JP2000319238A - Chromogen for oxidizable color-developing reagent, determination method using the chromogen and determination reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound useful as a chromogen for an oxidizable color-developing reagent forming a pigment having sufficiently high stability of color development and capable of suppressing the error of the measured value caused by the components coexisting in a biospecimen such as bilirubin, hemoglobin and ascorbic acid. SOLUTION: The objective compound is expressed by formula I (R1 is a 1-3C alkoxy; R2 is H or a 1-3C alkyl; R3 is a carboxyl-substituted 1-3C alkyl), e.g. N-(2-carboxyethyl)-N-ethyl-3-methoxyaniline. The compound of formula I can be produced e.g. by reacting a compound of formula II with a compound of formula C-R2 (X is a halogen), reacting the resultant compound of formula III with a compound of formula IV (R4 and R5 are each H or an alkyl; R6 is an alkyl), etc., in the presence of an alkali metal hydroxide, etc., to obtain a compound of formula V, etc., adding and reacting an aqueous solution of sodium hydroxide to the obtained compound, acidifying the product with hydrochloric acid and finally purifying the product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 3-alkoxyaniline derivative or a salt thereof, and a method and a reagent for measuring an oxidizing substance or a peroxidase-like substance using the same as a chromogen. The invention is particularly directed to chemistry,
It is useful in fields such as life science and clinical testing.

[0002]

2. Description of the Related Art Measuring biological components contained in biological samples such as blood and urine is very useful in diagnosing diseases, and enzymatic measurement methods have become widespread in clinical tests. Measurement methods have been developed. As such a method, for example, using an oxidizing enzyme, hydrogen peroxide which is an oxidizing substance is directly or indirectly generated from a biological component, and mixed with and contacted with peroxidase and an oxidizable coloring reagent. A method for measuring the amount of hydrogen peroxide generated by optically measuring a dye generated from an oxidizable color-forming reagent by an oxidative condensation reaction, which leads to a color forming system, and thereby measuring a biological component contained in a biological sample. Can be mentioned. For example, in measuring uric acid, glucose, and cholesterol, etc., uricase, glucose oxidase, and oxidizing enzymes such as cholesterol oxidase act to generate hydrogen peroxide, and the generated hydrogen peroxide is used as a peroxidase and an oxidizable color reagent. , And the mixture is brought into contact with a chromogenic system, and is measured by optically measuring a dye generated from an oxidizable color forming reagent by an oxidative condensation reaction, whereby each biological component can be accurately measured.

Conventionally, oxidizable color-forming reagents used in this method mainly include a coupler such as 4-aminoantipyrine and a chromogen such as phenol or a derivative thereof or an aniline derivative as a condensation target. Combinations have been used. However, these oxidizable color-forming reagents are poor in coloration stability due to large fading after color development, and include bilirubin and hemoglobin in biological samples.
Due to the influence of coexisting substances such as ascorbic acid, there are disadvantages such as easy measurement error, low sensitivity and insufficient measurement of trace components.Therefore, a better chromogen is required. Was.

Therefore, Japanese Patent Publication No. 57-27106,
JP-B-58-9094 and JP-B-2-1519
No. 8 proposes various aniline derivatives as chromogens to solve these problems. However, oxidizable color reagents using these aniline derivatives as chromogens have good water-solubility of the generated dye and high color-forming sensitivity, but have high color fading due to large fading of the generated dye. It is insufficient or easily affected by coexisting substances such as bilirubin, hemoglobin, and ascorbic acid in a biological sample, and is not always satisfactory for accurate measurement.

[0005] Japanese Patent Application Laid-Open Nos. 4-202164 and 5-262716 propose a p-fluoroaniline derivative in which a fluoro group is introduced into an aniline derivative as a chromogen. However, the synthesis of p-fluoroaniline derivatives is not common and is very complicated, and it is difficult to produce them at a high cost.

Further, recently, when a biological sample is measured using an oxidizable color reagent using these chromogens,
Sodium azide contained in other reagents as a preservative vaporizes as hydrogen azide and dissolves in the oxidizable color-forming reagent containing the chromogen in the vicinity, and despite the absence of the desired biological component Chromogenic N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (abbreviation: HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (abbreviation: HDAP)
S), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (abbreviation: DA
OS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (abbreviation: DAPS), N-ethyl-N
-(2-hydroxy-3-sulfopropyl) -3,5-
Dimethoxy-4-fluoroaniline, N-ethyl-N-
An aniline derivative having an electron donating group such as sulfopropyl-3,5-dimethoxy-4-fluoroaniline or N- (2-carboxyethyl) -N-ethyl-3,5-dimethoxyaniline (abbreviation: CEDB) is used. It is known that the color is developed and cannot be used for measurement.

