in the step ( a ), a sample containing hba 1 c is mixed with a protease in the presence of a composition of a cationic surfactant and a tetrazolium salt . preferably , the sample is an aqueous solution . the hba 1 c is decomposed by the protease to produce a glycated peptide . an example of the glycated peptide is a fructosyl peptide . an example of the fructosyl peptide is fructosyl - valine - histidine ( hereinafter , referred to as “ fru - val - his ”). in this way , obtained is an aqueous solution containing a glycated peptide . an example of the sample containing hba 1 c is blood derived from a human . the dilution of the blood derived from a human is also included in the sample containing hba 1 c . an example of the cationic surfactant is a quaternary ammonium salt , an alkylamine salt or a pyridine derivative . a preferable cationic surfactant is a quaternary ammonium salt , which is represented by the chemical formula r 1 r 2 r 3 r 4 n + x − . preferably , r 1 is an alkyl group having a carbon number of not less than 8 and not more than 18 . preferably , r 2 , r 3 and r 4 are lower alkyl groups independently . more preferably , r 2 , r 3 and r 4 are a methyl group or an ethyl group independently . a methyl group is still more preferable . x represents halogens . preferably , x represents chlorine or bromine . an example of the protease is thermolysin , papain , chymotrypsin , subtilisin , caspase , pepsin , or cathepsin d . thermolysin and papain are preferable . as demonstrated in the sample solutions a 8 - a 10 and b 8 - b 10 included in the test examples 1 and 2 , which are described later , the combination of the cationic surfactant and the tetrazolium salt significantly improves a decomposition rate of hba 1 c . in other words , the synergistic effect of the cationic surfactant and the tetrazolium salt significantly improves the decomposition rate of hba 1 c . see fig1 a , fig1 b , fig2 a and fig2 b . as demonstrated in the sample solutions a 1 and b 1 included respectively in the test examples 1 and 2 , an anionic surfactant such as sodium dodecyl sulfate ( hereinafter , referred to as “ sds ”) also improves the decomposition rate of hba 1 c . however , an anionic surfactant must not be used in the present invention . the reason is described in the description of the step ( b ). as demonstrated in the sample solutions a 2 and b 2 included respectively in the test examples 1 and 2 , the decomposition rate of hba 1 c is not improved when the cationic surfactant is used alone . similarly , as demonstrated in the sample solutions a 5 - a 7 and b 5 - b 7 included in the test examples 1 and 2 , the decomposition rate of hba 1 c is not improved when the tetrazolium salt is used alone . as demonstrated in the sample solutions a 3 , a 4 , b 3 and b 4 included in the test examples 1 and 2 , the decomposition rate of hba 1 c is not improved when a nonionic surfactant is used instead of the cationic surfactant . as demonstrated in the sample solutions a 11 - a 16 and b 11 - b 16 included in the test examples 1 and 2 , the decomposition rate of hba 1 c is not improved , even when a nonionic surfactant is used together with the tetrazolium salt ,. the glycated peptide aqueous solution obtained in the step ( a ) is mixed with fluctosylpeptide oxidase ( hereinafter , referred to as “ fpox ”). as disclosed in patent document 2 , the glycated peptide reacts with the fpox to produce hydrogen peroxide . in the step ( b ), the glycated peptide aqueous solution contains the composition of the cationic surfactant and the tetrazolium salt , similarly to the step ( a ). as demonstrated in the sample solutions c 8 - c 10 , d 8 - d 10 and e 8 - e 10 included in the test examples 3 - 5 , which are described later , the glycated peptide reacts with the fpox more rapidly in a case where the composition of a cationic surfactant and the tetrazolium salt is used , compared to the case where the cationic surfactant is used alone ( see sample solutions c 2 , d 2 , and e 2 ). an anionic surfactant improves the decomposition rate of hba 1 c . however , the anionic surfactants inactivate the fpox , as demonstrated in the sample solutions c 1 , d 1 and e 1 , which is included in the test example 3 , 4 and 5 , which are described later . for this reason , in case where the anionic surfactant is used , then the hba 1 c failed to be quantified . the amount of the hydrogen peroxide produced in the step ( b ) is proportional to the amount of the hba 1 c contained in the sample , as disclosed in patent document 4 ( see column 8 lines 6 - 37 ) and patent document 5 ( see column 10 line 63 - column 11 line 7 ). these patent documents are incorporated herein by reference . the following home page discloses that the amount of the hydrogen peroxide