Iodixanol, chemical structure, molecular formula, Reference Standards

Iodixanol

C35H44I6N6O15 1550.18
1,3-Benzenedicarboxamide,5,5¢-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]bis[N,N¢-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-.
5,5¢-[(2-Hydroxytrimethylene)bis(acetylimino)]bis[N,N¢-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide] [92339-11-2].

»Iodixanol contains not less than 98.6percent and not more than 101.0percent of C35H44I6N6O15,calculated on the anhydrous basis.

Packaging and storage— Preserve in well-closed,light-resistant containers.Store at 25

,excursions permitted between 15

and 30

USP Reference standards á11ñ— USP Iodixanol RS.USP Iohexol Related Compound B RS.USP Iodixanol Related Compound C RS.USP Iodixanol Related Compound D RS.USP Iodixanol Related Compound E RS.

Identification—

A:Infrared Absorption á197Kñ.

B: The retention times of the two principal peaks in the chromatogram of the Test solutioncorrespond to those in the chromatogram of Standard solution 2,as obtained in Related compounds,Test 2.[NOTE—Athird isomer may appear as a minor peak.]

C: Heat 0.5g in a crucible:violet vapors are evolved.

Specific rotation á781Sñ: between –0.5

and +0.5

Test solution: 50mg per mL.

Microbial limits á61ñ— Proceed as directed for Plate Methodunder Total Aerobic Microbial Count:not more than 100cfu per g.

Water,Method Iá921ñ: not more than 4.0%.

Heavy metals,Method Iá231ñ: 0.001%.

Free iodine— Transfer 2g to a glass-stoppered tube,add about 20mLof water,5mLof toluene,and 5mLof 2Nsulfuric acid,shake vigorously,and allow the phases to separate:the toluene layer shows no red or pink color.

Limit of free iodide— Transfer 5.0g to a suitable container,add about 30mLof water,and titrate with 0.001Nsilver nitrate VS,determining the endpoint potentiometrically.Each mLof 0.001Nsilver nitrate is equivalent to 126.9µg of iodine.Not more than 0.39mLof 0.001Nsilver nitrate is required:not more than 10µg of iodide per g is found.

Limit of free aromatic amine—

N-(1-Naphthyl)ethylenediamine dihydrochloride solution— Prepare a fresh solution of N-(1-naphthyl)ethylenediamine dihydrochloride (3in 1000)in a mixture of propylene glycol and water (70:30).

Blank solution— Add 15mLof water to a 25-mLvolumetric flask.

Standard stock solution— Dissolve an accurately weighed quantity of USP Iohexol Related Compound B RS,and quantitatively dilute with water to obtain a solution having a known concentration of about 10µg per mL.

Standard solution— Transfer 10.0mLof the Standard stock solutionand 5mLof water to a 25-mLvolumetric flask.

Test solution— Transfer about 200mg of Iodixanol,accurately weighed,to a 25-mLvolumetric flask,add 15mLof water,and mix.

Procedure— Treat the Standard solution,the Test solution,and the Blank solutionas follows.Place the flask in an ice bath for 5minutes.Add 1.5mLof 6Nhydrochloric acid,mix by swirling,add 1.0mLof sodium nitrite solution (2in 100),mix,and allow to stand in the ice bath for 4minutes.Remove the flask from the ice bath,add 1.0mLof 4%sulfamic acid solution,and swirl gently until gas evolution ceases.Add 1.0mLof N-(1-Naphthyl)ethylenediamine dihydrochloride solution,mix,dilute with water to volume,mix,and allow to stand for 5minutes.Transfer the solution obtained from the Test solutionand the solution obtained from the Standard solutionto separate color-comparison tubes.The solution obtained from the Test solutionis lighter than the solution obtained from the Standard solution:not more than 0.05%is found.If the solution obtained from the Test solutionis about the same color or darker than the solution obtained from the Standard solution,proceed as follows.Concomitantly determine the absorbances of the solution obtained from the Test solution,the solution obtained from the Standard solution,and the solution obtained from the Blank solutionin 5-cm cells,at the wavelength of maximum absorbance at about 495nm,using the solution obtained from the Blank solutionto zero the spectrophotometer.Calculate the percentage of free aromatic amine in the portion of Iodixanol taken by the formula:

