Assay—
Dilute phosphoric acid solution—
Prepare a solution of phosphoric acid in water (1 in 1000).
Mobile phase—
Prepare a filtered and degassed mixture of
Dilute phosphoric acid solution and acetonitrile (850:150). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Prepare a solution of
USP Norfloxacin RS in
Dilute phosphoric acid solution having a known concentration of about 0.06 mg per mL.
Resolution solution—
Prepare a solution of
USP Norfloxacin RS and pipemidic acid in
Dilute phosphoric acid solution having known concentrations of about 0.06 mg of each per mL.
Assay preparation—
Dilute an accurately measured volume of Ophthalmic Solution quantitatively and stepwise with Dilute phosphoric acid solution to obtain a solution having a concentration of about 0.06 mg of norfloxacin per mL.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 278-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The column temperature is maintained at 50
. The flow rate is about 0.5 mL per minute. Precondition the column for about 8 hours with 0.01 M monobasic sodium phosphate buffer adjusted with phosphoric acid to a pH of 4.0. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.8 for pipemidic acid and 1.0 for norfloxacin; and the resolution,
R, between the pipemidic acid peak and the norfloxacin peak is not less than 1.2. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor for the norfloxacin peak is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
[NOTE—Use peak areas where peak responses are indicated.
] Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of norfloxacin (C
16H
18FN
3O
3) in each mL of the Ophthalmic Solution taken by the formula:
(L / D)(C)(rU / rS),
in which
L is the labeled quantity, in mg per mL, of norfloxacin in the Ophthalmic Solution;
D is the concentration, in mg per mL, of norfloxacin in the
Assay preparation, based on the labeled quantity of norfloxacin in each mL of the Ophthalmic Solution and the extent of dilution;
C is the concentration, in mg per mL, of
USP Norfloxacin RS in the
Standard preparation; and
rU and
rS are the norfloxacin peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
