Analytical method using tetrazolium salt indicators having a reflectance plateau

• US 5,126,275 A
• Filed: 09/19/1990
• Issued: 06/30/1992
• Est. Priority Date: 09/19/1990
• Status: Expired due to Term

• Current Assignee: Precor Incorporated (Peloton Interactive, Inc.)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Lin, Nan-Horng, Wehling, Klaus, Kocher, Jurgen, Hatch, Robert P., Ruetten, Scott
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): DALEY, THOMAS
• Application Number:
  US07/584,907
• Time in Patent Office: 650 Days
• Field of Search: 435/26, 435/805, 435/25, 435/28, 422/56, 422/57, 436/163, 436/169, 436/164, 436/172, 436/518
• US Class Current: 436/169
• CPC Class Codes: C12Q 1/008 for determining co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: Y10S 435/805 Test papers

Abstract

General classes of 2-thiazolyl tetrazolium salt compounds have been found to be characterized by a reflectance spectrum exhibiting an extended plateau above about 600-650 nm. Such compounds are useful as chromogenic indicators for reducing substances such as NADH. The reflectance plateau confers improved accuracy to analytical assays, particularly for the determination of analytes of medical diagnostic significance, in which a colorimetric response on a reagent carrier matrix is measured by reflectance.

Claims

1. In a method for determining an analyte in a liquid sample wherein the sample is contacted with a test device comprising a solid carrier matrix incorporated with a test composition comprising (a) reagents which react with said analyte to produce a reducing substance and (b) an indicator compound which is reduced by said reducing substance to produce a chromogenic dye product, and where the chromogenic dye product is measured by irradiating the carrier matrix with a light source having a central wavelength above about 600 nm which wavelength will vary due to manufacturing variances and temperature dependence of the light source and sensing the reflectance of the carrier matrix by measuring reflected light with a detector element,

the improvement which comprises employing as said indicator compound a thiazolyl tetrazolium salt of the formula:

##STR20## wherein (a) R¹ and R² together form a benzo ring, said benzo ring being substituted or unsubstituted, or

(b) R¹ is carboxyl, carbalkoxy, carbaryloxy, carbomyl or cyano, and R² is alkyl or chloro, or

(c) R¹ is alkyl or aryl, and R² is carboxyl, carbalkoxy, carbaryloxy, carbamoyl or cyano, or

(d) R¹ is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R² is chloro, or

(e) one or both of R¹ and R² are substituted or unsubstituted phenyl, and if only one is substituted or unsubstituted phenyl, the other is hydrogen or alkyl; and

wherein R³ is aryl or styryl, R⁴ is aryl, and X^o is a counteranion wherein the tetrazolium salts are further characterized in that when they are reduced by the reducing substance to their corresponding formazan the reflectance of the formazan varies by less than 17% over a wavelength range of 50 nanometers at wavelengths of 600 nanometers or greater.

2. The method of claim 1 wherein R¹ and R² together form a benzo ring which is unsubstituted.

3. The method of claim 1 wherein R¹ and R² together form a benzo ring to give a benzothiazolyl residue having the formula:

##STR21## wherein (i) R⁵ and R⁶, or R⁶ and R⁷, or R⁷ and R⁸, together form a benzo or cyclohexyl ring that is unsubstituted or substituted with alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and wherein the others, which can be the same or different from each other, are hydrogen, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, provided that where R⁶ and R⁷ together form a benzo or cyclohexyl ring, R⁵ is not hydrogen, or

(ii) one or more of R⁵, R⁶, R⁷, and R⁸ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, cyano, halo, hydroxyl, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and the others, if any, are hydrogen.

4. The method of claim 1 wherein

(a) R¹ is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano, and R² is alkyl or chloro, or

(b) R¹ is alkyl or aryl, and R² is carboxyl, carbalkoxy, carbaryloxy, carbamoyl, or cyano, or

(c) R¹ is di- or trifluoroalkyl wherein the fluoro substituents are on the carbon adjacent to the thiazolyl residue in the formula, and R² is chloro.

5. The method of claim 1 wherein one or both of R¹ and R² are unsubstituted phenyl or phenyl substituted with, independently, alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, amino, carbamoyl, carbalkoxy, carbaryloxy, carboxy, cyano, halo, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and if only one is substituted or unsubstituted phenyl, the other is hydrogen, alkyl, or chloro.

