manual of methods for determining micronutrients in fortified foods

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B. Determination of vitamin A in foods by high-performance liquid chromatography I. References Horwitz W and GW Latimer (Eds.). AOAC Official Method 2001.13.Vitamin A (Retinol) in Foods.Vitamins and Other Nutrients.Chapter 45, p.53-56.Official Methods of Analysis of AOAC International. 18 th ed, Revision 2, 2007. Maryland. II. Principle Standards and samples are saponified in basic ethanol-water solution, neutralized, and diluted. This process converts fats to fatty acids, and retinyl esters to retinol and the corresponding fatty acids. Extract clean-up is carried out with a C18 cartridge and vitamin A is concentrated eluting with a smaller volume of isopropanol than the aliquot taken to clean. Retinol is quantified in an LC system, using UV detection at 326 nm. Concentration is calculated by comparison of peak heights or peak areas of retinol in test samples with those of standards. Recovery of vitamin A in Infant Formula 1849 (NIST) was 99% as measured in the CPHL Laboratory. III. Critical points and cautions Due to the labile nature of retinol, it is important to saponify the samples under a nitrogen atmosphere and in the presence of pyrogallic acid. Potassium hydroxide is extremely caustic and it can cause severe burns. Protect skin and eyes while performing this method. This method involves the use of flammable liquids. Perform behind a barrier when using hot water, steam or an electric heating mantle. Use an effective fume removal device to remove flammable vapors produced. Leave ample headroom in flask. Protect samples from light by covering the glassware containing the sample extracts with aluminum foil or a piece of black cloth, and work under subdue light. IV. Equipment and materials − − − − − HPLC system • Pump operating continuously at 1.0-2.0 mL/min with a flow precision of ± 1% or better • Injector. A manual injector or autosampling injector with a 100 μL fixed loop having a typical sampling precision of ±0.25% or better • Reverse-phase C18 column, 5 μm (4.6x250 mm) capable of separating cis and trans isomers of retinol with a resolution of 1.0 or greater. • Photometric detector monitoring absorbance at 326 nm. • Data collection system or integrator Sep-pak Cartridges C18 Vac 3cc (500 mg). Waters.or equivalent. Erlenmeyer flasks (125 mL) with neck adapted for connecting reflux condenser Hot plate Reflux condensers - 26 -
− Volumetric flasks (100 and 10 mL) − Nitrogen blanket apparatus 8 V. Reagents − − − − − − − − − − Certified vitamin A acetate concentrate (USP) or Retinyl palmitate, all-trans. Acetic acid glacial, AR Acetonitrile, AR Isopropanol, AR Methanol, HPLC grade Absolute ethanol AR Tetrahydrofuran (THF), AR grade Hexane (n-Hexane 95% for HPLC) Pyrogallic acid, crystal, AR grade VI. Solutions a. Mobile phase: Combine 890 mL methanol and 110 mL distilled water. Mix well. Stir overnight to degas or prior to use. b. THF-methanol [50+50]: Combine 500 mL tetrahydrofuran and 500 mL 95% ethanol. Mix well. c. Potassium hydroxide solution-50%: Slowly add 500 g of KOH pellets to 500 mL water contained in a 2L thick walled Erlenmeyer flask. The solution gives off substantial heat while KOH is dissolving. Add the KOH in 100 g portions while the flask is being cooled with cold water. Swirl the flask gently to aid in dissolution of the KOH. Store in glass container with cork stopper. d. Washing solution-acetonitrile-20% in water: Combine 80 mL water and 20 mLacetonitrile. Mix well. e. Vitamin A working standard (ca 5 µg retinol/mL) 1. Using USP standard: Weigh 50 mg retinyl acetate concentrate into a 100-mL volumetric flask. Record weight to nearest 0.1 mg. Record concentration in mg/g per USP certification. Add a small amount of acetone (less than 3 mL) to aid dissolution. Dilute to volume with absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks. 2. Using retinyl palmitate: Stock solution: Weigh 55 mg retinyl palmitate into 100-mL volumetric flask. Record weight to nearest 0.1 mg. Record purity per supplier certification or purity test. Add pea-sized piece of pyrogallic acid. Dissolve and dilute to volume with hexane. Working solution: Pipet 2 mL solution to second 100-mL flask and dilute to volume with absolute ethanol. Store at 4°C in dark. Solution is stable for two weeks. 8 A supply of nitrogen gas with appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during saponification. - 27 -
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: VIII. Qualitycontrol Run the flour
Page 29 and 30: VII. Procedure a. Preparation of sa
Page 31 and 32: c. Clean-up and concentration proce
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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