BLEND OF APPROPRIATE PRESERVATIVE AGENTS FOR ANALYSIS INSTRUMENTS WITH ION-SELECTIVE ELECTRODES

MELANGE D'AGENTS PRESERVATEURS APPROPRIES POUR INSTRUMENTS D'ANALYSE A ELECTRODES SELECTIVES POUR IONS

English Abstract

The inventive preservative agent for reagents intended for the diagnostic
determination of electrolytes by means of ion-selective electrodes contains a
compound which triggers the cleavage of formaldehyde, possibly combined with
chloracetamide. The present invention also relates to this blend of
preservative agents as well as to the preserved reagents.

French Abstract

L'agent préservateur proposé pour des réactifs utilisés aux fins de la détermination diagnostique d'électrolytes au moyen d'électrodes sélectives pour ions comprend un composé provoquant le clivage de formaldéhyde, éventuellement combiné avec du chloracétamide. La présente invention porte également sur l'utilisation de ce mélange d'agents préservateurs ainsi que sur les réactifs conservés.

Claims

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Claims
1. Preservative for reagents for the diagnostic
determination of electrolytes with the aid of
ion- selective electrodes,
wherein
it contains at least one compound which cleaves off
formaldehyde in combination with chloroacetamide as
the active substance, wherein the preservative does
not have an adverse effect on the performance of
the electrodes.
2. Preservative as claimed in claim 1,
wherein
the compound which cleaves off formaldehyde
corresponds to the following general formula:
<IMG>
in which R1 and R2 represent a branched or
unbranched alkyl, alkenyl or alkinyl group with 1
to 6 C atoms which can optionally contain
heteroatoms and
R3 represents H or a branched or unbranched alkyl,
alkenyl or alkinyl group with 1 to 6 C atoms where
one of R1 and R2 can optionally also form a ring
closure with R3.

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3. Preservative as claimed in claim 1 or 2,
wherein
the compound of formula (I) is Oxaban A or Oxaban E.
4. Preservative as claimed in claim 1,
wherein
it contains the active substance or active
substances at such a concentration that in the
reagents to be preserved chloroacetamide is present
in an amount of 0.1 to 0.5 mg/ml or/and Oxaban E is
present in an amount of 0.1 to 3 mg/ml or/and
Oxaban A is present in an amount of 0.2 to 1 mg/ml.
5. Preservative as claimed in claim 2,
wherein
in the reagents to be preserved chloroacetamide is
present in an amount of 0.1 to 0.4 mg/ml or/and
Oxaban E is present in an amount of 0.5 to 1.5 mg/ml
or/and Oxaban A is present in an amount of 0.3 to
0.8 mg/ml.
6. Use of a preservative as claimed in one of the
claims 1 to 5 for preserving reagents for the
diagnostic determination of electrolytes with the
aid of ion-selective electrodes.
7. Reagent for the diagnostic determination of
electrolytes with the aid of ion-selective
electrodes,
wherein
it contains a preservative mixture as claimed in
one of the claims 1 to 5.

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8. Reagent as claimed in claim 7,
wherein
it has a pH value of 7.0 to 8.2.
9. Use of a preservative containing at least one
compound which cleaves off formaldehyde optionally
in combination with chloroacetamide as the active
substance to improve the performance of
ion- selective electrodes for the diagnostic
determination of electrolytes.
10. Use as claimed in claim 9,
wherein
the compound which cleaves off formaldehyde and
chloroacetamide are used.

Description

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Suitable preservative mixture for analyzers with ion
selective electrodes
Description
The present invention concerns preservatives for
reagents for the diagnostic determination of
electrolytes with the aid of ion-selective electrodes,
their use for preserving the said reagents and reagents
to which the preservatives according to the invention
have been added.
The determination of electrolytes with the aid of ion-
selective electrodes is preferably carried out in flow-
through systems which are very vulnerable to any type of
microbial contamination. The growth of microorganisms
within the system leads to falsifications of the
analytical results and with increasing growth of the
microorganisms to an inactivation of the electrodes. It
is therefore desirable to preserve the reagents used in
such flow-through systems.
However, for several reasons it is difficult to preserve
the reagents used in the flow-through systems:
1) The diluent used to dilute the samples must not
contain any of the electrolytes to be determined or
substances that are co-detected by the electrodes
due to low selectivity. Unfortunately many biocides
cleave off for example C1- or Br' which falsifies
the results especially when this process proceeds
continuously.

