DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR ESTIMATION OF TIRZEPATIDE

The present study focuses on the development and validation of a simple, rapid, precise, accurate, and stability-indicating reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the quantitative estimation of Tirzepatide. It is a novel dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist widely used in the management of type 2 diabetes mellitus and obesity. Due to its peptide-based structure, the molecule presents significant analytical challenges, including susceptibility to hydrolytic and oxidative degradation, necessitating a robust and reliable analytical method for routine quality control and stability assessment. Chromatographic separation was achieved using a C18 column (250 mm × 4.6 mm, 5 µm) with a mobile phase consisting of acetonitrile and phosphate buffer (pH 3.0) in the ratio of 45:55 (v/v), delivered at a flow rate of 1.0 mL/min. Detection was performed at 230 nm using a UV detector. The method exhibited a well-resolved and symmetrical peak for Tirzepatide with a retention time of approximately 7.21 minutes. The developed method was validated in accordance with ICH Q2(R1) guidelines for specificity, linearity, precision, accuracy, robustness, limit of detection (LOD), and limit of quantification (LOQ). The method demonstrated excellent linearity over the concentration range of 10–60 µg/mL with a correlation coefficient (r²) of 0.9996. Precision studies showed %RSD values less than 2%, indicating high repeatability and reproducibility. Accuracy was confirmed through recovery studies at 80%, 100%, and 120% levels, yielding mean recoveries within 98–102%. The LOD and LOQ were found to be 0.073 µg/mL and 0.220 µg/mL, respectively, indicating good sensitivity of the method. Robustness evaluation revealed that minor deliberate variations in chromatographic conditions did not significantly affect the analytical performance. Tirzepatide showed maximum degradation under oxidative conditions, followed by alkaline and acidic hydrolysis, while thermal and humidity conditions resulted in comparatively lower degradation. The developed RP-HPLC method is simple, economical, reliable, and highly effective for routine quantitative analysis, quality control, and stability studies of Tirzepatide.