A: Dissolve 65 mg of phenylhydrazine hydrochloride in 100 mL of dilute sulfuric acid (3 in 5), add 5 mL of isopropyl alcohol, and mix. Heat 5 mL of this solution with 1 mL of Assay preparation (obtained as directed in the Assay) at 70 for 2 hours: a yellow color develops.

B: It responds to Identification test A under Prednisolone Sodium Phosphate.

pH 9 buffer with magnesium— Prepare as directed in the Assay under Prednisolone Sodium Phosphate.

Alkaline phosphatase solution— Prepare as directed in the Assay under Prednisolone Sodium Phosphate.

Standard preparation— Prepare as directed in the Assay under Prednisolone Sodium Phosphate.

Assay preparation— Pipet a volume of Injection, equivalent to about 100 mg of prednisolone phosphate, into a separator containing 20 mL of water. Wash the solution with two 10-mL portions of methylene chloride, and discard the washings. Transfer the aqueous layer to a 50-mL volumetric flask, dilute with water to volume, and mix.

Procedure— Proceed as directed for Procedure in the Assay under Prednisolone Sodium Phosphate. Calculate the quantity, in mg, of C21H29O8P in each mL of the Injection taken by the formula: in which C is the concentration, in µg per mL, of USP Prednisolone RS in the Standard preparation, V is the volume, in mL, of Injection taken, and AU and AS are the absorbances of the solution from the Assay preparation and the Standard preparation, respectively.