ANALYTICAL COMPOSITION, ELEMENT AND METHOD FOR THE DETERMINATION OF HYDROGEN PEROXIDE

Abstract: An analytical method for the determination of hydrogen peroxide utilizes a hydrazide or a hydrazine substituted with one or more electron withdrawing groups. The hydrazides or hydrazines react with phenolic color couplers in the presence of hydrogen peroxide and a peroxidative compound to form a dye detectable at a wavelength of 600 nm or greater. These materials can also be used to assay analytes, such as glucose and cholesterol, which can react to produce hydrogen peroxide.

Description: This invention relates to a novel composition, element and method using particular hydrazides and hydrazines to detect hydrogen peroxide or another analyte which can react to produce hydrogen peroxide. In particular, this invention relates to an assay of hydrogen peroxide or hydrogen peroxide-producing analytes in biological fluids. This invention is particularly useful in clinical chemistry. The detection and quantitative determination of hydrogen peroxide and compounds which yield hydrogen peroxide as a result of chemical or enzymatic reactions are of importance in many areas. For example, they are important in the determination of hydrogen peroxide produced in the enzymatic assay of various chemical or biological substances (herein identified as analytes), such as glucose, cholesterol, uric acid, triglycerides, creatine kinase or creatinine, in the presence of oxygen. The quantity of analyte present in a sample is determinable from the amount of hydrogen peroxide produced. Known compositions and methods for determining hydrogen peroxide generally comprise a substance having peroxidative activity, for example peroxidase, and a color dye former which undergoes a detectable change (for example, a color change) in the presence of hydrogen peroxide and peroxidase. Various materials which are known to undergo a detectable change in such conditions include monoamines, diamines, phenols and leuco dyes. Other hydrogen peroxide indicators include hydrogen donors (identified herein as color forming compounds) which react with color couplers to produce dyes. U.S. Patent 4,089,747 relates to assays for hydrogen peroxide or analytes which generate hydrogen peroxide using a combination of a hydrazone and a color coupler. Use of the hydrazones described in this reference in assaying whole blood is disadvantageous. Using the hydrazones, it is difficult to obtain detectable dyes which absorb electromagnetic radiation at relatively long wavelengths, that is, greater than 600 nm. Dyes formed with hydrazones generally absorb at shorter wavelengths and their detection is often hindered by various spectral interferents which are present in whole blood samples. The presence of these interferents would diminish the accuracy of the assay using hydrazones in testing whole blood or serum. The present invention overcomes the disadvantage noted above with an analytical composition which is useful for the determination of hydrogen peroxide or an analyte which will react to produce hydrogen peroxide. This composition comprises a phenolic color coupler, and a color-forming compound, the composition being characterized as further comprising a cationic mordant and the color-forming compound being represented by the structure wherein R is hydrogen, -SO₂R³ or -SONR³R⁴, R¹ is an electron withdrawing group, R², R³ and R⁴ are independently hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, n is an integer of 1 to 5, and Z¹ represents the carbon and nitrogen atoms necessary to complete a substituted or unsubstituted 5- to 10-membered aromatic carbocyclic or heterocyclic ring, provided that the color-forming compound is capable of reacting with the color coupler in the presence of the mordant to provide a dye detectable at a wavelength of 600 nm or greater. This invention also provides a dry analytical element for the determination of hydrogen peroxide or an analyte which will react to produce hydrogen peroxide. This element comprises an absorbent carrier material and contains a phenolic color coupler and a color-forming compound, the element being characterized as further comprising a cationic mordant and the color-forming compound shown above. A method for the determination of hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide comprises the steps of: A. contacting a sample of a liquid suspected of containing hydrogen peroxide or an analyte which will