A:Infrared Absorption á197Mñ.

B:Ultraviolet Absorption á197Uñ—

C: The principal spot obtained from the chromatogram of the Test preparationexhibits an RFvalue corresponding to that in the chromatogram of Standard solution 1,as obtained in the test for Related compounds.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Dissolve an accurately weighed quantity of Bumetanide in methanol to obtain a solution having a concentration of about 25mg per mL.

Standard solution 1— Dissolve an accurately weighed quantity of USP Bumetanide RSin methanol to obtain a solution having a known concentration of about 25mg per mL.

Standard solution 2— Dilute a volume of Standard solution 1quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.05mg per mL.

Standard solution 3— Dissolve an accurately weighed quantity of USP Bumetanide Related Compound B RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.05mg per mL.

Standard solution 4— Dissolve an accurately weighed quantity of USP Bumetanide Related Compound A RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.025mg per mL.

Standard solution 5— Dissolve an accurately weighed quantity of USP Butyl 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.025mg per mL.

Application volume: 20µLof each solution.

Developing solvent system: a mixture of chloroform,cyclohexane,glacial acetic acid,and methanol (80:10:10:2.5).

Procedure— Proceed as directed for Thin-Layer Chromatographyunder Chromatography á621ñ.After drying the application spots,place the plate in an unlined and unsaturated chromatographic chamber.Examine the plate under short-wavelength UVlight.Any secondary spots obtained from the chromatogram of the Test solutionhaving RFvalues corresponding to the RFvalues of the principal spots obtained from the chromatograms of Standard solutions 3,4,and 5are not larger or more intense than the principal spots obtained from the chromatograms of Standard solutions 3,4,and 5,respectively:not more than 0.2%of bumetanide related compound Bis found;not more than 0.1%of bumetanide related compound Ais found;and not more than 0.1%of butyl 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate is found.No other individual secondary spots obtained from the chromatogram of the Test solutionare larger or more intense than the principal spot obtained from the chromatogram of Standard solution 2:not more than 0.2%of any other individual impurity is found;and not more than 0.4%of the sum of all other impurities is found (excluding bumetanide related compound A,bumetanide related compound B,and butyl 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate).