Spontaneous aspecific coloration in so-called Trinder reagents which can alter subsequent colorimetric determinations is inhibited by adding compounds of the chelating agent class to the solution. The resultant compositions contain a peroxidase enzyme, a phenylpyrazone derivative, a compound of phenolic or aromatic amine structure and a stabilizer. Preferred stabilizers are ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).

BACKGROUND OF THE INVENTION 
The "Trinder reaction", named after its discoverer, is a reaction for 
detecting hydrogen peroxide evolved in a solution, by producing a colored 
compound. The colored compound which develops can then be determined 
spectrophotometrically. 
Color development by hydrogen peroxide is obtained if a peroxidase enzyme, 
phenylpyrazone derivative and a compound of phenolic or aromatic amine 
structure are present in the solution. It should be noted that the 
expression "Trinder reaction" has a wider meaning herein than the reaction 
originally conceived by Trinder (A., Clin. Biochem., 6, 24, 1969) in which 
a peroxidase, 4-aminoantipyrine and phenol were used. 
The Trinder reaction, when taken in its stated wider sense, is commonly 
used as an indicator reaction in clinical chemistry for determining 
numerous substrates, including uric acid, cholesterol, glucose, 
triglycerides etc. For example: 
reagent uric acid: Uricase.fwdarw.H.sub.2 O.sub.2 .fwdarw.Trinder 
reagent cholesterol: Cholesterol Oxidase.fwdarw.H.sub.2 O.sub.2 
.fwdarw.Trinder 
One of the biggest problems in the use of this reaction is the spontaneous 
aspecific coloration which occurs with time and which is accelerated by 
particular conditions such as storage at temperatures exceeding 
refrigerator temperatures or exposure to light. 
This increase in coloration has been a serious obstacle in the use of 
Trinder reagents after a certain period of time because it reduces the 
photometric linearity range or the wavelength used in determining the 
coloration. 
The availability of a preparation containing the Trinder reaction reagents 
with which the aforesaid drawbacks are absent is therefore of obvious 
importance. 
SUMMARY OF THE INVENTION 
The present invention therefore relates to a new method for inhibiting the 
spontaneous development of coloration in reagents for Trinder reactions, 
characterized by the use of chelating agents as stabilizers. 
The present invention also relates to a composition of the Trinder type for 
detecting hydrogen peroxide evolved in a solution, comprising: 
a) a phenylpyrazone derivative, 
b) a phenol or an aromatic amine, 
c) a peroxide enzyme, 
d) aqueous buffer, and 
e) a polyalkylaminepolyacetic acid or a salt thereof chelating agent as 
stabilizer. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a chelating agent added to the conventional 
components of a Trinder reagent used to detect hydrogen peroxide generated 
in solution. Those conventional components are 
a) a phenylpyrazone derivative, 
b) a phenol or an aromatic amine, 
c) a peroxide enzyme and 
d) aqueous buffer. Additional components may include an antimicrobial 
additive (e.g., sodium azide). 
Exemplary phenylpyrazone derivatives include 4-aminoantipyrine and those 
derivatives listed at col. 9, lines 51-65 of U.S. Pat. No. Re. 29,498 
(incorporated herein by reference). 
Exemplary phenols include phenol itself, 
3,5-dichloro-2-hydroxybenzenesulfonic acid and various other simple and 
complex phenols such as those described or listed in Table I, II and III 
and at col. 9, lines 1-35 of U.S. Pat. No. Re. 29,498 (incorporated herein 
by reference). Suitable compounds having cromatic amine structures 
include: 
1) N,N-dialkylaniline compounds, 
2) N,N-dialkyl-m-toluidine compounds 
3) toluidine and aniline sulphopropyl derivatives, 
4) N-ethyl-N-(3-sulphopropyl)-m-anisidine and 
5) N-ethyl-N-(2-hydroxyethyl)-m-toluidine. 
The peroxidase enzyme (POD) can be derived from a variety of microbial 
sources. 
The aqueous buffer can be phosphate buffer establishing a neutral pH at 
which POD activity is high (e.g., pH 7.3) or any of various organic 
buffers establishing a similar pH. Additives such as antimicrobials can 
also be used, particularly with phosphate buffer. 
A chelating agent is a compound containing donor atoms which can bond via 
coordination bonds to a single metal ion to form a cyclic structure known 
as a chelate complex or more simply a chelate (see for example Malatesta, 
"Chimica generale", 1980, 458). 
The descriptive term "chelate" derives in fact from the word "chela" (claw 
of a crustacean) and symbolizes the ring which forms between the chelating 
agent and the metal ion. 
The importance of the chelation phenomenon derives from the almost 
universal presence of metal ions of one species rather than another. 
Chelating agents therefore provide a means for checking the presence of 
metal ions by the formation of complexes which generally have properties 
which are markedly different from those of either the ions or the 
chelating agents themselves. 
It is this property which makes them so useful in applications in various 
fields. 
For example their ability to sequester metal ions is used for controlling 
the hardness of water in industrial water treatment, for recovering 
minerals from crude metals, for eliminating radioaction contaminants, for 
various syntheses, for metal passage from one phase to another in 
extraction and precipitation processes, etc. 
Although chelating agents are substances commonly used in various fields, 
including the chemical laboratory, it has only now been discovered that 
the use of such substances particularly in concentrations of between 0.005 
mM and 50 mm and preferably between 1 mM and 10 mm results in a Trinder 
reagent which does not develop the aforesaid undesired spontaneous 
aspecific coloration. 
By way of example, a description is given hereinafter of certain tested 
formulations which have given results enabling this important property of 
chelating agents of stabilizing Trinder reagents to be recognized. 
With regard to the chelating agents used, particularly significant results 
were obtained using ethylenediaminetetraacetic acid (EDTA) and 
diethylenetriaminepentaacetic acid (DTPA) or their salts. 
EDTA and DTPA are representative of the preferred class of chelating agents 
used as stabilizers: polyalkylaminepolyacetic acids and their salts. Other 
members of this preferred class have multiple amines linked by one or more 
alkylene moietics terminated by multiple acetic acid moieties which may be 
present in free acid or salt forms other known classes of chelating agents 
may also be useful, based upon corresponding effects on ions in the 
solution. 
Color development was evaluated by measuring the absorbance of the 
solutions during accelerated stability at 37 deg C. and 45 deg C. and at 
ambient temperature both in the dark and in the light. 
The test was carried out with two different Trinder systems with regard to 
the compound of phenolic or aromatic amine structure. In one case phenol 
was used; and in the other 
N-ethyl-N-(2l-hydroxy-3-sulphopropyl)-m-toluidine (TOOS) was used. The two 
formulations are as follows: