CA1155737A

CA1155737A - Process and diagnostic agents for the detection of redox reactions

Info

Publication number: CA1155737A
Application number: CA000372869A
Authority: CA
Inventors: Laszlo Koever; Walter Rittersdorf; Wolfgang Werner
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1980-03-29
Filing date: 1981-03-12
Publication date: 1983-10-25
Also published as: EP0037056B1; DE3012368C2; ZA812059B; JPS56151358A; MY8600707A; AU6864981A; HK82686A; US4957872A; JPH024861B2; DD157834A5; EP0037056A1; DE3160405D1; SG38686G; DE3012368A1; ATE3726T1; US4743559A; AU538332B2; CS231174B2; CS227181A2

Abstract

ABSTRACT

The present invention provides a process for the detection of redox reactions by introducing a redox reagent system into a test system, wherein a soluble iodate is additionally added to the test system in an amount which is in excess of the highest amount of disturbing reducing agents present in the test system.
The present invention also provides a diagnostic agent for the detection of redox reactions containing a redox reagent system, wherein the test system used additionally contains an iodate which is soluble therein in an amount which is in excess of the highest amount of disturbing reducing agents present in the test system.

Description

The present invention is concerned with a process and a diagnostic agent based upon redox reactions, and more especially with the use of an iodate for the avoidance of disturbances caused by reducing agents and especially by ascorbic acid.
In clinical and pharmaceutical chemistry, in biochemistry and in food stuff chemistry, redox systems are of great importance for determination methods for substrates and enzymes. There is a very large variety of photometric processes for such methods of determin-ation. However, the so-called rapid diagnostics are of especial importance, these being agents which contain all the reagents in dry form in absorbent carriers or in films. The agents are brought into contact with the liquids to be investigated and the resulting colours can be assessed visually or with a reflection photometer.
The materials to be investigated in the above-mentioned fields of chemical analy~is, for example, urine, blood, foodstuffs, pharmaceutical compositions and the like, frequently contain more or less large amounts of reducing agents, the commonest of which is ascorbic acid. It is clear that redox reactions can be considerably disturbed by strong reducing agents, such as ascorbic acid. Thus, it is known that, in the case of the detection of glucose with rapid diagnostics based upon the reaction of GOD-POD-redox indicators, falsely negative results can be brought about by ascorbic acid. The hydrogen peroxide result-ing from glucose with the help of GOD (glucose oxida~e), reacts with POD (peroxidase) on the ascorbic acid instead of on the indicator, with oxidation, and thus is removed from the determination.
Furthermore, it i8 known that rapid diagnostics for the detection of blood in urine al-~o give falsely negative results in the presence of a~corbic acid, the aacorbic acid apparently reducing the coloured material formed by oxidation catalysed by haemoglobin.
As an example of the falsely positive findings due to ascorbic acid, mention may be made of the determination of NADH or NADPH with the help of the reduction of tetrazolium salts to give coloured formazanes. Ascorbic acid here acts in the same manner and increase~ the measurement signal.
- Because of the special importance and the extent of the di~turbances due to reducing agents and e~pec-ially to ascorbic acid, attempts have been made to remove them from the fluids to be investigated or to develop processes and agents which are not disturbed by them. ~hus, for example, the following processes are known:
oxidation with iodine solution and removal of excess iodine with thiosulphate, oxidation with manganese dioxide and filtering off of the unused oxidation agent, .
. .

oxidation with alkaline hydrogen peroxide; treatment of the test solution with anion exchangers.
All these processes necessitate a laborious treatment of the sample solution. Furthermore, especially in the case of rapid diagnostic agents, an integrated solution to the problem is very laborious. Test papers are also known in which urine must first be chromato-graphed through a zone containing an anion exchanger (see Federal Republic of Germany Patent ~o. 15 98 008, Dahlquist, published April 13, 1972), in order then, upon running further, to be able to react without disturbance in the actual reagent zone. Tests with such ion exchanger zones are commercially available for glucose and galactose. However, they have a complicated construction and, due to the necessary chromatographing time, the analysis time is considerably increased in com-parison with conventional rapid tests.
Another possibility for reving ascorbic acid from liquids or for overcoming the disturbance of dia-gnostic agents depends upon the addition of ascorbateoxidase in optical tests and rapid diagnostics (see Federal Republic of Germany Patent ~o. 26 25 834, Danninger et al, published December 15, 1977).
Although this process is especially useful in the case of small ascorbic acid concentrations, the problem as a whole cannot be regarded as having been solved for the following reasons:

