JPH0418630B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION Field of the Invention The present invention is useful in general for analytical measurements of peroxide active substances in a sample, and in particular for such measurements,
The present invention relates to compositions, test means, test devices and test methods that are resistant to deleterious effects from ascorbic acid that may be present in a sample. BACKGROUND OF THE INVENTION A number of analytical methods are currently available for detecting the presence of peroxide active substances in biological samples, such as urine, fecal suspensions and gastrointestinal contents. For example, hemoglobin and its derivatives, the analytes measured by the occult blood test, represent peroxide-active substances because they behave similarly to peroxidase enzymes, and they are themselves also known as pseudoperoxidases. It is said. Peroxide active substances include peroxides or hydroperoxides and indicators that produce a detectable response such as a color change, such as benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7 - It is similar to an enzyme in that it catalyzes a redox reaction with diaminofluorene, etc. Therefore, most methods of determining the presence of occult blood in a sample rely on this pseudoperoxidase activity. A number of analytical methods have been developed to measure peroxide-active substances that rely on the enzyme-like catalysis of peroxide oxidation by colored indicators. First, these include wet chemistry or dissolution operations and so-called "dip and read" type reagent holding strip devices. A typical example of the former is Henry (RMHenry).
Clinical Chemistry Principles and Techniques
Principles and Techniques), 2nd edition 1124~
1125 (Hagerstown, MD: Harper
and Row, 1974). An example of this procedure is the use of glacial acetic acid (buffer), diphenylamine (indicator) and hydrogen peroxide. Although such wet chemistry methods have proven useful for analysis, these methods have a number of drawbacks, two of which are lack of reagent stability and inadequate sensitivity. There is a drawback. Another method of measuring peroxide actives, currently preferred by many clinical analysts, utilizes a so-called "dip-read" reagent strip device. A typical example of such a "dip-read" device is the one manufactured by Miles Laboratories, Inc.
It is commercially available from the Ames Division of Laboratories, Inc. under the trademark HEMASTIX. The test device consists of a porous paper matrix impregnated with a buffered mixture of organic hydroperoxide and indicator, secured to a plastic strip or handle. This matrix is called hemoglobin,
When immersed in a liquid containing myoglobin, red blood cells or other peroxide-active substances, ie pseudoperoxidase, a blue color appears in the matrix, the intensity of which is proportional to the concentration of the substance in the sample. By comparing the colors appearing in the matrix with a standard color chart, the analyst can semi-quantitatively determine the amount of analyte present in the sample. First, the advantages of such reagent strips over wet chemical methods are that 1) strips are easy to use and do not require reagent manufacturing or associated equipment; It becomes more stable and improves accuracy, sensitivity and economy. Regardless of the specific analysis for peroxide active species performed by either of the above methods, there are inherent problems with both. That is, interference generally occurs due to the presence of reducing agents and in particular ascorbic acid or ascorbate ions in the sample (hereinafter referred to as ascorbate interference). For example, in the case of urine analysis, the recent popularity of diets containing high doses of vitamin C (ascorbic acid) creates serious ascorbate interference problems when analyzing certain urine components, such as occult blood. As early as 1938, the harmful effects of reducing agents such as ascorbate were recognized. Corn (R.
Kohn and RMWatrous, Journal of Biological Chemistry, 124:163.
~168 (1938). Occult blood (pseudoperoxidase)
As evidenced by the 1979 proposal that when performing an assay, an ascorbate assay should also be performed to assess the accuracy of occult blood measurements.
The same problems still plague this field of diagnostic analysis. L.Nielsen, PJJorgensen and ACHansen
Author, Ugeskrift for Laeger, 141, 791-793
(1979). Numerous attempts to eliminate ascorbate interference on test systems, such as those containing glucose-sensitive reagents, have been reported in the literature. Various approaches exist for glucose sensitivity assays, ranging from filtering off ascorbate before it reaches the reagent to utilizing enzymes that degrade ascorbate in situ. Dahlqvist Canadian Patent No. 844564
A device for measuring glucose in urine or other media is disclosed that includes, in addition to a porous portion impregnated with a conventional glucose-responsive reagent, an additional portion for receiving a urine sample. The sample receiving portion contains an ion exchange material whose sole function in the measurement device is to adsorb ascorbate present in the urine sample. US Pat. No. 4,168,205 to Danninger et al. suggests incorporating the enzyme ascorbate oxidase into the test reagent formulation. In this case, ascorbate present in the sample is enzymatically oxidized by ascorbate oxidase to yield dehydroascorbate, a compound that does not adversely affect the desired analysis. Another approach to eliminating ascorbate interference is described in Fuji Organ Pharmaceutical Co., Ltd.'s Japanese Patent Application Publication No. 58-55757. The publication discloses the use of various ligands, such as metal chelates of ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, to pretreat samples and then analyze them for cholesterol, glucose or other components, such as uric acid. There is. Ku, U.S. Pat. No. 3,411,887, discloses the production of ascorbate with a reagent system that relies on enzyme oxidants, such as glucose oxidase, by using an ascorbate "trapping system." It is stated that it eliminates bait interference. "Trapping systems" relate to ionizable heavy metal compounds that, when ionized, drop the oxidation-reduction potential between the redox indicator dye and ascorbate. Suitable metals are, for example, cobalt, iron, mercury and nickel. No. 4,288,541, formally assigned to Magers et al., discloses the use of mercury ion complexes, such as sarcosine, to impart ascorbate resistance to glucose/glucose oxidase assay systems. It states that acid mercury is used. In addition to the above references, caution regarding ascorbate problems associated with glucose testing can be found in the following references: 1. H. Gifford et al., J. Amer. Med.
Assoc., 178, 149–150 (1961). 2 PO'Gorman et al., Brit.
Med.J., 603–606 (1960). 3 R.Brandt et al., Clin.Chem.
Acta, 51, 103–104 (1974). 4 R.Brandt et al., Am.J.Clin.
