GB2107863A - Colorimetric determination of peroxides - Google Patents
Colorimetric determination of peroxides Download PDFInfo
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- GB2107863A GB2107863A GB08229506A GB8229506A GB2107863A GB 2107863 A GB2107863 A GB 2107863A GB 08229506 A GB08229506 A GB 08229506A GB 8229506 A GB8229506 A GB 8229506A GB 2107863 A GB2107863 A GB 2107863A
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- peroxides
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- 150000002978 peroxides Chemical class 0.000 title claims abstract description 26
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- -1 aromatic aminoacid Chemical class 0.000 claims abstract description 11
- 235000001014 amino acid Nutrition 0.000 claims abstract description 9
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 5
- JJSHYECKYLDYAR-UHFFFAOYSA-N thozalinone Chemical compound O1C(N(C)C)=NC(=O)C1C1=CC=CC=C1 JJSHYECKYLDYAR-UHFFFAOYSA-N 0.000 claims abstract description 4
- RMMXTBMQSGEXHJ-UHFFFAOYSA-N Aminophenazone Chemical compound O=C1C(N(C)C)=C(C)N(C)N1C1=CC=CC=C1 RMMXTBMQSGEXHJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229960000212 aminophenazone Drugs 0.000 claims abstract description 3
- 150000001768 cations Chemical class 0.000 claims abstract description 3
- 229940120889 dipyrone Drugs 0.000 claims abstract description 3
- DJGAAPFSPWAYTJ-UHFFFAOYSA-M metamizole sodium Chemical compound [Na+].O=C1C(N(CS([O-])(=O)=O)C)=C(C)N(C)N1C1=CC=CC=C1 DJGAAPFSPWAYTJ-UHFFFAOYSA-M 0.000 claims abstract description 3
- ZGUHNWLPPVJTOG-UHFFFAOYSA-N 2-propyl-2h-phenazin-1-one Chemical compound C1=CC=C2N=C(C(C(CCC)C=C3)=O)C3=NC2=C1 ZGUHNWLPPVJTOG-UHFFFAOYSA-N 0.000 claims abstract 2
- 239000000203 mixture Substances 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 150000003839 salts Chemical class 0.000 claims description 14
- 229910001868 water Inorganic materials 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- ILPGQKHPPSSCBS-UHFFFAOYSA-N 2-phenyl-1h-pyrazol-3-one Chemical compound O=C1C=CNN1C1=CC=CC=C1 ILPGQKHPPSSCBS-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 5
- XFDUHJPVQKIXHO-UHFFFAOYSA-N 3-aminobenzoic acid Chemical compound NC1=CC=CC(C(O)=O)=C1 XFDUHJPVQKIXHO-UHFFFAOYSA-N 0.000 claims 1
- 125000001183 hydrocarbyl group Chemical group 0.000 claims 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 21
- 102000004190 Enzymes Human genes 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 6
- 239000013060 biological fluid Substances 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract description 5
- 239000001257 hydrogen Substances 0.000 abstract description 4
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract description 2
- 102000003992 Peroxidases Human genes 0.000 description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 description 9
- 239000000126 substance Substances 0.000 description 7
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 6
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 6
- 150000004982 aromatic amines Chemical class 0.000 description 6
- 229940116269 uric acid Drugs 0.000 description 6
- 239000007800 oxidant agent Substances 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 108010092464 Urate Oxidase Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 159000000000 sodium salts Chemical class 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 150000001491 aromatic compounds Chemical class 0.000 description 2
- 239000008366 buffered solution Substances 0.000 description 2
- GGSUCNLOZRCGPQ-UHFFFAOYSA-N diethylaniline Chemical compound CCN(CC)C1=CC=CC=C1 GGSUCNLOZRCGPQ-UHFFFAOYSA-N 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- MQIMNQOMJJXNDF-UHFFFAOYSA-N (1-amino-1-hydroxypropyl) acetate Chemical compound C(C)(=O)OC(CC)(O)N MQIMNQOMJJXNDF-UHFFFAOYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- ZGCHLAJIRWDGFE-UHFFFAOYSA-N 1-aminopropane-1,1-diol Chemical compound CCC(N)(O)O ZGCHLAJIRWDGFE-UHFFFAOYSA-N 0.000 description 1
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- NEGFNJRAUMCZMY-UHFFFAOYSA-N 3-(dimethylamino)benzoic acid Chemical compound CN(C)C1=CC=CC(C(O)=O)=C1 NEGFNJRAUMCZMY-UHFFFAOYSA-N 0.000 description 1