1 1557~7 -- 4 _ ascorbate oxidase only reacts with ascorbic acid itself but not with metabolites thereof, such as the glucuronide and the sulphate, or with other reducing agents;
the oxidation of ascorbic acid by ascorbate oxidase is relatively slow so that in the case of test fluids which can contain more than 100 mg. ascorbic acid/dl., uneconomi-cally large amounts of ascorbate oxidase must be used in order to ensure the overcoming of the disturbance to a reasonable extent, in certain cases, the enzyme ascorbate oxidase is destroyed relatively quickly by aggressive reagents. Thus, for example, the cumol hydroperoxide used in a urine blood test must be enclosed in microcapsules (see U.S. Patent ~o. 4,129,417, White, issued October 28, 1977). i Surprisingly, we have now found that a process and a diagnostic agent, especially a rapid diagnostic~
can be obtained which are not disturbed by ascorbic acid and its metabolites, even when these are present in relatively large amounts, when iodate is additionally added to known formulations or to the test system to be investigated.
Thus, according to one aspect of the present invention, there is provided a process for the detection of redox reactions by introducing a redox reagent system into a test system, wherein a soluble iodate is additionally added to the test system in an amcunt which is in excess of the highest amount of disturbing reducing agents present in the te~t system.
It is surprising that iodate oxidi~es the a~corbic acid sufficiently quickly but not the ~ubstrate~
important in clinical chemistry nor, furthermore, many of the redox indicators conventionally used in analy-qi 9 and their corresponding coloured reaction product~, as well as conventional adjuvants. Thus, for example, under the usual analysis conditions (pH 5 - 9) and within the u~ual analy~is times, the following are not attacked by iodate:
substrates: carbohydrates (glucose, galacto~e and the like), cholesterol, glycerol (from triglycerides), uric acid, NAD~, NADP~, etc.

