manual of methods for determining micronutrients in fortified foods

VII.
Procedure
a. Preparation of sample
1. Solid samples should be ground to pass a 40-mesh sieve. Liquid or wet samples should be blended to
homogeneity and stored at or below 4°. All samples should be stored in the dark.
b. Saponification and extraction of sample
2. Turn on hot plate to preheat. Start and adjust cooling water flow to precool reflux condensers. Reflux
system should be arranged as shown in the picture below.
Cooling water outlet
Connection to
nitrogen gas
Reflux condenser
Cooling water inlet
Flat-bottom flasks
with sample &
reagents covered
with aluminum foil
Water bath at 85ºC
3. Standards
• High standard: Pipet 4 mL vitamin A working standard into 125-mL Erlenmeyer flask. Add 25 mL
95% ethanol. Proceed to step 5.
• Intermediate standard: Pipet 3 mL vitamin A working standard into a second 125-mL Erlenmeyer
flask. Add 33 mL 95% ethanol. Proceed to step 5.
• Low standard 1: Pipet 2.0 mL vitamin A working standard into a third 125-mL Erlenmeyer flask. Add
35 mL 95% ethanol. Proceed to step 5.
• Low standard 2 (prepare this standard when running samples with vitamin A concentrations below 1.5
mg/kg): Pipet 1 mL vitamin A working standard into a 125-mL Erlenmeyer flask. Add 37.5 mL 95%
ethanol. Proceed to step 5.
IMPORTANT: For samples with vitamin A concentrations below 1.5 mg/kg, such as wheat flour, run
Intermediate standard, Low standard 1 and Low standard 2, only.
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