Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Clinical Metabolic Phenotyping Assays

Metabolic phenotyping of tissues
uses metabolomics and lipidomics
to measure the relative polar and nonpolar (lipid) metabolite levels
in biological samples. This approach aims to understand disease biochemistry
and identify biochemical markers of disease. Sample preparation methods
must be reproducible, sensitive (high metabolite and lipid yield),
and ideally rapid. We evaluated three biphasic methods for polar and
nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water,
and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method
for polar compound extraction (acetonitrile/methanol/water), and a
monophasic method for nonpolar compound extraction (isopropanol/water).
All methods were applied to mammalian heart, kidney, and liver tissues.
Polar extracts were analyzed by hydrophilic interaction chromatography
(HILIC) ultrahigh-performance liquid chromatography–mass spectrometry
(UHPLC–MS) and nonpolar extracts by C18 reversed-phase
UHPLC–MS. Method reproducibility and yield were assessed using
multiple annotated endogenous compounds (putatively and MS/MS annotated).
Monophasic methods had the highest yield and high reproducibility
for both polar (positive ion: median relative standard deviation (RSD)
< 18%; negative ion: median RSD < 28%) and nonpolar (positive
and negative ion: median RSD < 15%) extractions for heart, kidneys,
and liver. The polar monophasic method extracted higher levels of
lipid than biphasic polar extractions, and these lipids caused minimal
detection suppression for other compounds during HILIC UHPLC–MS.
The nonpolar monophasic method had similar or greater detection responses
of all detected lipid classes compared to biphasic methods (including
increased phosphatidylinositol, phosphatidylserine, and cardiolipin
responses). Monophasic methods are quicker and simpler than biphasic
methods and are therefore most suited for future automation.