JPS63309861A - Test composition for measuring peroxide activating material and testing means deposited with said composition - Google Patents

Abstract

PURPOSE:To measure peroxide activating materials (blood, etc.) with high sensitivity by incorporating a specific peroxide naphthyl compd. and oxidation color indicator into a compsn., thereby obtaining the titled compsn. CONSTITUTION:The text reagent compsn. for measuring the peroxide activating materials is obtd. by incorporating the peroxide naphthyl compd. expressed by formula and the oxidation color indicator therein. In formula, denotes a hydrogen atom., lower alkyl group, halogen group, nitro group. For example, the peroxide naphthyl compd. is 2-(alpha-hydroperoxyisopropyl)naphthalene, 2-(alpha- hydroperoxyisopropyl)-5-methyl naphthalene. The color indicator is orthotolidine, benzine, etc. Since this compsn. is used, the compsn. is stable with lapse of time and is capable of measuring the blood and hemoglobin which are the peroxide activating materials with the good detection sensitivity even after long-period storage.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a test composition for measuring a peroxide activator and a test device supporting the same. More specifically, the present invention relates to the above test composition and test device using a peroxide naphthyl compound as an organic hydroperoxide.

The compositions and test devices provided by the present invention are useful for detecting peroxide-activated substances such as blood or hemoglobin.

If blood or hemoglobin is contained in urine, feces, or vomit, it can be assumed that some disease such as inflammation or ulcer is progressing in the urinary or digestive system such as the kidneys, stomach, or intestines. .

Therefore, in order to promptly diagnose and treat these diseases, blood or hemoglobin (
It is important to accurately detect occult blood. The composition and test device of the present invention are suitably used for such occult blood tests.

[Prior art and its problems] A test device for detecting occult blood consists of a carrier impregnated with an organic hydroperoxide, a color indicator, and optionally a buffer, a wetting agent, an activator, and a stabilizer. The organic hydroperoxide is activated to produce nascent oxygen, which oxidizes the indicator and produces color. 2,5-dimethylhexane-2,5-dihydroperoxide and cumene hydroperoxide are conventionally known as organic hydroperoxides. Although these peroxides have been put into practical use, they are not stable over time, resulting in a significant drop in detection sensitivity.False negative results may occur if vitamin C is included in the urine sample. In the case of a multi-item test piece for detecting middle components, there are drawbacks such as discoloration of other adjacent test pieces, resulting in a decrease in performance, and low color sensitivity.

In recent years, compounds in which the benzene ring of cumene hydroperoxide is substituted with a Cl-8 alkyl group, CD, Br, I, No2, or a carboxyl group have been proposed as hydroperoxides that improve these drawbacks (Japanese Unexamined Patent Application Publication No. 59-1989).
Publication No. 0863). Although this peroxide was considerably improved over conventional ones, its stability over time was not sufficiently satisfactory.

[Means for Solving the Problems] The present invention aims to provide a test composition and a test device that do not have the above drawbacks, and the present invention consists of the following test composition and test device.

1) A test composition for measuring peroxide-activated substances containing a peroxide naphthyl compound represented by the general formula [in the formula, X represents a hydrogen atom, a lower alkyl group, a rogen atom or a nitro group] and an oxidized color indicator. thing.

2) The composition according to item 1, wherein the peroxide naphthyl compound is 2-(α-hydroperoxyisopropyl)naphthalene or 2-(α-hydroperoxyisopropyl)-5-methylnaphthalene.

3) The composition according to any one of items 1 to 3, wherein the oxidized color indicator is orthotolidine, benzidine, or leucomalachite green.

4) A composition containing a peroxide naphthyl compound represented by the general formula (1) [wherein X represents a hydrogen atom, a lower alkyl group, a rogen atom or a nitro group] and an oxidized color indicator is supported on a carrier. Test device for measuring peroxide activated substances.

5) The test device according to item 5, wherein the carrier is a nonwoven fabric made of filter paper, glass fiber, or a plastic material.

In the above formula (I), X is a hydrogen atom as described above,
Indicates a lower alkyl group, halogen atom or nitro group.