(C/W)[(AU–AB)/(AS–AB)],

in which Cis the concentration,in µg per mL,of USP Iohexol Related Compound B RSin the Standard solution;Wis the weight,in mg,of Iodixanol taken to prepare the Test solution;and AU,AB,and ASare the absorbances of the final solutions obtained from the Test solution,the Blank solution,and the Standard solution,respectively:not more than 0.05%is found.

Limit of calcium—

Internal standard solution— Accurately weigh about 3.067g of scandium oxide,and dissolve in 1Lof water (each mLcontains 1.000g of scandium).Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Blank solution— Transfer 5.0mLof Internal standard solutionto a 50-mLvolumetric flask,dilute with water to volume,and mix.

Standard solutions— Prepare a solution having a concentration of 10µg of calcium per mL.Add 0.5,2.5,5.0,and 10.0mLof this solution to separate 50-mLvolumetric flasks.To each flask,add 5.0mLof Internal standard solution,dilute with water to volume,and mix.

Test solution— Transfer about 2g of Iodixanol,accurately weighed,to a 20-mLvolumetric flask,add about 10mLof water,and mix.Add 2.0mLof Internal standard solution,dilute with water to volume,and mix.

Procedure— Concomitantly determine the absorbances of each Standard solutionand the Test solutionat 393.366nm,the calcium emission line,and at 361.38nm,the scandium emission line,with an atomic absorption spectrophotometer,using the Blank solutionas the blank.Plot a standard curve of the ratio of the calcium absorption to the scandium absorption versus the respective calcium concentrations.From the graph so obtained,determine the calcium concentration,C,in µg per mL,in the Test solution.Calculate the content of calcium,in µg per g,in the portion of Iodixanol taken by the formula:

20(C/W),

in which Cis as obtained above;and Wis the weight,in g,of Iodixanol taken to prepare the Test solution:not more than 5µg per g is found.

Limit of ionic compounds— [NOTE—Rinse all glassware with water.]Prepare a solution of Iodixanol in water (2in 100).The specific conductance of this solution is not greater than that of a sodium chloride solution having a concentration of 4µg per mL(equivalent to not more than 0.02%of ionic compounds,as NaCl).

Limit of methanol,isopropyl alcohol,and methoxyethanol—

Internal standard stock solution— Transfer about 500mg of secondary butyl alcohol to a 500-mLvolumetric flask,dilute with water to volume,and mix.

Internal standard solution— Transfer 1.0mLof Internal standard stock solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution— Transfer about 500mg of methanol,accurately weighed,and about 1000mg each of isopropyl alcohol and methoxyethanol,both accurately weighed,to a 500-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution and 1.0mLof Internal standard stock solutionto a second 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 1.0mLto a headspace vial,and seal the vial with a septum and a crimp cap.This solution contains about 0.005mg of methanol and about 0.01mg each of isopropyl alcohol and methoxyethanol per mL.

Test solution— Transfer about 250mg of Iodixanol,accurately weighed,to a headspace vial.Add 1.0mLof Internal standard solution,seal the vial with a septum and a crimp cap,and mix until dissolved.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a headspace injector and a 0.54-mm ×30-m capillary column coated with a 1-µm layer of phase G16.The carrier gas is helium,flowing at a rate of about 11mLper minute.The column temperature is maintained at 40

for 3.0minutes,then it is increased linearly at a rate of 8

per minute to 100

,and is maintained at 100

for 1minute.The temperatures of the headspace injector and the detector are maintained at 150

and 200

,respectively.The Standard solutionand the Test solutionare maintained at about 105

,and the needle temperature and transfer temperature are maintained at about 130

and 140

,respectively.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the elution order is methanol,isopropyl alcohol,secondary butyl alcohol,and methoxyethanol;the resolution,R,between methanol and isopropyl alcohol is not less than 1.0;and the relative standard deviation,determined from peak area ratios,for replicate injections is not more than 5%for methanol and isopropyl alcohol and not more than 10%for methoxyethanol.