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2) The performance as well as the life time of the
ion-selective electrodes is impaired by almost all
preservatives. The concentration that is tolerated
by the electrodes varies from electrode type to
electrode type. However, the concentration
tolerated by the electrodes is often no longer
adequate with regard to its biocidal effectiveness.
3) The spectrum of microorganisms which can occur in
such a system extends from bacteria, yeasts through
to fungi. Experience has therefore shown that a
single preservative is not sufficient to protect
the system against the entire spectrum of
microorganisms.
4) Preservatives that come into consideration can
often not be used worldwide due to country-specific
legal restrictions (such as formaldehyde,
antibiotics).
Hence the object of the present invention was to provide
preservatives which can be used worldwide and are
compatible for all electrodes and whose preservative
properties cover the widest possible spectrum of
microorganisms and minimize the formation of resistant
microorganisms.
This object is achieved according to the invention by a
preservative for reagents for the diagnostic
determination of electrolytes with the aid of ion-
selective electrodes which contain at least one compound
which cleaves off formaldehyde optionally in combination
with chloroacetamide as the active substance. Compounds
which cleave off formaldehyde have the major advantage

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over using formaldehyde itself that formaldehyde is only
formed in the reagent usually in low amounts which is in
turn consumed when it attacks the microbes and hence
there are always only low amounts of formaldehyde in the
solution. Especially in countries where the use of
formaldehyde is generally forbidden, it is possible in
this manner to nevertheless utilize the good
preservative action of formaldehyde although it is not
used directly and is formed in the reagent only in
extremely low amounts and is continuously consumed.
Hence one observes the preservative action of
formaldehyde although the substance itself is
practically only present in extremely small amounts.
According to the invention compounds which cleave off
formaldehyde are preferred which only slowly cleave off
formaldehyde in small amounts.
Particularly preferred compounds have the formula I
R~
N
R3
in which R1 and R2 represent a branched or unbranched
alkyl, alkenyl or alkinyl group with 1 to 6 C atoms
which can optionally contain heteroatoms and
R3 represents H or a branched or unbranched alkyl,
alkenyl or alkinyl group with 1 to 6 C atoms where one
of R1 and R2 can optionally also form a ring closure
with R3.
Within the scope of the present invention it has turned

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out that combinations with chloroacetamide can further
increase the biocidal action without adverse effects on
all three ion-selective electrodes.
Particularly preferred compounds within the scope of the
present invention are Oxaban A* and Oxaban E** (*4,4-
dimethyl-1,3-oxazolidine, **
Et
~~0
1 ~, N
Especially Oxaban E already has a very good preservative
action when used alone.
However, a combination with chloroacetamide can also in
the case of Oxaban E lead to an extension of the
spectrum of activities and increase the activity.
Surprisingly it has turned out that the preservatives
according to the invention at a concentration that can
be used without problems for the electrodes not only has
a biocidal action on a large spectrum of different
microorganisms but at the same time also does not have
an adverse effect on the life time of the electrodes. In
a preferred embodiment of the invention the preservative
contains the preserving substances at such a
concentration that, in the reagents to be preserved,
chloroacetamide is present at a concentration of 0.1 to
0.5 mg/ml or/and Oxaban E is present at a concentration
of 0.1 to 3 mg/ml or/and Oxaban A is present at a
concentration of 0.2 to 1 mg/ml. For this purpose it is
also possible to particularly advantageously use a
concentrate of the preservative with a ca. 10- to 100-
fold concentration of the ingredients. This concentrate
can than be introduced into the reagents to be preserved

