0.05M Ammonium acetateand Mobile phase— Prepare as directed in the Assayunder Physostigmine Salicylate Injection.

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

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

Chromatographic system (see Chromatography á621ñ)—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 2mLper minute.Chromatograph the Standard preparationand record the peak responses as directed for Procedure:the column efficiency determined from the analyte peak is not less than 1200theoretical plates,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Proceed as directed for Procedurein the Assayunder Physostigmine Salicylate Injection.Calculate the quantity,in mg,of C15H21N3O2·C7H6O3in each mLof the Ophthalmic Solution taken by the formula: in which the terms are as defined therein.