US4414327A
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Method for the estimation of N-acylated primary aromatic amines
Assignee: Microbiological Research Authority
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Filing: 1981-12-01
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Granted: 1983-11-08
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Expires: 2001-12-01
Abstract
Abstract A method for the estimation of an anilide in which the anilide is first hydrolyzed enzymatically to an aniline and then the quantity of the aniline produced is estimated spectrophotometrically preferably colorimetrically. The hydrolysis of the anilide may be catalyzed by any enzyme of the type, EC 3.5.1.13, known as aryl acylamidases. Preferably enzymes isolated from the cells of Pseudomonas fluorescens ATCC 39005 or Pseudomonas putida ATCC 39004 are employed. The aniline may be analyzed, for example, by conversion to an indamine, an indophenol or an indoaniline, followed by colorimetric analysis of the colored quinone-type compound produced. This conversion may take place in the presence of an oxidizing agent, such as a copper (II) salt, and/or a base, such as ammonia. A diagnostic kit to allow the routine use of the above method is also provided. In one embodiment of the kit, it comprises, (a) an aryl acylamidase, in solution or on a solid support, (b) an organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline, preferably in solution, and (c) a base, preferably in solution. The kit may further comprise an oxidizing agent, again preferably in solution. The above method of analysis and diagnostic kit may be particularly useful in the estimation of anilide drugs, such as paracetamol, dissolved in biological fluids.
Inventors
- 👤 Peter M. Hammond
- 👤 Christopher P. Price
- 👤 Michael D. Scawen
- 👤 Anthony Atkinson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 08:33
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/78; C12N9/80; C12N1/00; C12N1/20; C12N1/205; C12Q; C12Q1/00; C12Q1/34; C12R; C12R2001/00; C12R2001/01; C12R2001/38; C12R2001/39; G; G01; G01N; G01N2333/00; G01N2333/195; G01N2333/21; G01N2333/90; G01N2333/914; G01N2333/978; G01N2333/98; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S435/81; Y10S435/8215; Y10S435/822; Y10S435/874; Y10S435/876; Y10S435/877
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
64 words
In a method for the estimation of an anilide (N-acylated primary aromatic amine) comprising: (a) hydrolysing the anilide to an aniline, and (b) estimating the quantity of said aniline spectrophotometrically, wherein the improvement comprises conducting step (a) above in the presence of an aryl acylamidase enzyme defined as group EC3.5.1.13, said enzyme being able to catalyse the hydrolysis of the anilide to the aniline.
Claim 2
Dependent Claim
64 words
In a method for the estimation of an anilide (N-acylated primary aromatic amine) comprising: (a) hydrolysing the anilide to an aniline, and (b) estimating the quantity of said aniline spectrophotometrically, wherein the improvement comprises conducting step (a) above in the presence of an aryl acylamidase enzyme defined as group EC3.5.1.13, said enzyme being able to catalyse the hydrolysis of the anilide to the aniline.
Claim 3
Dependent Claim
42 words
A method according to claim 1 wherein the enzyme is an aryl acylamidase, EC 3.5.1.13, isolated from Pseudomonas fluorescens ATCC 39005 or aryl acylamidase producing mutants or variants thereof, or Pseudomonas putida ATCC 39004 or aryl acylamidase producing mutants or variants thereof.
Claim 4
Dependent Claim
47 words
A method according to claim 1 wherein the quantity of said aniline is estimated by: (a) converting the aniline to a substance selected from the group consisting of an indamine, an indophenol and an indoaniline, and (b) estimating the quantity of said indamine, indophenol or indoaniline spectrophotometrically.
Claim 5
Dependent Claim
27 words
A method according to claim 3 wherein the conversion of the aniline to the indamine, indophenol or indaniline is effected in the presence of an oxidising agent.
Claim 6
Dependent Claim
15 words
A method according to claim 4 wherein the oxidising agent comprises a copper (II) salt.
Claim 7
Dependent Claim
30 words
A method according to claim 4 wherein the oxidising agent comprises a salt selected from the group consisting of an Fe (III), a chromate, a dichromate and a permanganate salt.
Claim 8
Dependent Claim
26 words
A method according to claim 3 wherein the conversion of the aniline to the indamine, indophenol or indoaniline is effected in the presence of a base.
Claim 9
Dependent Claim
11 words
A method according to claim 7 wherein the base comprises ammonia.
Claim 10
Dependent Claim
19 words
A method according to claim 8 wherein the ammonia is in the form of an aqueous solution of ammonia.
Claim 11
Dependent Claim
33 words
A method according to claim 7 wherein the base comprises a base selected from the group consisting of an alkali metal hydroxide, a borate buffer and an alkaline solution of an organic amine.
Claim 12
Dependent Claim
39 words
A method according to claim 3 wherein the conversion of the aniline to the indamine, indophenol or indoaniline is effected by reacting the aniline with a substance selected from the group consisting of a phenol and a phenolic ether.
Claim 13
Dependent Claim
14 words
A method according to claim 11 wherein the phenol or phenolic ether is cresol.
Claim 14
Dependent Claim
14 words
A method according to claim 12 wherein the phenol or phenolic ether is ortho-cresol.
Claim 15
Dependent Claim
40 words
A method according to claim 3 wherein the conversion of the aniline to the indamine, indophenol or indoaniline is effected by reacting the aniline with a substance selected from the group consisting of a primary, secondary, and tertiary aryl amine.
Claim 16
Dependent Claim
16 words
A method according to claim 14 wherein the primary, secondary or tertiary aryl amine is phenylenediamine.
