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Analytical Challenges in Peptide Sequencing of Semaglutide The complex structure of Semaglutide presents several analytical challenges: Hydrophobic Modifications: The lipidation at Lys20 increases hydrophobicity, complicating chromatographic separation and mass spectrometric analysis. Non-Canonical Amino Acids: Incorporation of Aib and PEG-like spacers requires specialized detection and sequencing strategies. Sequence Confirmation: Ensuring complete sequence coverage, including modified residues, is critical for regulatory compliance. Addressing these challenges often requires integrated impurity and purity assessment

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