Vitamin C - Ascorbic acid

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Ascorbic acid.jpg
Figure 1. Ascorbic acid, C6H8O6, Mw = 176,12 g/mol.

L-ascorbic acid and its oxidation product L-dehydroascorbic acid show vitamin C activity. The D-isomer is often added to food as an antioxidant, but it is not a vitamin C active form.

Synonyms for vitamin C:

  • L-ascorbic acid, L-dehydroascorbic acid, L-threoascorbic acid, antiscorbutic factor

Summary



Golden standard

EN 14130:2003 Foodstuffs - Determination of vitamin C by HPLC


Method indicator

  • Name
  • Code

Scope

The European Standard EN 14130 specifies a method for the determination of vitamin C from foodstuffs by high performance liquid chromatography (HPLC). Vitamin C is expressed as the sum of L ( + )-ascorbic acid and dehydro-L ( + )-ascorbic acid.

Principle

Sample pretreatments include extraction with metaphosphoric acid solution and transformation of dehydro-L ( + )-ascorbic acid to L ( + )-ascorbic acid. The determination of total L(+)-ascorbic acid is performed by HPLC using UV-detection.

Key steps

Extraction

  • The sample is homogenised before extraction.
  • Metaphosphoric acid solution is added and sample solution is filtered.
  • NOTE:
    • Raw vegetables and fruits should be homogenised after addition of metaphosphoric acid.

Reduction

  • L-cysteine solution is added.
  • The pH of the sample is adjusted to 7,0-7,2.
  • The solution is stirred for 5 min, and then the pH is adjusted to 2,5-2,8
  • The sample solution is filtered.

HPLC

  • Liquid chromatographic system consisting of a pump, an injector, a UV detector (265 nm) and a data evaluation system. 
  • Reverse phase column: particle size 5 µm, diameter 4,0 mm, length 250 mm.
  • Mobile phase: potassium dihydrogen phosphate in water and N-acetyl-N,N,N-trimethylammonium bromide in methanol (9:1).
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of L-ascorbic acid and erythorbic acid from interferences.


Identification and detection


  • L-ascorbic acid is detected with UV-detector (265 nm)
  • Identification of L-ascorbic acid is done by the comparison of the retention time obtained with the standard test solution to that of the sample test solution.
    • Identification can also be done by adding the standard solution to the sample test solution.


Quantification and calculations

  • Quantification is done by external standard method by comparing the integrated peak areas or peak heights to the corresponding values of the standard substance. Also calibration curve may be used for quantification.
    • The linearity of the calibration must be checked.
  • The mass fraction of ascorbic acid in mg/100 g is calculated, and results are reported in milligrams of vitamin C per 100 g of the sample.


 



Remarks

  • This method can also be used for the determination of erythorbic acid.
  • All the solutions (sample and standard solutions) should be processed as soon as possible.
    • Solutions must be stored under 25 °C.
    • Analysis should be done within 8 hours.
  • Cysteine solution, dithiothreitol acid or diethylene triaminephosphoric acid may be added to metaphosphoric acid solution to increase the stability of the sample solution.
    • It should be considered that these stabilisers may influence the chromatography.
       

Criteria for analytical performance and analytical quality control

Certified Reference Materials/Standard Reference Material

Proficiency testing schemes 

Here are listed some completed, on-going and/or upcoming proficiency testing schemes concerning vitamin C:

LGC - Dairy Chemistry PT schemes (QDCS)

DLA - Dienstleistung Lebensmittel Analytik GbR: 33/2012 Food Supplement II: Biotin, Vitamin C, Niacin and Pantothenic Acid

Livsmedelsverket, National Food Agency of Sweden - Report 22 (2008) Vitamins in Foods - Round V-6

PTA - Proficiency Testing Australia: Report No. 651 Foor Proficiency Testing Program (Round 31) - Vitamins

Some upcoming proficiency testing schemes can be found in the EPTIS database.

Other methods available

  • AACC 86-10.01 Ascorbic Acid---Indophenol-Xylene Extraction Method
  • AOAC 967.22 Vitamin C (Total) in Vitamin Preparations
    • microfluorometric
  • AOAC 984.26 Vitamin C (Total) in Food
    • fluorometric, semiautomated
  • AOAC 985.33 Vitamin C (Reduced Ascorbic Acid) in Ready-To-Feed Milk-Based Infant Formula
    • titrimetric
  • ISO 6557-1:1986 Fruits, vegetables and derived products -- Determination of ascorbic acid -- Part 1: Reference method
  • ISO 6557-2:1984 Fruits, vegetables and derived products -- Determination of ascorbic acid content -- Part 2: Routine methods

 Literature

  •  see separate child page below

EuroFIR assistance to this method/guidelines