Salt of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate in colorimetric determination of hydrogen peroxide

• US 5,710,012 A
• Filed: 05/25/1994
• Issued: 01/20/1998
• Est. Priority Date: 08/25/1993
• Status: Expired due to Fees

• Current Assignee: ActiMed Laboratories, Inc.
• Original Assignee: ActiMed Laboratories, Inc.
• Inventors: Nikolyukin, Yuri A., Gibboni, David J.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US08/450,780
• Time in Patent Office: 1,336 Days
• Field of Search: 435/28, 435/810, 435/975, 436/135, 436/808, 436/904, 534/788, 548/161, 564/305, 564/309
• US Class Current: 435/28
• CPC Class Codes: C09B 26/02 Hydrazone dyes hydrazone-azo dyes C09B56/18
• CPC Class Codes: C09B 27/00 Preparations in which the azo group is formed in any way other than by diazotising and coupling, e.g. oxidation
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 2326/00 Chromogens for determinations of oxidoreductase enzymes
• CPC Class Codes: C12Q 2326/32 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate, i.e. MBTH
• CPC Class Codes: C12Q 2326/96 4-Amino-antipyrine
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

Water soluble salts of hydrazone compounds react quickly with aniline compounds in the presence of a peroxidase and hydrogen peroxide to provide a color which can be measured spectrophotometrically or visually, either in solution or when incorporated in a device for the measurement of analytes of interest. These water-soluble hydrazone salts are useful in determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide in an aqueous liquid, by, in the presence of a substance having peroxidative activity, physically contacting a sample of a liquid with a dye-forming composition comprising a water-soluble salt of a hydrazone compound and a substituted or unsubstituted aniline compound to produce a colored dye; and detecting the dye formed as a result of the presence of hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide.

Claims

1. Water soluble salts of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate.

2. The water soluble salts of claim 1 selected from the group consisting of sodium salt, potassium salt, and ammonium salt.

3. A dye formed from the reaction between a soluble salt of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate and a substituted or unsubstituted aniline dye.

4. A dye according to claim 3 wherein the aniline dye is selected from the compounds of the formula ##STR3## wherein R¹ and R² are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, C₁ -C₉ alkoxy;

NR³ R⁴ and R³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

F;

Cl;

Br;

I;

COOR⁵ wherein R⁵ is selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR⁶ R⁷ where R⁶ and R7 are selected from the group consisting of hydrogen and C₁ -C₉ aryl, alkyl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy; and

R¹¹, R¹², R¹³ and R¹⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl;

C₁ -C₉ alkoxy;

NR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR¹⁸ R¹⁹ where R¹⁸ and R¹⁹ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy;

n is an integer of from 0 to 10;

Z and Y are each selected from the group consisting of hydrogen;

OH, SH;

COOR⁸ where R⁸ is hydrogen, C₁ -C₉ alkyl, aryl or heteroaryl;

NR²⁰ NHR²¹ where R²⁰ and R²¹ are hydrogen, C₁ -C₉ alkyl, aryl, or heteroaryl; and

at least one of Z and Y is COOR⁸, CN'"'"'NR⁹ R¹⁰ or NR²⁰ NHR²¹.

5. A dye according to claim 4 wherein the aniline dye is selected from the group consisting of N,N-(biscarboxymethyl)aniline;

N,n-(biscarboxymethyl)-4-methoxyaniline;

N,N-(bis-beta-carboxyethyl)aniline;

N-ethyl-N-phenyl glycine;

N-ethyl-N-carboxyethylaniline;

N-phenylpiperidinyl succinate;

N-ethylanilinopropaneamine;

N-methylanilinopropaneamine;

N-methyl-N-carboxyethylaniline;

N,N-(bis-beta-carboxyethyl)2,5-dimethylaniline;

N-beta-carboxyethylaminobenzoic acid.

6. A dye-forming composition comprising a water soluble salt of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate, a substituted or unsubstituted aniline compound.

