Reagent and method for the analytic determination of hydrogen peroxide
First Claim
Patent Images
1. A method for producing a dye comprising contacting, in an aqueous medium at a temperature of from about 20°
- to about 45°
C., having a pH of from about 3 to about 9.5, and containing hydrogen peroxide, (1) peroxidase, (2) an amino-aromatic acid represented by the formula;
##STR7## wherein X is a mono- or dialkylamino group, and ZH is the carboxyl group or the sulfo group, and (3) a 4-aminoantipyrine represented by the formula;
##STR8## wherein each of R and R1, when taken separately, is alkyl, R2 is an aromatic moiety, each of R3 and R4, when taken separately, is hydrogen or alkyl, and Y is oxygen or sulfur, wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is in th range of from bout 0.1;
1 to about 25;
1.
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Accused Products
Abstract
A 3-aminobenzoic acid or a 3-aminobenzenesulfonic acid, when reacted with a 4-aminoantipyrine in aqueous medium in the presence of hydrogen peroxide forms a novel colored dye which is believed to be a charge-transfer complex of free radicals formed from the 3-aminoaromatic acid and the 4-aminoantipyrine. The dye and the process by which it is produced are useful in analytical procedures for hydrogen peroxide, as well as in enzymatic analyses involving the formation of hydrogen peroxide.
32 Citations
37 Claims
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1. A method for producing a dye comprising contacting, in an aqueous medium at a temperature of from about 20°
- to about 45°
C., having a pH of from about 3 to about 9.5, and containing hydrogen peroxide, (1) peroxidase, (2) an amino-aromatic acid represented by the formula;
##STR7## wherein X is a mono- or dialkylamino group, and ZH is the carboxyl group or the sulfo group, and (3) a 4-aminoantipyrine represented by the formula;
##STR8## wherein each of R and R1, when taken separately, is alkyl, R2 is an aromatic moiety, each of R3 and R4, when taken separately, is hydrogen or alkyl, and Y is oxygen or sulfur, wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is in th range of from bout 0.1;
1 to about 25;
1.
- to about 45°
-
2. A method according to claim 1 wherein said aminoaromatic acid is an aminobenzoic acid and said aminoantipyrine is one wherein R2 is phenyl and Y is oxygen.
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3. A method according to claim 2 wherein said aminoantipyrine is one wherein R and R1 each is methyl.
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4. A method according to claim 3 wherein said aminobenzoic acid is 3-(N,N-dimethylamino)benzoic acid.
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5. A method according to claim 4 wherein said aminoantipyrine is one wherein each of R3 and R4 is hydrogen.
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6. A method according to claim 4 wherein said aminoantripyrine is one wherein each of R3 and R4 is methyl.
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7. A method according to claim 5 wherein the molar ratio of said 3-(N,N-dimethylamino)benzoic acid to said aminoantipyrine is in the range of from about 8:
- 1 to about 12;
1.
- 1 to about 12;
-
8. A method for detecting the presence of hydrogen peroxide in an aqueous medium at a temperature of from about 20°
- C. to about 45°
C. and having a pH of from about 3 to about 9.5, which comprises;
(a) adding to said aqueous medium (1) peroxidase, (2) an aminoaromatic acid represented by the formula;
##STR9## wherein X is a mono-or dialkylamine group and ZH is the carboxyl group or the sulfo group, and (3) a 4-aminoantipyrine represented by the formula;
##STR10## wherein each of R and R1, when taken separately, is alkyl, R2 is an aromatic moiety, each of R3 and R4, when taken separately, is hydrogen or alkyl, and Y is oxygen or sulfur, wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is in the range of from about 0.1;
1 to about 25;
1; and
(b) thereafter measuring any resulting color change photometrically.
- C. to about 45°
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9. A method according to claim 8 wherein said aminoaromatic acid is an aminobenzoic acid and said aminoantipyrine is one wherein R2 is phenyl and Y is oxygen.
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10. A method according to claim 9 wherein said aminoantipyrine is one wherein each of R and R1 is methyl.
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11. A method according to claim 10 wherein said aminobenzoic acid is 3-(N,N-dimethylamino)benzoic acid.
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12. A method according to claim 11 wherein said aminoantipyrine is one wherein each of R3 and R4 is hydrogen.
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13. A method according to claim 11 wherein said aminoantipyrine is one wherein each of R3 and R4 is methyl.
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14. A method according to claim 12 wherein the molar ratio of said 3-(N,N-dimethylamino)-benzoic acid to said aminoantipyrine is in the range of from about 8:
- 1 to about 12;
1.
