Investigation Of Metabolic Disorder In Fish Induced By Typical Fluorine-containing Pollutants Using Internal Extractive Electrospray Ionization Mass Spectrometry

Synthetical fluorine-containing compounds play an indispensable role in human life and production.With the continuous increase of human demand,the production and usage of fluorine-containing compounds（such as perfluorinated compounds and fluorine-containing pesticides）have gradually increased,posing a great threat to human health and the ecological environment.In recent years,related studies have reported the metabolic effects of fluorine-containing compounds on organisms,revealing their toxic effects on organisms at the molecular level,which contributes to a better understanding of the potential hazards caused by fluorine-containing compounds.Due to the high sensitivity and stability in analyzing endogenous metabolites in biological samples,chromatography tandem mass spectrometry has been widely used to explore the metabolic effects of environmental pollutants on organisms.However,a series of pretreatment operations are required in biological sample analysis by using chromatography tandem mass spectrometry,which is not only time-consuming and laborious,but also may lead to the loss of active components in the sample,making the research results questionable.Internal extractive electrospray ionization mass spectrometry（i EESI-MS）is a typical ambient mass spectrometry technique that can quickly and directly acquire molecular information inside tissue samples without any sample pretreatment.In this study,based on i EESI-MS,the analytical method for direct endogenous metabolites detection in zebrafish and tilapia tissue samples was established and applied to explore the metabolic effects of typical fluorine-containing pesticides and perfluorinated compounds on fish.The main content of this paper includes the following two parts:（1）Investigating metabolic effects of fipronil and its derivatives on zebrafish based on i EESI-MS.Firstly,established the i EESI-MS method for untargeted metabolites analysis in the zebrafish liver sample through optimization experiments of ionization voltage and extraction solution flow rate.i EESI-MS analysis results showed that a variety of metabolites can be directly detected in zebrafish liver,including small metabolites,lysophosphatidylcholine（Lyso PC）,phosphatidylcholine（PC）etc.Finally,based on multivariate statistical analysis of i EESI-MS data obtained from mixed（fipronil and its three derivatives）exposure group,fipronil exposure group and control group,22 metabolites were employed as differential metabolites compared exposed groups with control group,including PC（34:1）,PC（40:8）,Lyso PC（16:0）etc.Moreover,by comparing the signal intensities of PCs,it was found that the mixed exposure leads to more obvious metabolic perturbation on 11 PCs in the liver of zebrafish than fipronil exposure.The above results showed that both fipronil exposure and mixed exposure can lead to metabolic disorders in zebrafish liver,and the mixed exposure showed more significant metabolic disturbance.（2）Exploring the effects of perfluorooctanoic acid（PFOA）on phospholipid metabolism in tilapia by using i EESI-MS.The i EESI-MS method was established for phospholipid species detection in tilapia tissue sample through extraction solution optimization experiment.i EESI-MS analysis results showed that i EESI-MS has a good performance on detecting seven categories phospholipid species in the liver and spleen tissues of tilapia by respectively using CH₃OH/H₂O（80:20,v/v）and CH₃OH/H₂O/NH₄OH（80:20:0.3,v/v/w）as extraction solution in the positive and negative ion detection model,including phosphatidyl cholines（PCs）,sphingomyelins（SMs）,phosphatidic acids（PAs）,phosphatidyl ethanolamines（PEs）,phosphatidyl glycerols（PGs）,phosphatidyl inositols（PIs）and phosphatidyl serines（PSs）.The results of multivariate statistical analysis of i EESI-MS data among high-concentration PFOA exposure group,low-concentration PFOA exposure group and control group showed that:20 and 18 phospholipid molecules were respectively considered as differential phospholipids in the liver and spleen of tilapia compared PFOA exposed groups with control group.Metabolic pathway analysis showed that PFOA exposure leads to significantly disorder in glycerophospholipid metabolic pathway in tilapia,indicating the potential harm of PFOA to fish.