A: It responds to Identificationtest Aunder Fluorescein Sodium.

B: The relative retention times of the major peaks in the chromatogram of the Assaycorrespond to those in the chromatograms of the Standard fluorescein sodium preparationand the Standard benoxinate hydrochloride preparationas obtained in the Assay.

Mobile phase— Dissolve 100mg of sodium 1-pentanesulfonate in 40mLof glacial acetic acid in a 2000-mLvolumetric flask.Add 600mLof acetonitrile and 10mLof triethanolamine,dilute with water to volume,and mix.Adjust with phosphoric acid to a pHof 3,and pass through a filter having a 0.5-µm or finer porosity.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard fluorescein sodium preparation— Transfer about 55mg of USP Diacetylfluorescein RS,accurately weighed,to a 50-mLvolumetric flask containing 5mLof alcohol.Add 1mLof 2.5Nsodium hydroxide,and heat on a steam bath at about the boiling temperature for 20minutes,with frequent swirling.Cool,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.This solution contains the equivalent of about 0.1mg of fluorescein sodium per mL.

Standard benoxinate hydrochloride preparation— Quantitatively dissolve an accurately weighed quantity of USP Benoxinate Hydrochloride RSin Mobile phase,and if necessary dilute quantitatively and stepwise with Mobile phaseto obtain a solution having a known concentration of about 0.1Jmg per mL,Jbeing the ratio of the labeled amount,in mg,of benoxinate hydrochloride to the labeled amount,in mg,of fluorescein sodium in each mLof Ophthalmic Solution.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 5mg of fluorescein sodium,to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard fluorescein sodium preparationand the Standard benoxinate hydrochloride preparation,and record the peak responses as directed for Procedure:the tailing factor for each analyte peak is not more than 2.0,and the relative standard deviation for replicate injections of each Standard preparationis not more than 2.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 25µL)of the Standard fluorescein sodium preparation,the Standard benoxinate hydrochloride preparation,and the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in mg,of fluorescein sodium (C20H10Na2O5)in each mLof Ophthalmic Solution taken by the formula: in which 376.28and 416.39are the molecular weights of fluorescein sodium and diacetylfluorescein,respectively;Wis the quantity,in mg,of USP Diacetylfluorescein RStaken to prepare the Standard fluorescein sodium preparation;Vis the volume,in mL,of Ophthalmic Solution taken;and rUand rSare the fluorescein peak responses obtained from the Assay preparationand the Standard fluorescein sodium preparation,respectively.Calculate the quantity,in mg,of benoxinate hydrochloride (C17H28N2O3·HCl)in each mLof Ophthalmic Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Benoxinate Hydrochloride RSin the Standard benoxinate hydrochloride preparation;Vis the volume,in mL,of Ophthalmic Solution taken,and rUand rSare the benoxinate peak responses obtained from the Assay preparationand the Standard benoxinate hydrochloride preparation,respectively.