Phosphate buffer solution— Dissolve about 13.8 g of monobasic sodium phosphate in 500 mL of water, adjust with 2.5 N sodium hydroxide to a pH of 8.0, and mix.

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and Phosphate buffer solution (5:4:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Dissolve about 5 mL of 2,6-dimethylaniline in 50 mL of hexane, and carefully add dropwise hydrochloric acid to precipitate the amine hydrochloride. Filter through a sintered-glass funnel, wash the solid precipitate with hexane, and allow the precipitate to dry. Transfer about 50 mg of the dried precipitate of 2,6-dimethylaniline hydrochloride to a 100-mL volumetric flask, add 10.0 mL of Phosphate buffer solution and 40 mL of water, and dilute with acetonitrile to volume.

Standard solution— Dissolve an accurately weighed quantity of USP Nordazepam RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with acetonitrile to obtain a solution having a known concentration of about 75 µg per mL. Transfer 4.0 mL of this solution to a 50-mL conical flask, add 4.0 mL of 0.7 M potassium carbonate, 2.0 mL of Internal standard solution, and 15.0 mL of water. Insert a stopper, and mix.

Test solution— Transfer an accurately weighed quantity of about 50 mg of Clorazepate Dipotassium to a 50-mL conical flask. Add 4.0 mL of 0.7 M potassium carbonate, and start stirring the solution. Add 2 mL of Internal standard solution and 19.0 mL of water. Stop stirring about 5 minutes after the addition of the 0.7 M potassium carbonate solution. [note—Prepare fresh immediately before each injection.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 232-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention time for 2,6-dimethylaniline is about 0.8 and 1.0 for nordazepam; the relative standard deviation of the peak area ratio of nordazepam to 2,6-dimethylaniline for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the percentage of nordazepam in the portion of Clorazepate Dipotassium taken by the formula: in which C is the concentration, in mg per mL, of USP Nordazepam RS in the Standard solution; W is the weight, in mg, of Clorazepate Dipotassium taken to prepare the Test solution; Ri is the peak area ratio of any impurity to 2,6-dimethylaniline obtained from the Test solution; and RS is the peak area ratio of nordazepam to 2,6-dimethylaniline obtained from the Standard solution: not more than 0.5% of nordazepam is found and not more than 0.1% of any individual impurity is found.

Diluent— Prepare a mixture of 0.001 N sodium hydroxide and acetonitrile (1:1).

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and a 1 M solution of tetrabutylammonium hydroxide in methanol (110:90:1), adjust with phosphoric acid to a pH of 7.7, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP 2-Amino-5-chlorobenzophenone RS in Diluent, and dilute quantitatively, and stepwise if necessary, with Diluent, to obtain a solution having a known concentration of about 0.0026 mg per mL.

Test solution— Transfer about 300 mg of Clorazepate Dipotassium, accurately weighed, to a glass test tube. Add 10.0 mL of Diluent, and vigorously mix on a vortex mixer for about 90 seconds. [note—Prepare fresh immediately before each injection.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 238-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation of the peak height for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each impurity in the portion of Clorazepate Dipotassium taken by the formula: in which C is the concentration, in mg per mL, of USP 2-Amino-5-chlorobenzophenone RS in the Standard solution; W is the weight, in mg, of sample taken; ri is the peak height of each impurity obtained from the Test solution; and rS is the peak height of 2-amino-5-chlorobenzophenone obtained from the Standard solution: not more than 0.1% of 2-amino-5-chlorobenzophenone is found, not more than 0.1% of any other individual impurity is found, and not more than 1.0% of total impurities in Test 1 and Test 2 is found.