Study On Analytical Method Of Tyrosine And Its Metabolites In Biological Samples And Its Application

Tyrosine (Tyr) is an important semi-essential amino acid. There are a variety of metabolic disorders like tyrosinaemia and alkaptonuria, which lead to altered tyrosine degradation in humans. Meanwhile, abnormal levels of Tyr-related metabolites were also found in patients with liver and kidney disease. Moreover, Tyr and its metabolites are related to several types of cancer, including colorectal, bladder, ovarian and breast tumors, malignant melanoma and hepatocellular carcinoma. Therefore, developing a simple, rapid and sensitive method for simultaneous determination of Tyr and its metabolites in biological fluid is important for clinical diagnosis. A deep study of Tyr was made and applied in clinical diagnosis, while studies about Tyr metobolites were relatively less. Methods about Tyr metabolites are only referred to one or two Tyr-related metabolites, and none of them were used for monitoring the levels of Tyr and its metabolites in biological fluid from normal subjects and cancer patients. Based on published researches, we established new methods to measure Tyr and its metabolites in biological sample. And main research results are as follows:1. A simple, rapid and sensitive method was developed for simultaneous determination of p-hydroxyphenyllactic acid (PHPLA),3,4-dihydroxyphenylalanine (DOPA) and2,5-dihydroxyphenylacetic acid (HGA) in this paper. Synchronous fluorescence (SFS) of PHPLA, DOPA and HGA were measured with wavelength interval of35nm in KH2PI4-NaOH (pH=6.00) buffer medium. The experimental conditions were investigated and the spectra from260nm to325nm were tested, which were processed by derivative partial least square (DPLS). The linear ranges for PHPLA, DOPA and HGA were0.06-3.00mg/L,0.06～3.00mg/L and0.08～3.00mg/L, respectively; and the limits of detection (LOD) for them were0.03mg/L,0.03mg/L and0.04mg/L, respectively. The present method was successfully applied to simultaneous determination of PHPLA, DOPA and HGA in culture fluid and there was no significant difference between the results obtained by SFS-DPLS and HPLC.2. A simple, selective, rapid and practical method for the simultaneous determination of tyrosine (Tyr),4-hydroxyphenylacetic acid (PHPAA),3,4-dihydroxyphenylalanine (DOPA),4-hydroxyphenyllactic acid (PHPLA),4-hydroxyphenethylamine (TA) and3-(4-hydroxyphenyl)propionic acid (PHPA) in human serum by ultra-performance liquid chromatography (UPLC) coupled with fluorescence detection (FLD) was proposed for the first time. The separation was carried on ZORBAX SB-C18Rapid Resolution HD column with isocratic elution using95:5(v/v) mixture of50mM ammonium formate buffer (pH=5.8, adjusted with formic acid) and acetonitrile at the flow rate of0.20mL/min. FLD was performed with excitation and emission wavelength of277and316nm, respectively. Under optimized conditions, all compounds were resolved within8min. The effectiveness of various protein precipitants was discussed for the pretreatment of serum samples and5%perchloric acid was selected. The calibration curves were indicative of good linearity (correlation coefficients>0.9996), and the limits of detections and quantifications for Tyr and its metabolites ranged from0.016to0.032mg/L and0.040to0.080mg/L, respectively. Recoveries of Tyr and its metabolites in serum were in the range of86.5%～102.5%and88.3%～107.3%, respectively, with RSDs of0.3～5.7%and0.5～4.3%, respectively. The present method was successfully applied to the determination of Tyr and its five metabolites in human serum from normal persons and cancer patients. The results of this study indicated the possibility of the serum level of Tyr and its five metabolites in estimations of cancer. Further studies are required to evaluate the clinical significance of Tyr and its metabolites in human serum as a marker. And this method for detecting Tyr and its five metabolites will probably be helpful for further investigation.3. A sensitive, rapid and practical method for determination of Tyrosine (Tyr),4-hydroxyphenyllactic acid (PHPLA), and4-hydroxyphenylacetic acid (PHPAA) in human urine by ultra-performance liquid chromatography (UPLC) coupled with fluorescence detection (FLD) has been developed. The Clean-Screen DAU cartridge was employed to purify Tyr, PHPLA and PHPAA from human urine. The seperation of Tyr and its metabolites from other substances was carried on ZORBAX SB-C18Rapid Resolution HD column with isocratic elution using95:5(v/v) mixture of50mM ammonium formate buffer (pH=5.8, adjusted with formic acid) and acetonitrile at the flow rate of0.20mL/min. Under the optimized condition, the limits of detections and quantifications for Tyr and its metabolites were0.016mg/L and0.040mg/L, respectively. Recoveries of Tyr and its metabolites in normal subjects and cancer patients were in the range of95.2%～107.8%and86.9%～107.9%, respectively, with RSDS of0.6%～2.8%and0.5%～2.5%, respectively. The present method was successfully applied to the determination of Tyr, PHPLA and PHPAA in human urine from normal persons and cancer patients.4. A simple, selective, rapid and reliable method for the simultaneous determination of3,4-dihydroxyphenylalanine (DOPA), tyrosine (Tyr),4-hydroxyphenyllactic acid (PHPLA) and4-hydroxyphenethylamine (TA) in human serum by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) was proposed for the first time. Under optimized conditions, all compounds were resolved within6min. The calibration curves were indicative of good linearity (correlation coefficients>0.9989), and the limits of detections and quantifications for Tyr and its metabolites ranged from0.0005to0.20mg/L and0.001to0.40mg/L, respectively. Recoveries of Tyr and its metabolites in breast cancer patient were in the range of85.2%～114.0%, with RSDS of0.7%～8.5%. The present method was successfully applied to the determination of DOPA, Tyr, PHPLA and TA in sera from breast cancer patients, which will probably be helpful for further investigation.