Procedure— Using a heated,gas-tight syringe,separately inject equal volumes (about 1mL)of the headspace of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas.Calculate the percentage of methanol,isopropyl alcohol,and methoxyethanol in the portion of Iodixanol taken by the formula:

100(C/W)(RI/RS),

in which Cis the concentration,in mg per mL,of the relevant analyte in the Standard solution;Wis the weight,in mg,of Iodixanol taken to prepare the Test solution;and RIand RSare the peak area ratios of the corresponding analyte to the internal standard obtained from the Test solutionand the Standard solution,respectively:not more than 50µg per g each of methanol,isopropyl alcohol,or methoxyethanol is found.

Related compounds—

TEST1—

Solution A— Prepare a solution containing 500mLof acetonitrile and 500mLof water,and degas.

Solution B— Use 1000mLof degassed water.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Blank solution— Use water.

Standard stock solution 1— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol RSin water to obtain a solution having a known concentration of about 12.5mg of anhydrous iodixanol per mL.

Standard stock solution 2— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol Related Compound C RSin water to obtain a solution having a known concentration of about 0.25mg of anhydrous iodixanol related compound Cper mL.

Standard stock solution 3— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol Related Compound D RSin water to obtain a solution having a known concentration of about 0.025mg of anhydrous iodixanol related compound Dper mL.

Standard solution 1— Dilute 2.0mLof Standard stock solution 1in water to 10.0mL,and mix.

Standard solution 2— Transfer 5.0mLof Standard stock solution 1,2.5mLof Standard stock solution 2,and 2.5mLof Standard stock solution 3to a 25-mLvolumetric flask,dilute with water to volume,and mix.

Test solution 1— Dissolve an amount of Iodixanol,equivalent to about 500mg of anhydrous iodixanol,in water,dilute with water to 20.0mL,and mix.

Test solution 2— Dilute 5.0mLof Test solution 1with water to 50.0mL.

Control solution— Dilute 5.0mLof Test solution 1and 2.5mLof Standard stock solution 2with water to 50mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1mLper minute.The chromatograph is programmed as follows.

Time (minutes)	Solution A (%)	Solution B (%)	Elution
0	6	94	equilibration (for about 20minutes)
0–30	6®20	94®80	linear gradient
30–70	20®100	80®0	linear gradient
70–80	100	0	isocratic
80–81	100®6	0®94	linear gradient
81–90	6	94	isocratic

Chromatograph the solutions as directed for Procedure,in the following injection sequence:Blank solution,Standard solution 1,Control solution,and at least three replicates of Standard solution 2.

The chromatogram obtained from Standard solution 1exhibits two or three principal unresolved peaks.If the chromatogram exhibits two principal peaks,their relative areas are about 60%and 40%.If the chromatogram exhibits three principal peaks,their relative areas are about 60%,38%,and 2%.The chromatogram obtained from Standard solution 2exhibits two resolved peaks due to iodixanol related compound Dthat elute before the iodixanol peaks and one iodixanol related compound Cpeak between the two principal iodixanol peaks.The area of the two iodixanol related compound Dpeaks is between 0.075%and 0.125%of the total area.[NOTE—Disregard any peak due to the solvent front and any peak corresponding to those obtained from the Blank solution.]Add the areas of the two isomer peaks for iodixanol related compound Dfrom each of the three injections of Standard solution 2,and calculate the relative standard deviation for the three summed areas:the relative standard deviation is not more than 5%.Measure the height of the iodixanol related compound Cpeak,and,if necessary,adjust the sensitivity of the amplifier to obtain a peak height between 80%and 100%of the full scale.Measure the height,A,above the baseline of the iodixanol related compound Cpeak and the height,B,above the baseline of the lowest part of the curve separating this peak from the first principal iodixanol peak:Ais not less than 1.3B.In the chromatogram obtained from the Control solution,iodixanol related compound Cexhibits a measurable peak.

