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

B: To 1mLof Injection add 0.1mLof 3Nhydrochloric acid and 0.5mLof potassium ferrocyanide TS:a dark blue precipitate is formed.

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

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

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

Assay preparation— Using a “to contain”pipet,transfer an accurately measured volume of Injection,equivalent to about 100mg of iron,to a 300-mL Kjeldahl flask,rinsing the pipet into the flask with several small portions of water.Add 10mLof nitric acid,10mLof sulfuric acid,and a few glass beads,and boil the solution gently until fumes of sulfur trioxide appear.Cool,add 3mLof 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 30minutes.Cool,cautiously add 100mLof water,and boil gently until solution is complete.Cool,transfer the solution to a 500-mLvolumetric flask,rinse the Kjeldahl flask with several small portions of water,dilute with water to volume,and mix.Transfer 5.0mLof the solution to a second 500-mLvolumetric flask,add 100mLof water and 1g of ascorbic acid,dilute with water to volume,and mix.

Procedure— Transfer 5.0-mLportions 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 3.0mLof a 1in 1500solution of 2,2¢-bipyridine in 0.6Nglacial acetic acid,mix,and allow to stand for 15minutes.Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 510nm,with a suitable spectrophotometer,using the blank to set the instrument.Calculate the quantity,in mg,of iron in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of the Standard preparation,Vis the volume,in mL,of Injection taken,and AUand ASare the absorbances of the solutions from the Assay preparationand Standard preparation,respectively.