A: To 1 mL of Injection add 5 mL of water and 1 mL of ammonium hydroxide: no precipitate is formed.

B: To 1 mL of Injection add 0.1 mL of 3 N hydrochloric acid and 0.5 mL of potassium ferrocyanide TS: a dark blue precipitate is formed.

C: To 1 mL of Injection in a separator add 4 mL of water and 10 mL of hydrochloric acid, and extract with three 15-mL portions of isopropyl ether. Dilute the aqueous layer with water to 25 mL, and mix. To 0.5 mL of this solution add 10 mL of water, 2 mL of 2 N sulfuric acid, and 10 mg of potassium periodate, allow to stand for 10 minutes, and then add 10 mL of 0.1 N sodium arsenite. When the solution is colorless, add 10 mL of a 1 in 250 solution of phenylhydrazine hydrochloride in 0.5 N hydrochloric acid. Allow to stand for 10 minutes, add 1 mL of potassium ferricyanide solution (1 in 20), allow to stand for 15 minutes, and then add 3 mL of hydrochloric acid: a wine-red color is produced (presence of sorbitol).

D: Dilute 1 mL of Injection with 50 mL of water, and to 4 mL of this solution add 1 mL of 6 N sulfuric acid and 0.5 mL of phosphoric acid, mix, and allow to stand for about 5 minutes or until decolorized. Add 1 mL of potassium bromide solution (1 in 10) and 1 mL of potassium permanganate solution (1 in 20). After 10 minutes, add hydrogen peroxide TS, dropwise, to discharge the pink color. Transfer the solution to a small separator, shake with 20 mL of solvent hexane, discard the water layer, and wash the hexane layer with 20 mL of water. To the washed hexane solution add 5 mL of a 1 in 25 solution of thiourea in sodium borate solution (1 in 50), and shake the mixture: the aqueous layer that separates shows a yellow color (presence of citric acid).

(Official January 1, 2007)

Standard preparation— Transfer an accurately weighed portion of ferrous ammonium sulfate, equivalent to about 100 mg of iron (Fe), to a 300-mL Kjeldahl flask, and proceed as directed under Assay preparation, beginning with “Add 10 mL of nitric acid,” to obtain a Standard preparation having a known concentration of about 2 µg of iron per mL.

Assay preparation— Using a “to contain” pipet, transfer an accurately measured volume of Injection, equivalent to about 100 mg of iron, to a 300-mL Kjeldahl flask, rinsing the pipet into the flask with several small portions of water. Add 10 mL of nitric acid, 10 mL of sulfuric acid, and a few glass beads, and boil the solution gently until fumes of sulfur trioxide appear. Cool, add 3 mL of nitric acid, and heat gently again until fumes of sulfur trioxide appear. Continue the addition of nitric acid, followed by gentle boiling, until the solution is clear and light yellow or green in color, and then boil for an additional 30 minutes. Cool, cautiously add 100 mL of water, and boil gently until solution is complete. Cool, transfer the solution to a 500-mL volumetric flask, rinse the Kjeldahl flask with several small portions of water, dilute with water to volume, and mix. Transfer 5.0 mL of the solution to a second 500-mL volumetric flask, add 100 mL of water and 1 g of ascorbic acid, dilute with water to volume, and mix.

Procedure— Transfer 5.0-mL portions of the Standard preparation, of the Assay preparation, and of water to serve as the blank to separate, clean, dry test tubes, and to each add 3.0 mL of a 1 in 1500 solution of 2,2¢-bipyridine in 0.6 N glacial acetic acid, mix, and allow to stand for 15 minutes. Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 510 nm, with a suitable spectrophotometer, using the blank to set the instrument. Calculate the quantity, in mg, of iron in each mL of the Injection taken by the formula: in which C is the concentration, in µg per mL, of the Standard preparation; V is the volume, in mL, of Injection taken; and AU and AS are the absorbances of the solutions from the Assay preparation and Standard preparation, respectively.