Development and Validation of an Assay Method for Levothyroxine Soft Gelatin Capsules Utilizing the RP‐HPLC Technique

A robust, precise, accurate, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative estimation of levothyroxine sodium in soft gelatin capsule dosage forms across a wide strength range (13 to 200 μg). The objective of this study was to establish a single, specific method capable of assessing both assay with high sensitivity, particularly at a low-test concentration of 1 ppm, while ensuring specificity toward all known impurities listed in the levothyroxine drug substance monograph. Chromatographic separation was achieved using an InertSustain AQ C18 column (250 × 4.6 mm, 5 μm) maintained at 60°C. The mobile phase system comprised 90% phosphate buffer and 10% acetonitrile (Mobile Phase A) and 90% acetonitrile with 10% water (Mobile Phase B), delivered at a flow rate of 1.2 mL/min. Detection was carried out at 225 nm with a total runtime of 25 min. The retention time of levothyroxine was approximately 7.1 min. The method was validated across a linearity range of 0.5 to 2.0 ppm (50% to 200% of the test concentration), yielding a correlation coefficient (r²) > 0.999. The method demonstrated excellent precision, with %RSD values of 0.2% for standard solutions and 0.8% for six sample preparations. Limits of detection (LOD) and quantification (LOQ) were determined to be 0.05 and 0.1 ppm, respectively. The method was found to be specific to all 14 known impurities, robust, accurate, and stability-indicating. This validated method is suitable for routine quality control analysis and regulatory submissions, meeting industrial standards for accuracy, precision, and specificity across all marketed strengths of levothyroxine sodium capsules.