A: Infrared Absorption 197K.

B: The RF value of the principal spot observed in the chromatogram of the Test preparation obtained as directed in Related compounds test A corresponds to that obtained from the Identification preparation.

A: Dissolve Lorazepam in chloroform to obtain a Test preparation containing 2 mg per mL. Dissolve USP Lorazepam RS in chloroform to obtain an Identification preparation having a known concentration of 2 mg per mL. Dissolve USP Lorazepam Related Compound A RS (7-chloro-5-(o-chlorophenyl)-1,3-dihydro-3-acetoxy-2H-1,4-benzodiazepin-2-one) in chloroform to obtain a Standard preparation having a known concentration of 20 µg per mL. Dilute portions of this Standard preparation quantitatively with chloroform to obtain solutions having concentrations of 10 µg per mL (Diluted standard preparation A) and 4 µg per mL (Diluted standard preparation B), respectively. Within 30 minutes after preparation, apply separately 50 µL of the Test preparation, the Identification preparation, the Standard preparation, the Diluted standard preparation A, and the Diluted standard preparation B to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture and previously washed with a mixture of chloroform, ethyl acetate, and methanol (2:1:1) and dried in air. Allow the spots to dry, and develop the chromatograms in a solvent system consisting of a mixture of chloroform, dioxane, and glacial acetic acid (91:5:4) until the solvent front has moved to within 2 cm to 3 cm from the top of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow to air-dry for about 30 minutes. Examine the plate under short-wavelength UV light. Compare the intensities of any secondary spots observed in the chromatogram of the Test preparation with those of the principal spots in the chromatograms of the Standard preparation and the Diluted standard preparations: the sum of the intensities of all secondary spots obtained from the Test preparation corresponds to not more than 1.0%.

B: Transfer 50.0 mg of Lorazepam to a 10-mL conical flask, add 2.5 mL of acetone, and shake. Allow any undissolved particles to settle, and use the supernatant as the Test preparation. Dissolve USP Lorazepam Related Compound B RS in acetone to obtain a Standard preparation having a known concentration of 10 µg per mL. Apply separately 50 µL of the Test preparation and 10 µL of the Standard preparation to a suitable thin-layer chromatographic plate (see test A under Related compounds). Allow the spots to dry, and develop the chromatograms in a solvent system consisting of a mixture of chloroform, dioxane, and glacial acetic acid (91:5:4) 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 0.01% of 2-amino-2¢,5-dichlorobenzophenone (lorazepam related compound B).

(Official January 1, 2007)