Levothyroxine Sodium

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

Levothyroxine Sodium is the sodium salt of l-3,3′,5,5′-tetraiodothyronine. It contains NLT 97.0% and NMT 103.0% of levothyroxine sodium (C₁₅H₁₀I₄NNaO₄), calculated on the anhydrous basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 〈197A〉or〈197K〉

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

C. Identification Tests—General 〈191〉, Chemical Identification Tests, Sodium

Sample solution: To 200 mg add 2 mL of 2 N sulfuric acid. Heat on a water bath and then carefully heat over an open flame, increasing the temperature gradually up to about 600°. [Note—Alternative procedures for igniting the material could also be used.] Continue the ignition until most of the particles have disappeared. Dissolve the residue in 2 mL of water.

Acceptance criteria: The Sample solution meets the requirements of test A.

3 ASSAY

Change to read:

Procedure

Mobile phase: Acetonitrile and water (4:6) that contains 0.5 mL of phosphoric acid in each 1000 mL

Solution A: 400 mg of sodium hydroxide in 500 mL of water. Cool and add 500 mL of methanol.

Levothyroxine stock solution: 0.4 mg/mL of USP Levothyroxine RS in Solution A

Liothyronine stock solution: 0.4 mg/mL of (USP 1-Aug-2020) USP Liothyronine RS in Solution A. Further dilute with Mobile phase to 0.004mg/mL. (USP 1-Aug-2020)

Standard solution: 10 μg/mL of USP Levothyroxine RS (USP 1-Aug-2020) from Levothyroxine stock solution and 0.2 μg/mL of USP Liothyronine RS (USP 1-Aug-2020) from Liothyronine stock solution in Mobile phase

Sample solution: 10 μg/mL of Levothyroxine Sodium in Mobile phase. [Note—A small amount of 0.01 M sodium hydroxide in methanol (USP 1-Aug-2020) can be used to facilitate the dissolution of the sample.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 225 nm

Column: 4.6-mm × 25-cm; 5-μm (USP 1-Aug-2020) packing L10

Flow rate: 1.5 mL/min

Injection volume: 100 μL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 5.0 between liothyronine and levothyroxine

Relative standard deviation: NMT 2.0% for levothyroxine

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of levothyroxine sodium (C₁₅H₁₀I₄NNaO₄) in the portion of Levothyroxine Sodium taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of levothyroxine from the Sample solution

r_S = peak response of levothyroxine from the Standard solution

C_S = concentration of USP Levothyroxine RS in the Standard solution (μg/mL)

C_U = concentration of Levothyroxine Sodium in the Sample solution (μg/mL)

M_r1 = molecular weight of levothyroxine sodium, 798.85

M_r2 = molecular weight of levothyroxine, 776.87

Acceptance criteria: 97.0%–103.0% on the anhydrous basis

4 IMPURITIES

[Note—On the basis of the synthetic route, perform either Organic Impurities, Procedure 1 or Organic Impurities, Procedure 2. Procedure 2 is recommended when related compounds listed in Table 3 may be present.]

Change to read:

Organic Impurities, Procedure 1

Diluent: Acetonitrile and water (1:1)

Solution A: Dilute 5 mL of phosphoric acid with Diluent to 100.0 mL.

Mobile phase: 1.0 g/L of (USP 1-Aug-2020) sodium 1-heptanesulfonate prepared as follows. Transfer 1 g of sodium 1-heptanesulfonate to a 1-L volumetric flask and dissolve (USP 1-Aug-2020) in 200 mL of water. Add 200 mL of acetonitrile, 400 mL of methanol, and 1.0 mL of phosphoric acid. Dilute with water to volume.

Levothyroxine stock solution: 0.25 mg/mL of USP Levothyroxine RS prepared as follows. (USP 1-Aug-2020) Transfer 25 mg of USP

Levothyroxine RS to a 100-mL volumetric flask containing (USP 1-Aug-2020) 50 mL of Diluent and 1 drop of 10 N sodium hydroxide, and sonicate until dissolved. Add 7 mL of Solution A and dilute with Diluent to volume.

