Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS)

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at molecular scale, requiring development bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled electrospray ionization mass spectrometry (ESI-MS) was separate synthetic multiphosphorylated cyclopeptides and their positional isomers physiological pH. We assessed (i) selectivity eleven HILIC columns, from manufacturers packed with diverse polar sorbents, (ii) effect mobile phase composition on separation selectivity. The best baseline resolution were achieved columns grafted by neutral sorbents amide diol. Furthermore, we investigated retention mechanism these peptides examining number phosphorylated residues peptide scaffold retention. behavior followed classical hydrophilic partitioning exclusively diol columns. This trend not fully respected bare hybrid silica due attractive/repulsive deprotonated surface silanol groups Arginine or Glutamate according sequence. position amino acid backbone also showed have an impact retention, making possible cyclic using HILIC.