A: Transfer a quantity of finely powdered Tablets,equivalent to not less than 1mg of digitoxin,to a suitable flask,add 20mLof chloroform,and sonicate.Filter,and evaporate the filtrate 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: 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.

Medium: dilute hydrochloric acid (3in 500);500mL.[NOTE—Use the same batch of Mediumthroughout the test.]

Apparatus 1: 120±5rpm.

Times: 30minutes;60minutes.

Standard stock solution— Weigh accurately about 30mg of USP Digitoxin RS,dissolve in a minimum amount of alcohol in a 500-mLvolumetric flask,add dilute alcohol (4in 5)to volume,and mix.

Standard solutions— Just prior to use,dilute 5.0mLof the Standard stock solutionwith Mediumto 500.0mL,and mix.Transfer aliquots (2.0to 10.0mL)of this solution to individual separators to prepare standards equivalent to 20,40,60,80,and 100%of the labeled amount of digitoxin in 500mL.Add Mediumto make 10mL,and proceed as directed for Procedure,beginning with “Extract with three 15-mLportions of chloroform.”

Procedure— Proceed as directed for Procedureunder Dissolution á711ñ.After 30minutes,accurately timed,withdraw a suitable aliquot of the solution under test from a point midway between the stirring shaft and the wall of the vessel,and approximately midway in depth.Filter the solution promptly after withdrawal,using a suitable membrane filter of not greater than 0.8-µm porosity,discarding the first 10mLof the filtrate.Without replacing the Mediumwithdrawn,continue to rotate the basket,and after an additional 30minutes,accurately timed,similarly withdraw and filter another aliquot.Treat each of these solutions as follows:Assuming dissolution of 100%of the labeled amount of digitoxin,transfer aliquots,equivalent to 6µg of digitoxin,to suitable separators.Extract with three 15-mLportions of chloroform,and combine the chloroform extracts in glass-stoppered flasks.Evaporate the combined extracts on a steam bath,with the aid of a current of air,to dryness.In a similar manner,prepare a blank using a suitable volume of Medium.

Measurement of fluorescence— Begin with the Standard solutions,and keep all flasks in the same sequence throughout,so that the elapsed time from addition of reagents to reading of fluorescence is the same for each set.Treat 1flask at a time as follows:Add 10mLof a solution freshly prepared by dissolving 35mg of ascorbic acid in 25mLof methanol and cautiously adding the solution to 100mLof hydrochloric acid.Mix,and add 1mLof a solution freshly prepared by diluting 1mLof 30percent hydrogen peroxide with water to 500mLand diluting 1volume of the resulting solution with 20volumes of water.Mix,and insert the stopper in the flask.After 45minutes,measure the fluorescence at about 575nm,the excitation wavelength being about 395nm.Correct each reading for the blank,and plot a standard curve of fluorescence versus percentage dissolution.By calculation from the standard curve,determine the percentage dissolution of digitoxin in each Tablet within 30minutes and the total percentage dissolution of digitoxin within 60minutes,taking into account the volume of the solution under test removed after the first 30minutes of the test.

Tolerances— Not less than 60%of the labeled amount of C41H64O13is dissolved within 30minutes for each Tablet tested,and not less than 85%of the labeled amount is dissolved within 60minutes for the average of the Tablets tested.

Procedure for content uniformity— Place 1Tablet in a suitable glass-stoppered conical flask.Add an accurately measured volume of Mobile phase(prepared as directed in the Assayunder Digitoxin)sufficient to obtain a solution containing about 10µg of digitoxin per mL,and shake by mechanical means until the Tablet has completely disintegrated (not less than 30minutes).Centrifuge,and use the clear supernatant as the test solution.Dissolve an accurately weighed quantity of USP Digitoxin RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a Standard solution having a known concentration of about 10µg per mL.Proceed as directed in the Assay.Calculate the quantity,in mg,of C41H64O13in the Tablet by the formula: in which Lis the labeled quantity,in mg,of digitoxin in the Tablet,Cis the concentration,in µg per mL,of USP Digitoxin RSin the Standard solution,Dis the concentration,in µg per mL,of digitoxin in the test solution based on the labeled quantity in the Tablet and the extent of dilution,and rUand rSare the digitoxin peak responses obtained from the test solution and the Standard solution,respectively.

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

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity of powder,equivalent to about 1mg of digitoxin,to a 25-mLvolumetric flask.Add 15mLof Mobile phase,and sonicate.Dilute with Mobile phaseto volume,and mix.Filter a portion of this solution,discarding the first few mLof the filtrate.The filtrate is the Assay preparation.

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