» Cyclophosphamide Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of anhydrous cyclophosphamide (C7H15Cl2N2O2P).
Packaging and storage Preserve in tight containers. Storage at a temperature not exceeding 25 is recommended. Tablets will withstand brief exposure to temperatures up to 30, but are to be protected from temperatures above 30.
USP Reference standards 11
A: Extract a portion of finely powdered Tablets, equivalent to about 50 mg of cyclophosphamide, with 25 mL of chloroform, filter about 2 mL of the chloroform solution, mix the filtrate with 500 mg 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 IR absorption spectrum of the potassium bromide dispersion so obtained exhibits maxima, between 6.5 and 14 µm, 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 preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Medium: water; 900 mL, deaerated.
Apparatus: 1:100 rpm.
Time: 45 minutes.
Determine the amount of C7H15Cl2N2O2P dissolved by employing the following method.
Mobile phase Prepare a suitable filtered and degassed mixture of water and acetonitrile (7:3). Make adjustments if necesary (see System Suitability under Chromatography 621).
Standard solution Dissolve an accurately weighed quantity of USP Cyclophosphamide RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration corresponding to that of the solution under test.
Test solution Use portions of the solution under test passed through a 0.8-µm filter.
Chromatographic system (see Chromatography 621) 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 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure Separately inject equal volumes (about 50 µL) of the Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peak. Calculate the amount of cyclophosphamide (C7H15Cl2N2O2P) dissolved by the formula:
900C(rU / rS)in which C is the concentration, in mg per mL, of USP Cyclophosphamide RS in the Standard solution; and rU and rS are the peak responses for cyclophosphamide obtained from the Test solution and Standard solution, respectively.
Tolerances Not less than 75% (Q) of the labeled amount of cyclophosphamide (C7H15Cl2N2O2P) is dissolved in 45 minutes.
Uniformity of dosage units 905: meet the requirements.
procedure for content uniformity
Perchloric acid solution Dissolve 23.5 mL of perchloric acid in water, and dilute with water to 1 L.
4-(p-Nitrobenzyl)pyridine solution Dissolve 1.5 g of 4-(p-nitrobenzyl)pyridine in 200 mL of ethylene glycol.
Sodium hydroxide solution Dissolve 20 g of sodium hydroxide in 1000 mL of diluted alcohol.
Procedure Place 1 Tablet in a volumetric flask of suitable size so that the final concentration is about 500 µg 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 10 mL of the filtrate. Place in separate 27-mm × 170-mm test tubes 2.0 mL of the filtrate, 2.0 mL of water to provide a blank, and 2.0 mL of the Standard solution, prepared by dissolving an accurately weighed quantity of USP Cyclophosphamide RS in water and diluting quantitatively and stepwise with water to obtain a solution having a known concentration of about 500 µg per mL. Treat each tube as follows. Add 0.7 mL of Perchloric acid solution, mix, and heat at 95 for 10 minutes. Cool, add 1.0 mL of sodium acetate TS, mix, add 1.6 mL of 4-(p-Nitrobenzyl)pyridine solution, mix, and heat at 95 for 10 minutes. Cool, add 8.0 mL of Sodium hydroxide solution, and mix. Within 4 minutes, determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at 560 nm, with a suitable spectrophotometer, against the blank. Calculate the quantity, in mg, of C7H15Cl2N2O2P in the Tablet taken by the formula:
(T/500)C(AU / AS)in which T is the labeled quantity, in mg, of anhydrous cyclophosphamide in the Tablet; C is the concentration, in µg per mL, of USP Cyclophosphamide RS, corrected for moisture by a titrimetric water determination, in the Standard solution; and AU and AS are 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 Assay under Cyclophosphamide.
Assay preparation Transfer not fewer than 10 Tablets to a volumetric flask of suitable size so that the final concentration is about 1 mg of anhydrous cyclophosphamide per mL. Fill about half full with water, shake for 30 minutes, dilute with water to volume, and mix. Filter through fast, fluted filter paper, discarding the first 40 to 50 mL of the filtrate. Pipet 25 mL of the filtrate and 5 mL of Internal standard solution into a 50-mL volumetric flask, dilute with water to volume, and mix.
Chromatographic system Proceed as directed for Chromatographic system in the Assay under Cyclophosphamide.
Procedure Proceed as directed for Procedure in the Assay under Cyclophosphamide. Calculate the quantity, in mg, of C7H15Cl2N2O2P per Tablet taken by the formula:
(2CV/N)(RU / RS)in which C is the concentration, in mg per mL, of anhydrous cyclophosphamide in the Standard preparation, as determined from the concentration of USP Cyclophosphamide RS corrected for moisture by a titrimetric water determination; V is the volume, in mL, of the volumetric flask to which the N Tablets were transferred; N is the number of Tablets taken; and RU and RS are the ratios of the peak responses of cyclophosphamide to those of the internal standard in the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2790Pharmacopeial Forum: Volume No. 30(4) Page 1186