produced in the step ( b ) is proportional to the amount of the hba 1 c contained in the sample . accordingly , the amount of the hba 1 c contained in the sample is calculated on the basis of the amount of the hydrogen peroxide with use of a standard curve obtained in advance . in this way , the hba 1 c contained in the sample is quantified . in other words , the concentration of the hba 1 c is measured . in the step ( d ), the sample containing hba 1 c is mixed with the protease and the fpox in the presence of the composition of the cationic surfactant and the tetrazolium salt to obtain hydrogen peroxide . preferably , the sample is an aqueous solution . the step ( e ) is performed after the step ( d ). in the step ( e ), hba 1 c is quantified on the basis of the quantity of the hydrogen peroxide obtained in the step ( d ) in the step ( d ), the step ( a ) and the step ( b ) are performed simultaneously . the step ( e ) is identical to the step ( c ). accordingly , the detailed descriptions of the step ( d ) and the step ( e ) are omitted . whole human blood ( available from biopredic international ) was diluted to make a ten - fold dilution thereof . in this way , a sample solution containing hemoglobin was prepared . hereinafter , this sample solution is referred to as “ sample solution p ”. the following sample solutions a 1 - a 16 were prepared . a comparative sample solution al was also prepared . the source of the used reagents is listed below . thermolysin : wako pure chemical industries , ltd . sds : wako pure chemical industries , ltd . ttab ( tetradecyl trimethyl ammonium bromide ): wako pure chemical industries , ltd . tritonx - 100 : wako pure chemical industries , ltd . tween20 : wako pure chemical industries , ltd . wst - 3 : dojindo laboratories wst - 4 : dojindo laboratories wst - 5 : dojindo laboratories ( sample solution a 1 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml sds pbs solution : 10 % by weight ( sample solution a 2 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight ( sample solution a 3 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight ( sample solution a 4 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight ( sample solution a 5 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml wst - 3 water solution : 2 % by weight ( sample solution a 6 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml wst - 4 pbs solution : 0 . 5 % by weight ( sample solution a 7 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml wst - 5 pbs solution : 2 % by weight ( sample solution a 8 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml ttab 10 %: by weight wst - 3 pbs solution : 2 % by weight ( sample solution a 9 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml ttab 10 %: by weight wst - 4 pbs solution : 0 . 5 % by weight ( sample solution a 10 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight wst - 5 pbs solution : 2 % by weight ( sample solution a 11 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight ( sample solution a 12 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 4 pbs solution : 0 . 5 % by weight ( sample solution a 13 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 5 pbs solution : 2 % by weight ( sample solution a 14 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight ( sample solution a 15 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight wst - 4pbs solution 0 . 5 %: by weight ( sample solution a 16 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight wst - 5 pbs solution : 2 % by weight ( comparative sample solution a 1 ) sample solution p thermolysin pbs solution : 150 , 000 u / ml the temperature of the sample solutions a 1 - a 16 and the comparative sample solution al was maintained at 37 degrees celsius for ten minutes and the reaction occurred . after the reaction , the sample solutions a 1 - a 16 and the comparative sample solution al were subjected to polyacrylamide gel electrophoresis ( page ). fig1 a and fig1 b show the electrophoresis patterns . the character “ m ” depicted in fig1 a and fig1 b indicates a protein maker . the degree of the hba 1 c decomposition is reflected in the color strength of the band near 64 . 5 kda , which corresponds to hemoglobin molecular region included in the electrophoresis pattern . if the color strength of the band near 64 . 