Pathol., 68, 592–594 (1977). Similar to the approach of the Ku patent mentioned above, other publications use cobalt to complex and oxidize ascorbate. For example, G. Bragagnolo, Ann.Chim.Applicata 31,
350-368 (1941) reported the air oxidation of solutions of ascorbic acid in the presence of metallic cobalt. In addition, the Journal of the Chemical Society of Japan (Journal of the Chemical Society of Japan)
Chemical Society of Japan), 63, 820-826
Similar activity for Co(NH ₃ ) ₆ CL ₃ was reported by Tomokichi Iwasaki in (1942). Although the aforementioned literature deals extensively with analytical systems for the measurement of glucose, it also addresses the problem of ascorbate interference associated with the measurement of peroxide-active substances such as peroxidase and pseudoperoxidase, e.g. occult blood (hemoglobin). No suggestions have been made to resolve the issue. Despite the disclosure in the above-mentioned Coo patent, the above literature indicates that metal ions such as Co() are in fact also pseudoperoxidases. For example, cobalt acetate () is used industrially for the catalytic decomposition of cumene hydroperoxide. [The Merck Index
Index), 9th edition 311 (1976). [K.Lohs.]
Monatsber.Deut.Akad.Wiss.Berlin, 8, 657~
659 (1966) (Chem. Abstracts, 67, 120383z,
A series of Co() complexes for the catalytic decomposition of peroxides have been reported by (1967). Therefore, the use of such metal complexes in typical analytical preparations for the determination of peroxide-active substances, i.e., analytical preparations containing organic hydroperoxides and indicators, may lead to harmful reactions with the hydroperoxides. It would be considered by one skilled in the art to cause the peroxide to react with the peroxide active substance in question, e.g. occult blood, and thus render it unusable for such measurements. It is clear that he is deaf. Indeed, efforts to use mercury complexes, such as mercuric sarcosinate, in occult blood testing have failed. U.S. Pat. No. 4,310,626 to Burkhardt et al., which is duly assigned, addresses this problem by adding ammonium Co to reduce ascorbate interference with compositions for measuring peroxide actives. () describes the use of a complex. This patent describes compositions comprising an organic hydroperoxide and a suitable indicator, such as 3,3',5,5'-tetramethylbenzidine, together with an ammonium Co() complex, such as Co( _NH3 ) _{6CL3 ,} among others. Disclosed. However, these complexes did not provide sufficient ascorbate resistance to be industrially advantageous for occult blood testing. Other approaches to ascorbate interference in the analytical determination of peroxide active substances include, for example, German Patent No. 2,907,628. This German patent relates to urine analysis in solution, in which a urine sample is pretreated with one or more oxidizing agents to remove ascorbate and then contacted with a suitable analytical reagent. The oxidizing agents disclosed are sodium iodate, sodium periodate, calcium hypochlorite, potassium triiodide, sodium hypochlorite, chloroamine and bromosuccinimide. In summary, various approaches to eliminate the interference problem caused by ascorbic acid in the determination of peroxide active substances include the use of various Co() ammonium complexes, pretreatment of the sample with oxidizing agents and the addition of alkalinity to the reagent composition. It included techniques such as direct addition of metal iodates. To elucidate the mechanism of action of natural peroxidases, such as those obtained from horseradish or potato, pseudoperoxidases, such as hemoglobin, are often studied as another peroxidase system. Ascorbic acid has long been known as a typical substrate for peroxidases, and ascorbic acid oxidation in the presence of metal chelates that act as pseudoperoxidases is a known phenomenon. In 1967 and 1968, M.Khan and A.
Martell) reported a kinetic study of the oxidation of ascorbic acid in the presence of several iron and copper chelates over the pH range of 1.8 to 3.45 [Kahn and Martell, J.Am.Chem.Soc. , 89, 4176
(1967); J.Am.Chem.Soc., 89, 7104 (1967); J.
Am.Chem.Soc., 90, 3386 (1968). ] In these studies, a large number of kinetic and thermodynamic parameters were studied. The results were the ranking of various chelates according to their ability to oxidize ascorbic acid. Of the four aminopolycarboxylic acids studied by these authors, N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) chelate of Fe ₊₃ was found to be the most rapid oxidizing agent. The work of Martell and even earlier work by Grinstead considered this ascorbic acid oxidizing activity to constitute a "model" peroxidase system [Grinstead, J. Am. Chem. Soc., 82, 3464]. 1960)]. Works such as those by Grinstead led to attempts to study peroxidase mechanisms using iron chelates whose structure is similar to that of the iron-containing heme found in the active site of the enzyme peroxidase. In fact, the authors repeatedly use the phrase "model peroxidase system" in their paper. However, since "model" peroxidase activity is shown by other substances similar in chelate and reactivity, such substances can be used in organic hydroperoxide/indicator systems, such as peroxidases or other peroxide activities. It would certainly not be expected that it could be incorporated into typical systems currently used in analytical reagent compositions and measuring devices for the determination of substances. Additionally, research conducted by the assignee of the present invention in connection with peroxidase activity has shown that indicators such as 3,3',5,5'-
Using tetramethylbenzidine (TMB) or o-tolidine (indicators typically used in analytical systems to detect the presence of peroxide actives), such activity reacts approximately 200 times faster than with ascorbic acid. It turned out that it was possible to predict what would happen. Therefore, if a "model" peroxidase, such as a peroxide-active metal chelate, readily oxidizes ascorbic acid (the assumption made by Kahn, Martell, and Grinstead), peroxidase with an indicator such as TMB The reaction proceeds at least as fast as ascorbic acid, if not 200 times faster (as suggested by the latter study done with horseradish). Obviously, when a highly reactive analyte is incorporated into a true reagent preparation configured to change color in the presence of that analyte,
It is expected that "false positive" results will be obtained. SUMMARY OF THE INVENTION Notwithstanding the foregoing teachings and suggestions, when certain peroxide-active metal chelates, and particularly metal chelates of polycarboxyalkylamines, are used in the method of the present invention, peroxide-active substances may be measured. Not only does it not produce the expected "false positive" results in compositions containing reagent systems, it actually produces unexpected results in terms of reliability, stability and sensitivity of the system in which analytes are to be measured in such systems. It's advantageous.
Furthermore, using metal chelates according to the invention,
It has been found to be particularly advantageous in overcoming inaccuracies caused by interferences, especially from ascorbate ions present in the sample. The present invention is therefore based on this discovery and, as mentioned above, generally provides a method for the analytical determination of peroxide active substances that is resistant to ascorbate interference and in particular provides a method for the analytical determination of peroxide active substances that are resistant to ascorbate interference, and in particular for organic hydroperoxides and redox compounds according to the present invention. indicator, such as o-tolidine or 3,
The present invention relates to a detection method utilizing a composition comprising 3',5,5'-tetramethylbenzidine and a peroxide-active metal chelate. In such detection, the peroxide-active analyte, similar to the enzyme peroxidase, catalyzes or participates in the reaction between the indicator and the organic hydroperoxide. This reaction produces a color or other detectable response, the intensity of which is indicative of the analyte concentration. Ascorbate ions, when present, pose significant interference problems. The presence of peroxide-active metal chelates in the composition would also be expected to interfere with analytical measurements of peroxide-active analytes.