Procedure— Separately inject equal volumes (about 10µL)of the Blank solution,Test solution 1,and Test solution 2.

HIGH-LOW CHROMATOGRAPHY— Where it is specified to proceed as directed for High-low chromatography,for the chromatogram obtained from Test solution 1,calculate the percentage of each specified related compound in the portion of Iodixanol taken by the formula:

(10X)/(0.1Y+Z),

in which Xis the peak area for each of the specified related compounds obtained from Test solution 1;Yis the total area of all the peaks eluted before and after iodixanol obtained from Test solution 1,disregarding any peaks due to injection noise or solvent;and Zis the sum of peak areas of iodixanol and any related compounds which are eluted together with,and between,the principal iodixanol peaks obtained from Test solution 2.

IOHEXOL— If iohexol is present,it exhibits two peaks,with retention times of about 0.37and 0.39relative to the main iodixanol peak,in the chromatogram obtained from Test solution 1.Draw a baseline at the height of the baseline obtained from the Blank solution.Calculate the total area of the two peaks and the percentage of iohexol in the portion of Iodixanol taken as directed for High-low chromatography.

IODIXANOL RELATED COMPOUND B1— If iodixanol related compound Bis present,it elutes as a single peak with a retention time of about 0.34relative to the main iodixanol peak,in the chromatogram obtained from Test solution 1.Draw a baseline at the height of the baseline obtained from the Blank solution.Calculate the area of the peak and the percentage of iodixanol related compound Bin the portion of Iodixanol taken as directed for High-low chromatography.

IODIXANOL RELATED COMPOUND C— If iodixanol related compound Cis present,only the first and larger peak,with a retention time of about 1.07relative to the main iodixanol peak,is seen between the two principal iodixanol peaks in the chromatogram obtained from Test solution 1;the second iodixanol related compound Cpeak co-elutes with iodixanol.The area of the first and larger peak corresponds to about 80%of the total area of iodixanol related compound C.Draw a vertical line through the minimum before the first and larger peak.Draw a horizontal baseline at the minimum after the first and larger peak.This encompasses the iodixanol related compound Cpeak area,X2.Calculate the percentage of iodixanol related compound Cin the portion of Iodixanol taken by the formula:

12.5X2/(0.1Y+Z),

in which Yand Zare as defined for High-low chromatography.

IODIXANOL RELATED COMPOUND F2— If iodixanol related compound Fis present,only the first and smaller peak with a retention time of about 0.8relative to the main iodixanol peak,can be seen in the chromatogram obtained from Test solution 1;the second peak co-elutes with iodixanol.The area of the first and smaller peak corresponds to about 25%of the total area of iodixanol related compound F.Draw the baseline at the height of the baseline obtained from the Blank solution.Calculate the percentage of iodixanol related compound Fin the portion of Iodixanol taken by the formula:

40X1/(0.1Y+Z),

in which X1is the actual observed area of the peak of iodixanol related compound Fobtained from Test solution 1;and Yand Zare as defined for High-low chromatography.

IODIXANOL RELATED COMPOUND G3— If iodixanol related compound Gis present,the second and larger peak,with a retention time of about 1.18relative to the last iodixanol peak,is seen in the chromatogram obtained from Test solution 1;the first peak co-elutes with iodixanol.The area of the second peak corresponds to about 85%of the total area of iodixanol related compound G.Draw the baseline at the height of the baseline obtained from the Blank solution.Calculate the percentage of iodixanol related compound Gin the portion of Iodixanol taken by the formula:

10X3/[0.85(0.1Y+Z)],

in which X3is the peak area of iodixanol related compound G;and Yand Zare as defined for High-low chromatography.