Liothyronine stock solution: 0.25 mg/mL of USP Liothyronine RS prepared as follows. (USP 1-Aug-2020) Transfer 25 mg of USP Liothyronine RS to a 100-mL volumetric flask containing (USP 1-Aug-2020) 50 mL of Diluent and 1 drop of 10 N sodium hydroxide, and sonicate until dissolved. Add 7 mL of Solution A and dilute with Diluent to volume.

System suitability solution: 0.0125 mg/mL each of USP Levothyroxine RS and USP Liothyronine RS prepared as follows. (USP 1-Aug-2020)

Transfer 5.0 mL each of Levothyroxine stock solution (USP 1-Aug-2020) and Liothyronine stock solution (USP 1-Aug-2020) to a 100-mL volumetric flask. Add 7 mL of Solution A, and dilute with Diluent to volume.

Standard solution: 0.0005 mg/mL of USP Levothyroxine RS and USP Liothyronine RS prepared as follows. (USP 1-Aug-2020) Transfer 4.0 mL of System suitability solution into a 100-mL volumetric flask. Add 7 mL of Solution A, and dilute with Diluent to volume.

Sample solution: 0.25 mg/mL of Levothyroxine Sodium prepared as follows. (USP 1-Aug-2020) Transfer 25 mg of Levothyroxine Sodium to a 100-mL volumetric flask. Add 50 mL of Diluent, and sonicate until dissolved. Add 7 mL of Solution A, and dilute with Diluent to volume.

Blank solution: Transfer 7 mL of Solution A to a 100-mL volumetric flask, and dilute with Diluent to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 225 nm

Column: 4.6-mm × 15-cm; 5-μm packing L7

Column temperature: 35°

Flow rate: 1.5 mL/min

Injection volume: 15 μL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 5.0 between levothyroxine and liothyronine, System suitability solution

Relative standard deviation: NMT 2.0% for the levothyroxine peak, Standard solution

Analysis

Samples: Standard solution, Sample solution, and Blank solution

[Note—Record the chromatograms for at least 6 times the retention time of the levothyroxine peak. Verify that no peaks elute in the Blank solution at the expected retention times for levothyroxine and related compounds.]

Calculate the area percentage of each impurity (USP 1-Aug-2020) in the portion of Levothyroxine Sodium taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of levothyroxine from the Standard solution

C_S = concentration of USP Levothyroxine RS in the Standard solution (mg/mL)

C_U = concentration of Levothyroxine Sodium in the Sample solution (mg/mL)

M_r1 = molecular weight of levothyroxine sodium, 798.85

M_r2 = molecular weight of levothyroxine, 776.87

(USP 1-Aug-2020)

Disregard peaks corresponding to those of the Blank solution. (USP 1-Aug-2020)

Acceptance criteria: See Table 1. The reporting threshold is 0.03%. (USP 1-Aug-2020)

Table 1

^a O-(4-Hydroxy-3,5-diiodophenyl)-3,5-diiodo-β-hydroxy-l-tyrosine.

^b 2-Hydroxy-2-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetic acid.

^c N-Formyl-O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-l-tyrosine.

^d 2-[4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetamide.

^e N-Acetyl-O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-l-tyrosine.

^f 2-[4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetic acid.

^g 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzaldehyde.

^h 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzoic acid.

Change to read:

Organic Impurities, Procedure 2

Solution A: 4.85 g/L of sulfamic acid and 0.75 g/L of sodium hydroxide in water. (USP 1-Aug-2020) Adjust with 2 N sodium hydroxide to a pH of 2.0.

Solution B: Acetonitrile

Mobile phase: See Table 2.

Table 2

Diluent 1: Methanol and Solution A (90:10)

Diluent 2: Acetonitrile and Solution A (30:70). Mix with Diluent 1 (1:1).

