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 1.0 mL of this stock solution into a 100-mL volumetric flask, dilute with Extracting solution to volume, and mix. Each mL of the Reference solution contains 1.0 µg of iodide. [note—Prepare this solution on the day of use.]

Test solution— Transfer 1.00 g, or proportionately less if the iodine content is greater than 0.2%, of Thyroid to a beaker, add 100.0 mL of 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 0.01%.

Mobile phase— Prepare a degassed and filtered mixture of water, acetonitrile, and phosphoric acid (650:350:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Reducing buffer solution— Freshly prepare a solution in 0.11 M sodium chloride that is 0.04 M with respect to tris(hydroxymethyl)aminomethane and 0.05 M with respect to methimazole. Adjust, if necessary, with 6 N hydrochloric acid or 0.1 N sodium hydroxide to a pH of 8.4 ± 0.05.

Proteolytic enzyme— Freshly prepare a solution containing 15 mg of bacterial protease* in each 5 mL of Reducing buffer solution.

Enzyme deactivating solution— Prepare a 1 in 100 mixture of phosphoric acid in acetonitrile.

Standard preparation— [note—Protect solutions from light.] Transfer accurately weighed quantities of about 9 mg of USP Liothyronine RS and about 38 mg of USP Levothyroxine RS to a 100-mL volumetric flask, add 50 mL of a mixture of water, acetonitrile, and ammonium hydroxide (500:500:1), and swirl to dissolve. Dilute with a mixture of water and acetonitrile (1:1) to volume, and mix (stock solution). On the day of use, pipet 5 mL of the freshly prepared stock solution into a 250-mL volumetric flask, dilute with Reducing buffer solution to volume, and mix to obtain a solution having known concentrations of about 1.8 µg of liothyronine per mL and about 7.6 µg of levothyroxine per mL. Pipet 5 mL of this solution into a screw-capped 16- × 125-mm culture tube. Pipet 2 mL of Enzyme deactivating solution into the tube, place the cap on the tube, and shake the mixture vigorously.

Assay preparation— Transfer an accurately weighed portion of finely powdered Thyroid, equivalent to about 38 µg of levothyroxine, to a screw-capped 16- × 125-mm culture tube that previously has been flushed with nitrogen. Taking precautions to avoid unnecessary exposure to air, pipet 5 mL of Proteolytic enzyme into the tube. Allow nitrogen to flow gently over the mixture for 5 minutes. Place the cap on the tube, mix to disperse the contents, and place in a covered water bath maintained at a temperature of 37 ± 1 for 28 hours. Protect the contents of the tubes from light. Examine occasionally, and mix as necessary to ensure dispersion. At the end of the incubation period, pipet 2 mL of Enzyme deactivating solution into the tube, place the cap on the tube, mix vigorously, and centrifuge at about 2000 rpm for 5 minutes. Filter the supernatant through a 0.45-µm porosity filter, discarding the first 1 mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 230-nm detector and a 4.6- × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factors for the liothyronine and levothyroxine peaks are not more than 1.8, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Assay preparation, and the Standard preparation, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg, of liothyronine (C15H12I3NO4) and levothyroxine (C15H11I4NO4) in the portion of Thyroid taken by the formula: in which C is the concentration, in µg per mL, of the corresponding USP Reference Standard in the Standard preparation, and rU and rS are the peak responses for the corresponding analytes obtained from the Assay preparation and the Standard preparation, respectively.