~ub-qtrate glucose oxidase, galactose oxidase, oxida~es:
cholesterol oxida~e, glycerol oxidase, uricase, etc.
indicators: benzidine derivatives (o-tolidine, 3,3',5,5'-tetramethylbenzidine), hetero-cyclic azine-~ (azino-bis-benzothiazolone--~ulphonic acid), formazanes obtained as reduction products of tetrazolium salts, etc.
peroxidases horse radish peroxidase, haemoglobin ~blood) adjuvants: aryl semicarbazides, which have been described as stabilisers for oxidation indicators (see Federal Republic of Germany Offenlegungsschrift (Publis~ed Patent Specification) No. 27 16 060, Rittersdorf et al, published October 28, 1977).
That this property of iodates is surprising and could not be deduced, for example, from the oxidation potential is shown by a comparison with other halogen compounds, the standard potentials of which are, according to Cotton-Wilkinson, ~Anorganische Chemie", pub~ Weinheim, 1970, 2nd edn., page 532, as follows:
iodate +0.26 V
periodate +0.39 V
iodine +0.54 V
bromate +0.61 V
chlorate +0.63 V
Whereas only periodate and free iodine, besides ascorbic acid, oxidise most indicators and especially the benzidine derivatives, bromate and chlorate, in spite of their higher oxidation potentials, are not able to oxidise ascorbic acid and thus are completely useless for the desired purpose. Furthermore, as far as iodates are con-cerned, it was previously assumed that they only oxidised ascorbic acid in acetic acid solution (see R. Indovina and D. Elia, Boll. Soc., Ital. Biol. sperm., 20, 390-393/1945;
Chem. Abs., 40, 6110 ~946).
According to a further aspect of the present invention, there is provided a diagnostic agent for llSS737 the detection of redox reactions containing a redox reagent system, wherein the test system used additionally contains an iodate which is soluble therein in an amount which is in excess of the highest amount of disturbing reducing agents present in the test system.
The diagnostic agent may conveniently be in the form of a device in which the redox reagent system and the iodate are supported by an inert carrier. For example, an absorbent strip impregnated with the redox reagent system and the iodate or a film incorporating the redox reagent system and the iodate, and which swells or dissolves in the test system to release the reagent system and iodate.
The employment of iodate in excess of the highest amount of disturbing reducing agents present in the test system provides an amount of iodate in excess of that required to oxidize the disturbing reducing agents.
The rapid diagnostic agents according to the pre-sent invention are practically not destroyed by ascorbic acid and are simple to produce by admixing an iodate with the formulations of known tests. Such known tests include, for example, the following:
test papers for the detection of blood in urine with organic hydroperoxides and o-tolidine (see Federal Republic of Germany AuSlege~chriffen Nos. 22 35 152, Rittersdorf et al, published January 10, 1974, and 12 42 905, Adams et al, published June 22, 1967, and Offenlegungsschrift 26 40 211, Adams et al, published June 22, 1967, and tetramethyl-benzidine (see Federal Republic of Germany Offenlegung--- 8 --sschriften Nos. 24 60 903, G~thlein et al, published June 24, 1976, and 27 16 060, Rittersdorf et al, published October 12, 197~, test papers for the detection of glucose in urine with GOD, POD and o-tolidine (see Federal Republic of Germany Auslegeschrift No. 24 15 257, Tiedemann et al, published July 31, 1975, and German Patent No. 11 21 847, Free et al, published January 11, 1976, and Austrian Patent Specification No. 19 88 96, Keston et al, issued July 25, 1958,) 3,3',5,5'-tetramethybenzidine (see Federal Republic of Germany Offenlegungsschrift No. 24 60 903, GMthlein et al, published June 24, 1976), substituted aminocarbazoles (see Federal Republic of Germany Offen-legungsschriften Nos. 22 05 733, Werner et al, published September 20, 1973, and 23 38 932 Werner et al, published February 13, 1975, and heterocyclic azines (see Federal Republic of Germany Auslegeschrift No. 16 48 840, Rey et al, published February 12, 1970, test papers for the detection of galactose in urine with galactose oxidase, POD and o-tolidine (See U.S. Patent No. 3,362,886, Chauncey Orvis Rupe, issued January 9, 1968~, test papers of diverse substrates with specific oxidases, POD and o-tolidine (see U.S. Patent No. 3,099,605, Free et al, issued July 30, 1963), 1 15~737 test films for the determination of glucose in blood with GOD, POD, o-tolidine (see Federal Republic of Germany Auslegeschrift ~o. 15 98 153, Rey et al, published May 3, 1973, and 3,3',5,5'-tetramethylbenzidine (see Federal Republic of Germany Offenlegungsschrift No.
24 60 903, Guthlein et al, published June 24, 1976 and test papers for the determination of NADH or NADH-forming sub~trates or enzymes with tetrazolium salts and diaphorase (see Federal Republic of Germany Offenlegungsschrift No.
24 52 283, Forgione et al, published, May 15, 1975.
Since most of the above-mentioned tests are carried out in aqueous solutions, water-soluble iodates are advantageously used. Practically all water-soluble salts of iodic acid with inorganic and organic cations can be used provided that they do no disturb the analytical process. These are, in particular, the alkali metal salts, which are easily obtainable and some of which are commercially available, as well as the alkaline earth metal salts, ammonium salts and the salts with simple amines, for example, piperidine, piperazine and the like.
Only in special cases are certain modifications of the conventional formulations necessary:
In the case of the large-scale production of test papers, amongst other things, relatively long impregnation times are necessary. In the course of these long impregnation times, under certain circumstances, a partial oxidation of the indicator can occur in the impregnation solution.
This is, for example, the case with the substituted amino-carbazoles used according to Federal Republic of GermanyOffenlegungsschrift No. 22 05 733, Werner et al, published, February 13, 1975.
In these cases, it is preferable to bring about a certain spatial separation of the reagents by first impregnating the test paper with all the other reagents and there-after post-impregnating with an appropriate iodate from an organic solvent which does not dissolve out the other components of the formulation. Examples of organo-soluble iodates include quaternary ammonium iodates, aswell as salts of iodic acid with comparatively long-chained amines.
The redox reagent and the iodate may be added separately or simultaneously to the test system.
In cases where, due to the use of iodate, stability pro -blems occur in the tests, use can be made of generally known measures for improving stability, such as a succes-sive impregnation from different solvents, possibly with the addition of appropriate separating agents, for example, polymers.
The use of iodates in rapid diagnostics is only expedient within the following pH limits:
At pH values below about 4.5, in the case of the reduction of iodate by ascorbic acid, free iodine is formed in increasing amounts which, as already stated, oxidises many indicators. Furthermore, in acidic media, iodate possesses an oxidation potential of +1.20 V (Cotton-Wilkinson, _. supra) and is, therefore, no longer com-patible with most indicators and other ~ormulationcomponents, Above pH 7 - 8, the oxidation of ascorbic acid by iodate becomes increasingly slower so that, for an effective removal of disturbances, excessively large amounts of iodate are necessary, which can give rise to difficulties in work-ing up and possibly to storage problems.
The redox reagent system is conveniently one that is effective to increase the pH value of the test system, particularly from 5 to 6 to 7 to 9.
The iodates are preferably used in 2 to 20 fold molar excess, referred to the amount of ascorbic acid pre-sent in the fluids to be investigated. Since the oxidation of the ascorbic acid must have taken place practically before the actual detection reaction, for detection reactions which proceed quickly, a larger excess of iodate is necessary than for slower reactions.
Since, as already mentioned above, the oxidation of ascorbic acid slows down at a high pH, in these cases, too, a larger excess of iodate must be used. In any case, the correct amount of iodate is easy to determine by a simple series of experiments.
In comparison with known disturbance-free or low disturbance rapid diagnostics, the rapid diagnostics according to the present invention have the following advantages:

