D'Hondt Matthias, Verbeke Frederick, Stalmans Sofie, Gevaert Bert, Wynendaele Evelien, De Spiegeleer Bart
Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
J Pharm Anal. 2014 Jun;4(3):173-182. doi: 10.1016/j.jpha.2013.09.001. Epub 2013 Sep 18.
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 B, caspofungin, daptomycin and gramicidin A), 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 -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 (/) 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.
脂肽目前正重新成为肽研究领域中一个有趣的亚组，在历史上曾用作抗菌和抗真菌剂，并且作为抗病毒、抗肿瘤、免疫调节和细胞穿透性化合物具有新的潜在应用。然而，由于其特定结构，色谱分析通常需要特殊的缓冲系统或使用三氟乙酸，这限制了质谱检测。因此，我们使用了一种基于水/乙腈的传统梯度系统，其中含有0.1%（m/v）的甲酸，以分离四种与药物相关的脂肽（多粘菌素B、卡泊芬净、达托霉素和短杆菌肽A），这些脂肽是基于层次聚类分析（HCA）和主成分分析（PCA）选择的。总共使用两种并行方法评估了四种不同的C18柱（包括一根超高效液相色谱柱）的性能。首先，使用Derringer合意函数，将六个单一和多个色谱响应值重新缩放到每根柱的一个总体值。使用这种方法，YMC Pack Pro C18柱被评为一般质谱兼容脂肽分离的最佳柱。其次，使用动力学图方法在不同流速范围内比较不同的柱。由于在最大压力下可获得最佳的动力学柱性能，因此使用长度伸长因子（/）来转换获得的实验数据（保留时间和峰容量）并构建动力学性能极限（KPL）曲线，从而可以对所选柱进行直接的直观且无偏的比较，据此YMC Triart C18超高效液相色谱柱和ACE C18柱表现最佳。最后，讨论了柱性能的差异以及两种方法的（优）缺点。