Identification solution: 0.2 mg/mL of USP Levothyroxine Peak Identification Mixture RS in Diluent 2

Standard stock solution: 0.1 mg/mL each of USP Levothyroxine RS and USP Liothyronine RS in Diluent 1

Standard solution: 0.002 mg/mL each of USP Levothyroxine RS and USP Liothyronine RS, from (USP 1-Aug-2020) Standard stock solution in Diluent 2

Sensitivity solution: 0.0002 mg/mL each of USP Levothyroxine RS and USP Liothyronine RS, from (USP 1-Aug-2020) Standard solution in Diluent 2

Sample solution: 1.0 mg/mL of Levothyroxine Sodium in Diluent 1. Further dilute a portion of this solution with Diluent 2 to 0.2 mg/mL.

(USP 1-Aug-2020)

Blank solution: Use Diluent 2.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 225 nm

Column: 4.0-mm × 15-cm; 3-μm packing L1

Flow rate: 1.0 mL/min

Injection volume: 25 μL

System suitability

Samples: Standard solution and Sensitivity solution

Suitability requirements

Resolution: NLT 5 between levothyroxine and liothyronine, Standard solution

Signal-to-noise ratio: NLT 5 for each peak, Sensitivity solution (USP 1-Aug-2020)

Analysis

Samples: Identification solution, Standard solution, Sample solution, and Blank solution

[Note—Identify the components on the basis of their relative retention times as listed in Table 3.]

Calculate the percentage of liothyronine sodium in the portion of Levothyroxine Sodium taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of liothyronine from the Sample solution

r_S = peak response of liothyronine from the Standard solution

C_S = concentration of USP Liothyronine RS in the Standard solution (mg/mL)

C_U = concentration of Levothyroxine Sodium in the Sample solution (mg/mL)

M_r1 = molecular weight of liothyronine sodium, 672.96

M_r2 = molecular weight of liothyronine, 650.97 (USP 1-Aug-2020)

Calculate the percentage of any other impurity in the portion of Levothyroxine Sodium taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of any other (USP 1-Aug-2020) impurity from the Sample solution

r_S = peak response of levothyroxine from the Standard solution

C_S = concentration of USP Levothyroxine RS in the Standard solution (mg/mL)

C_U = concentration of Levothyroxine Sodium in the Sample solution (mg/mL)

M_r1 = molecular weight of levothyroxine sodium, 798.85

M_r2 = molecular weight of levothyroxine, 776.87

 (USP 1-Aug-2020)

Disregard peaks corresponding to those of the Blank solution. (USP 1-Aug-2020)

Acceptance criteria: See Table 3. The reporting threshold is 0.03%. (USP 1-Aug-2020)

Table 3

^a (S)-2-Amino-3-[3-chloro-4-(4-hydroxy-3,5-diiodophenoxy)-5-iodophenyl]propanoic acid.

^b (S)-2-Amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]-N-methylpropanamide.

^c [4-(4-Hydroxy-3-iodophenoxy)-3,5-diiodophenyl]acetic acid.

^d (S)-2-Amino-3-[4-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenoxy]-3,5-diiodophenyl]propanoic acid.

^e 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodo-N-methylbenzamide. (USP 1-Aug-2020)

^f 2-(4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl)acetic acid.

^g 4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzoic acid. (USP 1-Aug-2020)

5 SPECIFIC TESTS

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: Equivalent to 30 mg/mL of anhydrous Levothyroxine Sodium in alcohol and 1 N sodium hydroxide (2:1)

Acceptance criteria: −5° to −6°

Water Determination 〈921〉, Method I: NMT 11.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers, protected from light. Store as stated in the labeling instructions.

Labeling: If a test for Organic Impurities other than Procedure 1 is used, the labeling states the test with which the article complies.

Change to read:

USP Reference Standards 〈11〉

USP Levothyroxine RS

O-(4-Hydroxy-3,5-diiodophenyl)-3,5-diiodo-l-tyrosine.

C₁₅H₁₁I₄NO₄ 776.87

USP Levothyroxine Peak Identification Mixture RS

Levothyroxine sodium spiked with liothyronine, triiodothyroacetic acid, and tetraiodothyroacetic acid in methanol.

USP Levothyroxine Sodium RS

USP Liothyronine RS

O-(4-Hydroxy-3-iodophenyl)-3,5-diiodo-l-tyrosine.

C₁₅H₁₂I₃NO₄ 650.97 (USP 1-Aug-2020)