Study On The Detection Of Metabolites And Drug Concentrations In Hepatocellular Carcinoma Blood

The hepatocellular carcinoma（HCC）with high incidence rate and low survival rate has been a common health problem of common concern in various countries.Early diagnosis with biomarkers and advanced prognosis are important means to solve this prblem.In the early stage of liver cancer,the traditional hepatocellular carcinoma biomarkers are not specific,and the method of diagnosis of markers is time-consuming and complicated,in the middle and late stages of liver cancer,the overuse of anti-rejection drugs can cause adverse drug reactions due to individual differences.It is important to develop a rapid,simple,accurate,and reliable method for the rapid detection of small molecule metabolites and liver transplantation drug concentrations in a large number of HCC blood samples.According to the requirements of clinical tests,a fast pretreatment method was selected to optimize the purification methods such as Qu ECh ERS,solid phase extraction,and solid phase support liquid-liquid extraction,and then combined with the optimized LC and LC-MS/MS triple quadrupole mass spectrometry techniques,The detection of liver transplant drugs in blood and the qualitative and quantitative detection of small molecule metabolites were established to achieve the simultaneous rapid detection of multiple components.The main contents and results of this study are as follows:1、A method to determinate 3 small-molecule metabolites in HCC blood sample simultaneously was established,it was developed by Qu ECh ERS coupled with LC-MS.Serum sample was extracted by acetonitrile,salted out by anhydrous sodium sulfate,purified with PSA,then analyzed by LC-MS.The result shows that the matrix effect of three small molecule metabolites is between 0.79 and 1.55,it has a good linear relationship in the range of 10～100 ng·m L^-1,with correlation coefficient more than 0.9917,the method recovery is between 77.5%and 105.1%in three spike levels with the relative standard deviation（n=5）less than 15%,the limits of detection or quantification are between 0.3 and 0.6 ng·m L^-1and 1 and 2 ng·m L^-1,respectively.It is a simple,accurate and reliable method,good for rapid detection of small-molecule metabolites in HCC blood sample of large quantity.2、A method to determine 6 small-molecule metabolites in HCC blood sample simultaneously was developed by solid phase extraction coupled with LC-MS.Serum sample was precipitated with 0.1 mol·L^-1Zn SO4 solution,the supernatant is mixed with acetonitrile of three times the volume,passed through solid phase extraction column,then analyzed by LC-MS.The result shows that the matrix effect of the 6 small-molecule metabolites is between 0.81 and 1.22,it has a good linear relationship with correlation coefficients more than 0.99904,the method recovery is between 76.7%and 116.8%in three spike levels with the relative standard deviation（n=6）less than 15%,the limits of detection and quantification are between 0.1 and 1.2 ng·m L^-1and 0.3 and 4.0 ng·m L^-1,respectively.This method is simple,fast,accurate and reliable,suitable for large-scale clinical detection.3、The method for the determination of mycophenolate mofetil and its metabolites by HPLC-diode array detector combined with solid supported liquid-liquid extraction was developed.The sample was precipitated with 0.1 mol·L^-1Zn SO4 solution,the supernatant was diluted with 0.1%formic acid water solution（V/V）of equal volume after passing through solid supported liquid-liquid extraction column and nitrogen blowing,it was reconstituted in methanol and then analyzed by HPLC.Matrix effects of MPAG、MMF and MPA are between 0.98～0.99,the linear relationship between MPAG,MMF,and MPA was good in the range of 1～40μg·m L^-1,the correlation coefficient is more than 0.9958,the recovery of the three levels（n=6）was between 87.0%～107.3%with the relative standard deviation less than 10%,the limits of detection and limits of quantification were between 0.1～0.3μg·m L^-1and 0.3～1μg·m L^-1.This method is sensitive,fast,accurate and convenient,and can be used to detect biological samples of large quantities.The established analytical methods not only the detection efficiency and accuracy will be improved,but also the application range of pretreatment technology is expanded,which will provide reference to detections of other small-molecule metabolites.