EP0053470A1
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EP
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Method for the estimation of N-acylated primary aromatic amines
Assignee: Microbiological Research Authority
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Filing: 1981-11-24
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Granted: —
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Expires: 2001-11-24
Abstract
Abstract A method for the estimation of an anilide in which the anilide is first hydrolysed enzymatically to an aniline and then the quantity of the aniline produced is estimated spectrophotometrically preferably colorimetrically. The hydrolysis of the anilide may be catalysed 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 analysed, for example, by conversion to an indamine, an indophenol or an indoaniline, followed by colorimetric analysis of the coloured quinone-type compound produced. This conversion may take place in the presence of an oxidising agent, such as a copper (11) 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 oxidising 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 Michael Hammond
- 👤 Christopher Philip Price
- 👤 Michael Denis Scawen
- 👤 Anthony Atkinson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 05:56
CPC Classifications
C; C12; C12N; C12N1/00; C12N1/20; C12N1/205; C12N9/00; C12N9/14; C12N9/78; C12N9/80; 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: 44
Claim 1
Independent Claim
37 words
A method for the estimation of an anilide (S-acylated primary aromatic amine) comprising: (a) hydrolysing the anilide to an aniline, and (b) estimating the quantity of said aniline spectrophotometrically, characterised in that the anilide is hydrolysed enzymatically.
Claim 2
Independent Claim
37 words
A method for the estimation of an anilide (S-acylated primary aromatic amine) comprising: (a) hydrolysing the anilide to an aniline, and (b) estimating the quantity of said aniline spectrophotometrically, characterised in that the anilide is hydrolysed enzymatically.
Claim 3
Dependent Claim
23 words
A method according to claim 1 characterised in that the anilide is enzymatically hydrolysed by an aryl acylamidase, EC 3.5.1.13 (as hereinbefore defined).
Claim 4
Dependent Claim
51 words
A method according to claim 2 characterised in that the anilide is enzymatically hydrolysed by 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 ATOC.39004. or aryl acylamidase producing mutants or variants thereof.
Claim 5
Independent Claim
42 words
A method according to any preceding claim characterised in that the quantity of said aniline is estimated by: (a) converting the aniline to an indamine, an indophenol or an indoaniline, and (b) estimating the quantity of said indamine, indophenol or indoaniline spectrophotometrically..
Claim 6
Dependent Claim
29 words
A method according to claim 4 characterised in that the conversion of the aniline to the indamine, indophenol or indoaniline is effected in the presence of an oxidising agent.
Claim 7
Dependent Claim
17 words
A method according to claim 5 characterised in that the oxidising agent comprises a copper (II) salt.
Claim 8
Dependent Claim
23 words
A method according to claim 5 characterised in that the oxidising agent comprises an Fe (III), a chlorate, adichromate or a permanganate salt.
Claim 9
Independent Claim
33 words
A method according to any one of claims 4 to 7 characterised in that the conversion of the aniline to the indamine, indophenol or indoaniline is effected in the presence of a base.
Claim 10
Dependent Claim
19 words
A method according to claim 8 characterised in that the base comprises ammonia, preferably an aqueous solution of ammonia.
Claim 11
Dependent Claim
25 words
A method according to claim 8 wherein the base comprises an alkali metal hydroxide, a borate buffer or an alkaline solution of an organic amine.
Claim 12
Independent Claim
35 words
A method according to any one of the claims 4 to 10 characterised in that the conversion of the aniline to the indamine, indophenol or indoaniline is effected by a phenol or a phenolic ether.
Claim 13
Dependent Claim
18 words
A method according to claim 11 characterised in that the phenol or phenolic ether is cresol, preferably ortho-cresol.
Claim 14
Independent Claim
35 words
A method according to any one of claims 4 to 10 characterised in that the conversion of the aniline to the indamine, indophenol or indoaniline is effected by a primary, secondary or tertiary aryl amine.
Claim 15
Dependent Claim
20 words
A method according to claim 13 characterised in that the primary, secondary or tertiary aryl amine is phenylenediamine, preferably meta-phenylenediamine.
Claim 16
Dependent Claim
18 words
A method according to claim 13 characterised in that the primary, secondary or tertiary aryl amine is para-hydroxyaniline.
Claim 17
Independent Claim
17 words
A method according to any preceding claim characterised in that the anilide is in a biological fluid.
Claim 18
Independent Claim
14 words
A method according to any preceding claim characterised in that the anilide is N-acetyl-4-hydroxyaniline.
