Optimization of Folch, Bligh-Dyer, and Matyash sample-to-extraction solvent ratios for human plasma-based lipidomics studies.

In order to profile the lipidome for untargeted lipidomics applications, analysis by ultra-high performance liquid chromatography - high resolution mass spectrometry (UHPLC-HRMS) typically requires the extraction of lipid content from sample matrices using matrix-specific conditions. The Folch, Bligh-Dyer, and Matyash extraction methods, while promising approaches, were originally tailored to specific matrices (brain tissue, fish muscle, and E. coli, respectively). Each of these methods have specific solvent ratios that must be adhered to achieve optimal extraction. Thus, the sample-to-solvent ratios for these methods should be optimized for the sample matrix of interest prior to employment. This study evaluated the appropriate sample-to-extraction solvent ratios for human plasma-based lipidomics studies. An advantage of employing biphasic lipid extractions is the ability to investigate both the aqueous and organic layers for increased analyte coverage in untargeted studies. Therefore, this work also evaluated the multi-omic capability of each lipid extraction method for plasma in an effort to provide a workflow capable of increasing analyte coverage in a single extraction, thus providing a more complete understanding of complex biological systems. In plasma, a decrease in sample-to-solvent ratios from 1:4, 1:10, 1:20, to 1:100 (v/v) resulted in a gradual increase in the peak area of a diverse range of metabolite (aqueous layer) and lipid (organic layer) species for each extraction method up to the 1:20(v/v) sample-to-solvent ratio. The Bligh-Dyer and Folch methods yielded the highest peak areas at every plasma sample-to-solvent ratios for both metabolite and lipid species. Depending on the lipid class of interest, the Folch or Bligh-Dyer method is best suited for analysis of human plasma at a 1:20 (v/v) sample to total solvent ratio.