JP2001008698A - Oxidative coloring reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject novel oxidative color-developing reagent that comprises a specific aniline derivative, has no self-coloring properties and is useful for producing an aqueous solution of oxidative color development for automated analyzers utilizing the enzymatic analyses of body compositions, for example, glucose, cholesterol, uric acid or the like. SOLUTION: This novel compound is represented by the formula (R1 is a sulfonic acid-substituted 3-4C alkyl group, a sulfonic acid-substituted 3-4C hydroxyalkyl group; R2 and R3 are each H, a 1-4C alkyl, a 1-4C alkoxy, a substituted or unsubstituted carbamoyl, an acyl-substituted amino group, a substituted or unsubstituted aryl, a halogen) or its salt and is useful for preparation of an aqueous solution of oxidative color-developing reagents for automated analyzer utilizing enzymatic assay. Particularly it is useful as an oxidative color-developing reagent for auto-analyzers of body composition, glucose, cholesterol and the like. This novel compound can be readily produced according to the procedures described in Examined Publication Number 57-27106 and Examined Publication Number 58-9094.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a specific oxidative chromogenic reagent for producing a stable oxidative chromogenic reagent aqueous solution for use in an automatic analyzer utilizing an enzymatic analysis of biological components such as glucose and cholesterol. is there.

[0002]

2. Description of the Related Art Conventionally, an enzymatic analysis method has been developed as a method for detecting various biological components contained in body fluids such as blood and urine, and has been widely used in daily clinical tests. For example, glucose, cholesterol,
For measurement of uric acid and uric acid, oxidases such as glucose oxidase, cholesterol oxidase, and uricase are used, respectively, and biological components serving as substrates are specifically degraded enzymatically. The amount of each biological component can be accurately determined by quantifying the hydrogen peroxide generated at this time using a means such as coloring.

In particular, phenol, which is an oxidative coloring reagent, and 4-aminoantipyrine, which is a coupling agent, are oxidatively coupled using an enzyme system of glucose oxidase and peroxidase, and the absorbance of the formed dye is measured. Method to determine the amount of hydrogen peroxide (Trinder, P., Ann. Clin. Biochem., 6, 24, 196
9; Barham, D. and Trinder, P., Analyst (London), 9
7, 142, 1972), enzymatic measurement methods using oxidative color reagents have been remarkably developed. Enzymatic analysis has the characteristics of higher specificity for the substrate and higher measurement accuracy than the measurement method using chemical reactions, and is easy to automate because the reaction is completed under mild conditions. It is also expected to become more widespread in the future because it has the advantage of

As the oxidative color reagent used in the above-mentioned enzymatic analysis method, an aniline derivative has been used in place of phenol used in the art, and an oxidative color reagent having improved water solubility and wavelength characteristics has been proposed. (Tamaoku, K., Murao, Y., et al., Pharmaceutical Society of Japan No. 101
Annual Meeting Abstracts, p.148, 1981; Tamaoku, K., Hirasak
i, K., et al., Proceedings of the 101st Annual Meeting of the Pharmaceutical Society of Japan, p.14
8, 1981; Tamaoku, K., et al., Anal.Clin. Acta, 13
6, 121, 1982; Tamaoku, K., et al., Chem. Pharm.Bul
l., 30, 2492, 1982; Johnson, KS, et al., Anal. C
him. Acta, 201, 83, 1987; Madsen, BC, et al., Ana
l. Chem., 56, 2849, 1984, etc.).

As an oxidative coloring reagent, Japanese Patent Publication No. 57-271
An N-sulfoalkylaniline derivative described in JP 06-A and an N-hydroxysulfoalkylaniline derivative described in JP-B-58-9094 have been proposed and put to practical use. These oxidative coloring reagents are characterized in that they react quantitatively with 4-aminoantipyrine in the presence of hydrogen peroxide and peroxidase very quickly and quantitatively to give a dye having excellent color stability.

In recent years, with the increase in the number of specimens and test items,
Methods for measuring large amounts of samples with automated analytical instruments are becoming increasingly important. Normally, reagents are stored in the instrument as a solution, and are weighed by automatic pipetting or the like and added to the sample solution.However, when an oxidative coloring reagent is stored as a solution in an automatic analyzer, light, air oxidation, etc. In some cases, self-coloring occurs, the background rises, and data fluctuates. For this reason, oxidative coloring reagents for automatic analyzers are generally prepared as aqueous solutions to which stabilizers have been added.

However, the presence of additives such as stabilizers may cause variations in sensitivity, and there is a problem that it is troublesome to add a stabilizer and uniformly prepare an aqueous solution of a reagent composition. Also used conventionally
Enzyme solution containing reagents such as 3,5-dimethoxy-N-ethyl-N- (2′-hydroxy-3′-sulfopropyl) aniline sodium (DAOS) is 25 ° C. or less in the absence of a preservative. For 2 weeks and 1 month at 2-10 ° C (for example, HA Test Wako, Cholesterol E-HA Test Wako Enzyme Solution A, Wako Pure Chemical Industries, Ltd.), and the storage stability is poor.