The lower alkyl group may further include a substituent that does not inhibit the color development of the color indicator, such as a halogen atom (CI).

Br, I), nitro group, hydroxyl group, sulfone group, carboxyl group, amide group, phenyl group, substituted phenyl group, etc. may be substituted.

There are no particular restrictions on the halogen atoms, but Cfl, B r
+I is preferred.

Typical peroxide naphthyl compounds represented by formula (1) used in the present invention include 2-(α-
hydroperoxyisopropyl) naphthalene and 2-
(α-hydroperoxyisopropyl)-5-methylnaphthalene is mentioned.

The peroxide naphthyl compound represented by the formula (I) of the present invention is a new compound, and is produced by oxidizing the α-hydroxyisopropyl naphthyl compound represented by the formula (n) with an aqueous hydrogen peroxide solution under acidic conditions. be done. Preferably, α-hydroperoxyisopropylnaphthyl compound (II) is dissolved in a suitable organic solvent such as ether, and a 30% or 50% aqueous hydrogen peroxide solution and a small amount of mineral acid, such as sulfuric acid or hydrochloric acid, are added to this solution and the mixture is stirred at room temperature. Let it react for several hours. After the reaction is complete, the desired product is collected from the reaction mixture in a conventional manner. For example, water is added to the reaction mixture, extracted with a suitable organic solvent such as ethyl acetate,
The desired product can be obtained by distilling off the solvent from the extract and purifying the residue by column chromatography or the like.

α-Hydroxyisopropylnaphthyl compound (II) is a naphthyl compound represented by the general formula [wherein X has the same meaning as above and Y has a halogen atom], n-butyllithium (or magnesium), It is then obtained by reacting with acetone. This reaction is carried out under the same conditions as the usual Grignard reaction. For example, tetrahydrofuran,
Compound (II) is obtained by reacting the re-compound in a suitable organic solvent such as diethyl ether at -78°C (in the case of n-butyllithium) or at room temperature to reflux (in the case of magnesium), and then adding acetone. It will be done.

As described above, the peroxide naphthyl compound (1) can be used as a peroxide in the measurement of peroxide-activated substances, and can be particularly advantageously used to detect occult blood in urine, feces, and vomit.

Therefore, the test composition for measuring a peroxide activator of the present invention comprises the above compound (T) and a color indicator.

As the indicator, so-called oxidized indicators that change color when oxidized are used, examples of which include orthotolidine, benzidine, leucomalachite green, and the like.

It may also contain a buffer, a wetting agent, an activator, a stabilizer, and a solvent if necessary.

Buffers are used to maintain a constant pi value directly above the test, such as when the test device, such as citrate, malate, or succinate, is immersed in the sample and the pi value is between 4 and 8. Preferably one that can be maintained within this range. Wetting agents are used to uniformly wet the test device with the sample liquid when the test device is immersed in the sample, and suitable surfactants include, for example, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium dioctyl sulfosuccinate. used for.

The activator is used to increase the sensitivity of the color reaction on the test device, and 3-aminoquinoline, quinine, isoquinoline, etc. are preferred. As the stabilizer, a thickening agent is used to prevent the test drug from flowing out of the test device, and polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol, gelatin, and gum arabic are preferred. The solvent may be one that easily dissolves the mixture of the above drugs;
Ethyl alcohol, acetone, benzene, toluene, chloroform and the like are preferably used.

This composition can be used by directly dropping it into a test liquid such as urine, or it can be used by dropping a test liquid such as urine into this composition.

Furthermore, the test device for measuring peroxide activating substances of the present invention is one in which the above composition is supported on a carrier.

The carrier is preferably a filter paper, glass fiber, or a nonwoven fabric made of a plastic material, as long as it does not dissolve or react with the solvent and absorbs the composition.

The amounts of the peroxide naphthyl compound and other reagents used in the test composition and test device are not particularly important, and are appropriately determined according to conventional methods. That is, an amount sufficient to react with the peroxide-activated substance to be detected and cause a color reaction is selected.