Nevertheless, new compositions, test means (and test devices) resistant to the interfering effects of ascorbic acid in a sample can be effectively prepared for detecting the presence of peroxide-active substances in a sample. There was found. This composition, test means (and test device)
Contains metal chelates of polycarboxyalkylamine derivatives, also known as "model peroxidases". Accordingly, the compositions of the present invention contain an organic hydroperoxide, a peroxide active agent and an indicator capable of producing a detectable response, such as a color change, in the presence of the peroxide, and furthermore a compound of the general formula: [In the formula, R ₁ represents hydrogen or a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, and R ₂ , R ₃ , Rx
and Ry are the same or different and represent a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, R ₁ , R ₂ ,
At least two of R ₃ , Rx or Ry are the aforementioned alkylcarboxylic acid groups, Rp and Rq are the same or different, and are straight or branched alkylene groups having 1 to 3 carbon atoms or 6 to 9 represents a divalent 1,2-alicyclic group having 1,2 carbon atoms, and n is 0 to 1
m is an integer having a numerical value from 0 to 2, and M is Fe ₊₃ ]. Preferred compounds are those in which m is 0, n is 0 or 1, and Rp is an ethylene group.
Metal chelates of polycarboxyalkylamine derivatives in which the alkylcarboxylic acid group is _-CH2COOH are particularly preferred. A preferred metal chelate is the iron chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid (Fe-HEDTA). In a preferred embodiment of the invention, the composition can be incorporated into a support matrix, absorbent paper, to form a test means, and the test means can be fixed to an inert support to form a test device. Furthermore, methods for manufacturing and testing test means (and test devices) are also provided by the present invention. The inclusion of metal chelates according to the present invention provides unexpected advantages, as can be confirmed experimentally not only by excellent resistance to ascorbate interference, but also by good storage and high temperature stability and lack of "false positive" results. Compositions, test means (and test devices) are provided that have a high stability. DETAILED DESCRIPTION OF THE INVENTION Early wet chemistry experiments during the development of this invention
Iron chelate of (2-hydroxyethyl)ethylenediaminetriacetic acid (hereinafter referred to as Fe-HEDTA),
This is used for convenience only and does not imply the presence of a covalent bond between the metal ion and the polycarboxyalkylamine derivative).
Ascorbic acid and a buffer were used to confirm the rapidity of ascorbic acid oxidation in the presence of this chelate, a result that would be expected from the Cahn and Martel report. Therefore, having seen how quickly TMB is oxidized in peroxidase-containing compositions by comparison with ascorbic acid oxidation by such compositions, organic hydroperoxides, oxidizable indicators, For example, compositions containing TMB and also metal chelates such as those described above were expected to be extremely unstable and rapidly produce "false positive" results. However, further experiments showed that containing such metal chelates, they are suitable for the detection of peroxide-active substances, are also compatible with the dry solid state format, and exhibit good reagent stability during manufacturing and storage. , “false positive” when in contact with known hemoglobin-negative urine
It has been found that compositions can be prepared that do not produce the same result. The present invention thus achieved provides that, as mentioned above, metal chelates, such as Fe-HEDTA, can be used as "model peroxidases" and thus used in compositions for determining the concentration of peroxide-active analytes. This contradicts the suggestion of the above-mentioned document, which suggests that it would be inappropriate for The test means (and test devices) that could be produced from the novel compositions of the present invention for detecting occult blood (OB), or hemoglobin, in biological fluids such as urine can detect abnormalities in urine. It was found to be resistant to high ascorbic acid concentrations. As mentioned above, inhibition by ascorbic acid is particularly important given that approximately 25% of urine samples can be expected to exhibit ascorbic acid concentrations higher than 10 mg/dL.
This is a serious problem. Conventional OB detectors that do not contain some type of ascorbate-interfering retardant typically contain 5
It was found to be inhibited (ie made less sensitive to the presence of hemoglobin) by ascorbic acid concentrations as low as mg/dL. However, the test device manufactured according to the present invention has a significant advantage over such conventional test devices in terms of resistance to ascorbate interference, and is capable of containing ascorbic acid at a relatively high concentration, for example, 50 mg/dL. It is possible to detect peroxide-active substances in liquids containing to a certain extent. Accordingly, the present invention provides compositions, test means (and test devices) for the detection of peroxide active substances in biological fluids such as urine. By means of the compositions, test means (and test devices) of the invention, other peroxide-active substances are detected in addition to hemoglobin, including, for example, peroxidase, myoglobin, red blood cells and other pseudoperoxidases. be able to. The present invention uses metal chelates which are derivatives of polycarboxyalkylamines and are recognized as "model peroxidases" for the purpose of reducing or eliminating the deleterious effects of ascorbic acid on analyzes performed on biological fluids. . In this respect, metal chelates act to promote the oxidation of ascorbate ions that may be present in such liquids. The composition, test means (and test device) of the present invention are
It was found to be substantially insensitive to ascorbate interference and to produce a visually or instrumentally detectable response, e.g., a color response, to the presence of traces of hemoglobin on the order of 0.03 mg/dL or less. . The organic hydroperoxide used in the compositions of the present invention can be selected from a number of well known organic hydroperoxides. However, it must be capable of reacting with the peroxide active agent in the presence of an indicator that produces a detectable response, such as a change in color or a change in the amount of light absorbed or reflected by the test composition. Particularly suitable hydroperoxides are cumene hydroperoxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-
2,5-dihydroperoxide, paramenthane hydroperoxide and other well known hydroperoxides suitable for oxidizing the indicator used, or mixtures of these compounds. Among the above compounds, cumene hydroperoxide is most preferred. Many indicators are suitable for use in the compositions of the present invention, so long as they are capable of reacting to produce a detectable response in the presence of the organic hydroperoxide and peroxide actives. Indicators are, for example, so-called "benzidine-type"compounds;benzidine;
o-tolidine;3,3',5,5'-tetra(loweralkyl)benzidine;2,7-diaminofluorene; and mixtures thereof or mixtures of various other indicators. As used herein, the term "lower alkyl" has 1 to 6 carbon atoms, including methyl, ethyl, n-propyl and isopropyl groups, and the various butyl, pentyl and hexyl isomers. number of alkyl groups. Particularly preferred is the indicator 3,3',5,5'-tetramethylbenzidine (TMB). The suitability of the particular metal chelate selected for use is determined not only by its ability to promote the oxidation of ascorbic acid, but also by its compatibility with the other components of the composition. It has therefore been found that such suitable chelates include metal chelates of polycarboxyalkylamine derivatives having the following general formula: Accordingly, metal chelates found suitable for use in the present invention include, for example, N-(2-hydroxyethyl)ethylenediamineferric triacetate chelate (Fe-HEDTA), ethylenediaminetetraacetic acid iron chelate (Fe-EDTA). , cyclohexylenediaminetetraacetic acid iron chelate (Fe-CDTA), nitrilotriacetic acid iron chelate (Fe-NTA), iminodiacetate iron chelate (Fe-IMDA), ethylenediaminediacetate iron dipropionate chelate (Fe-EDDP,
α-type and β-type), hydroxyethyliminodiacetic iron chelate (Fe-HIMDA), and mixtures thereof. Iron chelate forms are generally preferred, most preferably the compounds are Fe-HEDTA and Fe-EDTA, and most preferably Fe-HEDTA and Fe-EDTA.
