JPS6348311B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an improved diagnostic agent for detecting urobilinogen in body fluids, particularly urine. Urinary urobilinogen is essential for the diagnosis of various hemolytic diseases and liver dysfunction,
In particular, test strips are prized for their quickness. Hitherto, the so-called Ehrlichi reaction has been carried out, in which p-dimethylaminobenzaldehyde and urobilinogen are condensed with the active methylene in an acidic medium, but this method has obvious drawbacks. Namely: 1. Because the reaction is relatively slow, it takes more than 1 minute to judge the results. 2. Reaction rate is sensitive to temperature. 3. Reacts with indicane, skatole, porphobilinogen, 5-hydroxyindole acetic acid, aromatic amines excreted by metabolization of administered drugs, etc., which are present in the urine, and urea, which is present in large quantities in the urine. , the reaction color with urobilinogen is significantly disturbed. Incidentally, although it has been known that urobilinogen and diazonium salt undergo a significant reaction when bilirubin is measured by the azo coupling method, this reaction has little utility as a method for measuring urobilinogen. Recently, a method for measuring urobilinogen using the azo coupling method has been reported. The diazonium salts used in this reaction include benzene diazonium salts containing a polyatomic electron donating group with a mesomeric electron pair at the ortho or para position (Japanese Patent Application Laid-open No. 11093/1983), or aromatic compounds that are adjacent to each other. Examples include phenyl, pyrrole, and pyrazole diazonium salts that are ring-bonded or vinylogically bonded, as well as pyridine and pyrazole diazonium salts containing a polyatomic electron-donating group having a mesomerizable electron pair (Japanese Patent Application Laid-Open No. 49-44797). Furthermore, 4-
Fluoro-3-nitrobenzenediazonium salt (Japanese Patent Application Laid-Open No. 52-493) and biphenylbisdiazonium salt having a substituent (Japanese Patent Application Laid-open No. 49-99091) have also been reported to be effective as urobilinogen detection reagents. As a result of intensive research, the present inventors found that adjacent rings are bonded at positions that are not sterically hindered by saturated heterocyclic compounds,
In this case, it has been found that a phenyldiazonium salt capable of mesomerifying with at least one heteroatom of a saturated heterocyclic compound can be used particularly advantageously as a detection reagent for urobilinogen. The most important heteroatoms in the saturated heterocyclic compounds according to the invention are oxygen, nitrogen and sulfur, and may contain one or more of these. Such saturated heterocyclic compounds include tetrahydrofuran, tetrahydrothiophene, 1,3-dioxane, 1,4-dioxane, tetrahydropyran,
Examples include piperidine, morpholine, and pyrrolidine. The phenyl ring can have a substituent as long as it does not sterically hinder the coupling reaction. Suitable substituents include lower alkyl groups. A group of compounds that can be suitably used in the present invention is represented by the following general formula.