7 ~ 7 the use thereof is precisely the same as that of the previously used rapid diagnostics, some of which havæ
long been known, the production thereof is simple and is substantially the same as the conventional methods of production;
in comparison with rapid diagnostics which contain ascorbate oxidase, in some cases they bring about a higher degree of removal of disturbances and, in particular, they are much cheaper to produce.

The use of iodate is, in itself, not limited to an incorporation into rapid diagnostics. ThuY, for example, iodate can be added directly to the solution to be investigated and the solution thereby freed from disturbing reaction agents can be further investigated in known manner photometrically or with conventional rapid tests. However, it is to be pointed out that, in such a solution, tests, the substrates or reagents of which react with iodate, can no longer be carried out. Thus, for example, in the case of investigating urine with multi-tests, there are tests for nitrite and bile colouring materials (urobilinogen and bilirubin).
These materials are oxidised by iodates in the acidic media of the test paper even before the detection thereof. Furthermore, the aromatic amines employed for conventional nitrite tests are oxidised to give strongly coloured compounds.
Whether or not a particular test is disturbed by the addition of iodate can easily be ascertained by comparing standards with and without the addition of iodate.
~ n certain cases, for example in the case of analysis processes which are carried out at pH 5 to 6, where ascorbic acid is oxidised very quickly by iodate, it can be advantageous to add the iodate to the reagent composition or to parts thereof and thus substantially to simplify the course of the analysis. Thus, for example, in the case of the analysis of seru~, the iodate is added to the conventional, weakly acidic deproteinisation agent, for example uranyl acetate, a substantially undisturbed test solution thereby being obtained.
The diagnostic agent according to the present invention preferably contains 0.5 to 2 g. of iodate per 100 ml. of test system and preferably also has a pH value such that, together with the test system, a pH value of 5 to 9 is obtained.
A large variety of redox reagent systems can be present in the diagnostic agent according to the present invention, one preferred redox reagent system com-prising an oxidation indicator, a hydroperoxide, a peroxidase and conventional adjuvants and another preferred redox reagent system comprising a reduction indicator, a reducing agent and optionally an electron carrier.

