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 1.0mLof this stock solution into a 100-mLvolumetric flask,dilute with Extracting solutionto volume,and mix.Each mLof the Reference solutioncontains 1.0µg of iodide.[NOTE—Prepare this solution on the day of use.]

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

Mobile phase— Prepare a degassed and filtered mixture of water,acetonitrile,and phosphoric acid (650:350:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Reducing buffer solution— Freshly prepare a solution in 0.11Msodium chloride that is 0.04Mwith respect to tris(hydroxymethyl)aminomethane and 0.05Mwith respect to methimazole.Adjust,if necessary,with 6Nhydrochloric acid or 0.1Nsodium hydroxide to a pHof 8.4±0.05.

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

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

Standard preparation— [NOTE—Protect solutions from light.]Transfer accurately weighed quantities of about 9mg of USP Liothyronine RSand about 38mg of USP Levothyroxine RSto a 100-mLvolumetric flask,add 50mLof 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 5mLof the freshly prepared stock solution into a 250-mLvolumetric flask,dilute with Reducing buffer solutionto volume,and mix to obtain a solution having known concentrations of about 1.8µg of liothyronine per mLand about 7.6µg of levothyroxine per mL.Pipet 5mLof this solution into a screw-capped 16-×125-mm culture tube.Pipet 2mLof Enzyme deactivating solutioninto 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 38µg 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 5mLof Proteolytic enzymeinto the tube.Allow nitrogen to flow gently over the mixture for 5minutes.Place the cap on the tube,mix to disperse the contents,and place in a covered water bath maintained at a temperature of 37±1for 28hours.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 2mLof Enzyme deactivating solutioninto the tube,place the cap on the tube,mix vigorously,and centrifuge at about 2000rpm for 5minutes.Filter the supernatant through a 0.45-µm porosity filter,discarding the first 1mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— 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 1.5mLper 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 200µL)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 Cis the concentration,in µg per mL,of the corresponding USP Reference Standard in the Standard preparation,and rUand rSare the peak responses for the corresponding analytes obtained from the Assay preparationand the Standard preparation,respectively.