[expression] or (In the formula, Z is oxygen directly bonded to phenyl ring A,
represents a heteroatom selected from the group consisting of nitrogen and sulfur atoms; when Z is a nitrogen atom, this nitrogen atom may be substituted with an acetyl group; B is a saturated heteroatom having at least one heteroatom; represents a ring, R represents hydrogen or a lower alkyl group, and X represents a stabilizing anion. ) Stabilizing anions include chloride, sulfate, tetrafluoroborate, hexafluoroantimonate, arylsulfonate, tetrachlorotincate, and tribromide, with tetrafluoroborate being particularly advantageous due to its excellent thermal stability. These may be used alone or in combination. The compounds according to the invention react almost instantaneously with urobilinogen in the presence of acids and exhibit a very distinct red to reddish-purple color. On the other hand, it hardly reacts with urea, para-aminosalicylic acid, sulfur drugs, indole, skatole, and porphobilinogen. When large amounts of bilirubin are excreted in the urine, a blue or green reaction may occur, but this progresses very slowly and poses little problem in measuring urobilinogen. In some cases, reaction with bilirubin is also acceptable and can be used as a test tool for simultaneous detection with urobilinogen. This is achieved by increasing the electrophilicity of the diazonium salt through the choice of substituents. According to the present invention, the reaction between the diazonium salt and urobilinogen is desirably carried out under acidic conditions, particularly at pH 3 or below, and particularly strong color development is observed under this condition. Therefore, any organic or inorganic acid can be used as long as it satisfies this condition. Solid acids are advantageous when the test composition is contained in an absorbent carrier such as paper and used as a so-called test strip. Such acids include
Oxalic acid, maleic acid, succinic acid, citric acid, tartaric acid, sulfamic acid, sulfosalicylic acid, p-
Examples include toluenesulfonic acid, potassium hydrogen sulfate, and metaphosphoric acid, which may be used singly or in combination. Adducts of Lewis acids and Lewis bases are also acidic.
It can be used when giving a PH value. Since diazonium salts are generally thermally unstable, the addition of stabilizers is considered and is well known in the field of diazo chemistry. Such compounds include borofluoride salts, arylsulfonates, bisulfites, maleates, and borates. It is also possible to use surfactants, and anionic surfactants are particularly advantageous because they significantly increase the reactivity of the diazonium salt and urobilinogen and shift the color development toward the deep color side. Examples of anionic surfactants include sodium lauryl sulfate, sodium dodecylbenzenesulfonate,
Examples include sodium dioctyl sulfosuccinate. When suppressing the reaction with bilirubin, it is desirable to use a cationic surfactant. Examples of cationic surfactants include cetyltrimethylammonium bromide and laurylpyridinium chloride. When used as a so-called test piece, the addition of a nonionic surfactant is considered. This imparts wettability to the specimen and allows the reaction to proceed uniformly. A particularly advantageous test device is provided when the test composition according to the invention is impregnated into an absorbent carrier and kept dry. Examples of such absorbent carriers include paper, cotton, wood chips, polyester fleece, and polyamide fleece, with paper being preferred. When manufacturing test pieces, per 100ml of solution,
0.01 to 1 g, preferably 0.05 to 0.5 g, of the diazonium salt according to the invention, 1 to 30 g, preferably 5 to 15 g, of an organic or inorganic acid, 0.1 to 10 g, preferably 1 to 5 g, of a stabilizer, anionic interface 0.01-1 g, preferably 0.1-0.5 g of the activator is dissolved in water or a mixed solution of water and an organic solvent that does not react with the diazonium salt, impregnated onto an absorbent carrier such as paper and dried at below 40°C. Test pieces obtained by pre-impregnating an absorbent carrier with a diazonium salt and a stabilizer and drying it, then impregnating it with an organic acid in an organic solvent that does not dissolve them and drying it, show particularly good stability. . Some of the diazonium salts used in the present invention are known, but even unknown diazonium salts can be obtained from the corresponding amine by known methods. Instead of dissolving the isolated diazonium salt, it is also possible to directly impregnate the absorbent carrier with a diazonium salt solution formed from the corresponding amine. Furthermore, a method for obtaining a diazonium salt by directly introducing a diazonio group into a compound lacking an amino group is also known. The test strip thus obtained can be attached to a plastic sheet using adhesive tape and cut into squares for convenient use. The test results are determined by comparing the color tone produced by the reaction with urobilinogen with a standard color tone prepared in advance after a certain period of time. Further, the reflectance can be measured using a reflectometer, and the urobilinogen concentration can be estimated from the standard curve. In some cases, the concentration can also be determined from the amount of change in reflectance with respect to minute changes over time after a certain period of time. It is also possible to bring the test composition according to the present invention into contact with a test liquid in the form of a solution, and quantify urobilinogen spectroscopically at an appropriate wavelength. In this case, it is desirable to extract the resulting dye with an organic solvent such as chloroform. The following examples are provided to explain the present invention in detail, but the scope of the present invention is not limited thereby. Example 1 Paper (Watmann 3MM) was impregnated with a solution having the following composition and dried in a constant temperature bath at 40°C. 2,3-dihydrobenzofuran-5-diazonium tetrafluoroborate 0.3g Oxalic acid 10g Sodium lauryl sulfate 0.1g Methanol 10ml Purified water 90ml The test paper obtained is white and can be detected from the urine urobilinogen concentration of approximately 0.4mg/d. . Urine that normally contains urobilinogen turns pink after 10 to 20 seconds, and urine with a high concentration of urobilinogen turns dark red. Example 2 When using the equivalent mole of the following diazonium salt in place of the diazonium salt in Example 1, Example 1
A test strip with properties almost equivalent to that of the test strip can be obtained. 2,3-dihydrobenzothiophene-5-diazonium tetrafluoroborate 1,4-benzodioxane-6-diazonium tetrafluoroborate 2,3-dihydrobenzofuran-7-diazonium tetrafluoroborate 1-acetyl-2,3-dihydro Indore
5-Diazonium sulfate Benzomorpholine-6-diazonium tetrachlorotincate Example 3 300 mg of 6-amino-1,3-benzodioxane was dissolved in 75 ml of a 15% aqueous metaphosphoric acid solution and cooled.
Add 140mg of sodium nitrite. After about 2 hours, add 10 ml of a 1% methanol solution of sodium dodecylbenzenesulfonate to the solution, and fill with water to make a total volume of 100 ml. Paper (Watmann 3MM) is impregnated with this solution and dried at 40°C. This test paper has almost the same properties as the test paper obtained in Example 1. Example 4 A test paper obtained by using the following amine in an equimolar amount in place of the amine in Example 3 has properties substantially equivalent to those of the test paper in Example 1. 5-amino-1,3-benzodioxolane 6-amino-8-ethyl-1,3-benzodioxane 6-amino-5-methyl-1,3-benzodioxane 6-amino-3,4-dihydro-1, 2-benzopyran 6-amino-1-acetyl-1,2,3,4-
Tetrahydroquinoline Example 5 20 ml of 0.2% 1,3-benzodioxane-6-diazonium tetrafluoroborate solution in 10% P-toluenesulfonic acid aqueous solution is added to 4.0 ml of urine containing urobilinogen and mixed. After 30 minutes, add 4.0 ml of chloroform and shake thoroughly. After standing still, separate the chloroform layer and measure 505n using a spectrophotometer.
Measure the absorbance at m with chloroform as a control. Perform the same procedure using a 10% aqueous p-toluenesulfonic acid solution instead of the diazonium solution to determine the absorbance of the blank. The accurate amount of urobilinogen can be determined by comparing the absorbance obtained by subtraction with the standard absorbance.

Claims (1)

[Claims] 1 General formula [Formula] or (In the formula, Z is oxygen, nitrogen, or
and a sulfur atom; when Z is a nitrogen atom, this nitrogen atom may be substituted with an acetyl group; B represents a saturated heterocycle having at least one heteroatom where R represents hydrogen or a lower alkyl group, and X represents a stabilizing anion. 1. A test composition for detecting urobilinogen in body fluids, which contains a diazonium salt represented by the following formula and an organic acid or an inorganic acid as essential components.