JPS621220B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the determination of urea content in liquids and aqueous solutions, and more particularly to a test device for improving the stability of a rapid and accurate colorimetric test for the determination of urea. Urea is the main final product of protein metabolism in the human body. Therefore, the concentration of urea in the blood is an indicator of kidney function. An increase in urea concentration indicates failure of renal function. High concentrations of non-protein nitrogen or blood urea nitrogen are of concern to physicians because toxic substances are contained in the blood roughly in proportion to the level of urea. One of the most common causes of high blood urea levels (uremia) is kidney disease, either acute or chronic. Several inflammatory, degenerative, congenital, or neoplastic diseases that affect the kidneys can cause uremia, and the degree of uremia is a rough indicator of the severity of the current health condition. In practice, the concentration of urea in the blood is usually expressed by the specific term blood urea nitrogen (BUN).
This BUN value represents the amount of nitrogen present in urea and is approximately 1/2 of the total amount of urea. When one of the urea or nitrogen values is measured, the other value can be calculated therefrom. The normal range of BUN values for each person varies between 5 and 20 mg%. There is usually no problem even if the value is lower than that. However, elevated BUN values generally indicate an abnormal condition. The most common cause of increased blood urea nitrogen is insufficient excretion, usually due to kidney disease or urethral tract disorders. For example, in acute nephritis, the BUN value is 25mg% to 160mg
Varies between %. High concentrations of urea stagnate when substantial destruction of renal tissue develops, such as in pyelonephritis, aggravated nephrosclerosis, renal tuberculosis, central renal necrosis, malignant renal tumors, nephrogenic suppuration or chronic gout. BUN values can be as high as 400mg%,
Usually does not exceed 200mg%. Thus, a need exists today in the medical field for rapid and accurate analytical techniques to measure BUN. Traditionally, cumbersome wet chemical methods have been used to perform such analyses. Other methods have also been used, such as conduction methods, spectrophotometric methods, and colorimetric methods. Although these methods are generally accurate, they tend to be somewhat time consuming and require careful interpretation. In perhaps the most commonly used method for measuring urea in biological fluids, urea is hydrolyzed to ammonium carbonate by an enzyme, namely uriase, in the presence of a buffer. The amount of ammonia released is determined by colorimetric analysis. This first method is very specific and sensitive, but has the drawbacks of inhibiting and inactivating uriase, problems with reagent stability, and the additional drawback that this method measures endogenous ammonia. has. Another method utilizes uriase hydrolysis and requires special equipment not always available in routine laboratories.
Another method uses a direct colorimetric reaction of urea in a protein-free solution with an organic reagent such as diacetyl monooxime. This diacetyl reaction has the disadvantage that the resulting color is unstable, and there is also the fact that the color develops at about 95° C. and that the evaporated diacetyl has an odor that is objectionable to at least some people. U.S. Patent No. 4,074,942, issued to American Monitor Corporation, discloses a liquid test method for urea, which includes a biological fluid sample and an o- It involves the reaction of phthalaldehyde with an acidic reagent solution of 8-(4-amino-1-methylbutylamino)-6-methoxynoline. However, test means such as carrier matrices, let alone carrier matrices containing strong cation exchange resins in acid form, do not teach or suggest techniques for incorporating reagent solutions. moreover,
This patent does not disclose any method to overcome stability problems that occur when reagents in acidic reagent solutions are combined. Another problem with this disclosed test method is that the 470
The fact is that measurements have to be made at a wavelength of ~540 nm, which may result in reflection spectroscopy errors. In industrial areas, urea is an important additive for processing liquids such as plating baths, and a rapid means of determining the concentration of urea therein is of great value. Urea itself is used as a fertilizer, and rapid means of measuring this substance are important as control standards for its production and use. Therefore, there is a need for rapid determination of urea content in liquids for medical and industrial applications without requiring highly specialized equipment or skilled technicians to operate such equipment. . The first object of the present invention is to provide a test device for measuring the urea content in a liquid, which ensures the stability of the reagents used to make the test device. A second objective of the invention is to minimize interference by serum turbidity and the presence of bilirubin when measuring the urea content in serum. A third object of the invention is to provide a test device with a high degree of specificity and sensitivity over a range of concentrations for measuring urea in liquids. A fourth object of the present invention is to provide a colorimetric test device capable of detecting urea when the amount of liquid to be tested is limited. The fifth object of the present invention is to quickly perform BUN without requiring any special equipment.