OVERALKYLATED RELATED COMPOUNDS— These compounds elute after iodixanol related compound G,with a retention time greater than 1.18relative to the last iodixanol peak.Draw the baseline at the height of the baseline obtained from the Blank solution,and determine the peak areas.Calculate the percentage of overalkylated related compounds as directed for High-low chromatography.

UNSPECIFIED RELATED COMPOUNDS— Examine the chromatograms obtained from Test solution 1and the area of each peak eluting before or after iodixanol,other than those of iodixanol,specified related compounds,specified impurities,and overalkylated related compounds.Draw the baseline at the height of the baseline obtained from the Blank solution.Calculate the percentage of the largest of these peaks as directed for High-low chromatography.

OTHER UNSPECIFIED RELATED COMPOUNDS— Determine the area of any unspecified peak eluting between those of iodixanol.Draw the baseline between minima,and calculate the percentage as directed for High-low chromatography.

Not more than 0.2%of iodixanol related compound Bis found;not more than 0.4%of iodixanol related compound Cis found;not more than 0.2%of iodixanol related compound Fis found;not more than 0.2%of iodixanol related compound Gis found;not more than 0.6%of iohexol is found;not more than 1.0%of overalkylated related compounds is found;not more than 0.2%of any individual unspecified related compound is found;not more than 0.5%of total unspecified related compounds is found;and not more than 1.5%of total related compounds is found.

TOTAL RELATED COMPOUNDS— From each of the chromatograms obtained from Test solution 1,calculate the percentage of all related compounds as the sum of the results for the peaks appearing between the two principal iodixanol peaks,and the area percent obtained by the formula:

[100(Y–X1–X3+X1/0.25+X2/0.8+X3/0.85)]/10(0.1Y+Z),

in which the variables are as defined above.

TEST2—

Solution A— Use 1000mLof filtered and degassed acetonitrile.

Solution Band Mobile phase— Proceed as directed for Test 1.

Blank solution— Use water.

Standard stock solution 1— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol RSwith water to obtain a solution having a known concentration of about 12.5mg of anhydrous iodixanol per mL.

Standard stock solution 2— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol Related Compound D RS,equivalent to about 0.125g of anhydrous iodixanol related compound D,in water,and dilute with water to 100.0mL.Dilute 2.0mLof this solution with water to 100.0mL.

Standard stock solution 3— Quantitatively dissolve an accurately weighed quantity of USP Iodixanol Related Compound E RSin water to obtain a solution having a known concentration of about 2.5mg per mL.

Standard solution 1— Dilute 2.0mLof Standard stock solution 1with water to 10.0mL.

Standard solution 2— Transfer 5.0mLof Standard stock solution 1and 2.5mLof Standard stock solution 2to a 25-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution 3— Transfer 1.0mLof Standard solution 1and 1.0mLof Standard stock solution 3to a 10-mLvolumetric flask,dilute with water to volume,and mix.

Test solution— Dissolve an accurately weighed quantity of Iodixanol,equivalent to about 125mg of anhydrous iodixanol,in water,dilute with water to 50.0mL,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L8.The flow rate is about 2.5mLper minute.The chromatograph is programmed as follows.

Time (minutes)	Solution A (%)	Solution B (%)	Elution
0	85	15	equilibration
0–25	85®66	15®34	linear gradient

Chromatograph the solutions as directed for Procedure.The chromatogram obtained from Standard solution 1exhibits three principal unresolved peaks:the relative areas are about 62%,35%,and 3%;and the retention time of the last iodixanol peak is not more than 14minutes.

The chromatogram obtained from Standard solution 2exhibits two partially unresolved peaks due to iodixanol related compound D,with relative retention times of about 0.33and 0.39,that elute before the iodixanol peaks:the peak area of iodixanol related compound Dis between 0.075%and 0.125%of the total area.Disregard any peak due to the solvent.