~ 155737 The diagnostic agents are preferably rapid tests in which the reagent system is impregnated into an absorbent carrier which is insoluble in the test system or is incorporated into a film which swells in the test system, which film may be fixed on to a solid carrier, for example on to a synthetic resin film. The reaction is then, after moistening with the test system, deter-mined on the basis of the coloration which appears.
~However, the diagnostic agents can also be soluble in the test system and be present, for example, as a solution, lyophilisate or reagent tablet or can be incorporated into a test film which is soluble in the test system. The reagents are then mixed with the test system and possibly with further solvents and the reaction i8 determined photometrically in a cuvette.
By test systems, there are to be understood the samples to be investigated, possibly with the addition of appropriate solvents. By the reagent system, there is to be understood the totality of the reacting materials and all other adjuvants, such as buffer, wetting agent, viscosity-regulating materials, stabilising agents and contrast colouring agents, as well as possibly solvents and the li~e.
The following Examples are given for the purpose of illustrating the present invention:
Example 1.

Test pa~er for the deteclion of blood (erYthrocvtes) in urine.

, 115~737 Filter paper (Schleicher & Schull No.23 SL~ is successively impregnated with the following solutions and, after each impregnation, dried at 40 C.:
Solution 1.
1.2 molar citrate buffer, pH 5.25 35.0 ml ethylenediamine-tetraacetic acid, 0 1 disodium salt g-dioctyl sodium sulphosuccinate 0.5 g.

2,5-dimethylhexane-2,5-dihydro-1.6 g.
~peroxide (about 70O/o) phosphoric acid trimorpholide 12.7 g.
sodium iodate 0.5 g.
ethanol 30.0 ml distilled water ad 100.0 ml Solution 2.

3,3',5,5'-tetramethylbenzidine 0.3 g.
phenanthridine 0.2 g.
l-phenylsemicarbazide 0.02 g toluene/methanol (60:40 v/v)ad 100.0 ml The presence of 5 erythrocytes/mm3 can still be determined with this test paper in the presence of 150 to 200 mg. ascorbic acid/dl. In the case of an analogous test paper which, instead of the iodate, contains 3 x 10 U ascorbate oxidase, the positive reaction is only visible up to a concentration of 30 to 50 mg ascorbic acid/dl. and, in the case of a paper without additives, only up to a concentration of about 10 mg. ascorbic acid/dl. If the test paper according *supplier's designation to the present invention is heated for 3 days to 60C., then it ~till retains its sensitivity. The paper with ascorbate oxidase only reacts, after this stressing, like a paper without additives.
Exam~le 2.

Te~t Daper for the semiquantitative determination of ~lucose in urine.
Filter paper (Schleicher & Schull No.597 NF-Ind.
is successively impregnated with solutions of the following compositions and, after each impregnation, dried at 50C.:
Solution 1.
glucose oxidase (71 U/mg.) - 1.2 g. I
peroxidase (66 U/mg.) 0.2 g.
1.2M citrate buffer, pH 5 50.0 ml.

9-(y-dimethylaminopropyl)-6-chloro- 2 1 g 3-aminocarbazole dihydrochloride tartrazine 0.12 g.
laurol ~arco~ine 1.1 g.
distilled water ad 100.0 ml.
Solution 2.
tetramethylammonium iodate 1.4 g.
ethanol ad 100.0 ml.
A test paper is prepared in a similar manner but is only impregnated with Solution 1.
Urine samples were prepared containing 100, 300 and 1000 mg. glucose/dl., into each of which wa~ intro-duced 0, 50, 100 and 200 mg. ascorbic acid/dl.
* supplier's designation The test papers were dipped into the urine samples and then placed upon an absorbent substrate.
one minute after dipping in, their reaction colours were compared, the urine without ascorbic acid thereby being taken as an inner standard. As can be seen from the following Table, in which the values given are expressed in mg. glucose/dl., the disturbance due to the ascorbic acid is removed by the addition of iodate:

iodate amount of ascorbic ~ Icid/dl.

