A: Thin-Layer Chromatographic Identification Test á201ñ—

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.

Mobile phase— Prepare as directed in the Assay.

Resolution solution— Prepare a solution of USP Valrubicin RSand USP Related Compound A RSin methanol to obtain a solution having concentrations of about 0.2mg per mLand 0.05mg per mL,respectively.

Test solution— Use the Assay preparation,prepared as directed in the Assay.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector,a guard column,and a 5-mm ×10-cm analytical column that contains 4-µm packing L1.The flow rate is about 2.5mLper minute.Chromatograph the Resolution solution,and record the peak areas as directed for Procedure:the relative retention times are about 0.9for valrubicin related compound Aand 1.0for valrubicin;and the resolution,R,between valrubicin related compound Aand valrubicin is not less than 2.

Procedure— Inject a volume (about 10µL)of the Test solutioninto the chromatograph,record the chromatogram,and measure the areas for the major peaks.Calculate the percentage of each impurity in the portion of Intravesical Solution taken by the formula: in which riis the peak area for each impurity;and rsis the sum of the peak areas of all the peaks.Do not consider any peaks due to solvent or excipients.Not more than 0.5%of any impurity with a relative retention time of about 0.11is found;not more than 0.8%of any individual impurity with a relative retention time of 0.16,0.51or 0.71is found;not more than 0.5%of any other individual impurity is found;and the sum of all impurities is not more than 3.5%.

Mobile phase— Prepare a filtered and degassed mixture of 0.1Mammonium formate,previously adjusted with formic acid and acetonitrile (55:45)to a pHof 4.0.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Valrubicin RSin methanol,and quantitatively dilute with methanol to obtain a solution having a known concentration of about 0.2mg per mL.

Assay preparation— Transfer an accurately measured volume of Intravesical Solution,equivalent to about 20mg of valrubicin,to a 100-mLvolumetric flask,dissolve in methanol,dilute with methanol to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector,a guard column,and a 5-mm ×10-cm analytical column that contains 4-µm packing L1.The flow rate is about 2.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of valrubicin (C34H36F3NO13)in each mLof the Intravesical Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Valrubicin RSin the Standard preparation;Pis the specified percentage of valrubicin in USP Valrubicin RS;Vis the volume,in mL,of Intravesical Solution taken to prepare the Assay preparation;and rUand rSare the valrubicin peak responses obtained from the Assay preparationand the Standard preparation,respectively.