Determine the sum of the peak areas of the two isomers of iodixanol related compound Dfor each of the three chromatograms obtained from Standard solution 2:the relative standard deviation for replicate injections is not more than 5%.

The chromatogram obtained from Standard solution 3exhibits two unresolved peaks due to iodixanol related compound E,with relative retention times of about 0.67and 0.72,that elute before the iodixanol peaks.Adjust the sensitivity of the amplifier so that the peak heights are between 90%and 100%of full scale of the highest peak:the resolution,R,between the first and largest iodixanol related compound Epeak and the first principal iodixanol peak is not less than 5.0.

Procedure— Separately inject equal volumes (about 10µL)of Standard solution 1,three times Standard solution 2,Standard solution 3,and the Test solution.For the first chromatogram obtained from Standard solution 2,adjust the sensitivity of the amplifier to obtain a peak height of about 15%of the first and larger peak that corresponds to iodixanol related compound D.Use this sensitivity setting for the subsequent injections.

Compare the retention times of the peaks obtained from Standard solution 3to those obtained from the Test solution.Iodixanol related compound Eexhibits two peaks,the second of which may partly overlap with another peak;use only the area of the first and larger peak,which corresponds to about 60%of the total area of iodixanol related compound E.Draw a baseline at the height of the baseline obtained from the Blank solution.Calculate the percentage of iodixanol related compound Eby dividing the area obtained from the Test solutionby 0.6and using area percent.

Iodixanol related compound H4appears as a single peak,with a shoulder,on the tail of the iodixanol peak.Calculate the percentage of iodixanol related compound Hby using area percent.Not more than 0.3%of iodixanol related compound Eis found;and not more than 0.6%of iodixanol related compound His found.

Assay— Transfer about 500mg of Iodixanol,accurately weighed,to a glass-stoppered,125-mLconical flask,add 25mLof a sodium hydroxide solution (5in 100)and 500mg of powdered zinc,connect the flask to a reflux condenser,and reflux for 1hour.Cool the flask to room temperature,and rinse the condenser with 20mLof water,adding the rinsing to the refluxed solution.Filter the mixture,rinsing the flask and the filter with several small portions of water,and adding the filtered rinsings to the filtrate.Add 5mLof glacial acetic acid,titrate with 0.1Nsilver nitrate VS,and determine the endpoint potentiometrically.Each mLof 0.1Nsilver nitrate is equivalent to 25.84mg of C35H44I6N6O15.

1 5-(acetylamino)-N,N¢-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide

2 2-[[acetyl[3,5-bis[[(2,3-dihydroxypropyl)amino]carbonyl]-2,4,6-triiodophenyl]amino]methyl]-N,N¢-bis(2,3-dihydroxypropyl)-2,3-dihydro-5,7-diiodo-4H-1,4-benzoxazine-6,8-dicarboxamide

3 4-acetyl-2-[[acetyl[3,5-bis[[(2,3-dihydroxypropyl)amino]carbonyl]-2,4,6-triiodophenyl]amino]methyl]-N,N¢-bis(2,3-dihydroxypropyl)-2,3-dihydro-5,7-diiodo-4H-1,4-benzoxazine-6,8-dicarboxamide

4 5-[[3-[[3-[[[3-[[3-[[3,5-bis-[[[2,3-dihydroxypropyl]amino]carbonyl]-2,4,6-triiodophenyl](acetylimino)]-2-hydroxypropyl](acetylimino)]-5-[[[2,3-dihydroxypropyl]amino]carbonyl]-2,4,6-triiodophenyl]carbonyl]amino]-2-hydroxypropyl]oxy]-2-hydroxypropyl](acetylimino)]-N,N¢-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzendicarboxamide

Auxiliary Information— Staff Liaison:Andrzej Wilk,Ph.D.,Senior Scientific Associate

Expert Committee:(RMI)Radiopharmaceuticals and Medical Imaging Agents

USP28–NF23Page 1039

Pharmacopeial Forum:Volume No.29(6)Page 1908

Phone Number:1-301-816-8305