Development and Validation of a Novel Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for the Quantification of Related Substances of Vasopressin Injection: A Characterization and Comparative Analysis of Vasopressin Formulation Samples against Vasostrict (RLD) in Various Diluents.

Abstract

In the present study, we developed and validated a sensitive, accurate, robust and simple RP-HPLC method for quantification of related substances of Vasopressin (VPS) injection. The development phase prioritized the optimization of wavelength, mobile phase composition and column selection to enhance separation and sensitivity for the analytical evaluation of VPS injection. By considering peak symmetry, resolution and retention time, chromatographic conditions of VPS injection were established. The YMC PACK ODS AM (100 ×4.6) mm, 3µm was employed in the study. Mobile phase A was prepared with 0.113 M NaH₂PO₄.H₂O (pH 3.0), while mobile phase B was a 50:50 (v/v) ratio of acetonitrile and water, pumped through a flow rate of 1.0 mL/min. VPS injection was exposed to thermal, photolytic, acid, base and peroxide degradation conditions and these were analysed by the current method. The method was validated following the guidelines set by the International Council for Harmonization guideline (ICH). The linearity studies demonstrating that a correlation coefficient value is more than 0.999 for VPS and its related substances. The detection and quantification limits for all related substances were found to be 0.01% and 0.05%, respectively. All the impurities exhibited consistent recoveries ranging from 85 to 105%. The unknown impurities were eluting at RRT 1.23 and RRT 1.25 in the RS methodology (HPLC-UV) of VPS injection are identified and assessed the impurity levels. Furthermore, aggregate profiles and secondary structure analysis studies were carried out using FTIR and LC-HRMS, comparing the VPS formulation samples with the Reference Listed Drug (RLD) (Vasostrict) samples across various diluents.