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

2-Hydroxypropyltrimethyl ammonium chloride solution— Transfer 50.0mg of bethanechol chloride into a 50-mLvolumetric flask.Add about 40mLof 0.1Nsodium hydroxide,and sonicate until fully dissolved.Dilute with 0.1Nsodium hydroxide to volume,and allow to stand for five days to allow adequate time for conversion from bethanechol to 2-hydroxypropyltrimethyl ammonium chloride.Chromatograph as directed for Procedureto verify the presence and location of the peak for 2-hydroxypropyltrimethyl ammonium chloride.

Standard solution— Use the Standard preparation,prepared as directed in the Assay.

Test solution— Use the Assay preparation.

Procedure— Separately inject equal volumes (about 50µL)of the 2-Hydroxypropyltrimethyl ammonium chloride solution,the Standard solution,and the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the bethanechol and 2-hydroxypropyltrimethyl ammonium chloride peaks.Calculate the percentage of 2-hydroxypropyltrimethyl ammonium chloride in each mLof the Injection taken by the formula: in which CSis the concentration,in mg per mL,of USP Bethanechol Chloride RSin the Standard solution;Ciis the concentration,in mg per mL,of bethanechol chloride in the Test solution;riis the peak response for 2-hydroxypropyltrimethyl ammonium chloride obtained from the Test solution;and rSis the peak response for bethanechol obtained from the Standard solution.Not more than 4.0%is found.

Diluent— Transfer 10mg of calcium chloride and 10mg of magnesium chloride to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Mobile phase— Prepare a filtered and degassed solution of 20mMmethanesulfonic acid.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Transfer 25mg of USP Bethanechol Chloride RS,accurately weighed,to a 25-mLvolumetric flask,and add 15mLof water,2.0mLof the Diluent,and 0.5mLof 0.1Nsodium hydroxide.Dilute with water to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Bethanechol Chloride RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 1.0mg per mL.

Assay preparation— Dilute an accurately measured volume of Injection,if necessary,with water to obtain a solution having a concentration of about 1.0mg per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a conductivity detector and a 4-mm ×25-cm column that contains packing L53.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are 1.0for sodium,1.4for magnesium,1.6for calcium,2.0for 2-hydroxypropyltrimethyl ammonium chloride,and 2.8for bethanechol;the resolution,R,between the calcium ion and 2-hydroxypropyltrimethyl ammonium chloride is not less than 2.0;the column efficiency determined from the bethanechol peak is not less than 350theoretical plates;the tailing factor is not more than 4.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of bethanechol chloride (C7H17ClN2O2)in each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Bethanechol Chloride RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.