7. A reagent according to claim 6 wherein the aniline dye is selected from the group consisting of compounds of the formula ##STR4## wherein R¹ and R² are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, C₁ -C₉ alkoxy;

NR³ R⁴ where R³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

F;

Cl;

Br;

I;

COOR⁵ wherein R⁵ is selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR⁶ R⁷ where R⁶ and R⁷ are selected from the group consisting of hydrogen and C₁ -C₉ aryl, alkyl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy; and

R¹¹, R¹², R¹³ and R¹⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl;

C₁ -C₉ alkoxy;

NR¹⁵ R¹⁶ wherein R¹⁵ and R.sup. 16 are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR¹⁸ R¹⁹ where R¹⁸ and R¹⁹ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy;

n is an integer of from 0 to 10;

Z and Y are each selected from the group consisting of hydrogen;

OH, SH;

COOR⁸ where R⁸ is hydrogen, C₁ -C₉ alkyl, aryl or heteroaryl;

NR²⁰ NHR²¹ where R²⁰ and R²¹ are hydrogen, C₁ -C₉ alkyl, aryl, or heteroaryl; and

at least one of Z and Y is COOR⁸, CN'"'"'NR⁹ R¹⁰ or NR²⁰ NHR²¹.

8. A dye-forming composition according to claim 7 wherein the aniline dye is selected from the group consisting of N,N-(biscarboxymethyl)aniline;

N,n-(biscarboxymethyl)-4-methoxyaniline;

N,N-(bis-beta-carboxyethyl)aniline;

N-ethyl-N-phenyl glycine;

N-ethyl-N-carboxyethylaniline;

N-phenylpiperidinyl succinate;

N-ethylanilinopropaneamine;

N-methylanilinopropaneamine;

N-methyl-N-carboxyethylaniline;

N,N-(bis-beta-carboxyethyl)2,5-dimethylaniline;

N-beta-carboxyethylaminobenzoic acid.

9. A reagent for detection of hydrogen peroxide comprising the dye-forming composition of claim 6 and a peroxidase.

10. A diagnostic test kit for the determination of hydrogen peroxide, said kit comprising a container means containing:

a dye-forming composition according to claim 6 comprising;

(1) a water soluble salt of 6-carboxy-3-methyl benzothiazolone hydrazone hydrate; and

(2) a substituted or unsubstituted aniline dye which reacts with said water-soluble salt of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate to produce a colored dye.

11. A diagnostic test kit according to claim 10 wherein the aniline dye is selected from the group consisting of compounds of the formula ##STR5## wherein R¹ and R² are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, C₁ -C₉ alkoxy;

NR³ R⁴ where R³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

F;

Cl;

Br;

I;

COOR⁵ wherein R⁵ is selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR⁶ R⁷ where R⁶ and R⁷ are selected from the group consisting of hydrogen and C₁ -C₉ aryl, alkyl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy; and

R¹¹, R¹², R¹³ and R¹⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl;

C₁ -C₉ alkoxy;

NR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR¹⁸ R¹⁹ where R¹⁸ and R¹⁹ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy;

n is an integer of from 0 to 10;

Z and Y are each selected from the group consisting of hydrogen;

OH, SH;

COOR⁸ where R⁸ is hydrogen, C₁ -C₉ alkyl, aryl or heteroaryl;

NR²⁰ NHR²¹ where R²⁰ and R²¹ are hydrogen, C₁ -C₉ alkyl, aryl, or heteroaryl; and

at least one of Z and Y is COOR⁸, CN'"'"'NR⁹ R¹⁰ or NR²⁰ NHR²¹.

12. A diagnostic test kit according to claim 11 wherein the aniline dye is selected from the group consisting of N,N-(biscarboxymethyl)aniline;

N,n-(biscarboxymethyl)-4-methoxyaniline;

N,N-(bis-beta-carboxyethyl)aniline;

N-ethyl-N-phenyl glycine;

N-ethyl-N-carboxyethylaniline;

N-phenylpiperidinyl succinate;

N-ethylanilinopropaneamine;

N-methylanilinopropaneamine;

N-methyl-N-carboxyethylaniline;

N,N-(bis-beta-carboxyethyl)2,5-dimethylaniline;

N-beta-carboxyethylaminobenzoic acid.

13. A diagnostic test kit according to claim 10 further including a peroxidase.

14. A method for determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide in an aqueous liquid, said method comprising the steps of:

(a) in the presence of a substance having peroxidative activity, physically contacting a sample of a liquid with a dye forming composition according to claim 6, wherein if hydrogen peroxide or an analyte which reacts to form hydrogen peroxide in an aqueous liquid is present in the sample, the dye forming composition will react with said hydrogen peroxide or an analyte which reacts to form hydrogen peroxide in an aqueous liquid; and

(b) detecting dye formed from the reaction of the dye forming composition with the substance having peroxidative activity.