[0008]

It is an object of the present invention to provide a specific oxidative chromogenic reagent for producing a stable aqueous solution of an oxidative chromogenic reagent for an automatic analyzer. More specifically, it is an object of the present invention to provide a specific oxidative color-forming reagent for producing an aqueous solution of an oxidative color-forming reagent for an automatic analyzer, which is a stable aqueous solution that does not generate self-color over a long period of time. It is an issue of.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the aqueous solution of the oxidative color-forming reagent containing the compound represented by the following formula (I) was exposed to light or dissolved water. It is not affected by oxygen, and it exists stably without causing self-coloring for a long time even in the absence of a stabilizer, and these compounds have excellent properties as an oxidative color reagent for automatic analyzers. I found that. The present invention has been completed based on the above findings.

That is, the present invention provides the following general formula (I) for producing an oxidative color reagent aqueous solution for an automatic analyzer utilizing an enzymatic analysis method:

Embedded image (Wherein, R ¹ represents a sulfonic acid-substituted C _3-4 alkyl group or a sulfonic acid-substituted C _3-4 hydroxyalkyl group, and R ² and R ³ each independently represent a hydrogen atom, a C _1-4 alkyl group, _1-4 , a substituted or unsubstituted carbamoyl group, an acyl-substituted amino group, a substituted or unsubstituted aryl group, or a halogen atom), or a salt thereof.

Further, according to the present invention, there is provided an oxidative coloring reagent aqueous solution for an automatic analyzer utilizing an enzymatic analysis method, the aqueous solution containing the compound represented by the above general formula (I) or a salt thereof. . The aqueous solution is preferably provided in a form that does not include a stabilizer for the oxidative chromogenic reagent. Further, the present invention provides the use of the compound represented by the above general formula (I) or a salt thereof for the production of an oxidative coloring reagent aqueous solution for an automatic analyzer. The above general formula as an oxidative coloring reagent
The present invention also provides a method for analyzing a biological component such as glucose, cholesterol, and uric acid using an aqueous solution containing the compound represented by (I) or a salt thereof by an automatic analyzer using an enzymatic measurement method. You.

Further, the present invention relates to an oxidative chromogenic reagent for use in the production of an oxidative chromogenic reagent aqueous solution for an automatic analyzer utilizing an enzymatic analysis method.
An oxidative color reagent having an absorbance increase at a maximum absorption of 0.015 or less when stored as a mM solution at 37 ° C. for 15 days is provided. The oxidative coloring reagent having the stability defined above can be suitably used for producing an oxidative coloring reagent aqueous solution for an automatic analyzer using an enzymatic analysis method. Preferable examples of the oxidative coloring reagent are the compound represented by the above formula (I) or a salt thereof.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION R ¹ represents a sulfonic acid-substituted C _3-4 alkyl group or a sulfonic acid-substituted C _3-4 hydroxyalkyl group. In the present specification, the alkyl moiety in the alkyl group or the substituent containing the alkyl moiety may be linear or branched. In the case of a halogen atom, any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom may be used.

The substitution position of the sulfonic acid group in the sulfonic acid-substituted C _3-4 alkyl group or the sulfonic acid-substituted C _3-4 hydroxyalkyl group is not particularly limited, but is preferably substituted on the terminal carbon atom. For example, a 3-sulfopropyl group or a 4-sulfobutyl group can be used. The hydroxyl group in the sulfonic acid-substituted C _3-4 hydroxyalkyl group is preferably substituted on a carbon atom other than the terminal carbon atom. As the sulfonic acid-substituted C _3-4 alkyl group, for example, a 3-sulfopropyl group or a 4-sulfobutyl group can be used.
As the C _3-4 hydroxyalkyl group, for example, a 2-hydroxy-3-sulfopropyl group, a 2-hydroxy-4-sulfobutyl group, a 3-hydroxy-4-sulfobutyl group, or the like can be used.

The C _1-4 alkyl group represented by R ² and R ³ includes:
For example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, te
An rt-butyl group or the like can be used. Examples of the C _1-4 alkoxy group represented by R ² and R ³ include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, a sec-butoxy group, and a tert-butoxy group. be able to. As the substituted carbamoyl group, mono C
Examples thereof include a _1-4 alkyl group carbamoyl group and a di C _1-4 alkyl carbamoyl group.