- HLSJDSZSQGSLQO-UHFFFAOYSA-N 4-(diethylamino)-1,5-dimethyl-2-phenylpyrazol-3-one Chemical compound O=C1C(N(CC)CC)=C(C)N(C)N1C1=CC=CC=C1 HLSJDSZSQGSLQO-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-M 4-hydroxybenzoate Chemical compound OC1=CC=C(C([O-])=O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-M 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- UCTWMZQNUQWSLP-UHFFFAOYSA-N Adrenaline Natural products CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 241000147041 Guaiacum officinale Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229940102884 adrenalin Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000007859 condensation product Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 229940091561 guaiac Drugs 0.000 description 1
- 150000002391 heterocyclic compounds Chemical class 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- YQYUUNRAPYPAPC-UHFFFAOYSA-N n,n-diethyl-2-methylaniline Chemical compound CCN(CC)C1=CC=CC=C1C YQYUUNRAPYPAPC-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229960005382 phenolphthalein Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/96—4-Amino-antipyrine
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- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
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- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A reagent for use in the colorimetric determination of peroxides, more particularly the determination of hydrogen peroxide set free, from substrates present in biological fluids, by specific enzymes comprises (a) a component having at least one reactive group bonded to a structure derived from 1-phenyl-5- pyrazolinone and (b) an aromatic aminoacid having the formula: <IMAGE> wherein R and R1, which are the same or different, are hydrogen or a hydrocarbonaceous radical and X is hydrogen or amonovalent cation. Component (a) may be 4- aminoantipyrine, aminophenazone, sulphamylpyrine, dibupyrene, propylphenazone, thozalinone, methampyrone. <IMAGE>
Description
SPECIFICATION
Colorimetric determination of peroxides
This invention relates to a system of reagents for use in the determination of peroxides, for example those generated by enzymic and/or chemical reactions.
It is known that many reactions can be carried out in the presence of enzymes in order to achieve a higher reaction velocity and, in a few cases, to obtain a possibility of closer control. Many of the enzymic reactions themselves involve an interaction between the substrate and oxygen. One of the products is hydrogen peroxide, in an amount which is proportional to the amount of starting material.
There is, on the other hand, a wide range of reactions which are purely chemical reactions and which give, as an end product, a peroxide in an amount which is again proportional to the amount of starting material.
There is, on the other hand, a wide range of reactions which are purely chemical reactions and which give, as an end product, a peroxide in an amount which is again proportional to the amount of starting material.
In addition, it is known that hydrogen peroxide and peroxides in general can be used as substrates in reactions in which particular enzymes, such as peroxidase (POD) and catalase, are used. Such enzymes catalyse the oxidation of an appropriate substance at the expense of the peroxide, which is reduced (to water in the particular case of hydrogen peroxide). The oxidised substance is produced in an amount which is proportional to the amount of peroxide which is present and thus to the amount of the substrate which reacted to produce the peroxide.
A number of reagents have been suggested for the colorimetric determination of hydrogen peroxide, according to the following scheme:
Reduced acceptor (colourless)+H2O2 (POD)
Oxidized acceptor (coloured)+H2O.
As the reduced acceptor, various substances have been employed, for example o-dianisidine, otolidine, o-phenylenediamine, guaiac, adrenalin, phenolphthalein, ABTS (the ammonium salt of 2,2' azino-di-(3-ethylbenzothiazoline-6-sulphonic acid), and ferrocyanide.
From 1 937 to 1943, Eisenstaedt (and subsequently Emerson) showed that 4-aminoantipyrine, in the presence of various aromatic and heterocyclic compounds, resulted in the formation of coloured reaction products by the action of oxidizing agents such as ferrocyanide. These results were reexamined by Trinder, who provided a colorimetric technique for measuring glucose in biological fluids, according to the following scheme:: Glucose+H20+O2 Glucose
Oxidase
Gluconic acid+H202 H202+4-aminoantipyridine+phenol POD
quinone (coloured)+H2O Among the aromatic compounds capable of condensing with 4-aminoantipyrine in the presence of oxidising agents, Eisenstaedt indicated in 1 938 that aromatic amines were suitable for this purpose.