It is HEDTA. However, as will be apparent to one skilled in the art upon reading this specification, many suitable chelates in addition to those specifically listed above are within the scope of the present invention. Therefore, Fe-HEDTA and Fe-EDTA, especially
Fe-HEDTA gives excellent results in the compositions, test means (and test devices) of the present invention, produces the most satisfactory resistance to ascorbic acid interference in the occult bleeding test (hemoglobin is detectable), and provides good results before use. Experiments have shown that it exhibits good reagent stability and does not produce "false positive" results. Additionally, the other metal chelates mentioned, as well as numerous other chelates, will work satisfactorily. However, one can expect significant variation in the rate at which peroxide actives can be detected when using such other compounds. This is because the rate of oxidation of ascorbic acid varies. Accordingly, metal chelates suitable for use in the present invention may be selected from those which are polycarboxyalkylamine derivatives within the above class of compounds, all such compounds being capable of satisfactorily oxidizing ascorbate. It should be understood that this can be done and still be within the scope of the present invention. However, many in this respect act slowly and are therefore not very practical or preferred in industrial applications. Metal chelates suitable for use in the present invention include:
Aldrich Chemical
Co.), Sigma Chemical Company (Sigma
Chemical Company) or similar suppliers by routine experimentation using polycarboxyalkylamine derivatives. For example, the metal chelate Fe−
HEDTA is commercially available HEDTA and FeCl ₃ 6H ₂ O
can be prepared by mixing equimolar amounts of Fe-HEDTA in an aqueous solution to produce a 1:1 (mole:mole) Fe-HEDTA solution of iron:chelate. Other dissolved concentration ratios of metal:chelate can be easily adjusted simply by varying the respective concentrations of the mixing solutions. It has been found that the best results in overcoming ascorbic acid interference are obtained when the concentration of metal ion:polycarboxyalkylamine derivative in the chelate is in an approximately 1:1 (mole:mole) relationship. Preferred concentration ranges for a given metal chelate in various embodiments of the invention vary widely. For example, in the case of Fe-HEDTA, when used in a composition comprising an organic hydroperoxide and a tetra(lower alkyl)benzidine indicator, a preferred concentration range is about 0.5 millimolar (mM) to about 50 mM. This range was determined to be optimal for resisting ascorbate concentrations up to approximately 50 mg/dL in urine samples. Additionally, experiments have shown that hemoglobin can be detected relatively quickly using a suitable iron chelate at relatively low concentrations, whereas higher concentrations of the same or other chelates are less effective. Although these apparently anomalous results are discussed in detail below, these results demonstrate that there is no general correlation between chelate concentration and function or suitability in the compositions of the present invention. The majority of suitable metal chelates that work satisfactorily in the compositions of this invention have a structural alkylamine group or amine center group and a carboxylic acid group _-CH2
Has COOH. However, other chelates that do not have such characteristics, but which belong to the general compound range mentioned above, are generally effective in overcoming ascorbate interference, exhibit satisfactory sensitivity and stability, and are therefore satisfactory. used without. In use, the performance of a particular embodiment of a composition, test means (and test device) according to the general concepts of the present invention depends on a number of different factors.
A typical urine sample from a person who has a habit of ingesting large amounts of ascorbic acid (vitamin C) often contains 25-100 mg/dL or more of ascorbate. The reference ascorbate concentration for research is approximately 50
mg/dL was selected. Preferred embodiments of the ascorbate resistance compositions, test means (and test devices), and test methods of the present invention not only contain peroxide actives in such samples at a reference concentration of about 50 mg/dL; It has been found that various other ascorbate concentrations can be detected. In most cases, a delay in response time occurs when the ascorbate concentration is much higher than the chelate concentration. As described below, the "lag time", i.e., the time until an observable response occurs, was determined by the amount of chelate concentration that was tested for the ability to detect hemoglobin in urine in the presence of 50 mg/dL ascorbate. For preferred embodiments of the invention, experimentation has shown that it ranges from less than 1/3 minute to about 1/2 hour. In a preferred embodiment, the compositions of the invention are used to manufacture test means (and test devices) for the determination of peroxide active substances. In such preferred embodiments, the composition can be incorporated into a suitable support matrix to form the test means. The support matrix can take many forms, such as those disclosed in U.S. Pat. No. 3,846,247 (felt, porous ceramic strips and woven or matted glass fibers). It's good to be warm. The matrices described in US Pat. No. 3,552,928 (wood stakes, fabrics, sponge materials and clay materials) are also suitable. GB 1369139 suggests the use of synthetic resin fleece and glass fiber felt as the support matrix. GB 1349623 also proposes the use of a light-transparent network of fine filaments as a coating for the underlying paper matrix. French Patent No. 2170623 discloses polyamide fibers. Notwithstanding this disclosure, materials conventionally used as support matrices and which are particularly preferred and suitable for use in the present invention are water-absorbing materials such as filter paper and the like. However, the support matrix may be in a variety of physical and other forms as described above, and all such forms are suitable and intended for use in the present invention. In producing the test means of the invention, the components of the composition can be incorporated into the support matrix in a variety of ways. For example, the components can be dissolved or suspended in water or other suitable solvents, preferably organic solvents such as ethanol, acetone or dimethylformamide (DMF) and mixtures of these and other solvents.
