A: Ultraviolet Absorption á197Uñ—

Solution: 20µg per mL,USP Hydrocortisone Phosphate Triethylamine RSbeing used to prepare the Standard solution.

Medium: water.

B: Place 5mLof the Assay preparation,obtained as directed in the Assay,in a glass-stoppered,50-mLtube,and add 5mLof a solution prepared by dissolving 50mg of alkaline phosphatase enzyme in 50mLof pH9buffer with magnesiumprepared as directed in the Assayunder Hydrocortisone Sodium Phosphate.Allow to stand at room temperature for 2hours,with occasional mixing,and extract with 25mLof methylene chloride.Evaporate 15mLof the methylene chloride extract on a steam bath to dryness,and dissolve the residue in 0.5mLof methylene chloride.Apply 5µLof this solution and 5µLof a solution of USP Hydrocortisone RSin methylene chloride containing 300µg per mLto a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a tank completely lined with filter paper,using a solvent system consisting of a mixture of 50parts of chloroform,50parts of acetone,and 1part of water,until the solvent front has moved about three-fourths of the length of the plate.Remove the plate,mark the solvent front,and dry.Spray the plate with dilute sulfuric acid (1in 2),and heat at 105until brown or black spots appear:the RFvalue of the principal spot obtained from the solution under test corresponds to that obtained from the Standard solution.

Phenylhydrazine hydrochloride solution— Dissolve 65mg of phenylhydrazine hydrochloride in 100mLof dilute sulfuric acid (3in 5),add 50mLof isopropyl alcohol,and mix.Prepare this solution fresh daily.

Standard preparation— Dissolve a suitable quantity of USP Hydrocortisone Phosphate Triethylamine RS,accurately weighed,in water,and dilute quantitatively and stepwise with water to obtain a solution having a known concentration of about 110µg per mL.

Assay preparation— Pipet a volume of Injection,equivalent to about 100mg of hydrocortisone sodium phosphate,into a 100-mLvolumetric flask,and dilute with water to volume.Pipet 10mLof this solution into a separator,wash the solution with two 25-mLportions of methylene chloride,and discard the washings.Transfer the aqueous layer to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Procedure— Pipet 2mLeach of the Standard preparationand the Assay preparationinto separate glass-stoppered,50-mLconical flasks.To each flask,and to a similar flask containing 2.0mLof water to provide a blank,add 10.0mLof Phenylhydrazine hydrochloride solution,and mix.Place the flasks in a water bath maintained at a temperature of 60for 2hours,then cool the solutions to room temperature.Concomitantly determine the absorbances of the solutions from the Assay preparationand the Standard preparationat the wavelength of maximum absorbance at about 410nm,with a suitable spectrophotometer,using the blank to set the instrument.Calculate the quantity,in mg,of C21H30O5in each mLof the Injection taken by the formula: in which 0.667is the ratio of the molecular weight of hydrocortisone to that of hydrocortisone phosphate triethylamine;Cis the concentration,in µg per mL,of USP Hydrocortisone Phosphate Triethylamine RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.