Thyroid

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Thyroid is the cleaned, dried, and powdered thyroid gland previously deprived of connective tissue and fat. It is obtained from domesticated animals that are used for food by humans.

On hydrolysis it yields NLT 90.0% and NMT 110.0% each of the labeled amounts of Levothyroxine (C₁₅H₁₁I₄NO₄) and Liothyronine (C₁₅H₁₂I₃NO₄), calculated on the dried basis. It is free from iodine in inorganic or any form of combination other than that peculiar to the thyroid gland. It may contain a suitable diluent such as lactose, sodium chloride, starch, sucrose, or dextrose.

2 IDENTIFICATION

A. The retention times of the peaks for liothyronine and levothyroxine of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.

3 ASSAY

Change to read:

PROCEDURE

Mobile phase: Acetonitrile, water, and phosphoric acid (350:650:5), filtered and degassed

Reducing buffer solution: Freshly prepare 0.04 M tris(hydroxymethyl)aminomethane and 0.05 M methimazole in 0.11 M sodium chloride. 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 3 mg/mL of bacterial protease in Reducing buffer solution.1 Enzyme deactivating solution: Phosphoric acid in acetonitrile (1:99)

Standard stock solution: 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 acetonitrile, water, and ammonium hydroxide (500:500:1) and swirl to dissolve. Dilute with a mixture of acetonitrile and water (1:1) to volume, and mix. [NOTE—Protect solutions from light.]

Standard solution: Pipet 5 mL of the freshly prepared Standard 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/mL of liothyronine and 7.6 µg/mL of levothyroxine. 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. [NOTE—Prepare on the day of use.]

Sample solution: 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 has been flushed previously 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 min. 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 h. [NOTE—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 min. Pass the supernatant through a filter of 0.45-µm pore size, discarding the first 1 mL of the filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 230 nm

Column: 4.6-mm (ERR 1-Apr-2023) × 25-cm; packing L1

Flow rate: 1.5 mL/min

Injection volume: 200 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 1.8 for liothyronine and levothyroxine peaks

Relative standard deviation: NMT 2.0% for replicate injections

Analysis

Samples: Standard solution and Sample solution

Calculate the quantity, in µg, of levothyroxine (C₁₅H₁₁I₄NO₄) and liothyronine (C₁₅H₁₂I₃NO₄) in the portion of Thyroid taken:

Result = (V × C) × (r_U/r_S)

V = volume of Sample solution, 7 mL

C = concentration of the corresponding Reference Standard in the Standard solution (µg/mL)

r_U = peak response of the corresponding analytes from the Sample solution

r_S = peak response of the corresponding analytes from the Standard solution

Acceptance criteria: 90.0%–110.0% each of the labeled amounts of levothyroxine (C H I NO ) and liothyronine (C H I NO ) on the dried basis

4 IMPURITIES

LIMIT OF INORGANIC IODIDES

Extracting solution: Sulfuric acid and water (1:99)

Standard 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 Standard solution contains 1.0 µg of iodide. [NOTE—Prepare this solution on the day of use.]

Sample solution: Transfer 1.00 g, or proportionately less if the dry basis levothyroxine content is greater than 38 µg per grain, of Thyroid to a beaker. Add 100.0 mL of Extracting solution, and sonicate for 5 min.

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〉).

Analysis: Transfer the Standard solution to a beaker containing a magnetic stirring bar. Rinse and dry the electrodes, insert in the solution, stir for 5 min or until the reading stabilizes, and read the potential in mV. Repeat this process using the Sample solution.

Acceptance criteria: The requirements of the test are met if the Sample solution has a higher potential, in mV, than the Standard solution, the limit is 0.01%.

5 SPECIFIC TESTS

MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR SPECIFIED MICROORGANISMS 〈62〉: It meets the requirements of the tests for the absence of Salmonella species and Escherichia coli.

LOSS ON DRYING 〈731〉

Analysis: Dry under vacuum at 60° for 4 h. Acceptance criteria: NMT 6.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers.

USP REFERENCE STANDARDS 〈11〉

USP Levothyroxine RS USP Liothyronine RS