A: Infrared Absorption á197Kñ.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,both relative to the internal standard,as obtained in the Assay.

Standard sodium chloride solution— Transfer about 118.7mg of sodium chloride,accurately weighed,to a 200-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.This solution contains 360ppm of ionic chloride.

Procedure— Pipet 10mLof Standard sodium chloride solutioninto a beaker,and add 90mLof water and 10mLof acetic acid.Titrate with 0.01Nsilver nitrate VS(prepared fresh daily),determining the endpoint potentiometrically using silver and silver-silver chloride electrodes.Record the volume,V1,of 0.01Nsilver nitrate VSconsumed.Transfer about 2.0g of Ifosfamide,accurately weighed,into a beaker,and add 90mLof water and 10mLof acetic acid.Pipet 10mLof Standard sodium chloride solutioninto the beaker,and stir,if necessary,until solution is complete.Titrate with 0.01Nsilver nitrate VSas directed above,and record the volume,V2,of 0.01Nsilver nitrate VSconsumed.Calculate the difference in volume,V,of 0.01Nsilver nitrate VSconsumed between the two determinations by subtracting V1from V2:a difference of not more than 1.0mLcorresponding to not more than 0.018%of ionic chloride is found.

Ammonium molybdate solution— [NOTE—Prepare fresh on the day of use.]Dissolve 25g of ammonium molybdate in 300mLof water (Solution A).Cautiously add 75mLof sulfuric acid to 100mLof water,cool to room temperature,and dilute with water to 200.0mL(Solution B).Mix Solution Aand Solution Bto obtain Ammonium molybdate solution.

Hydroquinone solution— Dissolve 0.5g of hydroquinone in 100mLof water,and add one drop of concentrated sulfuric acid.[NOTE—When this solution darkens,discard it and prepare fresh.]

Sodium sulfite solution— Prepare a solution of sodium sulfite in water having a concentration of 200mg per mL.[NOTE—Prepare fresh at the time of use.]

Phosphorus stock solution— Transfer 0.1824g of monobasic potassium phosphate,accurately weighed,to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Phosphorus intermediate solution— Transfer 10.0mLof Phosphorus stock solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix.Prepare this solution fresh on the day of use.

Phosphorus standard solution— Transfer 10.0mLof Phosphorus intermediate solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix.

Test preparation— Transfer 1g of Ifosfamide,accurately weighed,to a 100-mLvolumetric flask,dissolve in 50mLof water,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a separatory funnel,and add 5mLof water.Add 15mLof chloroform,shake vigorously for 30seconds,allow the layers to separate and drain,and discard the lower chloroform layer.Repeat this extraction four times,each time with 15mLof chloroform,discarding the chloroform layer after each extraction.Transfer the aqueous portion to a conical flask,wash the separatory funnel with two 5-mLportions of water,and collect all the aqueous washings in the same flask.Add 3mLof sulfuric acid,and heat under a hood until white fumes appear.Remove the flask from the heat,and with swirling,add 0.6mLof hydrogen peroxide.Heat until white fumes reappear.If the solution is not colorless,repeat additions of hydrogen peroxide followed by heating until all color is gone.Cool to room temperature,add 25mLof water,and cautiously add 10mLof ammonium hydroxide solution.Cool to room temperature,add 2drops of phenolphthalein TS,and then add hydrochloric acid dropwise until all pink color has disappeared.Transfer the contents of the flask to a 100-mLflask,dilute with water to volume,and mix.

Blank solution— To 3mLof sulfuric acid in a second conical flask,adding 0.6mLof hydrogen peroxide,proceed as directed for the Test preparation,beginning with “Heat until white fumes reappear.”

Procedure— Transfer 15.0mLeach of the Test preparation,the Blank solution,and the Phosphorus standard solutionto three separate 25-mLvolumetric flasks.Add 2.5mLof Ammonium molybdate solutionto each of the flasks,swirl,and allow to stand for about 30seconds.To each of the three flasks in order,rapidly add 2.5mLeach of Hydroquinone solutionand Sodium sulfite solution.Dilute the contents of each flask with water to volume,mix,and allow the flasks to stand for 30minutes.Concomitantly determine the absorbances of the solutions obtained from the Test preparationand the Phosphorus standard solutionin 1-cm cells at the wavelength of maximum absorbance at about 730nm,with a suitable spectrophotometer,using the solution obtained from the Blank solutionas the blank.Calculate the percentage of chloroform-insoluble phosphorus in the portion of Ifosfamide taken by the formula: in which Cis the concentration,in µg per mL,of phosphorus in the Phosphorus standard solution,Wis the weight,in mg,of Ifosfamide taken,and AUand ASare the absorbances from the solutions obtained from the Test preparationand the Phosphorus standard solution,respectively:not more than 0.0415%is found.

Standard solution— Dissolve an accurately weighed quantity of 2-chloroethylamine hydrochloride in N,N-dimethylacetamide,and dilute quantitatively,and stepwise if necessary,with the same solvent to obtain a solution having a known concentration of about 0.025mg per mL.

Test solution— Transfer about 100mg of Ifosfamide,accurately weighed,to a flask,add 10.0mLof N,N-dimethylacetamide,and shake until dissolved.

Chromatographic system— The gas chromatograph is equipped with a flame-ionization detector and contains a 2-mm ×1.8-m column packed with 10%liquid phase G16containing 2%potassium hydroxide on 80-to 100-mesh support S1A.The injection port is maintained at a temperature of about 200,the detector is maintained at a temperature of about 300,the oven is maintained at a temperature of about 140,and nitrogen is used as the carrier gas at a flow rate of about 25mLper minute.

Procedure— Separately inject equal volumes (about 1.0µL)of the Test solutionand the Standard solutioninto the gas chromatograph,record the chromatograms,and measure the areas of the peaks due to 2-chloroethylamine hydrochloride.Calculate the percentage of 2-chloroethylamine hydrochloride in the portion of Ifosfamide taken by the formula: in which Cis the concentration,in mg per mL,of 2-chloroethylamine hydrochloride in the Standard solution,Wis the weight,in mg,of Ifosfamide taken,and rUand rSare the areas of the 2-chloroethylamine peaks obtained from the Test solutionand the Standard solution,respectively:not more than 0.25%of 2-chloroethylamine hydrochloride is found.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (70:30).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer about 50mg of ethylparaben,accurately weighed,to a 100-mLvolumetric flask,and add 25mLof alcohol to dissolve.Dilute with water to volume,and mix.

Standard preparation— Transfer about 15mg of USP Ifosfamide RS,accurately weighed,to a 25-mLvolumetric flask,add 1.0mLof Internal standard solution,dilute with water to volume,and mix.

Assay preparation— Transfer about 150mg of Ifosfamide,accurately weighed,to a 250-mLvolumetric flask,add 10.0mLof Internal standard solution,dilute with water to volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 195-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between ifosfamide and ethylparaben is not less than 6.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of C7H15Cl2N2O2Pin the portion of Ifosfamide taken by the formula: in which Cis the concentration,in mg per mL,of USP Ifosfamide RSin the Standard preparation;and RUand RSare the ratios of the responses of the ifosfamide peak to the ethylparaben peak obtained from the Assay preparationand the Standard preparation,respectively.