In an automatic analyzer, a component contained in a reagent such as an azide adheres to a reagent probe (reagent collection nozzle) of the automatic analyzer or the like, so that the reagent probe collects the next reagent. It is known that an azide or the like attached to a reagent probe is mixed into the reagent and causes the aniline derivative having an electron donating group such as the chromogen described above to develop a color, which cannot be used for measurement. I have.

[0008]

DISCLOSURE OF THE INVENTION In view of the above-mentioned problems in the prior art, the present invention has sufficient coloring stability of the dye to be produced, and has a bilirubin, hemoglobin, ascorbic acid, and the like in a biological sample. Oxidation that can suppress the occurrence of errors in measured values caused by coexisting substances, and also prevent reagent degradation and function degradation due to the dissolution of components contained in nearby reagents such as azide into the reagents It is an object of the present invention to provide a chromogen for a coloring reagent, a measuring method using the same, and a measuring reagent.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that a 3-alkoxyaniline derivative represented by the following chemical formula (1) or a salt thereof can be used as a chromogen. When used as a body and used in combination with a coupler, the coloration of the resulting dye is extremely stable,
In addition, it is possible to suppress the occurrence of errors in measured values caused by coexisting substances such as bilirubin, hemoglobin, and ascorbic acid in a biological sample. The present invention has been completed by confirming that it is possible to prevent the deterioration of the reagent and the decrease in the function due to the dissolution into the reagent and to accurately measure the oxidizing substance or the peroxidase-like substance.

Accordingly, the present invention provides the following chemical formula (1)

Embedded image (Wherein, R ₁ represents an alkoxy group having 1 to 3 carbon atoms;
₂ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,
R ₃ represents an alkyl group having 1 to 3 carbon atoms substituted with a carboxyl group. ), Or a 3-alkoxyaniline derivative or a salt thereof.

Further, the present invention is a method for measuring an oxidizing substance, wherein the chromogen and the coupler comprising the above-mentioned 3-alkoxyaniline derivative or a salt thereof are used as an oxidizable color-forming reagent.

Further, the present invention is a method for measuring a peroxidase-like substance, wherein the chromogen and coupler comprising the above-mentioned 3-alkoxyaniline derivative or a salt thereof are used as an oxidizable color-forming reagent. In the measurement method of the present invention, it is preferable that the coupler is 4-aminoantipyrine.

Further, the present invention is a reagent for measuring an oxidizing substance, which comprises the chromogen and the coupler comprising the 3-alkoxyaniline derivative or a salt thereof as an oxidizable color-forming reagent.

Further, the present invention is a reagent for measuring a peroxidase-like substance, comprising the chromogen and the coupler comprising the 3-alkoxyaniline derivative or a salt thereof as an oxidizable color-forming reagent.

In the measuring reagent of the present invention, the coupler is preferably 4-aminoantipyrine.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, in a 3-alkoxyaniline derivative represented by the chemical formula (1) or a salt thereof, R ₁ is, for example, a C ₁ to C 3 -carbon atom such as a methoxy group, an ethoxy group, and a propoxy group. And an alkoxy group of Here, this alkoxy group may be either straight-chain or branched. R ₂ is a hydrogen atom or a group having 1 to 3 carbon atoms such as a methyl group, an ethyl group, and a propyl group.
Alkyl group. Here, this alkyl group may be either straight-chain or branched. Examples of R ₃ include an alkyl group substituted with a carboxyl group having 1 to 3 carbon atoms such as a carboxymethyl group, a carboxyethyl group, and a carboxypropyl group. Here, carbon number 1
The alkyl group substituted with carboxyl groups of Nos. 1 to 3 may be either linear or branched, and the position of substitution of the carboxyl group is not particularly limited.

More specifically, R ₁ is OCH ₃ ,
OC ₂ H ₅ or _OC 3 _{H 7,} and examples of _{R 2} is _{H, CH 3, CH 2 CH} 3, CH 2 CH 2 CH 3 or C
H (CH ₃ ) ₂ and R ₃ is CH ₂ COO
H, CH ₂ CH ₂ COOH, CH ₂ CH ₂ CH ₂ COO
H, CH (CH ₃ ) CH ₂ COOH or CH ₂ CH (C
H ₃ ) COOH.

Examples of the salt of the 3-alkoxyaniline derivative represented by the chemical formula (1) include a metal salt such as a sodium salt, a potassium salt, and a calcium salt, an ammonium salt, and a triethylamine salt. .

In the present invention, the 3-alkoxyaniline derivative represented by the chemical formula (1) can be synthesized, for example, as follows. That is, the chemical formula (2)
(Wherein, R ₁ is the same as described above) and 1 to 3 mol of the chemical formula (3) (in the formula, X represents a halogen atom; R ₂ is the same as described above) based on 1 mol of the compound represented by the formula: The compound shown is reacted in a solvent such as an alcohol such as methanol, ethanol or isopropanol, or a cyclic ether such as 1,4-dioxane or tetrahydrofuran (THF) at 10 to 100 ° C. for 1 to 50 hours.

