| ObjectiveTarget-based metabolomics, the measurement of selected metabolites, is a valuable tool for assessing metabolic changes and providing both diagnostic and prognostic information. Unlike untargeted metabolomics, which is an intended comprehensive analysis of the entire set of chemical compounds in a sample, targeted metabolomics has a high sensitivity of quantitating selected metabolites. Thus, analysis of metabolites at low concentration and overlapping regions are well suited for this analysis. More importantly, such a targeted metabolomics platform might shed new light on the unexpected regulatory systems that associated with various diseases. In our study, targeted metabolomics analysis by ultra performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) was developed and validated. The methods were successfully applied to the analysis of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced Crohn’s disease rat model, to find out the time dependent alterations of the selected metabolites and the role of the interplaying networks. Moreover, it can provide both clinical, diagnostic and prognostic information of inherited metabolic and nutritional disorders.Methods1. Simultaneous determination of three aromatic amino acids (AAAs) and their four gut microbiota co-metabolites in rat plasma and urine by HPLC-MS-MSA HPLC-MS-MS method to determine three aromatic amino acids (AAAs) L-Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and their four gut microbiota co-metabolites Indole (I), p-Cresol (PC), Indoxyl sulfate (ILS) and p-Cresyl sulfate (PCS) in rat plasma and urine was developed. Then, chromatographic and mass spectrometric conditions were optimized. The linearity and sensitivity, precision, accuracy and recovery and stability were investigated.2. Simultaneous determination of three monoamine neurotransmitters, eight purines and pyrimidines and twenty amino acids in rat urine using hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-MS-MS)We have developed in the previous study quantitative method utilizing HILIC-MS/MS to detect 24 amino acids, four neurotransmitters and 8 purines and pyrimidines in rat serum and brain tissue. The developed method preformatted high throughput, highly selective and sensitivity. But it cannot be applied to urine samples. Therefore, A HILIC-MS-MS method to determine here monoamine neurotransmitters, eight purines and pyrimidines and twenty amino acids in rat urine was developed. Calibration curves of all 31 analytes in this study were prepared in acetonitrile. The analytical response differences between the urine and acetonitrile were evaluated by a recovery assessment to ensure that the calibration curve constructed in acetonitrile could be applied to quantify the biological samples. The recovery was also investigated.3. Development of a Crohn’s disease (CD) rat model induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) and application of the developed target-based metabolomics methods to the CD rat modelTNBS/ethanol was used as modeling reagent to establish CD rat model. And the developed target-based metabolomics methods were applied to analyze the real biological samples from CD model rats.Results1. A HPLC-MS-MS method to determine three aromatic amino acids (AAAs) L-Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and their four gut microbiota co-metabolites Indole (I), p-Cresol (PC), Indoxyl sulfate (ILS) and p-Cresyl sulfate (PCS) in rat plasma and urine was developed. The linearity, precision, accuracy and recovery and stability of the method were satisfactory. The method is sensitive, selectivity and high through-put.2. A HILIC-MS-MS method to determine three monoamine neurotransmitters, eight purines and pyrimidines and twenty amino acids in rat urine was developed. Recovery assessment and recovery study were satisfactory.3. Crohn’s disease (CD) rat model induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) was successfully developed.We have generated complex metabolite data and analyzed the data by multivariate statistical methods. We have found the change in biological matrix levels of the metabolites. Biomarkers have also found by the analysis.Conclusion:Targeted metabolomics analysis by ultra performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS) was developed and validated. The methods were successfully applied to the analysis of CD rat model. We have found out the biomarkers and time dependent alterations of the selected metabolites. |
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