0.05 M Ammonium acetate and Mobile phase— Prepare as directed in the Assay under Physostigmine Salicylate Injection.

Standard preparation— Dissolve an accurately weighed quantity of USP Physostigmine Salicylate RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with acetonitrile, to obtain a solution having a known concentration of about 30 µg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 3 mg of physostigmine salicylate, to a 100-mL volumetric flask, dilute with acetonitrile to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation and record the peak responses as directed for Procedure: the column efficiency determined from the analyte peak is not less than 1200 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Proceed as directed for Procedure in the Assay under Physostigmine Salicylate Injection. Calculate the quantity, in mg, of C15H21N3O2·C7H6O3 in each mL of the Ophthalmic Solution taken by the formula: in which the terms are as defined therein.