Embedded image

Embedded image Next, purification is carried out by a conventional method, and the chemical formula (4) (wherein R
₁ and _{R 2} are as defined above. ) Is obtained.

Embedded image

Next, 1 to 3 mol of the compound of the formula (5) (wherein R ₄
And R ₅ each represent a hydrogen atom or an alkyl group, and R ₆ represents an alkyl group. ), Alcohols such as methanol, ethanol or isopropanol,
Alternatively, 1,4-dioxane, tetrahydrofuran (TH
Dissolve in a mixture of cyclic ethers such as F) and water.

Embedded image 1 to 3 equivalents of an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide or alkali metal carbonate such as sodium carbonate or potassium carbonate for 1 equivalent of the compound represented by the chemical formula (4) After adding a salt and reacting at 10 to 100 ° C. for 1 to 50 hours, purification is carried out by a conventional method, and the chemical formula (6) (where R ₁ , R ₂ ,
R ₄ , R ₅ and R ₆ are the same as described above. ) Is obtained.

Embedded image

Next, an aqueous solution of 1 to 5 mol of sodium hydroxide is added to 1 mol of the compound represented by the chemical formula (6), and the mixture is reacted at room temperature for 1 to 10 hours. By purifying by the conventional method, 3
-An alkoxyaniline derivative or a salt thereof is obtained.

In the case of synthesizing a compound in which R ₂ in the chemical formula (1) is a hydrogen atom, only necessary steps among the reaction steps of the above-described synthesis method need to be performed.

The compound represented by the chemical formula (2) used for producing the 3-alkoxyaniline derivative or a salt thereof according to the present invention is widely commercially available, and therefore, a commercially available product may be used as it is.

The method and reagent for measuring an oxidizing substance according to the present invention use or contain a chromogen and a coupler comprising the 3-alkoxyaniline derivative of the above formula (1) or a salt thereof as an oxidizable coloring reagent. It is characterized by the following.

When the 3-alkoxyaniline derivative of the present invention or a salt thereof is used as a chromogen and used in combination with a coupler, a colorant whose coloration is extremely stable is produced. This dye can suppress the influence of a coexisting substance in a biological sample. Therefore, the 3-alkoxyaniline derivative or a salt thereof of the present invention is suitable as a chromogen of an oxidizable color-forming reagent in the measurement of an oxidizing substance or the measurement of a peroxidase-like substance.

The biological component which can be measured using the measuring method and the measuring reagent of the present invention is to measure by measuring an oxidizing substance such as hydrogen peroxide generated directly or indirectly by an enzymatic reaction. And a peroxidase-like substance. For example, uric acid, glucose, cholesterol, triglyceride, phospholipid, cholinesterase and the like can be mentioned.

Further, it is presumed that the sample in the measuring method and the measuring reagent of the present invention contains the above-mentioned biological component, and the presence or absence of the biological component in the sample or the measurement of the biological component concentration is performed. And the like. For example, human or animal blood, serum, plasma,
Body fluids such as urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, and amniotic fluid;
Human or animal pancreas, organs such as liver, hair, skin,
Extracts of nails, muscles, or nerve tissues, etc .; human or animal feces extracts or suspensions; cell extracts; plant extracts, and the like.

In the measuring method and the measuring reagent of the present invention, the 3-alkoxyaniline derivative of the present invention or a salt thereof, for example, 4-aminoantipyrine or a derivative thereof,
A conventionally known enzymatic measurement method and a measurement reagent, for example, except that a combination with a coupler such as phenylenediamine and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is used as an oxidizable color reagent. ,
Using an oxidase that generates hydrogen peroxide, directly generate hydrogen peroxide from a biological component, or by reacting an oxidase that generates hydrogen peroxide on a product obtained by an enzymatic reaction from the biological component, Indirectly producing hydrogen peroxide,
The resulting hydrogen peroxide is mixed with peroxidase and an oxidizable color reagent, brought into contact with the chromogenic system, and a biological component is measured by optically measuring a dye generated from the oxidizable color reagent by an oxidative condensation reaction. What is necessary is just to follow a measuring method and a measuring reagent. As the coupler in the measuring method and the measuring reagent of the present invention, 4-aminoantipyrine or a derivative thereof is particularly preferable.

In the measuring method and the measuring reagent of the present invention,
The peroxidase used is preferably derived from horseradish or a microorganism at a concentration of 20 units / L or more. The concentration of the oxidizable color forming reagent used is such that the 3-alkoxyaniline derivative of the present invention or a salt thereof as a chromogen preferably has a concentration in the range of 0.05 to 20 mM, particularly preferably 0.2 to 5 mM. Concentration range,
The couplers may be suitably combined in a concentration range of preferably 0.05 to 10 mM, particularly preferably 0.1 to 5 mM.

