Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS3627697 A
Publication typeGrant
Publication dateDec 14, 1971
Filing dateMar 20, 1970
Priority dateApr 9, 1969
Also published asCA919562A1, DE1917997A1, DE1917997B2
Publication numberUS 3627697 A, US 3627697A, US-A-3627697, US3627697 A, US3627697A
InventorsHans-Georg Rey, Peter Rieckmann, Hans Wielinger
Original AssigneeBoehringer Mannheim Gmbh
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Hydroperoxide diagnostic agents containing a chromogen indicator
US 3627697 A
Images(6)
Previous page
Next page
Description  (OCR text may contain errors)

11mm States Patenl Inventors Hans-Georg Rey;

Hans Wiellnger; Peter Rleckmann, all of Mannhelm-Waldhof, Germany Appl. No. 21,493 Filed Mar. 20, 1970 Patented Dec. 14, 1971 Assignee Boehringer Mannheim Gmbll Postfach, Germany Priority Apr. 9, 1969 Germany HYDROPEROXIDE DIAGNOSTIC AGENTS CONTAINING A CHROMOGEN INDICATOR 18 Claims, No Drawings 23 230 1;, 253 TP, 230 R; 195/1035; 424/7; 399 4 [56] References Cited UNITED STATES PATENTS 3,183,173 5/I965 Oakes .Q. 252/408 X Primary Examiner-John T. Goolkasian Assistant ExaminerM. E. McCamish AnorneyBu1-gess, Dinklage & Sprung ABSTRACT:

HYDROPEROXIDE DIAGNOSTIC AGENTS CONTAlNllNG A CHROMOGEN INDICATOR The present invention is concerned with process and diagnostic agent for the determination of hydroperoxides and of substances from which hydrogen peroxide or other hydroperoxides are liberated by a previous reaction, as well as for the determination of peroxidase and of other peroxidatively active substances.

The detection of glucose in urine, blood and serum is, in the case of, diabetes, of especial diagnostic interest, as is also the detection of peroxidatively active substances, such as hemoglobin, in urine and blood, and the detection of hydroperoxides in, for example, the milk industry, the cosmetics industry and in polymer chemistry.

A series of compounds is known which are oxidized to dyestuffs with hydrogen peroxide and peroxidase as catalyst. Preferred compounds of this type include, for example, the Nadi reagents, p-phenylene-diamine derivatives, carboazines, guaiac resins, aldazine and the like; Previously, it has been preferred to use benzidine, o-dianisidine and o-tolidine. However, some of these compounds are not very stable and, according to recent findings, can also be a health hazard so that their use does not appear to be free of danger.

We have now, surprisingly, found that the physiologically harmless compounds of the general formula:

1 %\/DNNA\NJ RZ wherein R, is lower alkyl; R is hydrogen or alkyl, preferably lower alkyl, or, together with Y, represents a fused benzene or napthalene nucleus; X is sulfur or oxygen or an alkylated imino group, preferably carrying lower alkyl groups, or a vinylene radical; and Y is a methine radical which, together with R,, can also form a benzene or naphthalene nucleus, are absolutely stable and are particularly suitable for the determination of hydrogen peroxide and of other hydroperoxides, notonly by means of an optical test but also with the help of reagent papers and reagent films.

it is known that some-of the compounds of the above-given general formula I can be oxidized by means of comparatively strong oxidizing agents, such as lead tetra-acetate, ceric sulfate and potassium persulfate, and thus give colored oxidation products (cf. S. Hunig et al. Liebigs Ann. Chem., 676, 32-65/1964; H. Lang et al. Z analyt. Chem., 201, 321/1964). Since, however, no reaction takes place with hydrogen peroxide alone, it could not be foreseen that reproducible and clear colorations can also be obtained in the presence of peroxidase or of peroxidatively active substances.

According to a further feature of the present invention, we have also found, surprisingly, that especially advantageous and clearly graduated color changes are obtained when the compounds of general formula I are used in admixture with reduction agents of the general formula:

wherein Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; \V is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.

These compounds of general formula II do not themselves give any coloration or, at most, only give very nonspecific colorations and are, therefore, generally not suitable for use as indicators. However, as already mentioned above, when these compounds of general formula ll are used together with the compounds of general formula I, there are obtained especially clear and well graduated color changes.