react to produce hydrogen peroxide with a peroxidative compound, a phenolic color coupler and a color-forming compound, and B. detecting the dye formed as a result of the presence of hydrogen peroxide or the analyte at a wavelength equal to or greater than 600 nm, the method characterized wherein the contacting is carried out in the presence of a cationic mordant and the color-forming compound shown above. The assay of the present invention has a number of advantages. It provides dyes in response to hydrogen peroxide which can be detected at a wavelength equal to or greater than 600 nm. Spectrophotometric detection of the dyes in this invention thereby avoids potential spectral interferents such as hemoglobin and bilirubin generally found in biological fluids. The color-forming compounds useful in the present invention are hydrazides or hydrazines substituted with one or more electron withdrawing groups which are represented by the structure: In this structure, R is hydrogen, -SO₂R³ or -SONR³R⁴. Preferably, R is hydrogen or -SO₂R³, and more preferably, it is -SO₂R³. R², R³ and R⁴ are independently hydrogen, substituted or unsubstituted alkyl (preferably of 1 to 6 carbon atoms, for example, methyl, chloromethyl, ethyl or isopropyl) or substituted or unsubstituted aryl (preferably of 6 to 10 carbon atoms, for example, phenyl, naphthyl or p -methylphenyl). Preferably, R² is hydrogen and R³ is hydrogen or substituted or unsubstituted phenyl. Also, n is an integer of 1 to 5, and preferably is 1 or 2. R¹ is an electron withdrawing group as defined in the art. Such a group generally has a positive Hamett sigma value which is calculated according to standard procedures described, e.g. in Steric Effects in Organic Chemistry , John Wiley and Sons, Inc., 1956, pp. 570-574 and Progress in Physical Organic Chemistry , Vol. 2, Interscience Publishers, 1964, pp. 333-339. Representative electron withdrawing groups include halo (for example, chloro, bromo or iodo), substituted or unsubstituted alkoxycarbonyl (for example, methoxycarbonyl or ethoxycarbonyl), nitro, cyano, substituted or unsubstituted arylsulfonyl (preferably of 6 to 12 carbon atoms, for example, phenylsulfonyl) and sulfamoyl. Nitro is a preferred electron withdrawing group. Z represents the carbon and nitrogen atoms necessary to complete a substituted or unsubstituted 5- to 10-membered aromatic carbocyclic or heterocyclic ring. For example, the completed ring could be a phenyl, pyridinyl, pyrazinyl, quinolyl or benzothiazolyl ring. Preferably, the ring is a substituted or unsubstituted phenyl or pyridinyl ring. Most preferably, the ring is a substituted or unsubstituted pyridinyl ring. Representative color forming compounds useful in the practice of this invention include: 2-(benzenesulfonylhydrazino)-5-nitropyridine, 2-hydrazino-5-nitropyridine, 2-benzenesulfonylhydrazino-(N-ethyl-N-phenylsulfamoyl)-3-chloropyridine, 2-chloro-3-hydrazinopyrazine and 2-hydrazino-3-chloro-5-sulfamoylpyridine. The first compound is a preferred one. The color-forming hydrazines and hydrazides useful in the practice of this invention are known compounds. They can be prepared using known procedures and generally available starting materials. The hydrazines can be prepared, for example, according to the procedure described in Org. Syn. Coll. , Vol. II , 228 (1943). The hydrazides can be prepared, for example, according to the procedure described in Org. Syn. , 40 , 93 (1960). The color-forming compounds described herein are used with a phenolic color coupler preferably represented by the structure: wherein Z¹ is as defined above, R⁵ is hydrogen or an electron donating group as known in the art, and p is an integer of 1 to 5. Electron donating groups generally have a negative Hammett sigma value as determined according to standard techniques described above. Representative electron donating groups include substituted or unsubstituted alkyl (preferably of 1 to 6 carbon atoms, for example, methyl, ethyl, aminomethyl or isopropyl), substituted or unsubstituted alkoxy (preferably of 1 to 6 carbon atoms, for example, methoxy, ethoxy or isopropoxy), substituted or unsubstituted dialkylamino (preferably having from 1 to 6 carbon atoms in the alkyl portion), hydroxy, sulfonamido and acetamido. Preferred electron donating groups are hydroxy, alkoxy or dialkylamino as defined above. Representative color couplers include 1,7-naphthalenediol, 2,6-dimethoxyphenol, m -dimethylaminophenol, 