_ 100 negative negative negative + 100 100 100 100 300 100 negative negative + 300 300 300 300 _ 1000 300 100 negative . + 1000 1000 1000 1000 - Exam~le 3.
Test ~a~er for the detection of qlucose in urine.
Filter paper (Schleicher & Schull No.597 NF) is impregnated with a solution of the following composition and dried at 50C.:
glucose oxidase (71 U~mg.) 0.38 g.
peroxidase (66 U/mg.) 0.02 g.
potas~i~m iodate 2.00 g.
tartrazine 0.08 g.

* supplier's designation o-tolidine 0.42 g.
ethanol 33.0 ml.
distilled water ad 100.0 ml.
Urine samples containing 50 mg. glucose/dl., into which has 'Deen introduced 0, 50, 100 and 200 mg.
ascorbic acid/dl., give practically the same green reaction colour with this test paper.
An analogous test paper but without iodate only gives a positive reaction in ascorbic acid-free urine.
ExamDle 4.

Test film for the determination of small qlucose contents in blood or serum.
Com~onent 9:
polyvinyl acetate propionate 45 0 dis~ersion (Propiofan 70 D3* g 1,85~ colution of ssdium alg~ate in o,5 m phosphate buffer, pH 5.5 35.0 g.

sodium nonyl sulphate, dissolved 0 75 g in 5.0 ml. water glucose oxidase ~71 U/mg.)) dissolved 0.2 g.
) in 10 ml.
peroxidase (66 U/mg.) ) water 0.25 g.

3,3',5,5'-tetramethyl~enzidine, 0.68 g.
dissolved in 5 ml. acetone sodium iodate 1,0 g.
The com~onents are well mixed, coated in a layer thickness of 200~ on to a synthetic film substrate and dried for 35 minutes at 60C.
Another film was produced in the same manner but without iodate.

* trade mark Sera containing 20 mg. glucose/dl. and 0, 2.5 and 5.0 mg. ascorbic acid/dl. were applied dropwise to the films which, after 1 minute, were wiped and, after a further 2 minutes, the coloration was measured with a commercially available remission photometer (Reflomat),*
with the use of a linear 0 to 100 scale. The following results were obtained:

serum with ascorbic acid test film 0 mg./dl. 2.5 mg./dl. 5.0 mg./dl.

without iodate 47 43 35 with iodate 46 45 45 Example 5.
Test pa~er for the detection of NADH.-Filter paper (Schleicher & Schull No. 23 S~; isimpregnated with a solution of the following composition and dried at 50C.:
iodonitrotriphenyltetrazolium 0.2 g.

sodium iodate 0.5 g.
nonylphenol polyglycol ether 0.2 g.
diaphorase ~32 U/mg.)0.05 g.
0.15M phosphate buffer, pH 7 40.0 ml.
distilled water ad 100.0 ml.

A test paper was produced in the same manner but without iodate.

** supplier's designation * trade ~ark Both papers reacted with aqueous solutions of NADH with the same red coloration. When ascorbic acid is added to the NADH solutions, the paper~ without iodate reacted more strongly.
Exam~le 6.
Determination of alucose in serum.
Solutions:
de~roteinisinq solution 1:
0.16% uranyl acetate in 0.9% sodium chloride solution de~roteinisinq solution 2:
0.16% uranyl acetate and 0.05% sodium iodate in 0.9%
sodium chloride solution reaqent solution:
POD 0.8 U/ml., GOD 10 U/ml., azino-bi~-benzthiazolone-sulphonic acid ammonium salt 1.0 mg./ml. in phosphate buffer, pH 7 (100 mMol/litre) Sample 1:
serum with 100 mg. glucose/dl.
Sam~le 2:
.
serum with 100 mg. glucose/dl. and 20 mg. a~corbic acid/dl.

