A: Infrared Absorption á197Kñ.

B: Thin-Layer Chromatographic Identification Test á201ñ—

Test solution: 5mg per mL,in acetone.

Developing solvent system: a mixture of ethyl acetate and n-heptane (1:1).

Procedure— Proceed as directed in the chapter except use an unsaturated developing chamber.

Change to read:

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

Standard solution— Dissolve accurately weighed quantities of USP Diazepam Related Compound B RS,USP28USP Diazepam Related Compound A RS,and USP Nordazepam RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having known concentrations of about 1µg per mL,0.1µg per mL,and 3µg per mL,respectively.

Test solution— Transfer about 10mg of Diazepam,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of diazepam related compound B,USP28diazepam related compound A,and nordazepam in the portion of Diazepam taken by the formula: in which CRis the concentration,in µg per mL,of USP Diazepam Related Compound B RS,USP28USP Diazepam Related Compound A RS,or USP Nordazepam RSin the Standard solution;Wis the weight,in mg,of Diazepam taken to prepare the Test solution;and rUand rSare the peak responses obtained from the Test solutionand the Standard solution,respectively:not more than 0.01%of diazepam related compound A,not more than 0.1%of diazepam related compound B,USP28and not more than 0.3%of nordazepam are found.

Solvent— Use dimethyl sulfoxide.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile,water,and methanol (2:2:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Dissolve suitable quantities of USP Nordazepam RSand USP Diazepam RSin methanol,using sonication if necessary,to obtain a solution containing about 0.1mg of each per mL.

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

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

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×15-cm column that contains packing L1.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 0.76for nordazepam and 1.0for diazepam;the resolution,R,between nordazepam and diazepam is not less than 4;the column efficiency is not less than 5000theoretical plates for the diazepam peak;the tailing factor for diazepam is not more than 2.0;and the relative standard deviation for the diazepam peak for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)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 C16H13ClN2Oin the portion of Diazepam taken by the formula: in which Cis the concentration,in mg per mL,of USP Diazepam RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.