Method for quantifying concentration of organic compound in test sample, and kit for quantifying concentration of organic compound

Abstract

To provide a determination method capable of accurately determining the concentration of an organic compound in a test sample by using a test paper colored by dehydrogenase reaction, and to provide a kit for determining the concentration of the organic compound.SOLUTION: The determination method of the present invention is a method for determining the concentration of organic compounds in a test sample, wherein the test sample is pre-treated using a pre-treatment buffer that has been adjusted to a level equal to or higher than pH8, and a test paper that is colored by a dehydrogenase reaction is brought into contact with the pre-treated test sample to determine the concentration by colorimetry.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying the concentration of an organic compound and a kit for quantifying the concentration of an organic compound. [Background technology]

[0002] A method for quantifying amino acids by color development using a dehydrogenase reaction is known as a solution method (see, for example, Patent Document 1). Also, a test paper using a dehydrogenase reaction has been proposed as a test paper for quantifying inosinic acid (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 08-242889 [Patent Document 2] Japanese Patent Application Publication No. 01-256397 Summary of the Invention [Problem to be solved by the invention]

[0004] When conventional test papers that develop color through a dehydrogenase reaction are used to measure the concentration of organic compounds in a test sample, poor color development occurs depending on the type of organic compound, making it impossible to accurately quantify the concentration. Therefore, an object of the present invention is to provide a quantitative determination method that can accurately determine the concentration of organic compounds in a test sample using test paper that develops color through a dehydrogenase reaction, and a kit for quantifying the concentration of organic compounds. [Means for solving the problem]

[0005] In order to achieve the above object, the inventors have conducted extensive research and have found that by pretreating a test sample with a predetermined buffer component before contacting the test sample with a test paper that develops color through a dehydrogenase reaction, the color of the test paper can be improved and the concentration of organic compounds in the test sample can be accurately quantified.

[0006] That is, the present invention is a method for quantifying the concentration of an organic compound in a test sample, which comprises pretreating the test sample with a pretreatment buffer component adjusted to a pH of 8 or higher, contacting the pretreated test sample with a test paper that develops color through a dehydrogenase reaction, and quantifying the concentration by colorimetric analysis.

[0007] The present invention also provides a concentration determination kit for quantifying the concentration of an organic compound in a test sample, the kit comprising a test paper that develops color through a dehydrogenase reaction and a pretreatment buffer component adjusted to a pH of 8 or higher for pretreating the test sample. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a quantitative determination method that can accurately determine the concentration of an organic compound in a test sample using a test paper that develops color through a dehydrogenase reaction, and a kit for quantifying the concentration of an organic compound. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below. The quantification method of the present invention is used to quantify the concentration of an organic compound in a test sample. Examples of organic compounds include those having an amino group and a molecular weight of 1,000 or less. The quantification method of the present invention is particularly suitable for quantifying amino acid concentrations. In this case, the test sample is preferably a liquid, and is not particularly limited as long as it is a sample suspected of containing amino acids. Examples include biological samples (urine, blood, saliva, tears, sweat, interstitial fluid, etc.), food and beverage products (foods, beverages, vegetables, fruits, processed foods, dishes, meat, etc.), seasonings, culture media, wastewater, river water, groundwater, seawater, soil, and fertilizer.

[0010] The amino acid concentration in the test sample may be a low concentration of less than 1 mM or a high concentration of 1 mM or more. Examples of amino acids include alanine (Ala), branched-chain amino acids (BCAAs), threonine (Thr), and lysine (Lys). Known branched-chain amino acids include valine, leucine, and isoleucine.

[0011] The quantitative method of the present invention uses a test paper that changes color due to a dehydrogenase reaction. Any test paper that changes color due to a dehydrogenase reaction can be used, but the test paper described below is preferably used in the quantitative method of the present invention. Specifically, the test paper includes a liquid-permeable carrier, and the carrier supports a dehydrogenase, a coenzyme, an electron mediator, and a reductive color-developing reagent.

[0012] As the liquid-permeable carrier, a filter paper or a porous membrane made of cellulose, glass fiber, etc. can be used. The shape and size of the carrier are not particularly limited and can be any shape and any size, for example, a small piece of cellulose fiber filter paper measuring 5 mm x 5 mm x 1 mm can be used.

