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

B: Dissolve USP Lorazepam RS in alcohol to obtain a solution having a concentration of 1 mg per mL. Transfer 10 mL of this solution to a suitable container. Transfer a volume of Injection, equivalent to about 10 mg of lorazepam, to a second container. Separately add 5 mL of hydrochloric acid to each container, heat each solution on a steam bath for 20 minutes, and cool. Transfer the solutions to separators, and add 8 mL of 10 N sodium hydroxide to each separator. Extract each solution with two 25-mL portions of ether, filtering the ether extracts through cotton plugs into suitable containers. Evaporate both ether extracts to about 2 mL, and add 8 mL of methanol to each. Apply separately 10 µL of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography 621) 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 15 cm. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a freshly prepared 1 in 80 solution of sodium nitrite in 0.5 N hydrochloric acid. Heat the plate at 100 for 5 minutes, allow to cool, and spray with a 1 in 1000 solution of N-(1-naphthyl)ethylenediamine dihydrochloride in alcohol: the RF value 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 phase having known concentrations of about 3.2 µg each of USP Lorazepam Related Compound C RS and USP Lorazepam Related Compound D RS per mL.

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

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Test preparation into 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 preparation that has a retention time shorter than that of the lorazepam related compound D peak 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 CS is the concentration, in µg per mL, of the corresponding component in the Standard preparation; CU is the concentration, in µg per mL, of Lorazepam in the Test preparation; ru is the peak response of lorazepam related compound C or lorazepam related compound D in the chromatogram obtained from the Test preparation; and rS is 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 preparation by the formula: in which ri is the peak response of the individual impurity; rT is the peak response of lorazepam obtained from the Test preparation; and CU is as defined above. The total of all impurities detected does not exceed 4.0%.

B: Transfer 5.0 mL of Injection to a suitable separator, and add 50 mL of 0.1 N sodium hydroxide. Extract with three 10-mL portions of chloroform, and collect the chloroform extracts in a second separator. Wash the chloroform extracts with 10 mL of 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 preparation having a concentration of 10 mg per mL. Dissolve USP Lorazepam Related Compound B RS in acetone to obtain a Standard preparation having a known concentration of 0.1 mg per mL. Apply separately 50 µL of the Test preparation and 5 µL of the Standard preparation to a suitable thin-layer chromatographic plate (see Chromatography 621) 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 10 cm 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 2 N sulfuric acid, dry at 105 for 15 minutes, and spray successively with sodium nitrite solution (1 in 1000), ammonium sulfamate solution (1 in 200), and N-(1-naphthyl)ethylenediamine dihydrochloride solution (1 in 1000), drying the plate with a current of air after each spraying. Observe the plate under visible light: the spot produced by the Test preparation is not greater in size or intensity than the principal spot produced at the corresponding RF value by the Standard preparation, corresponding to not more than 0.1% of 2-amino-2¢,5-dichlorobenzophenone (lorazepam related compound B).

(Official January 1, 2007)

Mobile phase— Prepare a mixture of methanol and 0.05 M monobasic ammonium phosphate (50:50). Adjust with ammonium hydroxide to a pH of 6.5, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

System suitability preparation— Prepare a solution of Lorazepam in Mobile phase containing about 0.04 mg of lorazepam per mL and about 0.032 mg each of lorazepam related compound C and lorazepam related compound D per mL.

Chromatographic system (see Chromatography 621)—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 2 mL per 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 0.7 for 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxylic acid (lorazepam related compound D), 1.0 for lorazepam, and 2.7 for 6-chloro-4-(o-chlorophenyl)-2-quinazolinecarboxaldehyde (lorazepam related compound C).

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of lorazepam (C15H10Cl2N2O2) in each mL of the Injection taken by the formula: in which C is the concentration, in mg per mL, of USP Lorazepam RS in the Standard preparation; V is the volume, in mL, of Injection taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.