Fluorescein Sodium and Proparacaine 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 Proparacaine Hydrochloride Ophthalmic Solution is a sterile aqueous solution of Fluorescein Sodium and Proparacaine Hydrochloride. It contains NLT 90.0% and NMT 110.0% of the labeled amounts of fluorescein sodium (C₂₀H₁₀Na₂O₅) and proparacaine hydrochloride (C₁₆H₂₆N₂O₃ · HCl). 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.

Solution A: 20 mg/mL of 2-naphthol in 1 N sodium hydroxide

Analysis: To 1 mL of Ophthalmic Solution in a test tube add 5 mL of diluted hydrochloric acid (1 in 100), and cool in an ice bath for 2 min. Add 1 mL of Solution A.

Acceptance criteria: A scarlet-red precipitate is formed. Add 5 mL of acetone. The precipitate does not dissolve.

3 ASSAY

3.1 Fluorescein Sodium

Standard stock solution: 1 μg/mL of fluorescein sodium from USP Diacetyl fluorescein RS in water prepared as follows. Dissolve USP Diacetyl fluorescein RS in 10 mL of alcohol contained in a 100-mL volumetric flask, add 2 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. Transfer a suitable aliquot to a volumetric flask, and dilute with water to volume. [Note—110.7 mg of anhydrous USP Diacetyl fluorescein RS is equivalent to 100.0 mg of fluorescein sodium.]

Standard solution: 0.03 μg/mL of fluorescein sodium in water prepared as follows. Transfer 3 mL of Standard stock solution to a 100-mL volumetric flask containing 20 mL of pH 9.0 alkaline borate buffer (see Reagents, Indicators, and Solutions-Buffer Solutions), and dilute with water to volume.

Sample stock solution: Nominally equivalent to 1 μg/mL of fluorescein sodium from Ophthalmic Solution in water

Sample solution: Nominally equivalent to 0.03 μg/mL of fluorescein sodium in water prepared as follows. Transfer 3.0 mL of Sample stock solution to a 100-mL volumetric flask containing 20 mL of pH 9.0 alkaline borate buffer (see Reagents, Indicators, and Solutions - Buffer Solutions), and dilute with water to volume.

3.1.1 Instrumental conditions

Mode: Fluorescence

Excitation wavelength: 485 nm

Emission wavelength: 515 nm

3.1.2 Analysis

Samples: Standard solution and Sample solution

Concomitantly determine the fluorescence intensities, I, of the Standard solution and the Sample solution.

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

Result = (I_U/I_S) × (C_S/C_U) × 100

I_U = fluorescence value of the Sample solution

I_S = fluorescence value of the Standard solution

C_S = concentration of fluorescein sodium in the Standard solution (μg/mL)

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

Acceptance criteria: 90.0% - 110.0%

3.2 Proparacaine Hydrochloride

Buffer: 6.8 g/L of monobasic potassium phosphate in 1000 mL of water. Add 5 mL of triethylamine, and adjust with 5 N potassium hydroxide to a pH of 7.5.

Mobile phase: Acetonitrile and Buffer (40:60)

Standard solution: 0.1 mg/mL of proparacaine in Mobile phase prepared as follows. Prepare 1 mg/mL of USP Proparacaine Hydrochloride RS in water, and dilute it with Mobile phase to 0.1 mg/mL. Use this solution within 6 h.

Sample solution: Nominally equivalent to 0.1 mg/mL of proparacaine hydrochloride in Mobile phase from Ophthalmic Solution. Use this solution within 6 h.

3.2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 270 nm

Column: 4.6-mm × 15-cm; 5-μm packing L10

Flow rate: 1.5 mL/min

Injection volume: 10 μL

3.2.2 System suitability

Sample: Standard solution

3.2.3 Suitability requirements

Column efficiency: NLT 3000 theoretical plates

Tailing factor: NMT 1.5

Relative standard deviation: NMT 2.0%

3.2.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of proparacaine hydrochloride (C₁₆H₂₆N₂O₃ · HCl) in the portion of Ophthalmic Solution

taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak area from the Sample solution

r_S = peak area from the Standard solution

C_S = concentration of USP Proparacaine Hydrochloride RS in the Standard solution (mg/mL)

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

Acceptance criteria: 90.0%–110.0%

4 SPECIFIC TESTS

Sterility Tests 〈71〉: It meets the requirements when tested as directed in Test for Sterility of the Product to Be Examined, Membrane Filtration.

pH 〈791〉: 4.0–5.2

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers, preferably of Type I amber glass, and store in a refrigerator.

Labeling: Label it to state that it is to be stored in a refrigerator before and after the container is opened. 

USP Reference Standards 〈11〉

USP Diacetylfluorescein RS

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

C₂₄H₁₆O₇ 416.39

USP Proparacaine Hydrochloride RS