A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,both relative to the internal standard,as obtained in the Assay.

B: Transfer a volume of Injection,equivalent to about 10mg of diazepam,to a separator,add 20mLof water,and shake.Add 20mLof chloroform,and shake vigorously for 2minutes.Filter the chloroform layer through about 5g of anhydrous granular sodium sulfate into a beaker.Wash the sodium sulfate with 20mLof chloroform,collecting the washing in the beaker.Evaporate the chloroform extract on a steam bath with the aid of a current of air to a volume of about 5mL.Remove the beaker from the steam bath,and evaporate the chloroform extract with the aid of a current of air to dryness.Dissolve the residue in 20mLof anhydrous ether,filter,and evaporate the filtrate to dryness using a current of air.Vigorously scrape the resulting oily film with a spatula,and dry in vacuum over phosphorus pentoxide at 60for 4hours:the IRabsorption spectrum of a potassium bromide dispersion of the residue exhibits maxima only at the same wavelengths as that of a similar preparation of USP Diazepam RS.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (65:35).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— [NOTE—Prepare fresh daily.]Prepare a solution of p-tolualdehyde in methanol containing about 0.3µLper 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 1mg per mL.Transfer 5.0mLof this solution to a 25-mLvolumetric flask,add 5.0mLof Internal standard solution,dilute with methanol to volume,and mix to obtain a Standard preparationhaving a known concentration of about 0.2mg of USP Diazepam RSper mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 10mg of diazepam,to a 50-mLvolumetric flask.Pipet 10mLof Internal standard solutioninto the flask,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 ×30-cm column that contains packing L1.The flow rate is about 1.4mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the relative retention times are about 0.5for p-tolualdehyde and 1.0for diazepam,the tailing factor for the diazepam peak is not more than 2.5,the resolution,R,between the p-tolualdehyde and diazepam peaks is not less than 3.5,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (between 10µLand 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 C16H13ClN2Oin each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Diazepam RSin the Standard preparation,Vis the volume,in mL,of Injection taken,and RUand RSare the ratios of the peak responses of diazepam to that of p-tolualdehyde obtained from the Assay preparationand the Standard preparation,respectively.