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. 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. 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.

中文翻译：

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碳酸氢铵缓冲液与混合表面技术色谱柱相结合，改善了强拖尾脂质的峰形


磷脂酸 (PA) 和心磷脂 (CL) 等脂质在反相液相色谱中存在强烈拖尾峰，导致检测能力较低。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的发展。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组覆盖范围。我们在传统 BEH C18 色谱柱和 HST-CSH C18 色谱柱上研究了碳酸氢铵 (ABC) 对峰不对称性和可检测性的影响，并与甲酸铵 (AFO) 进行比较。 2.5 mM ABC 缓冲液 pH 8 与 HST-CSH C18 色谱柱的组合可显着改善结果，将 PA 16:0/18:1 10% 峰高处的不对称因子从 8.4 降低至 1.6。此外，与 AFO 和 BEH C18 色谱柱相比，其 [M − H] 离子的峰高平均提高了 54 倍。我们证实了对其他强拖尾脂质的这种有益作用，这些脂质具有可接近的磷酸盐部分，例如心磷脂、磷酸磷脂酰肌醇、二磷酸磷脂酰肌醇、磷酸化神经酰胺和磷酸化鞘氨醇。此外，我们发现使用 HST-CSH C18 色谱柱时，在阴性模式下磷酸酯和鞘脂的检测能力提高了 28 倍。该方法成功应用于小鼠肝脏样本，通过改进的色谱分离可以分析以前未检测到的内源磷脂。总之，使用 2.5 mM ABC 显着改善了 PA 的峰形，并增强了 RPLC-ESI-Q-TOF-MS 系统在 BEH C18 和 HST-CSH C18 色谱柱上阴性模式下脂质组的检测能力。 该方法通过一次注射提供了更广泛的脂质组覆盖范围，用于未来负模式下的脂质组学应用。