Sample preparation optimization for metabolomics and lipid profiling from a single plasma and liver tissue based on...

Currently, the majorty of metabolomics studies rely on the use of a single analytical platform, either NMR or MS, which hampers the ability to conduct an unbiased and comprehensive analysis. However, the use of multiple platforms poses an additional challenge: the limited sample material. Consequently, it is highly desirable to develop comprehensive, effective and sample-conserving procedures for metabolite extraction. In this study, we presented pretreatment strategies enabling sequential NMR and multi-LC-MS (UHPLC-Q-Orbitrap MS, and UHPLC-QqQ MS) platform analysis using a single plasma and liver tissue sample. For the plasma sample, the biphasic CHCl₃/MeOH/H₂O method was suitable for polar and lipid extraction after NMR-based metabolomics analysis, in terms of the number of annotated metabolites, reproducibility, and the amount of sample needed. While for the liver sample, the two-step extraction involving CHCl₃/MeOH followed by MeOH/H₂O was recommended. In this method, resuspension of dried lipid extracts was used for lipidomics, and polar extracts were transferred for further untargeted metabolic profiles by UHPLC-Q-Orbitrap MS following an NMR-based metabolomics study. Finally, the proposed preparation protocols were evaluated for robustness, and the identification data were used to generate a comprehensive metabolic map for plasma and liver tissue. In summary, this study provides a unique sample preparation procedure for two biological specimens, allowing for multi-platform analysis using a single sample. By adopting this approach, comprehensive metabolic profiling can be conducted to detect metabolic alterations under different physiological or pathological conditions.