Biomarkers Study Of Errors Of Metabolism By Coupling Chromatography With MS

Disease diagnosis and health status evaluation by quantification ofbiomarkers has it own advantage: objectivity and reliability. Using biomarkeralso makes study of therapy strategy more convenient. In this thesis,information of biomarkers in gout, hyperuricemia and methylmalonic aciduriapatients was studied by newly developed analytical methods.A specific, simple and inexpensive HPLC-UV-MS/MS method wasestablished to analyze 10 purine compounds in serum. Reverse-phase HPLCwas combined with electrospray ionization (ESI), tandem mass spectrometry.Peaks were identified by auto MS2, all quantifications were done by UV at254nm. LOD of 10 compounds are among 0.132-0.317μmol/L. Theintraassay CVs of all compounds were below 10.9% and the interassay CVswere below 24.8%. The recoveries were between 84.5–98.4%. Serum of goutpatients, hyperuricemia patients and healthy people were analyzed by thismethod. The result showed that uric acid, xanthine and hypoxanthine in bothtwo kinds of patients have significant difference compared with healthypeople (p<0.001). Hypoxanthine (p<0.001) and xanthine (p<0.01) also havesignificant difference between two kinds of patients. Serum concentration ofuric acid, xanthien and hypoxanthine of two kinds of patients decreased afterallopurinol therapy. Although the difference was reduced, hypoxanthine(p<0.01) still has significant difference between two kinds of patients. Aconclusion was made that hypoxanthine and xanthine were good biomarkersto separate two kinds of patients and healthy people. Another HPLC fingerprint method using PCA and CHA was established to classify two kinds ofpatients and healthy people. This method takes more information of serumcompounds in consideration and reflects the status of disease morecomprehensively.As one of the diseases with most frequency, methylmalonic aciduria isstudied using GC/MS and ESI/MS/MS method. Information of almost allurinary metabolites can be obtained by GC/MS using urease pretreatment andstable isotpe dilution. We also develop a way to analyze a urine sample in 2minutes by ESI/MS/MS. Both the two methods have good recovery. Thecombination of the two methods can not only be used for high-risk screening,but also for newborn screening. The concentration of methylmalonic acid inmethylmalonic aciduria patients was about 5-10 times of healthy people. Themethod can also be used to value the therapy effect by analyzingmethylmalonic acid in a series.