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.

B: The residue remaining after the incineration of it responds to the tests for Sodium 191.

C: Place 1 drop of a solution (1 in 2000) upon a piece of filter paper: a yellow spot is produced and, when exposed while moist to the vapor of bromine for 1 minute and then to ammonia vapor, it becomes deep pink in color.

Standard preparation— Dissolve an accurately weighed quantity of USP Diacetylfluorescein RS in 10 mL of alcohol contained in a 100-mL volumetric flask. [note—110.7 mg of anhydrous USP Diacetylfluorescein RS is equivalent to 100.0 mg of fluorescein sodium.] 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. Transfer a suitable aliquot to a volumetric flask, and dilute with water to volume to obtain a solution having a concentration of 1 µg of fluorescein sodium per mL. Transfer a 3-mL aliquot 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. The concentration of fluorescein sodium in the Standard preparation is 0.03 µg per mL.

Assay preparation— Dissolve about 100 mg of Fluorescein Sodium, accurately weighed, in water, and dilute quantitatively and stepwise with water to obtain a solution having a concentration of 1 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 at 515 nm. Calculate the quantity, in mg, of C20H10Na2O5 in the Fluorescein Sodium taken by the formula: in which C is the concentration, in µg per mL, of fluorescein sodium in the Standard preparation; and IU and IS are the fluorescence values observed for the Assay preparation and the Standard preparation, respectively.