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

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.

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.

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.

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.

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.

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