manual of methods for determining micronutrients in fortified foods

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V. Reagents − − − − − − − Acetonitrile (CH 3 CN), mol wt. 41.05, HPLC grade. Calcium hydroxide. (Ca(OH) 2 ), mol wt. 74.09, p.a Ethanol. o-Phosphoric acid (H 3 PO 4 ), 85% purity, mol wt. 98.00, p.a. Oxalic acid (C 2 H 2 O 4 .2H 2 O), mol wt. 126.07, p.a. Sodium dodecylsulfate (CH 3 (CH 2 ) 11 OSO 3 Na), ≥ 99% purity for ion-pair chromatography, mol wt. 288.38. Fluka 71726. Mobile phase: Solution A (acidified water): Dilute 1.00 mL o-phosphoric acid in 600 mL water and filter through 0.45 um. Solution B: In a 500 mL beaker, mix 120 mL water and 230 mL acetonitrile. Dissolve 1.00 g sodium dodecyl sulfate in this solution. Bring total volume to 400 mL with water. Filter through 0.45 um. Mix solution A and B to make 1L mobile phase. Mix well. − − Stock niacin standard solution (2000 mg/L): Weigh 200 mg niacin and dissolve in ethanol:water (1+1) in a 100 mL volumetric flask. Bring to volume with the mixture ethanol:water (1+1) and mix well. This solution is stable at least 2 weeks at room temperature. Working Standard solution-2 mg/L: Take 5 mL of stock standard solution and dilute to 100 mL with water. Then take 2 mL of the dilution and dilute to 100 mL with water. VI. Procedure 1. Weigh 2 g flour in a 500 mL blender jar. The sample weight should contain about 0.04 mg niacin. 2. Add 198 mL water and add 10 g calcium hydroxide. 3. Standard treatment: Prepare three standard solutions with different concentrations. − − − Low standard: Pipet 10 mL of the working standard solution (2 mg/L) into 500 mL Erlenmeyer flask containing 190 mL water. Intermediate standard: Pipet 20 mL of the working standard solution into 500 mL Erlenmeyer flask containing 180 mL water. High standard: Pipet 40 mL of the working standard solution into 500 mL Erlenmeyer flask containing 160 mL water. Add 10 g calcium hydroxide and treat the standard as the sample starting from step 4. 4. Blend aprox. 30 seconds at high speed using a vertical stirrer or blender. 5. Autoclave 15 min at 121°C. 6. Cool in ice bath at least 30 min. - 48 -
7. Transfer the extract to a 250 mL volumetric flask and bring to volume with water. 8. Filter the cold solution through Whatman paper No. 2V, try to filter only the supernatant. Centrifuging the solution will help to obtain a clear filtrate. 9. Transfer 100 mL of the filtrate, measured with a volumetric pipette, to 250 mL Erlenmeyer flask containing 300 mg oxalic acid. 10. Mix well and adjust final pH to 6.5 to 7.0 by dropwise addition of filtrate or by addition of a few crystals of oxalic acid. 11. Centrifuge the filtrate at 5,000 rpm for 10 minutes. Filter slowly through 1 or 2 pieces of Whatman No. 42 paper so that clear filtrate is obtained. 12. Condition C18 cleanup cartridge with 10 mL ethanol and then pass 10 mL water. 13. Slowly pass 10 mL clear sample filtrate through cartridge. 14. Discard first 6 mL and collect next 3.5 mL in sample vial. 15. Add 1 drop 85% fosforic acid (H 3 PO 4 ) and mix well. 16. Inject 100 µL standard and sample solutions using the chromatographic conditions described below. Parameter Condition Column C18. Waters. 250 mm x 4.1mm ID Flow 1.5 mL/min Detector Ultraviolet Wavelength: 254 nm Injection volume 100 µL Note: The peak shape and retention time may be modified by adding acetonitrile (95% mobile phase: 5% acetonitrile). VII. Calculations 1. Calculate the equation of the standard curve using height (y) vs. concentration-mg/L (x) for the three standard concentrations injected. 2. The correlation coefficient (r) should be 0.999 or above. 3. Calculate the niacin concentration (mg/L) in the injected solution using the regression equation. 4. Using the concentration of sample in mg/L, calculate niacin concentration in the sample using the following equation. Where: Parameter Explanation Value C s Concentration of niacin in the sample (mg/L) obtained from the regression equation. ? V i Initial volume (mL) 250 W s Sample weight (g). Around 2 g. ? - 49 -
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 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: C. Determination of niacin in flour
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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