Fluorescein
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332.31Spiro[isobenzofuran-1(3H),9¢-[9H]xanthen-3-one, 3¢,6¢-dihydroxy-.
Fluorescein
[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.
Packaging and storage—
Preserve in tight containers.
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Identification, Infrared Absorption 
197K
:
previously dried over silica gel for 16 hours.
197K:
previously dried over silica gel for 16 hours.
Water, Method I 921:
not more than 1.0%.
921:
not more than 1.0%.
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.
Acriflavine—
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.
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 diacetylfluorescein 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.
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 C20H12O5 in the Fluorescein taken by the formula:
(332.31 / 416.39)(3333C)(IU / IS)
in which 332.31 and 416.39 are the molecular weights of fluorescein and diacetylfluorescein, respectively; C is the concentration, in µg per mL, of USP Diacetylfluorescein RS in the Standard preparation; and IU and IS are the fluorescence values observed for the Assay preparation and the Standard preparation, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Feiwen Mao, M.S.
Senior Scientific Liaison 1-301-816-8320 |
(SM32010) Monographs - Small Molecules 3 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 3229