JPH04188068A - Test piece for detecting bilirubin - Google Patents

Abstract

PURPOSE:To improve detecting sensitivity by containing diazonium compound which reacts with bilirubin in body fluid and displays colorating, a sufficient amount of an acid component for reaction and the compound of 4-phenyl-2H- pyrido(1,2-a)pyrimidine-2-one or the like as essential components. CONSTITUTION:As an accelerating agent, the compound expressed by the general formula (R1 represents a hydron atom, an alkyl group, an alkoxy group, an amino group and a halogen atom, and R2 and R3 represent a hydrogen atom, an alkyl group, an aryl group, a hydroxyl group, a halogen group and alkoxy carbonyl group) and acid are dissolved in refined water. Then, diazonium salt is dissolved. Thus immersion liquid is obtained. The solution is impregnated in a carrier such as filter paper, and dried with hot air. The obtained carrier is stuck to a supporting body made of polystyrene or the like and the material is used. For the detection of the bilirubin in urine, the test piece is immersed into the urine to be detected and lifted, and the color which has appeared after the specified time is compared with a standard color.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a diagnostic agent for measuring bilirubin in body fluids. More specifically, the present invention provides an improved test strip for detecting bilirubin in body fluids, particularly urine.

[Prior Art] Detecting bilirubin in urine is important in the diagnosis of liver and intestinal diseases.

Red blood cells destroyed in the intracellular system of the spleen and other organs release hemoglobin, which liberates heme and becomes bilirubin, which binds to albumin in the bloodstream and is transported to the liver in the form of indirect bilirubin.

In the liver, it undergoes glucuronidation by the action of an enzyme called glucuronyltransferase and is directly converted to bilirubin.

In the intestinal tract, bilirubin is reduced to urobilin by the enzyme action of intestinal bacteria, and most of it is excreted in the feces, but some is absorbed in the intestinal tract and returns to the liver, and some is excreted in the urine. We parted ways.

Clinically, the phenomenon in which the blood level of pharyngeal phlegmon increases due to disturbances in bilirubin metabolism is called jaundice.
There are cases where the amount of unconjugated bilirubin increases in the blood and cases where the amount of conjugated bilirubin increases. In the latter case, conjugated bilirubin is water-soluble and is excreted in the urine. Detection of bilirubin in urine is an indicator of increased conjugated bilirubin in the blood due to hepatocyte damage or biliary tract disease.

Therefore, the urine bilirubin test is very effective as a screening test for liver diseases and the like.

Urinary bilirubin testing methods include observing the green color of biliverdin produced by oxidizing bilirubin (rosin method,
Damerin method, etc.) and a method that observes the azo dye produced by the coupling reaction between bilirubin and diazonium salt (azo coupling method).

Today, the test strip method, which is an application of the latter method to test strips, has become popular as the simplest semi-quantitative method for measuring bilirubin, and its importance is increasing.

In the azo coupling method, sulfanilic acid, 2,
4-dichloroaniline, 2,5-dichloroaniline, 2
Diazonium salts derived from amines such as , 6-dichloroaniline and 4-nitroaniline are used. However, the reaction between these diazonium salts and bilirubin has problems in reaction rate and sensitivity, making it difficult to detect bilirubin at low concentrations.

[Problems to be Solved by the Invention] Various reaction accelerators have been studied to solve the above problems. For example, Japanese Patent Publication No. 53-28119 discloses a test composition containing certain phosphoric acid diesters to promote the reaction with bilirubin. Further, Japanese Patent Publication No. 52-43493 discloses an addition compound of a ureido compound and a sulfonic acid as an accelerator.

However, according to these methods, although the reaction between bilirubin and diazonium salt is promoted to some extent, it is affected by the interfering reaction between diazonium salt and 5-hydroxyindoleacetic acid, indican, or urobilinogen, etc. that coexist in urine, and the reaction between bilirubin and diazonium salt is promoted to a certain extent. Concentrations of bilirubin were difficult to detect.