2,7-naphthalenediol, 1-naphthol-8-sulfonic acid, 8-amino-1-naphthol, 1-naphthol and 2,3-dimethylphenol. Preferred couplers include 1,7-naphthalenediol, 2,6-dimethoxyphenol and 1-naphthol. In the practice of this invention, the color-forming compounds and color couplers described herein are used in combination with a cationic mordant in order to provide a dye detectable at a wavelength of 600 nm or greater. Such a mordant is a polymeric material having one or more positive charges placed along the polymeric backbone directly thereon or on side chains. Cationic mordants are known in the art and described, for example, in U.S. Patents 3,958,995, 4,069,017, 4,124,386 and 4,247,615 and Research Disclosure , publication 12045 (April, 1974). Procedures for making such mordants are described in these and other published references. Representative useful mordants include: poly(styrene- co -N-benzyl-N,N-dimethyl-N-vinyl-benzylammonium chloride- co -divinylbenzene), poly(N,N,N-trimethyl-N-vinylbenzylammonium chloride), poly(N,N,N-trioctyl-N-vinylbenzylphosphonium chloride) and poly(styrene- co -N-vinylbenzyl-N,N,N-trihexylammonium chloride). The analytical composition of this invention can be used in both solution and dry assays, and comprises the color-forming compound, phenolic color coupler and cationic mordant described above. The composition is used with, and preferably includes, a substance having peroxidative activity. Substances having peroxidative activity are also known as peroxidative substances and are capable of catalyzing the oxidation of another substance by means of hydrogen peroxide and other peroxides. Such substances include natural and synthetic peroxidases, cytochromes, hemin, forms of hemoglobin, alkaline hematin, iron sulfocyanate, iron tannate, chromic salts and the like. Peroxidase is a particularly useful peroxidative substance. Substantially any buffer can be used with the composition of this invention. Buffers are generally used to provide a pH in the assay which is conductive to dye formation. Generally, the pH is kept within the range of from 4 to 9, but a specific pH will depend to some extent on the particular analyte being assayed. Useful buffers include carbonates, borates, phosphates, glutarates, the tris materials, for example, tris(hydroxymethyl)aminomethane and others known in the art. The compositions of this invention can be prepared for use in a solution assay be mixing the components of the analytical composition in water or a buffer. If the color-forming compounds, phenolic couplers and mordants have limited solubility in water, they can be dissolved in a water-miscible solvent, such as an alcohol or N,N-dimethylformamide, prior to mixing. The peroxidative substance can be mixed in as well. The details of preparing representative analytical compositions are given in Examples 1-5 below. When the compositions of this invention are used in solution assays, generally the color-forming compound is present in a concentration of at least 1, and preferably from 5 to 50, µg/ml of final solution. The color coupler is generally present in a concentration of at least 1, and preferably from 5 to 50, µg/ml of solution. The cationic mordant is generally present in an amount of at least 0.1, and preferably from 0.5 to 1, µg/ml of final solution. The peroxidative substance can be present in an amount sufficient to catalyze the dye-forming reaction. For example, peroxidase can be generally present in an amount of at least 0.01 and preferably from 1 to 10, I.U./ml. The amounts of other addenda generally used (for example, buffer or surfactant) and of the interactive composition (described below) are within the skill of a worker in the art. The compositions of this invention can be used to determine hydrogen peroxide or an analyte which is capable of producing hydrogen peroxide, that is, an analyte which can participate in one or more reactions which produce hydrogen peroxide. To determine such analytes, the analytical composition described above is combined with an interactive composition with which the analyte will react to produce hydrogen peroxide in one or more reactions. Analytes which can be determined in this manner include glucose, triglycerides, uric acid, cholesterol, galactose, amino acids, creatine kinase, creatinine, and others known to one skilled in the clinical chemistry art. Specific interactive compositions for a given analyte can be readily determined by one of ordinary skill in the