Examples of the acyl-substituted amino group include an alkanoylamino group such as an acetylamino group, a substituted alkanoylamino group such as a trifluoroacetylamino group, and an aroylamino group such as a benzoylamino group. Examples of the aryl group include a phenyl group,
A naphthyl group or the like can be used, and the type, number, and substitution position of the substituents present on the ring of the substituted aryl group are not particularly limited. Examples of the substituted aryl group include a chlorophenyl group, a methoxyphenyl group, and a toluyl group. Etc. can be used. As R ² and R ³ , a hydrogen atom, a methyl group, or a methoxy group is preferable, and a compound in which both are hydrogen atoms or a compound in which both are methoxy groups is preferable.

Preferred examples of the compound represented by the formula (I) include N- (3-sulfopropyl) aniline (compound A) and N- (3-sulfopropyl) -3-methylaniline (compound B) N- (3-sulfopropyl) -3-methoxyaniline (compound C); N- (3-sulfopropyl) -3,5-dimethylaniline (compound D); N- (3-sulfopropyl) -3, 5-dimethoxyaniline (compound E); N- (2-hydroxy-3-sulfopropyl) aniline (compound F); N- (2-hydroxy-3
-Sulfopropyl) -3-methylaniline (Compound G); N-
(2-hydroxy-3-sulfopropyl) -3-methoxyaniline (Compound H); N- (2-hydroxy-3-sulfopropyl) -3,
5-dimethylaniline (Compound I); and N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (Compound J).

The compound represented by the above formula (I) is, for example,
It can be easily produced according to the methods described in JP-B-57-27106 and JP-B-58-9094. In the production of the oxidative coloring reagent aqueous solution, in addition to the free form compound represented by the above formula (I), a salt of the above compound can be used. As the salt, for example, in addition to a sodium salt, a potassium salt, a magnesium salt and the like, an organic amine salt such as an ammonium salt and a triethylamine salt and the like can be used. In addition, in the production of the oxidative coloring reagent, a hydrate or a solvate thereof can be used in addition to the compound in a free form or a salt thereof.

The compound represented by the above formula (I) or a salt thereof is used for producing an oxidative coloring reagent aqueous solution for an automatic analyzer using an enzymatic measurement method. Many automatic analyzers using enzymatic measurement methods are known, for example, glucose, cholesterol, or various types of analyzers for measurement of biological components such as uric acid have been developed, The aqueous solution containing the compound represented by the formula (I) or a salt thereof can be used in any analytical instrument. The aqueous solution containing the compound represented by the above formula (I) or a salt thereof can exist stably without being affected by dissolved oxygen or light, and generally, it is not necessary to add a stabilizer.
As used herein, the term "automated analytical instrument" includes instruments in which part or all of the measurement is automated and should not be construed as limiting in any way.

The concentration of the compound represented by the above formula (I) or a salt thereof contained in the aqueous oxidizing color reagent solution for an automatic analyzer is not particularly limited. The concentration of the compound represented by the above formula (I) or a salt thereof is about 0.0001 to 0.1 mol / L, preferably 0.0
It is desirable to prepare it to be about 01 mol / L. Usually, about 0.0005 to 0.002 mol / L, preferably 0.001 mol
It is desirable to prepare an oxidative coloring reagent aqueous solution so as to be about / L and set it in an automatic analyzer. The aqueous solution of the present invention may contain, in addition to a buffer, a pH adjuster, and a preservative, a transition metal ion such as an iron ion in order to promote a reaction with a coloring agent. The addition amount of these components can be appropriately selected by those skilled in the art.

The type of the color former used in the automatic analyzer is not particularly limited. For example, 3-methyl-2-benzothiazolinone hydrazone (MBTH) or 4-aminoantipyrine can be used. When using 3-methyl-2-benzothiazolinone hydrazone as a color former, the concentration of the color former in the reaction solution prepared in the automatic analyzer is 0.001 to
It may be adjusted so as to be about 0.1 mol / L, preferably about 0.01 mol / L. The pH of the reaction solution is about 7.0 to 10.0, preferably about 7.9 to 9.1, and the reaction may be performed at 15 to 35 ° C, preferably 30 ° C for 1 to 10 minutes, preferably about 5 minutes.
Methods for producing a dye compound by reacting the compound represented by the formula (I) or a salt thereof with a color former in the presence of hydrogen peroxide are well known in the art, and the concentration of each reactive species, Appropriate conditions can be easily selected for the reaction solution pH and the like.