Standard:

9.1 mg. glucose/100 ml. water De~roteinisation Pipette (ml.) and centrifuge according to the following scheme:

-21_ deproteinisation 1.00 1.00 _ solution 1 deproteinisation _ _ 1.00 1.00 .

sample 1 0.10 _ 0.10 sample 2 _ 0.10 _ 0.10 g ves supernatant 1,l _ 2.1 2.2 Analysis:
Pipette (ml.) according to the following scheme, incubated for 30 minutes at 25C. and measure the extinction at 436 nm ~d = 1 cm.).

supernatant blank standard i-l ¦1-2 ¦2-1 ¦2-2 di tilled 0.1 _ _ _ _ _ standard _ 0.1 _ _ _ _ supernatant _ _ 0.1 0.1 0.1 0.1 reagent 5.0 5.0 5.0 5.0 5.0 5.0 E - 0.115 0.425 0.424 0.366 0.426 0.423 E-E (blank) _ 0.310 0.309 0.251 0,311 0.303 Results:
Calculated from C = 100 x E(sample)/E(standard) ., 115~737 supernatant 1.1 1.2 2.1 2.2 ascorbic acid in sample _ + _ +
iodate in deprotein- _ _ +
isation solution result ~mg./dl.) 99.7 89.0 100.3 99.4 The disturbance brought about by ascorbic acid in Sample 2 is thus completely removed by iodate.
Exam~le 7.
Determination of L-alutamic acid.
Solutions Reaqent solution 1:
1.2 ml. Triton X 100*
30 U diaphorase 10 mg. NAD
60 mg. iodonitrotriphenyltetrazolium chloride in 100 ml. O.lM potassium phosphate/triethanolamine buffer, pH 8.6 Reaqent solution 2:
90,000 U glutamate dehydrogenase in 100 ml. water Sample 1:
100 mg. L-glutamic acid in 100 ml. water Sam~le 2:
100 mg. L-glutamic acid and 40 mg. ascorbic acid in 100 ml. water *trade mark - 1 15~737 Iodate solution:
_ 200 mg. sodium iodate in 100 ml. water Preparation of samPles:
Pipette (ml.) according to the following scheme and leave to stand for 15 minutes at ambient temperature:

sample 1 1.0 1.0 _ sample 2 _ _ 1.0 1.0 NaIO3 solution _ 1.0 _ 1.0 distilled water 1.0 _ 1.0 gives mixture No.1.1 1.2 2.1 2.2 Analysis:
Pipette (ml.) according to the following scheme, after 2 minutes measure the initial extinction El at 492 nm td = 1 cm.), start with reagent 2 and after 15 minutes measure the end extinction E2:

1 15~737 l mixture blank 1 1 1 1.2 ¦ 2.1 ¦ 2.2 reagent 1 1.0 1 1.0 1 1.0 1,0 1.0 water 2.0 1.8 1.8 1.8 1.8 mixture _ 0.2 0.2 0.2 0.2 El 0.058 0.058 0.053 creeps 0.041 solution 2 0.03 0.03 0.03 0.03 0.03 E2 0.062 0.490 0.499 creeps 0.503 (~ ) 0.004 0.432 0.446 _ 0.454 (blank) _ 0.428 0.442 _ 0.452 Results:

Calculated from C = 224 x (~E-~E(blank)) .
mixture No. 1.1 1.2 2.1 2.2 ascorbic acid in _ _ + +
sample iodate treatment _ + _ +

result (mg./dl.) 95.9 99.0 not 101.2 , _ ~ readable Ihe creeps of the extinction caused by the ascorbic acid (slow reduction of the tetrazolium salt), which prevents an exact measurement, can be prevented by pre-incubation of the sample solution with iodate, without the excess iodate disturbing the analysis.

115~7~7 In a particular embodiment iodate is first added to the test sample, and this produces a lowering of the pH value to 5 to 6, by reason of the redox potential. Then the redox reagent system is added, whereby the test system comes into being. The pH of the total test system should be 5 to 9, preferably 7 to 9.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the detection of a redox reaction in a test system in the presence of ascorbic acid as an interfering reducing agent, which method comprises adding to the test system a redox reagent system and a soluble iodate in an amount which is in excess of the highest amount of ascorbic acid present in the test system.

