 PLN |
| Homogenizer | IKA Ultra-Turrax T25, Kinematica PT 3100 | 5 000–15 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| L-Ascorbic acid (standard) | 50-81-7 | Purity ≥99%, certified CRM for calibration |
| Metaphosphoric acid HPO₃ | 37267-86-0 | 3–5% solution, vitamin C stabilizer, prevents oxidation |
| Dithiothreitol (DTT) | 3483-12-3 | Reductant DHAA → ascorbic acid, 40 mg/mL solution |
| Potassium dihydrogen phosphate KH₂PO₄ | 7778-77-0 | Mobile phase: 50 mmol/L, pH 2.5 (acidified with H₃PO₄) |
| Methanol HPLC grade | 67-56-1 | Mobile phase additive (2–5%) |
Health and safety (OHS)
- Metaphosphoric acid — irritant, gloves and safety glasses required
- Methanol — toxic, flammable. Work in fume hood.
- DTT — irritant, unpleasant odor. Work under extraction.
- HPLC waste — collect separately (organic waste)
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 14130:2004 — “Foodstuffs — Determination of vitamin C by HPLC”.
How does this method work?
The sample is homogenized with metaphosphoric acid (HPO₃) solution to prevent oxidation of vitamin C. After centrifugation, the supernatant is treated with DTT (dithiothreitol) to reduce DHAA to ascorbic acid.
What is the measuring range and accuracy?
Measurement range: 1–1000 mg/100 g; Limit of detection: 0.5 mg/100 g; Detection wavelength: 245 nm; Precision (CV): 2–5%.
How long does the test take?
The times given for the individual stages add up to approximately 1 h 25 min. The procedure comprises 6 steps.
What equipment is required?
HPLC system with UV/DAD detector, C18 column, Autosampler, Laboratory centrifuge, Homogenizer. Indicative cost of the core instrument (HPLC system with UV/DAD detector): 200 000–400 000 PLN.
Where is this test used?
Food labeling control — vitamin C declaration (EU Reg. 1169/2011); Analysis of fruit and vegetable juices; Analysis of fortified foods and dietary supplements; Quality control of infant food; Stability testing — vitamin C degradation over time; Analysis of fresh and frozen fruits and vegetables.
What safety precautions apply?
Metaphosphoric acid — irritant, gloves and safety glasses required; Methanol — toxic, flammable. Work in fume hood.; DTT — irritant, unpleasant odor. Work under extraction.; HPLC waste — collect separately (organic waste).
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN 14130.