Fluorescein Sodium

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

C₂₀H₁₀Na₂O₅ 376.27

Benzoic acid, 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl), sodium salt (1:2);

Fluorescein disodium salt CAS RN®: 518-47-8; UNII: 93X55PE38X.

1 DEFINITION

Fluorescein Sodium contains NLT 90.0% and NMT 102.0% of fluorescein sodium (C₂₀H₁₀Na₂O₅), calculated on the anhydrous basis.

2 IDENTIFICATION

A. A solution 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. Identification Tests - General, Sodium〈191〉: The residue remaining after incineration meets the requirements.

C. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 Procedure

Buffer: 0.61 g/L of monobasic potassium phosphate. Adjust with phosphoric acid to a pH of 2.0.

Mobile phase: See Table 1.

Table 1

Diluent: Acetonitrile and Buffer (30:70)

Standard stock solution: [Note—1.1 mg of USP Diacetyl fluorescein RS is equivalent to 1 mg of fluorescein sodium.] 1.0 mg/mL of fluorescein sodium is prepared as follows. Transfer USP Diacetyl fluorescein RS to a suitable volumetric flask. Add 2.5 N sodium hydroxide to fill 2% of the final volume and alcohol to fill 10% of the final volume. Heat on a water bath for 20 min, mixing frequently. Cool and dilute with water to volume.

Standard solution: 0.02 mg/mL of fluorescein sodium in Diluent from Standard stock solution

Sample stock solution: 1.0 mg/mL of Fluorescein Sodium in Diluent

Sample solution: 0.02 mg/mL of Fluorescein Sodium in Diluent from Sample stock solution

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 25-cm; 5-μm packing L7

Column temperature: 35°

Flow rate: 1.0 mL/min

Injection volume: 20 μL

3.3 System suitability

Sample: Standard solution

3.4 Suitability requirements

Tailing factor: NMT 1.5

Relative standard deviation: NMT 0.73%

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of fluorescein sodium (C₂₀H₁₀Na₂O₅) in the portion of Fluorescein Sodium taken:

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

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

C_U = concentration of Fluorescein Sodium in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–102.0% on the anhydrous basis

4 IMPURITIES

4.1 Zinc

Sample solution: 10 mg/mL of Fluorescein Sodium in a saturated solution of sodium chloride

Analysis: To 10 mL of Sample solution add 2 mL of 3 N hydrochloric acid. Shake well, filter, and add 1 mL of potassium ferrocyanide TS to the filtrate.

Acceptance criteria: No turbidity is produced.

4.2 Acriflavine

Sample solution: 2 mg/mL of Fluorescein Sodium in water. Swirl the mixture, and filter.

Analysis: To the Sample solution add a few drops of 100-mg/mL sodium salicylate solution.

Acceptance criteria: No precipitate is formed.

4.3 Organic Impurities

Buffer, Mobile phase, Diluent, and Chromatographic system: Proceed as directed in the Assay.

Standard stock solution: Proceed as directed in the Assay for Standard solution.

Standard solution A: 0.005 mg/mL of fluorescein sodium in Diluent from Standard stock solution

Standard solution B: 0.005 mg/mL each of USP Resorcinol RS, USP Phthalic Acid RS, and USP Fluorescein Related Compound C RS in Diluent

Sample solution: Use Sample stock solution from the Assay.

System suitability

Sample: Standard solution B

Suitability requirements

Resolution: NLT 1.5 between resorcinol and phthalic acid

Analysis

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

Calculate the percentage of resorcinol, phthalic acid, and fluorescein related compound C in the portion of Fluorescein Sodium taken:

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

r_U = peak response of resorcinol or phthalic acid or fluorescein related compound C from the Sample solution

r_S = peak response of resorcinol or phthalic acid or fluorescein related compound C from Standard solution B

C_S = concentration of USP Resorcinol RS or USP Phthalic Acid RS or USP Fluorescein Related Compound C RS in Standard solution B (mg/mL)

C_U = concentration of Fluorescein Sodium in the Sample solution (mg/mL)

Calculate the percentage of unspecified impurity in the portion of Fluorescein Sodium taken:

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

r_U = peak response of unspecified impurity from the Sample solution

r_S = peak response from Standard solution A

C_S = concentration of fluorescein sodium in Standard solution A (mg/mL)

C_U = concentration of Fluorescein Sodium in the Sample solution (mg/mL)

Acceptance criteria: See Table 2. Disregard any impurity peaks less than 0.05%.

Table 2

5 SPECIFIC TESTS

Water Determination, Method I〈921〉: NMT 17.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

USP Reference Standards 〈11〉

USP Diacetylfluorescein RS C₂₄H₁₆O₇ 416.39

USP Fluorescein Related Compound C RS

2-(2,4-Dihydroxybenzoyl)benzoic acid.

C₁₄H₁₀O₅ 258.23

USP Phthalic Acid RS C₈H₆O₄ 166.13

USP Resorcinol RS C₆H₆O₂ 110.11