5 kda decreases , the more amounts of hba 1 c is decomposed . as is clear from the electrophoresis patterns shown in fig1 a and fig1 b , the more amount of hba 1 c was decomposed in the sample solutions a 1 , a 8 , a 9 and a 10 , compared to the comparative sample solution a 1 . the more amount of hba 1 c was decomposed in the sample solutions a 8 , a 9 and a 10 , compared to the sample solutions a 2 , a 5 , a 6 , and a 7 . sds ( sample solution a 1 ), the composition of ttab and wst - 3 ( sample solution a 8 ), the composition of ttab and wst - 4 ( sample solution a 9 ) and the composition of ttab and wst - 5 ( sample solution a 10 ) improve the decomposition rate of hba 1 c using thermolysin . the composition of ttab and wst - 3 ( sample solution a 8 ) improves the decomposition rate of hba 1 c using thermolysin , compared to the case where ttab was used alone ( sample solution a 2 ) and the case where wst - 3 was used alone ( sample solution a 5 ). the composition of ttab and wst - 4 ( sample solution a 9 ) improves the decomposition rate of hba 1 c using thermolysin , compared to the case where ttab was used alone ( sample solution a 2 ) and the case where wst - 4 was used alone ( sample solution a 6 ). the composition of ttab and wst - 5 ( sample solution a 10 ) improves the decomposition rate of hba 1 c using thermolysin , compared to the case where ttab was used alone ( sample solution a 2 ) and the case where wst - 5 was used alone ( sample solution a 7 ). the following sample solutions b 1 - b 16 were prepared . a comparative sample solution b 1 was also prepared . the source of the used reagents is listed below . papain : roche diagnostics k . k . sds : wako pure chemical industries , ltd . ttab ( tetradecyl trimethyl ammonium bromide ): wako pure chemical industries , ltd . tritonx - 100 : wako pure chemical industries , ltd . tween20 : wako pure chemical industries , ltd . wst - 3 : dojindo laboratories wst - 4 : dojindo laboratories wst - 5 : dojindo laboratories ( sample solution b 1 ) sample solution p papain pbs solution : 300 u / ml sds pbs solution : 10 % by weight ( sample solution b 2 ) sample solution p papain pbs solution : 300 u / ml ttab : 10 % by weight ( sample solution b 3 ) sample solution p papain pbs solution : 300 u / ml tritonx - 100 pbs solution : 10 % by weight ( sample solution b 4 ) sample solution p papain pbs solution : 300 u / ml tween20 pbs solution : 10 % by weight ( sample solution b 5 ) sample solution p papain pbs solution : 300 u / ml wst - 3 water solution : 2 % by weight ( sample solution b 6 ) sample solution p papain pbs solution : 300 u / ml wst - 4 pbs solution : 0 . 5 % by weight ( sample solution b 7 ) sample solution p papain pbs solution : 300 u / ml wst - 5 pbs solution : 2 % by weight ( sample solution b 8 ) sample solution p papain pbs solution : 300 u / ml ttab 10 %: by weight wst - 3 pbs solution : 2 % by weight ( sample solution b 9 ) sample solution p papain pbs solution : 300 u / ml ttab 10 %: by weight wst - 4 pbs solution : 0 . 5 % by weight ( sample solution b 10 ) sample solution p papain pbs solution : 300 u / ml ttab : 10 % by weight wst - 5 pbs solution : 2 % by weight ( sample solution b 11 ) sample solution p papain pbs solution : 300 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight ( sample solution b 12 ) sample solution p papain pbs solution : 300 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 4 pbs solution : 0 . 5 % by weight ( sample solution b 13 ) sample solution p papain pbs solution : 300 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 5 pbs solution : 2 % by weight ( sample solution b 14 ) sample solution p papain pbs solution : 300 u / ml tween 20 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight ( sample solution b 15 ) sample solution p papain pbs solution : 300 u / ml tween 20 pbs solution : 10 % by weight wst - 4 pbs solution 0 . 5 %: by weight sample solution p papain pbs solution : 300 u / ml tween20 pbs solution : 10 % by weight wst - 5 pbs solution : 2 % by weight ( comparative sample solution b 1 ) sample solution p papain pbs solution : 300 u / ml the temperature of the sample solutions b 1 - b 16 and the comparative sample solution b 1 was maintained at 37 degrees celsius for ten minutes and the reaction occurred . after the reaction , the sample solutions b 1 - b 16 and the comparative sample solution b 1 were subjected to polyacrylamide gel electrophoresis ( page ). fig2 a and fig2 b show the electrophoresis patterns . the character “ m ” depicted in fig2 a and fig2 b indicates a protein maker . the degree of the hba 1 c decomposition is reflected in the color strength of the band near 64 . 5 kda , which corresponds to hemoglobin molecular region included in the electrophoresis pattern . if the color strength of the band near 64 . 