art. The present invention is adaptable to both solution and dry assays. In a solution assay, generally the analytical composition, peroxidative substance and interactive compositions, if included, are contacted with a liquid test sample suspected of containing hydrogen peroxide or another analyte by mixing in a suitable container (for example, test tube, petri dish, beaker or cuvette). The resulting solution is incubated for a relatively short time at a temperature of up to 45°C. The sample is then evaluated by measuring the amount of dye provided as a result of the presence of hydrogen peroxide. The amount of dye can then be correlated to the amount of hydrogen peroxide either initially present in the sample, or produced as a result of the presence of an analyte. Such an evaluation can be done visually or with suitable colorimetric detection equipment and procedures at a wavelength greater than or equal to 600 nm. Alternatively, the method of this invention can be practiced with a dry analytical element which is comprised of an absorbent carrier material, that is, self-supporting absorbent sheet or pressed material, such as filter paper or strips, which contains the analytical composition and optionally, any other desired reagents, for example, the peroxidative substance. Such elements are known in the art as test strips, diagnostic elements, dip sticks or diagnostic agents. When employed in dry elements, the composition of this invention can be incorporated into a suitable carrier material by imbibition, impregnation, coating or another suitable technique. Useful carrier materials are insoluble and maintain their structural integrity when exposed to water or physiological fluids such as urine or serum. Useful elements can be prepared from paper, porous particulate structures, cellulose, glass fibers, or woven and nonwoven fabrics (synthetic and nonsynthetic). A useful dry analytical element is made by imbibing a solution of the analytical composition into the material and drying. Useful materials and procedures for making such elements are well known in the art as exemplified in U.S. Patents 3,092,465, 3,802,842, 3,915,647, 3,917,453, 3,936,357, 4,248,829, 4,255,384 and 4,270,920 and U.K. Patent 2,052,057. Preferably, the dry analytical elements of this invention have at least one porous spreading zone. This zone can be a self-supporting carrier material as described above, but preferably it is carried on a separate nonporous support. Such a support can be any suitable dimensionally stable, and preferably, transparent material which transmits electromagnetic radiation of a wavelength between 200 and 900 nm. A support of choice for a particular element should be compatible with the intended mode of detection (reflection, transmission or fluorescence spectroscopy). Useful support materials include paper, metal foils, polystyrene, polyesters, polycarbonates or cellulose esters. The porous spreading zone can be prepared from any suitable fibrous or non-fibrous material or mixtures of either or both. The void volume and average pore size of this zone can be varied depending upon the type of liquid sample to be applied. Useful spreading zones can be prepared as described, for example, in U. S. Patents 4,292,272, 3,992,158, 4,258,001 and 4,430,436 and Japanese Patent Publication 57(1982)-101760. It is desirable that the spreading zone be isotropically porous, meaning that the porosity is the same in each direction in the zone as created by interconnected spaces or pores between particles, fibers or polymeric strands. The elements can have more than one zone, for example, one or more reagent zones, spreading zones, registration zones, mordant zones, radiation-blocking or filter zones, subbing zones, barrier zones or buffer zones. The zones are generally in fluid contact with each other meaning that fluids, reagents and reaction products can pass between superposed regions of adjacent zones. Preferably, the zones are separately coated superposed layers, although multiple zones can be in a single layer. The components of the analytical composition of this invention, as well as the peroxidative substance, interactive composition or buffer can be incorporated in any of the element zones. The location of individual components is within the skill of a worker in the clinical chemistry art. In the elements of this invention, the amount of the composition components can be varied widely. Generally the color-forming compound