Assay—
Mobile phase—
Dissolve 990 mg of sodium 1-heptanesulfonate in 890 mL of water, add 10 mL of glacial acetic acid and 1100 mL of methanol, mix, pass through a suitable filter having a 1-µm or finer porosity, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Levobunolol Hydrochloride RS in
Mobile phase to obtain a solution having a known concentration of about 0.1 mg per mL.
Assay preparation—
Dilute an accurately measured volume of Ophthalmic Solution quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution containing about 0.1 mg of levobunolol hydrochloride per mL.
Chromatographic system
(see
Chromatography 621)—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 1.5 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 1000 theoretical plates; the capacity factor,
k¢, for levobunolol is between 1.0 and 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 30 µL) of the
Standard preparation and the
Assay preparation into 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 mL of the Ophthalmic Solution taken by the formula:
(L / D)(C)(rU / rS),
in which
L is the labeled amount, in mg, of levobunolol hydrochloride in each mL of the Ophthalmic Solution;
D is the concentration, in mg per mL, of levobunolol hydrochloride in the
Assay preparation, based on the labeled quantity per mL and the extent of dilution;
C is the concentration, in mg per mL, of
USP Levobunolol Hydrochloride RS in the
Standard preparation; and
rU and
rS are the peak area responses obtained from the
Assay preparation and the
Standard preparation, respectively.
