Iron Sucrose Injection
»Iron Sucrose Injection is a sterile,colloidal solution of ferric hydroxide in complex with Sucrose in Water for Injection.It contains not less than 95.0percent and not more than 105.0percent of the labeled amount of iron.Sodium Hydroxide may be added to adjust the pH.It contains no antimicrobial agent,chelating agent,dextran,gluconate,or other added substances.
Packaging and storage— Preserve in single-dose containers of Type Iglass.Store at controlled room temperature.Do not freeze.
Labeling— Label it to state that it is for intravenous use only.Label it to indicate that when administered by intravenous infusion,the Injection must be diluted with 0.9%Sodium Chloride Injection to a concentration of 0.5to 2.0mg of elemental iron per mL.Label it also to state the total osmolarity of the solution expressed in mOsmol per L.
Identification—
A: Iron—To 2.5mLof Injection add 17.5mLof water and 5mLof hydrochloric acid,mix,and heat for 5minutes in a boiling water bath.Cool,add dropwise 13.5Nammonium 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 2Nhydrochloric acid,and add sufficient water to make a volume of 20mL.To 3mLof the solution so obtained add 1mLof 2Nhydrochloric acid and 1mLof potassium thiocyanate TS:the resulting solution(Solution 1)is red.To 1mLofSolution 1add 5mLof amyl alcohol or ethyl ether,shake,and allow to stand:the organic layer is pink.To a separate 1-mLaliquot ofSolution 1add 2mLof mercuric chloride TS:the red color is discharged [iron (III)salts].
B: Sucrose—The retention time of the major peak in the chromatogram of theAssay preparation corresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay for sucrose.
C: Molecular weight determination—
Mobile phase— Dissolve 7.12g of dibasic sodium phosphate dihydrate,5.52g of monobasic sodium phosphate,and 0.40g of sodium azide in 2Lof water.
System suitability solution— Dissolve 200mg of high molecular weight dextran and 100mg of glucose in 20mLofMobile phase.
Standard solutions— Transfer about 20mg of each polysaccharide molecular weight standard (5,000–400,000Da),accurately weighed,to separate 5-mLvolumetric flasks.Add 4mLofMobile phase to each flask,and allow each aliquot to stand at or below 25for a minimum of 12hours.After the agglomerate particles of eachStandard solution have swelled to their fullest extent,gently swirl eachStandard solution until dissolved.[NOTE—The chromatograms of freshly preparedStandard solutions regularly show a small,unidentified secondary peak following the main peak.Discard theStandard solutions if the secondary peak reaches half the height of the main peak.]
Test solution— Transfer 5.0mLof Injection to a 10-mLvolumetric flask,dilute withMobile phase to volume,and mix.
Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a refractive index detector maintained at a constant temperature of 45and two 7.8-mm ×30-cm columns set up in series that contain packing L39with pore sizes of 1000Åand 120Å,respectively.The column temperatures are maintained at 45±2and the flow rate is about 0.5mLper minute.Chromatograph theSystem suitability solution,and measure the peak areas as directed forProcedure:the resolution,R,between dextran and glucose is not less than 4.0.Chromatograph theStandard solutions,and measure the peak areas as directed forProcedure.Using a suitable program,plot the retention times of theStandard solutions and their molecular weights to generate a third order (cubic)calibration curve.The correlation coefficient obtained is not less than 0.98.
Procedure— Separately inject equal volumes (about 25µL)of eachStandard solution,theSystem suitability solution,and theTest solution into the chromatograph,record the chromatograms,and measure the retention times and peak areas.The molecular weight of the complex is calculated from the calibration curve.The molecular weight distribution curve of the sample is sliced into fractions.Calculate the weight-average molecular weight,MW,as follows:
S(ATMT)/SAT,
and the number-average molecular weight,MN,as follows:
S(AT)/S(AT/MT),
in which ATis the area of each fraction of the sample distribution;and MTis the corresponding mean molecular weight of each fraction as determined from its retention time on the calibration curve.The molecular weight distribution curve obtained for the Injection conforms to the following parameters:

