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

Test solution— Dissolve an accurately weighed quantity of Clonazepam in acetone to obtain a solution having a concentration of 25mg per mL.

Standard solution— Dissolve an accurately weighed quantity of USP Clonazepam Related Compound C RSin acetone to obtain a solution having a known concentration of 50µg per mL.

Application volume: 20µL.

Developing solvent system: a mixture of acetone and n-heptane (3:2).

Procedure— Proceed as directed for Thin-Layer Chromatographyunder Chromatography á621ñ.After air-drying the plate,heavily spray the plate with 2Msulfuric acid,and dry at 105for 15minutes.Successively spray the plate with 0.01Msodium nitrite,9mMammonium sulfamate,and N-(1-naphthyl)ethylenediamine dihydrochloride TS,and dry the plate with a current of air.Compare the intensities of any secondary spots observed in the chromatogram of the Test solutionwith that of the principal spot in the chromatogram of the Standard solution:no secondary spot from the chromatogram of the Test solutionis larger or more intense than the principal spot obtained from the Standard solution(0.2%).

Buffer solution,Mobile phase,Diluent,System suitability solution,Standard preparation,and Chromatographic system— Proceed as directed in the Assay.

Test preparation— Use the Assay preparation.

Procedure— Inject a volume (about 50µL)of the Test preparationinto the chromatograph,record the chromatogram,and measure the responses for all of the peaks.Calculate the percentage of each impurity in the portion of Clonazepam taken by the formula: in which Pis the relative response factor,which is 1.84for clonazepam related compound A,0.94for clonazepam related compound B,and 1for all other impurities;riis the peak response for each impurity obtained from the Test preparation;and rCis the peak response for clonazepam in the Test preparation:not more than 0.1%of clonazepam related compound Aor of clonazepam related compound Bis found,not more than 0.2%of any other impurity is found,and the sum of all other impurities is not more than 0.3%.

Solvent— Use dimethyl sulfoxide.

Buffer solution— Transfer about 6.6g of anhydrous dibasic ammonium phosphate to a 1-Lvolumetric flask,dissolve in 950mLof water,adjust with 1Nphosphoric acid or 1Nsodium hydroxide to a pHof 8.0,dilute with water to volume,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution,methanol,and tetrahydrofuran (60:52:13).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Prepare a mixture of water,methanol,and tetrahydrofuran (60:52:13).

Standard preparation— Dissolve an accurately weighed quantity of USP Clonazepam RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 0.1mg per mL.

System suitability solution— Dissolve suitable quantities of USP Clonazepam Related Compound A RS,USP Clonazepam Related Compound B RS,and USP Clonazepam RSin Diluentto obtain a solution containing about 0.04mg per mLof each Reference Standard.

Assay preparation— Transfer about 10mg of Clonazepam,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×15-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 2.2for clonazepam related compound A,2.5for clonazepam related compound B,and 1.0for clonazepam;and the resolution,R,between clonazepam related compound Aand clonazepam related compound Bis not less than 2.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.5,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)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 C15H10ClN3O3in the portion of Clonazepam taken by the formula: in which Cis the concentration,in mg per mL,of USP Clonazepam RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.