» Bumetanide Injection is a sterile solution of Bumetanide in Water for Injection, prepared with the aid of Sodium Hydroxide. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of bumetanide (C17H20N2O5S).
Packaging and storage Preserve in single-dose or multiple-dose containers, preferably of Type I glass, protected from light.
USP Reference standards 11
USP Endotoxin RS
A: The relative retention time of the major peak in the chromatogram of the Assay preparation corresponds 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 solution exhibits an RF value corresponding to that of the Identification solution, as obtained in the test for Related compounds.
Bacterial endotoxins 85 It contains not more than 350 USP Endotoxin Units per mg of bumetanide.
pH 791: between 6.8 and 7.8.
Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.
Test solution Pipet a volume of Injection, equivalent to 5 mg of bumetanide, into a 125-mL separator, and adjust with 0.1 N sodium hydroxide to a pH of 12. Extract with two 20-mL portions of ethyl ether, discard the ethyl ether extracts, and adjust the aqueous layer with 1 N acetic acid to a pH of 4. Extract with two 20-mL portions of ethyl ether, passing the extracts through anhydrous sodium sulfate. Wash the sodium sulfate with about 5 mL of ethyl ether. Evaporate the combined ethyl ether extracts with the aid of a stream of nitrogen to dryness, and dissolve the residue in 0.5 mL of methanol.
Identification solution Dissolve USP Bumetanide RS in methanol to obtain a solution having a concentration of about 10 mg 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 0.08 mg of USP Bumetanide RS per 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 RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.02 mg per mL.
Application volume: 50 µL.
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. Examine the plate under short-wavelength UV light. Any secondary spot obtained from the chromatogram of the Test solution having an RF value corresponding to the RF value of the principal spot obtained from the chromatogram of Standard solution 6 is 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 A is 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 1 through 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).
Other requirements It meets the requirements under Injections 1.
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 Suitability under Chromatography 621).
Internal standard solution Transfer about 50 mg of 4-ethylbenzaldehyde to a 100-mL volumetric flask. Dissolve in and dilute with methanol to volume, and mix. Transfer 10.0 mL of the resulting solution to a 100-mL volumetric flask, add 10.0 mL of tetrahydrofuran and 4.0 mL of glacial acetic acid, dilute with methanol to volume, and mix.
Standard preparation Dissolve an accurately weighed quantity of USP Bumetanide RS in Internal standard solution, and quantitatively dilute with Internal standard solution to obtain a solution having a known concentration of about 250 µg per mL. Transfer 5.0 mL of the resulting solution to a 10-mL volumetric flask, dilute with water to volume, and mix to obtain a solution having a known concentration of about 125 µg of USP Bumetanide RS per mL.
Assay preparation Transfer an accurately measured volume of Injection, equivalent to about 0.25 mg of bumetanide, to a flask. Add an equal volume of Internal standard solution, accurately measured, insert the stopper, and mix.
Chromatographic system (see Chromatography 621) 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 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for 4-ethylbenzaldehyde and 1.0 for 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 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C17H20N2O5S in each mL of the Injection taken by the formula:
(2C / V)(RU / RS)in which C is the concentration, in mg per mL, of USP Bumetanide RS in the Standard preparation; V is the volume, in mL, of Injection taken; and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2397Pharmacopeial Forum: Volume No. 27(6) Page 3252