The purpose is to provide testing equipment. According to the invention, a test device for detecting urea present in a test liquid consists of an acid-modified carrier member containing reagents separated from each other. One reagent is o-phthalaldehyde and the other reagents are 3-hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline, 3-acetoxy-1,
2,3,4-tetrahydrobenzo[h]quinoline or their dihydrochlorides. Advantageously, these reagents are separated from each other by a polymeric substance. The test device can be used by momentarily dipping into the test sample or otherwise introducing the test sample into the carrier matrix and observing the detectable color change that occurs. In this application, the term “liquid” refers to serum,
It will be understood that body fluids such as plasma, urine, spinal fluid, and also aqueous solutions containing urea are meant. Although the most preferred embodiment of the test device of the present invention is for use with body fluids such as serum, it will be understood that the test device can be applied to industrial liquids as well. According to the invention, unique reagent strips are prepared by incorporating the above-mentioned reagents into a carrier matrix, separated for example by a polymeric barrier material. The expression "carrier matrix" refers to absorbable and non-absorbable matrices that are insoluble and maintain their structure intact when in contact with water or other physiological fluids. Suitable absorbent matrices that can be used include paper, cellulose, wood, synthetic resin fleece, woven and non-woven fabrics, and the like. Non-absorbable matrices include glass fibers, organic plastics such as polypropylene. For convenient use, this matrix is advantageously applied by suitable means, such as double-sided adhesive tape, to an insoluble support material such as an organic plastic strip, for example polystyrene. The reagents contained in the carrier matrix react with the urea under acidic conditions to produce a dye that absorbs light above about 550 nm, typically between about 550 and 700 nm. Quantification can be achieved by measuring the change in reflectance at about 620 nm after introducing or contacting a sample. It was found that in order to obtain the desired acidity of the test conditions it was necessary to modify or impregnate the carrier matrix with acidic substances. One method for preparing acid-modified carrier matrices includes impregnating the carrier matrix with about 5 to about 10 g% of an acidic material such as benzenesulfonic acid, benzenedisulfonic acid, polystyrene sulfonic acid, or the like. Another method of preparing acid-modified carrier matrices includes incorporating a strong cation exchange resin, preferably in acid form, into the carrier matrix. Amberlite IR120 [Rohm & Haas, Philadelphia, Pennsylvania]
Pennsylv-ania)] or Dowex 50 (Dowex, Midland, Michigan)
Dow Chemical Co.
A sulfonic acid-modified carrier matrix is particularly preferred when using serum, especially when using serum. Specific examples of these ion exchange resins include sulfonic acid-modified polystyrene crosslinked with various amounts of divinylbenzene. from about 25 to about 50% by weight, preferably about 35% by weight in each carrier matrix.
An ion exchange resin is included in an amount of ~50% by weight. Prepare the test device using two reagents. One reagent is o-phthalaldehyde and the other reagent is 3-hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline, 3-acetoxy-1,
2,3,4-tetrahydrobenzo[h]quinoline or their dihydrochlorides, and 3-hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline is particularly preferred. According to the invention, the two reagents must be applied separately to the carrier matrix, but the order in which the reagents are applied is not critical. Thus any reagent can be applied first. The sulfonic acid material can be applied to the carrier matrix simultaneously with the first reagent;
Also, carrier matrices containing ion exchange resins can be used. Thus, in a preferred method, the first step consists of applying at least one reagent to the carrier matrix. The second step usually consists of applying a polymer to the carrier matrix, which polymer is present in a solvent that does not dissolve the first reagent or sulfonic acid material. Examples of polymers include methylcellulose, cellulose acetate, polyethylene oxide, styrene-butadiene copolymer, and styrene-butadiene copolymer.
Isoprene copolymers, polystyrene, styrene-acrylonitrile copolymers, and the like can be used.
Finally, the third or final step consists in applying another reagent to the carrier matrix, which reagent is present in a solvent that does not dissolve the polymeric substance applied in the second step. Usually the reagent strip may be dried between each step. Suitable solvents for o-phthalaldehyde include water, methanol, acetone and hexane. When the reagent o-phthalaldehyde is applied in the first step, water, methanol or mixtures thereof are the preferred solvents. The reagent o-
When using phthalaldehyde in the third step,
Acetone or hexane are preferred solvents.
