A: Mix a portion of finely powdered Tablets, equivalent to about 0.1 mg of liothyronine, with an equal amount of anhydrous potassium carbonate, and transfer to a crucible. Ignite the crucible at 700 for 5 minutes, and cool. Add 5 mL of water to the crucible, heat on a steam bath for 5 minutes, cool, and filter. To the filtrate add 1 mL of chloroform, 1 mL of dilute phosphoric acid (1 in 2), and 1 mL of sodium nitrite solution (1 in 100), shake vigorously, and allow to separate: the chloroform layer is colored purple.

B: Shake a portion of finely powdered Tablets, equivalent to 0.1 mg of liothyronine, with 15 mL of water for 1 minute. Add 2 drops of hydrochloric acid and 10 mL of butyl alcohol, and shake for 1 minute. Centrifuge the mixture for 5 minutes. Remove as much as possible of the clear, upper layer by means of a pipet, and evaporate it on a steam bath until the odor of butyl alcohol is no longer present. Add 3 drops of methanol to the residue, and rotate the container to wet the contents thoroughly. Transfer, with the aid of a capillary tube, as much of the methanol as possible to a small area on a filter paper. When the filter paper is dry, spray it with diazotized sulfanilamide, prepared by mixing 5 mL of a 1 in 100 solution of sulfanilamide in dilute hydrochloric acid (1 in 10) with 5 mL of sodium nitrite solution (1 in 20) for 1 minute, adding butyl alcohol to make 50 mL, shaking for 1 minute, allowing to stand for 4 minutes, and decanting the butyl alcohol layer to be used as the spraying solution. Dry the filter paper in a stream of air, and spray it with sodium carbonate solution (1 in 10): pink color is produced on the paper in the area where the test specimen was applied.

Medium: pH 10.0 ± 0.05 alkaline borate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions); 250 mL.

Apparatus 3 (see Drug Release 724): 30 dips per minute, using 20-mesh screen on the top and 40-mesh screen on the bottom of the glass reciprocating cylinder.

Time: 45 minutes.

Ammoniated solution— Add 0.05 mL of ammonium hydroxide to 200 mL of water.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (55:45) that contains 1 mL of phosphoric acid in each 1000 mL of solution. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Liothyronine RS in Ammoniated solution, and dilute quantitatively, and stepwise if necessary, with Ammoniated solution to obtain a solution having a known concentration of about 10 µg of USP Liothyronine RS per mL. Dilute a portion of this solution quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.5 µg of USP Liothyronine RS per mL.

Test solution— Transfer 20 mL of the solution under test to a centrifuge tube, and centrifuge until a clear supernatant is obtained.

Resolution solution— Prepare a solution of USP Liothyronine RS and USP Levothyroxine RS in Ammoniated solution having known concentrations of about 10 µg of each USP Reference Standard per mL. Dilute with water to obtain a concentration of about 0.5 µg of each USP Reference Standard per mL.

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 2 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between liothyronine and levothyroxine is not less than 3.0. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount of C15H12I3NO4 dissolved.

Tolerances— Not less than 70% (Q) of the labeled amount of C15H12I3NO4 is dissolved in 45 minutes.

Medium: pH 10.0 ± 0.05 alkaline borate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions); 250 mL.

Apparatus 3: 30 dips per minute, using 20-mesh screen on the top and 40-mesh screen on the bottom of the glass reciprocating cylinder.

Time: 45 minutes.

Ammoniated solution— Add 0.05 mL of ammonium hydroxide to 200 mL of water.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (55:45) that contains 1 mL of phosphoric acid in each 1000 mL of solution. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Liothyronine RS in Ammoniated solution, and dilute quantitatively, and stepwise if necessary, with Ammoniated solution to obtain a solution having a known concentration of about 10 µg of USP Liothyronine RS per mL. Dilute a portion of this solution quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.5 µg of USP Liothyronine RS per mL.

Test solution— Transfer 20 mL of the solution under test to a centrifuge tube, and centrifuge until a clear supernatant is obtained.

Resolution solution— Prepare a solution of USP Liothyronine RS and USP Levothyroxine RS in Ammoniated solution having known concentrations of about 10 µg of each USP Reference Standard per mL. Dilute with water to obtain a concentration of about 0.5 µg of each USP Reference Standard per mL.

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 2 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between liothyronine and levothyroxine is not less than 3.0. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount of C15H12I3NO4 dissolved.

Tolerances— Not less than 70% (Q) of the labeled amount of C15H12I3NO4 is dissolved in 45 minutes.

(Official April 1, 2006)

(Official January 1, 2007)

Mobile phase, Standard preparation, and Chromatographic system— Proceed as directed in the Assay under Liothyronine Sodium.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 µg of liothyronine sodium, to 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.

Procedure— Proceed as directed in the Assay under Liothyronine Sodium. Calculate the quantity, in µg, of liothyronine (C15H12I3 NO4) in the portion of Tablets taken by the formula: in which 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 Assay preparation and the Standard preparation, respectively.