Therefore, there is a need for a means to weaken the reaction color with these coexisting substances and to strengthen the reaction color between bilirubin and diazonium salt. Eight patent applications were filed for promoters that increase reactivity (Japanese Patent Publication No. 56950/1983). However, when detecting bilirubin in urine using a bilirubin measurement test piece containing this accelerator, urobilinogen coexisting in urine has no effect at low concentrations, but when it coexists at high concentrations, Due to its yellowish-brown color, it may interfere with the detection of bilirubin.

In addition, since the accelerator has an active methylene group in its molecule, it has had problems such as the need to maintain the pH of the system as acidic as possible in order to suppress side reactions with the diazonium salt. .

[Means for Solving the Problems] As a result of repeated studies on the above-mentioned problems, the present inventors have determined that, in the reaction between bilirubin and diazonium salt, the following general formula is used as an accelerator: (wherein R2 is a hydrogen atom) , alkyl group, alkoxy group,
It represents an amino group or a halogen atom, and Rz and Ri each represent a hydrogen atom, an alkyl group, an aryl group, a hydroxyl group, a halogen atom, or an alkoxycarbonyl group. The present invention was completed based on the discovery that containing a compound represented by the following formula is less susceptible to the effects of 5-hydroxyindoleacetic acid, indican, and urobilinogen, and also significantly enhances the reaction with bilirubin.

The compound of general formula [I] is described in the Journal of the American Chemical Society [J.

Am, Chem, Soc, ] 74.5943 (19
52), it has been reported that the compound has a resonance structure as shown in the following formula [II].

For example, 2H-pyrido(1,2-a)pyrimidine-2-
On is a colorless solid, has a melting point of 250°C, is stable to heat, and has excellent solubility when 80 g or more is dissolved in 100 ml of water.

The compounds used in the present invention are known except for some,
The synthesis method is generally known, for example,
Earl Lappin, Journal of Organic Chemistry [G, R, Lappin.

J,Org,Chem,] 26.2350 (196
1), Etchenu Alsharow, Aini Albiaty, Journal on Heterocyclic
Chemistry [H,N, Al-Jallo and r
,A,AI-Biatyj,Heterocyclic
, Chem,] 15.801 (1978), and the like.

Examples of compounds used in the present invention and synthesis examples thereof are shown below, but the present invention is not limited thereto.

Synthesis Example [Ia] 9.4 g of 2-aminopyridine dissolved in 50 ml of ether and 8.4 g of methyl propiolate dissolved in 10 ml of ether are stirred at room temperature for 48 hours. After the reaction, the product was filtered and the collected crystals were extracted using a Soxhe extractor to obtain 3.1 g of colorless crystals of [Ial.

The melting point was 248°C to 250°C.

Synthesis Example [Ib] 11.8g of ethyl phenylpropiolate and 7.1g of 2-aminopyridine were dissolved in 30ml of ethanol,
Heat in an oil bath at 130°C to 140°C. After 20 hours, ethanol is removed using an evaporator, and the remaining crystals are washed with ether and filtered.

The obtained yellow crystals were recrystallized with acetone to obtain 11.3 g of [Ib] as colorless needle crystals.

The melting point was 220°C to 221°C.

It is not clear what is the basis of the promoting effect in the reaction of bilirubin and diazonium salt when the compound of the general formula [I] according to the present invention is present, but it is clear that the compound containing only an acid in addition to the diazonium salt and the general formula [■ ] The test piece that does not contain the compound reacts extremely slowly with bilirubin, so
The good results provided by the present invention are truly surprising. Furthermore, a major feature of the present invention is that it is less susceptible to interfering substances such as 5-hydroxyindoleacetic acid, indican, and urobilinogen that coexist in urine.