For polymers, suitable solvents include chloroform, acetone benzene, toluene, and the like. Suitable solvents for other reagents include water, methanol and mixtures of methanol and acetone. Water is also the preferred solvent when the reagent is applied in the first step, and either methanol or a methanol-acetone mixture is the preferred solvent when the reagent is applied in the third step. These reagents can be used at various concentrations. Usually o-phthalaldehyde is about 300mg%~
Present in an amount of 800mg%, preferably about 500mg% to 600mg%. Another reagent is typically present in an amount of about 200 mg% to about 500 mg% when using the dihydrochloride;
About 600 mg% to about 1200 mg when using the free base
Present in an amount of %. The polymer substance is approximately 1g% to 3g
Preferably, it is present in an amount of %. Although the preferred method of preparing the test device is a three-step impregnation and drying method, the test devices of the present invention can be fabricated using other methods such as printing on one or both sides of the matrix or applying the reagent separately to both sides of the substrate or matrix. It can also be made by any suitable technique. The test device according to the invention can be advantageously used by immersing it momentarily in a test sample or by separately introducing the test sample onto the carrier matrix and observing the resulting detectable color change. can. Example 1 Preparation of the test device is not described. Reeve Engiel of Clifton, New Jersey
Angel, Clifton, New Jersey).
It has an exchange capacity of about 4 millieequivalents per gram of dry resin, which is 10-20%
Paper SA-2 containing cation exchange resin, previously washed with hydrochloric acid of
This SA-2 paper is a paper containing a sulfonated ion exchange resin (Amberlite IR120) to provide ion exchange properties. The resin comes in the form of sodium and the paper contains 45-50% resin. First Impregnation Step The paper was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Ingredients Water 100ml Maleic anhydride-methyl vinyl ether 100mg Ether copolymer (stabilizer) Boric acid (color stabilizer) 2.5g o-phthalaldehyde 300mg 7-(2,3-dihydroxypropyl)-theophylline (stabilizer) After impregnation, the paper was dried at 60°C for 30 minutes. Second Impregnation Step The dried impregnated paper obtained in the first step was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Ingredients Toluene 100 ml Polystyrene pellets (Styron, Dow Chemical Co., Midland, Mich.) 2 g Third impregnation step Then the dried impregnated paper obtained in the second step The samples were impregnated by immersion in a solution containing the indicated amounts of the following materials: Materials (1) and (4) are first dissolved, and then material (2) and
A solution was prepared by separately dissolving (3) and then adding it to materials (1) and (4). Amount of materials (1) Methanol 30ml (2) Acetone 70ml (3) Hydroxypropyl cellulose 0.5g (stabilizer) (4) 3-Hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline hydrochloride 300mg Next, The paper was dried at 60° C. for 30 minutes and attached to a polystyrene support using double-sided adhesive tape. The polystyrene support provides a suitable handle for dipping the resulting reagent-impregnated paper test device into the solution in order to determine the urea content in the solution. The color intensity of the urea nitrogen reagent strips for various urea concentrations is shown in Table 1 below. Serum sample 1 containing the indicated amount of urea nitrogen
1 part was diluted with 2 parts of water, and 0.030 ml of the solution was applied to the surface of the test device prepared in Example 1. After incubating for 1 minute at 37°C, the reflectance of the reagent strip against a white reflective surface of barium sulfate was measured at 620 nm.

