Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R&D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control (QC) revealed a large discrepancy between the purity levels as stated on the supplier’s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid(s) in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

Lipopeptides are currently re-emerging as an interesting subgroup in the peptide research field, having historical applications as antibacterial and antifungal agents and new potential applications as antiviral, antitumor, immune-modulating and cell-penetrating compounds. However, due to their specific structure, chromatographic analysis often requires special buffer systems or the use of trifluoroacetic acid, limiting mass spectrometry detection. Therefore, we used a traditional aqueous/acetonitrile based gradient system, containing 0.1% (m/v) formic acid, to separate four pharmaceutically relevant lipopeptides (polymyxin B1, caspofungin, daptomycin and gramicidin A1), which were selected based upon hierarchical cluster analysis (HCA) and principal component analysis (PCA).
In total, the performance of four different C18 columns, including one UPLC column, were evaluated using two parallel approaches. First, a Derringer desirability function was used, whereby six single and multiple chromatographic response values were rescaled into one overall D-value per column. Using this approach, the YMC Pack Pro C18 column was ranked as the best column for general MS-compatible lipopeptide separation. Secondly, the kinetic plot approach was used to compare the different columns at different flow rate ranges. As the optimal kinetic column performance is obtained at its maximal pressure, the length elongation factorλ(Pmax/Pexp) was used to transform the obtained experimental data (retention times and peak capacities) and construct kinetic performance limit (KPL) curves, allowing a direct visual and unbiased comparison of the selected columns, whereby the YMC Triart C18 UPLC and ACE C18 columns performed as best. Finally, differences in column performance and the (dis)advantages of both approaches are discussed.

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quan-tification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono-and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. De-pending on the used iodination method, iodination yields varied from low (2%) to high (57%).