Research on Metabolomics Analysis Method for Fish Gills Target Organs Based on UPLC£­QTOF MS

KeyWord:metabolomics  sample pretreatment  fish  gill  ultra-high performance liquid chromatography£­quadrupole time-of-flight mass spectrometry£¨UPLC£­QTOF MS£©

Abstract:

      In view of the lack of accurate and efficient high-throughput detection techniques in metabolomics research of gill target organs in fish£¬an analysis method based on ultra-high performance liquid chromatography£­quadrupole time-of-flight mass spectrometry£¨UPLC£­QTOF MS£© was established and optimized£®In order to improve the extraction effect of metabolites in the sample£¬the total peak area£¬number of metabolites£¬relative standard deviations£¨RSDs£© of relative peak area£¬and response values of glucose£¬L-leucine£¬citric acid£¬and other three representative endogenous metabolites with different species and polarities were used as indicators£¬and the effects of different experimental conditions on the extraction coverage and stability of metabolites in gill tissue were investigated£®The results showed that the best extraction effect could be obtained by adding pre-cooled methanol£­water£¨4¡Ã1£© at a sample£­reagent ratio£¨mg¡Ã¦ÌL£© of 1¡Ã16 to the gill tissue ground at low temperature£¬and holding for 30 min at £­20 ¡æ£¬then centrifuged to extract gill tissue samples£®Meanwhile£¬the chromatographic and mass spectrometry parameters in the data acquisition process were optimized£¬further improving the separation effect and signal intensity of metabolites£®Methodological investigations were carried out by using quality control£¨QC£© samples interspersed in the assay sequence£®The results showed that this metabolomics analysis posessed good instrumental precision£¬method precision£¬and sample stability£®The RSDs for the relative peak areas of the six representative metabolites with different polarities were lower than 10%£¬which meets the requirements for metabolomics research of fish gill target organs£®The results provide an technical support for the metabolomic study of fish gill target organs£®