Investiga The Correlation Of Metabolites In Human Serum And Urine With Two Cancers Using UPLC-MS/MS

In this thesis,quantitative analysis of metabolites in human blood and urine is performed by UPLC-MS/MS.It was demonstrated that the content of metabolites between normal people and cancer patients is obviously different.It is very important for early diagnosis,reducing the psychological pain and easing the financial stress of patients.Firstly,the method for the determination of a biomarker(8-OHdG)of DNA oxidative damage in human urine by UPLC-MS/MS was established.The urine samples were purified by HLB solid-phase extraction column and analyzed by positive ion mode electrospray ionization and multiple reaction monitoring mode.The linear range of 8-OHdG was 1-300 nmol/L.And the LOD and LOQ was 0.2 and 0.6 nmol/L,respectively.The intra-day precision was 0.7%-1.1%.The inter-day precision was 0.5%-0.7%.The recovery of 8-OHdG was between 99.8-111.4%.The 8-OHdG levels in the urine of 65 normal subjects(21 smokers and 44 non-smokers)and 61 patients with colorectal cancer(17 smokers and 44 non-smokers)were determined by the proposed method.The 8-OHdG content of smokers and patients with colorectal cancer were significantly higher than that of the control group.Secondly,the serum samples were pretreated using a protein precipitation method.The quantitive analysis of GCA in serum was performed by a UPLC-MS/MS method with negative ion mode ESI in a multiple reaction mode.The calibration curve coverage range of GCA ranged from 0.2 to 400 ng/ml.LOD and LOQ were 0.01 ng/mL and 0.05 ng/mL,respectively.The intra-day(2.3-6.1%)and inter-day(2.4-4.6%)precisin was examined.The recovery was 103.7-114.3%.The established method has been successfully applied to the measurement of serum GCA in 32 healthy volunteers,10 hepatocellular carcinoma patients and 14 other cancer patients.GCA levels in serum were higher in patients with hepatocellular carcinoma,and there was no significant difference in GCA between healthy controls and other cancer groups.Finally,the analytical method for the simultaneous determination of DNA,RNA methylation and hydroxymethylation metabolites: 5-methyl-2'-deoxycytidine(5-mdC),5-hydroxymethyl-2'-deoxycytidine(5-hmdC),5-methylcytidine(5-mrC),5-hydroxymethylcytidine(5-hmrC)in human urine were developed.The four cytidine metabolites in urine were enriched and purified by MCX solid phase extraction column,then the types of additives in UPLC mobile phase were optimized.0.06 mmol/L malic acid was used as a mobile phase additive.And signal for 5-hmrC was increased by 9.4 times.This is the first time that 5-hmrC has been detected and quantified in human urine.The linear range was 0.1-100.0 nmol/L.The intra-day precision was 0.5%-2.6%,and the inter-day precision varied in the range of 0.2%-1.4%.The measured recoveries ranged from 91.9% to 115.8%.Finally,the urine samples of 90 patients with colorectal cancer and 90 healthy volunteers were measured.Compared with healthy controls,the levels of 5-mdC,5-hmdC,5-mrC and 5-hmrC were significantly reduced in patients with colorectal cancer.