This solution or suspension can then be used to impregnate a water-absorbing filter paper, eg, as an ink printed with the reagents onto a suitable matrix. Alternatively, the support matrix can be dipped into the composition or coated with the composition using, for example, a doctor blade. A currently preferred method of incorporating the components of the composition into the support matrix is to impregnate a water-absorbing filter paper with a solution or suspension of two or more of the components. Impregnation is accomplished by dipping a piece of filter paper into such a solution or suspension two or more times and drying the soaked paper in an oven after each dipping. The test means thus formed is laminated to one side of a piece of double-sided adhesive tape, the laminate is cut into strips, each strip is fixed to a long sheet of plastic backing material (e.g. polystyrene), which is then parallel to the short dimension. to form a rectangular test device with the impregnated paper on one end and the other end serving as the handle. The test device thus formed consists of a twice-dried, impregnated test strip secured at one end to one flat side of a long plastic support which constitutes a convenient handle. A preferred method of making the test means of the invention is to introduce, for example, Fe-HEDTA together with an organic hydroperoxide into the filter paper in a first aqueous dip before addition of the indicator. That is, first, a filter paper is mixed with Fe-coated with one or more suitable solvents and/or buffers, such as triethanolamine borate and tris(hydroxymethyl)aminomethane-malonate (hereinafter referred to as tris-malonate).
It is impregnated with an aqueous solution of HEDTA and hydroperoxide, dried, impregnated again with a second dip solution of the indicator in a suitable solvent, such as ethanol, and dried a second time. It has been found that this "double-dip" method, in which the paper is first impregnated with the metal chelate and then with other active reagents, yields a test device that exhibits excellent ascorbate resistance and storage stability. did. A particularly preferred method of preparing the test means of the invention is to introduce reagents other than the metal chelate and the indicator into the filter paper by soaking it in a first solution of the reagents as described above and then drying the paper. ,after that,
dry paper with indicator and thickener in a suitable solvent;
For example, the indicator can be added by dipping into a solution of polyvinylpyrrolidone, followed by a second drying. In addition to the test composition reagents and other ingredients described above, other ingredients such as various thickening agents, wetting agents, buffering agents,
Emulsifiers and well-known auxiliaries can also be added to the compositions, test means (and test devices) of the invention. For example, in addition to or in place of polyvinylpyrrolidone, various materials can be used as thickeners, such as gelatin, algin, carrageenan, casein, albumin, methylcellulose, and the like. As wetting agent it is preferred to use sodium dodecyl sulfate, but it is also possible to use long-chain organic sulfates and sulfonates, such as sodium dioctyl sulfosuccinate or sodium dodecylbenzenesulfonate. In addition to triethanolamine borate and tris-malonate, tartrates, phosphates, phthalates, citrates, acetates, succinates or other buffers can be used in the buffer system. The composition has a pH of approximately 6.0-7.0
It is preferable to buffer the value. As the emulsifier, polyvinyl alcohol, gum arabic, carboxyvinyl polymer, etc. can be used. Organic solvents useful for suspending the indicator are mostly non-reactive organic volatile solvents such as ethanol, acetone, DMF, chloroform, ethylene dichloride,
Includes benzene, ethyl acetate, etc. Of course, it is possible for a person skilled in the art to select other suitable solvents. When using a test means (or test device), it must be immersed in a liquid suspension of the liquid to be tested or the material to be tested and immediately removed, or a liquid, solid or semi-solid sample must be placed in the test means. (or test device)
It can be applied to The presence of peroxide actives in the sample causes the test composition to produce a color change or other detectable response. If the response is a color, this can be compared to a pre-calibrated color standard to assess the quantitative amount of peroxide active contained in the sample. Intact peroxide active substances, such as intact red blood cells, appear as colored spots on an otherwise uncolored matrix. The hemolyzed peroxide active can uniformly color the matrix. In addition to visual comparison, a variety of instrumental measurements can be used to measure the amount of color or other response exhibited, increasing the accuracy of the test by eliminating the subjective measurements of the human eye. be able to. The following examples are merely illustrative of the concepts and advantages of the invention and are not intended to limit the scope of the invention. Such limitations are made only by the scope of the claims appended hereto. Example A Example Test Composition ... Fe-HEDTA Experiments were carried out to prepare compositions of the present invention capable of determining the presence of peroxidase or another peroxide-active substance and in particular hemoglobin in a sample. The composition uses N-(2-
It contains a 1:1 (mol:mol, M:M) iron chelate (Fe-HEDTA) of hydroxyethyl)ethylenediaminetriacetic acid.
Dissolve 0.278g of HEDTA in 100ml of distilled water,
By making a 10mMHEDTA solution and dissolving 0.270g of FeCl ₃ 6H ₂ O in the 10mMHEDTA solution.
Fe-HEDTA chelate was produced. Ascorbic acid at a concentration of 5mM was added to the composition in an amount sufficient to yield a 50 micromolar concentration in the final volume of solution. The components of the composition and ascorbic acid were mixed in the order and amounts shown in the table below. The final composition solution contains Fe-HEDTA at a concentration of 100 micromolar (μM), which is a significantly lower concentration than used in similar compositions for incorporation into solid test means, as well as the concentrations of other components present. It was. 0.2M Sodium Citrate Buffer 10mM Fe-HEDTA 9.5ml 10g/dL Sodium Dodecyl Sulfate 0.1ml 1M Cumene Hydroperoxide 0.1ml 10mM 3,3',5,5'-Tetramethylbenzidine 0.1ml 5mM Ascorbic Acid 0.1 ml The composition of the invention thus prepared was observed to form a blue color when hydrated blood was added to give a final concentration of 0.139 mg per deciliter in solution, which is due to the ascorbate content of the sample. It is shown that the composition is able to detect hemoglobin present even though the concentration is 50 μM. Example: Fe-EDTA The example experiment was repeated except that a 10 mM solution of iron chelate of ethylenediaminetetraacetic acid (Fe-EDTA) was used instead of Fe-HEDTA. Fe-EDTA was prepared substantially as in the example by dissolving 0.292 g of EDTA in 100 ml of distilled water and adding FeCl ₃ .6H ₂ O as described in the example.
was manufactured. The composition thus produced formed a blue color in the presence of 0.139 mg/dL hemoglobin and 50 μM ascorbate, as in the example. Example: Fe-CDTA Instead of Fe-HEDTA, iron chelate of cyclohexylenediaminetetraacetic acid (Fe-CDTA)
The experiment of Example 1 was repeated except that a 10 mM solution was used. A Fe-CDTA solution was prepared by dissolving 0.346 g of CDTA in 100 ml of distilled water and adding FeCl ₃ .6H ₂ O as described in the example.