is present in a coverage of at least 10, and preferably from 30 to 600, mg/m². The phenolic color coupler is generally present in a coverage of at least 10, and preferably from 30 to 600, mg/m². The cationic mordant is generally present in a coverage of at least 1, and preferably from 5 to 25, mg/m². The peroxidative substance can be present in a coverage generally of at least 25,000, and preferably from 50,000 to 100,000 I.U./m² for peroxidase. A variety of other desirable, but optional reagents and addenda can be present in the element in amounts known to one skilled in the art. A variety of different elements, depending on the method of assay, can be prepared in accordance with the present invention. Elements can be configured in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips. The assay of this invention can be manual or automated. In general, in using the dry elements, hydrogen peroxide or analyte determination is made by taking the element from a supply roll, chip packet or other source and physically contacting it with a sample (up to 200 µl) of the liquid to be tested such that the sample mixes with the reagents within the element. Such contact can be accomplished in any suitable manner, for example, by dipping or immersing the element into the sample or, preferably, by spotting the element by hand or machine with a drop of the sample using a suitable dispensing means. After sample application, the element is exposed to any conditioning, such as incubation, heating or the like, that may be desirable to quicken or otherwise facilitate obtaining any test result. Determination of hydrogen peroxide or an analyte is achieved when the color-forming compound and phenolic coupler react to form a dye. This dye can be detected with the unaided eye or with suitable spectrophotometric means and procedures at a wavelength greater than or equal to 600 nm. Generally, the dyes formed in the practice of this invention have a λ max , or an absorption maximum, equal to or greater than 600 nm. As used in the context of this disclosure, I.U. represents the International Unit for enzyme activity defined as one I.U. being the amount of enzyme activity required to catalyze the conversion of 1 micromole of substrate per minute under standard pH and temperature conditions for the enzyme. Examples 1- 7: Solution Assay of Hydrogen Peroxide with Various Couplers These examples illustrate the practice of this invention to determine hydrogen peroxide in solution assays using a particular color-forming compound and various phenolic couplers. Analytical compositions of this invention were prepared, each comprised of: the color-forming compound 2-(benzenesulfonylhydrazino)-5-nitropyridine (0.1 ml of 0.025% solution in methanol), a color coupler as noted in Table I below (0.1 ml of 0.025% solution in methanol), 0.2 ml of a 1.8% latex dispersion of the mordant poly(styrene- co -N-benzyl-N,N-dimethyl-N-vinylbenzyl ammonium chloride- co -divinylbenzene) (99:99:2 molar ratio), and phosphate buffer (pH 8) to 4 ml. Control compositions were similarly constituted but absent the mordant latex. Peroxidase (0.1 ml of 0.2% in pH 7 phosphate buffer) and a test sample (1 drop) containing 3% hydrogen peroxide were added to each solution. Each resulting assay solution was shaken and the hue and intensity (optical density, O.D.) of the resulting dye were observed after color formation was complete (0.5 to 5 minutes). The Control assays provided dyes having little or no absorption above 600 nm as noted in Table I below. The compositions of this invention, however, provided dyes which had significant absorption at or above 600 nm as noted in Table I below. Dye densities were measured against a blank containing all of the components and invention compositions except hydrogen peroxide. Examples 8-14 : Solution Assay for Hydrogen Peroxide Using Various Color-Forming Compounds These examples are like Examples 1-7 except the same color coupler (1-naphthol) was used at two concentrations with several color-forming compounds as shown in Table II below. The assays were performed as described in Examples 1-7. The assays provided dyes which have significant absorption at or greater than 600 nm as noted in Table II below. It was observed that the Control solutions (without mordant) had some turbidity which contributed to most of the optical density. Little or no turbidity was observed in the solutions containing mordant. In Examples 10, 12 and 14, color