Assay—
Mobile phase—
Dissolve 990mg of sodium 1-heptanesulfonate in 890mLof water,add 10mLof glacial acetic acid and 1100mLof methanol,mix,pass through a suitable filter having a 1-µm or finer porosity,and degas.Make adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Levobunolol Hydrochloride RSin
Mobile phaseto obtain a solution having a known concentration of about 0.1mg per mL.
Assay preparation—
Dilute an accurately measured volume of Ophthalmic Solution quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution containing about 0.1mg of levobunolol hydrochloride per mL.
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 preparation,and record the peak responses as directed for
Procedure:the column efficiency determined from the analyte peak is not less than 1000theoretical plates;the capacity factor,
k¢,for levobunolol is between 1.0and 1.4;the tailing factor for the analyte peak is not more than 2.6;and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure—
Separately inject equal volumes (about 30µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in mg,of C
17H
25NO
3·HCl in each mLof the Ophthalmic Solution taken by the formula:
(L/D)(C)(rU/rS),
in which
Lis the labeled amount,in mg,of levobunolol hydrochloride in each mLof the Ophthalmic Solution;
Dis the concentration,in mg per mL,of levobunolol hydrochloride in the
Assay preparation,based on the labeled quantity per mLand the extent of dilution;
Cis the concentration,in mg per mL,of
USP Levobunolol Hydrochloride RSin the
Standard preparation;and
rUand
rSare the peak area responses obtained from the
Assay preparationand the
Standard preparation,respectively.