manual of methods for determining micronutrients in fortified foods

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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. - 30 -
c. Clean-up and concentration procedure Note: The assembly shown in the picture below is helpful for processing the samples. 15. Condition the C18 cartridge with 10 mL ethanol, then pass 10 mL water. 16. Pass 10 mL of the sample extract through the C18 cartridge, controlling the flow in order to allow interaction between the extract and the resin. 17. Wash the cartridge with 5 mL washing solution (acetonitrile-20% in water). 18. Wash again the cartridge with 5 mL washing solution (acetonitrile-20% in water). The picture below shows the difference between the cartridge after the sample has been loaded (right) and after the cartridge has been washed to eliminate sample impurities other than vitamin A (left). 19. Elute vitamin A with 1.5 mL isopropanol. - 31 -
Page 1 and 2: MANUAL OF METHODS FOR DETERMINING M
Page 3 and 4: - 3 -
Page 5 and 6: I. Introduction Food fortification
Page 7 and 8: A. Qualitative method to determine
Page 9 and 10: VII. Interpretation • Unfortified
Page 11 and 12: − − − − − − Refrigerato
Page 13 and 14: 3. Stir with a magnetic stirrer for
Page 15 and 16: IX. Calculations 1. Plot a graph of
Page 17 and 18: − 1,10-phenanthroline-monohydrate
Page 19 and 20: . Preparation of the ash solution 1
Page 21 and 22: III. Methods to determine vitamin A
Page 23 and 24: IV. Equipment and materials − 50
Page 25 and 26: VIII. Qualitycontrol Run the flour
Page 27 and 28: − Volumetric flasks (100 and 10 m
Page 29: VII. Procedure a. Preparation of sa
Page 33 and 34: VIII. Calculations Results can be c
Page 35 and 36: IV. Methods to determine water-solu
Page 37 and 38: A. Method to determine riboflavin i
Page 39 and 40: VI. Standard solutions 1. Riboflavi
Page 41 and 42: 8. Pipette exactly 4.0 mL of the fi
Page 43 and 44: B. Determination of thiamin in flou
Page 45 and 46: In a 25-mL beaker weigh 0.9602 g so
Page 47 and 48: C. Determination of niacin in flour
Page 49 and 50: 7. Transfer the extract to a 250 mL
Page 51 and 52: IV. Equipment − Analytical balanc
Page 53 and 54: 6. Ammonium hydroxide-0.1M: In a 10
Page 55 and 56: VIII. Procedure A. Strain propagati
Page 57 and 58: Note: Prior to start analyzing samp
Page 59 and 60: IX. Calculations 1. Calculate the a
Page 61 and 62: B. Verification 1. In an Erlenmeyer
Page 63 and 64: V. References • Bowman BA and Rus

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