Next, the present invention will be explained in more detail by showing Examples, Reference Examples, and Test Examples.

(Left below) Reference Example 1 Preparation of 2-(α-hydroperoxyisopropyl)naphthalene Under argon atmosphere, 1.25 g of 2-bromonaphthalene
(8,04+mol) of dry tetrahydrofuran (50
ml) solution of 1.80M hexane solution (4.53ml 1.7.25 Iomol) of n-butyllithium at -78°C.
After addition, the mixture was reacted for 30 minutes. After adding 5.00 ml (88.1 mmol) of acetone to the solution and reacting at -78°C for 10 minutes, a saturated aqueous ammonium chloride solution was added and extraction was performed with ethyl acetate. After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. Elution with dichloromethane-methanol (100:1) gave 2-(α-hydroxyisopropyl)naphthalene 868 rag (4,65 mmo+).

- 1 - 1 1 - 866 mg (4,65 mmo) of the above hydroxy compound
l) 5 ml of ether, 20 ml of 30% hydrogen peroxide aqueous solution
After adding 0.50 ml of concentrated sulfuric acid and reacting at room temperature for 18 hours, water was added and extraction was performed with ethyl acetate. After washing the organic layer with water, the resulting residue was separated and purified using silica gel column chromatography. By elution with dichloromethane, 696 mg of 2-(α-hydroperoxyisopropyl)naphthalene (3
, 44+nmol) was obtained.

NMR (ppm, CDCΩ3) 8.03 (s, 111), 7.98-7.18 (m,
711), 1.55 (s, 0H) IR (c+n-1.CHCΩ) 3530.3
320 (blank below) -13= -1z - Reference Example 2 Preparation of 2-(α-hydroperoxyisopropyl)-5-methylnaphthalene Under argon atmosphere, 1.38 g (8.24 mmol) of 2-bromo-5-methylnaphthalene of n-butyllithium in dry tetrahydrofuran (50 ml)
0M hexane solution (4.68ml 1.7.45mmol)
was added at -78°C, and then reacted for 30 minutes. Add 5.00ml of acetone (68,11Iliol) to the solution.
) and reacted for 10 minutes at -78°C, then a saturated aqueous ammonium chloride solution was added and extraction was performed with ethyl acetate.

After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. By elution with dichloromethane-methanol (100:1), 959 g of 2-(α-hydroxyisopropyl)-5-methylnaphthalene (4,7 buttons mo
l) was obtained.

959 mg (4,79 mmo) of the above hydroxy compound
l) 5 ml of ether, 20 ml of 30% hydrogen peroxide aqueous solution
After adding 0.50 ml of concentrated sulfuric acid and reacting at room temperature for 16 hours, water was added and extraction was performed with ethyl acetate. After washing the organic layer with water, the resulting residue was separated and purified using silica gel column chromatography. By elution with dichloromethane, 2-(α-hydroperoxyisopropyl)-5-methylnaphthalene 818
mg (3.78 mmol) was obtained.

NMR (ppm, CDCN3) 8, old (s, I
II), 7.96-7.15 (m, 61(), 2.
63 (s, 311), 1.57 (s, 611) 1R (
ν mark, CHC, Q 3) 3530.3330 (margin below) Example test paper manufacturing method Solution I Peroxidenaphthyl compound (I) Molecular weight X 4.48 X 10-3g p-Toluenesulfonyl-N-diethylamide 5, Og Sodium dioctyl sulfosuccinate 1.5g ethanol
A 100 ml filter paper is thoroughly wetted with solution I and dried in a drying oven at 40° C. for 20 minutes.

Solution
10g polyethylene glycol
10g trisodium citrate dihydrate 9
g Citric acid-hydrate 1g Saponin
100mgEDTA-2Na 30mg Water 10
The paper which had been dried with 0 ml of Solution I was thoroughly wetted with Solution (I') and dried in a drying oven at 40 DEG C. for 50 minutes.

