A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

C: The principal spot obtained from the chromatogram of the Test preparation exhibits an RF value 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 25 mg per mL.

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

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

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

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

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

Application volume: 20 µL of 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 Chromatography under Chromatography 621. After drying the application spots, place the plate in an unlined and unsaturated chromatographic chamber. Examine the plate under short-wavelength UV light. Any secondary spots obtained from the chromatogram of the Test solution having RF values corresponding to the RF values of the principal spots obtained from the chromatograms of Standard solutions 3, 4, and 5 are 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 B is found; not more than 0.1% of bumetanide related compound A is 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 solution are 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).

(Official January 1, 2007)