Thus, according to the present invention, there is provided a process for the determination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide, as well as of peroxidase and of peroxidatively active substances, with the help of a chromogen, using the reaction of the hydroperoxides with peroxidase or peroxidatively active substances, by evaluation, in known manner, and of the coloration, wherein a compound of general formula I is used as chromogen, preferably in admixture with a compound of general formula ll.

The evaluation of the coloration can be carried out, for example, by optical measurement in a spectrophotometer or, in the case of the use of test paper strips and test films, by comparison of the color strength with blank samples of known composition or with color charts.

The determination of hydroperoxides by the process according to the present invention is particularly useful for coupled and uncoupled enzyme reactions, for example, for the determination of glucose, galactose, amino acids, uric acid, peroxides, hemoglobin, peroxidase or other peroxidatively active substances, as well as of enzyme activities. Because of their outstanding importance, the routine determination of substrates of this type is now an essential feature of clinical chemistry and of foodstuff chemistry.

In the case of the determination of glucose, the latter is, for example, oxidized by glucose-oxidase to gluconic acid, atmospheric oxygen thereby being reduced to hydrogen peroxide. By means of peroxidase or of a peroxidatively active substance, the hydrogen peroxide then oxidizes the indicator used according to the present invention to give the corresponding color dyestuff.

Further examples of analytically useful enzyme systems of this type, which react with the liberation of hydrogen peroxide, include L-amino acid oxidase L-amino acids, uricase uric acid, xanthine oxidase hypoxanthine or xanthine, glycine oxidase glycine, monoamine oxidase monoamine (such as adrenaline, mescaline and the like), diamine oxidase diamine (such as histamine), luciferase luciferin, D-aspartic acid oxidase D-aspartic acid, liver aldehyde oxidase aldehyde, galactose oxidase galactose, Edsons flavine enzyme +lactic acid.

According to a further feature of the present invention, there is provided a diagnostic agent for the determination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide, which comprises peroxidase or a peroxidatively active substance and a chromogen of general formula I alone or in admixture with a compound of general formula ll.

According to yet another feature of the present invention, there is provided a diagnostic agent for the determination of hemoglobin and of other peroxidatively active substances, which comprises hydrogen peroxide or a substance forming hydrogen peroxide and a chromogen of general formula I alone or in admixture with a compound of general formula ll.

it is to be understood that, within the context of the present invention, the expression a substance forming hydrogen peroxide" is intended to mean not only a single compound, such as an organic peroxide, but also a mixture, such as glucose and glucose-oxidase.

It is to be understood that the new diagnostic agents according to the present invention can be prepared in the form of solutions in appropriate solvents, if necessary with the addition of conventional adjuvants, such as buffers. Alternatively, the new diagnostic agents can be prepared in the form of test papers by the impregnation of suitable absorbent materials, such as filter paper, with solutions of the components of the diagnostic reagents or can be used for making reagent films. Here again, in the case of such test papers and test films, it is frequently advantageous for them to contain conventional adjuvants, such as buffers.

Finally, mention should also be made of the fact that the process according to the present invention can, in addition, he used for the determination of the chromogens of general formula l, as well as of the compounds of general formula II, with the help of hydroperoxides and of peroxidatively active substances, which is very useful for control purposes in the preparation of these diagnostic materials.

The compounds of general formula I can be prepared in known manner, for example, by the reaction of 2-haloor 2- merapto-quatemary salts with an appropriate N-alkylated hydrazone salt, with the addition of an amine, preferably of triethylamine, in a polar solvent, for example methanol, at a temperature of l 5l00 C. Depending upon the reactivity of the individual reaction components, the reaction is finished after a period of from minutes to 2 hours (see S. Hi'Inig et al. Liebigs Ann. Chem., 676, 32-65/1964 The preparation of the hydrazones used as starting materials, as well as of the N-quaternized salts, can be carried out in a manner analogous to that described by S. Hnnig (Ann. Chem., 609, l60l80/l957 and by H. Balli (Liebigs Ann Chem., 647, 1-8! 1961 A more comprehensive understanding of the invention may be obtained by reference to the following illustrative examples which are not intended, however, to be unduly limitative of the present invention.

