Test specimen— Stir a portion of finely powdered Tablets, equivalent to about 15 mg of lorazepam, with 40 mL of acetone for 5 minutes. Pass through very retentive filter paper pre-washed with acetone. Evaporate the filtrate on a steam bath with the aid of a current of air to dryness. Dissolve the residue in 1 mL of acetone, and add 20 mL of 2,2,4-trimethylpentane. Heat the solution on a hot plate to a gentle boil, and evaporate to a volume of about 10 mL. Remove the solution from the hot plate, and evaporate with the aid of a current of air to dryness. Dry the residue in vacuum at 60 for 1 hour.

Diluent, Mobile phase, and Chromatographic system— Prepare as directed in the Assay.

Standard solution— Prepare as directed for Standard preparation in the Assay.

Test solution— Place 1 Tablet in a volumetric flask of appropriate size, based on the labeled quantity, in mg, of lorazepam in the Tablet, to obtain a solution having a concentration of about 0.1 mg of lorazepam per mL. Add a volume of Diluent equal to about 50% of the volume of the flask, sonicate for 10 minutes, and shake by mechanical means for 20 minutes. Dilute with Diluent to volume, mix, and centrifuge a portion of the solution for 10 minutes at 2000 rpm.

Procedure— Separately inject equal volumes (about 20 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of lorazepam (C15H10Cl2N2O2) in the Tablet taken by the formula: in which T is the labeled quantity, in mg, of lorazepam in the Tablet; C is the concentration, in mg per mL, of USP Lorazepam RS in the Standard solution; D is the concentration, in mg per mL, of lorazepam in the Test solution, based on the labeled quantity per Tablet and the extent of dilution; and rU and rS are the lorazepam peak responses obtained from the Test solution and the Standard solution, respectively.

Standard preparation	Dilution	Concentration (µg of each RS per mL)	Percentage (%, for comparison with test specimen)
A	(1 in 25)	40	2.0
B	(1 in 50)	20	1.0
C	(1 in 100)	10	0.5

Test preparation— Transfer a quantity of finely powdered Tablets, equivalent to 4.0 mg of lorazepam, to a sintered-glass funnel. Extract with two 1-mL portions of chloroform followed by two 1-mL portions of methanol, collecting the filtrate in a centrifuge tube. Evaporate the filtrate with the aid of a stream of nitrogen at room temperature to dryness. Dissolve the residue in 2.0 mL of chloroform, and centrifuge. Use the clear supernatant as the Test preparation.

Procedure— Within 30 minutes after preparation, apply separately 50 µL of the Test preparation and 50 µL of each Standard preparation 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 preparations: [NOTE—The RF value and the intensity of the spot for the USP Lorazepam Related Compound E RS in the Standard preparations correspond closely, but not necessarily precisely, to those observed for one of the secondary spots observed in the chromatogram of the Test preparation.] The sum of the intensities of all secondary spots obtained from the Test preparation corresponds to not more than 4.0%.

100(C / 20)(VU / V)(rU / rS),