A Review on Analytical Method Development and Validation for the Simultaneous Estimation of Levothyroxine and Liothyronine in Combined Pharmaceutical Dosage Form by RP-HPLC

The simultaneous estimation of Levothyroxine (T4) and Liothyronine (T3) in combined pharmaceutical dosage formsis crucial for effective hypothyroidism management.Due totheir lowconcentrations,structural similarity, and high potency, method development poses analytical challenges. Reversed-phase high-performance liquid chromatography (RP-HPLC) stands out as a preferred technique owing to its high sensitivity, resolution, and reproducibility across a wide range of compound polarities. This review outlines RP-HPLC principles and highlights method development strategies such as columns election, mobile phase optimization, detection wavelength, and sample preparation. It also discusses essential method validation parameters in line with ICH guidelines, including accuracy, precision, specificity, linearity, LOD, LOQ, robustness, and system suitability. Literature-based insights into validated methods for routine analysis, stability studies, and pharmacokinetic evaluations are presented. Advancements such as gradient elution, stability-indicating approaches, and mass spectrometry integration are explored for their impact on method performance. The review concludes with clinical and regulatory perspectives, reinforcing RP-HPLC as a reliable tool for the quantitative analysis of thyroid hormones in pharmaceutical formulations.