A: Extract a portion of finely powdered Tablets,equivalent to about 50mg of cyclophosphamide,with 25mLof chloroform,filter about 2mLof the chloroform solution,mix the filtrate with 500mg of potassium bromide,evaporate the chloroform,carefully removing the last trace of solvent in a small vacuum flask,and use the residue to prepare a potassium bromide dispersion:the IRabsorption spectrum of the potassium bromide dispersion so obtained exhibits maxima,between 6.5and 14µm,only at the same wavelengths as that of a similar preparation of USP Cyclophosphamide RS.

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

Procedure for content uniformity—

Perchloric acid solution— Dissolve 23.5mLof perchloric acid in water,and dilute with water to 1liter.

4- (p-Nitrobenzyl)pyridine solution—Dissolve 1.5g of 4-(p-nitrobenzyl)pyridine in 200mLof ethylene glycol.

Sodium hydroxide solution— Dissolve 20g of sodium hydroxide in 1000mLof diluted alcohol.

Procedure— Place 1Tablet in a volumetric flask of suitable size so that the final concentration is about 500µg per mL.Fill the flask about two-thirds full of water,shake until the Tablet is completely disintegrated,dilute with water to volume,and filter,discarding the first 10mLof the filtrate.Place in separate 27-mm ×170-mm test tubes 2.0mLof the filtrate,2.0mLof water to provide a blank,and 2.0mLof the Standard solution,prepared by dissolving an accurately weighed quantity of USP Cyclophosphamide RSin water and diluting quantitatively and stepwise with water to obtain a solution having a known concentration of about 500µg per mL.Treat each tube as follows.Add 0.7mLof Perchloric acid solution,mix,and heat at 95for 10minutes.Cool,add 1.0mLof sodium acetate TS,mix,add 1.6mLof 4-(p-Nitrobenzyl)pyridine solution,mix,and heat at 95for 10minutes.Cool,add 8.0mLof Sodium hydroxide solution,and mix.Within 4minutes,determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at 560nm,with a suitable spectrophotometer,against the blank.Calculate the quantity,in mg,of C7H15Cl2N2O2Pin the Tablet taken by the formula: in which Tis the labeled quantity,in mg,of anhydrous cyclophosphamide in the Tablet,Cis the concentration,in µg per mL,of USP Cyclophosphamide RS,corrected for moisture by a titrimetric water determination,in the Standard solution,and AUand ASare the absorbances of the solution from the Tablet and the Standard solution,respectively.

Mobile phase,Internal standard solution,and Standard preparation— Prepare as directed in the Assayunder Cyclophosphamide.

Assay preparation— Transfer not fewer than 10Tablets to a volumetric flask of suitable size so that the final concentration is about 1mg of anhydrous cyclophosphamide per mL.Fill about half full with water,shake for 30minutes,dilute with water to volume,and mix.Filter through fast,fluted filter paper,discarding the first 40to 50mLof the filtrate.Pipet 25mLof the filtrate and 5mLof Internal standard solutioninto a 50-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system— Proceed as directed for Chromatographic systemin the Assayunder Cyclophosphamide.

Procedure— Proceed as directed for Procedurein the Assayunder Cyclophosphamide.Calculate the quantity,in mg,of C7H15Cl2N2O2Pper Tablet taken by the formula: in which Cis the concentration,in mg per mL,of anhydrous cyclophosphamide in the Standard preparation,as determined from the concentration of USP Cyclophosphamide RScorrected for moisture by a titrimetric water determination;Vis the volume,in mL,of the volumetric flask to which the NTablets were transferred;Nis the number of Tablets taken;and RUand RSare the ratios of the peak responses of cyclophosphamide to those of the internal standard in the Assay preparationand the Standard preparation,respectively.