| Objective: Using Ultrafast Liquid Chromatography–Ion Trap–Time of Flight Tandem Mass Spectrometry(UFLC-IT-TOF MS)technology to establish a new method for the analysis of small molecule metabolites in serum of patients with liver cancer,which is applied to the determination of serum metabolites in different stages in HBV infected patients and healthy volunteers.It can analysis the differences and variation,explore the metabolic characteristics,which is to reveal the pathogenesis to provide reference information of metabolism.Method:1 Using UFLC-IT-TOF MS technology to establish a new methodSerum samples were collected from 80 patients with primary hepatocellular carcinoma and 20 healthy volunteers as the control group in the same period.Fasting peripheral venous blood was collected and placed in a disposable blood sampling tube,and was centrifuged.Then the blood was sealed in a 1.5 ml EP tube,marked,and frozen by keeping in a –80 ?refrigerator.According to the corresponding values of the mass spectra of the standard substance to determine the ion mode of each substance detection,which was used in comparison of different pretreatment methods and methodological investigation.2-Chlorophenylalanine was prepared into 0.1mg/ml as internal standard solution.The ratio of the volume of solvent to serum and the temperature were optimized..Observation of acetonitrile and methanol two mobile phase,comparison of the mobile phase effect of adding0.2% formic acid or acetic acid on the shape and the ionization efficiency,which was to determine the appropriate flow rate and column temperature.Amixed standard solution was added to the mixed serum samples of the patients with hepatocellular carcinoma,and the precision,stability and reliability of the samples were examined according to the above treatment and the conditions of the instrument.Examination of the samples and data analysis2 Analysis of metabolic marker with potential clinical application valueRandomly selected 197 cases in different stages of HBV infection.In addition,58 cases of healthy volunteers were selected as healthy control group,255 participants were selected.The diagnostic criteria,each sample serum collection method was the same as the previous.Every serum sample of255 patients was thawed at room temperature before experiments.The best solution obtained by the above steps was detected by UFLC-IT-TOF MS technique,then the components and contents of the corresponding compounds were obtained.IBM SPSS 21 software was used to analyze the data.Using ROC curve,AUC value calculation,to get the best definition of sensitivity and specificity.Result:1 Optimization of the detection methodThe volume ratio of precipitant to serum in the organic solvent protein precipitation method was optimized.2 We got the fingerprint of metabolites,which was further screened potential metabolic biomarkers in hepatocellular carcinoma patients,including glycocholic acid,glycerol phosphate,fourteen amide,acetyl phenylalanine,oleamide,acetyl carnitine,lysophosphatidylcholine,glycochenodeoxycholic acid.3 The serum of 255 patients were detected by using the above method,with 4 potential metabolic markers were found,including the contents of lysophosphatidylcholine,oleamide,acetyl-L-carnitine,and glycocholic acid.Conclusion:1 A method based on the UFLC-IT-TOF MS technique was established to study the serum pretreatment of patients with liver cancer,which can satisfy the high throughput requirement of metabonomics,and is suitable formetabonomics research with large batches of biosamples.2 Lysophosphatidylcholine,acetyl-L-carnitine and oleamide were indentified as potential metabolic biomarkers of hepatocellular carcinoma.The levels of Lysophosphatidylcholine in the blood of the patients with liver cancer,compared to other groups increased significantly.Compared with other2 metabolites,Lysophosphatidylcholine has the maximum AUC value,the best diagnostic performance,which can provide reference information for clinical and scientific research. |
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