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Cholecalciferol D3.jpg Ergocalciferol D2.jpg                                                       
Figure 1. Cholecalciferol (vitamin D3) C27H44O, Mw = 384,64 g/mol    Figure 2. Ergocalciferol (vitamin D2) C28H44O, Mw = 396,65 g/mol

Different forms of vitamin D in foods include cholecalciferol (vitamin D3), which is the predominant form of vitamin D in foods, and ergocalciferol (vitamin D2).

Synonyms for vitamin D:

  • cholecalciferol, ergocalciferol, calciferol, activated 7-dehydrocholesterol, calciol


Golden standard

  • EN 12821:2009 Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2)

Method indicator

  • Name
  • Code


The European Standard EN 12821 describes a method for the determination of vitamin D in foodstuffs by high performance liquid chromatography (HPLC).

This method can be used for determination of either vitamin D3 or vitamin D2 but not for samples which contain both of these forms. This European Standard cannot be used for determination of 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D which are metabolites of vitamin D3 and vitamin D2.


Pretreatments include saponification and extraction of vitamin D3 and vitamin D2. Semi-preparative normal phase HPLC followed by analytical reverse phase HPLC is used for determination. Only one form of vitamin D (vitamin D3 or vitamin D2) is determined at a time and the other form is used as an internal standard. Detection is carried out by ultraviolet spectrometry.

Key steps


  • The sample must be homogenised prior to saponification.
  • Saponification is carried out by adding ethanol, water, an antioxidant and potassium hydroxide solution. 
  • The internal standard is added
    • When determining vitamin D3 content of the sample, vitamin D2 is used as an internal standard.
    • The amount of internal standard should be equal to the amount of determined form of vitamin D expected in the sample.
    • There should also be a sample without added internal standard to exclude a possibility of matrix interference.
  • Saponification includes heat treatment.
    • 70-100 °C, 20-45 min
    • Saponification can also be carried out at room temperature overnight.


  • The ratio of alcohol to water should be 1:1 before extraction to avoid emulsions.
  • Suitable solvent is added to extract vitamin D, and extracts are washed with water.
  • Solvent is evaporated and the sample is redissolved using solvent that is suitable for semi-preparative HPLC system.
  • After semi-preparative HPLC, the solvent is evaporated and redissolved using solvent that is suitable with analytical HPLC system.


  • Semi-preparative HPLC system
    • Liquid chromatographic system consisting of a pump, an injector, a UV detector (265 nm), a system for collecting a certain portion of the eluate, and a recorder or integrator.
    • Normal phase column: particle size 5 µm, diameter 4,0-8,0 mm, length 250-300 mm. 
    • Semi-preparative HPLC is used for separation of vitamin D from tocopherols and other interfering compounds.
    • The vitamin D fraction is collected via a band-cut.
      • Suitable time for band-cut is determined using vitamin D standard.
  • Analytical HPLC system
    • 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 4,0-4,6 mm, length 250 mm.
  • Alternative HPLC conditions with suitable mobile phases are described in the Standard.

Identification and detection

  • Vitamin D is detected using a UV detector (265 nm).
  • Identification of vitamin D3 and vitamin D2 is done by the comparison of the retention time obtained with the standard test solution to that of the sample test solution.
  • The sample extracts may be detected using different UV detector wavelengths to ensure the peak identity and the purity of the vitamin D peak.

Quantification and calculations

  • Standard solutions of vitamin D3 and vitamin D2 are used for calibration of semi-preparative HPLC system and analytical HPLC system.
  • Quantification is done by internal standard method.
    • The response factor of vitamin D3 to vitamin D2 is calculated using the standards with known concentration.
  • The mass fraction of vitamin D3 in µg/100 g of the sample is calculated taking the response factor into account.



  • Vitamin D is sensitive to UV radiation and to oxidizing agents.
    • Exposure to light should be minimised.
    • Antioxidants should be added to the sample solutions.
  • Alternative HPLC conditions are described.
  • Old data
    • biological assay
    • colorimetry
    • GC
    • HPLC
    • radio-immunoassay
    • Older methods give unreliable data.
  • Review
    • Blake CJ. 2007. Status of methodology for the determination of fat-soluble vitamins in foods, dietary supplements, and vitamin premixes. JAOAC International, 90:897-910

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 D:

DEQAS - Vitamin D External Quality Assessment Scheme

PTA - Proficiency Testing Australia: Report No. 651, Food Proficiency Testing Program, Round 31 - Vitamins

DLA - Dienstleistung Lebensmittel Analytik GbR: 32/2012 Food Supplement I: Vitamins A, D3, E, K1 and beta-Carotin

http://www.lgcpt.com/PTCatalogue/en-GB/PTCatalogue.pdfLGC - Dairy Chemistry PT schemes (QDCS)

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

Other methods available

  • AOAC 936.14 Vitamin D in milk, vitamin preparations, and feed concentrates
    • rat bioassay
  • AOAC 932.16 Vitamin D in poultry feed supplements
    • chick bioassay
  • AOAC 975.42 Vitamin D in vitamin preparations
    • colorimetric
  • AOAC 979.24 Vitamin D in vitamin preparations
    • liquid chromatographic
  • AOAC 980.26 Vitamin D multivitamin preparations
    • liquid chromatographic
  • AOAC 981.17 Vitamin D in fortified milk and milk powder
    • liquid chromatographic
  • AOAC 982.29 Vitamin D in mixed feeds, premixes, and per foods
    • liquid chromatographic
  • AOAC 985.27 Vitamin D in vitamin AD concentrates
    • liquid chromatographic
  • AOAC 992.26 Vitamin D3 (cholecalciferol) in ready-to-feed milk-based infant formula (Codex-AOAC method)
    • liquid chromatographic
  • AOAC 995.05 Vitamin D in infant formula and enteral products
    • liquid chromatographic
  • AOAC 2002.05 Determination of cholecalciferol (vitamin D3) in selected foods (Joint AOAC-NMKL method)
    • liquid chromatographic
  • ISO 14892:2002 Dried skimmed milk -- Determination of vitamin D content using high-performance liquid chromatography


  •  see separate child page below

EuroFIR assistance to this method/guidelines