A: Infrared Absorption á197Mñ.

B: Dissolve about 50mg in 2mLof water,add 0.1mLof cobaltous chloride solution (1in 100),then add 0.1mLof potassium ferrocyanide TS:an emerald-green color is produced,and almost entirely fades in 5to 10minutes (distinction from choline chloride,which gives the same reaction but the color does not fade).

C: To 1mLof a solution (1in 100)add 0.1mLof iodine TS:a brown precipitate is formed,and it rapidly changes to a dark olive-green color.

D: Asolution of it responds to the tests for Chloride á191ñ.

Buffer solution— Transfer about 0.48g of methanesulfonic acid to a 1000-mLvolumetric flask.Dissolve in and dilute with water to volume.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand acetonitrile (95:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Bethanechol Chloride RSin Mobile phaseand dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 1µg of USP Bethanechol Chloride RSper mL.

Test solution— Transfer about 25mg of Bethanechol Chloride,accurately weighed,to a 250-mLvolumetric flask.Dissolve in and dilute with Mobile phaseto volume,and mix.

System suitability solution— Transfer about 25mg of Bethanechol Chloride,accurately weighed,to a 250-mLvolumetric flask.Add 10mLof 0.1Nsodium hydroxide,and allow to stand for about 15minutes.Add 10mLof 0.1Nhydrochloric acid.Dissolve in and dilute with Mobile phaseto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a conductivity detector and a 3.9-×150-mm column containing packing L55.The flow rate is about 1.0mLper minute.The detector and column temperatures are maintained at 35and 30,respectively.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention time is about 0.9for 2-hydroxypropyltrimethyl ammonium chloride and 1.0for bethanechol;the resolution,R,between 2-hydroxypropyltrimethyl ammonium chloride and bethanechol is not less than 1.5.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 10.0%for bethanechol chloride.

Procedure— Separately inject equal volumes (about 50µL)of the Mobile phase,the Standard solution,and the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses for all the peaks.Calculate the percentage of each impurity in the portion of Bethanechol Chloride taken by the formula: in which Cis the concentration,in mg per mL,of USP Bethanechol Chloride RSin the Standard solution;Fis the relative response factor and is equal to 0.79for 2-hydroxypropyltrimethyl ammonium and 1.0for any other impurity;riis the peak response for any impurity in the Test solution;rSis the peak response of USP Bethanechol Chloride RSin the Standard solution;and Wis the weight,in mg,of Bethanechol Chloride taken to prepare the Test solution.Not more than 1.0%of 2-hydroxypropyltrimethyl ammonium is found;not more than 0.1%of any other impurity is found;and the sum of all the impurities is not more than 1.5%.

Buffer solution— Transfer about 29mg of edetate disodium to a 1000-mLvolumetric flask,and dissolve in 500mLof water.Add 300µLof nitric acid to the volumetric flask,and dilute with water to volume.Pass through 0.45-µm nylon membrane filter.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand acetonitrile (95:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Transfer about 25mg of Bethanechol Chloride,accurately weighed,to a 250-mLvolumetric flask.Add 10mLof 0.1Nsodium hydroxide,and allow to stand for about 15minutes.Add 10mLof 0.1Nhydrochloric acid.Dissolve in and dilute with Mobile phaseto volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Bethanechol Chloride RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.1mg of USP Bethanechol Chloride RSper mL.

Assay preparation— Transfer about 25mg of Bethanechol Chloride,accurately weighed,to a 250-mLvolumetric flask.Dissolve in and dilute with Mobile phaseto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a conductivity detector and a 3.9-×150-mm column containing packing L55.The flow rate is about 1.0mLper minute.The detector and column temperatures are maintained at 35and 30,respectively.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.9for 2-hydroxypropyltrimethyl ammonium chloride and 1.0for bethanechol;and the resolution,R,between 2-hydroxypropyltrimethyl ammonium chloride and bethanechol is not less than 1.5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 3.5;and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in mg,of C7H17ClN2O2in the portion of Bethanechol Chloride 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 bethanechol chloride peak responses obtained from the Assay preparationand the Standard preparation,respectively.