Mobile phase— Mix 300mLof a 1in 50solution of ammonium hydroxide in isopropyl alcohol and 700mLof n-hexane.Filter through a 0.5-µm filter before using.

Standard preparation— Using an accurately weighed quantity of USP Pilocarpine Hydrochloride RS,prepare a solution having a known concentration of about 1.6mg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 80mg of pilocarpine hydrochloride,to a 50-mLvolumetric flask.Dilute with methanol to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×25-cm column that contains packing L3.The flow rate is about 2mLper minute.Chromatograph three replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph by means of a suitable microsyringe or sampling valve,record the chromatograms,and measure the responses for the major peaks.The retention time is about 16minutes for pilocarpine hydrochloride.Calculate the quantity,in mg,of C11H16N2O2·HCl in each mLof the Ophthalmic Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Pilocarpine Hydrochloride RSin the Standard preparation,Vis the volume,in mL,of Ophthalmic Solution taken,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.