The composition thus produced formed a blue color in the presence of 0.139 mg/dL hemoglobin and 50 μM ascorbate, as in the example. Example: Fe-IMDA The example experiment was repeated except that a 10 mM solution of iron chelate of iminodiacetic acid (Fe-IMDA) was used instead of Fe-HEDTA.
A Fe-IMDA solution was prepared by dissolving 0.133 g of IMDA in 100 ml of distilled water and adding FeCl ₃ .6H ₂ O as described in the example. The composition thus produced, as in the example, contains hemoglobin
A blue color was formed in the presence of 0.139 mg/dL and 50 μM ascorbate. Example: Fe-NTA The example experiment was repeated except that a 10mM solution of iron chelate of nitrilotriacetic acid (Fe-NTA) was used instead of Fe-HEDTA.
A Fe-NTA solution was prepared by dissolving 0.191 g of NTA in 100 ml of distilled water and adding FeCl ₃ .6H ₂ O as described in the example. The composition produced in this way had 0.139 mg of hemoglobin/
A blue color was formed in the presence of dL and 50 μM ascorbate. Example ...Fe-EDDP〓 Instead of Fe-HEDTA, iron chelate of α-ethylenediamine diacetic acid dipropionic acid (Fe-
except using a 10mM solution of EDDPα).
The example experiment was repeated. Dissolve 0.320 g of EDDP in 100 ml of distilled water and prepare as described in the example.
By adding FeCl ₃ 6H ₂ O
An EDDPα solution was prepared. The composition thus produced formed a blue color in the presence of 0.139 mg/dL of hemoglobin and 50 μM of ascorbate, as in the example. Example ...Fe-EDDP〓 Instead of Fe-HEDTA, iron chelate of α-ethylenediamine diacetic acid dipropionic acid (Fe-
Except using a 10mM solution of EDDP〓)
The example experiment was repeated. Dissolve 0.320 g of EDDP in 100 ml of distilled water and prepare as described in the example.
By adding FeCl ₃ 6H ₂ O
An EDDP solution was prepared. The composition thus produced formed a blue color in the presence of 0.139 mg/dL hemoglobin and 50 μM ascorbate, as in the example. Example: Fe-HIMDA Instead of Fe-HEDTA, iron chelate of hydroxyethyliminodiacetic acid (Fe-HIMDA)
The example experiment was repeated except that a 10 mM solution was used. A Fe-HIMDA solution was prepared by dissolving 0.177 g of HIMDA in 100 ml of distilled water and adding FeCl ₃ .6H ₂ O as described in the example. The composition thus produced contained hemoglobin 0.139 mg/dL and ascorbate as in the example.
A blue color was formed in the presence of 50 μM. Example - In each example, 9.4 ml of 0.2 M sodium citrate buffer was used instead of 9.5 ml, and each iron chelate was
Experiments were carried out essentially as described in Examples to prepare compositions according to the invention, except that 0.2 ml was used instead of 0.1 ml. This is in each composition
This resulted in the presence of iron chelate at a concentration of 200 μM. In each case, when a composition containing 200 μM iron chelate was tested as described above, it was observed that the composition formed a blue color in the presence of 0.139 mg/dL hemoglobin and 50 μM ascorbate;
This indicates that hemoglobin can be detected even if ascorbate is present in the sample. However, compared to the color formation observed when the 100 μM iron chelate composition of Example ~ was similarly tested,
Differences were observed between the times required for color formation. The color formation time (referred to as "lag time") in each case is shown in the table below. This time period is determined by the concentration of one of the metal chelates, e.g. Indicates that there is no general relationship or correlation between

[Table] B Test Device Example An experiment was conducted to produce a solid-state test device according to the present invention. The test device consists of a paper support matrix incorporating the composition of the invention as described in the previous example, except that the concentrations of the ingredients were varied to suit the solid state test device. Incorporation of the composition into the matrix and formation of the test device was performed using the following procedure. N-(2-hydroxyethyl)ethylenediaminetriacetic acid was prepared by dissolving 1.39g of N-(2-hydroxyethyl)ethylenediaminetriacetic acid in 100ml of distilled water and then adding _1.35g of _FeCl3.6H2O to this solution. A 50 mM solution of 1:1 (mol:mol) iron chelate (Fe-HEDTA) in acetic acid was prepared. Two types of solutions (resulting in compositions) were manufactured. Approximately 15.24 cm (6 inches) x
A piece of Whatman 3MM filter paper having dimensions of 10.16 cm (4 inches) was impregnated with the reagent solution and
The paper was made fully saturated with the reagent composition after the second immersion. The operation used was to impregnate the paper by dipping it in the first solution, drying the impregnated paper, and then further dipping the dried paper in the second solution to impregnate it and finally dry it. It is. Drying is done in a forced air dryer at 105℃ for about 8 minutes after the first impregnation.
After the second impregnation, it was carried out at 50°C for about 5 minutes. The first reagent solution was prepared by mixing the following components. Distilled water 74.0ml 1M Tris-malonate buffer PH6.5 10.0ml 5.5M cumene hydroperoxide 4.0ml 10g/dL sodium dodecyl sulfate 2.0ml 50mM Fe-HEDTA (prepared as above)
A 10.0 ml second reagent solution was prepared by mixing the following components. Ethanol 79.4ml 6-methoxyquinoline, free form 0.6ml 20% (w/v) polyvinylpyrrolidone (hydrated)
(Molecular weight 40000) 20.0ml 3,3',5,5'-tetramethylbenzidine 0.6g The dried and impregnated paper was washed at 3M Company (St.
It was laminated to one side of a piece of double-sided adhesive transfer tape commercially available from Paul, Minn. 55144). Next, cut the laminate into approximately 15.24 cm (6 inches) x 0.51 cm (0.2 inches).