6. The method of any one of claims 1 to 5 wherein R³ is selected from:

##STR22## wherein Q is a bond or --CH═

CH--, and wherein

(i) Y¹ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, or hydrogen, Y² is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, alkylthio, amino, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y³ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carbaryloxy, carboxyl, cyano, halo, hydrogen, hydroxyl, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, and Y⁴ is alkoxy, halo, or hydrogen, or

(ii) Y² and Y³ together form methylenedioxy or imidazo and Y¹ and Y⁴ are hydrogen,

(b₁) 2, 3, or 4-pyridyl,

(c₁) 2 or 3-thienyl, and

(d₁) 2 or 3-furanyl;

and wherein R⁴ is selected from;

##STR23## wherein Y⁵ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, halo, hydrogen, nitro, or ureido, Y⁶ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido, Y⁷ is alkoxy, aryloxy, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, hydrogen, hydroxyl, nitro, phenylazo, sulfo, sulfonamido sulfamoyl, or ureido, and Y⁸ is alkoxy, aryloxy, alkyl, halo, hydrogen or nitro, ##STR24## wherein Y⁹ is alkoxy, aryloxy, alkyl, amido, alkylamido, arylamido, alkylthio, arylthio, carbamoyl, carbalkoxy, carboxyl, cyano, halo, hydrogen, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, trialkylammonio, or ureido,

(c₂) 2, 4, 6, or 8-quinolyl, or 2-methylquinolyl, and

(d₂) anthranyl

7. The method of any one of claim 1 to 5 wherein R³ is selected from:

##STR25## wherein (i) Y², Y³, and Y⁴ are each C_1-4 alkoxy,

(ii) Y⁴ is hydrogen and Y² and Y³ are both C_1-4 alkoxy or together form methylenedioxy, or

(iii) Y² and Y⁴ are both hydrogen and Y³ is C_1-4 alkoxy, C_1-4 alkyl, C_1-4 alkylamido, C_1-4 alkylthio, carbamoyl, carb(C_1-4)-alkoxy, carboxyl, cyano, halo, hydrogen, nitro, tri(C_1-4)alkylammonio, or ureido, and

(b₁) 2 or 3-thienyl;

and wherein R⁴ is selected from;

##STR26## wherein (i) Y⁵ is hydrogen and each of Y⁶, Y⁷, and Y⁸ is C_1-4 alkoxy,

(ii) Y⁵ Y⁸ are both hydrogen and Y⁶ and Y⁷ are both C_1-4 alkoxy or together form methylenedioxy,

(iii) Y⁵, Y⁶ and Y⁸ are each hydrogen and Y⁷ is C_1-4 alkoxy, C_1-4 alkylamido, C_1-4 -alkylthio, benzamido, carbamoyl, carb(C_1-4)alkoxy, carboxyl, cyano, nitro, phenylazo, sulfo, sulfonamido, sulfamoyl, or ureido,

(iv) Y⁵ is alkoxy or alkyl, Y⁶ and Y⁸ are both hydrogen, and Y⁷ is C_1-4 alkoxy, C_1-4 alkylamido, C_1-4 alkylthio, benzamido, carbamoyl, carb(Cl-4)alkoxy, carboxyl, cyano, hydrogen, nitro, phenylazo, or ureido,

(v) Y⁵ and Y⁸ are C alkoxy, or

(vi) Y⁵ and Y⁸ are C_1-4 alkoxy and Y⁷ is C_1-4 -alkylamido or benzamido;

##STR27## wherein Y⁹ is C_1-4 alkoxy, C_1-4 alkyl, C_1-4 -alkylamido, C_1-4 alkylthio, benzamido, cyano, halo, hydrogen, nitro, sulfo, sulfonamido, or ureido, and

(c₂) 8-quinolyl.

8. The method of any one of claims 1 to 5 wherein R³ is selected from:

3,4,5-trimethyoxyphenyl,

3,4-dimethoxyphenyl,

3,4-methylenedioxyphenyl,

4-methoxyphenyl,

4-acetamidophenyl, or

4-methylthiophenyl,

and wherein R⁴ is selected from;

3,4,5-trimethyoxyphenyl,

3,4-dimethoxyphenyl,

2,4-dimethoxyphenyl,

3,4-methylenedioxyphenyl,

4-methoxyphenyl,

4-acetamidophenyl,

4-methylthiophenyl,

4-carboxyphenyl,

4-nitrophenyl,

2-methoxyphenyl,

2-methoxy-4-carboxyphenyl,

2,5-dimethoxyphenyl,

2,5-dimethoxy-4-benzamidophenyl

1-naphthyl,

4-nitro-1-naphthyl,

4-methoxy-1-naphthyl, or

8-quinolyl.

9. The method of claim 1 wherein the light which reaches the detector element consists of a narrow wavelength band whose central wavelength is susceptible to variations of over about ±

5 nm.

10. The method of claim 9 wherein said light source is an LED.

11. The method of claim 1 wherein said reducing substance is NADH.