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: 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.