| L-tyrosine is one of important amino acids which to constitute the proteins. The literature shows that the change of tyrosine and its metabolites in human body fluid is connected with some diseases. Therefore, developing a simple, rapid and sensitive method for determination of tyrosine and its metabolites in human body fluid is important for clinical diagnosis. This paper we studied tyrosine and its metabolites in human serum sample and urine sample by anion-exchange chromatography combined with different detectors. And main research results are as follows:1. A sensitive, practical and environmental friendly method for the determination of 4-hydroxyphenyllactic acid in human serum by anion-exchange chromatography(AEC) coupled with integrated pulsed amperometric detector(IPAD) was proposed for the first time. The separation of 4-hydroxyphenyllactic acid was carried out on IonPac AS18 column with isocratic elution using 0.023 mol/L NaOH at the flow rate of 0.25 mL/min. Waveform in IPAD exhibited adsorption / desorption catalytic features at gold electrode surface, and potentials of the waveform were optimized in this study. Under optimized conditions, the linear range was 0.03- 2 mg/L and the limit of detection for 4-hydroxyphenyllactic acid was computed to be 0.010 mg/L with the signal-to-noise ratio of 3. Recoveries were in the range of 92.0% ~ 102.0%, with relative standard derivations(RSDs) of 2.78% ~ 3.79% respectively. The results indicate this method is suitable for the actual sample testing. In addition, the present method was successfully applied to the determination of 4-hydroxyphenyllactic acid in human serum from normal persons and gastric cancer patients.2. A novel, simple, rapid and accurate method was proposed for the simultaneous determination of Tyrosine(Tyr), p-hydroxyphenyllactic acid(PHPLA) and 3-p-hydroxyphenylpropionic acid(PHPA) in human serum by anion exchange chromatography- fluorescence detection. The deproteinized serum samples by acetonitrile were separated and determined. Dionex IonPac AS21(250 mm×2 mm) analytical column and IonPac AG21(50 mm×2 mm) guard column were used for the separation of the target analyte. The mobile phase of ACE was 0.004 mol/L NaOH with the flow rate of 0.45 mL/min. Fluorescent detector worked at an excitation wavelength of 277 nm and emission wavelength of 340 nm. The concentrations of Tyr,PHPLA and PHPA in human serum were determined by ACE-FLD. The results of separation were satisfied(R>1.5) and the retention time was within 20 min. The linear ranges for Tyr, PHPLA and PHPA were 0.30~25 mg/L, 0.025~2 mg/L and 0.025~2 mg/L, respectively; and the detection limits were 0.024 mg/L, 0.020 mg/L and 0.019 mg/L, respectively. In the three spiked levels, the average recoveries were in the range of 72.6% ~99.1%, with relative standard derivations(RSDs) less than 6.62%. The results indicate this method is suitable for the actual sample testing.3. A method of magnetic solid-phase extraction was proposed for the preconcentration of 4-hydroxyphenyllactic acid(PHPLA) in human urine by anion-exchange chromatography(AEC) coupled with fluorescence detection(FLD). The separation of PHPLA was carried out on IonPac AS21 column with isocratic elution using 0.003 mol/L Na2CO3 at the flow rate of 0.45 mL/min. Fluorescent detector worked at an excitation wavelength of 277 nm and emission wavelength of 340 nm. The separation parameters and pretreatment conditions of urine were optimized in this study. Under optimized conditions, the linear range was 0.05 ~ 10 mg/L and the limit of detection for PHPLA was 0.016 mg/L with the signal-to-noise ratio of 3. Recoveries of PHPLA in normal subjects and cancer patients were in the range of 98.5% ~ 105.5% and 86.5% ~ 95.0%, respectively, with RSDS of 1.66% ~ 4.12% and 0.81% ~ 3.36%. In addition, the present method was successfully applied to the determination of PHPLA in human urine from normal persons and breast cancer patients. |
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