Claim 17
Dependent Claim
16 words
A method according to claim 15 wherein the primary, secondary or tertiary aryl amine is meta-phenylenediamine.
Claim 18
Dependent Claim
16 words
A method according to claim 14 wherein the primary, secondary or tertiary aryl amine is para-hydroxyaniline.
Claim 19
Dependent Claim
14 words
A method according to claim 1 wherein the anilide is in a biological fluid.
Claim 20
Dependent Claim
11 words
A method according to claim 1 wherein the anilide is N-acetyl-4-hydroxyaniline.
Claim 21
Dependent Claim
11 words
A method according to claim 1 wherein the anilide is N-acetyl-4-ethoxyaniline.
Claim 22
Dependent Claim
12 words
A method according to claim 1 wherein the aniline is estimated colorimetrically.
Claim 23
Dependent Claim
67 words
A diagnostic kit for use in the estimation of an anilide by a method according to claim 1 comprising: (a) an aryl acylamidase enzyme, defined as EC 3.5.1.13, suitable for catalysing the hydrolysis of the anilide to an aniline, and (b) an organic compound suitable for the conversion of the aniline to a substance selected from the group consisting of an indamine, an indophenol and an indoaniline.
Claim 24
Dependent Claim
49 words
A diagnostic kit according to claim 22 wherein the enzyme is an aryl acylamidase, EC 3.5.1.13, isolated from cells of one of the strains Pseudomonas fluorescens ATCC 39005 or aryl acylamidase producing mutants or variants thereof, or Pseudomonas putida ATCC 39004 or aryl acylamidase producing mutants or variants thereof.
Claim 25
Dependent Claim
39 words
A diagnostic kit according to claim 22 wherein the organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline is selected from the group consisting of a phenol and a phenolic ether.
Claim 26
Dependent Claim
15 words
A diagnostic kit according to claim 24 wherein the phenol or phenolic ether is cresol.
Claim 27
Dependent Claim
15 words
A diagnostic kit according to claim 25 wherein the phenol or phenolic ether is ortho-cresol.
Claim 28
Dependent Claim
40 words
A diagnostic kit according to claim 22 wherein the organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline is selected from the group consisting of a primary, secondary and tertiary aryl amine.
Claim 29
Dependent Claim
17 words
A diagnostic kit according to claim 27 wherein the primary, secondary or tertiary aryl amine is phenylenediamine.
Claim 30
Dependent Claim
17 words
A diagnostic kit according to claim 28 wherein the primary, secondary or tertiary aryl amine is meta-phenylenediamine.
Claim 31
Dependent Claim
17 words
A diagnostic kit according to claim 27 wherein the primary, secondary or tertiary aryl amine is para-hydroxyaniline.
Claim 32
Dependent Claim
12 words
A diagnostic kit according to claim 22 additionally comprising an oxidising agent.
Claim 33
Dependent Claim
16 words
A diagnostic kit according to claim 31 wherein the oxidising agent comprises a copper (II) salt.
Claim 34
Dependent Claim
31 words
A diagnostic kit according to claim 31 wherein the oxidising agent comprises a salt selected from the group consisting of an Fe (III), a chromate, a dichromate and a permanganate salt.
Claim 35
Dependent Claim
11 words
A diagnostic kit according to claim 22 additionally comprising a base.
Claim 36
Dependent Claim
12 words
A diagnostic kit according to claim 34 wherein the base comprises ammonia.
Claim 37
Dependent Claim
34 words
A diagnostic kit according to claim 34 wherein the base comprises a base selected from the group consisting of an alkali metal hydroxide, a borate buffer and an alkaline solution of an organic amine.
Claim 38
Dependent Claim
16 words
A diagnostic kit according to claim 22 wherein the enzyme is retained on a solid support.
Claim 39
Dependent Claim
17 words
A diagnostic kit according to claim 22 wherein the enzyme is dissolved in one or more solvents.
Claim 40
Dependent Claim
19 words
A diagnostic kit according to claim 38 wherein at least one of the one or more solvents comprises glycerol.
Claim 41
Dependent Claim
29 words
A diagnostic kit according to claim 39 wherein the one or more solvents comprises, in admixture, between 10 and 70% (v/v) glycerol and between 90 and 30% (v/v) water.
Claim 42
Dependent Claim
30 words
A diagnostic kit according to claim 22 wherein the organic compound suitable for converting the aniline to an indamine, an indophenol or anindoaniline is dissolved in one or more solvents.
Claim 43
Dependent Claim
18 words
A diagnostic kit according to claim 31 wherein the oxidising agent is dissolved in one or more solvents.
Claim 44
Dependent Claim
17 words
A diagnostic kit according to claim 34 wherein the base is dissolved in one or more solvents.
Claim 45
Dependent Claim
22 words
A diagnostic kit according to claim 43 wherein the solution of the base is combined with a solution of the oxidising agent.
Claim 46
Dependent Claim
41 words
A diagnostic kit according to claim 44 wherein the solution of the base, the solution of the oxidising agent and a solution of the organic compound suitable for converting the aniline to an indamine, an indophenol or an indoaniline, are combined.
Claim 47
Dependent Claim
12 words
A diagnostic kit according to claim 44 wherein the solvent is water.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-11-07 | 1987-05-09 | 1987-11-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-11-07 | 1991-05-09 | 1991-11-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-11-07 | 1995-05-09 | 1995-11-08 | $7700.00 | Window Closed / Lapsed |