formation was low at low concentrations of 1-naphthol. Example 15 : Dry Assay of Glucose Using Dry Analytical Element This example illustrates the assay of glucose in a test liquid using a dry analytical element having the format and components illustrated below. Samples (10 µl) of test liquids containing various amounts of either hydrogen peroxide or glucose were applied to the spreading layer of the element. The reflection density (D R ) of the resulting dye in the element was measured at 650 nm after incubation at 37°C for 5 minutes using a commercially available spectrophotometer. The resulting densities are recorded in Table III below. Dye density was proportional to the amount of analyte in the test liquid. A Control liquid containing no analyte was similarly tested. Hydrogen Peroxide Glucose Concentration (mmolar) D R Concentration (mg/dl) D R 0 0.56 0 0.72 2.5 1.15 12.5 1.17 5 1.61 25 1.41 10 2.02 50 1.7 20 2.22 100 2.05

Claims: An analytical composition for the determination of hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide comprising a phenolic color coupler and a color-forming compound, characterized in that the composition further comprises a cationic mordant and the color-forming compound being represented by the structure wherein R is hydrogen, -SO₂R³ or -SONR³R⁴, R¹ is an electron withdrawing group, R², R³ and R⁴ are independently hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, n is an integer of 1 to 5, and Z¹ represents the carbon and nitrogen atoms necessary to complete a substituted or unsubstituted 5- to 10-membered aromatic carbocyclic or heterocyclic ring, provided the color-forming compound is capable of reacting with the color coupler in the presence of the mordant to provide a dye detectable at a wavelength of 600 nm or greater. A dry analytical element for the determination of hydrogen peroxide or an analyte which will react to produce hydrogen peroxide comprising an absorbent material and containing a phenolic color coupler and a color-forming compound, characterized in that the element further comprises a cationic mordant and the color-forming compound as defined in claim 1. The analytical composition or the dry analytical element as claimed in either of claims 1 or 2 wherein R is hydrogen or -SO₂R³, R² is hydrogen, R³ is hydrogen or substituted or unsubstituted phenyl, n is 1 or 2, and Z¹ represents the carbon and nitrogen atoms necessary to complete a substituted or unsubstituted 6-membered phenyl or pyridyl ring. The analytical composition or the dry analytical element as claimed in claim 3 wherein R¹ is nitro and Z represents the carbon and nitrogen atoms necessary to complete a substituted or unsubstituted pyridyl ring. The analytical composition or the dry analytical element as claimed in any of claims 1 to 4 further comprising a substance having peroxidative activity. The analytical composition or the dry analytical element as claimed in claim 5 wherein the substance having peroxidative activity is peroxidase. The analytical composition or the dry analytical element as claimed in any of claims 1 to 6 wherein the phenolic color coupler is represented by the structure wherein Z¹ is as defined in claim 1, R⁵ is hydrogen or an electron donating group, and p is an integer of 1 to 5. The analytical composition or the dry analytical element as claimed in any of claims 1 to 7 further comprising an interactive composition which produces hydrogen peroxide upon interaction with an analyte. A method for the determination of hydrogen peroxide or an analyte which will react to produce hydrogen peroxide comprising the steps of: A. contacting a sample of a liquid suspected of containing hydrogen peroxide or an analyte which will react to produce hydrogen peroxide with a substance having peroxidative activity, a phenolic color coupler, and a color-forming compound, and B. detecting the dye formed as a result of the presence of hydrogen peroxide or the analyte at a wavelength equal to or greater than 600 nm, characterized in that the contacting is carried out in the presence of a cationic mordant and the color-forming compound as defined in claim 1. The method as claimed in claim 9 for the determination of glucose with an interactive composition comprising glucose oxidase.

Inventors: EVANS, STEVEN, SUTHERLAND, JOHN WILLIAM H.
Applicants: EASTMAN KODAK COMPANY (A NEW JERSEY CORPORATION)
Application year: 1987
Main classification: G01N33
IPC codes: C12Q1, G01N31, G01N33