Acetylcholine Chloride for Ophthalmic Solution

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Acetylcholine Chloride for Ophthalmic Solution is a sterile mixture of Acetylcholine Chloride with Mannitol or other suitable diluent, prepared by freeze-drying. Each container contains NLT 90.0% and NMT 115.0% of the labeled amount of acetylcholine chloride (C₇H₁₆ClNO₂).

2 IDENTIFICATION

A.

Standard solution: 10 mg/mL of USP Acetylcholine Chloride RS

Sample solution: 10 mg/mL of acetylcholine chloride

Chromatographic system

Adsorbent: 0.25-mm layer of aluminum oxide

Application volume: 2 μL

Developing solvent system: Mix butyl alcohol, glacial acetic acid, and water (40:10:50). Allow the layers to separate completely. Use the upper layer.

Spray reagent A: Freshly prepared solution of 5 mg/mL of cobaltous chloride prepared as follows. Dissolve the required amount of cobaltous chloride in 50% of the final volume of water, and dilute with 50% alcohol. [Note—This solution is freshly prepared.]

Spray reagent B: Freshly prepared potassium ferrocyanide solution prepared as follows. Dissolve 1.0 g of potassium ferrocyanide in 100mL of water, and dilute with 50 mL of alcohol.

Analysis

Samples: Standard solution and Sample solution

Develop the chromatogram, without delay, in a vapor-saturated chamber containing the Developing solvent system. Allow the solvent front to move about 10 cm beyond the initial spotting line. Dry the plate with a current of warm air. Immediately spray the plate with Spray reagent A. Dry the plate as before, and immediately spray the plate with Spray reagent B. Dry the plate with a current of warm air.

Acceptance criteria: The R value and color of the principal spot from the Sample solution correspond to those from the Standard solution.

B.

Sample solution: Nominally 10 mg/mL of acetylcholine chloride

Analysis: To 2 mL of Sample solution add 1 drop of nitric acid and 1 mL of silver nitrate TS.

Acceptance criteria: A curdy, white precipitate, soluble in an excess of 6 N ammonium hydroxide, is formed.

3 ASSAY

Procedure

Mobile phase: Add 1.03 g of sodium 1-heptanesulfonate to a mixture of 900 mL of water and 10 mL of methanol. Adjust with ammonium hydroxide or glacial acetic acid to a pH of 4.0. Add 50 mL of acetonitrile. Dilute with water to 1 L. [Note—A slight variation of the amount of acetonitrile may be required to improve resolution or adjust retention time.]

Standard solution: A quantity of USP Acetylcholine Chloride RS in Mobile phase, to obtain a solution having a known concentration equal to that of the acetylcholine chloride in the Sample solution

Sample solution: Transfer the contents of 1 container of Acetylcholine Chloride for Ophthalmic Solution to a 10-mL volumetric flask with the aid of Mobile phase, and dilute with Mobile phase to volume.

System suitability solution: 0.2% each of acetylcholine chloride and Choline chloride

Chromatographic system

Mode: LC

Detector: Refractive index

Column: 3.9-mm × 30-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 50 μL

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 2.0 between acetylcholine chloride and choline chloride, System suitability solution

Relative standard deviation: NMT 3.5%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of acetylcholine chloride (C₇H₁₆ClNO₂) in the container taken:

Result = (r_u/r_s) × C_s× V × (1/L) × 100

r_u= peak response from the Sample solution

r_s = peak response from the Standard solution

C_s = concentration of USP Acetylcholine Chloride RS in the Standard solution (mg/mL)

V = volume of the Sample solution, 10 mL

L = label claim (mg/vial)

Acceptance criteria: 90.0%–115.0%

4 PERFORMANCE TESTS

Uniformity of Dosage Units 〈905〉: Meets the requirements

5 SPECIFIC TESTS

Sterility Tests 〈71〉: Meets the requirements

Acidity

Analysis: Dissolve an amount of Acetylcholine Chloride for Ophthalmic Solution equivalent to 100 mg of acetylcholine chloride in 10 mL of recently boiled water. Add at once 1 drop of bromothymol blue TS.

Acceptance criteria: NMT 0.50 mL of 0.010 N sodium hydroxide is required to produce a color change.

Water Determination, Method I〈921〉

Analysis: Perform the titration in the original container, observing precautions against contact with water or moist atmosphere. Adjust the concentration of the reagent so that the titration volume approaches but does not exceed the capacity of the container. Titrate to an amber color that persists for 15 s after mixing.

Acceptance criteria: NMT 1.0%

Constituted Solution: At the time of use, it meets the requirements for Injections and Implanted Drug Products 〈1〉, Specic Tests, Completeness and clarity of solutions.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers as described in Packaging and Storage Requirements 〈659〉, Injection Packaging, Packaging for constitution, and store at controlled room temperature.

USP Reference Standards 〈11〉

USP Acetylcholine Chloride RS