The application of peptide sequencing for characterization of cetrorelix and its related impurities

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the structure of cetrorelix and the related impurities generated throughout the solid phase peptide synthesis (SPPS) reactions by Fmoc strategy. It was concluded that these by-products are formed as a result of incomplete removal of the protecting groups placed on side chains during synthesis. The impurity with 56 Da mass difference from cetrorelix was determined to be the result of failure in deprotection of L-serine residue and the impurity at 252 Da higher m/z than cetrorelix was due to the incomplete removal of Pbf from L-arginine. Peptide sequencing using ESI-MS allowed for rapid and reliable identification and characterization of the target peptide and its impurities. Furthermore, the information obtained from this method can offer a significant advantage for purity assessment of synthetic peptides which are used as therapeutic or research tools and it can also contribute a significant amount toward designing more efficient synthesis pathways for these molecules.