Standard preparation— Dissolve about 110mg of USP Diacetylfluorescein RS,accurately weighed,in 10mLof alcohol contained in a 100-mLvolumetric flask.Add 2mLof 2.5Nsodium hydroxide,and heat on a steam bath at about the boiling temperature for 20minutes,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 1.1µg of diacetylfluorescein per mL.Transfer 3.0mLof this solution to a 100-mLvolumetric flask containing 20mLof pH9.0alkaline borate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions),dilute with water to volume,and mix.

Assay preparation— Dissolve about 90mg of Fluorescein,accurately weighed,in 10mLof alcohol contained in a 100-mLvolumetric flask.Add 2mLof 2.5Nsodium hydroxide,and heat on a steam bath at about the boiling temperature for 20minutes,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 0.9µg per mL.Transfer 3.0mLof this solution to a 100-mLvolumetric flask containing 20mLof pH9.0alkaline borate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions),dilute with water to volume,and mix.

Procedure— Concomitantly determine the fluorescence intensities,I,of the Standard preparationand the Assay preparationin a fluorometer at an excitation wavelength of 485nm and an emission wavelength of 515nm.Calculate the quantity,in mg,of C20H12O5in the Fluorescein taken by the formula: in which 332.31and 416.39are the molecular weights of fluorescein and diacetylfluorescein,respectively;Cis the concentration,in µg per mL,of USP Diacetylfluorescein RSin the Standard preparation;and IUand ISare the fluorescence values observed for the Assay preparationand the Standard preparation,respectively.