A: Ignite about 50 mg in a platinum dish over a flame: it decomposes and liberates iodine vapors. [note—Cool the residue obtained, and reserve it for use in Identification test D.]

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.

C: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

D: To the residue retained from Identification test A, add a 1 N potassium hydroxide solution dropwise until the residue is dissolved: the solution meets the requirements of the flame test for Sodium 191.

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).

Diluent— Prepare a mixture of equal volumes of water and acetonitrile.

Phosphoric acid solution— Prepare by diluting 5 mL of phosphoric acid with Diluent to 100 mL.

Mobile phase— Dissolve 1.0 g of sodium 1-heptanesulfonate in about 200 mL of water; add 200 mL of acetonitrile, 400 mL of methanol, and 1.0 mL of phosphoric acid; dilute with water to 1 L; and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Levothyroxine standard stock solution— Transfer about 25 mg of USP Levothyroxine RS, accurately weighed, to a 100-mL volumetric flask. Add approximately 50 mL of Diluent and 1 drop of 10 N sodium hydroxide, and sonicate until dissolved. Add 7 mL of Phosphoric acid solution, dilute with Diluent to volume, and mix well.

Liothyronine standard stock solution— Transfer about 25 mg of USP Liothyronine RS, accurately weighed, to a 100-mL volumetric flask. Add approximately 50 mL of Diluent and 1 drop of 10 N sodium hydroxide, and sonicate until dissolved. Add 7 mL of Phosphoric acid solution, dilute with Diluent to volume, and mix.

Resolution solution— Pipet 5.0 mL of the Levothyroxine standard stock solution and 5.0 mL of the Liothyronine standard stock solution into a 100-mL volumetric flask. Add 7 mL of Phosphoric acid solution, and dilute with Diluent to volume.

Standard solution— Pipet 4.0 mL of the Resolution solution into a 100-mL volumetric flask. Add 7 mL of Phosphoric acid solution, and dilute with Diluent to volume.

Blank solution— Add 7 mL of Phosphoric acid solution to a 100-mL volumetric flask, and dilute with Diluent to volume.

Test solution— Transfer about 25 mg of Levothyroxine Sodium, accurately weighed, to a 100-mL volumetric flask. Add approximately 50 mL of Diluent, and sonicate until dissolved. Add 7 mL of Phosphoric acid solution, dilute with Diluent to volume, and mix well.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L7. The column temperature is maintained at about 35, and the flow rate is about 1.5 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between levothyroxine and liothyronine is not less than 5.0. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections of the Standard solution is not more than 2.0% for the levothyroxine peak.

Procedure— Separately inject equal volumes (about 15 µL) into the chromatograph in the following order: Blank solution, Standard solution, and Test solution. Record the chromatograms for at least six times the retention time of the levothyroxine peak, and measure the peak area responses. Verify that no peaks elute in the Blank solution at the expected retention times for levothyroxine and related compounds. Calculate the percentage of each related compound in the portion of Levothyroxine taken by the formula: in which 798.85 and 776.87 are the molecular weights of levothyroxine sodium and levothyroxine, respectively; CS is the concentration, in mg per mL, of levothyroxine in the Standard solution; CU is the concentration, in mg per mL, of levothyroxine sodium in the Test solution; rU is the peak area for each impurity obtained from the Test solution; rS is the peak area for levothyroxine obtained from the Standard solution; and L is the percentage of water in Levothyroxine Sodium, as determined separately in the test for Water 921. [note—The relative response factor for the impurities listed in Table 1 is 1.00. Any unspecified impurity peaks should be assigned a relative response factor of 1.00.] Disregard peaks corresponding to those obtained from the Blank solution, and disregard peaks corresponding to less than 0.03%.

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— Prepare a solution of Levothyroxine Sodium in Mobile phase having a known concentration of about 10 µg per mL. [note—A small amount of 0.01 M Methanolic sodium hydroxide can be used to facilitate the dissolution of the sample.]

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 percentage 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; CS is the concentration, in µg per mL, of USP Levothyroxine RS in the Standard preparation; CT is the concentration, in µg per mL, of levothyroxine sodium in the Assay preparation; and rU and rS are the levothyroxine peak responses obtained from the Assay preparation and the Standard preparation, respectively.