Fluorescein Sodium and Benoxinate Hydrochloride Ophthalmic Solution

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

1 DEFINITION

Fluorescein Sodium and Benoxinate Hydrochloride Ophthalmic Solution is a sterile aqueous solution of fluorescein sodium and benoxinate hydrochloride. It contains NLT 90.0% and NMT 120.0% of the labeled amounts of fluorescein sodium (C₂₀H₁₀Na₂O₅) and benoxinate hydrochloride (C₁₇H₂₈N₂O₃·HCI). It contains a suitable preservative.

2 IDENTIFICATION

A. A solution of it is strongly fluorescent, even in extreme dilution. The fluorescence disappears when the solution is made acid, and reappears when the solution is again made alkaline.

B. The relative retention times of the major peaks of the Sample solution correspond to those of Standard solution A and Standard solution B, as obtained in the Assay.

3 ASSAY

3.1 PROCEDURE

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, and dilute with water to volume. Adjust with phosphoric acid to a pH of 3.

Standard stock solution A: Transfer 55 mg of USP Diacetylfluorescein RS 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 the boiling temperature for 20 min, with frequent swirling. Cool, and dilute with water to volume.

Standard solution A: 0.1 mg/mL of fluorescein sodium in Mobile phase prepared as follows. Transfer 10.0 mL of Standard stock solution A to a 100-mL volumetric flask, and dilute with Mobile phase to volume.

Standard solution B: 0.1J mg/mL of USP Benoxinate Hydrochloride RS in Mobile phase, where J is 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

Sample solution: Nominally equivalent to 0.1 mg/mL of fluorescein sodium in Mobile phase from Ophthalmic Solution

3.2 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4-mm × 30-cm; packing L1

Flow rate: 1.5 mL/min

Injection volume: 25 µL

3.3 System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Tailing factor: NMT 2.0 for each analyte peak, Standard solution A and Standard solution B

Relative standard deviation: NMT 2.0% for each analyte peak, Standard solution A and Standard solution B

3.4 Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Calculate the percentage of the labeled amount of fluorescein sodium (C₂₀H₁₀Na₂O₅) in the portion of Ophthalmic Solution taken:

Result = (r_U/r_S) x (C_S/C_U) x (M_r1/M_r2) x 100

r_U = peak area of fluorescein from the Sample solution

r_S = peak area of fluorescein from Standard solution A

C_S = concentration of USP Diacetylfluorescein RS in Standard solution A (mg/mL)

C_U = nominal concentration of fluorescein sodium in the Sample solution (mg/mL)

M_r1 = molecular weight of fluorescein sodium, 376.28

M_r2 = molecular weight of diacetylfluorescein, 416.39

Calculate the percentage of the labeled amount of benoxinate hydrochloride (C₁₇H₂₈N₂O₃·HCI) in the portion of Ophthalmic Solution taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U = peak area of benoxinate from the Sample solution

r_S = peak area of benoxinate from Standard solution B

C_S = concentration of USP Benoxinate Hydrochloride RS in Standard solution B (mg/mL)

C_U = nominal concentration of benoxinate hydrochloride in the Sample solution (mg/mL)

Acceptance criteria

Fluorescein sodium: 90.0%-120.0%

Benoxinate hydrochloride: 90.0%-120.0%

4 SPECIFIC TESTS

4.1 STERILITY TESTS (71)

It meets the requirements when tested as directed in Test for Sterility of the Product to Be Examined, Membrane Filtration.

4.2 PH (791)

4.3-5.3

5 ADDITIONAL REQUIREMENTS

5.1 PACKAGING AND STORAGE

Preserve in tight, light-resistant containers.

5.2 USP REFERENCE STANDARDS (11)

USP Benoxinate Hydrochloride RS

USP Diacetylfluorescein RS

Spiro(isobenzofuran-1(3H), 9'-(9H)xanthen)-3-one, 3',6'-bis(acetyloxy)-.

C₂₄H₁₆O₇ 416.39