In the measuring method and the measuring reagent of the present invention,
The pH of the measurement reaction solution is preferably in the range of pH 4.0 to 10.0, and particularly preferably in the range of pH 6.0 to 8.0. As the buffer, it is preferable to appropriately use a buffer having a buffering capacity in the above-mentioned pH range. Such buffers include, for example, phosphate buffer, Tris,
Imidazole, glycylglycine, MES, Bis-T
ris, ADA, ACES, Bis-Tris propane, PIPES, MOPSO, MOPS, BES, HE
PES, TES, DIPSO, TAPSO, POPS
O, HEPPS, HEPPSO, Tricine, Bi
cine, TAPS and the like.

The measuring method and the measuring reagent of the present invention may further contain a stabilizer, an activator, a preservative, or other reagent components.

As the stabilizing agent and the activating agent, for example, a coenzyme, a surfactant, an alkali metal ion and the like may be contained.

As a preservative, for example, benzoic acid, a synthetic antibacterial agent or an antibiotic may be added.

In the measuring method and the measuring reagent of the present invention,
The oxidizing substance refers to a substance that undergoes the action of peroxidase together with the chromogen and the coupler to generate a dye. Such substances include, for example, hydrogen peroxide, periodic acid and the like.

The 3-alkoxyaniline derivative of the present invention or a salt thereof can be used for measurement of a peroxidase-like substance in combination with a coupler. Here, the peroxidase-like substance refers to a substance that acts on the oxidizing substance, the chromogen, and the coupler to generate a dye. For example, peroxidase, hemoglobin, other heme compounds and the like can be mentioned. Further, the 3-alkoxyaniline derivative or a salt thereof of the present invention may be, for example, an enzyme immunoassay using peroxidase as a labeling compound, or an oxidizing substance such as hydrogen peroxide or sodium periodate in hemoglobin in serum. It can also be used in the case of using and measuring.

In the measuring reagent of the present invention, the shape of the reagent may be any shape such as a liquid reagent, a lyophilized preparation, a test piece or a film type carrier.

[0037]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Synthesis of N- (2-carboxyethyl) -N-ethyl-3-methoxyaniline [compound of the present invention] 34.1 g of 60% sodium hydride was suspended in 720 mL of 1,4-dioxane. And heated to 80 ° C., to which 100 g of m-anisidine was added dropwise over 1 hour, and then 92.7 g of ethyl bromide was added dropwise over 1 hour.
The reaction was performed at 100 ° C. for 8 hours. After completion of the reaction, the mixture was allowed to cool, 5 L of water was added, and the mixture was extracted with 3 L of ethyl acetate and concentrated.

Next, distillation was performed under reduced pressure to obtain oily N-ethyl-3.
-92 g of methoxyaniline were obtained. Next, 59.2 g of methyl acrylate and 5.5 g of acetic acid were added to 90 g of the N-ethyl-3-methoxyaniline, and the mixture was refluxed at 110 ° C. for 20 hours. After concentration, a silica gel column (ethyl acetate:
Hexane = 1: 9 using an elution solvent) to give oily N- (2-carboxyethyl) -N-ethyl-3-
90 g of methoxyaniline methyl ester were obtained.

This N- (2-carboxyethyl) -N-
After adding 280 mL of 2N sodium hydroxide to 90 g of ethyl-3-methoxyaniline methyl ester, the mixture was refluxed for 2 hours. This was cooled on ice, and 60 mL of concentrated hydrochloric acid was added thereto to adjust the pH to 4.
0 was set. Then, extraction and concentration were performed using 1 L of ethyl acetate, and N- (2-carboxyethyl) -N-ethyl-
3-methoxyaniline [CEMO; compound of the present invention] 26
g was obtained.

[Example 2] Confirmation of color stability in uric acid measurement reagent N- (2-carboxyethyl)-synthesized in Example 1
A first reagent for measuring uric acid containing N-ethyl-3-methoxyaniline [CEMO; the compound of the present invention] or another aniline derivative as a chromogen, and a first reagent for measuring uric acid containing 4-aminoantipyrine as a coupler. A uric acid measurement reagent composed of the second reagent was prepared, and the stability of coloration at the time of measurement was confirmed.

(1) Preparation of Reagent Preparation of the First Reagent for Uric Acid Measurement of the Present Invention: The following reagent components for measurement were dissolved in pure water so as to have the respective concentrations described below.
The pH was adjusted to 7.0 (20 ° C). Reagent components for measurement Concentration 3- (N-morpholine) propanesulfonic acid [MOPS] 50 mM N- (2-carboxyethyl) -N-ethyl-3-methoxyaniline [CEMO / chromogen] 1 mM peroxidase 1000 units / L ascorbin Acid oxidase 1500 units / L surfactant 0.2%

Control 1: Preparation of first reagent for uric acid measurement: Chromogen was converted to N- (2-carboxyethyl) -N-ethyl-3
Preparation was performed using the same reagent components and concentrations as those of the above-mentioned first reagent for measuring uric acid of the present invention, except that -methylaniline (abbreviation: CEMB) was used.