Claim 19
Independent Claim
17 words
A method according to anyone of claims 1 to 16 characterised in that the anilide is N-acetyl-4-ethoxyaniline.
Claim 20
Independent Claim
15 words
A method according to any preceding claim characterised in that the aniline is estimated colorimetrically.
Claim 21
Dependent Claim
52 words
A diagnostic kit for use in the estimation of an anilide by a method according to claim 1 comprising: (a) an enzyme suitable for the hydrolysis of the anilide to an aniline, and (b) an organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline.
Claim 22
Dependent Claim
21 words
A diagnostic kit according to claim 20 characterised in that the enzyme is an aryl acylamidase, EC 3.5.1.13 (as hereinbefore defined).
Claim 23
Dependent Claim
51 words
A diagnostic kit according to claim 21 characterised in that 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 24
Dependent Claim
40 words
A diagnostic kit according to any one of claims 20 to 22 characterised in that the organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline is a phenol or a phenolic ether.
Claim 25
Dependent Claim
19 words
A diagnostic kit according to claim 23 characterised in that the phenol or phenolic ether is cresol, preferably ortho-cresol.
Claim 26
Dependent Claim
41 words
A diagnostic kit according to any one of claims 20 to 22 characterised in that the organic compound suitable for the conversion of the aniline to an indamine, an indophenol or an indoaniline is a primary, secondary or tertiary aryl amine.
Claim 27
Dependent Claim
21 words
A diagnostic kit according to claim 25 characterised in that the primary, secondary or tertiary aryl amine is phenylenediamine, preferably meta-phenylenediamine.
Claim 28
Dependent Claim
19 words
A diagnostic kit according to claim 25 characterised in that the primary, secondary or tertiary aryl amine is para-hydroxyaniline.
Claim 29
Dependent Claim
17 words
A diagnostic kit according to any one of claims 20 to 27 additionally comprising an oxidising agent.
Claim 30
Dependent Claim
18 words
A diagnostic kit according to claim 28 characterised in that the oxidising agent comprises a copper (II) salt.
Claim 31
Dependent Claim
24 words
A diagnostic kit according to claim 28 characterised in that the oxidising agent comprises an Fe (III), a chlorate, adichromate or a permanganate salt.
Claim 32
Dependent Claim
16 words
A diagnostic kit according to any one of claims 20 to 30 additionally comprising a base.
Claim 33
Dependent Claim
14 words
A diagnostic kit according to claim 31 characterised in that the base comprises ammonia.
Claim 34
Dependent Claim
28 words
A diagnostic kit according to claim 31 characterised in that the base comprises an alkali metal hydroxide, a borate buffer or an alkaline solution of an organic amine.
Claim 35
Dependent Claim
23 words
A diagnostic kit according to any one of claims 20 to 33 characterised in that the enzyme is retained on a solid support.
Claim 36
Dependent Claim
24 words
A diagnostic kit according to any one of claims 20 to 33 characterised in that the enzyme is dissolved in one or more solvents.
Claim 37
Dependent Claim
21 words
A diagnostic kit according to claim 35 characterised in that at least one of the one or more solvents comprises glycerol.
Claim 38
Dependent Claim
23 words
A diagnostic kit according to claim 35 or 36 characterised in that at least one of the one or more solvents comprises water.
Claim 39
Dependent Claim
33 words
A diagnostic kit according to claim 37 characterised in that the one or more solvents comprises, in admixture, between about 10 and 70% (v/v) glycerol and between about 90 and 30% (v/v) water.
Claim 40
Dependent Claim
39 words
A diagnostic kit according to any one of claims 20 to 38 characterised in that the organic compound suitable for converting the aniline to an indamine, an indophenol or anindoaniline is dissolved in one or more solvents, preferably water.
Claim 41
Dependent Claim
26 words
A diagnostic kit according to any one of claims 28 to 39 characterised in that oxidising agent is dissolved in one or more solvents, preferably water.
Claim 42
Dependent Claim
26 words
A diagnostic kit according to any one of claims 31 to 40 characterised in that the base is dissolved in one or more solvents, preferably water.
Claim 43
Dependent Claim
24 words
A diagnostic kit according to claim 41 characterised in that the solution of the base is combined with a solution of the oxidising agent.
Claim 44
Dependent Claim
43 words
A diagnostic kit according to claim 42 characterised in that the solution of the base, the solution of the oxidising agent and the solution of the organic compound suitable for converting the aniline to an indamine, an indophenol or an indoaniline, are combined.
References Cited (Backward Citations)
FR2353855A1
Cited
GB1527346A
Cited
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