Cut to size. One of these was bonded to a polystyrene sheet approximately 3.5 in. 8.89 with impregnated paper at one end and the other end serving as a handle
A test device was formed consisting of a cm rectangular polystyrene strip. A test device prepared according to the procedure of the example was tested with urine samples containing various concentrations of hemoglobin and 50 mg/dL of ascorbate, producing easily distinguishable blue levels corresponding to the various hemoglobin concentrations; It was found that the test device was able to detect hemoglobin present even though it contained high concentrations of ascorbate. Example Instead of Fe-HEDTA in the first reagent solution,
Except for using 10.0ml of 50mM Fe-EDTA solution.
The example experiment was repeated. Test devices prepared according to the example were tested as described above with urine samples containing various concentrations of hemoglobin and 50 mg/dL of ascorbate, producing easily distinguishable blue levels corresponding to the various hemoglobin concentrations. Ta. Example Instead of Fe-HEDTA in the first reagent solution,
Except for using 10.0ml of 50mM Fe-CDTA solution.
The example experiment was repeated. Test devices prepared according to the example were tested as described above with urine samples containing various concentrations of hemoglobin and 50 mg/dL of ascorbate, producing easily distinguishable blue levels corresponding to the various hemoglobin concentrations. Ta. Example Instead of Fe-HEDTA in the first reagent solution,
Except for using 10.0ml of 50mM Fe-IMDA solution.
The example experiment was repeated. Test devices prepared according to the example were tested as described above with urine samples containing various concentrations of hemoglobin and 50 mg/dL of ascorbate, producing easily distinguishable blue levels corresponding to the various hemoglobin concentrations. Ta. Example XI In the first reagent solution, instead of Fe-HEDTA,
Except for using 10.0ml of 50mM Fe-NTA solution.
The example experiment was repeated. The test device prepared according to Example occured. C. Ascorbate Interference Resistance and Stability of the Test Device The ability of the test device manufactured as described in the previous example to detect hemoglobin in urine in the presence of ascorbate after stress simulating long-term storage Further experiments were conducted for evaluation.
Experiments were conducted on newly manufactured test devices and test devices that had been stored for long periods of time under high temperature conditions.
In particular, the test devices were tested and compared for performance immediately after manufacture at ambient temperature (23°C) and after 10 and 28 days of "heat stress" at 50°C in an oven.
Test urine solutions containing various hemoglobin concentrations were prepared. Two hemoglobin solutions containing ascorbate at a concentration of 50 mg/dL were also prepared. Whole blood with distilled water, hemoglobin per 100ml of water
A stock solution containing 15.4 mg/dL of hemoglobin was prepared by diluting to a concentration of 15.4 mg. The hemoglobin content of whole blood was previously determined using conventional techniques. Pooled, prescreened urine samples negative for hemoglobin and ascorbic acid were collocated in a blinded fashion. Test solutions were then prepared by pipetting the blood solution into the pooled urine to produce urine solutions containing 0.015, 0.031, 0.062, and 0.139 mg/dL of hemoglobin.
Urine solutions containing 0.031 and 0.062 mg/dL hemoglobin were separated into separate containers and ascorbic acid was added to bring the ascorbate concentration of the solution to 50% immediately before testing.
mg/dL. A test device manufactured in the same manner as in the example and Fe-
Control test devices made as described in that example, but without HEDTA, were tested blind and with each hemoglobin/urine solution. The test device was momentarily immersed in each solution, removed, and after 1 minute, color formation on the test device was observed. The color formed was visually compared to another test device and a standard color chart for relative intensity one minute after immersion. Color is colorless (blind test)
Ranges from dark green-blue to 0.139 mg/dL hemoglobin solution. The results of this test, as shown in the table below, show that the test device according to the invention was tested on two hemoglobin samples containing ascorbate, freshly prepared and after 50
It is shown that testing after 10 days of exposure to elevated temperatures of 0.degree. C. produces a color response to the presence of hemoglobin similar to the response of the test device used to test ascorbate-free urine samples. Fe−
Similar testing of a control device without HEDTA showed that the response of the control device was almost completely impaired by ascorbate in the sample. However, test devices made in accordance with this preferred embodiment of the invention showed that even in the presence of 50 mg/dL ascorbate, 0.031 and 0.062 mg/dL
It was demonstrated that hemoglobin at a concentration of . Therefore, the presence of Fe-HEDTA dramatically reduced ascorbate interference.

TABLE The results shown in the table above demonstrate that there is little difference in reactivity between freshly manufactured test devices of the invention and test devices stored for 10 or 28 days at 50°C. This result is in line with the predicted result that the reaction of iron chelate and hydroperoxide in the same test device would reduce the reactivity of the test device at a faster rate under heat stress than when stored at ambient temperature. contradicts. This demonstrates the good stability and advantageous "shelf life" of the compositions and test devices of the invention. As seen from the data, organic hydroperoxide and Fe−
There was no or little apparent incompatibility between HEDTA, Fe-HEDTA and the indicator, or these materials and other strip components, even after long periods of storage at elevated temperatures. Furthermore, test devices containing metal chelate (Fe-HEDTA) and Fe-HEDTA
Test devices similarly manufactured without ascorbate are substantially similar in sensitivity to the presence of hemoglobin in the ascorbate-free test solution;
There were no "false positive" results, indicating excellent compatibility of the reagents. However, test devices made without Fe-HEDTA were actually completely hindered by the presence of ascorbate in the test solution, whereas Fe-HEDTA
- The strips of the invention containing HEDTA are hardly disturbed by ascorbate and in fact
It was possible to respond to hemoglobin concentrations of the order of 0.031 and 0.062 mg/dL by the appearance of a color discernible to the naked eye after 1 minute. In order to further demonstrate the advantages of the present invention, tests similar to those described above were carried out using test devices manufactured in the same manner as in the example, ie containing Fe-HEDTA. However, rather than using naked eye techniques, they tracked color formation using a device known as a "Rapid Scanner." This device is a scanning reflectance spectrophotometer with an intervening laboratory microcomputer. This device is
Used to quickly measure reflection spectra in the visible region. A computer allows storage of spectral data and performs calculations. Rapid
Measuring the performance of a reagent strip in a scanner has the following advantages over, for example, visually observing the same strip: 1. The light source and the conditions surrounding the sample are held constant. In visual observation, the light source can vary not only in wavelength but also in the position of the observed strip. 2 Detector characteristics are held constant. In visual observation, the detector (ie, the observer's eyes) varies from person to person, and even from day to day for the same person. 3 Rapid scanners can quantify data more precisely than visual observation, which allows results to be compared more objectively. The Rapid Scanner instrument was manufactured by Ames Division, Miles Laboratories, Inc. of Elkhart, Indiana, from which complete information regarding its construction and performance characteristics is obtained. MAGenshaw and RWRogers, Anal.Chem.Vol.53:
See pages 1949-1952 (1981). Using the tristimulus values from the rapid scanner, the
Addendum to L′Eclairage (Paris, France)
2Publication No.15, Colorimeter, (E.−1.3.1) 1971”
The color difference value (ΔE) was calculated using the conventional method included in . Therefore, the data from this device is recorded below as ΔE or color difference units. Test strips according to the invention containing Fe-HEDTA were tested for their ability to detect hemoglobin concentrations of 0.031 mg/dL and 0.062 mg/dL using the procedure described above. Some test devices were tested with urine samples containing 50 mg/dL of ascorbate, some were tested with similar samples without ascorbate,
Some items were tested after being newly manufactured at ambient temperature, and some items were tested after being newly manufactured at ambient temperature and 50℃ for 11 days.
