A: Apply 2-µL portions of a test solution, containing about 10 mg of acetylcholine chloride per mL, and of a Standard solution, containing about the same concentration of USP Acetylcholine Chloride RS, to a line 2 cm from the bottom edge of a thin-layer chromatographic plate coated with a 0.25-mm layer of aluminum oxide. Develop the chromatogram, without delay, in a vapor-saturated chamber, using a solvent system consisting of the upper layer obtained by mixing water, butyl alcohol, and glacial acetic acid (100:80:20) and allowing to separate completely. Allow the solvent front to move about 10 cm beyond the initial spotting line, remove the plate, and dry it with the aid of a current of warm air. Immediately spray it with a solution freshly prepared by dissolving 250 mg of cobaltous chloride in 50 mL of water and diluting with alcohol to 100 mL. Dry the plate as before, and immediately spray it with a solution prepared by dissolving 1.0 g of potassium ferrocyanide in 100 mL of water and diluting with 50 mL of alcohol. Dry the plate as before: the RF value and color of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.

B: Dissolve a portion, equivalent to about 20 mg of acetylcholine chloride, in about 2 mL of water, add 1 drop of nitric acid and 1 mL of silver nitrate TS: a curdy, white precipitate, soluble in an excess of 6 N ammonium hydroxide, is formed.

Mobile phase— Add 1.03 g of sodium 1-heptanesulfonate to a mixture of 900 mL of water and 10 mL of methanol. Mix, then add sufficient glacial acetic acid and ammonium hydroxide, if necessary, to adjust the solution to a pH of 4.0. Add 50 mL of acetonitrile, then add water to make 1000 mL, and mix. Slight variation of the amount of acetonitrile may be required to improve resolution or adjust retention time. Degas the solution.

Standard preparation— Dissolve an accurately weighed quantity of USP Acetylcholine Chloride RS in Mobile phase, and dilute quantitatively and stepwise with Mobile phase to obtain a solution having a known concentration about equal to that of the acetylcholine chloride in the Assay preparation.

Assay preparation— Transfer the contents of 1 container of Acetylcholine Chloride for Ophthalmic Solution to a 10-mL volumetric flask with the aid of Mobile phase, add Mobile phase to volume, and mix.

Chromatographic system (seeChromatography 621)— Use a liquid chromatograph fitted with a 3.9-mm × 30-cm stainless steel column packed with packing L1, and a refractive index detector. The flow rate is about 2 mL per minute. Chromatograph replicate 50-µL injections of the Standard preparation, and record the peak response: the relative standard deviation is not more than 3.5%. Chromatograph a solution containing about 0.2% each of acetylcholine chloride and choline chloride: the resolution, R, is not less than 2.0.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses. Calculate the quantity, in mg, of C7H16ClNO2 in the container taken by the formula: in which C is the concentration, in mg per mL, of USP Acetylcholine Chloride RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.