Department of Chemistry, Faculty of Science, University of Hradec Králové, Rokitanského 62, 50003 Hradec Králové, Czech Republic.
Department of Chemistry, Faculty of Science, University of Hradec Králové, Rokitanského 62, 50003 Hradec Králové, Czech Republic.
J Chromatogr A. 2022 May 10;1670:462975. doi: 10.1016/j.chroma.2022.462975. Epub 2022 Mar 15.
Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.
由于天然脂质组的极端复杂性，脂质组学分析需要强大的色谱技术。在过去的几年中，超高效超临界流体色谱（UHPSFC）已被证明是传统 LC 方法进行综合脂质组学分析的良好替代品。本工作的目的是研究根据脂肪酸组成对 UHPSFC 类内脂质分离。广泛研究和讨论了柱化学、流动相组成和梯度、流速、背压、温度和柱耦合对脂质类内分离的影响。一般来说，UHPSFC 表现出类间选择性，同时根据其双键总数和酰基链长度对脂质进行类内分离。此外，在某些情况下还实现了二酰基甘油和溶血磷脂酰胆碱的区域异构体分离。固定相的性质对 UHPSFC 类内选择性的影响最为显著，而其他色谱条件则用于部分提高脂质种类的分辨率。十八烷基固定相对非极性脂质种类（包括三酰甘油等异构体）具有出色的分离效果；然而，它对极性脂质的峰形和保留时间重现性较差。二醇和 1-氨基蒽基柱对大多数脂质提供了最佳的类间和类内分辨率。UHPSFC 分离复杂脂质样品的主要优点是脂质的酰基链/双键类内分离与出色的脂质类别选择性相结合，这可以促进质谱检测和痕量物质的定量，而不会产生离子抑制效应。
