Measurement of all-trans-retinol and 13-cis-retinol

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Golden standard

EN 12823-1:2000 Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 1: Measurement of all-trans-retinol and 13-cis-retinol

  • draft prEN 12823-1:2012

Summary

Method indicator

  • Name
  • Code

Scope

The European Standard EN 12823-1 describes a method for the determination of vitamin A from foodstuffs by high performance liquid chromatography (HPLC). This part 1 of the EN 12823 specifies a method for the measurement of all-trans-retinol and 13-cis-retinol.

Principle

Sample pretreatments include saponification and extraction of retinol. The measurement is carried out by HPLC using either fluorometric or ultra-violet detection.  

Key steps

Saponification and extraction

  • The samples should be homogenised prior to saponification and extraction.
  • Saponification is performed by adding methanol or ethanol, antioxidant and potassium hydroxide solution to the sample solution.
    • The solution is heated to 80-100 °C for 15-45 min or kept in room temperature overnight. 
  • Extraction is carried out with suitable solvent, and the extracts are washed with water.
  • The extract is evaporated and dissolved using mobile phase.   

HPLC

  • Liquid chromatographic system consisting of a pump, an injector, a UV-VIS (325 nm) or fluorescence detector (excitation wavelength 325 nm, emission wavelength 475 nm) and a data evaluation system.
  • Normal phase column: particle size 5 µm, diameter 4 mm, length 125-250 mm.
  • Mobile phase: n-hexane:n-butanol, 98:2 (v/v)
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of all-trans-retinol and 13-cis-retinol from interferences.


Identification and detection

  • All-trans-retinol and 13-cis-retinol are detected either fluorometrically (excitation wavelength 325 nm, emission wavelength 475 nm) or with a UV detector (325 nm).
  • Identification of all-trans-retinol and 13-cis-retinol 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 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.
    • Also internal standard method may be used if the similar behaviour of the internal standard and the analyte itself during the analysis has been tested.
  • The concentrations of all-trans-retinol and 13-cis-retinol in mg/100 g of the sample are calculated.




Remarks

  • After saponification, the extract can be used for the determination of beta-carotene using a method described in EN 12823-2.
  • The concentration of calibration solution must be checked spectrometrically.
  • The samples should be protected from excessive UV radiation, light, acidity and heat treatments.
    • Vitamin A is sensitive especially to UV radiation and oxidising agents.
    • The standard solutions must be stored at below 4 °C and protected from light.
  • When analysing samples with high starch content, treatment with taka-diastase prior to saponification may be needed.

Criteria for analytical performance and analytical quality control

Certified Reference Materials/Standard Reference Material

EuroFIR assistance to this method/guidelines