| Domestic and international studies for many years suggest that pteridines and amino acids play important roles in metabolic process, the levels of which have significant importance in clinical diagnosis and monitoring of various diseases including cancer. In this paper, the author reviewed pteridines and amino acids through sample pretreatment technologies, analysis methods and clinical applications. We established new methods to measure the concentrations of pteridines and amino acids in biological samples, and compared the experiment results, the experiments laid a solid foundation for further study of cancer pathogenesis and finding new tumor markers. In general, main research results are as follows:1. The determination of pteridines in biological samples by HPLC-FLD was studied. the mobile phase was H2O-CH3OH (90:10, v/v), the flow rate was 1.0 mL/min, the excitation and emission wavelengths were 360 nm and 445 nm, respectively. The linear ranges of neopterin, biopterin, pterin and sepiapterin were 0.005～1.50μg/mL, 0.05～2.00μg/mL,0.01～1.00μg/mL and 0.3～10.0μg/mL, respectively. The limits of detection were 0.0030μg/mL,0.0020μg/mL,0.0010μg/mL and 0.15μg/mL, respectively. The urine was oxidised by I2-KI under acidic condition for 30 min and mixed with ascorbic acid, then the sample was diluted 5 times, filtered by 0.45μm membrane and analyzed by HPLC-FLD. Cell cultures were filtered by 0.45μm membrane and analyzed directly. The average recoveries were in the range of 84.8～120.0%, with relative standard deviations equal to or lower than 7.34%. The concentrations of neopterin, biopterin, pterin and sepiapterin in biological samples were determined and compared, which showed satisfactory results.2. The method for the determination of tyrosine and tryptophan in human urine by HPLC-FLD was presented. The mobile phase was H2O- CH3CN (80:20, v/v), the flow rate was 1.0 mL/min, the excitation and emission wavelengths were 260 nm and 340 nm, respectively. The linear ranges of tyrosine and tryptophan were 0.10～6.0μg/mL and 0.050～6.0μg/mL, respectively. The linear regression equations were y=12.219x+0.6694 (r=0.9991) and y=83.612x+6.7351 (r=0.9993). The limits of detection were 0.05μg/mL and 0.02μg/mL. The urine sample was centrifuged, filtered by 0.45μm membrane, then diluted 5 times and analyzed by HPLC-FLD. The average recoveries were in the range of 86.1～122.2%, with relative standard deviations equal to or lower than 8.51%. The concentrations of tyrosine and tryptophan in urines were detected and compared between controls and cancer patients. The concentration of tryptophan in cancer group was lower than that in control groups, the levels of which increased after operation.3. A liquid chromatography-tandem mass spectrometry method was established for the qualitative and quantitative analysis of pteridines in urine samples. The chromatographic separation was achieved on a C18 column, the column temperature was 25℃, the mobile phase was CH3OH-0.1%HCOOH(10:90, v/v), the flow rate was 0.4 mL/min, the analysis was realized by multiple reaction-monitoring modes. Under optimal conditions, The linear ranges of neopterin, biopterin, pterin and sepiapterin were 0.000704～1.99,0.000700～1.01,0.000705～2.03,0.00506～1.01μg/mL and the limits of detection were 0.103,0.102,0.0156,0.0366 ng/mL. The average recoveries for four pteridines(spiked at three different levels) ranged from 78.6 to 123.9%, the RSD was below 6.87%. The levels of neopterin and biopterin in healthy persons' urine samples were found to be 0.0230～0.335μg/mL,0～0.530μg/mL, while were 0.0570～2.21μg/mL,0～0.193μg/mL in cancer persons' urine. The concentration of neopterin in cancer group increased more than that in control groups, while the concentration of biopterin in cancer group decreased, the results were consistent with the findings before. This method can provide simple sample preparation, rapid analysis and high sensitivity.4. A method for identification and qualification of tyrosine and tryptophan in urine samples by HPLC/MS/MS was developed. The chromatographic separation was achieved on a Agilent XDB-C18 column, the column temperature was 25℃, the mobile phase was CH3CN-0.1%HCOOH(10:90, v/v), the flow rate was 0.3 mL/min, the analysis was realized by multiple reaction-monitoring modes. Under optimal conditions, The linear ranges of tyrosine and tryptophan were 0.001～2.00,0.001～2.03μg/mL, The linear regression equations were Y=636.2003X+19176.5238, Y=2259.5523X+4005.8170, and the limits of detection were 0.00010 and 0.00025 ng/mL. The average recoveries for tyrosine and tryptophan(spiked at three different levels) ranged from 74.0 to 124.0%, the RSD was below 7.97%. The results were good when the method was applied to urine samples. The t test results showed us there was no significant difference between HPLC-FLD and HPLC-MS/MS. |
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