A: Iron—To 2.5 mL of Injection add 17.5 mL of water and 5 mL of hydrochloric acid, mix, and heat for 5 minutes in a boiling water bath. Cool, add dropwise 13.5 N ammonium hydroxide until no further precipitation of ferric hydroxide occurs, and filter. Wash the precipitate with water to remove excess ammonium hydroxide, dissolve the precipitate in a minimum volume of 2 N hydrochloric acid, and add sufficient water to make a volume of 20 mL. To 3 mL of the solution so obtained add 1 mL of 2 N hydrochloric acid and 1 mL of potassium thiocyanate TS: the resulting solution (Solution 1) is red. To 1 mL of Solution 1 add 5 mL of amyl alcohol or ethyl ether, shake, and allow to stand: the organic layer is pink. To a separate 1-mL aliquot of Solution 1 add 2 mL of mercuric chloride TS: the red color is discharged [iron (III) salts].

B: Sucrose—The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for sucrose.

C: Molecular weight determination—

Supplementary electrolyte solution— Dissolve 15.0 g of sodium acetate in 100 mL of water, and adjust with 0.1 N acetic acid to a pH of 7.0.

Procedure— Transfer a suitable amount of Supplementary electrolyte solution to a polarographic cell equipped with a mercury drop electrode. With the electrode submerged in the liquid, bubble nitrogen through the liquid for 5 minutes. Avoiding any undue exposure to air, immediately transfer a volume of Injection, accurately measured, equivalent to a concentration of about 20 to 120 µg of elemental iron per mL, to the polarographic cell. [note—The sample must be analyzed immediately upon opening the container.] Record the polarogram from 0 mV and –1700 mV. The iron (III)/iron (II) peak is detected at –750 ± 50 mV and the iron (II)/iron (0) peak is detected at –1400 ± 50 mV. Measure the iron (II)/iron (III) peak responses obtained from the polarogram, and perform a blank determination. Calculate the iron (II) content, in % w/v, in the volume of Injection taken by the formula: in which R is the peak response ratio of iron (II) to iron (III); and Fe is the total iron concentration, in % w/v, of the Injection. Not more than 0.4% (w/v) of iron (II) is found.

Mobile phase— Prepare a mixture of acetonitrile and water (79:21).

Standard preparations— Dissolve an accurately weighed quantity of USP Sucrose RS in water, and quantitatively dilute with water to obtain solutions having known concentrations of about 13, 16, 18, 21, and 23 mg of sucrose per mL.

Assay preparation— Transfer about 1.875 g of Injection, accurately weighed, into a 25-mL flask, add 1.25 mL of water, and mix. Add 1.25 mL of a monobasic sodium phosphate solution, prepared by dissolving 30 g in 50 mL, and mix. Allow the resulting solution to stand for 10 minutes to precipitate the colloidal ferric hydroxide. Dilute with water to volume, and mix. Centrifuge this solution at 3000 rpm for 15 minutes. Pass the resulting solution through a filter, discarding the first 2 mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a column compartment and a refractive index detector each maintained at a controlled temperature between 20 and 25 (±2) and a 4-mm × 25-cm column that contains packing L8. The flow rate is about 2.0 mL per minute. Chromatograph the Standard preparations, and measure the peak areas as directed for Procedure. The correlation coefficient obtained from the linear regression of the Standard preparations is not less than 0.998. [note—The retention time for sucrose is about 8 minutes.]

Procedure— Separately inject equal volumes (about 20 µL) of each Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Plot the peak area for each Standard preparation versus concentration, in mg per mL, of sucrose, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, in mg per mL, of sucrose in the Assay preparation. Calculate the quantity, in mg, of sucrose in each mL of Injection taken by the formula: in which C is the concentration, in mg per mL, of sucrose in the Assay preparation; D is the dilution volume of the Assay preparation; G is the density, in g per mL, of Injection taken; and W is the weight, in g, of Injection taken. It contains not less than 260 mg and not more than 340 mg of sucrose per mL.

Iron stock solution— Transfer about 350 mg of ferrous ammonium sulfate, accurately weighed, to a 1000-mL volumetric flask, add water to dissolve, dilute with water to volume, and mix to obtain a solution having a concentration of about 50 µg of iron per mL.

Calcium chloride solution— Transfer 2.64 g of calcium chloride to a 1000-mL volumetric flask, add 500 mL of water, and swirl to dissolve. Add 5.0 mL of hydrochloric acid, and dilute with water to volume.

Standard preparations— To separate 50-mL volumetric flasks transfer 2.0, 4.0, 6.0, 8.0, and 10.0 mL of Iron stock solution. Dilute each flask with Calcium chloride solution to volume, and mix to obtain Standard preparations having known concentrations of about 2.0, 4.0, 6.0, 8.0, and 10.0 µg of iron per mL.

Assay preparation— Using a “to contain” pipette, transfer 2.0 mL of Injection to a 100-mL volumetric flask. Rinse the pipette several times with Calcium chloride solution. Add 5 mL of hydrochloric acid, and swirl until the solution turns yellow. After the solution has cooled to room temperature, dilute with Calcium chloride solution to volume, and mix. Pipet 2.0 mL of this solution to a 100-mL volumetric flask, dilute with Calcium chloride solution to volume, and mix to obtain a solution with a theoretical concentration of about 8.0 µg of iron per mL.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay preparation at the iron emission line at 248.3 nm with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with an iron hollow-cathode lamp and air–acetylene flame, using Calcium chloride solution as a blank. Plot the absorbances for each Standard preparation versus concentration, in µg per mL, of iron and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, in µg per mL, of iron in the Assay preparation. 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 iron in the Assay preparation; and V is the volume of Injection taken.