Advanced unified monophasic lipid extraction protocol with wide coverage on the polarity scale optimized for...

Lipid extraction is a critical step in sample preparation of lipidomics studies. Biphasic liquid-liquid extraction protocol with methyl tert-butyl ether (MTBE)/methanol (MeOH) as organic solvents are widely adopted by researchers nowadays as an eco-friendly replacement of classic Folch, and Bligh&Dyer protocols. Yet, it has some limitations such as suboptimal performance for the most polar lipids (e.g. acylcarnitines), complicated handling as it requires phase separation, and is therefore non-ideal for large-scale clinical studies. To advance the extraction protocol for large-scale clinical lipidomics, in this study we explored i) 6 different extraction solvent systems, ii) distinct processing procedures (sonication, mechanical cell lysis and bead homogenizer), and iii) also 7 different reconstitution solvents. The extraction systems investigated included biphasic systems MTBE/MeOH/H₂O and Hexane/2-propanol (IPA)/1 M acetic acid, and monophasic systems MTBE/MeOH/CHCl₃, IPA/H₂O (90% IPA), MeOH/MTBE/IPA, and IPA/H₂O/MTBE as solvent system for lipid extraction of human platelets. Extraction recovery was investigated by repeated extraction cycles. Subcellular extraction efficiency was assessed by the mitochondria-specific cardiolipins. It turned out that monophasic extraction with MeOH/MTBE/IPA (1.3:1:1, v/v/v), bead homogenizer for cell disruption and MeOH/MTBE (1:1, v/v) as reconstitution solvent provide optimal cellular and subcellular extraction efficiencies for both polar (e.g. acylcarnitines) and apolar lipids (e.g. triglycerides). It is simplified (no phase separation), eco-friendly (reduced solvent consumption and no halogenated ones), fast (5 min for 24 samples in parallel), and can be easily adapted for cells, plasma, and tissue. Therefore, it has great potential for large-scale clinical lipidomics studies.