Analysis of short-chain bioactive peptides by unified chromatography-electrospray ionization mass spectrometry. Part I. Method development.

A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was developed with a unified chromatography (UC) analysis, including a wide mobile phase gradient ranging from supercritical fluid to near-liquid conditions, with UV and electrospray ionization mass spectrometry detection (ESI-MS). Four stationary phases and three mobile phase compositions were examined. Ten model peptides were first selected to identify the best operating conditions, including five linear tripeptides and five cyclic pentapeptides, with log P values ranging from -5.9 to 3.6, and including isomeric species. Derringer desirability functions were designed to identify optimal operating conditions based on 7 criteria, namely the number of peaks detected (including all impurities resolved), the proportion of the chromatogram occupied by target peaks, the least favourable resolution observed between the main peptide and impurities, peak shape features (asymmetry and peak width at half height), and finally the signal-to-noise ratio observed both with UV (210 nm) and ESI-MS in positive ionization mode. The optimum conditions were obtained on Ascentis Express OH5 stationary phase, with a mobile phase composed of carbon dioxide and methanol, comprising 2% water and 20 mM ammonium hydroxide. The final gradient program ranged from 5 to 80% co-solvent in CO2, with a reversed flow rate gradient ranging from 3.0 to 1.5 mL/min. Back-pressure was set at 120 bar and the column oven temperature at 60°C. Optimal conditions were applied to a large set of 76 peptides (34 linear tripeptides and 42 cyclic pentapeptides) and provided adequate scattering of the peaks in the retention space, together with some separation of isomeric species, particularly for the cyclic peptides.