Identification—
Dilute a volume of Ophthalmic Solution with water to obtain a test solution containing about 3 mg of ciprofloxacin per mL. Prepare a Standard solution of
USP Ciprofloxacin Hydrochloride RS in water containing about 3.5 mg per mL. Proceed as directed in
Identification test
B under
Ciprofloxacin Hydrochloride, beginning with “Separately apply, as 1-cm bands,” except to use 3 µL each of the test solution and the Standard solution instead of 5 µL. The specified result is obtained.
Assay—
Mobile phase—
Prepare 0.005
M tetrabutylammonium phosphate, and adjust with phosphoric acid to a pH of 2.0. Prepare a degassed and filtered mixture of this solution and methanol (750:250). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Ciprofloxacin Hydrochloride RS in water to obtain a solution having a known concentration of about 0.14 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 6 mg of ciprofloxacin, to a 50-mL volumetric flask, dilute with water to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column containing packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the
Resolution solution, and record the responses as directed under
Procedure: the relative retention times are about 0.8 for the ciprofloxacin ethylenediamine analog and 1.0 for ciprofloxacin, and the resolution,
R, between the ciprofloxacin ethylenediamine analog peak and the ciprofloxacin peak is not less than 1.5. Chromatograph the
Standard preparation, and record the responses as directed under
Procedure: the capacity factor,
k¢, for the ciprofloxacin peak is between 1.5 and 6, the column efficiency is not less than 500 theoretical plates, the tailing factor for the analyte peak is not less than 0.9 and not more than 2.0, and the relative standard deviation for replicate injections is not more than 2%.
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 ciprofloxacin peaks. Calculate the quantity, in mg, of ciprofloxacin (C
17H
18FN
3O
3) in each mL of the Ophthalmic Solution taken by the formula:
(331.34 / 367.81)(50C / V)(rU / rS),
in which 331.34 and 367.81 are the molecular weights of ciprofloxacin and anhydrous ciprofloxacin hydrochloride, respectively;
C is the concentration, in mg per mL, of
USP Ciprofloxacin Hydrochloride RS in the
Standard preparation, calculated on the anhydrous basis;
V is the volume, in mL, of Ophthalmic Solution taken; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
