A: Transfer a volume of Injection,equivalent to 125mg of hydroxyprogesterone caproate,to a 60-mLseparator containing 10mLof solvent hexane,8mLof methanol,and 2mLof water.Insert the stopper,shake for 2minutes,and allow the phases to separate.To 3mLof the lower layer add sulfuric acid dropwise until a color develops,then add 3mLof methanol:a purple color develops,and the solution,when viewed under long-wavelength UVlight,exhibits a pale yellow fluorescence.

B: Evaporate 4mLof the Assay preparation,obtained as directed in the Assay,on a water bath to dryness,and dissolve the residue in 0.5mLof chloroform.Apply 10µLof this solution and 10µLof a solution of USP Hydroxyprogesterone Caproate RSin chloroform,containing 400µg per mL,to a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture,on a line about 2.5cm from the bottom edge and about 2cm apart.Place the plate in a developing chamber that contains and that has been equilibrated with a mixture of 3volumes of chloroform and 1volume of ethyl acetate.Develop the plate until the solvent front has moved to about 10cm above the points of application.Remove the plate,mark the solvent front,and dry.Spray the plate with a mixture of 1volume of sulfuric acid and 3volumes of alcohol,and heat in an oven at 105for 5minutes:the RFvalue of the principal yellowish green spot obtained from the solution under test corresponds to that obtained from the Standard solution.

Isoniazid reagent— Dissolve 375mg of isoniazid and 0.47mLof hydrochloric acid in 500mLof methanol.

Standard preparation— Dissolve a suitable quantity of USP Hydroxyprogesterone Caproate RS,accurately weighed,in methanol,and dilute quantitatively and stepwise with methanol to obtain a solution having a known concentration of about 50µg per mL.

Assay preparation— Transfer to a 250-mLvolumetric flask an accurately measured volume of Injection,equivalent to about 250mg of hydroxyprogesterone caproate,add methanol to volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,add methanol to volume,and mix.

Procedure— Pipet 5mLof Assay preparationinto a glass-stoppered,50-mLconical flask.Pipet 5mLof Standard preparationinto a similar flask.To each flask,add 10.0mLof Isoniazid reagent,mix,and allow to stand in a water bath at 30for about 45minutes.Concomitantly determine the absorbances of both solutions at the wavelength of maximum absorbance at about 380nm,with a suitable spectrophotometer,using as a blank a mixture of 5mLof methanol and 10mLof Isoniazid reagent.Calculate the quantity,in mg,of hydroxyprogesterone caproate (C27H40O4)in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydroxyprogesterone Caproate 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.