A: Apply 2-µLportions of a test solution,containing about 10mg of acetylcholine chloride per mL,and of a Standard solution,containing about the same concentration of USP Acetylcholine Chloride RS,to a line 2cm 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 10cm 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 250mg of cobaltous chloride in 50mLof water and diluting with alcohol to 100mL.Dry the plate as before,and immediately spray it with a solution prepared by dissolving 1.0g of potassium ferrocyanide in 100mLof water and diluting with 50mLof alcohol.Dry the plate as before:the RFvalue 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 20mg of acetylcholine chloride,in about 2mLof water,add 1drop of nitric acid and 1mLof silver nitrate TS:a curdy,white precipitate,soluble in an excess of 6Nammonium hydroxide,is formed.

Mobile phase— Add 1.03g of sodium 1-heptanesulfonate to a mixture of 900mLof water and 10mLof methanol.Mix,then add sufficient glacial acetic acid and ammonium hydroxide,if necessary,to adjust the solution to a pHof 4.0.Add 50mLof acetonitrile,then add water to make 1000mL,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 RSin Mobile phase,and dilute quantitatively and stepwise with Mobile phaseto 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 1container of Acetylcholine Chloride for Ophthalmic Solution to a 10-mLvolumetric flask with the aid of Mobile phase,add Mobile phaseto volume,and mix.

Chromatographic system (seeChromatography á621ñ)— 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 2mLper minute.Chromatograph replicate 50-µLinjections 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 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses.Calculate the quantity,in mg,of C7H16ClNO2in the container taken by the formula: in which Cis the concentration,in mg per mL,of USP Acetylcholine Chloride RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.