5 kda decreases , the more amounts of hba 1 c is decomposed . as is clear from the electrophoresis patterns shown in fig2 a and fig2 b , the more amount of hba 1 c was decomposed in the sample solutions b 1 , b 8 , b 9 and b 10 , compared to the comparative sample solution b 1 . the more amount of hba 1 c was decomposed in the sample solutions b 8 , b 9 and b 10 , compared to the sample solutions b 2 , b 5 , b 6 , and b 7 . sds ( sample solution b 1 ), the composition of ttab and wst - 3 ( sample solution b 8 ), the composition of ttab and wst - 4 ( sample solution b 9 ) and the composition of ttab and wst - 5 ( sample solution b 10 ) improve the decomposition rate of hba 1 c using papain . the composition of ttab and wst - 3 ( sample solution b 8 ) improves the decomposition rate of hba 1 c using papain , compared to the case where ttab was used alone ( sample solution b 2 ) and the case where wst - 3 was used alone ( sample solution b 5 ). the composition of ttab and wst - 4 ( sample solution b 9 ) improves the decomposition rate of hba 1 c using papain , compared to the case where ttab was used alone ( sample solution b 2 ) and the case where wst - 4 was used alone ( sample solution b 6 ). the composition of ttab and wst - 5 ( sample solution b 10 ) improves the decomposition rate of hba 1 c using papain , compared to the case where ttab was used alone ( sample solution b 2 ) and the case where wst - 5 was used alone ( sample solution b 7 ). the following sample solutions c 1 - c 12 were prepared . a comparative sample solution c 1 was also prepared . the source of the used reagents is listed below . fpox - ce : kikkoman corporation sds : wako pure chemical industries , ltd . peroxidase : wako pure chemical industries , ltd . kn - 111 ( color forming dye ): dojindo laboratories ttab ( tetradecyl trimethyl ammonium bromide ): wako pure chemical industries , ltd . tritonx - 100 : wako pure chemical industries , ltd . tween20 : wako pure chemical industries , ltd . wst - 3 : dojindo laboratories wst - 4 : dojindo laboratories wst - 5 : dojindo laboratories ( sample solution c 1 ) sds pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase 20 u / ml kn - 111 : 2 mm ( sample solution c 2 ) ttab : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase 20 u / ml kn - 111 : 2 mm ( sample solution c 3 ) tritonx - 100 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 4 ) tween 20 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 5 ) wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 6 ) wst - 4 pbs solution : 0 . 5 % by weight fpox - ce pbs solution : 28 u / ml peroxidase 20 u / ml kn - 111 : 2 mm ( sample solution c 7 ) wst - 5 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 8 ) ttab : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 9 ) ttab : 10 % by weight wst - 4 pbs solution : 0 . 5 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 10 ) ttab : 10 % by weight wst - 5 pbs solution : 2 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 11 ) tritonx - 100 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution c 12 ) tween20 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( comparative sample solution c 1 ) fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm the temperature of the sample solutions c 1 - c 12 and the comparative sample solution c 1 was maintained at 37 degrees celsius for ten minutes and the reaction occurred . after the reaction , five milliliter of fru - val - his 1 mm pbs solution ( available from peptide institute inc .) was each added to the sample solution c 1 - c 12 and comparative sample solution c 1 ( 95 microliter ). in this way , mixture solutions c 1 - c 12 and a comparative mixture solution c 1 were obtained . absorbance of the mixture solutions c 1 - c 12 and the comparative mixture solution c 1 at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer ( available from tecan group inc ., under the trade name of infinite 200 pro ). fig3 shows the results . the glycated peptide fru - val - his reacts with the fpox to generate hydrogen peroxide . hydrogen peroxide reacts with the color forming dye kn - 111 to change the color forming dye kn - 111 into red . accordingly , the activity of the fpox is evaluated by measuring the absorbance of the color forming dye kn - 111 at a wavelength of 660 nanometers ( red ). as shown in fig3 , an absorbance change was observed in all of the mixture solutions c 2 - c 12 other than the mixture solution c 1 and the comparative sample solution c 1 . among them , it was observed that the absorbance changes were similar to that of the comparative mixture solution c 1 , other than the mixture solution c 2 . it was observed that the absorbance change of the mixture solution c 2 was more gradual than that of the comparative mixture solution c 1 . on the other hand , in the sample solution c 1 , the absorbance change was not observed at all . in this way , sds , which is an anionic surfactant , inactivates fpox ( see the result of the mixture solution c 1 ). therefore , sds is not allowed to be used for the present quantification method of hba 1 c using the fpox . on the other hand , a cationic