Targeted Metabolomics Analysis Based On Liquid Chromatography–Tandem Mass Spectrometry And The Application In Obesity Rat Model

ObjectiveEndogenous metabolites play an important role in maintaining the balance of organisms,and their concentration changes are often closely related to various diseases.Developing the targeted metabolomics analytical platform for these metabolites and monitoring their dynamic changes will help to elucidate the relationship between metabolic variations and diseases.Here,we developed the targeted metabolomics analysis of multiple important metabolites in different metabolic pathways by high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）.Moreover,the methods were applied to monitor the global changes of these metabolites in obesity.This study aimed to establish a reliable and practical targeted metabolomics analytical platform for endogenous metabolites,which would be expected to be a valuable tool for clinical researches.Methods1.Targeted metabolomics analysis of nine gut microbiota-host co-metabolites in rat serum,urine and feces by LC-MS/MSA novel LC-MS/MS method for the analysis of nine gut microbiota-host co-metabolites in rat serum,urine and feces was developed and validated.The compounds were gradient eluted on a reversed-phase C₁₈ column with a solvent system consisting of methanol and water（containing 0.05%formic acid）in 7 min running time.The validated parameters were linearity,precision,accuracy,extraction recovery,matrix effect,and stability.In addition,this method was applied to monitor the dynamic changes of these nine co-metabolites in the obesity rats induced by high-fat diet.2.Targeted metabolomics for simultaneous determination of eighteen bile acids in rat serum and feces by LC-MS/MSBased on LC-MS/MS,a simple and fast method for the targeted metabolomics analysis of 18 bile acids in biological samples was developed.The developed method enabled the rapid quantification of 18 bile acids within 10 min.The compounds were separated on a Kinetex Phenyl-Hexyl column with a mobile phase consisting of acetonitrile and water（containing 10 mmol/L ammonium acetate and 0.05%formic acid）.Serum and feces were extracted by protein precipitation.After method validation,this method was applied in obese rats to investigate the reliability and practicability.In the process,the dynamic changes of bile acids in obese rats could be depicted.3.Targeted metabolomics for the quantitative measurement of 12 carbohydrate metabolites in rat serum,urine and feces by LC-MS/MSA targeted metabolomics approach was developed to simultaneous determine 12carbohydrate metabolites in biological samples.In the method,the 12 targeted metabolites were chromatographically separated on the ACQUITY BEH Amide column by gradient elution with a solvent system consisting of acetonitrile and water（containing 0.025%NH₄OH）,and the run time was 6 min.Linearity,precision,accuracy,extraction recovery,matrix effect,and stability were investigated.Finally,the concentration of these metabolites in the biological samples of obese rats was quantitatively measured by the developed method.Results1.A targeted metabolomics analysis of 9 gut microbiota-host co-metabolites in rat serum,urine and feces by LC-MS/MS was developed.The method is precise,accurate and reliable.With the simple protein precipitation procedure and rapid LC–MS/MS quantification,the method is suitable for high-throughput quantitative analysis.Moreover,this method was successfully applied in obesity model.It was found that gut microbiota-host co-metabolism markedly changed in obese rats,and the changes increased with the increase of high-fat diet feeding time.2.An original LC-MS/MS method for simultaneous determination of 18 bile acids in rats serum and feces was developed and validated.Linearity,lower limit of quantification,precision,accuracy,extraction recovery,matrix effect and stability met the requirements of biological sample analysis.This approach was successfully applied to monitor the concentration variation of bile acids in obese rats,and the results showed that the constituents of the model group on the last day of acclimatization were completely separated from the last day of 4,8 and 12 weeks after high-fat diet induced.3.A targeted metabolomics approach based on LC-MS/MS for quantitatively determination of 12 carbohydrate metabolites was developed.In the method,all of the calibration curves exhibited good linear relationships.With high sensitivity,accuracy and precision,this method is suitable for quantitative analysis of these metabolites in biological samples.Additionally,this method was successfully applied in the dynamic monitoring of targeted metabolites in obese rats model.The results showed that the metabolic trajectories of rats diverged from their initial metabolic position after 12-week high-fat diet induced,and these metabolic changes might be closely related to obesity and its complications.ConclusionThrough the development of three targeted metabolomics analysis,the gut microbiota-host metabolites,bile acids and carbohydrate metabolites could be quantitatively measured and monitored dynamic changes in obese rats.The targeted metabolomics analytical platform for endogenous metabolites will provide a more comprehensive understanding of the metabolic variation in diseases,and may become an important tool in the diagnosis,treatment evaluation and drug development of diseases.