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Prepared at the 53rd JECFA (1999) and published in FNP 52 Add 7 (1999), superseding specifications prepared at the 41st JECFA (1993), published in FNP 52 Add 2 (1993). Considered "safe when used in supervised food fortification programmes in response to a need for iron supplementation in a population as determined by public health officials" at the 53rd JECFA in 1999.
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Water-insoluble matter
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Not more than 0.1%
Weigh accurately 5 g of sample and transfer into 100 ml water. Stir until dissolved. Place a filter paper (1 �m porosity, maximum) in a Gooch crucible (3.5-4.0 cm) and seat the paper by applying vacuum while washing with water. Dry the crucible at 175� for 15 min, cool in a desiccator, and weigh. Pour sample solution through the crucible and wash with three successive 10 ml portions of water. Dry the crucible at 110� for one hour. Cool in a desiccator and weigh the crucible. Calculate as percentage.
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Nitrilotriacetic acid
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Mobile Phase Add 10 ml of a 1 in 4 solution of tetrabutylammonium hydroxide in methanol to 200 ml of water, and adjust with 1 M phosphoric acid to a pH of 7.5 � 0.1. Transfer the solution to a 1000-ml volumetric flask, add 90 ml of methanol, dilute with water to volume, mix, filter through a membrane filter (0.5-�g or finer porosity), and degas.
Ammonium hydroxide blank solution Add 0.5 ml of ammonium hydroxide to a 10 ml volumetric flask. Dilute with water to volume.
Stock Standard Solution Transfer about 100 mg of nitrilotriacetic acid, accurately weighed, to a 10-ml volumetric flask, and add 0.5 ml of ammonium hydroxide, and mix. Dilute to volume, and mix.
Standard Preparation Transfer 1.0 g of the sample to a 100-ml volumetric flask. Add 100 �l of the Stock Standard Solution, dilute with water to volume, and mix. Sonicate, if necessary, to achieve a complete solution.
Test Preparation Transfer 1.0 g of the sample to a 100-ml volumetric flask. Add 100 �l of Ammonium hydroxide blank solution and dilute with water to volume, and mix. Sonicate, if necessary, to achieve complete solution.
Chromatographic System See High-Pressure Liquid Chromatography (FNP 5). The chromatograph is equipped with a 254-nm detector and a 4.6-nm � 15-cm column that contains 5- 10-�m porous microparticles of silica to which is bonded octylsilane (Zorbax 8 or equivalent). The flow rate is about 2 ml/min. Chromatograph three replicate injections of the Standard Preparation, and record the peak responses as directed under Procedure. The relative standard deviation is not more than 2.0%, and the resolution factor between nitrilotriacetic acid and sodium iron EDTA is not less than 4.0.
Procedure Separately inject equal volumes (about 50 �l) of the Standard Preparation and the Test Preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The retention times are about 3.5 min for nitrilotriacetic acid and 11.9 min for sodium iron EDTA. The response of the nitrilotriacetic acid peak of the Test Preparation does not exceed the difference between the nitrilotriacetic acid peak responses obtained from the Standard Preparation and the Test Preparation.
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METHOD OF ASSAY
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Iron
Dissolve the sample (approx. 0.5 g accurately weighed) in distilled water (40 ml) in an iodine flask. Add concentrated hydrochloric acid (20 ml) mix, add potassium iodide (3 g) and then allow to stand for 5 min.
Titrate the liberated iodine with standardised 0.1 M sodium thiosulfate, using starch solution as indicator. Avoid vigorous mixing during the titration.
Perform a blank, omitting the sample.
where
Ts = sample titre in ml
Tb = blank titre in ml
0.05585 = atomic weight of iron � 10-3
M = molarity of sodium thiosulfate
W = sample weight (g)
EDTA
Reagents
(1) 0.25 M calcium acetate solution, standardized - Weigh and transfer 44.0 g reagent grade calcium acetate monohydrate to a 1 L volumetric flask; add water to dissolve and fill to the mark. Weigh accurately 2.0 to 2.1 g of reagent grade EDTA acid into each of three 250-ml conical flasks. Add 150 ml water and adjust to pH 11-12 (pH paper may be used) with 50% sodium hydroxide solution. Add about 30 mg of hydroxynaphthol blue indicator and titrate with calcium acetate to a sharp red endpoint.
(2) Triethanolamine, reagent grade. (3) Hydroxynaphthol Blue indicator. (4) 50 % sodium hydroxide solution.
Procedure
Accurately weigh 0.8 - 1.0 g of sample into a 250-ml beaker. Add 75 ml of distilled water to dissolve. Adjust the pH to 9.0 by dropwise addition of triethanolamine. Then adjust to pH 12.5 - 13.0 by addition of 50% aqueous NaOH. The solution should be clear and colourless. Add about 30 mg hydroxynaphthol blue indicator and titrate with 0.25 M calcium acetate solution to a red endpoint.
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