surfactant and a non - ionic surfactant do not inactivate fpox ( see the results of the mixture solutions c 2 - c 12 and the comparative mixture solution c 1 ). as is clear from fig3 , the fpox reacts more rapidly with the glycated peptide in the case where the composition of ttab and wst - 3 , the composition of ttab and wst - 4 , or the composition of ttab and wst - 5 is used ( mixture solutions c 8 - c 10 ), compared to the case where ttab is used alone ( mixture solution c 2 ). the following sample solutions d 1 - d 12 were prepared . a comparative sample solution d 1 was also prepared . the source of the used reagents is listed below . fpox - ce : kikkoman corporation thermolysin : wako pure chemical industries , ltd . sds : wako pure chemical industries , ltd . peroxidase wako pure chemical industries , ltd . kn - 111 ( color forming dye ): dojindo laboratories ttab ( tetradecyl trimethyl ammonium bromide ): wako pure chemical industries , ltd . tritonx - 100 : wako pure chemical industries , ltd . tween20 : wako pure chemical industries , ltd . wst - 3 : dojindo laboratories wst - 4 : dojindo laboratories wst - 5 : dojindo laboratories ( sample solution d 1 ) thermolysin pbs solution : 150 , 000 u / ml sds pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 2 ) thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 3 ) thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 4 ) thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 5 ) thermolysin pbs solution : 150 , 000 u / ml wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 6 ) thermolysin pbs solution : 150 , 000 u / ml wst - 4 pbs solution : 0 . 5 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 7 ) thermolysin pbs solution : 150 , 000 u / ml wst - 5 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 8 ) thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 9 ) thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight wst - 4 pbs solution : 0 . 5 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 10 ) thermolysin pbs solution : 150 , 000 u / ml ttab : 10 % by weight wst - 5 pbs solution : 2 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 11 ) thermolysin pbs solution : 150 , 000 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution d 12 ) thermolysin pbs solution : 150 , 000 u / ml tween20 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( comparative sample solution c 1 ) thermolysin pbs solution : 150 , 000 u / ml fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm the temperature of the sample solutions d 1 - d 12 and the comparative sample solution d 1 was maintained at 37 degrees celsius for ten minutes and the reaction occurred . after the reaction , five milliliter of fru - val - his 1 mm pbs solution ( available from peptide institute inc .) was each added to the sample solution d 1 - d 12 and comparative sample solution d 1 ( 95 microliters ). in this way , mixture solutions d 1 - d 12 and a comparative mixture solution dl were obtained . absorbance of the mixture solutions d 1 - d 12 and the comparative mixture solution d 1 at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer ( available from tecan group inc ., under the trade name of infinite 200 pro ). fig4 shows the results . the glycated peptide fru - val - his reacts with the fpox to generate hydrogen peroxide . hydrogen peroxide reacts with the color forming dye kn - 111 to change the color forming dye kn - 111 into red . accordingly , the activity of the fpox is evaluated by measuring the absorbance of the color forming dye kn - 111 at a wavelength of 660 nanometers ( red ). as shown in fig4 , an absorbance change was observed in all of the mixture solutions d 2 - d 12 other than the mixture solution d 1 and the comparative sample solution d 1 . among them , it was observed that the absorbance changes were similar to that of the comparative mixture solution d 1 , other than the mixture solution d 2 . it was observed that the absorbance change of the mixture solution d 2 was more gradual than that of the comparative mixture solution d 1 . on the other hand , in the sample solution d 1 , the absorbance change was not observed at all . in this way , sds , which is an anionic surfactant , inactivates fpox ( see the result of the mixture solution d 1 ). therefore , sds is not allowed to be used for a quantification method of hba 1 c using the fpox . on the other hand , a cationic surfactant and a non - ionic surfactant do not inactivate fpox ( see the results of the mixture solution d 2 - d 12 and the comparative mixture solution c 1 ). thermolysin also does not inactivate the fpox . as is clear from fig4 , the fpox reacts more rapidly with the glycated peptide in the case where the composition of ttab and wst - 3 , the composition of ttab