Surprisingly, when the compound of general formula '[I] is present, the color tone of bilirubin and diazonium salt shifts to the hypsochromic side compared to when the compound of general formula [I] is not present. In addition, the color tone of urobilinogen and diazonium salt, which coexist in urine, shifts to the hypochromic side, which apparently removes the color of the reaction between urobilinogen and diazonium salt. It is also used at a concentration of 1 to 15% in the impregnating liquid, and can be used singly or in combination.

As the diazonium compound contained in the test piece, well-known diazonium compounds can be used, but aryl diazoniums containing halogen and nitro groups in the molecule are particularly preferred, such as 2,4-dichlorobenzenediazonium, 2.4 -dibromobenzenediazonium, 4-nitrobenzenediazonium, 2.5-dichlorobenzenediazonium, 2,4.5-1-lichlorobenzenediazonium, and the like.

Furthermore, in order to stabilize the diazonium compound, it is advantageous to use known salts, such as sulfates, tetrafluoroporates, arylsulfonates, and the like. In particular, tetrafluoroborate and aryl sulfonate are thermally stable and therefore have excellent retention and are optimal.

! , in the present invention, ・diazoium salt Cma・0・O
It can be used in the range of v2 to 2%, preferably 0.
The concentration is between 0.05 and 1%.

In addition, in order for bilirubin and diazonium salt to react, it is advantageous to add an acid component to maintain the pH in a strongly acidic range, particularly pH 1 to 3, and a solid acid that can maintain this pH is used. . Examples include oxalic acid, maleic acid, citric acid, sulfosalcylic acid, p-toluenesulfonic acid, metaphosphoric acid, etc., preferably at a concentration of 5 to 25%.

In order to stabilize the composition, the addition of stabilizers known from diazo chemistry may also be considered, such as borofluoride salts, arylsulfonates, sodium metaphosphates, etc., used in concentrations of 0 to 5%. be done.

It is also possible to use a surfactant to impart wettability to the test piece, and any cationic, anionic, or nonionic surfactant may be used, but especially anionic surfactants may be used. Ionic surfactants are advantageously used in the present invention because they enhance the action of the compound represented by general formula [I].

Examples of anionic surfactants include sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium dioctylsulfosuccinate. Surfactants are used in concentrations of 0.1-2%, preferably 0.2-0.8%.

Examples of the carrier used in the present invention include filter paper, cotton, wood chips, and nonwoven fabric, and filter paper is particularly preferred.

The test piece of the present invention is manufactured, for example, as follows.

The compound represented by the general formula [I] and the acid are dissolved in purified water, and if necessary, a surfactant and a stabilizer dissolved in water or a water-miscible solvent are added thereto, and then the diazonium salt is dissolved. and use it as an immersion liquid.

A carrier such as filter paper is impregnated with the solution thus obtained and dried with hot air at 40 to 60°C. The obtained carrier is cut to an appropriate size and used by attaching it to a support such as polystyrene using double-sided adhesive tape.

When detecting bilirubin in body fluids, especially urine, using this test piece, it is carried out, for example, as follows.

Immerse the test piece in the urine sample and remove it immediately.The color that appears after a certain period of time is compared to the standard color (prepared in advance) at various concentrations.
Estimate the density by comparing it with the color tone table). It is also possible to measure the reflection intensity from the color obtained after a certain period of time using a spectroscopic reflectometer and determine the concentration from the calibration curve.

[Examples] Next, in order to explain the present invention in detail, the following examples are shown, but the present invention is not limited thereto.

Example 1 Production of a test piece for detecting bilirubin according to the present invention A filter paper (No. 52 manufactured by Toyo Roshi ■) was added to an immersion liquid in which the ingredients of the following formulation were dissolved.
5) and dried at 50°C for 30 minutes.

