Mobile phase— Prepare a filtered and degassed solution of water and acetonitrile (67:33). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Bethanechol Chloride RS in Mobile phase, and dilute quantitatively with Mobile phase to obtain a solution having a known concentration of about 500 µg per mL.

Assay preparation— Agitate the container of Oral Solution for 30 minutes on a rotating mixer, remove a 10-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 2.0 mL of the sample into a 20-mL volumetric flask, and dilute with Mobile phase to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm × 25-cm analytical column that contains 5-µm packing L11. The flow rate is about 0.7 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the retention time is about 3 minutes, and the relative standard deviation for replicate injections is not more than 3.1%.

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 bethanechol chloride (C7H17ClN2O2) in the volume of Oral Solution taken by the formula: in which C is the concentration, in µg per mL, of USP Bethanechol Chloride RS in the Standard preparation; V is the volume, in mL, of Oral Solution taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.