In the light of the above, many authors have prepared chromogens in which there are used, together with 4-aminoantipyrine, aromatic amines which contain substituents in place of the hydrogen atom or atoms (e.g., N,N-dimethylaniline and N,N-diethylaniline) and amines which are substituted on the aromatic ring (e.g. N,N-diethyltoluidine and N-ethyl-N-ethylacetamide-m-toluidine).
According to the present invention, there is provided a chromogenic composition adapted to the determination by colorimetric methods of peroxides, comprising (a) a compound containing at least one reactive group bonded to a structure derived from 1 -phenyl-pyrazolin-5-one, and (b) an aromatic aminoacid of the formula:
wherein R and R1, which are the same or different, are each hydrogen or hydrocarbonaceous radical, and X is hydrogen or a monovalent cation.
We have found, in accordance with the present invention, that a number of aromatic aminoacids (or their salts) are capable of condensing with 4-aminoantipyrine or the like compounds in the presence of oxidising agents to give coloured products. Such aminoacids and their salts, in comparison to the aromatic amines mentioned above, have a number of advantages. Firstly, the coloured condensation products which are obtained have molar extinction coefficients higher than the coloured products which are obtained with the corresponding aromatic amines. Secondly, the aromatic aminoacids and their salts are solid, so that they can be included in solid mixtures, in contrast to the corresponding aromatic amines which are liquid. Thereby, the aromatic aminoacids and their salts are not toxic, contrary to the corresponding aromatic amines.
In our Patent No. 1,564,467, there is claimed a dry composition which, when dissolved in water, forms a reagent solution suitable for use in the determination of peroxides, which composition comprises (a) a 4-hydroxybenzoate, and (b) a compound containing at least one reactive group bound to a structure derived from 1 -phenyl-pyrazolin-5-one. In the determination of the hydrogen peroxide which is set free enzymatically in biological fluids, this composition permits satisfactory results to be obtained. However, in many cases, it is essential for the chromogen to have a greater sensitivity, for example when determining the hydrogen peroxide which is set free enzymically by substances and metabolites which are present in very small concentrations in biological fluids.The chromogenic system of the present invention, which may have a sensitivity about four times more than that of
Patent No. 1,564,467, makes it possible to use small samples of biological fluids (while the dosage of the substance concerned remains sufficiently accurate and reliable), with the attendant advantage of reducing in the reaction mixture the concentration of potentially disturbing substances which may be simultaneously present in the sample being tested.
The chromogenic composition of the present invention is adapted to the determination of peroxides, more particularly to the determination of hydrogen peroxide which is set free enzymically from substrates which are contained in biological fluids.
Preferred components (b) of the composition are o-aminobenzoic acid or a salt thereof, maminobenzoic acid or a salt thereof, N-alkyl-m-aminobenzoic acids or salts thereof, and N,N-dialkyl-maminobenzoic acids or salts thereof, the m-substituted derivatives being most preferred. Among the latter, the most prominent efficiency has been exhibited by acids or salts which are wholly substituted on the nitrogen atom.
Examples of component (a) of the composition, in addition to 4-aminoantipyrine, are:
(A) aminophenazone (i.e. 4-diethylamino-2,3-dimethyl-1 -phenyl-3-pyrazolin-5-one); (B) sulphamipirine or sulphamylpyrine (i.e. the sodium salt of 2,3-dimethyl-1 -phenyl-5-pyrazolone4-aminomethanesulphonic acid;
(C) dibupyrene (i.e. the sodium salt of (1-phenyl-2,3-dimethyl-5-pyrazolon-4-yl) isobutylamino methanesulphonate);
(D) propylephenazone (i.e. 4-isopropyl-2,3-dimethyl-1 -phenyl-3-pyrazolin-3-one); (E) thozalinone (i.e. 2-dimethylamino-5-phenyl-2-oxazoline-4-one);
(F) methampyrone (i.e. the sodium salt of 1-phenyl-2,3-dimethyl-5-pyrazolone-4methylaminomethane sulphonate).