Prescription 2. 4-dichlorobenzene 87゛diazonium tetrafluoroborate 0.08g
1'['tsu, uya 1゜
82H-pyrido(1,2-a) pyrimidin-2-one 4.01 g Sodium dodecylbenzenesulfonate 0.3 g Methanol 10 ml Purified water
Total amount: 100 ml The thus obtained test paper was cut into 5 mm square pieces and attached to one end of a 5 mm x 80 mm polystyrene sheet using double-sided adhesive tape. When this test piece was immersed in test urine, urine that did not contain bilirubin showed a light yellow color, but urine that contained bilirubin showed a reddish-orange to red color after 10 to 20 seconds, and the sensitivity limit was 0.3 mg/ It was dl.

A test strip of the same composition but without 2H-pyrido(1,2-a)pyrimidin-2-one required 1-2 minutes to react and had a sensitivity limit of 1-2 mg/di.

Example 2 Comparison of the influence of urobilinogen The test piece for detecting bilirubin of the present invention obtained in Example 1,
1, 4°8, and 12 mg/di, respectively, and a test piece manufactured using a conventional accelerator for comparison.
The test piece was immersed in a pressure containing urobilinogen at a concentration of , immediately removed, and the reaction color produced after a certain period of time was examined. The results are shown in Table 1.

The test piece for comparison was 2H in the formulation of Example 1.
-In place of pyrido(1,2-a)pyrimidin-2-one, 3,4-dihydro-2H-pyrido(1,2-a)pyrimidin-2-one [manufactured by Tokyo Kasei Kogyo ■: trade name: Example 1 except that equimolar amounts of Acid Cabter H] were used.
It was manufactured in the same manner.

From the results in Table 1, it was confirmed that the test piece according to the present invention was less susceptible to the influence of urobilinogen than the test piece using a conventional accelerator.

Example 3 Influence of 5-hydroxyindoleacetic acid and indicane The test piece obtained in Example 1 (test piece of the present invention) and the accelerator in the formulation of Example 1, namely 2H-pyrid (1, 2-a) Using a test piece manufactured in the same manner as in Example 1 except for the formulation excluding pyrimidin-2-one, reactivity with a test solution of 5 ff 1 g/di of 5-hydroxyindoleacetic acid was determined. I looked into it. Immerse the test piece in the test solution, pull it out immediately, and evaluate the degree of color development at each elapsed time of 15, 30, 60, and 120 seconds.
Table 2 shows the results, which are expressed as numbers assigned according to the intensity of color development when color development was observed.

Table 3 shows the results of similarly examining the reactivity with a test solution containing 5 mg/dl of indican.

Table 1: Comparison of effects of urobilinogen 5-hydroxyindoleacetic acid (5mg/di aqueous solution)
Reaction with Table 3 Reaction with indicane (5 mg/di aqueous solution) Example 4゜In place of 2H-pyrido(1,2-a)pyrimidin-2-one in the formulation of Example 1, various general formulas [ Table 4 shows the results of investigating the sensitivity limit and reaction time when equimolar amounts of the compound shown in [I] were used.

Table 4 with blank space below [Effects of the Invention] The test piece for detecting bilirubin of the present invention is less susceptible to interference reactions caused by coexisting substances in the urine sample, and the reaction with bilirubin is significantly accelerated. It also makes it possible to detect with high sensitivity.

Furthermore, even when urobilinogen coexists in urine at a high concentration, its influence is significantly reduced, and the specificity of the reaction with bilirubin is therefore significantly improved, which is an excellent effect.

Claims (1)

[Claims] (1) A diazonium compound that can react with bilirubin in body fluids to develop a color, an acid component in an amount sufficient for the reaction, and a general formula, ▲Mathematical formulas, chemical formulas, tables, etc.▼...[I] (In the formula, R_1 represents a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a halogen atom; R_2, R
Each of _3 represents a hydrogen atom, an alkyl group, an aryl group, a hydroxyl group, a halogen atom, or an alkoxycarbonyl group. ) A test piece for detecting bilirubin in body fluids, which is characterized by containing the compound shown in () as an essential component.