- 1 to about 12;
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15. In a method for the enzymatic determination of the amount of a component in a body fluid by admixing said fluid with (a) an enzyme capable of promoting the oxidation of said component and forming hydrogen peroxide, (b) peroxidase, and (c) a chromagen capable of being oxidized by peroxidasecatalyzed reaction with hydrogen peroxide to form a chromophore, and measuring the resulting color change, the improvement wherein said chromogen is a mixture of (1) an aminoaromatic acid represented by the formula:
- ##STR11## wherein X is a mono- or dialkyl amine group and ZH is the carboxyl or the sulfo group and (2) a 4-aminoantipyrine represented by the formula;
##STR12## wherein each of R and R1, when taken separately, is alkyl, R2 is an aromatic moiety, each of R3 and R4, when taken separately, is hydrogen or alkyl, and Y is oxygen or sulfur, wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is in the range of from about 0.1;
1 to about 25;
1, said method being conducted at a temperture of from about 20°
C. to about 45°
C., and a pH of from about 3 to about 9.5.
- ##STR11## wherein X is a mono- or dialkyl amine group and ZH is the carboxyl or the sulfo group and (2) a 4-aminoantipyrine represented by the formula;
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16. A method according to claim 15 wherein said aminoaromatic acid is an aminobenzoic acid and said aminoantipyrine is one wherein R2 is phenyl and Y is oxygen.
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17. A method according to claim 16 wherein sid aminoantipyrine is one wherein each of R and R1 is methyl.
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18. A method according to claim 17 wherein said aminobenzoic acid is 3-(N,N-dimethylamino)benzoic acid.
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19. A method according to claim 18 wherein said aminoantipyrine is one wherein each of R3 and R4 is hydrogen.
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20. A method according to claim 18 wherein said aminoantipyrine is one wherein each of R3 and R4 is methyl.
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21. A method according to claim 19 wherein the molar ratio of said 3-(N,N-dimethylamino)-benzoic acid to said aminoantipyrine is in the range of from about 8:
- 1 to about 12;
1.
- 1 to about 12;
-
22. A method according to claim 15 wherein said enzyme capable of promoting oxidation of said component is uricase.
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23. A method according to claim 15 wherein said enzyme capable of promoting oxidation of said component is glucose oxidase.
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24. A method according to claim 15 wherein said enzyme capable of promoting oxidation of said component is cholesterol oxidase.
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25. A reagent system for detecting the presence of hydrogen peroxide in an aqueous medium comprising:
- (1) peroxidase, (2) an aminoaromatic acid represented by the formula;
##STR13## wherein X is a mono- or dialkylamino group and ZH is the carboxyl group or the sulfo group, and (3) a 4-aminoantipyrine represented by the formula;
##STR14## wherein each of R and R1, when taken separately, is alkyl, R2 is an aromatic moiety, each of R3 and R4, when taken separately, is hydrogen or alkyl, and Y is oxygen or sulfur, wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is from about 0.1;
1 to about 25;
1.
- (1) peroxidase, (2) an aminoaromatic acid represented by the formula;
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26. A system according to claim 25 additionally including a buffer to buffer an aqueous solution of said peroxidase, aminoaromatic acid and aminoantipyrine to a pH in the range of from about 3 to about 9.5.
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27. A system according to claim 26 additionally including an enzyme capable of supporting oxidation of a component of a body fluid to form hydrogen peroxide.
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28. A system according to claim 27 wherein said enzyme is capable of supporting the oxidation of uric acid, cholesterol or glucose to form hydrogen peroxide.
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29. A system according to claim 27 wherein ZH is carboxyl, R2 is phenyl and Y is oxygen.
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30. A system according to claim 29 wherein X is aminoalkyl, R is methyl and R1 is methyl.
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31. A system according to claim 30 wherein X is dimethylamino and R3 and R4 each is methyl.
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32. A system according to claim 30 wherein X is dimethylamino and R3 and R4 each is hydrogen.
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33. A system according to claim 32 wherein the molar ratio of said aminoaromatic acid to said aminoantipyrine is in the range of from about 8:
- 1 to about 12;
1.
- 1 to about 12;
-
34. A system according to claim 33 wherein said enzyme capable of supporting oxidation is uricase.
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35. A system according to claim 33 wherein said enzyme capable of supporting oxidation is glucose oxidase.
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36. A system according to claim 33 wherein said enzyme capable of supporting oxidation is cholesterol oxidase.
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37. The system according to claim 25 in kit form.
Specification