The variety of lipid compounds and the complexity of biological sample matrices require lipidomics analysis to utilize advanced separation techniques and detection methods. At present, various chromatographic techniques are mainly used, including thin-layer chromatography (TLC), gas chromatography (GC), capillary electrophoresis (CE), supercritical fluid chromatography (SFC), high-performance liquid chromatography (HPLC) and various mass spectrometry (MS) techniques, as well as chromatography-mass spectrometry coupling techniques. Nuclear magnetic resonance (NMR), infrared spectroscopy and Raman spectroscopy are less used, mainly because the separation capacity of these techniques is limited, the sensitivity for the detection of lipid compounds is not yet satisfactory, and the qualitative capacity is not strong. Lipidomics analysis mainly includes untargeted lipidomics analysis, target lipidomics analysis, and imaging analysis.
Untargeted lipidomic analysis is the isolation and identification of all lipid compounds and their metabolites in biological samples in order to screen biomarkers from them.
In the early days, TLC (and TLC-MS) and GC (and GC-MS) were mostly used. However, the former has low separation efficiency, and the latter requires derivatization of lipid compounds, which is a complex and time-consuming analytical step. Nowadays, LC-MS is increasingly used for lipidomics analysis.
LC has many modes and is suitable for the separation of complex lipid compounds without derivatization. Moreover, the rapid development of MS technology and the commercialization of various high-resolution MS and tandem MS (often using MS/MS), as well as interfaces to LC (mainly electrospray), have enabled LC-MS to provide excellent separation and identification capabilities.
In addition, shotgun lipidomics, which uses MS for direct analysis, is also used for non-targeted lipidomics analysis. It is characterized by fast analysis speed and high identification ability. However, the shortcomings are also obvious: 1) the instrument is expensive; 2) the information of lipid composition is missing. In the birdshot method, whole lipids are ionized in the ion source without chromatographic separation, resulting in the ionization of low-abundance and difficult-to-ionize lipid components being significantly inhibited, causing non-detection of relevant lipids; 3) the lack of information on lipid structure. The role of isomers in organisms may vary significantly, and accurate identification becomes particularly important. It is difficult to distinguish certain lipid isomers (e.g. positional and enantiomeric isomers) by the birdshot method of MS alone.
Targeted lipidomics analysis is the analysis of several, one or a few classes of lipid compounds (e.g. biomarkers). Targeted analysis requires high speed, qualitative and quantitative accuracy. Direct MS analysis and RPLC are commonly used methods, especially ultra-high performance LC (UHPLC) coupled with MS for high separation efficiency and fast analysis.
Imaging analysis is to visually analyze the distribution of lipid compounds in biological samples to obtain dynamic data. The lipidomics imaging analysis mainly includes fluorescence imaging and MS imaging. The former has high sensitivity, but it requires derivatization of the sample, and only one or several lipid compounds can be obtained in one imaging. The latter can obtain information on many types of lipid compounds in one imaging, and does not require derivatization of the sample, but the detection sensitivity is limited.
Among the various MS imaging techniques, MALDI-MS imaging is the most advantageous. However, MALDI-MS still has sensitivity limitations and is difficult to use for in vivo or in situ imaging analysis. Secondary ion mass spectrometry (SIMS) with high spatial resolution has been used for single-cell imaging analysis of lipid compounds.