Control 2: Preparation of First Reagent for Uric Acid Measurement The chromogen was N- (2-hydroxy-3-sulfopropyl)
-N-ethyl-3,5-dimethylaniline [abbreviation: MA
OS] was prepared using the same reagent components and concentrations as those of the first reagent for measuring uric acid of the present invention described above.

Control 3 Preparation of First Reagent for Uric Acid Measurement The chromogen was converted to N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline [abbreviation: TOOS]
Except for changing to the above, the first method for measuring uric acid of the present invention described above.
Preparation was performed using the same measurement reagent components and concentrations as the reagents.

Control 4 Preparation of First Reagent for Uric Acid Measurement The chromogen was converted to N- (2-hydroxy-3-sulfopropyl)
-3,5-dimethoxyaniline sodium [abbreviation: HD
AOS] was prepared using the same reagent components and concentrations as those of the above-mentioned first reagent for measuring uric acid of the present invention, except for changing to AOS].

Control 5 Preparation of First Reagent for Uric Acid Measurement The chromogen was converted to N- (2-sulfopropyl) -N-ethyl-3.
Preparation was performed using the same reagent components and concentrations as in the above-described first reagent for measuring uric acid of the present invention, except that -methoxyaniline (abbreviation: ADPS) was used.

The compounds used as chromogens in the present invention and the control first reagent for uric acid measurement are shown in Table 1.

[0049]

[Table 1]

Preparation of Second Reagent for Uric Acid Measurement The following reagent components for measurement were dissolved in pure water so as to have the concentrations described, respectively, and adjusted to pH 7.0 (20 ° C.). Measurement reagent components Concentration 3- (N-morpholine) propanesulfonic acid [MOPS] 50 mM 4-aminoantipyrine (coupler) 2.5 mM uricase 1000 units / L surfactant 0.2%

(2) Confirmation of Color Stability 6 mg of lithium carbonate was dissolved in 90 mL of pure water, and 15 mg of uric acid was added to completely dissolve it. This was made up to 100 mL with pure water to prepare a sample having a uric acid concentration of 15 mg / dL. The uric acid level in this sample was measured with each of the first and second reagents for measuring uric acid of the present invention and Controls 1 to 5 prepared in (1) above, and the respective reagents were used. The stability of the coloring was confirmed.

The measurement of uric acid in this sample was carried out by an automatic analyzer 7150 manufactured by Hitachi, Ltd., and 320 μL of a first reagent for uric acid measurement was added to 5 μL of the sample, and reacted at 37 ° C. for 5 minutes. The reaction was started at 37 ° C. by adding 80 μL of the two reagents. Main wavelength 600 nm and sub wavelength 700
The absorbance in nm at 2 minutes after the addition of the second reagent (35 points) and the absorbance at 5 minutes after the addition of the second reagent (50 points) were measured.

(3) Results Table 2 shows the results concerning the stability of coloration of each uric acid measurement reagent.

[0054]

[Table 2]

As can be seen from the table, when the compound of the present invention was used as a chromogen, the absorbance hardly changed. In contrast, when the first reagent for uric acid measurement of Controls 1 to 3 was used,
In each case, it can be seen that the absorbance has decreased.
From these, it was confirmed that the uric acid measurement reagent containing the chromogen and the coupler comprising the compound of the present invention as an oxidizable color-forming reagent has extremely high color stability.

Example 3 Confirmation of the Influence of Coexisting Substances in the Sample at the Time of Uric Acid Measurement The first reagent for measuring uric acid containing the compound of the present invention or another aniline derivative as a chromogen, and a coupler 4
A uric acid measurement reagent composed of a second reagent for uric acid measurement containing aminoantipyrine was prepared, and the effect of coexisting substances contained in the sample was examined.

(1) Reagent The first reagent for measuring uric acid of the present invention: The first reagent for measuring uric acid prepared in Example 2 was used as it was.

Control 1: Preparation of first reagent for uric acid measurement: The first reagent for uric acid measurement prepared in Example 2 was used as it was.

Control 2 Preparation of First Reagent for Uric Acid Measurement The first reagent for uric acid measurement prepared in Example 2 was used as it was.

Control 3 Preparation of First Reagent for Uric Acid Measurement The first reagent for uric acid measurement prepared in Example 2 was used as it was.

Preparation of second reagent for uric acid measurement The second reagent for uric acid measurement prepared in Example 2 was used as it was.

