Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Dissolve an accurately weighed quantity of Clonazepam in acetone to obtain a solution having a concentration of 25 mg per mL.

Standard solution— Dissolve an accurately weighed quantity of USP Clonazepam Related Compound C RS in acetone to obtain a solution having a known concentration of 50 µg per mL.

Application volume: 20 µL.

Developing solvent system: a mixture of acetone and n-heptane (3:2).

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. After air-drying the plate, heavily spray the plate with 2 M sulfuric acid, and dry at 105 for 15 minutes. Successively spray the plate with 0.01 M sodium nitrite, 9 mM ammonium sulfamate, and N-(1-naphthyl)ethylenediamine dihydrochloride TS, and dry the plate with a current of air. Compare the intensities of any secondary spots observed in the chromatogram of the Test solution with that of the principal spot in the chromatogram of the Standard solution: no secondary spot from the chromatogram of the Test solution is larger or more intense than the principal spot obtained from the Standard solution (0.2%).

Buffer solution, Mobile phase, Diluent, System suitability solution, Standard preparation, and Chromatographic system— Proceed as directed in the Assay.

Test preparation— Use the Assay preparation.

Procedure— Inject a volume (about 50 µL) of the Test preparation into the chromatograph, record the chromatogram, and measure the responses for all of the peaks. Calculate the percentage of each impurity in the portion of Clonazepam taken by the formula: in which P is the relative response factor, which is 1.84 for clonazepam related compound A, 0.94 for clonazepam related compound B, and 1 for all other impurities; ri is the peak response for each impurity obtained from the Test preparation; and rC is the peak response for clonazepam in the Test preparation: not more than 0.1% of clonazepam related compound A or of clonazepam related compound B is found, not more than 0.2% of any other impurity is found, and the sum of all other impurities is not more than 0.3%.

Buffer solution— Transfer about 6.6 g of anhydrous dibasic ammonium phosphate to a 1-L volumetric flask, dissolve in 950 mL of water, adjust with 1 N phosphoric acid or 1 N sodium hydroxide to a pH of 8.0, dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution, methanol, and tetrahydrofuran (60:52:13). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Prepare a mixture of water, methanol, and tetrahydrofuran (60:52:13).

Standard preparation— Dissolve an accurately weighed quantity of USP Clonazepam RS in Diluent, and dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of about 0.1 mg per mL.

System suitability solution— Dissolve suitable quantities of USP Clonazepam Related Compound A RS, USP Clonazepam Related Compound B RS, and USP Clonazepam RS in Diluent to obtain a solution containing about 0.04 mg per mL of each Reference Standard.

Assay preparation— Transfer about 10 mg of Clonazepam, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 2.2 for clonazepam related compound A, 2.5 for clonazepam related compound B, and 1.0 for clonazepam; and the resolution, R, between clonazepam related compound A and clonazepam related compound B is not less than 2.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.5, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 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 C15H10ClN3O3 in the portion of Clonazepam taken by the formula: in which C is the concentration, in mg per mL, of USP Clonazepam RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.