Apparatus— Use a submicron laser light-scattering instrument.1

Standards— Use 90- and 270-nm NIST-traceable polystyrene monospheres.

Standard dilutions— Transfer 3 mL of 0.2-µm filtered water to each of two clear acrylic cuvettes. Add a sufficient amount of the 90-nm monospheres to one of the cuvettes and a sufficient amount of the 270-nm monospheres to the other cuvette to make the dilutions slightly turbid, place in the Apparatus, and then measure the particle sizes: the particle size in the Standards is within 10% of the certified diameter.

Procedure— Transfer about 40 µL of the Injection to a clear acrylic cuvette, and add 3 mL of 0.2-µm filtered water. Cover, invert to mix (without shaking), place in the Apparatus, and measure the intensity-weighted effective diameter. [NOTE—Several minutes are necessary for the sample to reach equilibrium.] The intensity-weighted effective diameter is between 100 and 250 nm.

Apparatus: a calibrated magnetic susceptibility balance.

Standard solutions— Transfer 118.85 g of nickel chloride hexahydrate, accurately weighed, to a 500-mL volumetric flask, dilute with water to volume, and mix to obtain 1 M nickel chloride. Transfer 356.55 g of nickel chloride hexahydrate, accurately weighed, to a separate 500-mL volumetric flask, dilute with water to volume, and mix to obtain 3 M nickel chloride.

Balance constant— [NOTE—Each magnetic susceptibility balance has a Balance constant, K, that is determined each time the balance is moved to a new location. The Balance constant is referenced to the known magnetic susceptibility of nickel chloride hexahydrate.] Measure the length, L, in cm, of the large-bore susceptibility tube, from the bottom of the inside of the tube to the bottom of the black band. Tare the susceptibility tube on a balance, fill it with water to the bottom of the black band, and weigh again. Record the weight, W, in g, of the water in the tube, zero the susceptibility balance, and then place the tube containing water in the susceptibility balance. Zero the balance again. Rinse the tube with 1 M nickel chloride, and then fill the tube with fresh 1 M nickel chloride. Insert the tube into the susceptibility balance, and record the magnetic susceptibility, R, in cgs units, of 1 M nickel chloride. Calculate the Balance constant, K, by the formula: in which 4.24 × 10–6 is the magnetic susceptibility, in cgs units, of nickel chloride hexahydrate; and the other terms are as defined above.

Tube constant— [NOTE—For each susceptibility tube, a tube constant, CT , is determined.] Measure the length of the tube, L, in cm, from the bottom of the inside of the tube to the bottom of the black band. Tare the tube on a balance, fill it with water to the bottom of the black band, and weigh again. Record the weight, W, in g, of the water in the tube. Calculate the tube constant, CT , by the formula: in which K is the Balance constant as obtained above; and L and W are as defined above.

Diluted sample— Transfer about 0.2 g of the Injection, accurately weighed, to a 10-mL volumetric container, dilute with water to volume, mix, and accurately weigh the contents.

Procedure— [NOTE—Use the same susceptibility tube throughout the procedure.] Fill the susceptibility tube with water to the black band. Insert the tube into the susceptibility balance, and adjust to zero. Remove the tube from the balance, pour the water from the tube, rinse the tube with 1 M nickel chloride, and then fill the tube with fresh 1 M nickel chloride. Insert the tube into the balance, and record the magnetic susceptibility in cgs units. Similarly, measure the magnetic susceptibility of 3 M nickel chloride. The balance readings are within 5% of the expected values, which are (4.24 × 10–6)/CT for 1 M nickel chloride and (12.72 × 10–6)/CT for 3 M nickel chloride. Rinse the tube with well-mixed Injection, then fill the tube with the Injection to the black band, and weigh. Insert the tube into the susceptibility balance, and proceed as directed for the Standard solutions. Calculate the magnetic susceptibility, in cgs units, per g of iron in the Injection by the formula: in which CT is the Tube constant as obtained above; R is the balance reading, in cgs units; T is the weight, in g, of the Diluted sample; W is the weight, in g, of the ferumoxides in the Diluted sample; and I is the concentration of iron, in mg per g, in the Injection, obtained by using specific gravity to convert the concentration of iron, in mg per mL, as determined in the Assay for iron, to mg per g. The magnetic susceptibility is not less than 17,100 × 10–6 in cgs units per g of iron.

(Official January 1, 2007)

Iron standard solution— Use a NIST-traceable iron standard containing 1000 µg per mL (1000 ppm).

Standard preparations— Pipet 5.0, 10.0, 15.0, and 20.0 mL of the Iron standard solution into separate 1000-mL volumetric flasks, dilute each with water to volume, and mix to obtain solutions having known concentrations of 5 µg per mL, 10 µg per mL, 15 µg per mL, and 20 µg per mL, respectively.

Assay preparation— Accurately weigh 100 µL of the Injection, and transfer to a test tube. Add 2 mL of hydrochloric acid, and mix. [NOTE—The Injection dissolves to yield a medium yellow solution.] Add 2 mL of water, and then transfer the contents of the tube to a 100-mL volumetric flask, dilute with water to volume, and mix.