(2) Preparation of sample Bilirubin-added sample Bilirubin concentration of 200 mg / dL was added to 9 mL of human serum.
Interference check A ・ Bilirubin C (manufactured by Kokusai Reagents) 1m
L was mixed to obtain a bilirubin-added sample to which bilirubin 20 mg / dL was added. Ascorbic acid-added sample Ascorbic acid concentration of 2000 mg in 9 mL of human serum
/ DL of an aqueous solution of ascorbic acid (1 mL) was mixed to obtain an ascorbic acid-added sample to which ascorbic acid (200 mg / dL) was added. Hemoglobin-added sample A hemoglobin concentration of 5000 mg /
dL interference check A hemoglobin (manufactured by International Reagents)
1 mL was mixed to obtain a hemoglobin-added sample to which 500 mg / dL of hemoglobin was added. Control Sample 9 mL of human serum was mixed with 1 mL of pure water to prepare a control sample. The uric acid concentration in the control sample is the same as the uric acid concentration in the bilirubin-added sample, ascorbic acid-added sample, and hemoglobin-added sample. Uric acid standard solution 6 mg of lithium carbonate was dissolved in 90 mL of pure water, and 15 mg of uric acid was further added to completely dissolve it. 10 volumes with pure water
The volume was adjusted to 0 mL to obtain a uric acid standard solution having a uric acid concentration of 15 mg / dL.

(3) Confirmation of the effect of the coexisting substance in the sample on the measured value The four types of samples (bilirubin-added sample, ascorbic acid-added sample, hemoglobin-added sample,
The uric acid value in the control sample) was determined using the first reagent for uric acid measurement of the present invention and Controls 1 to 3 prepared in (1) above, and
The measurement was performed with each of the reagents.
The effect of the coexisting substance in the sample when using the above reagent was confirmed.

The measurement of uric acid in the sample (2) was carried out using an automatic analyzer 7150 manufactured by Hitachi, Ltd.
After adding 320 μL of a uric acid measurement first reagent to the mixture and reacting at 37 ° C. for 5 minutes, 80 μL of a uric acid measurement second reagent was added and the reaction was started at 37 ° C. The main wavelength of 600 nm and the sub-wavelength are the absorbance at the time of measuring the uric acid standard solution of a known concentration from the increase in absorbance at 800 nm immediately before the addition of the second reagent (24 points) and 5 minutes after the addition of the second reagent (50 points). The uric acid value was determined by proportional calculation with The absorbance obtained when pure water was used as the sample was used as the reagent blank value, and the absorbance obtained by subtracting the reagent blank value from the absorbance immediately before the addition of the second reagent and the absorbance 5 minutes after the addition of the second reagent was used to calculate the uric acid value. Using.

(4) Results Using the first reagent for measuring uric acid of the present invention, the first reagent for measuring uric acid of Controls 1 to 3, and the second reagent for measuring uric acid, the four types of samples described above (bilirubin-added sample, Table 3 shows the measurement results when uric acid in the ascorbic acid-added sample, hemoglobin-added sample, and control sample) was measured. In the table, the upper part shows the measured value of uric acid obtained by the measurement, and the lower part shows the percentage of the measured value of uric acid with respect to the measured value of the control sample.

[0066]

[Table 3]

From this table, it is found that the measurement reagents of Controls 1 to 3
While the coexisting substances (bilirubin, ascorbic acid, hemoglobin) in the sample are affected, the measurement reagent of the present invention is not affected by the coexisting substances in the sample at all, and an accurate uric acid measurement value can be obtained. It was confirmed that.

Example 4 Confirmation of Influence of Coexisting Substances in Sample on Total Cholesterol Measurement Reagent A first reagent for measuring total cholesterol containing the compound of the present invention or another aniline derivative, and a coupler 4
A total cholesterol measuring reagent composed of a second reagent for measuring total cholesterol containing aminoantipyrine was prepared, and the effect of coexisting substances contained in the sample was examined.

(1) Preparation of Reagent Preparation of First Reagent for Measurement of Total Cholesterol The following reagent components for measurement were dissolved in pure water so as to have the concentrations described respectively, and the pH was adjusted to 7.0 (20 ° C.). Measurement reagent components Concentration N- (2-acetamido) iminodiacetic acid [ADA] 100 mM 4-aminoantipyrine (coupler) 0.44 mM cholesterol esterase 400 units / L ascorbate oxidase 300 units / L surfactant 0.1%

Preparation of Second Reagent for Measurement of Total Cholesterol of the Present Invention The following reagent components for measurement were dissolved in pure water so as to have the concentrations described respectively, and adjusted to pH 7.0 (20 ° C.). Measurement reagent components Concentration N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid [TES] 20 mM N- (2-carboxyethyl) -N-ethyl-3-methoxyaniline [CEMO / chromogen] 1 mM cholesterol Oxidase 300 units / L Peroxidase 1600 units / L Sodium azide 0.01% Surfactant 0.1%

Preparation of Control / Second Reagent for Measurement of Total Cholesterol The chromogen was converted to N- (2-sulfopropyl) -N-ethyl-3.
Preparation was performed using the same measurement reagent components and concentrations as those of the above-mentioned second reagent for measuring total cholesterol of the present invention, except that -methoxyaniline (abbreviation: ADPS) was used.

