A: Place 5 mL of the Assay preparation obtained as directed in the Assay, in a glass-stoppered, 100-mL volumetric flask, mix with 5 mL of Alkaline phosphatase solution prepared as directed in the Assay, and add 50 mL of methylene chloride. Insert the stopper, and allow to stand, with occasional gentle inversion (about once every 15 minutes), for 2 hours. Filter the methylene chloride layer through a dry paper, and evaporate 25 mL of the filtrate to dryness: the residue so obtained responds to Identification test A under Prednisolone.

B: The residue from the ignition of about 20 mg of it responds to the tests for Sodium 191 and for Phosphate 191.

Test solution: 10 mg per mL, in a mixture of pH 7.0 phosphate buffer and carbon dioxide-free water (9:1).

Standard phosphate solution— Dissolve 143.3 mg of dried monobasic potassium phosphate, KH2PO4, in water to make 1000.0 mL. This solution contains the equivalent of 0.10 mg of phosphate (PO4) in each mL.

Phosphate reagent A— Dissolve 5 g of ammonium molybdate in 1 N sulfuric acid to make 100 mL.

Phosphate reagent B— Dissolve 350 mg of p-methylaminophenol sulfate in 50 mL of water, add 20 g of sodium bisulfite, mix to dissolve, and dilute with water to 100 mL.

Procedure— Dissolve about 50 mg of Prednisolone Sodium Phosphate, accurately weighed, in a mixture of 10 mL of water and 5 mL of 2 N sulfuric acid contained in a 25-mL volumetric flask, by warming if necessary. Add 1 mL each of Phosphate reagent A and Phosphate reagent B, dilute with water to 25 mL, mix, and allow to stand at room temperature for 30 minutes. Similarly and concomitantly, prepare a standard solution, using 5.0 mL of Standard phosphate solution instead of the 50 mg of the substance under test. Concomitantly determine the absorbances of both solutions in 1-cm cells at 730 nm, with a suitable spectrophotometer, using water as the blank. The absorbance of the test solution is not more than that of the standard solution. The limit is 1.0% of phosphate (PO4).

(Official January 1, 2007)

pH 9 buffer with magnesium— Mix 3.1 g of boric acid and 500 mL of water in a 1-liter volumetric flask, add 21 mL of 1 N sodium hydroxide and 10 mL of 0.1 M magnesium chloride, dilute with water to volume, and mix.

Alkaline phosphatase solution— Transfer 250 mg of alkaline phosphate enzyme to a 25-mL volumetric flask, dissolve by adding pH 9 buffer with magnesium to volume, and mix. Prepare this solution fresh daily.

Standard preparation— Dissolve a suitable, accurately weighed quantity of USP Prednisolone RS in methylene chloride, and dilute quantitatively and stepwise with methylene chloride to obtain a solution having a known concentration of about 16 µg per mL. Pipet 100 mL of the solution into a glass-stoppered, 100-mL cylinder, and add 1.0 mL of Alkaline phosphatase solution and 1.0 mL of water. Allow to stand, with occasional gentle inversion, for 2 hours.

Assay preparation— Dissolve about 100 mg of Prednisolone Sodium Phosphate, accurately weighed, in water that has been saturated with methylene chloride, to make 50.0 mL, and mix. Pipet 10 mL of this solution into a 125-mL separator, and shake with two 25-mL portions of water-washed methylene chloride, discarding the methylene chloride layers.

Procedure— Pipet 1 mL of the Assay preparation into a glass-stoppered, 100-mL cylinder, add 1.0 mL of Alkaline phosphatase solution and about 50 mL of methylene chloride, insert the stopper, and allow to stand, with occasional gentle inversion (about once every 15 minutes), for 2 hours. Add methylene chloride to volume, mix, and allow to stand until the methylene chloride layer is clear (about 20 minutes). Concomitantly and without delay, determine the absorbances of the methylene chloride solution obtained from the Assay preparation and the Standard preparation at 241 nm, with a suitable spectrophotometer, using methylene chloride as the blank. Calculate the quantity, in mg, of C21H27Na2O8P in the portion of Prednisolone Sodium Phosphate taken by the formula: in which 1.344 is the ratio of the molecular weight of prednisolone sodium phosphate to that of prednisolone, C is the concentration, in µg per mL, of USP Prednisolone RS in the Standard preparation, and AU and AS are the absorbances of the solution from the Assay preparation and the Standard preparation, respectively.