[Table] Example 2 A method for preparing another test device is described. Dowex 50W in acid form cross-linked with 8% divinylbenzene (Dow Chemical Co., Midland, Michigan)
Paper containing 40% by weight of was impregnated with the following solution in three steps. First impregnation step In a solution containing the indicated amounts of the following materials:
The paper was dipped to impregnate it. Materials : Water 100ml Polyethylene oxide 50mg O-phthalaldehyde 300mg 7-(2,3-dihydroxycypropyl)-theophylline (stabilizer) 10g Boric acid 2.5g After impregnation, the paper was dried at 60°C for 30 minutes. Second Impregnation Step The dried impregnated paper obtained in the first step was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Materials : Toluene 100ml Styrene-acrylonitrile copolymer 2g After impregnating with the styrene copolymer, the paper was dried at 60°C for 15 minutes. Third Impregnation Step The dried impregnated paper obtained in the second step was impregnated by immersion in a solution containing the following materials in the indicated amounts. First dissolve materials [1] and [4],
Materials [2] and [3] were dissolved separately and then added to materials [1] and [4] to form a solution. Amount of materials (1) Methanol 30ml (2) Acetone 70ml (3) Hydroxypropyl cellulose 0.5 (4) 3-acetoxy-1,2,3,4-tetrahydrobenzo [h] Quinoline hydrochloride 300mg Next, this paper was heated to 60℃. for 10 minutes and attached to a polystyrene support with double-sided adhesive tape. The polystyrene support provides a suitable handle for dipping the resulting reagent-impregnated paper, or test device, into the solution in determining the urea content in the solution. Cross-linked with 2% and 16% cyvinylbenzene
Another test device was prepared by the same method as this Example 2 using Dowex 50W. It has been found that the intensity of the color produced upon immersion in a urea sample can be adjusted by varying the amount of crosslinking. The color intensity varied inversely with the degree of crosslinking. The ratio of color formation of reagent strips for urea nitrogen measurement prepared using carriers containing ion exchange resins with varying degrees of crosslinking of divinylbenzene (DVB) is shown in Table 2 below. . Reagent strips were prepared as in Example 2 using paper loaded with ion exchange resins containing Dowex 50 resin crosslinked with 2%, 8% and 16% divinylbenzene. Then dilute the urea sample and incubate at 37 °C.
After incubating for 1 minute, measurements were made using a spectrophotometer in the same manner as in Table 1.

[Table] The color formation rates of reagent strips for urea nitrogen measurement prepared using carriers containing various amounts of ion exchange resin are shown in Table 3 below. Reagent strips were prepared as in Example 1 using paper loaded with ion exchange resins containing 25%, 35% and 50% by weight of ion exchange resin crosslinked with 8% divinylbenzene. The urine samples were diluted, incubated at 37°C for 1 minute, and then measured with a spectrophotometer as shown in Table-1.

Table: Example 3 A test device was prepared using paper containing an ion exchange resin as described in Example 2 using the following three step method. First Impregnation Step The paper was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Materials Water 25ml Polyethylene oxide 100mg 7-(2,3-dihydroxypropyl)-theophylline 10g Ethylene dinitrilotetraacetic acid dipotassium salt 1.5g Sodium lauryl sulfate 100mg Methanol 75ml Glycerin 0.2ml Boric acid 4.5g O-phthalaldehyde 600mg After impregnation, The paper was dried at 50°C for 20 minutes. 2nd impregnation process Material amount Toluene 100ml Polystyrene pellets 3g After styrene polymer impregnation, the paper was heated at 50℃.
Let dry for 10 minutes. Third Impregnation Step The dried impregnated paper obtained in the second step was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Combine ingredients (2), (3) and (4) separately,
Next, material (1) was added to prepare a solution. Amount of materials (1) Methanol 25ml (2) Acetone 75ml (3) Hydroxypropyl cellulose 0.75g (4) 3-Hydroxy-1,2,3,4-tetrahydrobenzo [h] Quinoline 900mg This paper was heated at 50℃ for 5 minutes. Dry. The resulting test device can be used with undiluted serum or plasma, as was required for the test devices of Examples 1 and 2 for urea nitrogen concentrations up to 70 mg%. It will be appreciated that the use of the stabilizer hydroxypropyl cellulose is not essential to the present invention. Additionally, it will be appreciated that stabilizers such as caffein can be replaced with daifilin, which is used in the following examples. It has been found that daphyllin or its substitute should be used in amounts greater than 5g%. Example 4 For comparison, another test device was prepared by the following method. First Step Whatmann 3MM paper was acid modified by immersing it in a solution containing the following materials in the indicated amounts. Materials : Water 100ml Benzenedisulfonic acid 10g N-naphthylethylenediamine dihydrochloride 400mg Daifilin 10g Maleic anhydride-methylvinyl 0.1g Ether copolymer (stabilizer) After impregnation, the