(kla' dri been).
» Cladribine contains not less than 98.0 percent and not more than 102.0 percent of C10H12ClN5O3, calculated on the anhydrous basis.
Packaging and storage Preserve in well-closed containers, and protect from light. Store between 2 and 8.
USP Reference standards 11
USP Cladribine Related Compound A 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.
Specific rotation 781S: between 17.0 and 21.0.
Test solution: 10 mg per mL, in dimethylformamide.
Water, Method I 921: not more than 4.0%.
Residue on ignition 281: not more than 0.1%.
Heavy metals, Method II 231: 0.002%.
Buffer, Diluent, Mobile phase, System suitability solution, and Chromatographic system Proceed as directed in the Assay.
Test solution Use the Assay preparation.
Chromatographic system (see Chromatography 621) Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between cladribine and cladribine related compound A is not less than 1.5; and the tailing factor is not more than 2.0.
Procedure Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of each impurity in the portion of Cladribine taken by the formula:
100(ri / rs)in which ri is the peak response of each individual impurity, and rs is the sum of the responses of all the peaks. Refer to Table 1 for the impurity limits.
Buffer Dissolve 9.96 g of triethylamine phosphate, accurately weighed, in 500 mL of water, and add another 500 mL of water. Adjust with potassium hydroxide to a pH of 6.1.
Diluent Prepare a mixture of water and methanol (90:10).
Mobile phase Prepare a filtered and degassed mixture of Buffer and methanol (78:22). Make adjustments if necessary (see System Suitability under Chromatography 621).
System suitability solution Prepare a solution of USP Cladribine RS and USP Cladribine Related Compound A RS in Diluent to obtain known concentrations of about 0.02 mg per mL each.
Standard preparation Dissolve an accurately weighed quantity of USP Cladribine RS in Diluent to obtain a concentration of 0.5 mg per mL.
Assay preparation Transfer about 25 mg of Cladribine, accurately weighed, to a 50-mL volumetric flask, dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability solution and the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between cladribine and cladribine related compound A is not less than 1.5; the tailing factor for the cladribine peak in the System suitability solution is not more than 2.0; and the relative standard deviation for replicate injections of the Standard preparation is not more than 2.0%.
Procedure Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C10H12ClN5O3 in the portion of Cladribine taken by the formula:
50C(rU / rS)in which C is the concentration, in mg per mL, of USP Cladribine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2690Pharmacopeial Forum: Volume No. 33(1) Page 49