and wst - 4 , or the composition of ttab and wst - 5 is used ( mixture solutions d 8 - d 10 ), compared to the case where ttab is used alone ( mixture solution d 2 ). the following sample solutions e 1 - e 12 were prepared . a comparative sample solution e 1 was also prepared . the source of the used reagents is listed below . fpox - ce : kikkoman corporation papain : roche diagnostics k . k . sds : wako pure chemical industries , ltd . peroxidase : wako pure chemical industries , ltd . kn - 111 ( color forming dye ): dojindo laboratories ttab ( tetradecyl trimethyl ammonium bromide ): wako pure chemical industries , ltd . tritonx - 100 : wako pure chemical industries , ltd . tween20 : wako pure chemical industries , ltd . wst - 3 : dojindo laboratories wst - 4 : dojindo laboratories wst - 5 : dojindo laboratories ( sample solution e 1 ) papain 300 u / ml sds pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase 20 u / ml kn - 111 : 2 mm ( sample solution e 2 ) papain : 300 u / ml ttab : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 3 ) papain : 300 u / ml tritonx - 100 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 4 ) papain : 300 u / ml tween20 pbs solution : 10 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 5 ) papain : 300 u / ml wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 6 ) papain : 300 u / ml wst - 4 pbs solution : 0 . 5 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 7 ) papain : 300 u / ml wst - 5 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 8 ) papain : 300 u / ml ttab : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 9 ) papain : 300 u / ml ttab : 10 % by weight wst - 4 pbs solution : 0 . 5 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 10 ) papain : 300 u / ml ttab : 10 % by weight wst - 5 pbs solution : 2 % by weigh fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 11 ) papain : 300 u / ml tritonx - 100 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm ( sample solution e 12 ) papain : 300 u / ml tween20 pbs solution : 10 % by weight wst - 3 pbs solution : 2 % by weight fpox - ce pbs solution : 28 u / ml peroxidase 20 u / ml kn - 111 : 2 mm ( comparative sample solution c 1 ) thermolysin pbs solution : 150 , 000 u / ml fpox - ce pbs solution : 28 u / ml peroxidase : 20 u / ml kn - 111 : 2 mm the temperature of the sample solutions e 1 - e 12 and the comparative sample solution el was maintained at 37 degrees celsius for ten minutes and the reaction occurred . after the reaction , five milliliter of fru - val - his 1 mm pbs solution ( available from peptide institute inc .) was each added to the sample solution e 1 - e 12 and comparative sample solution e 1 ( 95 microliters ). in this way , mixture solutions e 1 - e 12 and a comparative mixture solution e 1 were obtained . absorbance of the mixture solutions e 1 - e 12 and the comparative mixture solution e 1 at a wavelength of 660 nanometers was measured every minute with an absorption spectrometer ( available from tecan group inc ., under the trade name of infinite 200 pro ). fig5 shows the results . the glycated peptide fru - val - his reacts with the fpox to generate hydrogen peroxide . hydrogen peroxide reacts with the color forming dye kn - 111 to change the color forming dye kn - 111 into red . accordingly , the activity of the fpox is evaluated by measuring the absorbance of the color forming dye kn - 111 at a wavelength of 660 nanometers ( red ). as shown in fig5 , an absorbance change was observed in all of the mixture solutions e 2 - e 12 other than the mixture solution e 1 and the comparative sample solution e 1 . among them , it was observed that the absorbance changes were similar to that of the comparative mixture solution e 1 , other than the mixture solution e 2 . it was observed that the absorbance change of the mixture solution e 2 was more gradual than that of the comparative mixture solution e 1 . on the other hand , in the sample solution e 1 , the absorbance change was not observed at all . in this way , sds , which is an anionic surfactant , inactivates fpox ( see the result of the mixture solution e 1 ). therefore , sds is not allowed to be used for a quantification method of hba 1 c using the fpox . on the other hand , a cationic surfactant and a non - ionic surfactant do not inactivate fpox ( see the results of the mixture solution e 2 - e 12 and the comparative mixture solution c 1 ). papain also does not inactivate the fpox . as is clear from fig5 , the fpox reacts more rapidly with the glycated peptide in the case where the composition of ttab and wst - 3 , the composition of ttab and wst - 4 , or the composition of ttab and wst - 5 is used ( mixture solutions e 8 - e 10 ), compared to the case where ttab is used alone ( mixture solution e 2 ).