A: Mix a portion of finely powdered Tablets,equivalent to about 0.1mg of liothyronine,with an equal amount of anhydrous potassium carbonate,and transfer to a crucible.Ignite the crucible at 700for 5minutes,and cool.Add 5mLof water to the crucible,heat on a steam bath for 5minutes,cool,and filter.To the filtrate add 1mLof chloroform,1mLof dilute phosphoric acid (1in 2),and 1mLof sodium nitrite solution (1in 100),shake vigorously,and allow to separate:the chloroform layer is colored purple.

B: Shake a portion of finely powdered Tablets,equivalent to 0.1mg of liothyronine,with 15mLof water for 1minute.Add 2drops of hydrochloric acid and 10mLof butyl alcohol,and shake for 1minute.Centrifuge the mixture for 5minutes.Remove as much as possible of the clear,upper layer by means of a pipet,and evaporate it on a steam bath until the odor of butyl alcohol is no longer present.Add 3drops of methanol to the residue,and rotate the container to wet the contents thoroughly.Transfer,with the aid of a capillary tube,as much of the methanol as possible to a small area on a filter paper.When the filter paper is dry,spray it with diazotized sulfanilamide,prepared by mixing 5mLof a 1in 100solution of sulfanilamide in dilute hydrochloric acid (1in 10)with 5mLof sodium nitrite solution (1in 20)for 1minute,adding butyl alcohol to make 50mL,shaking for 1minute,allowing to stand for 4minutes,and decanting the butyl alcohol layer to be used as the spraying solution.Dry the filter paper in a stream of air,and spray it with sodium carbonate solution (1in 10):pink color is produced on the paper in the area where the test specimen was applied.

Medium: pH10.0±0.05alkaline borate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions);250mL.

Apparatus 3 (see Drug Release á724ñ):30dips per minute,using 20-mesh screen on the top and 40-mesh screen on the bottom of the glass reciprocating cylinder.

Time: 45minutes.

Ammoniated solution— Add 0.05mLof ammonium hydroxide to 200mLof water.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (55:45)that contains 1mLof phosphoric acid in each 1000mLof solution.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Liothyronine RSin Ammoniated solution,and dilute quantitatively,and stepwise if necessary,with Ammoniated solutionto obtain a solution having a known concentration of about 10µg of USP Liothyronine RSper mL.Dilute a portion of this solution quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 0.5µg of USP Liothyronine RSper mL.

Test solution— Transfer 20mLof the solution under test to a centrifuge tube,and centrifuge until a clear supernatant is obtained.

Resolution solution— Prepare a solution of USP Liothyronine RSand USP Levothyroxine RSin Ammoniated solutionhaving known concentrations of about 10µg of each USP Reference Standard per mL.Dilute with water to obtain a concentration of about 0.5µg of each USP Reference Standard per mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×25-cm column that contains packing L10.The flow rate is about 2mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the resolution,R,between liothyronine and levothyroxine is not less than 3.0.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 200µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the amount of C15H12I3NO4dissolved.

Tolerances— Not less than 70%(Q)of the labeled amount of C15H12I3NO4is dissolved in 45minutes.

Mobile phase ,Standard preparation,and Chromatographic system—Proceed as directed in the Assayunder Liothyronine Sodium.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 100µg of liothyronine sodium,to a centrifuge tube,add 2glass beads,pipet 10mLof Mobile phaseinto the tube,and mix using a vortex mixer for 3minutes.Centrifuge to obtain a clear supernatant,filtering if necessary.

Procedure— Proceed as directed in the Assayunder Liothyronine Sodium.Calculate the quantity,in µg,of liothyronine (C15H12I3NO4)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Liothyronine RSin the Standard preparation;and rUand rSare the liothyronine peak responses obtained from the Assay preparationand the Standard preparation,respectively.