Table 1 Analytical factors affecting lipid analysis.

Different fates of fatty acids with different chemical structures: Metabolic flux is dependent on a fatty acid’s characteristics. For example, the oxidation of saturated fatty acids decreases with increasing carbon length.

Different lipid species: Lipidome diversity constrains the ability to analyze several lipids using a single methodology. This also increases m/z ratio overlap in chromatograms. Different lipids require different separation techniques.

Synthesis interrelationships: Increases in the synthesis of one lipid species may affect the synthesis of a related species, resulting in complex patterns of isotopic labeling.

Choice of internal standard: Different internal standards are needed when lipids are labeled at multiple sites.

Biological matrices: Lipids can be found in different tissues and associated with different lipoproteins (e.g., VLDL, IDL, LDL, and HDL).

Lipid aggregation: At high concentrations, lipids are likely to form aggregates (e.g., dimers, oligomers, or micelles), hindering lipid detection by mass spectrometry.

Low abundance: Lipids may serve as intermediates and signaling molecules present in low abundance in biological systems.

Isotopic labeling protocols: Different molecules and pathways will require different protocols.