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: Accurately weigh a quantity of Capsule contents,equivalent to 10mg of diazepam,place in a 50-mLcentrifuge tube,and add 2mLof acetone.Place the centrifuge tube in an ultrasonic bath for 5minutes,remove from the ultrasonic bath,and centrifuge.Using 100µLof the supernatant as the test solution,and 100µLof a solution of USP Diazepam RSin acetone containing 5mg per mLas the Standard solution,proceed as directed in Identificationtest Bunder Diazepam.

Medium: simulated gastric fluid TS,prepared without enzymes;900mL.

Apparatus 1: 100rpm.

Times: 1hour;4hours;8hours;12hours.

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

Buffer solution— Dissolve 77.1g of ammonium acetate in water to make 1000mLof solution,and adjust with ammonium hydroxide to a pHof 8.7.

Standard preparation— Dissolve an accurately weighed quantity of USP Diazepam RSin Dissolution Medium,dilute quantitatively with Dissolutionto obtain a solution having a known concentration of about 0.15mg per mL,and mix.Transfer 2.0-,5.0-,8.0-,and 10.0-mLaliquots of this solution to separate 100-mLvolumetric flasks,add Dissolution Mediumto volume,and mix.Pipet 1.0mLof each solution and 1.0mLof Buffer solutioninto individual small vials,mix,and allow to stand at room temperature for about 10minutes.

Test preparation— Wrap each Capsule in a coil made from a 10-cm piece of 18-gauge copper wire weighing approximately 750mg,so that the wire encircles the Capsule 4times.The Capsule enclosed in the coil remains at the bottom of the basket (it should not float).Filter a portion of the solution under test,obtained at each time interval,through a suitable 0.6-µm porosity filter.Pipet 1.0mLof each solution and 1.0mLof Buffer solutioninto individual small vials,mix,and allow to stand at room temperature for about 10minutes.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and an 8-mm ×10-cm column that contains packing L1.The flow rate is about 5.0mLper minute.Chromatograph the appropriate Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not greater than 1.7;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Determine the amount of C16H13ClN2Odissolved from peak responses of diazepam obtained from the Test preparationand the Standard preparation.

Tolerances— The percentage of the labeled amount of C16H13ClN2Odissolved is within the range stated at each of the following times.

Mobile phase and Chromatographic system—Prepare as directed in the Assayunder Diazepam Capsules.

Internal standard solution— Transfer about 300mg of ethylparaben to a 200-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Diazepam RSin methanol,dilute quantitatively with methanol to obtain a solution having a known concentration of about 1mg per mL,and mix.Transfer 15.0mLof this solution and 5.0mLof Internal standard solutionto a 100-mLvolumetric flask,dilute with methanol to volume,and mix.

Assay preparation— Weigh and mix the contents of not less than 20Capsules.Transfer an accurately weighed portion of the mixture,equivalent to about 15mg of diazepam,to a 100-mLvolumetric flask.Add 5.0mLof Internal standard solutionand about 45mLof methanol.Shake by mechanical means for 30minutes,dilute with methanol to volume,and mix.Centrifuge about 30mLof this solution for 5minutes,and filter.

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.The relative retention times are about 0.5for ethylparaben and 1.0for diazepam.Calculate the quantity,in mg,of C16H13ClN2Oin the portion of Capsules 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 response ratios obtained from the Assay preparationand the Standard preparation,respectively.