[0013] The dehydrogenase catalyzes the dehydrogenation reaction of the amino acid to be measured, and can be selected depending on the type of amino acid. When the target to be measured is alanine, L-alanine dehydrogenase is used. The loading amount of L-alanine dehydrogenase is 5300 to 49000 kU / m 3 is preferred, and 11,000 to 21,000 kU / m 3 When the measurement target is leucine, a type of BCAA, L-leucine dehydrogenase is used as the dehydrogenase. The amount of L-leucine dehydrogenase supported is 3300 to 33000 kU / m 3 is preferred, and 6700 to 13000 kU / m 3 is more preferred.

[0014] When the measurement target is threonine, L-threonine dehydrogenase is used. The amount of L-threonine dehydrogenase supported is 4000 to 40000 kU / m3 is preferred, and 8000 to 16000 kU / m 3 When the measurement target is lysine, L-lysine dehydrogenase is used. The amount of L-lysine dehydrogenase supported is 133 to 1330 U / m 3 is preferred, and 266 to 530 kU / m 3 kU / m 3 is more preferred.

[0015] Coenzymes promote the dehydrogenase reaction. Examples of coenzymes include nicotinamide adenine dinucleotide (NAD + ), nicotinamide adenine dinucleotide phosphate (NADP + ) and NAD + NAD is preferred. + The loading amount is 44 to 440 g / m 3 is preferable, and 89 to 220 g / m 3 is more preferred.

[0016] The electron mediator promotes the reaction of the reductive color-developing reagent. Examples of the electron mediator include diaphorase and 1-methoxy-5-ethylphenazinium ethyl sulfate (1-methoxy PES), with diaphorase being preferred. The amount of diaphorase supported is 4400 to 44000 kU / m 3 It is preferable that the concentration is 8900 to 18000 kU / m 3 is more preferred.

[0017] As the reducing color-developing reagent, a tetrazolium salt can be used, and specific examples thereof include nitrotetrazolium blue (Nitro-TB), (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC), WST-1, WST-3, and WST-8. The loading amount of Nitro-TB is 67 to 560 g / m. 3 is preferable, and 110 to 330 g / m 3 is more preferred.

[0018] In order to support the dehydrogenase, coenzyme, electron mediator, and reductive color-developing reagent on the carrier, these are dissolved in a solvent at a predetermined concentration to prepare a staining solution. The solvent for the staining solution can be water.

[0019] The concentration of dehydrogenase in the staining solution is determined depending on the type of dehydrogenase. The concentration of L-alanine dehydrogenase is preferably about 24,000 to 220,000 U / L, more preferably about 48,000 to 96,000 U / L. The concentration of L-leucine dehydrogenase in the staining solution is preferably about 15,000 to 150,000 U / L, more preferably about 30,000 to 60,000 U / L. The concentration of L-threonine dehydrogenase in the staining solution is preferably about 8000 to 180000 U / L, more preferably about 36000 to 72000 U / L.The concentration of L-lysine dehydrogenase in the staining solution is preferably about 600 to 6000 U / L, more preferably about 1200 to 2400 U / L. The concentration of the coenzyme in the staining solution is preferably about 0.2 to 2 g / L, and more preferably about 0.4 to 1 g / L.

[0020] The concentration of the electron mediator in the staining solution is preferably about 20,000 to 200,000 U / L, more preferably about 40,000 to 80,000 U / L.The concentration of the reducing color-developing reagent in the staining solution is preferably about 0.037 to 0.306 mM, more preferably about 0.061 to 0.183 mM.

[0021] In addition to the above-mentioned components, the dye solution may contain optional components such as a pH buffer and a reaction accelerator. The pH buffer is used to maintain the pH in the staining solution at a value suitable for the target enzymatic reaction. An example of the pH buffer is N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid. The concentration of the pH buffer in the staining solution is preferably about 0.033 to 0.308 mM.

[0022] Examples of reaction accelerators include polyethylene glycol mono-p-isooctylphenyl ether (trade name Triton X-100, Dow Chemical Company, etc.) and polyoxyethylene (20) sorbitan monooleate (trade name Tween 80, Imperial Chemical Industries, etc.). The accelerator accelerates the oxidative deamination of amino acids, which has the advantage of enabling determination in a short time. The concentration of accelerator in the staining solution is preferably about 0.1 to 1%.

[0023] Test paper can be prepared by impregnating a carrier with a staining solution containing predetermined components. The staining solution can be prepared by adding a dehydrogenase, a coenzyme, an electron mediator, a reductive color-developing reagent, and optional components, at predetermined concentrations, to water and mixing uniformly. The resulting staining solution is placed in a container, and a carrier such as paper is immersed in it for approximately 0.01 to 1 minute to impregnate the carrier with the staining solution. A glass beaker, a stainless steel tank, or the like can be used as the container. The carrier impregnated with the staining solution is dried in an air dryer or drum dryer at 30 to 50°C for 10 to 45 minutes to prepare a test paper in which the dehydrogenase, coenzyme, electron mediator, and reductive color-developing reagent are supported on the carrier.

[0024] The organic compound concentration quantification kit of the present invention can be used to quantify the concentration of organic compounds in a test sample. The organic compound concentration quantification kit includes a test paper that develops color through a dehydrogenase reaction and a pretreatment buffer component adjusted to a pH of 8 or higher for pretreating the test sample. The pretreatment buffer component is preferably a pretreatment buffer component adjusted to a pH of 8 to 11, and examples thereof include components containing Tris hydrochloride. More specifically, examples of the pretreatment buffer component include Tris hydrochloride, glycine sodium hydroxide, and glycine potassium hydroxide, with Tris hydrochloride being particularly preferred. The pretreatment buffer component may be used in either a liquid or solid form.

[0025] In quantifying the concentration of organic compounds, the test sample is first pretreated using a pretreatment buffer component. The pretreatment buffer component can be brought into direct contact with the test sample. If the pretreatment buffer component is in liquid form, it is pretreated by mixing it with the test sample. For example, when Tris hydrochloride is used as the pretreatment buffer component, it can be used as an approximately 1 M aqueous solution. This aqueous solution is added to the test sample in a volume ratio of 5:95 to 95:5 (test sample:pretreatment buffer component) and mixed uniformly. The test sample mixed with the pretreatment buffer component is alkaline. If the test sample is in solid form, it can be pretreated by suspending it in an aqueous solution of the pretreatment buffer component.

[0026] The pretreatment buffer component may be used in a solid form by drying it in a container such as a tube. Specifically, the pretreatment buffer component can be dried using a centrifugal concentrator. The test sample is supplied to a container containing the solid pretreatment buffer component, and the pretreatment buffer component is dissolved in the test sample, thereby pretreating the test sample. The test sample in which the pretreatment buffer component is dissolved is also alkaline. When the pretreatment buffer component is used in a solid form, there is an advantage that the pretreatment process can be easily carried out, as there is no need to mix the test sample and the pretreatment buffer component in an accurate ratio.

[0027] A test paper that develops color upon dehydrogenase reaction is immersed in the pretreated test sample for approximately 1 to 30 seconds and then removed. The temperature of the test sample is not particularly limited and can be approximately 20 to 30°C. The immersion time in the test sample is preferably approximately 1 to 10 seconds, more preferably approximately 1 to 2 seconds. Excess test sample is shaken off, and after 10 to 600 seconds, preferably 60 to 180 seconds, the test paper is visually compared with the color chart. The color chart is prepared in advance using a solution with a known amino acid concentration. Specifically, a color chart can be prepared using L-alanine for Ala test paper, L-leucine, L-isoleucine, L-valine, or a mixture thereof for BCAA test paper. Furthermore, a color chart can be prepared using L-threonine for Thr test paper, and L-lysine for Lys test paper.

[0028] Depending on the structure of the test paper, it may be possible to pretreat the test sample on the test paper. In this case, the pretreatment buffer component is attached to or supported on the carrier so as not to affect the quantitative component supported on the test paper. However, it is necessary to configure the test paper so that when immersed in the test sample, the pretreatment buffer component comes into contact with the test sample before the quantitative component. For example, it is conceivable to attach the pretreatment buffer component to one end of the carrier to form a pretreatment section. The remaining area of ​​the carrier is conceivable to support the quantitative component and form a quantitative section. Alternatively, a multilayer structure in which the pretreatment section and quantitative section are stacked on top of each other is conceivable.

