the l - tyrosine is a substance that generates a neurotransmitter such as adrenaline , norepinephrine ( noradrenaline ) , or dopamine and becomes the thyroid hormone that controls the metabolism and is a precursor of melanin [ 1 , 2 ] .
therefore , the development of a method to determine small amounts of l - tyrosine is important in the medical and pharmacology fields .
moreover , the intake of l - tyrosine is also necessary for the preservation of health and is contained in nutrition supplements .
recently , the guarantee of its quality and analysis has become especially important problems in the nutritional science field . 

for example , ultraviolet absorptiometry at the wavelength of 280  nm   and the lowry method using the phenol reagent are known as a general determination analysis methods , but both have too low a sensitivity .
they are as follows : spectrophotometry using the phenylalanine ammonia - lyase enzyme reaction , fluorimetry using the 1,5-bis(4,6-dichloro-1,3,5-triazinyl - amino ) synthesis reaction   or using the mo(vi)-phenyl - fluorone quenching reaction , chemiluminescence ( cl ) using the oxidation reaction of k3fe(cn)6 and kmno4 [ 79 ] , cyclic voltammetry using the multiwalled carbon nanotube/4-amino - benzenesulfonic acid film - coated glassy carbon electrode oxidation reaction , uv detection of microcolumn electrophoresis , and hplc - fl .
however , only a few rapid , simple , and highly sensitive analyses of l - tyrosine are known .
the setup is simple and has the advantage that the measurement time is short . the cl analysis of various samples is performed in many fields such as biochemistry , clinical chemistry , and environmental chemistry for this reason .
however , the absolute number of high sensitive and high functional cl substance is a few although the cl system using luminol or peroxyoxalate has been many reported [ 13 , 14 ] . therefore , the appearance of a high cl substance is strongly desired . up to now , the authors have reported about the chlorophyll cl   and the iron - chlorophyllin complex cl . in these cls , each compound itself has multifunctions such as a luminescence substrate , a catalyst , and an energy donor .
moreover , in the iron - chlorophyllin complex cl , the luminescence was quenched when l - ascorbic acid was made to coexist in an oxidation reaction with hydrogen peroxide .
the quenching - cl determination of 10  m for l - ascorbic acid was developed .
on the other hand , the cl phenomenon with respect to the iron - phthalocyanine tetrasulfonic acid ( fe - pts ) was reported [ 18 , 19 ] .
the fe - pts complex had multi - cl functions similar to the iron - chlorophyllin complex , because the fe - pts as a luminescence substrate is the analog of an iron - chlorophyllin complex . in this paper , as a result of examining the fe - pts cl system , the coexistence of l - tyrosine significantly influenced the cl signal intensity .
the quenching - cl method of l - tyrosine with fe - pts was then developed , especially , the determination of tyrosine in soft drink and dietary supplement were examined .
iron - phthalocyanine tetrasulfonic acid was obtained from aldrich ( milwaukee , wi , usa ) .
sodium tetraborate ( borax ) was obtained from the kanto chemical co. ( tokyo , japan ) .
the l - tyrosine was obtained from the tokyo chemical industry co. ( tokyo , japan ) .
the practical samples were obtained from a soft drink ( amino supli , kirin beverage co. , tokyo , japan ) and a dietary supplement ( amino body , orihiro , gunma , japan ) .
a genelight200s ( the spectra range of pmt : 400570  nm ; microtec niti - on , chiba , japan ) was used for the cl measurements .
the distilled water was obtained using a swac-500 ( shimadzu co. , kyoto , japan ) .
a 300  l sample of water containing l - tyrosine was injected into a glass cell using a microsyringe . a 100  l ph buffer solution and 50  l iron - phthalocyanine complex solution
next , the cell was then placed into the cell holder of the cl detection equipment .
a 50  l aliquot of hydrogen peroxide was then injected using a microsyringe and the cl signal was measured .
the cl = cl0cls , where cl is the difference in the cl signal intensity , cl0 is the intensity of the l - tyrosine additive - free sample , and cls is the intensity of a certain amount of l - tyrosine contained in the sample .   a 1.0  ml soft drink was diluted to the marked line with distilled water in a 50  ml volumetric flask .
next , 5  ml of a 6.7  10  m l - tyrosine solution was added to 5  ml of this soft drink .
the measurement was done using the standard procedure concerning the determination of l - tyrosine .
a 0.25  g sample of the tablet ( the content of l - tyrosine is 6.00  mg ) was dissolved in 15  ml of a 1.0  m sodium hydroxide solution and then was diluted to the marked line with distilled water in a 250  ml volumetric flask .
a 1.4  ml of this solution was diluted to the marked line with distilled water in a 10  ml of volumetric flask , and the cl signal was measured using the standard procedure concerning the determination of l - tyrosine .
l - tyrosine is a reducing agent , naturally , authors think that it may work as h2o2 scavenger . in a previous paper , it is because the similar quenching - cl phenomenon by l - ascorbic acid was observed in iron - chlorophyllin / h2o2/l - ascorbic acid .
it was examined whether l - tyrosine works as a ligand of fe - pts .
