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

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

Standard dilutions— Transfer 3mLof 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 Standardsis within 10%of the certified diameter.

Procedure— Transfer about 40µLof the Injection to a clear acrylic cuvette,and add 3mLof 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 100and 250nm.

Apparatus: a calibrated magnetic susceptibility balance.

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

Balance constant— [NOTE—Each magnetic susceptibility balance has aBalance constant,K,that is determined each time the balance is moved to a new location.TheBalance 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 1Mnickel chloride,and then fill the tube with fresh 1Mnickel chloride.Insert the tube into the susceptibility balance,and record the magnetic susceptibility,R,in cgs units,of 1Mnickel chloride.Calculate theBalance constant,K,by the formula: in which 4.24×10–6is 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 whichKis theBalance constant as obtained above;andLandWare as defined above.

Diluted sample— Transfer about 0.2g of the Injection,accurately weighed,to a 10-mLvolumetric 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 1Mnickel chloride,and then fill the tube with fresh 1Mnickel chloride.Insert the tube into the balance,and record the magnetic susceptibility in cgs units.Similarly,measure the magnetic susceptibility of 3Mnickel chloride.The balance readings are within 5%of the expected values,which are (4.24×10–6)/CTfor 1Mnickel chloride and (12.72×10–6)/CTfor 3Mnickel 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 whichCTis theTube constant as obtained above;Ris the balance reading,in cgs units;Tis the weight,in g,of theDiluted sample;Wis the weight,in g,of the ferumoxides in theDiluted sample;andIis 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 theAssay for iron,to mg per g.The magnetic susceptibility is not less than 17,100×10–6in cgs units per g of iron.

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

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

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

Procedure— Using an atomic absorption spectrophotometer (seeSpectrophotometry and Light-Scattering á851ñ)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 theStandard preparation containing 20µg per mLat the iron emission line of 296.7nm.Concomitantly determine the absorbances of theStandard preparations.Calculate the iron concentration,in µg per mL,of eachStandard preparation by the formula: in whichASis the absorbance of the relevantStandard preparation.The reading for eachStandard preparation is within 0.3µg per mLof its nominal concentration.Measure the absorbance of theAssay preparation,and calculate the content of iron,in mg per mL,in the Injection by the formula: in whichAUis the absorbance of theAssay preparation;A20is the absorbance of theStandard preparation containing 20µg per mL;Sis the specific gravity of the Injection;andWis the weight,in g,of the volume of Injection taken to prepare theAssay preparation.

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

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

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

Procedure— To each of four test tubes,separately add 0.2mLof theAssay preparation,theStandard preparation,theControl preparation,and water (to be used as the blank).Add 0.2mLof a 5%phenol solution to each test tube.Mix each tube briefly on a vortex mixer,rapidly add 1.0mLof 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 15minutes.[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 theStandard preparation,theControl preparation,and theAssay preparation against the blank at the wavelength of maximum absorbance at about 490nm.Calculate the percent recovery of dextran in theControl preparation by the formula: in which 1.11is a correction factor (to account for dextrose being a monomer of dextran);Cis the concentration,in mg per mL,of USP Dextran T-10RSin theStandard preparation;CDis the concentration,in mg per mL,ofUSP Dextrose RSin theControl preparation;andACandASare the absorbances of the solutions from theControl preparation and theStandard 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 whichSis the specific gravity of the Injection;Cis the concentration,in mg per mL,of USP Dextran T-10RSin theStandard preparation;Wis the weight,in g,of the portion of the Injection taken to prepare theAssay preparation;andAUandASare the absorbances of the solutions from theAssay preparation and theStandard preparation,respectively.

Mobile phase— Prepare a filtered and degassed 0.0375Nsodium hydroxide solution.Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

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

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

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

Assay preparation— Transfer about 0.5mLof the Injection,accurately weighed,to a test tube,add 0.2mLof hydrochloric acid and about 9mLof water,and mix.Remove the free iron from this solution by passing the solution through a cation-exchange column2into a 25-mLvolumetric flask.Rinse the column with about 9mLof water,collecting the washings in the flask,dilute with water to volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph3is equipped with an ion detector with suppressed conductivity at 30µS,a 4-mm ×25-cm separator column4that contains 15-µm packing L48,a 4-mm ×50-mm guard column,5and integrators.The flow rate is about 1mLper minute.Chromatograph theSystem suitability solution,and record the peak responses as directed forProcedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 10µL)of theStandard preparations and theAssay preparation into the chromatograph,record the chromatograms for about 16minutes,and measure the responses for the major peaks.Prepare a standard curve by plotting the conductivities of theStandard preparations versus their concentrations,in µg per mL.Determine the concentration,C,in µg per mL,of citrate in theAssay 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 whichSis the specific gravity of the Injection;andWis the weight,in g,of the volume of Injection taken to prepare theAssay preparation.