A: Ignite about 50mg in a platinum dish over a flame:it decomposes and liberates iodine vapors.

B: To about 0.5mg add 7.5mLof acid sodium chloride solution (prepared by mixing 300mLof water,250mLof alcohol,100mLof 1Nsodium hydroxide,and 100mLof hydrochloric acid)and 1mLof sodium nitrite solution (1in 100).Allow to stand in the dark for 20minutes,and add 1.25mLof ammonium hydroxide:a pink color is produced.

Test solution: an amount equivalent to 30mg of anhydrous Levothyroxine Sodium per mL,in a mixture of alcohol and 1Nsodium hydroxide (2:1).

Extracting solution— Prepare a 1in 100solution of sulfuric acid in water.

Reference solution— Dissolve an accurately weighed quantity of potassium iodide in water to obtain a stock solution containing 0.131mg,equivalent to 0.100mg of iodide per mL.Transfer 0.6mLof this stock solution into a 1000-mLvolumetric flask,dilute with the Extracting solutionto volume,and mix.Each mLof the Reference solutioncontains 0.06µg of iodide.[NOTE—Prepare this solution on the day of use.]

Test solution— Transfer 7.5mg of Levothyroxine Sodium to a beaker,add 100mLof the Extracting solution,and sonicate for 5minutes.

Electrode system— Use an iodide-specific,ion-indicating electrode and a silver-silver chloride reference electrode connected to a pHmeter capable of measuring potentials with a minimum reproducibility of ±1mV(see pHá791ñ).

Procedure— Transfer the Reference solutionto a beaker containing a magnetic stirring bar.Rinse and dry the electrodes,insert in the solution,stir for 5minutes or until the reading stabilizes,and read the potential,in mV.Repeat this process using the Test solution.The requirements of the test are met if the Test solutionhas a higher potential,in mV,than the Reference solution:the limit is 0.08%.

Mobile phase,and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Prepare as directed for Standard preparation in the Assay.

Test solution— Proceed as directed for the Assay preparation.

Procedure— Proceed as directed in the Assay.Calculate the quantity,in µg,of liothyronine sodium (C15H11I3NNaO4)in the sample taken by the formula: in which 672.96and 650.98are the molecular weights of liothyronine sodium and liothyronine,respectively,Cis the concentration,in µg per mL,of USP Liothyronine RSin the Standard preparation,and rUand rSare the liothyronine peak responses obtained from the Test solutionand the Standard solution,respectively:not more than 2.0%of liothyronine is found.

Mobile phase— Prepare a degassed and filtered mixture of water and acetonitrile (60:40)that contains 0.5mLof phosphoric acid in each 1000mL.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 0.2µg of liothyronine per mL.

Assay preparation— Transfer an accurately weighed portion of about 100µg of Levothyroxine Sodium into 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.

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 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the liothyronine and levothyroxine peaks is not less than 5.0,and the relative standard deviation for replicate injections is not more than 2.0%for levothyroxine.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in µg,of C15H10I4NNaO4in the portion of Levothyroxine Sodium taken by the formula: in which 798.85and 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.