A: When diluted to a concentration of about 1 mg of carbachol per mL, it responds to Identification test A under Carbachol.

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

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

(Official January 1, 2007)

Hypochlorite reagent— Dilute 1 volume of sodium hypochlorite TS with water to 15 volumes, allow to stand for 30 minutes, then mix equal volumes of the resulting solution and 1 N sodium 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 100 µg per mL.

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

Procedure— Transfer 2.0-mL portions each of the Assay preparation and the Standard preparation, and of water to provide a blank, to separate 50-mL conical flasks. To each flask add 1.0 mL of 0.1 N hydrochloric acid, and mix. Treat each as follows. Add 4.0 mL of Hypochlorite reagent, rinsing the inner walls of the flask with small portions of water, mix, and allow to stand for 15 minutes, accurately timed. Add 2.0 mL of phenol solution (1 in 200), rinsing the walls of the flask with the solution and with additional small portions of water. Mix, and allow to stand for 5 minutes. Add 2.0 mL of 3.5 N hydrochloric acid, washing the sides of the flask upon addition. Rinse the flask sparingly with 0.1 N hydrochloric acid to assure complete acidification of all contents, then mix. Add 1.0 mL of potassium iodide solution (3 in 1000), mix, and allow to stand for 5 minutes. Add 3.0 mL of starch TS, mix, transfer the solutions to 50-mL volumetric 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 preparation and the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 590 nm, with a suitable spectrophotometer, against the blank. Calculate the quantity, in mg, of C6H15ClN2O2 in each mL of the Ophthalmic Solution taken by the formula: in which C is the concentration, in µg per mL, of USP Carbachol RS in the Standard preparation, D is the dilution factor used in the Assay preparation, and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.