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.
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 C
6H
15ClN
2O
2 in each mL of the Ophthalmic Solution taken by the formula:
0.001CD(AU / AS),
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.