Over the past few decades, peptide analysis has aroused growing interest due to the increasing use of peptides as drugs or disease biomarkers as well as their monitoring in the field of proteomics.

The chromatographic behaviour of peptides is totally different from that of small molecules and therefore a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC-MS is strongly recommended for a successful development of qualitative or quantitative analytical methods. Miniaturised chromatographic systems often involve the use of proportionally very large sample volumes compared to system size. In most cases, however, sample composition is surprisingly neglected in method development.  

In this study, a design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a mixture of nine peptides covering a wide range of molecular weights, isoelectric points and hydropathy indices. First, a screening design was developed to identify the significant factors with respect to mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity. Then, after selection of the experimental domain and the significant factors, a full factorial design was used to further investigate the role of these factors and possible interaction effects. As expected, ion-pair formation had a tremendous effect on retention and response intensity. Optimal generic LC conditions leading to adequate peptide retention without band splitting as well as good MS sensitivity could be selected. These chromatographic conditions can be used as a starting point for rapid method development for peptide analysis using classical solvents and ion-pairing reagents.