A: Place 5mLof the Assay preparationobtained as directed in the Assay,in a glass-stoppered,100-mLvolumetric flask,mix with 5mLof Alkaline phosphatase solutionprepared as directed in the Assay,and add 50mLof methylene chloride.Insert the stopper,and allow to stand,with occasional gentle inversion (about once every 15minutes),for 2hours.Filter the methylene chloride layer through a dry paper,and evaporate 25mLof the filtrate to dryness:the residue so obtained responds to Identificationtest Aunder Prednisolone.

B: The residue from the ignition of about 20mg of it responds to the tests for Sodium á191ñand for Phosphate á191ñ.

Test solution: 10mg per mL,in a mixture of pH7.0phosphate buffer and carbon dioxide-free water (9:1).

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

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

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

Procedure— Dissolve about 50mg of Prednisolone Sodium Phosphate,accurately weighed,in a mixture of 10mLof water and 5mLof 2Nsulfuric acid contained in a 25-mLvolumetric flask,by warming if necessary.Add 1mLeach of Phosphate reagent Aand Phosphate reagent B,dilute with water to 25mL,mix,and allow to stand at room temperature for 30minutes.Similarly and concomitantly,prepare a standard solution,using 5.0mLof Standard phosphate solutioninstead of the 50mg of the substance under test.Concomitantly determine the absorbances of both solutions in 1-cm cells at 730nm,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).

pH9buffer with magnesium— Mix 3.1g of boric acid and 500mLof water in a 1-liter volumetric flask,add 21mLof 1Nsodium hydroxide and 10mLof 0.1Mmagnesium chloride,dilute with water to volume,and mix.

Alkaline phosphatase solution— Transfer 250mg of alkaline phosphate enzyme to a 25-mLvolumetric flask,dissolve by adding pH9buffer with magnesiumto volume,and mix.Prepare this solution fresh daily.

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

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

Procedure— Pipet 1mLof the Assay preparationinto a glass-stoppered,100-mLcylinder,add 1.0mLof Alkaline phosphatase solutionand about 50mLof methylene chloride,insert the stopper,and allow to stand,with occasional gentle inversion (about once every 15minutes),for 2hours.Add methylene chloride to volume,mix,and allow to stand until the methylene chloride layer is clear (about 20minutes).Concomitantly and without delay,determine the absorbances of the methylene chloride solution obtained from the Assay preparationand the Standard preparationat 241nm,with a suitable spectrophotometer,using methylene chloride as the blank.Calculate the quantity,in mg,of C21H27Na2O8Pin the portion of Prednisolone Sodium Phosphate taken by the formula: in which 1.344is the ratio of the molecular weight of prednisolone sodium phosphate to that of prednisolone,Cis the concentration,in µg per mL,of USP Prednisolone RSin the Standard preparation,and AUand ASare the absorbances of the solution from the Assay preparationand the Standard preparation,respectively.