Chromatographic solvent— Equilibrate,by shaking in a separator,95mLof alcohol,5mLof water,and 50mLof chromatographic n-heptane.Allow the layers to separate.

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

Standard preparation— Dissolve a suitable quantity of USP Testosterone Enanthate RS,accurately weighed,in methanol,and dilute quantitatively and stepwise with methanol to obtain a solution having a known concentration of about 40µg per mL.

Assay preparation— Transfer to a 10-mLvolumetric flask an accurately measured volume of Injection,equivalent to about 100mg of testosterone enanthate,add chromatographic n-heptane to volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,add chromatographic n-heptane to volume,and mix.

Procedure— Mix in a beaker 3g of silanized chromatographic siliceous earth and 3mLof the upper layer of the Chromatographic solvent.Pack the mixture into a 250-×25-mm chromatographic tube that contains a small pledget of glass wool above the stem constriction.Mix in a beaker 3g of silanized chromatographic siliceous earth and 2.0mLof Assay preparation,transfer the mixture to the tube,and pack.Dry-wash the beaker with 1g of silanized chromatographic siliceous earth,and transfer to the tube.Place a small pad of glass wool above the column packing.Pass 35mLof the lower layer of the Chromatographic solventthrough the column,and collect the eluate in a 50-mLvolumetric flask.Add alcohol to volume,and mix.Pipet 10mLof the resulting solution into a glass-stoppered,50-mLconical flask,and evaporate on a water bath to dryness.Pipet 5mLof methanol into the flask,and swirl to dissolve the residue.Pipet 5mLof Standard preparationinto a similar flask.To each flask add 10.0mLof Isoniazid reagent,mix,and allow to stand for 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 5mLof methanol that has been treated similarly with Isoniazid reagent.Calculate the quantity,in mg,of C26H40O3in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Testosterone Enanthate 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.