Measurement of β-carotene

From Metrofood Wiki
Jump to: navigation, search

Golden standard


EN 12823-2:2000 Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurements of Beta-carotene


Summary


Method indicator


  • Name
  • Code


Scope


The European Standard EN 12823-2 specifies a method for the determination of total β-carotene in foodstuffs by high performance liquid chromatography (HPLC).


Principle


Sample pretreatments include saponification and extraction. Extraction is done similarly as when determining all-trans-retinol and 13-cis-retinol (EN 12823-1) and the extract obtained according to EN 12823-1 may be used also for quantification of β-carotene. Determination of the sum of β-carotene isomers is performed by HPLC with spectrometric detection.


Key steps

Extraction

  • The samples should be homogenised prior to saponification and extraction.
  • Saponification is performed by adding methanol or ethanol, water, antioxidant and potassium hydroxide solution to the sample solution.
    • The solution is heated to 80-100 °C for 15-40 min.
  • The ratio of alcohol to water should be 1:1 before extraction to avoid emulsions.
  • Extraction is carried out with suitable solvent, and the extracts are washed with water.
  • The extract is evaporated and dissolved using the solvent mixture, which is also used when preparing the standard solutions. 
    • The mobile phase should be used for dissolution if possible.

HPLC

  • Liquid chromatographic system consisting of a pump, an injector, a UV-VIS detector (450 nm)  and a data evaluation system.
  • Reverse phase column (e.g. C18), particle size 5 µm, diameter 4,0-4,6 mm, length 250 mm.
  • Mobile phase: acetonitrile + methanolic ammonium acetate solution + dichloromethane (75 + 20 + 5) with butylated hydroxytoluene and triethylamine.
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of all-trans-α-carotene and all-trans-β-carotene from interferences.



Identification and detection


  • β-carotene is detected with a UV-VIS detector using 450 nm as a wavelength. 
  • Identification of β-carotene 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 small amounts of the standard substances to the sample test solution.




Quantification and calculations


  • If quantification is done by external standard method, peak areas or heights obtained with sample solutions are compared to the corresponding values of the standard substance.
  • The linearity of the calibration should be checked.
  • The mass fraction of total β-carotene in mg/100 g of the sample is calculated.




Remarks


  • The purity of a-carotene and β-carotene standard substances can vary, and therefore the concentration of the stock solution should be checked spectrometrically.
  • The samples should be protected from excessive UV radiation, light, acidity and heat treatments.
    • β-carotene is sensitive especially to UV radiation and oxidising agents.
    • The standard solutions must be stored at below 4 °C and protected from light.

Criteria for analytical performance and analytical quality control


Certified Reference Materials/Standard Reference Material







EuroFIR assistance to this method/guidelines