the absorbance of fe - pts was decreased with increasing l - tyrosine concentration . from this result
, it is thought that l - tyrosine works as a ligand of fe - pts .  
it was examined whether l - tyrosine might work as a quencher ( energy transfer type ) .
stern - volmer plot from 2.0  10 to 2.0  10  m in the range of concentrations of l - tyrosine became linear .
therefore , stern - volmer plot showed that l - tyrosine worked as a quencher .   in conclusion ,
the function of l - tyrosine in the cl phenomenon is presumed form the above to work compositely and to happen .
based on the difference between the cl intensity of the blank reaction and the cl intensity for the 2.0  10  m l - tyrosine concentration that was the highest , each condition was optimized .
the concentration of the fe - pts was varied in the concentration range of 1.0  10  m to 5.0  10  m. the cl intensity increased with the increasing concentration of fe - pts .
the optimal concentration of fe - pts was 4.0  10  m according to the procedure optimization .  
the concentration of the hydrogen peroxide was varied in the range of 1.6  10  m to 8.0  10  m. as a result , the maximum cl0 intensity could be measured at 6.4  10  m hydrogen peroxide .
however , the maximum cl could be obtained at 3.2  10  m hydrogen peroxide according to the procedure optimization .
the ph was varied within the range of 9 to 12 . the maximum cl intensity according to the ph change was observed at ph 10 .
the cl intensity quantitatively decreased when the concentration of the coexisting l - tyrosine increased .
the cl signal for various l - tyrosine concentrations is shown in figure 3 . in the calibration curve of l - tyrosine ,
the relationship obtained between the concentration range of 2.0  10  m to 2.0  10  m of l - tyrosine , and the difference in the cl intensity ( cl ) was y = 2544 ln(x ) + 39864 , where y is cl and x is the l - tyrosine concentration [ m ] .
the correlation coefficient was 0.981 , the rsd was 1.63% ( n = 5 ) at a 2.0  10  m l - tyrosine concentration , and the detection limit ( 3 ) was 1.81  10  m. moreover , the result of determination of tyrosine of enantiomer is shown in table 1 .
as for the allowable limit , the change in time was within 5% based on the difference in the cl intensity with the blank when no foreign substances were added .
, l - ascorbic acid could be present up to 5-fold without affecting the results of the test .
also , although ni and co could be present up to equivalent without affecting the results of the test and cu could be present up to 0.5-fold without affecting the results of the test , they could be masked up to 10-fold with the addition of edta as a masking reagent .
also , l - cysteine could be present up to 0.5-fold without affecting the results of the test .
it is reported that l - cysteine and zn form a complex under neutral and alkaline conditions . as a result of the experiment , l - cysteine
the proposed method can then be comparatively said to be selective as an analytical method for l - tyrosine .
the proposed method and other methods were compared . the proposed method ( procedure time : approximately 3  min )
was proved to be rapid and simple , but the detection limits ( detection limits : 1.81  10  m ) are poor for fluorophotometry by the synthesis reaction with 1,5-bis(4,6-dichloro-1,3,5-triazinyl - amino ) ( detection limits : 6.8  10  m , procedure time : 30  min ) . also , the proposed method was rapid , simple , and highly sensitive compared to spectrophotometry using the phenylalanine ammonia - lyase enzyme reaction ( detection limits : 5.0  10  m , procedure time : 2  h )   and quenching - fluorophotometry using mo(vi)-phenyl - fluorone ( detection limits : 5.2  10  m , procedure time : 5  min ) .  
the determined concentration of l - tyrosine was 1.96  10  m for 2.00  10  m , and the recovery was 98% .
the indicated value of the components in the supplement was measured by the japan food research laboratories , and the various amino acids were measured by the amino acid analytical method using ninhydrin reaction .
the experimental value that had been obtained by the measurement was 2.04  10  m for the tabulated one of 2.00  10  m l - tyrosine .
in this study , rapid , simple , and highly sensitive analysis method of trace amounts of l - tyrosine based on the cl reaction of fe - pts was developed . in the future
, a new fia cl - detection will be developed for application to multisamples containing trace amounts of l - tyrosine .
 the chemiluminescence ( cl ) signal immediately appeared when a hydrogen peroxide solution was injected into an iron - phthalocyanine tetrasulfonic acid ( fe - pts ) aqueous solution . 
 moreover , the cl intensity of fe - pts decreased by adding l - tyrosine . based on these results , 
 the determination of trace amounts of l - tyrosine was developed using the quenching - chemiluminescence . 
 the calibration curve of l - tyrosine was obtained in the concentration range of 2.0  107  m to 2.0  105  m. moreover , the relative standard deviation ( rsd ) was 1.63 % ( 
 n = 5 ) for 2.0  106  m l - tyrosine , and its detection limits ( 3 ) were 1.81  107  m. the spike and recovery experiments for l - tyrosine were performed using a soft drink . furthermore , the determination of l - tyrosine was applied to supplements containing various kinds of amino acids . 
 each satisfactory relative recovery was obtained at 98 to 102% . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion