A: When diluted to a concentration of about 1mg of carbachol per mL,it responds to Identificationtest Aunder Carbachol.

B: Evaporate a volume of Ophthalmic Solution,equivalent to about 500mg of carbachol,on a steam bath to dryness:the residue responds to Identificationtest Bunder Carbachol.

C: Evaporate a volume of Ophthalmic Solution,equivalent to about 100mg of carbachol,on a steam bath to dryness:the residue responds to Identificationtest Dunder Carbachol.

Hypochlorite reagent— Dilute 1volume of sodium hypochlorite TSwith water to 15volumes,allow to stand for 30minutes,then mix equal volumes of the resulting solution and 1Nsodium hydroxide.Prepare fresh daily.

Standard preparation— Dissolve a suitable quantity of USP Carbachol RS,accurately weighed,in water,and dilute quantitatively and stepwise with water to obtain a solution having a known concentration of about 100µg per mL.

Assay preparation— Dilute an accurately measured volume of Ophthalmic Solution quantitatively and stepwise with water to obtain a solution containing about 100µg of carbachol per mL.

Procedure— Transfer 2.0-mLportions each of the Assay preparationand the Standard preparation,and of water to provide a blank,to separate 50-mLconical flasks.To each flask add 1.0mLof 0.1Nhydrochloric acid,and mix.Treat each as follows.Add 4.0mLof Hypochlorite reagent,rinsing the inner walls of the flask with small portions of water,mix,and allow to stand for 15minutes,accurately timed.Add 2.0mLof phenol solution (1in 200),rinsing the walls of the flask with the solution and with additional small portions of water.Mix,and allow to stand for 5minutes.Add 2.0mLof 3.5Nhydrochloric acid,washing the sides of the flask upon addition.Rinse the flask sparingly with 0.1Nhydrochloric acid to assure complete acidification of all contents,then mix.Add 1.0mLof potassium iodide solution (3in 1000),mix,and allow to stand for 5minutes.Add 3.0mLof starch TS,mix,transfer the solutions to 50-mLvolumetric flasks with the aid of several small portions of water,and dilute each solution with water to volume.Concomitantly determine the absorbances of the solutions from the Assay preparationand the Standard preparationin 1-cm cells at the wavelength of maximum absorbance at about 590nm,with a suitable spectrophotometer,against the blank.Calculate the quantity,in mg,of C6H15ClN2O2in each mLof the Ophthalmic Solution taken by the formula: in which Cis the concentration,in µg per mL,of USP Carbachol RSin the Standard preparation,Dis the dilution factor used in the Assay preparation,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.