EXAMPLE 1 Reagent Film for the Detection of Glucose in Blood Forty-Five g. Propiofan (BASF), 35 g. of a solution of 37 g. algipon in 2 liters of a 0.5 M phosphate or citrate buffer of pH 5.7, l g. Texapon P (Henkel, Dusseldorf), 10 ml. water, 75

give a homogeneous slurry. To this solution there was added 8 ml. of a concentrated methanolic solution of l -[l-methylbenMflquinoline-2-one]-2-[3-ethyl-benzthiazol-2-one1- azine, as well as 4 ml. of a 2 percent solution of p-aminosallcylic acid. A foil was coated with the mixture prepared in this manner with a layer thickness of 300 p. and then dried. When glucose-containing blood is'dropped onto the reagent strip thus produced, left to react therewith and the blood then again removed, there was obtained a red-brown color reaction.

When, to the indicator mixture, instead of a 2 percent solution of p-arninosalicylic acid, there was added a solution of (1- arninornaph'thol-8 )-2,4 disulfonic acid, then a green color reaction was obtained. When naphthyl-l-amine-6-sulfonic acid or m-aminophenol was added, then the reagent film, by the action of glucose-containing blood, produced color graduations from pink to red-brown, depending upon the glucose concentration.

EXAMPLE 2 To a mixture of Propiofan, algipon, buffer, Texapon P, water, peroxidase and glucose oxidase prepared in the manner described in example I, there were added 8 ml. of a concentrated methanolic solution of l-[ l-methyl-benzo(f)quinoline- 2-one]-2-[ 1ethyl-quinoline-2-onel-azine, as well as 4 ml. of a 2 percent solution of p-aminosalicylic acid. A foil was coated with the mixture prepared in this manner with a layer thickness of 300 u and then of When glucose-containing blood was dropped onto the reagent film thus produced, left to react therewith and the blood then again removed, there was obtained a pink color reaction.

EXAMPLE 3 Detection of Hydrogen Peroxide in Liquids Three ml. of a 0.005 percent methanolic solution of 1-H methyl-benzo-(f)quinoline-2-one]-2-[3-ethyl-benzoxazol-2- oneI-azine, 0.05 ml. of a 1 percent solution of peroxidase in a 0.l M phosphate buffer of pH 5.7 and 0.05 ml. of a solution of hydrogen peroxide were pipetted together, any precipitate formed then centrifuged off and the supernatant measured at a wavelength of 443 nm. The hydrogen peroxide concentration was subsequently determined with the help of a previously prepared calibrated curve.

EXAMPLE 4 A filter paper (Schleicher & Schilll 23 S) was impregnated with a solution of 0.25 ml. Texapon P (Henkel, Dusseldorf) and mg. sodium alginate in 100 ml. of a 0.1M phosphate buffer of pH 5.7 and then dried. The buffered paper thus prepared was further impregnated with a 0.01 percent methanolic solution of l-[l-methyl-benzo-(f)quinoline-2- one]-2 -[1-ethyl-quinoline-2-one]-azine and a 0.01 percent methanolic solution of p-aminosalicylic acid and again dried. When a drop of a body fluid which contained blood was applied to a test paper strip prepared in this manner, together with a drop of a 3 percent hydrogen peroxide solution, then the paper became blue-violet colored.

EXAMPLE 5 Dection of Glucose in Urine .Eil QKBEI P .l9hlll3[.&?hiil wa pr n with a solution of 75 mg. peroxidase, 143 mg. glucose oxidase and 0.25 ml. Texapon P in 100 ml. ofa 0.1M phosphate buffer of pH 5.6 and dried. The enzyme-buffer paper thus prepared was subsequently impregnated with a 0.01 percent solution of 1-[ l-methyl-benzo(f)quinoline-2-one]-2-[ 3-ethyl-benzoxazol-Lonel-azine in chloroform and with a 0.0] percent methanolic solution of p-aminosalicylic acid and again dried. When a reagent paper prepared in this manner was dipped into a urine with a pathological glucose concentration, then the reagent paper assumed a red-brown coloration.

