Fluorescein

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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C₂₀H₁₂O₅ 332.31

Spiro[isobenzofuran-1(3H),9′-[9H]xanthen-3-one, 3′,6′-dihydroxy-.

Fluorescein CAS RN®: 2321-07-5.

Fluorescein contains not less than 97.0 percent and not more than 102.0 percent of C₂₀H₁₂O₅ calculated on the anhydrous basis.

1 Packaging and storage

Preserve in tight containers.

USP Reference standards 〈11〉

USP Diacetylfluorescein RS C₂₄H₁₆O₇ 416.39

USP Fluorescein RS

Change to read:

2 Identification

Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020) : previously dried over silica gel for 16 hours.

Water Determination, Method I 〈921〉: not more than 1.0%.

3 Zinc

Suspend 100 mg in 10 mL of a saturated solution of sodium chloride, add 2 mL of 3 N hydrochloric acid, mix, filter, and add 1 mL of potassium ferrocyanide TS to the filtrate: no turbidity is produced.

4 Acrifiavine

Suspend 10 mg in 5 mL of water, swirl the mixture, and filter. To the filtrate add a few drops of sodium salicylate solution (1 in 10): no precipitate is formed.

5 Assay

Standard preparation-Dissolve about 110 mg of USP Diacetylfluorescein RS, accurately weighed, 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 about the boiling temperature for 20 minutes, with frequent swirling. Cool, dilute with water to volume, and mix. Dilute quantitatively and stepwise with water to obtain a solution having a known concentration of about 1.1 μg of diacetyl fluorescein per mL. Transfer 3.0 mL of this solution to a 100-mL volumetric flask containing 20 mL of pH 9.0 alkaline borate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions), dilute with water to volume, and mix.

Assay preparation - Dissolve about 90 mg of Fluorescein, accurately weighed, 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 about the boiling temperature for 20 minutes, with frequent swirling. Cool, dilute with water to volume, and mix. Dilute quantitatively and stepwise with water to obtain a solution having a concentration of 0.9 μg per mL. Transfer 3.0 mL of this solution to a 100-mL volumetric flask containing 20 mL of pH 9.0 alkaline borate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions), dilute with water to volume, and mix.

6 Procedure

Concomitantly determine the fluorescence intensities, I, of the Standard preparation and the Assay preparation in a fluorometer at an excitation wavelength of 485 nm and an emission wavelength of 515 nm. Calculate the quantity, in mg, of C H O in the Fluorescein taken by the formula:

(332.31/416.39)(3333C)(I_U/I_S)

in which 332.31 and 416.39 are the molecular weights offluorescein and diacetyl fluorescein, respectively; C is the concentration, in μg per mL, of USP Diacetyl fluorescein RS in the Standard preparation; and I_U and I_S are the fluorescence values observed for the Assay preparation and the Standard preparation, respectively.