Characterisation of monophasic solvent-based tissue extractions for detection of polar metabolites and lipids applying UHPLC-MS clinical metabolic phenotyping assays

ABSTRACT:

Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and non-polar (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 non-polar compound extraction (chloroform/methanol/water; dichloromethane/methanol/water; methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water) and a monophasic method for non-polar compound extraction (isopropanol/water). All methods were applied to sheep heart, kidney, and liver tissue. Polar extracts were analysed by hydrophilic interaction chromatography (HILIC) ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) and non-polar extracts by C18 reversed phase UHPLC-MS. Method reproducibility and yield was 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 RSD<18%; negative ion: median RSD<28%) and non-polar (positive and negative ion: median RSD<15%) extractions for heart, kidney, 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 non-polar 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 therefore most suited for future automation.

INSTRUMENT(S): Q Exactive

SUBMITTER: Andrew Southam  Ralf Weber 

PROVIDER: MTBLS1707 | MetaboLights | 2020-09-28

REPOSITORIES: MetaboLights