15. A method according to claim 14 wherein the aniline dye is selected from the group consisting of compounds of the formula ##STR6## wherein R¹ and R² are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, C₁ -C₉ alkoxy;

NR³ R⁴ where R³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

F;

Cl;

Br;

I;

COOR⁵ wherein R⁵ is selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR⁶ R⁷ where R⁶ and R⁷ are selected from the group consisting of hydrogen and C₁ -C₉ aryl, alkyl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy; and

R¹¹, R¹², R¹³ and R¹⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl;

C₁ -C₉ alkoxy;

NR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR¹⁸ R¹⁹ where R¹⁸ and R¹⁹ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy;

n is an integer of from 0 to 10;

Z and Y are each selected from the group consisting of hydrogen;

OH, SH;

COOR⁸ where R⁸ is hydrogen, C₁ -C₉ alkyl, aryl or heteroaryl;

NR²⁰ NHR²¹ where R²⁰ and R²¹ are hydrogen, C₁ -C₉ alkyl, aryl, or heteroaryl; and

at least one of Z and Y is COOR⁸, CN'"'"'NR⁹ R¹⁰ or NR²⁰ NHR²¹.

16. A method according to claim 15 wherein the aniline dye is selected from the group consisting of N,N-(biscarboxymethyl) aniline;

N,N-(biscarboxymethyl)-4-methoxyaniline;

N,N-(bis-beta-carboxyethyl)aniline;

N-ethyl-N-phenyl glycine;

N-ethyl-N-carboxyethylaniline;

N-phenylpiperidinyl succinate;

N-ethylanilinopropaneamine;

N-methylanilinopropaneamine;

N-methyl-N-carboxyethylaniline;

N,N-(bis-beta-carboxyethyl)2,5-dimethylaniline;

N-beta-carboxyethylaminobenzoic acid.

17. A method for determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide in an aqueous liquid comprising the method according to claim 14 wherein the dye forming composition further includes a peroxidase.

18. In a method for determining hydrogen peroxide or an analyte which reacts to produce hydrogen peroxide, the improvement comprising using as a color-forming agent a combination of 6-carboxy-3-methylbenzothiazolone hydrazone hydrate and an aniline dye.

19. The method according to claim 18 wherein the aniline dye is selected from the group consisting of compounds of the formula ##STR7## wherein R¹ and R² are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, C₁ -C₉ alkoxy;

NR³ R⁴ where R³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

F;

Cl;

Br;

I;

COOR⁵ wherein R⁵ is selected from the group consisting of hydrogen, C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR⁶ R⁷ where R⁶ and R⁷ are selected from the group consisting of hydrogen and C₁ -C₉ aryl, alkyl, and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy; and

R¹¹, R¹², R¹³ and R⁴ are selected from the group consisting of hydrogen, C₁ -C₉ alkyl;

C₁ -C₉ alkoxy;

NR¹⁵ R¹⁶ wherein R¹⁵ and R¹⁶ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl, and heteroaryl;

CN;

CONR¹⁸ R¹⁹ where R¹⁸ and R¹⁹ are selected from the group consisting of hydrogen;

C₁ -C₉ alkyl, aryl and heteroaryl;

aryl;

aryloxy;

heteroaryl;

heteroaryloxy;

n is an integer of from 0 to 10;

Z and Y are each selected from the group consisting of hydrogen;

OH, SH;

COOR⁸ where R⁸ is hydrogen, C₁ -C₉ alkyl, aryl or heteroaryl;

NR²⁰ NHR²¹ where R.sup.° and

R²¹ are hydrogen, C₁ -C₉ alkyl, aryl, or heteroaryl; and

at least one of Z and Y is COOR⁸, CN'"'"'NR⁹ R¹⁰ or NR²⁰ NHR²¹.

20. A method according to claim 18 wherein the aniline dye is selected from the group consisting of N,N(biscarboxymethyl)aniline;

N,N-(biscarboxymethyl)-4-methoxyaniline;

N,N-(bis-beta-carboxyethyl)aniline;

N-ethyl-N-phenyl glycine;

N-ethyl-N-carboxyethylaniline;

N-phenylpiperidinyl succinate;

N-ethylanilinopropaneamine;

N-methylanilinopropaneamine;

N-methyl-N-carboxyethylaniline;

N,N-(bis-beta-carboxyethyl) 2,5 -dimethylaniline;

N-beta-carboxyethylaminobenzoic acid.