Evaluation of two-step liquid-liquid extraction protocol for untargeted metabolic profiling of serum samples to achieve broader metabolome coverage by UPLC-Q-TOF-MS

Untargeted metabolomics studies aim to extract a broad coverage of metabolites from biological samples, which largely depends on the sample preparation protocols used for metabolite extraction. The aim of this study was to evaluate a comprehensive sample pretreatment strategy using two-step liquid-liquid extraction to achieve broader metabolome coverage by ultra-high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (UPLC-Q-TOF-MS). We compared four protocols: (A) methanol protein precipitation, (B) Ostro 96-well plates, (C) two-step extraction protocol of CHCL3-MeOH followed by MeOH-H2O, and (D) two-step extraction protocol of CH2CL2-MeOH followed by MeOH-H2O. The number of extracted features, reproducibility and recovery were the major criteria for evaluation. Our results demonstrated that Protocols B, C and D, with approximately similar number of features, extracted more features than Protocol A. Protocols C and D appeared to have similar extraction reproducibility (low coefficient of variation < 30%) and Protocol D enabled an acceptable recovery of serum metabolites. The two-step extraction Protocol D (CH2CL2-MeOH followed by MeOH-H2O) resulted in the greatest improvement in metabolite coverage, satisfactory extraction reproducibility, acceptable recovery and environmental safety. The selected protocol was applied to an obesity metabolomics study to obtain different metabolites between participants with obesity and the controls, and to investigate complex metabolic alterations in obesity during a 2-h oral glucose-tolerance test. Our results suggested that this protocol was useful for analyzing serum metabolome changes in obese individuals in the fasting and postprandial state.