Although shot-gun lipidomics can answer many of the needs associated with sample lipid profiling, some workflows do require chromatographic separation before MS analysis. For example, the quantification of isobaric lipids can only be done if the lipids are somehow separated before entering the mass spectrometer. In these cases, we would choose a column that is suitable for the separation of your lipids of interest and use ultrahigh-performance gas/liquid chromatography equipped with different mass spectrometry systems to analyze the lipidomics.
Fig1. The protocol workflow of GC/LC-MS lipidomics (Creative Proteomics)
Gas chromatography (GC) has a niche application and is the most suitable method for the analytics of fatty acids and cholesterol, due to its highly efficient separation of volatile compounds at low temperatures without thermal decomposition. Since temperatures up to 300°C are required to transfer lipids of high molecular weight via the GC column, this method is not suitable for polyunsaturated, acyl-substituted lipids or intact large lipids such as triglycerides. The elevated temperature increases the risk for thermal decomposition of the lipids in the GC column even before the lipids pass the mass spectrometer. A further limitation to GC-MS is the need for fatty acids to be derivatized prior to the GC-MS measurement, because fatty acids cannot be evaporated in their native form.
In contrast, liquid chromatography (LC) is universally applicable for all lipid classes, both volatile and non-volatile. Due to its high separation efficiency and selectivity, LC methods are the most frequently used separation methods in lipidomics. The wide choice of mobile and stationary phase results in a high selectivity and resolution for the different lipid classes.
Compared to GC, LC methods such as HPLC can process all classes of native lipids without the need for derivatization and purificationThe major downside of HPLC is cost due to high acquisition costs, large volumes of mobile phase and the budget needed for LC columns.
In the TOF mass spectrometer ions are accelerated to the same high kinetic energy (Ek) and thus ions of different m/z have different velocities (v). The m/z values of ions are deduced from their flight time (t) through a tube with a fixed length (L) under high vacuum. TOF and hybrid TOF instruments, such as ion mobility-TOF, TOF-TOF, Qq-TOF, and QIT-TOF, have all been used for lipid analysis.
The orbitrap is a newer member of the ion-trap family of mass analyzers. It consists of an axial spindle-like central electrode and a coaxial barrel-like outer electrode. The trapped ions undergo rotation and harmonic oscillations along the central electrode. The m/z values of the trapped ions are related to the frequencies of their harmonic oscillations along the central axis. Mass analysis is performed by measuring the image current that is induced in the outer electrode by the motions of the ions and converting the time-domain signal into mass spectra using fast Fourier transforms. Orbitrap mass spectrometers offer high mass accuracy and resolving power. Hybrid LIT-orbitrap instruments have been employed for high-quality lipidome identification and quantification.
The advantages of triple-quadrupole mass spectrometers in lipidomics research include their ability to perform precursor ion scanning and neutral loss scanning and their unequalled ability to provide quantitative analyses of high precision and accuracy using the multiple-reaction monitoring (MRM) mode. These characteristics are suitable for the analysis of various lipid classes.
Bou Khalil, Maroun.; et al. Lipidomics era: accomplishments and challenges. Mass spectrometry reviews. 2010, 29.6: 877-929.