🍎 Vitamin C — HPLC

Food Chemistry PN-EN 14130

Overview

STATUS (as of 03.11.2026). PN-EN 14130:2004 — Polish version (published on 17.12.2004), implements EN 14130:2003 [IDT] and replaced the English version PN-EN 14130:2003. The standard was WITHDRAWN on 21.11.2010 and the PKN catalogue card names no replacing standard; CEN withdrew EN 14130:2003 on 05.08.2010, also without a successor. The description gives the content of the withdrawn standard, with its clause numbering.

SCOPE AND PRINCIPLE (clauses 1 and 3). The standard describes an HPLC method for the determination of vitamin C in foodstuffs; vitamin C is the sum of L(+)-ascorbic acid and dehydro-L(+)-ascorbic acid. Vitamin C is extracted from the sample with a metaphosphoric acid solution, a reducing solution converts dehydroascorbic acid into L(+)-ascorbic acid, and the total L(+)-ascorbic acid content is determined by HPLC with UV detection at 265 nm.

SAMPLE PREPARATION AND REDUCTION (6.1–6.3). Standard and sample solutions are processed as soon as possible, kept below 25 °C and not analysed after 8 h. The sample is homogenised (coarse material is ground) and analysed immediately after homogenisation. A test portion, e.g. 3 g for a vitamin C content of about 50 mg/100 g, is placed in a 100 ml volumetric flask with 80 ml of metaphosphoric acid solution 20 g/l, made up to the mark with that solution and filtered; raw vegetables and fruit (e.g. 2–10 g) are comminuted directly in a beaker containing the metaphosphoric acid solution. 10 ml of L-cysteine solution 40 g/l are added to 20 ml of the extract, the pH is adjusted to 7.0–7.2 with trisodium phosphate solution and the mixture is stirred for exactly 5 min; the pH is then lowered to 2.5–2.8 with metaphosphoric acid solution 200 g/l, the solution is transferred to a 50 ml flask, made up with water and filtered through a membrane filter. If the sample contains thickeners or solidifying agents, 1 ml of methanol is added to 4 ml of the reduced solution, which is filtered again. Stabilisers of the ascorbic acid solution (e.g. dithiothreitol) can influence the chromatography and were not used in the interlaboratory test.

CHROMATOGRAPHIC CONDITIONS (4.2.11, 5.4, 6.4). The interlaboratory test used an endcapped RP-18 reversed-phase column (5 µm, 250 mm × 4.0 mm), a mobile phase of 900 ml of a solution of 13.6 g of potassium dihydrogen phosphate in water and 100 ml of a solution of 1.82 g of N-cetyl-N,N,N-trimethylammonium bromide in methanol, a flow rate of 0.7 ml/min, an injection volume of 30 µl and detection at 265 nm. The performance criterion for the column is baseline resolution of L-ascorbic acid and erythorbic (isoascorbic) acid; the same procedure can quantify erythorbic acid, which is not counted as vitamin C. The standard also gives other suitable conditions: a mobile phase of 10 % acetonitrile and 90 % water containing 13.6 g/l of potassium dihydrogen phosphate and 2 g/l of cetrimide, a C8 type column (10 µm, 250 mm × 4.6 mm) and a flow rate of (1.0 ± 0.1) ml/min.

CALCULATION (6.5, clause 7). Appropriate volumes of the standard and sample solutions (not more than 50 µl) are injected, peak areas or peak heights are integrated and compared with the standard or with a calibration graph whose linearity is checked. The result is given as the mass fraction of ascorbic acid in mg/100 g of sample.

REGULATIONS. Under Regulation (EU) No 1169/2011, the mandatory nutrition declaration may be supplemented with the vitamins listed in Annex XIII Part A point 1 that are present in significant amounts (Article 30(2)(f)); the reference intake of vitamin C for adults is 80 mg.

WHAT WE DO NOT GIVE HERE. The precision of the method (clause 8 and Annex B) and the example chromatogram (Annex A) — they are outside the available text of the standard. Measuring range, limit of detection and analysis time — the available text of the standard does not give them.

Method principle

Vitamin C is extracted from the sample with a metaphosphoric acid solution. Dehydro-L(+)-ascorbic acid is reduced to L(+)-ascorbic acid with an L-cysteine solution in a neutral medium, after which the solution is acidified again with metaphosphoric acid. The reduction is carried out at pH 7.0–7.2 for 5 min and the solution is acidified to pH 2.5–2.8. The total L(+)-ascorbic acid content is determined by HPLC on a reversed-phase column with UV detection at 265 nm and external calibration with ascorbic acid solutions of 5–50 µg/ml.

Applications

• Determination of vitamin C (sum of ascorbic and dehydroascorbic acid) in foodstuffs (clause 1)
• Raw vegetables and fruit — comminution directly in the metaphosphoric acid solution (6.2, 6.3.1)
• Products with thickeners or solidifying agents — precipitation with methanol before chromatography (6.3.2)
• Determination of erythorbic (isoascorbic) acid by the same procedure, without counting it as vitamin C (6.4)
• Nutrition declaration — vitamin C as a supplementary component (Regulation (EU) No 1169/2011, Article 30(2))

Key parameters

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

1. Sample preparation

   Homogenise the sample, grind coarse material and mix again; analyse immediately after homogenisation. Keep sample and standard solutions below 25 °C and do not analyse them after 8 h (6.1, 6.2).

2. Extraction

   Weigh to the nearest 1 mg an appropriate amount of sample (e.g. 3 g at about 50 mg of vitamin C per 100 g) into a 100 ml flask, add 80 ml of metaphosphoric acid 20 g/l, shake, make up to the mark with this solution, shake and filter. Comminute raw vegetables and fruit (e.g. 2–10 g) directly in a beaker with metaphosphoric acid and transfer quantitatively into a 100 ml flask (6.3.1).

3. Reduction

   Add 10 ml of L-cysteine solution 40 g/l to 20 ml of the extract, adjust the pH to 7.0–7.2 with trisodium phosphate solution while stirring and stir for exactly 5 min. Lower the pH to 2.5–2.8 with metaphosphoric acid solution 200 g/l, transfer quantitatively to a 50 ml flask, make up with water and filter through a membrane filter (6.3.2).

4. Samples with thickeners

   If the sample contains thickeners or solidifying agents, add 1 ml of methanol to 4 ml of the reduced solution and filter through a membrane filter (6.3.2).

5. Chromatography

   Identify L-ascorbic acid by its retention time against the standard or by adding the standard to the sample solution. Inject appropriate volumes of standard and sample (not more than 50 µl); in the interlaboratory test: RP-18 column, 0.7 ml/min, 30 µl, detection at 265 nm (6.4, 6.5).

6. Calculation

   Integrate peak areas or heights and compare with the standard or with the calibration graph (check linearity); report the result as mg of ascorbic acid per 100 g of sample (6.5, clause 7).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard warns that its use can involve hazardous materials, operations and equipment and that it does not address all safety problems — health and safety practices and regulatory limitations are for the user to establish (foreword)
• Methanol — in the classification notified to ECHA: highly flammable (H225), toxic if swallowed, in contact with skin and if inhaled (H301, H311, H331), causes damage to organs (H370)

Frequently asked questions