Mobile phase— Prepare a filtered and degassed solution of water, methanol, and acetonitrile (4:3:3). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock preparation— Dissolve an accurately weighed quantity of USP Clonazepam RS in acetonitrile to obtain a concentration of about 0.5 mg per mL.

Standard preparation— Dilute the Standard stock preparation with acetonitrile to obtain a solution having a known concentration of 25 µg per mL.

Assay preparation— Agitate the container of Oral Suspension for 30 minutes on a rotating mixer, remove a 5-mL sample, and store in a clear glass vial at –70 until analyzed. At the time of analysis, remove the sample from the freezer, allow it to reach room temperature, and mix with a vortex mixer for 30 seconds. Pipet 2.5 mL of the sample solution into a 10-mL volumetric flask, and dilute with acetonitrile to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 10-cm analytical column that contains 5-µm packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the retention time of clonazepam is about 7 minutes; and the relative standard deviation for replicate injections is not more than 1.8%.

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 clonazepam (C15H10ClN3O3) in the volume of Oral Suspension taken by the formula: in which C is the concentration, in mg per mL, of USP Clonazepam RS in the Standard preparation; V is the volume, in mL, of Oral Suspension taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.