Abstract：
BACKGROUND: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column.
RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation.
CONCLUSIONS: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.
RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation.
CONCLUSIONS: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.
摘要：
背景：脂质如磷脂酸（PAs）和心磷脂（CLs）在反相液相色谱中存在强烈的拖尾峰，这需要低可检测性。它们通常通过亲水相互作用液相色谱（HILIC）进行分析，这阻碍了高通量脂质组学。因此，非常需要改进的分析方法,以便在单一色谱方法中获得更广泛的脂质凝集覆盖率。我们研究了碳酸氢铵（ABC）对峰不对称性和可检测性的影响，与常规BEHC18柱和HST-CSHC18柱上的甲酸铵(AFO)相比。
结果：2.5mMABC缓冲液pH8与HST-CSHC18柱的组合产生了显着改善的结果，将PA16:0/18:1的10%峰高的不对称因子从8.4降低到1.6。此外，平均而言,与AFO和BEHC18柱相比，[M-H]-离子的峰高增加了54倍。我们证实了这种对其他强烈拖尾脂质的有益作用，具有可接近的磷酸盐部分，例如，心磷脂,磷脂酰肌醇磷酸酯，磷脂酰肌醇双磷酸酯，磷酸化神经酰胺和磷酸化鞘氨醇。此外，我们发现,当使用HST-CSHC18色谱柱时,在阴性模式下,磷脂和鞘脂的可检测性增加了28倍.该方法已成功应用于小鼠肝脏样本，以前未检测到的内源性磷脂可以用改进的色谱分离进行分析。
结论：结论：在BEHC18和HST-CSHC18色谱柱上的RPLC-ESI-Q-TOF-MS系统上,使用2.5mMABC显著改善了PAs的峰形,并增强了脂质体在阴性模式下的可检测性.该方法通过一次单次注射提供了更广泛的脂质体覆盖，以阴性模式用于未来的脂质体应用。
结果：2.5mMABC缓冲液pH8与HST-CSHC18柱的组合产生了显着改善的结果，将PA16:0/18:1的10%峰高的不对称因子从8.4降低到1.6。此外，平均而言,与AFO和BEHC18柱相比，[M-H]-离子的峰高增加了54倍。我们证实了这种对其他强烈拖尾脂质的有益作用，具有可接近的磷酸盐部分，例如，心磷脂,磷脂酰肌醇磷酸酯，磷脂酰肌醇双磷酸酯，磷酸化神经酰胺和磷酸化鞘氨醇。此外，我们发现,当使用HST-CSHC18色谱柱时,在阴性模式下,磷脂和鞘脂的可检测性增加了28倍.该方法已成功应用于小鼠肝脏样本，以前未检测到的内源性磷脂可以用改进的色谱分离进行分析。
结论：结论：在BEHC18和HST-CSHC18色谱柱上的RPLC-ESI-Q-TOF-MS系统上,使用2.5mMABC显著改善了PAs的峰形,并增强了脂质体在阴性模式下的可检测性.该方法通过一次单次注射提供了更广泛的脂质体覆盖，以阴性模式用于未来的脂质体应用。
