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

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

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

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

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

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

Electrode system— Use an iodide-specific, ion-indicating electrode and a silver-silver chloride reference electrode connected to a pH meter capable of measuring potentials with a minimum reproducibility of ±1 mV (see pH 791).

Procedure— Transfer the Reference solution to a beaker containing a magnetic stirring bar. Rinse and dry the electrodes, insert in the solution, stir for 5 minutes 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 solution has 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 672.96 and 650.98 are the molecular weights of liothyronine sodium and liothyronine, respectively; C is the concentration, in µg per mL, of USP Liothyronine RS in the Standard preparation; and rU and rS are the liothyronine peak responses obtained from the Test solution and the Standard solution, respectively: not more than 2.0% of liothyronine is found.

(Official January 1, 2007)

Mobile phase— Prepare a degassed and filtered mixture of water and acetonitrile (60:40) that contains 0.5 mL of phosphoric acid in each 1000 mL. 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 1:100 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 0.2 µg of liothyronine per mL.

Assay preparation— Transfer an accurately weighed portion of about 100 µg of Levothyroxine Sodium into a centrifuge tube, add 2 glass beads, pipet 10 mL of Mobile phase into the tube, and mix using a vortex mixer for 3 minutes. Centrifuge to obtain a clear supernatant, filtering if necessary.

Chromatographic system (see Chromatography 621)— 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 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between liothyronine and levothyroxine 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 100 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg, of C15H10I4NNaO4 in the portion of Levothyroxine Sodium taken by the formula: in which 798.85 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.