Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry

• Sample and mobile phase composition were optimised for RP-LC–MS peptide analysis.
• Multivariate analysis was used for experimental condition optimisation.
• Peak splitting can be avoided using appropriate sample composition.
• Sample dissolution medium influences sensitivity.
• Generic conditions lead to high peak efficiency and sensitivity for most peptides.

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC–MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.