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,both relative to the internal standard,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.

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

Test solution— Pipet a volume of Injection,equivalent to 5mg of bumetanide,into a 125-mLseparator,and adjust with 0.1Nsodium hydroxide to a pHof 12.Extract with two 20-mLportions of ethyl ether,discard the ethyl ether extracts,and adjust the aqueous layer with 1Nacetic acid to a pHof 4.Extract with two 20-mLportions of ethyl ether,passing the extracts through anhydrous sodium sulfate.Wash the sodium sulfate with about 5mLof ethyl ether.Evaporate the combined ethyl ether extracts with the aid of a stream of nitrogen to dryness,and dissolve the residue in 0.5mLof methanol.

Identification solution— Dissolve USP Bumetanide RSin methanol to obtain a solution having a concentration of about 10mg per mL.

Standard solutions— Dilute a volume of the Identification solution quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.08mg of USP Bumetanide RSper mL.Quantitatively dilute with methanol to obtain Standard solutions having 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 0.02mg per mL.

Application volume: 50µL.

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 á621ñ.Examine the plate under short-wavelength UVlight.Any secondary spot obtained from the chromatogram of the Test solution having 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— Prepare a filtered and degassed mixture of methanol,water,tetrahydrofuran,and glacial acetic acid (50:45:5:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer about 50mg of 4-ethylbenzaldehyde to a 100-mLvolumetric flask.Dissolve in and dilute with methanol to volume,and mix.Transfer 10.0mLof the resulting solution to a 100-mLvolumetric flask,add 10.0mLof tetrahydrofuran and 4.0mLof glacial acetic acid,dilute with methanol to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Bumetanide RSin Internal standard solution,and quantitatively dilute with Internal standard solutionto obtain a solution having a known concentration of about 250µg per mL.Transfer 5.0mLof the resulting solution to a 10-mLvolumetric flask,dilute with water to volume,and mix to obtain a solution having a known concentration of about 125µg of USP Bumetanide RSper mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 0.25mg of bumetanide,to a flask.Add an equal volume of Internal standard solution,accurately measured,insert the stopper,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for 4-ethylbenzaldehyde and 1.0for bumetanide;the resolution,R,between the analyte and internal standard peaks is not less than 1.5,the tailing factor for the analyte peak is not more than 1.4,and the relative standard deviation for replicate injections is not more than 2.0%.

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.Calculate the quantity,in mg,of C17H20N2O5Sin each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Bumetanide RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.