The following reaction illustrates the oxidation of the chromogens described herein, hydrogen peroxide being used in the presence of peroxidase as the oxidising agent for the chromogen (although other peroxides and appropriate oxidising agents are suitable for the purpose):
The invention will now be illustrated by the following Examples.
Example 1
Determination of hydrogen peroxide and other peroxides
The principle of the determination is as follows. A solution which contains a peroxide in an appropriate concentration is admixed with a buffered solution which contains one of the chromogens described herein and a peroxide. When the colour is completely evolved, a photometric reading is taken at the appropriate wavelength. The colour which is evolved is proportional to the concentration of peroxide.
The reagent can be formulated as a powder reagent containing (a) a buffer system which can
consist of phosphates, TRIS, aminopropanediol acetate and other derivatives or another equivalent, (b) an antiseptic and mould killing agent, (c) a chromogenic system consisting of 4-aminoantipyrine or the
like, and an aromatic aminoacid, and (d) peroxidase.
Example 2
Determination of uric acid manually or automatically (with a continuous flow apparatus or by the use of separate samples), by taking an end reading.
The principle of the determination is as follows. The material to be tested is mixed, manually or automatically, with the reagent, which contains enzymes (uricase and peroxidase) and the chromogen, the whole being in a buffered solution. After the uric acid has been oxidised by the enzyme and the colour of the reaction has developed, a photometric reading is taken, either manually or automatically, at an appropriate wavelength. The evolved colour is proportional to the quantity of uric acid which is present in the sample.
For use in such a reaction, the formulation for the reagent can be as follows. In a single mixture there are present all of the substances which are necessary for the enzymic and chromogenic reactions, i.e. the enzymes uricase and POD, a buffer (e.g. phosphate, TRIS, acetate, aminopropanediol and derivatives and others), the aromatic aminoacid, 4-aminoantipyrine (or the like compound), an antibacterial agent, a capillary-active agent and a compound adapted to avoid potential interference.
Example 3
Results obtained in the manual determination of uric acid in human blood sera, using a particular
reagent composition
The reagents and concentrations thereof in a reagent system ready for use were: phosphate
buffer (100 mM, pH 7.4), 4-aminoantipyrine (0.05 mM), N,N-dimethyl-m-aminobenzoic acid (1 mM),
peroxidase (2000 U/litre), and uricase (50 u/litre). The results obtained using the reagent system were as follows.
Linearity The correlation between A at 550 nm and the concentration of uric acid was linear up to a
concentration of 20 mg/dl of uric acid in the sample.
Comparison
A comparison with the method by Fossati, P., Prencipe, L. (Quad. Sclavo Diagn. 14, 2, 1978) gave the following results (y=Fossati method): y=1 .001, x=0.036 mg/dl: r=0.985.
Accuracy in the "series"
No. 20, average 4.6 mg/dl SD 0.08, mg/dl C.V.%=1.84.
Accuracy "among series"
No. 20, average 4.7 mg/dl, S.D. 0.12 mg/dl C.V.%=2.84.
Claims (11)
1. A composition suitable for use in the chromogenic determination of peroxides, which composition comprises (a) a compound containing at least one reactive group bound to a structure derived from 1 -phenylpyrazolin-5-one, and (b) an aromatic aminoacid or salt having the general formula:
wherein R and R1, which are the same or different, are each a hydrogen atom or a hydrocarbyl group, and X is a hydrogen atom or a monovalent cation.
2. A composition as claimed in claim 1, wherein component (b) is o-aminobenzoic acid or a salt thereof, m-aminobenzoic acid or a salt thereof, an N-alkyi-m-aminobenzoic acid or a salt thereof, or an
N,N-dialkyl-m-aminobenzoic acid or a salt thereof.
3. A composition as claimed in claim 1 or 2, further comprising a chromogenic agent.
4. A composition as claimed in claim 3, wherein the chromogenic agent is a ferrocyanide.
5. A composition as claimed in any of claims 1 to 4, wherein component (a) is 4-aminoantipyrine, aminophenazone, sulphamylpyrine, dibupyrene, propylphenazone, thozalinone or methampyrone.
6. A composition as claimed in any of claims 1 to 5, in the form of a reagent solution suitable for use in the chromogenic determination of peroxides.