(2) Preparation of sample Bilirubin-added sample Bilirubin concentration of 500 mg / dL was added to 9 mL of human serum.
Interference check A ・ Bilirubin C (manufactured by Kokusai Reagents) 1m
L was mixed to obtain a bilirubin-added sample to which bilirubin 50 mg / dL was added.

Control Sample 9 mL of human serum was mixed with 1 mL of pure water to prepare a control sample. The total cholesterol concentration in the control sample is the same as the total cholesterol concentration in the bilirubin-added sample.

(3) Confirmation of the influence of the coexisting substance in the sample on the measured value The total cholesterol value in the two types of samples (bilirubin-added sample and control sample) prepared in the above (2) was determined by the above (1). The measurement was performed using the first reagent for measuring total cholesterol and the second reagent for measuring total cholesterol of the present invention and the control prepared in the above, and the measurement reagent of the present invention and the control measuring reagent were used. The effect of coexisting substances in the sample was confirmed.

The measurement of the total cholesterol in the sample of the above (2) was carried out by an automatic analyzer 7150 manufactured by Hitachi, Ltd., and the first reagent for total cholesterol measurement was added to 4 μL of the sample.
After adding 00 μL and reacting at 37 ° C. for 5 minutes, 100 μL of a second reagent for measuring total cholesterol was added and the reaction was started at 37 ° C. Main wavelength 600nm and sub wavelength 700n
m from the increase in absorbance immediately before the addition of the second reagent (24 points) and 5 minutes after the addition of the second reagent (50 points),
The total cholesterol value was determined by proportional calculation with the absorbance when measuring a known concentration of the total cholesterol standard solution.
The absorbance obtained when pure water was used as a sample was used as the reagent blank value, and the absorbance obtained by subtracting the reagent blank value from the absorbance immediately before the addition of the second reagent and the absorbance 5 minutes after the addition of the second reagent was used to calculate the total cholesterol value. It was used for.

(4) Results Using the first reagent for measuring total cholesterol, the second reagent for measuring total cholesterol of the present invention, and the second reagent for measuring total cholesterol as a control, total cholesterol in the two kinds of samples described above was used. Table 4 shows the results of the measurement. The upper row in the table shows the measured values obtained by measuring the total cholesterol, and the lower row shows the percentage of the total cholesterol measured value to the measured value of the control sample.

[0077]

[Table 4]

From the table, it can be seen that the negative error due to bilirubin is as high as 5% in the control measurement reagent, whereas it is only 2% in the measurement reagent of the present invention.

From the above, the measurement of total cholesterol using the first reagent for measuring total cholesterol and the second reagent for measuring total cholesterol of the present invention was hardly affected by bilirubin, and an accurate total cholesterol value was obtained. It was confirmed that it could be done.

Example 5 Demonstration of the Effect of Preventing the Deterioration of the Reagent by Dissolution of Gas The use of the sodium azide-containing reagent for vaporizing azide and the measurement reagent of the present invention, The degree of color development and deterioration due to penetration was confirmed.

(1) Preparation of Reagents Reagents containing sodium azide The following measurement reagent components were dissolved in pure water so as to have the respective concentrations described, and adjusted to pH 5.2 (20 ° C.). Measurement reagent component Concentration N- (2-hydroxyethylpiperazine) -N'-2-ethanesulfonic acid [HEPES] 100 mM Sodium azide 10 mM

Preparation of the First Reagent for Uric Acid Measurement of the Present Invention The following reagent components for measurement were dissolved in pure water so as to have the respective concentrations described below, and adjusted to pH 7.0 (20 ° C.). Measurement reagent components Concentration N- (2-hydroxyethylpiperazine) -N'-2-ethanesulfonic acid [HEPES] 100 mM N- (2-carboxyethyl) -N-ethyl-3-methoxyaniline [CEMO / chromogen ] 2 mM peroxidase 3000 units / L ascorbate oxidase 3000 units / L surfactant 0.2%

Control 1—Preparation of First Reagent for Uric Acid Measurement The chromogen was N- (2-hydroxy-3-sulfopropyl)
-3,5-dimethoxyaniline sodium [abbreviation: HD
AOS] was prepared using the same measurement reagent components and concentrations as the uric acid measurement first reagent of the present invention, except for changing to AOS].

Control 2 Preparation of First Reagent for Uric Acid Measurement The chromogen was converted to N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline [abbreviation: D
AOS] was prepared using the same measurement reagent components and concentrations as the uric acid measurement first reagent of the present invention, except for changing to AOS].

