Development and validation of a high-performance liquid chromatography method for levothyroxine sodium quantification in plasma for pre-clinical evaluation of long-acting drug delivery systems.

ABSTRACT

Levothyroxine (LEVO) sodium is an FDA-approved drug that is used to treat underactive thyroid (hypothyroidism) and other conditions. It is generally used as a thyroid-stimulating hormone administered orally. However, this approach has some drawbacks such as this drug should be taken every day 30 min to 1 h prior to breakfast with an empty stomach, moreover, some food interactions must be monitored. Thus, alternative innovative approaches capable of providing sustained LEVO release should be developed. Our research was designed to establish a simple quantitative determination method for LEVO in rat plasma for pre-clinical evaluation of long acting formulations using a high-performance liquid chromatography method, to validate the analytical method according to ICH guidelines and to characterise its pharmacokinetic behavior in rats. After simple protein precipitation with acetonitrile, LEVO was eluted on a Xselect CSH™ C18 column (Waters, 3.0 × 150 mm) with a particle size of 3.5 µm using a mobile phase of water and acetonitrile at a ratio of 65 35% v/v, including 0.1% v/v of trifluoracetic acid. The calibration standards used for plasma ranged between 0.5-1000 ng mL-1 with a correlation coefficient (r2) of ≥0.998. The limit of detection was 0.44 ng mL-1 and the lower limit of quantitation was 1.33 ng mL-1. The extraction recovery of LEVO in rat plasma samples by this method was between 80 and 85%. The method was selective, sensitive, accurate and precise for detecting and quantifying LEVO in a pharmacokinetic study carried out in rats for pre-clinical evaluation of long acting formulations. The validated HPLC method meets the ICH established requirements and therefore offers a wide range of potential applications in pre-clinical therapeutic drug monitoring, pharmacokinetics and toxicology.