7. A composition as claimed in any of claims 1 to 5, in the form of a dry composition which when dissolved in water forms a reagent solution suitable for use in the chromogenic determination of peroxides.
8. A reagent solution for use in the chromogenic determination of peroxides, formed by dissolving in water a composition as claimed in claim 7.
9. A method for the determination of a peroxide, which comprises (1) reacting the peroxide with a reagent solution as claimed in claim 6 or 8, either directly (when the solution contains a chromogenic agent) or after a preliminary reaction of the peroxide with a chromogenic agent (when the solution does not contain a chromogenic agent), and (2) determining the resulting intensity of colour which is proportional to the original peroxide content.
10. A composition as claimed in claim 1, substantially as described in the Examples.
11. A method as claimed in claim 9, substantially as described in the Examples.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT24634/81A IT1168043B (en) | 1981-10-22 | 1981-10-22 | CHROMOGEN FOR THE COLORIMETRIC DETERMINATION OF PEROXIDES AND METHOD USING THE SAME |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| GB2107863A true GB2107863A (en) | 1983-05-05 |
Family
ID=11214225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08229506A Withdrawn GB2107863A (en) | 1981-10-22 | 1982-10-15 | Colorimetric determination of peroxides |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS5880556A (en) |
| DE (1) | DE3238339A1 (en) |
| ES (1) | ES517130A0 (en) |
| FR (1) | FR2515356A1 (en) |
| GB (1) | GB2107863A (en) |
| IT (1) | IT1168043B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7276379B2 (en) | 2001-05-29 | 2007-10-02 | Nihon Trim Co., Ltd. | Detection method and quantitative analysis method for hydrogen radical |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3720506A1 (en) * | 1987-06-20 | 1988-12-29 | Draegerwerk Ag | METHOD FOR DETECTING GASEOUS SUBSTANCES BY AN ENZYMATIC REDOX REACTION |
| JP6266574B2 (en) | 2015-09-10 | 2018-01-24 | 株式会社日立ハイテクサイエンス | X-ray inspection method and X-ray inspection apparatus |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL7801413A (en) * | 1977-03-09 | 1978-09-12 | Hoffmann La Roche | PEROXYDASE DETERMINATION. |
| JPS5425892A (en) * | 1977-07-29 | 1979-02-27 | Wako Pure Chem Ind Ltd | Quantitative determination of hydrogen peroxide |
| JPS5520471A (en) * | 1978-08-01 | 1980-02-13 | Kyowa Hakko Kogyo Co Ltd | Hydrogen peroxide quantifying method |
| US4247631A (en) * | 1979-01-31 | 1981-01-27 | Millipore Corporation | Reagent and method for the analytic determination of hydrogen peroxide |
| DE3032421A1 (en) * | 1980-08-28 | 1982-04-01 | Behringwerke Ag, 3550 Marburg | Peroxide determn. e.g. in glucose or uric acid enzymatic analysis - using chromogen consisting of 5-hydroxy- or 5-amino-1H-pyrazole and aniline or 2-amino-pyridine, -pyrimidine or -triazine |
-
1981
- 1981-10-22 IT IT24634/81A patent/IT1168043B/en active
-
1982
- 1982-10-15 GB GB08229506A patent/GB2107863A/en not_active Withdrawn
- 1982-10-15 DE DE19823238339 patent/DE3238339A1/en not_active Ceased
- 1982-10-21 FR FR8217652A patent/FR2515356A1/en active Pending
- 1982-10-22 JP JP57185890A patent/JPS5880556A/en active Pending
- 1982-10-22 ES ES517130A patent/ES517130A0/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7276379B2 (en) | 2001-05-29 | 2007-10-02 | Nihon Trim Co., Ltd. | Detection method and quantitative analysis method for hydrogen radical |
Also Published As
| Publication number | Publication date |
|---|---|
| ES8401255A1 (en) | 1983-12-01 |
| JPS5880556A (en) | 1983-05-14 |
| DE3238339A1 (en) | 1983-05-11 |
| FR2515356A1 (en) | 1983-04-29 |
| IT1168043B (en) | 1987-05-20 |
| IT8124634A0 (en) | 1981-10-22 |
| ES517130A0 (en) | 1983-12-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |