| Cancer is one of fatal danger for people's life. Early detection has an important role in the treatment of cancer, for it can reveal predictive information for cancer. Xanthopterin, isoxanthopterin, pterin, neopterin, biopterin, pterin-6-carboxylic, etc., all belong to the pteridine family, which are well distributed in nature in living organisms. Since pteridines are very important cofactors in the process of cell metabolism, the levels of which have significant importance in clinical diagnosis. Among them, due to neopterin has been classical considered to be the most important pteridine cancer marker, most of works are focused on neopterin detection, however, the study about other pteridines were still sparse.In our study, three new methods were developed for the simultaneous determination of xanthopterin and isoxanthopterin in human urine by chromatography analytical technique. Choosing one of analytical methods was applied to determine xanthopterin and isoxanthopterin in many of healthy and cancer urine samples. The T test results displayed that the average levels of isoxanthopterin and xanthopterin were significantly elevated in urine samples of cancer patients, compare with healthy people. Meanwhile, a new assay of identifying urine was developed based on fingerprint of three-dimensional fluorescence spectrum. The technique of principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to differentiate and evaluate the character of urines' three-dimensional Fluorescence spectra. In general, main research results as follows:1. The high-performance liquid chromatography (HPLC) with fluorescence detection method was developed for the determination of xanthopterin and isoxanthopterin in human urine. The mobile phase was phosphate buffer (pH=7.5)-methanol (98:2, v:v), the flow rate was 1.0 mL/min, the excitation and emission wavelengths were 345 nm and 420 nm, respectively. The linear ranges of xanthopterin and isoxanthopterin were 0.0013μg/mL～0.945μg/mL and 0.00017μg/mL～0.118μg/mL, respectively. The linear regression equations were y=3.0×106x+28959 (r=0.9999) and y=1.0×108x-162133 (r=0.9996), respectively. The detection limits were 0.5 ng/mL and 0.05 ng/mL, respectively. For xanthopterin and isoxanthopterin in human urine, the recoveries were in the range of 83.9%～106.0%. The proposed method was successfully applied to the determination of xanthopterin and isoxanthopterin in human urine.2. A novel method was developed for the simultaneous determination of xanthopterin and isoxanthopterin in human urine using liquid chromatography-mass spectrometry. Urine samples were centrifuged and filtered, then analyzed by electrospray ionization tandem mass spectrometry. The linear ranges of xanthopterin and isoxanthopterin were 0.0204μg/mL～2.04μg/mL and 0.0202μg/mL～2.02μg/mL, respectively. The linear regression equations were y=342622x-7848 (r2=0.9995) and y=309694x-3573 (r2=0.9997), respectively. The detection limits were 0.007μg/mL and 0.006μg/mL, respectively. For xanthopterin and isoxanthopterin in human urine, the recoveries were in the range of 88.0%～110.0% The proposed method was successfully applied to the determination of xanthopterin and isoxanthopterin in human urine with satisfactory result.3. A sensitive, simple and practical method for the determination of isoxanthopterin in human urine by high-performance anion-exchange chromatography with integrated pulsed amperometric detector has been developed. The double-column solid phase extraction was employed to purify isoxanthopterin from human urine. The seperation of isoxanthopterin from other substances was carried on IonPac(?) AS21 anion-exchange column with mobile phase of NaOH (0.10 mol/L)-H2O (20:80, v:v) at the flow rate of 0.40 mL/min. Under optimized condition, the detection limit (S/N=3) for isoxanthopterin was 3μg/L, and the linear range was 0.005μg/mL～0.200μg/mL. The spiked recoveries at the concentration of 2.00μg/mL and 5.00μg/mL were 95.4%～96.8%.4. The levels of two pteridines were measured in 34 randomly selected urine samples from healthy persons,29 gastric cancers,14 kinds of cancer patients. It was found that the average levels of xanthopterin and isoxanthopterin were significantly elevated in cancer patients by T tests and may be important factors for the diagnosis of cancer and pre-cancer screening, and they are probably to be the potential makers for cancers. 5. A new assay of identifying urine was developed based on fingerprint of three-dimensional Fluorescence spectrum, and 51 urine samples from 24 gastric cancer patients and 27 healthy people were measured. The techniques of principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to differentiate and evaluate the character of urines'three- dimensional fluorescence spectra. The results showed that there are some differences in urine from patients and healthy people, and the PCA and HCA can be of better separation of samples for identification. Thereby the method could provide a certain basis for the clinical diagnosis.
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