Solvent system— Place a suitable volume of tert-amyl alcohol in a separator, add an equal volume of 3 N ammonium 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 1 hour before using.

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

Standard preparations— Prepare a solution of about 15 mg of USP Levothyroxine RS, accurately weighed, in 100 mL of a mixture of methanol and 3 N ammonium hydroxide (1:1). Prepare a solution of about 4 mg of USP Liothyronine RS, accurately weighed, in the same solvent mixture. Dilute 20.0 mL of each solution with the same solvent to 100.0 mL.

Test preparation— Shake a quantity of powdered Tablets, equivalent to about 60 µg of levothyroxine sodium and 15 µg of liothyronine sodium, with 2 mL of a mixture of methanol and 3 N ammonium hydroxide (1:1) in a centrifuge tube for 10 minutes, and centrifuge.

Procedure— Apply 10-µL volumes of the Test preparation and 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 10 cm beyond the point of application, air-dry, and spray the plate with Detection reagent: the chromatogram of the Test preparation shows blue spots corresponding in RF value 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 2 mL of trifluoroacetic acid in each 1000 mL of solution. Make adjustments if necessary (see System Suitability under Chromatography 621).

0.01 M Methanolic sodium hydroxide— Dissolve 400 mg of sodium hydroxide in 500 mL of water. Cool, add 500 mL of methanol, and mix.

Levothyroxine stock solution— Dissolve an accurately weighed quantity of USP Levothyroxine RS in 0.01 M Methanolic sodium hydroxide to obtain a solution having a known concentration of about 0.4 mg of levothyroxine per mL.

Liothyronine stock solution— Dissolve an accurately weighed quantity of USP Liothyronine RS in 0.01 M Methanolic sodium hydroxide to obtain a solution having a known concentration of about 0.4 mg of liothyronine per mL. Make a 1:10 dilution of this solution using Mobile phase.

Standard preparation— Transfer appropriate volumes of Levothyroxine stock solution and Liothyronine stock solution to a suitable container, and dilute quantitatively and stepwise, if necessary, with Mobile phase to obtain a solution having known concentrations of about 10 µg of levothyroxine per mL and 2.5 µg of liothyronine per mL.

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

Chromatographic system— Proceed as directed in the Assay under Levothyroxine Sodium.

Procedure— Proceed as directed for Procedure in the Assay under Levothyroxine Sodium. Calculate the quantity, in µg, of levothyroxine sodium (C15H10I4NNaO4) in the portion of Tablets taken by the formula: in which 798.86 and 776.87 are the molecular weights of levothyroxine sodium and levothyroxine, respectively; C is the concentration, in µg per mL, of USP Levothyroxine RS in the Standard preparation; and rU and rS are the levothyroxine peak responses obtained from the Assay preparation and the Standard preparation, respectively.

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