A: To a portion of Injection,equivalent to about 1mg of digitoxin,add 10mLof water,and extract with 10mLof chloroform.Evaporate the chloroform extract on a steam bath with the aid of a current of air to dryness.Dissolve the residue in 2mLof a solution prepared by mixing 0.3mLof ferric chloride TSand 50mLof glacial acetic acid,and underlay with 2mLof sulfuric acid:at the zone of contact of the two liquids a brown color,which gradually changes to light green,then to blue,is produced,and finally the entire acetic acid layer acquires a blue color.

B: To a portion of Injection,equivalent to about 0.2mg of digitoxin,add 10mLof water,and extract with 10mLof chloroform.Evaporate the chloroform extract on a steam bath with the aid of a current of air to dryness.Add 2mLof a freshly prepared 1in 100solution of m-dinitrobenzene in alcohol,and allow to stand for 10minutes with frequent shaking.Add 2mLof a 1in 200solution of tetramethylammonium hydroxide in alcohol,and mix:a red-violet color develops slowly and then fades.

C: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the major peak in the chromatogram of the Standard preparationas obtained in the Assay.

Mobile phase,Standard preparation,System suitability preparation,andChromatographic system— Prepare as directed in the Assayunder Digitoxin.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 1mg of digitoxin,to a 25-mLvolumetric flask.Dilute with Mobile phaseto volume,and mix.

Procedure— Proceed as directed for Procedurein the Assayunder Digitoxin.Calculate the quantity,in µg,of C41H64O13in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Digitoxin RSin the Standard preparation,Vis the volume,in mL,of Injection taken,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.