2. A process according to claim 1, wherein the redox reagent system and the iodate are added separately to the test system.

3. A process according to claim 2, wherein the iodate is added before the redox reagent system.

4. A process according to claim 1, wherein the redox reagent system and the iodate are added simultaneously to the test system.

5. A process according to claim 1, wherein the addition of the redox reagent system is effective to increase the pH value of the test system.

6. A process according to claim 5, wherein the pH
value is increased from 5 to 6 to 7 to 9.

7. A process according to claim 1 or 4, wherein said iodate is an alkali metal or ammonium iodate.

8. A process according to claim 1 or 4, wherein said iodate is a water soluble alkali metal or ammonium iodate present in a 2 to 20 fold molar excess of the ascorbic acid content of said test system, said iodate together with said test system establishing a pH of 5 to 9.

9. A process according to claim 1 or 2, wherein said iodate is a lower alkyl quaternary ammonium iodate pre-sent in 2 to 20 fold molar excess of the ascorbic acid content of said test system, said iodate together with said test system establishing a pH of 5 to 9.

10. A diagnostic agent for the detection of a redox reaction in the presence of ascorbic acid as an inter-fering reducing agent, comprising a redox reagent system and, additionally, an iodate soluble therein in an amount which is in excess of the highest amount of ascorbic acid present in the test system to be analyzed.

11. A diagnostic agent according to claim 10, con-taining 0.5 to 2 g. of iodate per 100 ml. of test system.

12. A diagnostic agent according to claim 10 or 11, the pH value of which is such that, together with the test system, a pH value of 5 to 9 is obtained.

13. A diagnostic agent according to claim 10 or 11, wherein the redox reagent system comprises an oxidation indicator, a hydroperoxide and a peroxidase.

14. A diagnostic agent according to claim 10, wherein the redox reagent system comprises a reduction indicator and a reducing agent.

15. A diagnostic agent according to claim 14, wherein said redox reagent system includes an electron carrier.

16. A diagnostic agent according to claim 10 or 11, which is present in an absorbent carrier which is insoluble in the test system.

17. A diagnostic agent according to claim 10 or 11, which is contained in a film which is swellable in the test system.

18. A diagnostic agent according to claim 10 or 11, which is contained in a film which is swellable in the test system, said film being supported on a solid carrier.

19. A diagnostic agent according to claim 10 or 11, which is in the form of a solution lyophilisate or soluble reagent tablet.

20. A diagnostic agent according to claim 10 or 11, contained in a film which is soluble in the test system, said film being on or in a solid carrier.

21. A diagnostic test device for the determination of a fluid sample containing a disturbing reducing agent which comprises ascorbic acid comprising:

a redox reagent system and an iodate, soluble in said fluid sample, supported by an inert carrier, said iodate being present in an amount to provide an iodate content in said fluid sample, in excess of that required to oxidise said disturbing reducing agent, such that said disturbing reducing agent is oxidised to prevent interference with the redox reagent system.

22. A diagnostic test device according to claim 21, wherein said inert carrier comprises a test paper, said redox reagent system and iodate being impregnated in said paper.

23. A diagnostic test device according to claim 21, wherein said inert carrier comprises a film supported on a support strip, said film being swellable in said fluid to release said redox reagent system and iodate.

24. A diagnostic test device according to claim 21, 22 or 23 wherein said iodate is an alkali metal, ammonium or lower alkyl quaternary ammonium iodate present in a 2 to 20 fold molar excess of the ascorbic acid content of said fluid sample, said test device being effective to establish a pH of 5 to 9 in said fluid sample.

25. In a method of determining a fluid sample con-taining ascorbic acid as a disturbing reducing agent, with a redox reagent system, the improvement comprising oxidi-zing said ascorbic acid with an excess of an iodate soluble in said fluid sample, such that said ascorbic acid does not interfere with said redox reagent system.

26. A method according to claim 25, wherein said iodate is employed in an amount of 0.5 to 2 g per 100 ml of fluid sample.