Optimization of Pretreatment Methods for Cerebrospinal Fluid Metabolomics Based on Ultrahigh Performance Liquid Chromatography/mass Spectrometry

Sample pretreatment of cerebrospinal fluid (CSF) in metabolomics plays an important role in metabolic profiling study, especially for samples related to central nervous system diseases. However, there is few study about optimization of CSF metabolomics pretreatment. Therefore, it is an urgent need to optimize CSF pretreatment in order to promote the extraction efficiency of metabolites. In this study, CSF samples were separately subjected to nine different protein precipitation solvents and five different reconstitution solvents to establish the most effective pretreatment method before hydrophilic interaction (HILIC) and reverse-phase (RP) ultrahigh performance liquid chromatography mass spectrometry (UPLC/MS) analysis. The optimal conditions for different sample pretreatment methods were analyzed based on coverage (number of detected potential metabolites), stability (the relative standard deviation (RSD) distribution of metabolites) and the reproducibility of the data. Our results suggested that using EtOH or MeOH-EtOH-ACN (1:1:1, v/v/v) as the protein precipitation solvents and H2O-MeOH-ACN (2:1:1, v/v/v) as the reconstitution solvent were optimal methods for T3 column analysis. For HILIC column analysis, using EtOH to precipitate protein and H2O-MeOH-ACN (2:1:1, v/v/v) to reconstitute or MeOH to precipitate and 5 %ACN to reconstitute performed best. This developed UPLC/MS pretreatment method could provide better protein precipitation solvents and reconstitution solvents for global CSF metabolic analysis, potentially facilitating the comprehensive understanding of many central nervous system diseases.