Identification—
A:
Thin-Layer Chromatographic Identification Test 
201
—
Test solution—
Dilute a portion of Ophthalmic Solution with a mixture of chloroform and methanol (1:1) to obtain a solution having a concentration of about 0.3 mg of ofloxacin per mL.
Standard solution—
Dissolve an accurately weighed quantity of
USP Ofloxacin RS in a mixture of chloroform and methanol (1:1) to obtain a solution having a concentration of 3.0 mg per mL. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, add 5 mL of water, dilute with a mixture of chloroform and methanol (1:1) to volume, and mix.
Application volume:
2 µL.
Developing solvent system:
a mixture of chloroform, methanol, and a solution (1 in 30) of ammonium hydroxide (150:75:15). Saturate a paper-lined chromatographic chamber with this mixture.
B:
The retention time of the ofloxacin peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of sodium dodecyl sulfate (0.24% aqueous solution), acetonitrile, and glacial acetic acid (580:400:20). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
0.05 N Hydrochloric acid—
Add 4.0 mL of hydrochloric acid to 500 mL of water, dilute with water to 1000 mL, and mix.
Resolution solution—
Prepare a solution of about 0.1 mg of
USP Ofloxacin RS and about 2.4 mg of propylparaben in each mL of acetonitrile.
Standard preparation—
Quantitatively dissolve an accurately weighed quantity of
USP Ofloxacin RS in
0.05 N Hydrochloric acid to obtain a solution having a known concentration of about 0.06 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 3 mg of ofloxacin, to a 50-mL volumetric flask, dilute with 0.05 N Hydrochloric acid to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 294-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. The column temperature is maintained at a constant temperature of about 35
. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the resolution,
R, between propylparaben and ofloxacin is not less than 2. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 3; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of ofloxacin (C
18H
20FN
3O
4) in each mL of the Ophthalmic Solution taken by the formula:
50(C / V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Ofloxacin RS in the
Standard preparation; V is the volume, in mL, of Ophthalmic Solution taken to prepare the
Assay preparation; and
rU and
rS are the ofloxacin peak areas obtained from the
Assay preparation and the
Standard preparation, respectively.