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without substantially changing the concentration of the
compounds contained therein. Using the said final
concentrations for the reagents to be preserved no
adverse effects whatsoever on the electrodes are to be
expected. Hence the advantages of the preservatives
according to the invention are:
- No influence on the performance of all three ion-
selective electrodes over their life time. This is
important since it is only possible to use all three
electrodes simultaneously.
- Adequate preservative properties at the concentration
to be used.
- Effective biocidal properties at an optimal pH value
for the electrodes of pH 7.0 to 8.2.
- Better avoidance of resistance development by the
preferred use of two biocides (formaldehyde cleaver
and chloroacetamide).
- Avoidance of legal restrictions in various countries.
In the preservative according to the invention the
substances are particularly preferably present in such
an amount that chloroacetamide is present in the
reagents to be preserved in an amount of 0.1 to
0.4 mg/ml or/and Oxaban E is present in an amount of 0.5
to 1.5 mg/ml or/and Oxaban A is present in an amount of
0.3 to 0.8 mg/ml.
As already mentioned the preservative according to the

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invention is advantageous since it ensures a very
effective preservation without influencing the ion-
selective electrodes. Hence a further subject matter of
the present invention is the use of the preservative
according to the invention to preserve reagents for the
diagnostic determination of electrolytes with the aid of
ion-selective electrodes.
Yet a further subject matter of the present invention is
a reagent for the diagnostic determination of
electrolytes with the aid of ion-selective electrodes
which contains a preservative according to the
invention. The reagent preserved according to the
invention preferably has a pH value of 7.0 to 8.2. The
electrodes are not adversely effected within this pH
range with a concurrent optimal action of the preserving
agents.
The following example in conjunction with the figures
further elucidates the invention.
Example:
Various preservatives were tested for their ability to
preserve reagents for the determination of electrolytes
with the aid of ion-selective electrodes. In doing so
particular attention was paid to possible impairment of
the electrodes. The following table 1 shows that only
chloroacetamide, Oxaban A and Oxaban E have no effects
whatsoever on all three types of electrodes.
The preservative capability of 4,4-dimethyl-1,3-
oxazolidine (Oxaban A) and chloroacetamide (CAA) and
Oxaban E was examined in further investigations. As a

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comparison the microbial growth of a non-preserved
composition at pH 7.94 is shown in figure 1.
Figure 2 shows the same composition but with addition of
0.5 mg/ml Oxaban A pH 7.6 and 0.2 mg/ml CAA. It can be
clearly seen from figure 2 that fungi as well as both
examined types of bacteria are reduced to zero within 6
weeks in the solution. Hence the stated concentration
does not only have the ability to prevent growth of
germs already present in the solution but severely
reduces the germs within a short period.
Figure 3 shows a further investigation in which again
the same solution was preserved with 0.3 mg/ml Oxaban A
and 0.12 mg/ml CAA. The pH value of this solution was
8.06. This also shows that bacteria and fungi are in
some cases very strongly reduced and in the case of the
type 2 bacteria it was at least observed that the number
of germs only increased to a negligible extent.
Finally figure 4 shows an even lower concentration of
the two preservatives which also prevents growth of the
germs.
Figure 5 shows, in comparison to this, preservation with
only one of the two preserving components i.e. with
0.5 mg Oxaban A. Whereas a very strong germ-reducing
effect was observed with this concentration of Oxaban in
combination with CAA in figure 2, such a strong effect
is not detected with Oxaban A alone at a concentration
of 0.5 mg/ml.
Figures 6 to 14 show the effect of various preservatives
on the three types of electrodes. It can be seen that

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Oxaban A as well as CAA and Oxaban E do not have any
effects whatsoever on the electrodes in the functional
range preferably used according to the invention whereas
other preservatives which are also listed in table 1
have adverse effects at least in the functional ranges
required for these preservatives.
Table 1
O - no adverse effect
O - small adverse effect
O - large adverse effect
sodium potassium chloride
electrode electrode electrode
bromo-nitro-dioxane (BND) O O
methylisothiazolone (MIT) O O
hydroxypyridine oxide (HPO) O O O
imidazolidinyl urea (IZU) ~ O O
chloroacetamide (CAA) O O O
1,2-dibromo-2,4-dicyano- O O
butane and phenoxyethanol
(Euxyl K400)
4,4-dimethyl-1,3- O O O
oxazolidine (Oxaban A)
Oxaban E O O O
Figures 15 to 19 show the preservative action of Oxaban
E. at various concentrations and in combination with CAA.
From this it can be seen that Oxaban E even alone
already has a very good preservative action.