Automated derivatization with 6-aminoquinolyl-N-hydroxysccinimidyl carbamate for the enantioselective amino acid analysis of neurotensin synthesized by liquid phase peptide synthesis

This study presents an automated derivatization protocol utilizing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for enantioselective amino acid analysis of peptides synthesized via liquid phase peptide synthesis, as exemplified by neurotensin. The primary aim was to enhance operational efficiency to manage the derivatization of large sample sets and reduce human error in routine enantioselective amino acid analysis of peptide therapeutics. The chromatographic method based on Chiralpak QN-AX demonstrated enantio- and chemoselectivity for all proteinogenic amino acids (except D-Leu/D-Ile and Glu/pGlu), with quantitative analysis achieved by HPLC-ESI-MS/MS with MRM acquisition through external calibration using stable isotope-labeled internal standards. The goal was to test for racemization of amino acids during peptide synthesis and process optimization, respectively. The results confirmed varying susceptibility to racemization among amino acids during peptide synthesis and cleavage of protection groups, with specific amino acids exhibiting higher levels of D-enantiomer formation. The developed protocol effectively assessed the amino acid composition and stereointegrity of the liquid phase synthesized neurotensin. This research and application highlights the critical role of automation in optimizing peptide analysis workflows and sets the foundation for future improvements in peptide synthesis and chromatographic conditions to enhance specificity, particularly for challenging amino acid pairs. Ultimately, the findings contribute to advancing laboratory practices in peptide chemistry, ensuring the quality and efficacy of peptide-based therapeutics.