MW=34,000–60,000Da,

MN=not less than 24,000Da,and

MW/MN=not more than 1.7.
Specific gravity á841ñ: not less than 1.135and not more than 1.165at 20.
Bacterial endotoxins á85ñ: not more than 3.7USP Endotoxin Units per mg of iron contained in the Injection.
Alkalinity— Transfer 5mLof Injection to a suitable vessel,and titrate with 0.1Nhydrochloric acid VSwith constant stirring to a pHof 7.4.Record the volume of 0.1Nhydrochloric acid VSconsumed,and calculate the alkalinity of the Injection as the volume of acid,in mL,consumed per mLof Injection.Not less than 0.5mLand not more than 0.8mLof 0.1Nhydrochloric acid VSis consumed per mLof Injection.
pHá791ñ: between 10.5and 11.1at 20.
Osmolarity á785ñ: not less than 1150mOsmol per Land not more than 1350mOsmol per Lfor the Injection.The solution for test is prepared by diluting the Injection 1in 10.
Absence of low-molecular weight Fe(II)and Fe(III)complexes— In the polarograms obtained in the test forLimit of iron (II),no additional peaks are found.
Turbidity— Transfer 0.5g of Injection to a 150-mLbeaker,add 100mLof water,and with constant stirring adjust with 0.1Nhydrochloric acid VSto a pHof about 6.0.Remove the pHelectrode from the solution.Adjust a light source such that the beam hits the beaker at a parallel angle about 2cm below the surface of the liquid.The light must shine through to the surface,and the solution must not have any turbidity.Measurement must be carried out in as dark a room as possible.Slowly add 0.1Nhydrochloric acid VS,dropwise,until a slight but lasting turbidity develops.Record the pHof the solution as the turbidity point of the Injection:not less than 4.4and not more than 5.3.
Particulate matter á788ñ— Prepare a solution of Injection (1in 40)using water that has been passed through a filter having a 1.2-µm or finer porosity:meets the requirements forLight Obscuration Particle Count Test for small-volume injections.
Limit of iron (II)—
Supplementary electrolyte solution— Dissolve 15.0g of sodium acetate in 100mLof water,and adjust with 0.1Nacetic acid to a pHof 7.0.
Procedure— Transfer a suitable amount ofSupplementary 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 5minutes.Avoiding any undue exposure to air,immediately transfer a volume of Injection,accurately measured,equivalent to a concentration of about 20to 120µg of elemental iron per mL,to the polarographic cell.[NOTE—The sample must be analyzed immediately upon opening the container.]Record the polarogram from 0mVand –1700mV.The iron (III)/iron (II)peak is detected at –750±50mVand the iron (II)/iron (0)peak is detected at –1400±50mV.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:
[1–(2/R)]×Fe
in which Ris 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.
Content of chloride— Transfer about 12g of Injection,accurately weighed,into a 50-mLbeaker.Add 40mLof water,0.3mLof 65%nitric acid,and,while stirring,titrate with 0.01Nsilver nitrate VS,determining the endpoint potentiometrically with silver–glass electrodes.Calculate the chloride content,in mg,of Injection taken.Each mLof 0.01Nsilver nitrate consumed is equal to 0.3545mg of chloride (Cl).The chloride content of the Injection is not less than 0.012%and not more than 0.025%.
Other requirements— It meets the requirements under Injections á1ñ.
Assay for sucrose—
Mobile phase— Prepare a mixture of acetonitrile and water (79:21).
Standard preparations— Dissolve an accurately weighed quantity of USP Sucrose RSin water,and quantitatively dilute with water to obtain solutions having known concentrations of about 13,16,18,21,and 23mg of sucrose per mL.
Assay preparation— Transfer about 1.875g of Injection,accurately weighed,into a 25-mLflask,add 1.25mLof water,and mix.Add 1.25mLof a monobasic sodium phosphate solution,prepared by dissolving 30g in 50mL,and mix.Allow the resulting solution to stand for 10minutes to precipitate the colloidal ferric hydroxide.Dilute with water to volume,and mix.Centrifuge this solution at 3000rpm for 15minutes.Pass the resulting solution through a filter,discarding the first 2mLof the filtrate.
Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a column compartment and a refractive index detector each maintained at a controlled temperature between 20and 25(±2)and a 4-mm ×25-cm column that contains packing L8.The flow rate is about 2.0mLper minute.Chromatograph theStandard preparations,and measure the peak areas as directed forProcedure.The correlation coefficient obtained from the linear regression of theStandard preparations is not less than 0.998.[NOTE—The retention time for sucrose is about 8minutes.]
Procedure— Separately inject equal volumes (about 20µL)of eachStandard preparation and theAssay preparation into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Plot the peak area for eachStandard 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 theAssay preparation.Calculate the quantity,in mg,of sucrose in each mLof Injection taken by the formula:
CDG/W,
in which Cis the concentration,in mg per mL,of sucrose in theAssay preparation;Dis the dilution volume of theAssay preparation;Gis the density,in g per mL,of Injection taken;and Wis the weight,in g,of Injection taken.It contains not less than 260mg and not more than 340mg of sucrose per mL.
Assay for iron—
Iron stock solution— Transfer about 350mg of ferrous ammonium sulfate,accurately weighed,to a 1000-mLvolumetric flask,add water to dissolve,dilute with water to volume,and mix to obtain a solution having a concentration of about 50µg of iron per mL.
Calcium chloride solution— Transfer 2.64g of calcium chloride to a 1000-mLvolumetric flask,add 500mLof water,and swirl to dissolve.Add 5.0mLof hydrochloric acid,and dilute with water to volume.
Standard preparations— To separate 50-mLvolumetric flasks transfer 2.0,4.0,6.0,8.0,and 10.0mLofIron stock solution.Dilute each flask withCalcium chloride solution to volume,and mix to obtainStandard preparations having known concentrations of about 2.0,4.0,6.0,8.0,and 10.0µg of iron per mL.
Assay preparation— Using a “to contain”pipette,transfer 2.0mLof Injection to a 100-mLvolumetric flask.Rinse the pipette several times withCalcium chloride solution.Add 5mLof hydrochloric acid,and swirl until the solution turns yellow.After the solution has cooled to room temperature,dilute withCalcium chloride solution to volume,and mix.Pipet 2.0mLof this solution to a 100-mLvolumetric flask,dilute withCalcium chloride solution to volume,and mix to obtain a solution with a theoretical concentration of about 8.0µg of iron per mL.
Procedure— Concomitantly determine the absorbances of theStandard preparations and theAssay preparation at the iron emission line at 248.3nm with a suitable atomic absorption spectrophotometer (seeSpectrophotometry and Light-Scattering á851ñ)equipped with an iron hollow-cathode lamp and air–acetylene flame,usingCalcium chloride solution as a blank.Plot the absorbances for eachStandard 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 theAssay preparation.Calculate the quantity,in mg,of iron in each mLof the Injection taken by the formula:
5C/V,
in which Cis the concentration,in µg per mL,of iron in theAssay preparation;andVis the volume of Injection taken.
Auxiliary Information— Staff Liaison:Andrzej Wilk,Ph.D.,Senior Scientific Associate
Expert Committee:(PA5)Pharmaceutical Analysis 5
USP28–NF23Page 1064
Pharmacopeial Forum:Volume No.28(4)Page 1130
Phone Number:1-301-816-8305