Evaluation of LC-MS and LCxLC-MS in analysis of zebrafish embryo samples for comprehensive lipid profiling

In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18xHILIC, HILICxC18, and HILICxPFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18xHILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILICxC18 and C18xHILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18xHILIC was much higher. A comparison of the HILICxC18 and the HILICxPFP methods demonstrated that the HILICxPFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILICxPFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18xHILIC was selected for further studies.