Abstract
An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. Here we present a novel three-phase lipid extraction (3PLE) technique using a single-step liquid-liquid extraction (LLE) that allows both extraction and fractionation of lipids by polarity. 3PLE is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesteryl esters), while the middle organic phase contains the major glycerophospholipids. Thin-layer chromatography, radioactive labeling, and MS were used to confirm lipid partitioning. 3PLE efficiency was demonstrated for bovine liver, human pooled plasma, mouse liver, mouse brain, and mouse white adipose tissue. Compared with the gold-standard Bligh/Dyer LLE, 3PLE showed significant advantages. For direct-infusion workflows, there was a decrease in ion suppression with a corresponding increased number of lipid species identified. For LC/MS workflows, increased signal intensities were observed for lower-abundance lipid species such as phosphatidic acid and phosphatidylserine. 3PLE also proved to be a valuable tool for fatty acid profiling by GC/MS, allowing for the separate identification of neutral and polar fatty acids.
Highlights
An unbiased sample preparation free of interferents is critical to any lipid MS workflow
We present a three-phase liquid extraction (3PLE) method that extracts and separates lipids by polarity in a single liquid-liquid extraction (LLE)
The TLC results showed that cholesteryl ester (CE) species are present in the 3PLE upper phase (UP) but absent from the middle phase (MID) (Fig. 1A)
Summary
An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. By partitioning the neutral and polar lipid content between two organic phases, ion suppression decreases, resulting in an increase in sensitivity and subsequently increased the number of lipid species identified by direct-infusion and LC/MS techniques. LC/MS revealed increased signal intensities for some lipid species in 3PLE extracts compared with mBD, most notably in phospholipid profiling in the negative ionization mode (Fig. 4A).
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