| An ultra performance liquid chromatography-mass spectrometry (UPLC/MS) methodwas developed for the metabonomics study of effect of acarbose on type Ⅱ diabete rats.Urine from three groups of rats were analyzed，including control group，model group andtype Ⅱ diabete rats treated with acarbose group，and the data were analyzed by the method ofprincipal component analysis (PCA) and partial least squares discriminant analysis(PLS-DA)．The change of urine metabolic profiling was analyzed and some potentialbiomarkers about type Ⅱ diabete were found and identified according to the multi-stagemass spectrum information. The UPLC/MS/MS Method was established for thedetermination of some potential biomarkers in urine samples. The pathway about thesepothential biomakers have been analyzed. New methods based on metabonomics to diagnoseearly type Ⅱ diabete and screen drugs were established.Firstly, high fat and sugar diet plus STZ-induced type Ⅱ diabetes in rats as the researchobject, the urine of control group，model group and type Ⅱ diabete rats treated with acarbosegroup was detected by UPLC/MS/MS to generate metabolic fingerprints. The LC/MS/MSdata was imported SEIVE software, after the peak extraction, peak matching, and datapreprocessing, the detected compounds were analyzed by PCA and PLS-DA with SIMCA-Psoftware. The PCA score plot showed that the three groups were significantly differentbetween the metabolic profile. According to the results of PLS-DA,16kinds of endogenousmetabolites were identified as potential biomarkers，which satisfied the condition of VIP>1and P value﹤0.05in PLS-DA loading plot. The LC/MS/MS data information of thebiomakers were matched in Human Metabolome Database (HMDB) or Mass Bank. Jp, andthe structure of9kinds of the endogenous metabolites was initially identified. The structureof four biomakers, including glucose acid, creatinine, phenylacetylglycine and hippuric acidwere determined by using standard article of tandem mass spectrum information verification.The metabolic pathways of the preliminary identification biomarkers were describedaccording to relevant literature.The UPLC/MS/MS methods were established for the determination of glucose acid,creatinine, phenylacetylglycine and hippuric acid in rat urine. Methods: An ACQUITY DEH -C18(2.7×50mm，1.7μm）was used with the mobile phase of acetonitrile (containing0.1%formic acid) and ultra pure water (containing0.1%formic acid). Creatinine, phenylace-tylglycine and hippuric acid used positive ions mode and N,N-Dimethyl-L-phenylalanineacid was used as internal standard. Glucose acid used negative ion mode with5-fluorouracilas internal standard. Multiple reaction monitoring (MRM) mode was employed, and thetransition of m/z was m/z114.0→86.1for creatinine, m/z194.1→91.0forphenylacetylglycine, m/z180.0→105.0for hippuric acid, m/z194.1→148.1for N,N-dimethyl-L-phenylethylamine acid, m/z194.9→128.8for D-glucose acid and m/z129.0→42.0for5-fluorouracil. Results: The calibration curves of creatinine, phenylacetylglycineand hippuric acid were linear in the range of0.01~10.00μg·mL-1and glucose acid was inthe range of0.01~30μg·mL-1. RSDs of the inter-day and intra-day precisions of low, mediumand high concentrations were all less than15%. Conclusion: The method is sensitive,specific,repeatable and accurate for the determination of glucose acid, creatinine,phenylacetylglycine and hippuric acid in rat urine. The results of determination ofbiomarkers in the three group showed that the trend of quantitative data was consistent withthe half quantitative data with LTQ. The changes of concentration between health group andmodel group had significant difference, which were glucose acid (p <0.001), creatinine (p <0.001), phenylacetylglycine (p <0.001) and hippuric acid (p <0.05) in rat urine.The changeof phenylacetylglycine (p <0.001) between model group and type Ⅱ diabete rats treated withacarbose group had significant difference in rat urine. The study results provide the scientificbasis for pharmacology and clinical research of the treatment of typeⅡdiabetes withacarbose. |
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