Assay—
Mobile phase—
Dissolve 100 mg of sodium 1-pentanesulfonate in 40 mL of glacial acetic acid in a 2000-mL volumetric flask. Add 600 mL of acetonitrile and 10 mL of triethanolamine, dilute with water to volume, and mix. Adjust with phosphoric acid to a pH of 3, and pass through a filter having a 0.5-µm or finer porosity. Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard fluorescein sodium preparation—
Transfer about 55 mg of
USP Diacetylfluorescein RS, accurately weighed, to a 50-mL volumetric flask containing 5 mL of alcohol. Add 1 mL of 2.5 N sodium hydroxide, and heat on a steam bath at about the boiling temperature for 20 minutes, with frequent swirling. Cool, dilute with water to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with
Mobile phase to volume, and mix. This solution contains the equivalent of about 0.1 mg of fluorescein sodium per mL.
Standard benoxinate hydrochloride preparation—
Quantitatively dissolve an accurately weighed quantity of
USP Benoxinate Hydrochloride RS in
Mobile phase, and if necessary dilute quantitatively and stepwise with
Mobile phase to obtain a solution having a known concentration of about 0.1
J mg per mL,
J being the ratio of the labeled amount, in mg, of benoxinate hydrochloride to the labeled amount, in mg, of fluorescein sodium in each mL of Ophthalmic Solution.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 5 mg of fluorescein sodium, to a 50-mL volumetric flask, dilute with
Mobile phase to volume, and mix.
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 fluorescein sodium preparation and the
Standard benoxinate hydrochloride preparation, and record the peak responses as directed for
Procedure: the tailing factor for each analyte peak is not more than 2.0, and the relative standard deviation for replicate injections of each
Standard preparation is not more than 2.0%.
Procedure—
[NOTE—Use peak areas where peak responses are indicated.
] Separately inject equal volumes (about 25 µL) of the
Standard fluorescein sodium preparation, the
Standard benoxinate hydrochloride preparation, and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantity, in mg, of fluorescein sodium (C
20H
10Na
2O
5) in each mL of Ophthalmic Solution taken by the formula:
(376.28 / 416.39)(W / 10V)(rU / rS),
in which 376.28 and 416.39 are the molecular weights of fluorescein sodium and diacetylfluorescein, respectively;
W is the quantity, in mg, of
USP Diacetylfluorescein RS taken to prepare the
Standard fluorescein sodium preparation; V is the volume, in mL, of Ophthalmic Solution taken; and
rU and
rS are the fluorescein peak responses obtained from the
Assay preparation and the
Standard fluorescein sodium preparation, respectively. Calculate the quantity, in mg, of benoxinate hydrochloride (C
17H
28N
2O
3·HCl) in each mL of Ophthalmic Solution taken by the formula:
50(C / V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Benoxinate Hydrochloride RS in the
Standard benoxinate hydrochloride preparation; V is the volume, in mL, of Ophthalmic Solution taken; and
rU and
rS are the benoxinate peak responses obtained from the
Assay preparation and the
Standard benoxinate hydrochloride preparation, respectively.
