Identification and Quantification of Structurally Related Peptide Impurity in Linaclotide by Liquid Chromatography–High Resolution Mass Spectrometry

ABSTRACT Rationale Linaclotide is an important peptide drug used to treat irritable bowel syndrome with constipation (IBS‐C). However, structurally related impurities in peptide drugs can be generated during synthesis, storage, or transport, which compromise the quality and safety. Therefore, developing a highly sensitive analytical method for impurity detection is of great significance. Methods A liquid chromatography–high resolution mass spectrometry (LC‐HRMS) method was developed. Linaclotide samples were directly analyzed using an Orbitrap mass spectrometer with an electrospray ionization (ESI) source operated in positive ion mode. Tandem mass spectrometry with collision‐induced dissociation was utilized. The method leveraged high mass accuracy to identify and quantify structurally related peptide impurities in linaclotide samples from different manufacturers. Results The method was applied to characterize the peptide impurities in linaclotide study materials. In manufacturer A's material, [Des‐Tyr 14 ]‐linaclotide was found at 0.453 mg/g. The same impurity was found in manufacturer B's material at 0.768 mg/g, as well as another impurity [endo‐Ala 9 ]‐linaclotide at 0.555 mg/g. Conclusions The developed LC‐HRMS method successfully addresses the challenge of determining structurally related peptide impurities in linaclotide. Its ability to characterize specific impurities provides a valuable complement to existing quality control strategies for peptide therapeutics.