paper was dried at 60°C for 20 minutes. Second Step The dried impregnated paper from the first step was impregnated by immersing it in a solution containing the following materials in the indicated amounts. Materials : Polyethylene oxide 1g Chloroform 70ml Acetone 30ml The paper was then dried at 60°C for 20 minutes. Third Step The dried impregnated paper obtained in the second step was then impregnated by immersion in a solution containing the indicated amounts of the following materials. Materials Hexane 100 ml O-phthalaldehyde 300 mg The paper was then dried at 60° C. for 20 minutes as before. The resulting dry impregnated paper was adhered to a polystyrene support using double-sided adhesive tape. The resulting reagent strip can be used for quantitative analysis of the deproteinized sample. Undesirable precipitation occurs when attempting to quantify samples containing proteins. This fact indicates that the discovery made is of great importance. From the foregoing, it will be appreciated that the present invention is well suited for achieving the results and objectives set forth above along with other advantages that are obvious and inherent to the system. Three specific problems are solved by preparing reagent strips according to the present invention. First, under normal acidic test conditions,
Serum proteins precipitate and interfere with pigment (chromogen) production. The present invention eliminates serum protein precipitation;
On the one hand, the use of paper containing strong cation exchange resins provides a suitable acidic matrix. Second, while the reagents used are typically unstable, the present invention provides a method for incorporating the reagents into the paper matrix in such a manner that the reagents are stabilized. The third problem solved according to the invention is minimizing the measurement error of the spectrophotometer when measuring urea content in serum. The present invention
Utilizes coupling agents that produce dyes detectable at wavelengths above 550 nm, thus causing problems caused by the yellow sky reaction that occurs in the strips and bilirubin and/or in serum observed by spectrophotometer or spectrophotometer. overcome the interfering colors caused by turbidity. 3-Hydroxy-
The use of 1,2,3,4-tetrahydrobenzo[h]quinoline or its dihydrochloride minimizes the occurrence of blank reactions. Conversely, when primaquine diphosphate [8-(4-amino-1-methylbutylamino)-6-methoxyquinoline] or N-naphthylethylenediamine dihydrochloride is used, the resulting dye is near the absorption maximum of the vacant reaction. Must be measured in wavelength. Therefore 3-
By using hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline or its dihydrochloride, it becomes possible to measure at a wavelength at which the color due to empty reaction is minimized. Furthermore, the present invention encapsulates all reagents within a single matrix without the use of corrosive liquid reagents. The present invention is of great importance for the early detection of physiological disorders by measuring the urea content in body fluids as shown here, but the concentration of urea is within a limited range. It is also used to accurately measure urea in industrially processed water that needs to be controlled. A device for measuring urea-containing liquids, whether industrial or physiological, is advantageous enough that the test device is fast, reliable, and simple enough to be easily mastered by technicians. If so, it's worth it. Furthermore, in the case of medical diagnosis, the test device must be accurate enough to reflect changes in the patient's health status. This objective is achieved by using the present invention. Obviously, many other modifications and variations of the invention may be made without departing from the spirit and scope of the invention as described above.

Claims (1)

[Claims] 1. A carrier matrix containing an acid type strong cation exchange resin contains reagents o-phthalaldehyde and 3-
Hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline, 3-acetoxy-1,2,
and any one reagent selected from the group consisting of 3,4-tetrahydrobenzo[h]quinoline and their dihydrochlorides, both of which are separated from each other by a polymeric substance. A test device for detecting urea. 2. The test device according to claim 1, wherein 3-acetoxy-1,2,3,4-tetrahydrobenzo[h]quinoline is present. 3. The test device according to claim 1, wherein 3-hydroxy-1,2,3,4-tetrahydrobenzo[h]quinoline is present. 4 Reagent o-phthalaldehyde is approximately 300 mg% to approximately
800 mg% and the other reagents are present in an amount of about 200 mg% to about 500 mg% when using the dihydrochloride;
4. The test device of claim 2 or 3, wherein the free base is present in an amount of about 600 mg% to about 1200 mg%. 5. The test device according to claim 1, wherein the carrier matrix is a water-absorbing cellulose paper matrix. 6. The test device according to claim 5, wherein the carrier matrix contains about 25% to about 50% by weight of an ion exchange resin. 7. The test device of claim 6, wherein the ion exchange resin is a resin crosslinked with about 2% to about 16% divinylbenzene.