EXAMPLE 6 Detection of Glucose with and without a Modifier Substance of General Formula ll One hundred mg. amounts of the indicators set out in the following table were slurried in 10 ml. methanol, which, if desired, contained 0.1 percent p-aminosalicylic acid as moditier, 2 ml. ofa solution of mg. glucose oxidase and 75 mg. peroxidase in 100 ml. of a 0.1M phosphate buffer of pH 5.6 and l ml. glucose solution are mixed with 1 ml. of the indicator slurry and stirred up. There were obtained the color reactions set out in the following table.

qulnoline] What is claimed is:

1. Diagnostic agent for use in the analytical determination of (a) hydroperoxide and substances which react with the liberation of hydroperoxide or (b) peroxidase and peroxidatively active substances which agent comprises (a) a member selected from the group consisting of peroxidase and peroxidatively active substances; or (b) a member selected from the group consisting of hydroperoxide and substances which react with the liberation of hydrogen peroxide; respectively, and a chromogen of the formula wherein R is lower alkyl; R is hydrogen or alkyl and, together with Y, can represent a fused benzene or napthalene nucleus; X is sulfur, oxygen, an alkylated imino group, or a vinylene radical; and Y is a methine radical which, together with R,, can also form a benzene or naphthalene nucleus.

2. Diagnostic agent as claimed in claim 1 for use in the determination of hydroperoxide or a substance which reacts with the liberation of hydrogen peroxide, which agent comprises (l) a member selected from the group consisting of peroxidase and a peroxidatively active substance, and (b) a chromogen as defined in claim 1.

3. Diagnostic agent as claimed in claim 2 additionally containing a modifier compound having the formula:

wherein Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V' is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as'with the alkali metal salts thereof.

4. Diagnostic agent as claimed in claim 1 for use in the determination of peroxidase or peroxidatively active substances which agent comprises (1) a member selectedfrom the group consisting of hydrogen peroxide and substances forming hydrogen peroxide and (2) a chromogen as defined in claim 1.

5. Diagnostic agent as claimed in claim 4 additionally containing a modifier compound having the formula:

wherein Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.

6. Diagnostic agent as claimed in claim 1 in the form of a test paper impregnated with said diagnostic agent.

7. Diagnostic agent as claimed in claim 1 in the form of a test film impregnated with said diagnostic agent.

8. Diagnostic agent as claimed in claim 1 in the form of a solution thereof.

9. Diagnostic agent as claimed in claim 1 additionally containing a modifier compound having the formula:

wherein Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.

10. Diagnostic agent as claimed in claim 9 wherein said modifier compound is o-amino-phenol-p-sulfonic acid.

11. Diagnostic agent as claimed in claim 9 wherein said modifier compound is p-amino-salicylic acid.

12. Process for the analytical determination of (a) hydroperoxide and a substance which reacts with the liberation of hydroperoxide or (b) peroxidase and a peroxidatively active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 9, and producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.

13. Diagnostic agent as claimed in claim 1 wherein said chromogen (l) is l-methyl-benzo(f)quinoline-2-one]-2-[3- ethyl-benzthiazol-2one]-azine.

14. Diagnostic agent as claimed in claim 1 wherein said chromogen (l) is l-[ l-methyl-benzo-(f)quinoline-2-one1-2- [l-eth l-quinoline-Z-onel-azine.

l5. iagnostic agent as claimed in claim 1 wherein said chromogen (l) is l-[ l-methylbenzo-(f)quinoline-2-one1-2- [3-ethyl-benzoxazol-2-one l-azine.

16. Diagnostic agent as claimed in claim 1 wherein said chromogen (l) is l-[ l-methyl-benzo(f)quinoline-2-one]-2- l ,3-diethyl-benzimidazol-2-onel-azine.

17. Diagnostic agent as claimed in claim 1 wherein said chromogen (l) is 2,2'-azino-di-[1-methyl-benzo(f)quinoline].

18. Process for the analytical determination of (a) hydroperoxide and a substance which reacts with the liberation of hydroperoxide or (b) peroxidase and a peroxidatively active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 1 and producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.

