Solvent system— Place a suitable volume of tertiary amyl alcohol in a separator,add an equal volume of 3Nammonium hydroxide,and shake to equilibrate.Discard the lower layer,and transfer the upper layer to the developing chamber,cover the chamber,and allow to stand for 1hour before using.

Detection reagent— Add 65mLof 2Nhydrochloric acid to 50mLof a 1in 10solution of sodium arsenite in 1Nsodium hydroxide,with vigorous stirring.Mix 1volume of this solution with 5volumes of a 27in 1000solution of ferric chloride in 2Nhydrochloric acid and 5volumes of freshly prepared potassium ferricyanide solution (35in 1000).

Standard preparations— Prepare a solution of about 15mg of USP Levothyroxine RS,accurately weighed,in 100mLof a mixture of equal volumes of methanol and 3Nammonium hydroxide.Prepare a solution of about 4mg of USP Liothyronine RS,accurately weighed,in the same solvent mixture.Dilute 20.0mLof each solution with the same solvent to 100.0mL.

Test preparation— Shake an amount of powdered Tablets,equivalent to about 60µg of levothyroxine sodium and 15µg of liothyronine sodium,with 2mLof a mixture of equal volumes of methanol and 3Nammonium hydroxide in a centrifuge tube for 10minutes,and centrifuge.

Procedure— Apply 10-µLvolumes of the Test preparationand each of the Standard preparations,respectively,to a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic microcrystalline cellulose containing a fluorescent indicator.Develop the plate until the solvent front has moved not less than 10cm beyond the point of application,air-dry,and spray the plate with Detection reagent:the chromatogram of the Test preparationshows blue spots corresponding in RFvalue to the chromatograms from the levothyroxine and liothyronine Standard preparations,respectively.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (65:35)that contains 2mLof trifluoroacetic acid in each 1000mLof solution.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer accurately weighed quantities of USP Levothyroxine RSand USP Liothyronine RSto a suitable container,dissolve in and dilute quantitatively and stepwise with Mobile phaseto obtain a solution having known concentrations of about 10µg of levothyroxine per mLand 2.5µg of liothyronine per mL.

Assay preparation— Transfer 20Tablets to a 200-mLvolumetric flask,add 180mLof Mobile phase,and sonicate for 15minutes,occasionally swirling the flask to accelerate the disintegration of the Tablets.Cool to room temperature and dilute with Mobile phaseto volume.Transfer a portion of the solution to a centrifuge tube,and centrifuge for 10minutes at 5000rpm.Dilute a portion of the clear supernatant quantitatively with Mobile phaseto obtain concentrations of about 10.0µg of levothyroxine sodium per mLand 2.5µg of liothyronine sodium per mL.

Chromatographic system— Proceed as directed in the Assayunder Levothyroxine Sodium.

Procedure— Proceed as directed for Procedurein the Assayunder Levothyroxine Sodium.Calculate the quantity,in µg,of C15H10I4NNaO4in the portion of Tablets taken by the formula: in which 798.86and 776.87are the molecular weights of levothyroxine sodium and levothyroxine,respectively,Cis the concentration,in µg per mL,of USP Levothyroxine RSin the Standard preparation,and rUand rSare the levothyroxine peak responses obtained from the Assay preparationand the Standard preparation,respectively.

(672.96/650.98)(10C)(rU/rS),