Control 3 Preparation of First Reagent for Uric Acid Measurement The chromogen was converted to N- (2-carboxyethyl) -N-ethyl-
The preparation was carried out with the same measuring reagent components and concentrations as the above-mentioned first reagent for measuring uric acid of the present invention, except that 3,5-dimethoxyaniline [abbreviation: CEDB] was used.

Preparation of Second Uric Acid Measurement Reagent The following measurement reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.). Measurement reagent components Concentration N- (2-hydroxyethylpiperazine) -N'-2-ethanesulfonic acid [HEPES] 100 mM 4-aminoantipyrine 2.5 mM uricase 1000 units / L Surfactant 0.2%

(2) Storage of Reagents for Stability Study The sodium azide-containing reagent prepared in the above (1), the first reagent for uric acid measurement of the present invention and Controls 1 to 3, and the second reagent
Reagents were placed in test tubes (volume 10 mL,
Dispense into 105 mm length, 13 mm inside diameter), put in a plastic bag (135 mm x 85 mm; with a zipper) in the following combination, close and seal the zipper, and seal at 25 ° C for 30
Saved for days. In addition, the mouth of each test tube was not plugged. 1) The present invention: the first reagent for measuring uric acid, the second reagent and the reagent containing sodium azide 2) the control 1 the first reagent for measuring uric acid, the second reagent and the reagent containing sodium azide 3) the control 2 for the measurement of uric acid First reagent, second reagent and sodium azide-containing reagent 4) Control 3-first reagent, second reagent and sodium azide-containing reagent for uric acid measurement

(3) Judgment of stability of stored reagents At the start of storage, and after 1 day, 5 days, 12 days, and 17 days
After one day and 30 days, it was visually determined whether or not the first reagents for measuring uric acid of the present invention and Controls 1 to 3 were colored.

(4) Results of Judgment of Stability Table 5 shows the results of judgment of color development regarding the stability of the reagent.

[0090]

[Table 5]

From this table, it is found that the first to third uric acid measurements of Controls 1 to 3 were performed.
It can be seen that the reagent has already developed color 5 days after storage, whereas the first reagent for uric acid measurement of the present invention has not developed color even after 30 days of storage.

Thus, in the measuring reagent of the present invention using the compound of the present invention as a chromogen, even if the azide vaporized from the sodium azide-containing reagent dissolves in the measuring reagent of the present invention, the deterioration and the function of the reagent are prevented. It has been confirmed that the decrease of the temperature can be prevented.

[0093]

According to the present invention, the 3-alkoxyaniline derivative represented by the above-mentioned chemical formula (1) or a salt thereof is used as a chromogen and the measuring method and the measuring reagent used in combination with a coupler produce a dye. Is extremely stable. In addition, it is possible to suppress the occurrence of an error in the measured value caused by the coexisting substance such as bilirubin, hemoglobin, and ascorbic acid in the biological sample, and furthermore, the component contained in a reagent such as azide or the like in the vicinity. Can be prevented from deteriorating and reducing the function of the reagent due to dissolution in the reagent, whereby the oxidizing substance or the peroxidase-like substance can be accurately measured.

[Brief description of the drawings]

FIG. 1 is a drawing showing a method of storing reagents for studying stability.

 ──────────────────────────────────────────────────続 き Continuing on the front page F term (reference) 2G054 AA06 AA07 AB02 CA10 CA26 CA28 CE01 EA04 EB02 JA11 4B063 QA01 QA19 QQ02 QQ03 QQ08 QQ09 QQ22 QQ65 QQ76 QQ79 QQ89 QR41 QR66 QS02 QX01 4H006 AA01 AB30 AB10 AB03 AB

Claims (7)

[Claims] 1. The following chemical formula (1): (Wherein, R ₁ represents an alkoxy group having 1 to 3 carbon atoms;
₂ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,
R ₃ represents an alkyl group having 1 to 3 carbon atoms substituted with a carboxyl group. A) 3-alkoxyaniline derivative or a salt thereof. 2. A method for measuring an oxidizing substance, comprising using the chromogen and coupler comprising the 3-alkoxyaniline derivative or a salt thereof according to claim 1 as an oxidizable color-forming reagent. 3. A method for measuring a peroxidase-like substance, comprising using the chromogen and coupler comprising the 3-alkoxyaniline derivative or a salt thereof according to claim 1 as an oxidizable color-forming reagent. 4. The method according to claim 2, wherein the coupler is 4-aminoantipyrine. 5. A reagent for measuring an oxidizing substance, comprising a chromogen and a coupler comprising the 3-alkoxyaniline derivative or a salt thereof according to claim 1 as an oxidizable color developing reagent. 6. A reagent for measuring a peroxidase-like substance, comprising a chromogen and a coupler comprising the 3-alkoxyaniline derivative or a salt thereof according to claim 1 as an oxidizable color-forming reagent. 7. The measuring reagent according to claim 5, wherein the coupler is 4-aminoantipyrine.