Absolute Quantitation of Coeluting Impurities in Peptide Drugs Using High Resolution Mass Spectrometry: Glucagon a Case Study in Pharmaceutical Development

Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (<i>p</i>-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly⁴ glucagon and des-Thr⁵ glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (<i>R</i>² > 0.99) across a concentration range of 0.25-25 μg/mL. The limits of detection (LODs) for des-Gly⁴-glucagon and des-Thr⁵-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, <i>p</i>-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.