It was tested after being stored for 28 days. The color difference units (ΔE) provided by the rapid scanner correspond to various hemoglobin concentrations. Test device containing Fe-HEDTA at 0.031mg/dL
and 0.062 mg/dL of hemoglobin, with or without ascorbate, and the results are shown in the table below.

【table】

【table】

【table】

TABLE The above experiment presents instrumental data that corroborates the data from the above macroscopic experiment. This data demonstrates the stability of the test devices of the present invention and the significant elimination of ascorbate interference in such test devices even after long-term storage and storage at elevated temperatures. Further visual testing was performed on test devices made according to the invention, ie containing Fe-EDTA, as described in the previous examples. After manufacturing the test device, the test device can be used to collect urine samples containing various concentrations of hemoglobin and urine samples without hemoglobin;
Two hemoglobin concentration samples were tested, including 50 mg/dL ascorbate. Some test devices were tested immediately after manufacture and some after 28 days of storage at 50° C. in a desiccator. The results of this test are shown in the table below, where the numbers correspond to the visual color values obtained in comparison to the HemaStix standard color chart described above. All testing of these test devices was conducted as described above.

The results of these latter tests confirm the stability, lack of false positive results, and significant resistance to ascorbate interference of the test devices produced according to this embodiment of the invention. Obviously, many modifications and variations may be made from the particularly disclosed preferred embodiments of the invention without departing from the spirit and scope of the invention.
Accordingly, the invention is limited only by the scope of the claims that follow.

Claims (1)

Claims: 1. A composition for detecting the presence of occult blood in a sample, comprising an organic hydroperoxide and a peroxide-active substance and an indicator capable of producing a detectable response in the presence of peroxide, further comprising: formula [In the formula (a) R ₁ represents hydrogen or a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, R ₂ , R ₃ , Rx
and Ry are the same or different and represent a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms,
At least 2 of R ₁ , R ₂ , R ₃ , Rx or Ry
is an alkylcarboxylic acid group as defined above; (b) Rp and Rq are the same or different and are a straight or branched alkylene group having 1 to 3 carbon atoms or 2 having 6 to 9 carbon atoms; Value of 1,2-
Represents an alicyclic group; (c) n is an integer having a numerical value of 0 to 1, m is 0 to 2
and if m is greater than 0, the repeated Rp and repeated Rq groups may be the same or different, and (d) M is Fe ₊₃ . 1. A composition for detecting the presence of occult blood in a sample, comprising a trivalent iron ion chelate of a polycarboxyalkylamine derivative. 2. The composition according to claim 1, wherein (a) m is 0 and (b) Rp is an ethylene group. 3. The composition according to claim ₁ , wherein at least two of R1, _R2 , _R3 , Rx and Ry are alkylcarboxylic acid groups _-CH2COOH . 4 The indicator is benzidine, o-tolidine, 3,
The composition according to claim 1, which is 3',5,5'-tetra(lower alkyl)benzidine, 2,7-diaminofluorene or a mixture thereof. 5 Iron chelate is N-(2-hydroxyethyl)
Ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid, cyclohexylenediaminetetraacetic acid, nitrilotriacetic acid, iminodiacetic acid, α-ethylenediaminediacetic acid dipropionic acid, β-ethylenediaminediacetic acid dipropionic acid, hydroxyethylaminodiacetic acid and mixtures thereof. A composition according to claim 1, wherein the composition is selected from the group consisting of iron chelates. 6 Iron chelate is N-(2-hydroxyethyl)
A composition according to claim 1, which is an iron chelate of ethylenediaminetriacetic acid. 7. The composition according to claim 1, wherein the iron chelate is an iron chelate of ethylenediaminetetraacetic acid. 8 The organic hydroperoxide is cumene hydroperoxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5
2. The composition of claim 1, wherein the composition is selected from the group consisting of -dimethylhexane-2,5-dihydroperoxide, paramenthane hydroperoxide, and mixtures thereof. 9. The composition according to claim 1, wherein the organic hydroperoxide is cumene hydroperoxide and the indicator is 3,3',5,5'-tetramethylbenzidine. 10 The indicator is 3,3',5,5'-tetramethylbenzidine, the iron chelate is an iron chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid, and the organic hydroperoxide is cumene hydroperoxide. A composition according to claim 1. 11 containing an organic hydroperoxide and a peroxide active substance and an indicator capable of producing a detectable response in the presence of the peroxide, and further having the general formula [In the formula (a) R ₁ represents hydrogen or a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, R ₂ , R ₃ , Rx
and Ry are the same or different and represent a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms,
At least 2 of R ₁ , R ₂ , R ₃ , Rx or Ry
is an alkylcarboxylic acid group as defined above; (b) Rp and Rq are the same or different and are a straight or branched alkylene group having 1 to 3 carbon atoms or 2 having 6 to 9 carbon atoms; Value of 1,2-
Represents an alicyclic group; (c) n is an integer having a numerical value of 0 to 1, m is 0 to 2
and if m is greater than 0, the repeated Rp and repeated Rq groups may be the same or different, and (d) M is Fe ₊₃ . A test device for detecting the presence of occult blood in a sample comprising a support matrix incorporating a composition for detecting the presence of occult blood comprising a trivalent iron ion chelate of a polycarboxyalkylamine derivative. 12. The test device according to claim 11, wherein (a) m is 0 and (b) Rp is an ethylene group.