Levothyroxine and Liothyronine Sodium Analysis by HPLC - AppNote
May 1, 2012
/
/
/
Date: 1-MAY-2012 Last Update: 13-AUGUST-2026
Superior Resolution, Reproducibility, & Peak Shapes Compared to USP MethodCogent™ Phenyl Hydride HPLC Column Catalog Numbers, Available Configurations, Product Specifications, and Ordering Information
The USP assay method for Levothyroxine requires that a resolution of not less than 5.0 must be demonstrated between Levothyroxine and related compound Liothyronine. A chromatogram obtained from following the USP method using a Type-B Silica based L10 Column is shown in
Figure B
.
The average resolution between the two compounds over five runs is 2.8, which does not satisfy the system suitability for resolution for this assay. Figure A shows the five-run overlay obtained from a method developed with the Cogent Phenyl Hydride Column. The average resolution in this case was 5.3.
Peaks:
1. Liothyronine Sodium,
2. Levothyroxine Sodium
Columns:
— Fig. A: Cogent Phenyl Hydride ™, 4 µm, 100 Å
— Fig. B: Standard Silica Based Column, 5 µm, 100 Å
Catalog Nos.:
— Fig. A: 69020-7.5P
— Fig. B: N/A
Dimensions:
— Fig. A: 4.6 x 75 mm
— Fig. B: 4.6 x 250 mm
Mobile Phase:
- Fig. A:
-- A: DI Water / 0.1% Formic Acid (v/v)
-- B: 97% Acetonitrile / 3% DI Water / 0.1% Formic Acid (v/v)
- Fig. B: 60% DI Water / 40% Acetonitrile / 0.05% Phosphoric Acid
Gradient:
| Time (minutes) | %B |
|---|---|
| 0 | 20 |
| 6 | 50 |
| 7 | 20 |
-- Fig. A: 35˚C
-- Fig. B: Ambient
Injection vol.:
-- Fig. A: 2 µL
-- Fig. B: 100 µL
Flow rate:
— Fig. A: 1.0 mL / minutes
— Fig. B: 1.5 mL / minutes
Sample Preparation: Mix of Levothyroxine and Liothyronine standards.
- - Stock Solution: 0.4 mg Levothyroxine or Liothyronine dissolved with 1 mL 10 mM NaOH in 50:50 DI Water: Methanol.
- Working Solution: Fig. A: Aliquots of stock solutions were mixed and diluted with 50:50 A:B to obtain concentrations of 40 mg / L and 4 mg / L for Levothyroxine and Liothyronine respectively.
- Working Solution: Fig. B: Aliquots of stock solutions were mixed and diluted with the Mobile Phase to obtain concentrations of 10 mg /L and 0.2 mg / L for Levothyroxine and Liothyronine respectively.
Note: Levothyroxine is the L-isomer of the main thyroid hormone Thyroxine (T4). It is used as a replacement for the Thyroxine that is deficient in patients with hypothyroidism. Liothyronine is the L-isomer of another thyroid hormone, Triiodothyronine (T3). T3 is produced from T4 and is the metabolically active form of the hormone.
Levothyroxine Assay pdf 0.3 Mb Download File