[0029] When using a test paper equipped with a quantification section and a pretreatment section, the pretreatment section to which the pretreatment buffer component is attached is immersed in the test sample. The temperature and immersion time of the test sample can be the same as those described above. The test sample is pretreated by contacting the test sample with the pretreatment buffer component. After pretreatment, the test sample penetrates the test paper and reaches the quantification section in an alkaline state. After a predetermined time, the color of the test paper is compared with the color chart as described above to quantify the organic compound concentration.

[0030] When using a test paper with a quantification section and a pretreatment section superimposed on each other, the test sample is brought into direct contact with the test paper. The test sample is pretreated by contacting the test sample with the pretreatment buffer component. After pretreatment, the test sample penetrates the test paper and reaches the quantification section in an alkaline state. After a predetermined time, the color of the test paper is compared with the color chart as described above to quantify the organic compound concentration.

[0031] In the quantitative determination method of the present invention, the test sample is pretreated with a predetermined pretreatment buffer component before contacting it with a test paper that develops color through a dehydrogenase reaction. Since the pretreatment buffer component is effective at a pH of 8 or higher, the test solution after pretreatment is alkaline and is used for quantification. This avoids poor color development due to interfering substances, resulting in good color development on the test paper, enabling accurate quantification of the concentration of organic compounds in the test sample. [Example]

[0032] Examples are given below to specifically describe the present invention. Materials, contents, mixing ratios, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention should not be construed in a limited manner by the specific examples shown below.

[0033] <Preparation of Ala Test Paper> The following components were dissolved in 100 mL of water as a solvent to prepare a staining solution. L-alanine dehydrogenase 7200 U NAD + 66.3 mg (1 mM) Diholase 6000 U Nitro-TB 81.8 mg (1 mM) 0.1 M TAPS-NaOH (pH 7.7) 100 mL TritonX-100 0.3 g (0.3%)

[0034] As the carrier, qualitative filter paper No. 2 (300 mm × 300 mm) made by Toyo Roshi Kaisha, Ltd. was prepared. The staining solution was placed in a metal bath, and the carrier was immersed therein for 5 seconds to impregnate the carrier with the staining solution. Next, the carrier impregnated with the staining solution was taken out and dried at 40 °C for 30 minutes using a hair dryer manufactured by Toyo Seisakusho Co., Ltd. to remove the solvent. Thus, an Ala test paper in which dehydrogenase, coenzyme, electron mediator, and reducing system chromogenic reagent were supported on qualitative filter paper as a carrier was prepared. The obtained test paper also supports a pH buffer and a reaction accelerator as optional components. [[ID=зо]]

[0035] <Example 1> Using the Ala test paper immediately after production, the concentration of alanine in the test sample was quantified. As the test samples, five types of liquids (a1 to a5) with different alanine concentrations were prepared. The alanine concentrations were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM.

[0036] First, the test sample was pretreated using Tris hydrochloride as a buffer component for pretreatment. Pretreatment was carried out by drying 1M Tris hydrochloride (pH 9) by centrifugal concentration and dissolving this in the test sample. One end of the test paper was immersed in the pretreated test sample for 2 seconds and then taken out. Excess test sample was shaken off, and the color development after 120 seconds was visually compared with the color scale.

[0037] In the case of any test sample with an alanine concentration of 0.05 - 1 mM, the color tone of the test paper after 120 seconds was comparable to the color scale. It was confirmed that the alanine concentration could be accurately quantified by pretreating the test sample before contacting it with the test paper.

[0038] <Comparative Example 1> The alanine concentration in the test samples (a1 - a5) was quantified in the same manner as in Example 1, except that the test samples were not pretreated. As a result, in the case of any test sample with an alanine concentration of 0.05 - 1 mM, the color tone of the test paper after 120 seconds did not match the color scale, and the alanine concentration could not be accurately quantified.

[0039] <Preparation of BCAA Test Paper> The following components were dissolved in 100 mL of water as a solvent to prepare a staining solution. L-Leucine dehydrogenase 4800 U NAD + 66.3 mg (1 mM) Diholase 6000 U Nitro-TB 81.8 mg (1 mM) 0.1 M TAPS - NaOH (pH 7.7) 100 mL TritonX-100 0.3 g (0.3%)

[0040] A BCAA test paper on which dehydrogenase, coenzyme, electron mediator, and reducing system color reagent were supported was prepared on qualitative filter paper as a carrier in the same manner as the above Ala test paper, except that the obtained staining solution was used. The obtained test paper also supports a pH buffer and a reaction accelerator as optional components.

