A: Evaporate 15mLof a methylene chloride solution of it,prepared as directed under Procedurein the Assay,on a steam bath to dryness,and dissolve the residue in 1mLof methylene chloride.Proceed as directed in Identificationtest Bunder Hydrocortisone Sodium Phosphate Injection,beginning with “Apply 5µLof this solution.”

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

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 Hydrocortisone 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).

1000WU(1-L/100)/WS,

Specific rotation á781Sñ :between +121and +129,determined in a solution prepared by weighing accurately 5.0mLof Test preparationand diluting with pH7.0phosphate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions)to 50.0mL.

pHá791ñ :between 7.5and 10.5,in a solution prepared by diluting a portion of Test preparationwith 9volumes of carbon dioxide-free water.

Free hydrocortisone— Dilute 1mLof Test preparationwith carbon dioxide-free water to 100mL.Pipet 5mLof this solution into a glass-stoppered,50-mLtube,add 25.0mLof methylene chloride,insert the stopper,and mix by gentle shaking.Prepare a 1in 500,000solution of USP Hydrocortisone RSin methylene chloride.Similarly,shake 25mLof this solution with 5mLof water.Allow to stand until the methylene chloride layers are clear (about 5minutes).Determine the absorbances of the methylene chloride solutions in 1-cm cells at 239nm,with a suitable spectrophotometer,using methylene chloride as the blank.The absorbance of the Test preparationdoes not exceed that of the Standard solution (1.0%).

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 phosphatase enzyme to a 25-mLvolumetric flask,and dissolve by diluting with pH9buffer with magnesiumto volume.Prepare this solution fresh daily.

Standard preparation— Dissolve about 50mg of USP Hydrocortisone Phosphate Triethylamine RS,accurately weighed,in carbon dioxide-free water to make 25.0mL.

Assay preparation— Weigh accurately,in g,2.0mLof the Test preparation,prepared as directed under Specific rotation,pH,and Free hydrocortisone,into a tared 100-mLvolumetric flask,and dilute with carbon dioxide-free water that has been saturated with methylene chloride to volume.Pipet 10mLof this solution into a 125-mLseparator,and extract with two 25-mLportions of water-washed methylene chloride,discarding the washings.

Procedure— Weigh accurately 1.0mLeach of the Standard preparation(WS)and the Assay preparation(WA)into separate tared 100-mLvolumetric flasks.To each flask,and to a similar flask containing 1.0mLof water to provide a blank,add 1.0mLof Alkaline phosphatase solutionand then 50mLof methylene chloride,and insert the stopper.Allow the flasks to stand at room temperature (not below 25)for 2hours with gentle mixing about every 15minutes.Add 1mLof dilute hydrochloric acid (1in 10)to each flask,and mix gently.Add methylene chloride to each flask until the interfaces are at the 100-mLmarks,and mix gently.Remove the aqueous layers by aspiration.Determine the absorbances of the methylene chloride solutions obtained from the Standard preparationand the Assay preparationat 239nm,with a suitable spectrophotometer,using the methylene chloride solution blank to set the instrument.Calculate the percentage of C21H29Na2O8P,on the dried basis,taken by the formula: in which AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively;CAand CSare the corresponding concentrations,in mg per mL,of those preparations;and 0.895is the ratio of the molecular weight of hydrocortisone sodium phosphate to that of hydrocortisone phosphate triethylamine.