manual of methods for determining micronutrients in fortified foods

4. Samples
• Low fat (less than 40% fat). Weigh sample (not more than 5 g) to give approximately 50 μg vitamin
A into 125-mL Erlenmeyer flask. For samples high in sugar, add 3 mL water and disperse sample as
slurry. Add 40 mL 95% ethanol.
• High-fat. Weigh sample (not more than 2 g) to give approximately 50 μg vitamin A into 125-mL
Erlenmeyer flask. Add 40 mL 95% ethanol.
• Wheat flour: Weigh 10 g sample, considering the vitamin A concentration is lower than 1.5 mg/kg.
Add 40 mL 95% ethanol.
5. Add a pea-sized piece (approximately 50 mg) of pyrogallic acid (antioxidant) to each standard and
sample flask. Add a glass bead to promote even boiling.
6. Swirl all flasks to ensure that all samples are thoroughly dispersed in the solution.
7. Turn on nitrogen flow and ensure a nitrogen atmosphere for all flasks while refluxing.
8. Pipet 10 mL 50% KOH solution into each flask and immediately place flask on hot plate under reflux
condenser. Swirl.
9. Reflux 45 min. Swirl flasks every 10 min.
10. Remove reflux flasks from hot plate, stopper with corks, and quickly cool flasks to room temperature,
using cold water or ice water.
11. Pipet 10 mL glacial acetic acid solution into each flask to neutralize the KOH. Mix well and let flasks
cold again to room temperature.
12. Quantitatively transfer solution in each flask to 100 mL volumetric flaks, using 50:50 THF:ethanol.
Dilute to volume with same.
13. Stopper and invert volumetric flasks 10 times.
14. Allow samples to set for at least 1 hour at room temperature and preferably overnight in refrigerator
to allow fatty acid salts formed during saponification to precipitate. In some cases, centrifugation may
be helpful to reduce settling time. The color of solution will be dark (see picture below) and a whitish
precipitate will form in samples at the bottom of the flask along with the solids. The higher the fat
content in the sample, the more precipitate will form.
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