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

B: Dissolve USP Lorazepam RSin alcohol to obtain a solution having a concentration of 1mg per mL.Transfer 10mLof this solution to a suitable container.Transfer a volume of Injection,equivalent to about 10mg of lorazepam,to a second container.Separately add 5mLof hydrochloric acid to each container,heat each solution on a steam bath for 20minutes,and cool.Transfer the solutions to separators,and add 8mLof 10Nsodium hydroxide to each separator.Extract each solution with two 25-mLportions of ether,filtering the ether extracts through cotton plugs into suitable containers.Evaporate both ether extracts to about 2mL,and add 8mLof methanol to each.Apply separately 10µLof the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Allow the spots to dry,and develop the chromatograms in toluene until the solvent front has moved about 15cm.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Spray the plate with a freshly prepared 1in 80solution of sodium nitrite in 0.5Nhydrochloric acid.Heat the plate at 100for 5minutes,allow to cool,and spray with a 1in 1000solution of N-(1-naphthyl)ethylenediamine dihydrochloride in alcohol:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

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

Standard preparation— Prepare a solution in Mobile phasehaving known concentrations of about 3.2µg each of USP Lorazepam Related Compound C RSand USP Lorazepam Related Compound D RSper mL.

Test preparation— Prepare as directed for Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the peak responses of any peaks observed.Do not include as an impurity any peak observed in the chromatogram of the Test preparationthat has a retention time shorter than that of the lorazepam related compound Dpeak in the Standard preparation.Calculate the percentage of 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxaldehyde (lorazepam related compound C)and the percentage of 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxylic acid (lorazepam related compound D)by the formula: in which CSis the concentration,in µg per mL,of the corresponding component in the Standard preparation;CUis the concentration,in µg per mL,of Lorazepam in the Test preparation;ruis the peak response of lorazepam related compound Cor lorazepam related compound Din the chromatogram obtained from the Test preparation;and rSis the peak response of the corresponding component in the Standard preparation.Calculate the quantity,in µg per mL,of any other impurity detected in the chromatogram of the Test preparationby the formula: in which riis the peak response of the individual impurity;rTis the peak response of lorazepam obtained from the Test preparation;and CUis as defined above.The total of all impurities detected does not exceed 4.0%.

B: Transfer 5.0mLof Injection to a suitable separator,and add 50mLof 0.1Nsodium hydroxide.Extract with three 10-mLportions of chloroform,and collect the chloroform extracts in a second separator.Wash the chloroform extracts with 10mLof water,and transfer the chloroform extracts to a centrifuge tube.Evaporate the chloroform extracts with the aid of a current of air to dryness,and dissolve the residue in acetone to obtain a Test preparationhaving a concentration of 10mg per mL.Dissolve USP Lorazepam Related Compound B RSin acetone to obtain a Standard preparationhaving a known concentration of 0.1mg per mL.Apply separately 50µLof the Test preparationand 5µLof the Standard preparationto 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 chromatograms in a solvent system consisting of a mixture of chloroform,n-heptane,and alcohol (10:10:1)until the solvent front has moved not less than 10cm from the origin.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Lightly spray the plate with 2Nsulfuric acid,dry at 105for 15minutes,and spray successively with sodium nitrite solution (1in 1000),ammonium sulfamate solution (1in 200),and N-(1-naphthyl)ethylenediamine dihydrochloride solution (1in 1000),drying the plate with a current of air after each spraying.Observe the plate under visible light:the spot produced by the Test preparationis not greater in size or intensity than the principal spot produced at the corresponding RFvalue by the Standard preparation,corresponding to not more than 0.1%of 2-amino-2¢,5-dichlorobenzophenone (lorazepam related compound B).

Mobile phase— Prepare a mixture of methanol and 0.05Mmonobasic ammonium phosphate (50:50).Adjust with ammonium hydroxide to a pHof 6.5,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Lorazepam RSin methanol to obtain a solution having a known concentration of about 1.0mg per mL.Transfer 4.0mLof this solution to a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.16mg per mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 4mg of lorazepam,to a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

System suitability preparation— Prepare a solution of Lorazepam in Mobile phasecontaining about 0.04mg of lorazepam per mLand about 0.032mg each of lorazepam related compound Cand lorazepam related compound Dper mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 240-nm detector and 4.6-mm ×10-to 15-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:the resolution,R,between any of the major peaks is not less than 1.2;and the relative retention times are about 0.7for 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxylic acid (lorazepam related compound D),1.0for lorazepam,and 2.7for 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxaldehyde (lorazepam related compound C).

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 lorazepam (C15H10Cl2N2O2)in each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Lorazepam RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.