Fluorescein Sodium and Benoxinate Hydrochloride Ophthalmic Solution
» Fluorescein Sodium and Benoxinate Hydrochloride Ophthalmic Solution is a sterile aqueous solution of Fluorescein Sodium and Benoxinate Hydrochloride. It contains not less than 90.0 percent and not more than 120.0 percent of the labeled amounts of fluorescein sodium (C20H10Na2O5) and benoxinate hydrochloride (C17H28N2O3·HCl). It contains a suitable preservative.
Packaging and storage—
Preserve in tight, light-resistant containers.
USP Reference standards 
11
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11—
USP Benoxinate Hydrochloride RS 
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USP Diacetylfluorescein RS
C24H16O7 416.39
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C24H16O7 416.39
Identification—
A:
It responds to Identification test A under Fluorescein Sodium.
B:
The relative retention times of the major peaks in the chromatogram of the Assay correspond to those in the chromatograms of the Standard fluorescein sodium preparation and the Standard benoxinate hydrochloride preparation as obtained in the Assay.
Sterility 71—
It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the Product to be Examined.
71—
It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the Product to be Examined.
pH 791:
between 4.3 and 5.3.
791:
between 4.3 and 5.3.
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).
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 0.1J 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%.
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 (C20H10Na2O5) 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 (C17H28N2O3·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.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Feiwen Mao, M.S.
Senior Scientific Liaison 1-301-816-8320 |
(SM32010) Monographs - Small Molecules 3 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
|
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 3231