Analytical development of seamless procedures on cation-exchange chromatography and ion-pair chromatography with high-precision mass spectrometry for short-chain peptides

We report comprehensive analytical procedures for characterization of underivatized short-chain peptides by wet chemical treatment and ion-pair liquid chromatography with electrospray ionization–mass spectrometry (LC/ESI–MS). The chromatographic separation via a porous graphitic carbon stationary column eluted using an acetonitrile solvent gradient revealed a positive correlation between the retention time and the degree of hydrophobicity reflected in a distinct relationship between methylene (–CH₂–) elongation of amino acids. We also validated the chromatographic separation utilizing an InertSustain PFP column and high-resolution mass spectrometry of the Orbitrap system, which supports the analytical accuracy for identification of monoisotopic mass. Using the quantitative analysis on a Corona charged aerosol detector, the recovery of the underivatized short-chain peptide fraction was >94.0 ± 4.9% (n = 10) after purification via cation-exchange chromatography. By eliminating the inorganic and organic matrix effect, we can avoid ion suppression and enhancement to demonstrate accurate analytical evaluations with robust mass spectroscopic responses. The developed method provides new insight into short-chain peptide chemistry that can be utilized for numerous interdisciplinary research applications.