[0041] <Example 2> Using the BCAA test paper immediately after preparation, the concentration of BCAA in the test sample was quantified. Five types of liquids (b1 to b5) with different concentrations of leucine as BCAA were prepared. The concentrations of leucine were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM.

[0042] The test sample was pretreated using tris hydrochloride as a buffer component for pretreatment in the same manner as in Example 1. 1 M tris hydrochloride (pH 9) was dried by centrifugal concentration and dissolved in the test sample to perform the pretreatment. One end of the test paper was immersed in the pretreated test sample for 2 seconds and then taken out. Excess test sample was shaken off, and the color development after 120 seconds was visually compared with the color scale.

[0043] In the case of any test sample with a leucine concentration of 0.05 to 1 mM, the color tone of the test paper after 120 seconds was comparable to the color scale. It was confirmed that the leucine concentration could be accurately quantified by pretreating the test sample before contacting it with the test paper.

[0044] <Comparative Example 2> The leucine concentrations in the test samples (b1 to b5) were quantified in the same manner as in Example 2, except that the test samples were not pretreated. As a result, in the case of any test sample with a leucine concentration of 0.05 to 1 mM, the color tone of the test paper after 120 seconds did not match the color scale, and the leucine concentration could not be accurately quantified.

[0045] <Preparation of Thr Test Paper> The following components were dissolved in 100 mL of water as a solvent to prepare a staining solution. L-threonine dehydrogenase 5400 U NAD + 66.3 mg (1 mM) Diholase 6000 U<0\000212>Nitro-TB 81.8 mg (1 mM) 0.1 M TAPS - NaOH (pH 7.7) 100 mL Triton X-100 0.3 g (0.3%)

[0046] Except for using the obtained staining solution, a Thr test paper in which dehydrogenase, coenzyme, electron mediator, and a reducing system chromogenic reagent were supported on qualitative filter paper as a carrier was prepared in the same manner as the above-described Ala test paper. The obtained test paper also supports a pH buffer and a reaction accelerator as optional components.

[0047] <Example 3> Using the Thr test paper immediately after preparation, the concentration of threonine in the test sample was quantified. As the test samples, five types of liquids (c1 to c5) with different threonine concentrations were prepared. The threonine concentrations were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM.

[0048] In the same manner as in Example 1, the test sample was pretreated using tris hydrochloride as a buffer component for pretreatment. 1 M tris hydrochloride (pH 9) was dried by centrifugal concentration and dissolved in the test sample to perform the pretreatment. One end of the test paper was immersed in the pretreated test sample for 2 seconds and then taken out. Excess test sample was shaken off, and the color development after 120 seconds was visually compared with the color scale.

[0049] In the case of any test sample with a threonine concentration of 0.05 to 1 mM, the color tone of the test paper after 120 seconds was comparable to the color scale. It was confirmed that the threonine concentration could be accurately quantified by pretreating the test sample before contacting it with the test paper.

[0050] <Comparative Example 3> The threonine concentration in the test samples (c1 to c5) was quantified in the same manner as in Example 3, except that the test samples were not pretreated. As a result, in the case of any test sample with a threonine concentration of 0.05 to 1 mM, the color tone of the test paper after 120 seconds did not match the color scale, and the threonine concentration could not be accurately quantified.

[0051] <Preparation of Lys Test Paper> The following components were dissolved in 100 mL of water as a solvent to prepare a staining solution. L-lysine dehydrogenase 186U NAD + 66.3mg(1mM) Diaphorase 6000U Nitro-TB 81.8mg (1mM) 0.1M TAPS-NAOH (pH 7.7) 100mL Triton X-100 0.3g (0.3%)

[0052] Lys test paper was prepared in the same manner as the Ala test paper described above, except that the staining solution was used. The test paper contained a dehydrogenase, a coenzyme, an electron mediator, and a reductive color-developing reagent, and also contained an optional pH buffer and a reaction accelerator.

