A: The relative 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.

B: The principal spot obtained from the chromatogram of the Test solutionexhibits an RFvalue corresponding to that of the Identification solution,as obtained in the test for Related compounds.

Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

pH2.9Glycine buffer— Dissolve 7.505g of glycine and 5.85g of sodium chloride in water to make 1000mL(stock solution).Dilute 80.0mLof the stock solution and 20.0mLof 0.1Nhydrochloric acid with water to 1000mL.Adjust,if necessary,with 0.1Nhydrochloric acid or 0.1Nsodium hydroxide to a pHof 2.9.

Procedure— Determine the amount of C17H20N2O5Sdissolved,by employing a suitable fluorometer having an excitation wavelength of about 350nm and a fluorescence emission of about 450nm on filtered portions of the solution under test,suitably diluted with pH2.9Glycine buffer,in comparison with a Standard solution having a known concentration of USP Bumetanide RSin the same Medium.

Tolerances— Not less than 85%(Q)of the labeled amount of C17H20N2O5Sis dissolved in 30minutes.

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

Test solution— Transfer an accurately weighed portion of finely powdered Tablets,equivalent to 10mg of bumetanide,to a 50-mLcentrifuge tube,add 20mLof acetone (spectrophotometric or HPLCquality),and shake by mechanical means for 10minutes.Centrifuge for 10minutes,decant the supernatant into a glass-stoppered,25-mLconical flask,and evaporate with the aid of a stream of nitrogen to dryness.Dissolve the residue in 0.5mLof methanol.

Identification solution— Dissolve USP Bumetanide RSin methanol to obtain a solution having a concentration of about 20mg per mL.

Standard solutions— Dilute a volume of the Identification solutionquantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.16mg of USP Bumetanide RSper mL.Quantitatively dilute with methanol to obtain Standard solutionshaving the following compositions.

Standard solution 6— 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 0.04mg per mL.

Application volume: 25µL.

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 ChromatographyunderChromatography á621ñ.Examine the plate under short-wavelength UVlight.Any secondary spot obtained from the chromatogram of the Test solutionhaving an RFvalue corresponding to the RFvalue of the principal spot obtained from the chromatogram of Standard solution 6is not larger or more intense than the principal spot obtained from the chromatogram of Standard solution 6:not more than 0.2%of bumetanide related compound Ais found.For all other secondary spots obtained from the chromatogram of the Test solution,compare the intensity of each spot with the principal spots obtained from the chromatograms of Standard solutions 1through 5:not more than 0.2%of any individual other impurity is found;and not more than 0.8%of the sum of all other impurities is found (excluding bumetanide related compound A).

Mobile phase,Internal standard solution,Standard preparation,andChromatographic system— Prepare as directed in the Assayunder Bumetanide Injection.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 0.5mg of bumetanide,to a 10-mLvolumetric flask,add 2.0mLof Internal standard solution,and sonicate for 5minutes.Add 2.0mLof water,and mix.Cool,and filter,discarding the first 1mLof the filtrate.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.7for 4-ethylbenzaldehyde and 1.0for bumetanide.Calculate the quantity,in mg,of bumetanide (C17H20N2O5S)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Bumetanide RSin the Standard preparation;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.