Fluorescein Injection

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

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

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1 DEFINITION

Fluorescein Injection is a sterile solution, in Water for Injection, of Fluorescein prepared with the aid of Sodium Hydroxide. It contains the equivalent of NLT 90.0% and NMT 110.0% of the labeled amount of Fluorescein Sodium (C₂₀H₁₀Na₂O₅). It may contain Sodium Bicarbonate.

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.

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B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay (USP 1-May-2019)

3 ASSAY

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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 Diacetylfluorescein RS is equivalent to 1 mg of fluorescein sodium.]1.0 mg/mL of fluorescein sodium prepared as follows. Transfer USP Diacetylfluorescein RS to a suitable volumetric flask. Add 2.5 N sodium hydroxide to fill 2% of the final volume and add 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: Nominally 1.0 mg/mL of fluorescein sodium in Diluent prepared as follows. Transfer a portion of Injection equivalent to 10.0 mg of fluorescein sodium into a 10-mL volumetric flask. Add 1 mL of alcohol. Dilute with Diluent to volume and mix well.

Sample solution: Nominally 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

Suitability requirements

Tailing factor: NMT 1.5

Relative standard deviation: NMT 1.0%

3.4 Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U/r_S) x (C_S/C_U) x 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 = nominal concentration of fluorescein sodium in the Sample solution (mg/mL)

(USP 1-May-2019)

Acceptance criteria: 90.0%-110.0%

4 IMPURITIES

Add the following:

4.1 ORGANIC IMPURITIES

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

Standard stock solution A: Use the Standard stock solution from the Assay.

Standard stock solution B: 0.05 mg/mL of fluorescein sodium in Diluent from Standard stock solution A

Standard stock solution C: 0.05 mg/mL each of USP Resorcinol RS, USP Phthalic Acid RS, and USP Fluorescein Related Compound C RS in Diluent

Standard solution: 0.001 mg/mL each of fluorescein sodium, USP Resorcinol RS, USP Phthalic Acid RS, and USP Fluorescein Related Compound C RS in Diluent from Standard stock solution B and Standard stock solution C

Sample solution: Use the Sample stock solution from the Assay.

4.2 System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.5 between resorcinol and phthalic acid

Relative standard deviation: NMT 5.0% for the resorcinol, phthalic acid, fluorescein related compound C, and fluorescein peaks

4.3 Analysis

Samples: Standard solution and Sample solution

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

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

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

r_S = peak response of the corresponding Reference Standard from the Standard solution

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

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

Calculate the percentage of any individual unspecified impurity in the portion of Injection taken:

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

r_U = peak response of each unspecified impurity from the Sample solution

r_S = peak response of fluorescein sodium from the Standard solution

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

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

Acceptance criteria: See Table 2.

Table 2

(USP 1-May-2019)

5 SPECIFIC TESTS

5.1 PYROGEN TEST (151)

Meets the requirements, the test dose being the equivalent of 250 mg/kg of fluorescein sodium

5.2 PH (791)

8.0-9.8

5.3 INJECTIONS AND IMPLANTED DRUG PRODUCTS (1)

Meets the requirements

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE

Preserve in single-dose containers, preferably of Type I glass.

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6.2 USP REFERENCE STANDARDS (11)

USP Diacetylfluorescein RS

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

C₂₄H₁₆O₇ 416.39

USP Fluorescein Related Compound C RS

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

C₁₄H₁₀O₅ 258.23

USP Phthalic Acid RS

Phthalic acid.

C₈H₆O₄ 166.13

USP Resorcinol RS

Resorcinol.

C₆H₆O₂ 110.11

(USP 1-May-2019)