Department of Medicinal Chemistry, Uppsala Biomedical Centre, Uppsala University, Box 574, 75123 Uppsala, Sweden.
Department of Medicinal Chemistry, Uppsala Biomedical Centre, Uppsala University, Box 574, 75123 Uppsala, Sweden; Instituto de Biomedicina y Genética Molecular (IBGM), CSIC-Universidad de Valladolid, C/ Sanz y Forés 3, 47003, Valladolid, Spain.
Anal Chim Acta. 2024 Aug 8;1316:342811. doi: 10.1016/j.aca.2024.342811. Epub 2024 Jun 8.
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.
磷脂，如磷脂酸（PA）和心磷脂（CL），在反相液相色谱中呈现强烈拖尾峰，这导致其检测灵敏度较低。它们通常通过亲水相互作用液相色谱（HILIC）进行分析，但这会阻碍高通量脂质组学的发展。因此，为了在单一色谱方法中获得更广泛的脂质组覆盖范围，非常需要改进分析方法。我们研究了在常规 BEH C18 柱和 HST-CSH C18 柱上，与甲酸铵（AFO）相比，碳酸氢铵（ABC）对峰不对称性和检测灵敏度的影响。
使用 2.5 mM ABC 缓冲液 pH 8 与 HST-CSH C18 柱相结合，可显著改善结果，使 PA 16:0/18:1 在 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 柱相比，其[M-H]离子的峰高平均增强了 54 倍。我们在其他具有可及磷酸部分的强烈拖尾脂质（例如心磷脂、磷脂酰肌醇磷酸盐、磷脂酰肌醇二磷酸盐、磷酸化神经酰胺和磷酸化鞘氨醇）上证实了这种有益效果。此外，我们发现当使用 HST-CSH C18 柱时，负模式下磷酯和鞘脂的检测灵敏度提高了 28 倍。该方法成功应用于小鼠肝样品，可分析以前未检测到的内源性磷脂，同时具有更好的色谱分离效果。
总之，在 BEH C18 和 HST-CSH C18 柱上的 RPLC-ESI-Q-TOF-MS 系统中，使用 2.5 mM ABC 可显著改善 PA 的峰形，并增强负模式下脂质组的检测灵敏度。该方法为未来的负模式脂质组学应用提供了一种更广泛的脂质组覆盖范围，只需单次进样即可完成。