FZ-PF (5/09) r\' '1' r1 1 n u 1- I CERLIIJLCAHL Ol'. COhlLLCllON Patent No, 97 Dated December 1971 Hans-Georg Rey, Hans Wielinger and Peter Rieckmann Invcntor(s) I It is certified that error appears in the above-identified patent:

and that said Letters Patent are hereby corrected as shovm below:

fiitle Page Abstract, consists only of a formula. should read:

HYDROPEROXIDE DIAGNOSTIC AGENTS CONTAINING A CHROMOGEN INDICATOR ABSTRACT OF THE DISCLOSURE Diagnostic agents suitable for use in carrying out id analytical determinations of the presence and/or concentration of hydroperoxides, substances which react with the liberation of hydrogen peroxide or hydroperoxide, peroxidase or peroxidatively;ctive substances,comprising an indicator, i.e. chromogen, which is oxidized by hydrogen droperoxide in the presence of peroxidase or,

peroxide or peroxidatively-active substance to form a dyestuff, the color intensity of which is dependent on the quantity of peroxide,

- peroxidase or peroxidatively-active substance present in the test sample, wherein the chromogen is a compound having the formula:

wherein R is lower alkyl; R is hydrogen or alkyl, preferably lower alkyl, or, together with Y, represents a T fused benzene or naphthalene nucleus; X is sulfur or oxygen i E or an alkylated imino group, preferably carrying lower I l alkyl groups, or a vinylene radical; and Y is a :nethina radical which, together with R2, can also form a benzene or naphthalene nucleus,

l 4 CERTIFICATE OF CORRECTION Patent. 3,627,697 r Dntcd' December 14, 1971' Hans-Georg Rey, Hans Wielinger and Peter Rieelenann Inventor(s) a If is certified that error appears in t he above-identified patent and tht said Letters Patent: are hereby corrected as shown below:

- PAGE 2 Coi. 3, line 10 "merapto" ghoul be "dereapm Col. 3 .line 63 "of". (second occurrence) should read --dried.-- I

' Claim 13 line 2 "1-methy1" should read --1-[1 -m ethy1-- Claim 18, line 1 I Delete close parenthesis at erid of line Signed and sealed this 11th'de of July 1972.

(SEAL) Attest: v v EDWARD MJ LETCHER, JR. ROBE-HT GOTTSCHAL K 3 .Attesting Officer Commissioner of Patents e

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3183173 *Jun 10, 1963May 11, 1965Miles LabTest composition for detecting hydrogen peroxide
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4017261 *Oct 16, 1975Apr 12, 1977Lachema, Narodni PodnikBiological diagnostic test strip and method of producing same
US4175923 *Jun 26, 1978Nov 27, 1979Friend William GApplying developing solution to an absorbent material impregnated with a guaiac reagent
US4385114 *Feb 23, 1981May 24, 1983Boehringer Mannheim GmbhDetermination of hydrogen peroxide or peroxidase
US4956300 *Oct 16, 1984Sep 11, 1990Helena Laboratories CorporationAdsorbent carrier having guaiac that will react with hemoglobin fresent to form a blue dye, oxidizers and binders
US5081040 *Jun 6, 1989Jan 14, 1992Helena Laboratories CorporationComposition and kit for testing for occult blood in human and animal excretions, fluids, or tissue matrixes
US5196167 *May 9, 1991Mar 23, 1993Helena Laboratories CorporationFecal occult blood test product with positive and negative controls
US5217874 *May 9, 1991Jun 8, 1993Helena Laboratories CorporationFecal occult blood test product with positive and negative controls
US5273888 *Apr 29, 1988Dec 28, 1993Helena Laboratories CorporationChemical test kit and method for determining the presence of blood in a specimen and for verifying the effectiveness of the chemicals
US5702913 *Jun 12, 1989Dec 30, 1997Helena Laboratories CorporationUsed for fetal specimens for occult blood
US5885789 *Mar 20, 1997Mar 23, 1999Stc Technologies IncorporatedSpectrophotometry; useful for qualitative or quantitative analysis of hemoglobin obtained from bodily fluid
Classifications
U.S. Classification436/135, 436/66, 435/14, 435/28, 422/400
International ClassificationG01N31/22, C12Q1/28, C12Q1/54
Cooperative ClassificationC12Q1/54, C12Q2326/00, C12Q1/28
European ClassificationC12Q1/28, C12Q1/54