[0053] Example 4 The lysine concentration in the test samples was quantified using the Lys test paper immediately after preparation. Five types of liquids (d1 to d5) with different lysine concentrations were prepared as test samples. The lysine concentrations were 0 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM.

[0054] The test sample was pretreated using Tris hydrochloride as a pretreatment buffer component in the same manner as in Example 1. 1 M Tris hydrochloride (pH 9) was evaporated to dryness by centrifugal concentration, and the dried solution was then dissolved in the test sample to perform the pretreatment. One end of the test paper was dipped into the pretreated test sample for 2 seconds, then removed. Excess test sample was shaken off, and the color developed after 120 seconds was visually compared with the color chart.

[0055] For all test samples with lysine concentrations between 0.05 and 1 mM, the color tone of the test paper after 120 seconds was comparable to that of the color chart. This demonstrates that the lysine concentration can be accurately determined by pretreating the test sample before contacting it with the test paper.

[0056] <Comparative Example 4> The lysine concentrations in the test samples (d1 to d5) were quantified in the same manner as in Example 4, except that the test samples were not subjected to pretreatment. As a result, for all test samples with lysine concentrations of 0.05 to 1 mM, the color tone of the test paper after 120 seconds did not match the color chart, and the lysine concentration could not be quantified with high accuracy.

Claims

1. 1. A method for quantifying the concentration of an organic compound in a test sample, comprising: The test sample is pretreated using a pretreatment buffer component adjusted to a pH of 8 or higher, The pretreated test sample is brought into contact with a test paper that develops color through a dehydrogenase reaction, A quantitative method that uses colorimetry.

2. 2. The method according to claim 1, wherein the pretreatment buffer component is adjusted to a pH of 8 to 11.

3. 2. The method according to claim 1, wherein the pretreatment buffer component contains Tris hydrochloride.

4. The quantitative method according to claim 1 , wherein the test sample is selected from biological samples.

5. 5. The method according to claim 4, wherein the organic compound has an amino group and a molecular weight of 1,000 or less.

6. 6. The method according to claim 5, wherein the organic compound is an amino acid.

7. 7. The method according to claim 6, wherein the amino acid is alanine, a branched-chain amino acid, threonine, or lysine.

8. 8. The method according to claim 7, wherein the branched-chain amino acid is valine, leucine, or isoleucine.

9. 2. The method according to claim 1, wherein the test paper includes a liquid-permeable carrier, and a dehydrogenase, a coenzyme, an electron mediator, and a reduction-based color-developing reagent are supported on the carrier.

10. 10. The method according to claim 9, wherein the dehydrogenase in the test paper is L-alanine dehydrogenase, L-leucine dehydrogenase, L-threonine dehydrogenase, or L-lysine dehydrogenase.

11. The amount of L-alanine dehydrogenase carried on the test paper is 5,300 to 49,000 kU / m ３ The quantitative method according to claim 10,

12. The amount of L-leucine dehydrogenase carried on the test paper is 3,300 to 33,000 kU / m ３ The quantitative method according to claim 10,

13. The amount of L-threonine dehydrogenase supported is 4,000 to 40,000 kU / m ３ The quantitative method according to claim 10,

14. The amount of L-lysine dehydrogenase supported is 133 to 1330 kU / m ３ The quantitative method according to claim 10,

15. 10. The method according to claim 9, wherein the coenzyme in the test paper is nicotinamide adenine dinucleotide.

16. The amount of the coenzyme carried on the test paper is 44 to 440 g / m ３ The quantitative method according to claim 15,

17. 10. The method according to claim 9, wherein the electron mediator in the test paper is diaphorase.

18. The amount of diaphorase carried on the test paper is 4400 to 44000 kU / m ３ The quantitative method according to claim 17,

19. 10. The method according to claim 9, wherein the reducing color-developing reagent in the test paper is a tetrazolium salt.

20. 20. The method according to claim 19, wherein the reducing color-developing reagent is nitrotetrazolium blue.

21. The amount of nitrotetrazolium blue carried on the test paper is 67 to 560 g / m ３ The quantitative method according to claim 20,

22. A concentration determination kit for determining the concentration of an organic compound in a test sample, comprising: a test paper that changes color due to a dehydrogenase reaction; a pretreatment buffer component adjusted to pH 8 or higher for pretreatment of the test sample; A kit for quantifying the concentration of an organic compound.