[0022]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to the following examples. The compound names in the examples correspond to the compound names exemplified as preferred compounds above. Example 1 Compounds A to J were dissolved at a concentration of 10 mM in 50 mM HEPES Buffer (pH 7.0) and stored at 37 ° C. After 15 days, the absorption spectra of the respective solutions were measured, and the changes appeared over time. The absorbance of the maximum absorption (λmax) was compared with the absorbance immediately after dissolution, and the increase in absorbance was calculated (ΔAbs). In addition, as a control, the compounds having an ethyl group instead of a hydrogen atom on the amino group of Compounds A to J were measured in the same manner, and the maximum absorption (N-
Etλmax) and the increase in absorbance (N-EtΔAbs) were determined.
To J, the ratio of increase in absorbance (N-Et / NH) was calculated. This value indicates how many times the oxidative color reagent of the present invention is more stable than the oxidative color reagent having an ethyl group on the nitrogen atom. All of the oxidative color-forming reagents of the present invention are superior in stability to the N-ethyl form, and it has been revealed that they can be suitably used for preparing an oxidative color-forming reagent aqueous solution for an automatic analyzer.

[0023]

[Table 1]

Example 2: Determination of cholesterol Preparation of reagent solution 1 1.2 mmol of compound J in 46 ml of 50 mM MES buffer (pH 6.1)
/ L, ascorbate oxidase (from pumpkin) at 0.
Reagent solution 1 was prepared by dissolving to 29 units / mL. In addition, cholesterol esterase (derived from the genus Pseudomonas) was added to 46 ml of 50 mM MES buffer (pH 6.1) at 7.6 units / m2.
L, cholesterol oxidase (CO) (derived from the genus Streptomyces) 1.7 units / ml, peroxidase (derived from horseradish) 5.2 units / mL, 4-aminoantipyrine 4.0 mmol / ml
The resulting solution was dissolved in L to prepare a reagent solution 2.

The principle of measurement is as follows. When reagent solutions 1 and 2 act on the sample, cholesterol esters in the sample are hydrolyzed by the action of cholesterol esterase and decomposed into free cholesterol and fatty acids. The generated cholesterol is oxidized by the action of cholesterol oxidase together with the free cholesterol contained in the sample, and simultaneously generates hydrogen peroxide. The produced hydrogen peroxide quantitatively oxidizes and condenses compound J and 4-aminoantipyrine by the action of peroxidase (POD) to produce a blue dye. By measuring the absorbance of the blue dye, the total cholesterol concentration in the sample can be determined.

Automatic biochemical analyzer CL-700 manufactured by Shimadzu Corporation
Using 0, sample 5μL (cholesterol 200 mg / dL) at 37 ° C
Is reacted with reagent solution 1 (360 μL), and after 5 minutes, reagent solution 2 (120 μL) is added.
The absorbance was measured at a subwavelength of 700 nm, and the cholesterol concentration was measured. The measurement conditions of the automatic analysis are the Test Master Table and Calibration Table Set U described in the package insert of cholesterol measurement reagents (HA Test Wako, Cholesterol E-HA Test Wako, CL-7000) manufactured by Wako Pure Chemical Industries.
Determined with reference to p. Next, after storing the reagent solution 1 at 25 ° C. for 6 months, the same sample was measured by setting the reagent solution 1 in the above automatic analyzer. Reagent solution 2 was prepared just before measurement in the same manner as above. As a result, the correlation coefficient r = 0.997 (n = 40)
Was able to measure cholesterol accurately.

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Claims (7)

[Claims] 1. The following general formula (I) for use in the production of an oxidative color reagent aqueous solution for an automatic analyzer using an enzymatic analysis method: (Wherein, R ¹ represents a sulfonic acid-substituted C _3-4 alkyl group or a sulfonic acid-substituted C _3-4 hydroxyalkyl group, and R ² and R ³ each independently represent a hydrogen atom, a C _1-4 alkyl group, _1-4 alkoxy group, substituted or unsubstituted carbamoyl group, acyl-substituted amino group, substituted or unsubstituted aryl group, or halogen atom) or a salt thereof. 2. An aqueous solution of an oxidative coloring reagent for an automatic analyzer utilizing an enzymatic analysis method, wherein the aqueous solution contains the compound represented by the general formula (I) or the salt thereof according to claim 1. 3. The aqueous solution according to claim 2, which does not contain a stabilizer for the oxidative color-forming reagent. 4. The use of the compound represented by the general formula (I) or a salt thereof according to claim 1, for the production of an oxidative coloring reagent aqueous solution for an automatic analyzer. 5. An aqueous solution containing the compound represented by the general formula (I) according to claim 1 or a salt thereof as an oxidative color-forming reagent, and a biological component is analyzed by an automatic analyzer using an enzymatic measurement method. How to analyze. 6. An oxidative chromogenic reagent for use in the production of an oxidative chromogenic reagent aqueous solution for an automatic analyzer using an enzymatic analysis method, which is a 10 mM solution in 50 mM HEPES buffer (pH 7.0) at 37 ° C. An oxidative coloring reagent which exhibits an increase in absorbance at the maximum absorption of 0.015 or less when stored for 15 days. 7. The oxidative coloring reagent according to claim 5, which is the compound represented by the general formula (I) according to claim 1 or a salt thereof.