Solution ■ Orthotrisine
1.20g3-aminoquinoline 0,
5g benzene
The filter paper dried with 100 ml of solution (2) is sufficiently wetted with solution (2) and dried in a drying oven at 40°C for 10 minutes. This is used as a test paper for performance evaluation.

Test Example 1 A test piece obtained as shown in the above test paper manufacturing example is immersed in a sample for 1 second. The color development of the test piece is compared with a predetermined color tone table over time, and the judgment code written on the color tone table is visually read. The concentration of occult blood in the sample is determined from the degree of coloration using the color tone table. The correlation between the initial determination number and hemoglobin concentration is shown below.

(The following is a blank space) Table 1 The color tone that correlates with the hemoglobin concentration in the color tone table is for the test piece prepared using the peroxide 2,5-dimethylhexane-2,5-dihydroperoxide by the method shown in the production example above. It shows the color after 60 seconds of judgment time.

As a result, 2-(α-hydroperoxyisopropyl)naphthalene had a coloration corresponding to the color table in a determination time of about 15 seconds. On the other hand, it takes about 25 minutes for a test paper using cumene hydroperoxide to develop a color corresponding to the color tone table.
It took seconds. Note that 2,5-dimethylhexane-2,5-dihydroberoxide used to create the color table requires 60 seconds.

From the above, the 2-(α-hydroperoxyisopropyl)naphthalene of the present invention is a peroxide 2,5-dimethylhexane-2,5- It was found to have higher sensitivity than dihydroperoxide and cumene hydroperoxide.

Test Example 2 Tables 2 and 3 show the results of the aging test at 60°C.

(The following is a blank space) Tables 2 and 3 show that 2-(α-hydroperoxyisopropyl)naphthalene has excellent stability over time.

Test Example 3 It is known that when a test piece for measuring occult blood and a test piece for measuring glucose are adjacent to each other on a stick, the glucose test paper becomes discolored.

A test piece prepared by the method shown in the production example above using 2-(α-hydroperoxyisopropyl)naphthalene as the peroxide and 2,5-dimethylhexane-2,5
- test pieces using dihydroperoxide were pasted on separate sticks, and glucose test pieces were pasted on adjacent parts of each stick and stored at 40°C for one month. The results showed that while there was no discoloration of the glucose test piece in the former case, discoloration occurred in the latter case. From this, the peroxide of the present invention has little influence on other adjacent urine test items.

[Effects of the Invention] The test composition and test device of the present invention are effectively used for detecting peroxide-activated substances, particularly blood or hemoglobin. That is, when the peroxide activator compound (I) of the present invention is used as the organic hydroperoxide in a peroxide activator test composition and test device consisting of an organic hydroperoxide and a color indicator, the test composition has the following characteristics. can get.

(1) It is stable over time and can maintain good detection sensitivity even after long-term storage.

(2) In the case of a multi-item test piece for detecting components in urine, other test pieces adjacent to the test piece, such as the glucose test piece, will not be discolored, and performance will not deteriorate.

(3) The rate of color reaction is faster and the color sensitivity is higher than that of conventional test compositions.

As described above, the test composition and test device of the present invention have excellent properties in the peroxide activator test.

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Claims (1)

[Claims] 1) A peroxide naphthyl compound represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) [In the formula, X represents a hydrogen atom, a lower alkyl group, a halogen atom or a nitro group] A test composition for measuring peroxide-activated substances containing an oxidized color indicator. 2) Claim 1 in which the peroxide naphthyl compound is 2-(α-hydroperoxyisopropyl)naphthalene or 2-(α-hydroperoxyisopropyl)-5-methylnaphthalene.
The composition described in Section. 3) Claim 1, wherein the oxidized color indicator is orthotolidine, benzidine or leucomalachide green.
The composition according to any one of Items 1 to 3. 4) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, X represents a hydrogen atom, a lower alkyl group, a halogen atom, or a nitro group] Contains a peroxide naphthyl compound and an oxidized color indicator A test device for measuring a peroxide activating substance, in which a carrier supports a composition for activating a peroxide. 5) The test device according to claim 5, wherein the carrier is a nonwoven fabric made of filter paper, glass fiber, or a plastic material.