Procedure— Using an atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering 851) equipped with an iron hollow-cathode lamp and an air–acetylene flame, set the instrument to zero with water, and measure the absorbance, A20, of the Standard preparation containing 20 µg per mL at the iron emission line of 296.7 nm. Concomitantly determine the absorbances of the Standard preparations. Calculate the iron concentration, in µg per mL, of each Standard preparation by the formula: in which AS is the absorbance of the relevant Standard preparation. The reading for each Standard preparation is within 0.3 µg per mL of its nominal concentration. Measure the absorbance of the Assay preparation, and calculate the content of iron, in mg per mL, in the Injection by the formula: in which AU is the absorbance of the Assay preparation; A20 is the absorbance of the Standard preparation containing 20 µg per mL; S is the specific gravity of the Injection; and W is the weight, in g, of the volume of Injection taken to prepare the Assay preparation.

Control preparation— Transfer about 50 mg of USP Dextrose RS, accurately weighed, to a 1000-mL volumetric flask, dilute with water to volume, mix, and filter.

Standard preparation— Transfer about 50 mg of USP Dextran T-10 RS, accurately weighed, to a 1000-mL volumetric flask, dilute with water to volume, mix, and filter.

Assay preparation— Transfer 0.25 g of the Injection, accurately weighed, to a test tube, add about 0.25 mL of hydrochloric acid, then add about 9 mL of water, and mix. Remove the free iron from this solution by passing it through a cation-exchange column into a 25-mL volumetric flask. Rinse the column with about 9 mL of water, collecting the washings in the 25-mL volumetric flask, dilute with water to volume, and mix.

Procedure— To each of four test tubes, separately add 0.2 mL of the Assay preparation, the Standard preparation, the Control preparation, and water (to be used as the blank). Add 0.2 mL of a 5% phenol solution to each test tube. Mix each tube briefly on a vortex mixer, rapidly add 1.0 mL of sulfuric acid to each test tube, and again mix briefly on a vortex mixer. [Caution—Reaction is exothermic. ] Cover the test tubes, and allow to stand at room temperature for at least 15 minutes. [NOTE—The resultant solution is orange-yellow in color and free of any solid material.] Mix each tube on a vortex mixer. Using a suitable spectrophotometer, determine the absorbances of the solutions from the Standard preparation, the Control preparation, and the Assay preparation against the blank at the wavelength of maximum absorbance at about 490 nm. Calculate the percent recovery of dextran in the Control preparation by the formula: in which 1.11 is a correction factor (to account for dextrose being a monomer of dextran); C is the concentration, in mg per mL, of USP Dextran T-10 RS in the Standard preparation; CD is the concentration, in mg per mL, ofUSP Dextrose RS in the Control preparation; and AC and AS are the absorbances of the solutions from the Control preparation and the Standard preparation, respectively: not less than 90% to 110% is found. Calculate the quantity, in mg per mL, of dextran in the volume of Injection taken by the formula: in which S is the specific gravity of the Injection; C is the concentration, in mg per mL, of USP Dextran T-10 RS in the Standard preparation; W is the weight, in g, of the portion of the Injection taken to prepare the Assay preparation; and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a filtered and degassed 0.0375 N sodium hydroxide solution. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Transfer 0.7776 g of trisodium citrate dihydrate to a 100-mL volumetric flask, dilute with water to volume, filter, and refrigerate. This solution contains 5000 µg of citrate per mL (5000 ppm).

Standard preparations— Transfer 10 mL of Standard stock solution to a 100-mL volumetric flask, dilute with water to volume, and mix. Transfer 1.0, 2.0, and 4.0 mL of this solution to separate 100-mL volumetric flasks, add 0.8 mL of hydrochloric acid to each flask, dilute with water to volume, and mix to obtain solutions having known concentrations of 5 µg per mL, 10 µg per mL, and 20 µg per mL.

System suitability solution— Use the filtered Standard preparation containing 5 µg per mL.

Assay preparation— Transfer about 0.5 mL of the Injection, accurately weighed, to a test tube, add 0.2 mL of hydrochloric acid and about 9 mL of water, and mix. Remove the free iron from this solution by passing the solution through a cation-exchange column2 into a 25-mL volumetric flask. Rinse the column with about 9 mL of water, collecting the washings in the flask, dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph3 is equipped with an ion detector with suppressed conductivity at 30 µS, a 4-mm × 25-cm separator column4 that contains 15-µm packing L48, a 4-mm × 50-mm guard column,5 and integrators. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparations and the Assay preparation into the chromatograph, record the chromatograms for about 16 minutes, and measure the responses for the major peaks. Prepare a standard curve by plotting the conductivities of the Standard preparations versus their concentrations, in µg per mL. Determine the concentration, C, in µg per mL, of citrate in the Assay preparation by extrapolation from the standard curve. Calculate the quantity, in mg per mL, of citrate in the volume of Injection taken by the formula: in which S is the specific gravity of the Injection; and W is the weight, in g, of the volume of Injection taken to prepare the Assay preparation.