A: Apply a measured volume of Injection containing 0.025mg of metronidazole and a measured volume of a solution of USP Metronidazole RScontaining 0.025mg of metronidazole to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of chloroform,methanol,water,and ammonium hydroxide (70:28:4:2)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots on the plate by viewing under short-wavelength UVlight:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

B: The retention time of the major peak obtained from the Assay preparationcorresponds to that of the Standard preparationobtained as directed in the Assay.

Mobile phase— Prepare a suitable filtered and degassed mixture of monobasic potassium phosphate,prepared by dissolving 0.68g of monobasic potassium phosphate in 930mLof water,and methanol (930:70),and adjust with 1Mphosphoric acid to a pHof 4.0±0.5.

Standard preparation— Transfer about 25mg of USP Metronidazole RS,accurately weighed,to a 25-mLvolumetric flask,dissolve in methanol,dilute with methanol to volume,and mix.Pipet 2mLof this solution into a 10-mLvolumetric flask containing 2mLof water,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 25mg of metronidazole,to a 25-mLvolumetric flask,dilute with water to volume,and mix.Pipet 2mLof this solution into a 10-mLvolumetric flask containing 2mLof methanol,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 320-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 2.0mLper minute.Chromatograph five replicate injections of the Standard preparation,and record the peak responses as directed under Procedure:the relative standard deviation is not more than 2.0%,and the tailing factor is not greater 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 metronidazole (C6H9N3O3)in each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Metronidazole RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and rUand rSare the peak responses for metronidazole obtained from the Assay preparationand the Standard preparation,respectively.