Derringer desirability and kinetic plot LC-column comparison approach for MS-compatible lipopeptide analysis

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 factor λ（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.

Reversed-phase fused-core HPLC modeling of peptides

Different fused-core stationary phase chemistries（C18,Amide,Phenyl-hexyl and Peptide ES-C18） were used for the analysis of 21 structurally representative model peptides.In addition,the effects of the mobile phase composition（ACN or MeOH as organic modifier;formic acid or acetic acid,as acidifying component） on the column selectivity,peak shape and overall chromatographic performance were evaluated.The RP-amide column,combined with a formic acid-acetonitrile based gradient system,performed as best.A peptide reversed-phase retention model is proposed,consisting of 5 variables：log SumAA,log Sv,clog P,log nHDon and log nHAcc.Quantitative structure-retention relationship（QSRR） models were constructed for 16 different chromatographic systems.The accuracy of this peptide retention model was demonstrated by the comparison between predicted and experimentally obtained retention times,explaining on average 86% of the variability.Moreover,using an external set of 5 validation peptides,the predictive power of the model was also demonstrated.This peptide retention model includes the novel in-silico calculated amino acid descriptor,AA,which was calculated from log P,3D-MoRSE,RDF and WHIM descriptors.

Implementation of a single quad MS detector in routine QC analysis of peptide drugs

A newly developed single quad mass spectrometry （MS） detector was coupled to a ultra-high perfor- mance liquid chromatography （UPLC） system and implemented in the routine quality control （QC） and impurity analysis of four therapeutic peptides, namely bleomycin sulfate, tyrothricin, vancomycin HCl and bacitracin, which were selected given their multi-component drug nature and their closely struc- turally related impurity profiles. The QC and impurity profiling results obtained using the ultra-high performance liquid chromatography ultraviolet/mass spectrometry （UPLC-UV/MS） detection system were analyzed against the results obtained using traditional high performance liquid chromatography- ultraviolet detection （HPLC-UV） methods derived from pharmacopoeial methods. In general, the used stationary phases of sub-2 μm particle （UPLC） technology resulted in lower limits of detection and higher resolution separations, which resulted in more detected impurities and shorter overall run times con- trasting the traditional HPLC columns. Moreover, online coupling with a single quad MS detector allowed direct peak identification of the main compounds as well as small impurities, hereby increasing the information content without the need of reference standards.