2, 6-Dibromobenzoequinone-4-Chloroimide as a Reagent for Determination of Dimcthoate, Monoalkyl-Aryl-Phosphorothionates and Some Organic Sulfides by J. H. V. STENERSEN Norwegian Plant Protection Institute, VoUebekk
In studies of enzymatic formation and degradation of the monoalkyl derivatives of the thiophosphorus insecticides,
we had a great need for a simple and rapid
spectrophotometric method for monoalkyl-aryl-phosphorothionates.
The possible use of 2,6-dibromobenzoequinone-
4-chloroimide
(DQC) as a reagent for these compounds was
therefore investigated. I~C has been very successfully used as a chromatographic spray for detection of phosphorothionates Rowlands
(i-3).
(4) has used the reagent both for qualitative
and quantitative determinations
of organophosphorus
insecticides and their degradation products.
364 Bulletin of Environmental Contamination & Toxicology, Voh 2, No. 6, 1967, published by Springer-Verlag New York, Inc.
His
quantitative method involves extraction,
extensive puri-
fications and heating to obtain the colour reaction. The heating step is very critical and difficult to reproduce.
Kamiya
(5) has found that a great number of aroma-
tic and aliphatic thioketones which can enolize, coloured products with DQC in ethanol.
give
Cuzoni and Lissi
(6) have used DQC for qualitative and quantitative determination of aromatic and aliphatic disulfides.
The
coloured products were found to be quinone-sulfeneimines (7). In this paper the use of DQC for the determination of monoalkyl-aryl-phosphorothionates interesting sulfhydryl compounds
and some biological
is reported.
EXPERIMENTAL Reagents:
0,4% W/V of DQC (Fluka) in: A) acetic acid B) acetic acid - ethyl ether
(i:i) and
C) absolute ethanol adjusted to pH i~5 with 1 N
HCl
The reagents are stable for some days if kept in dark bottles
in refrigerator.
Standard solutions:
0,1% w/v of the compounds
in acetone
or water according to their solubilities. The compounds tried were: Parathion-methyl, demeton-methyl, ronat,
dimethoate,
fenitrothion,
bromophos
bromophos,
diazinon,
monomethyl-parathion,
trichlo-
monomethyl-
(sodium salt), monoethyl-bromophos, 365
parathion,
monoethyl-
trichloronat thionate),
(2,
4,
5-trichlorophenyl-ethyl-phosphono-
monomethyl-fenitrothion,
glutathione
oxidized,
glutathione
reduced,
cysteinhydrochloridemonohydrate,
2-mercapto-ethanol. Procedure:
O, 10,
were transferred pipette. added.
30 a n d 5 0 ~ 1
to test
3 ml o f t h e When t h e
of
tubes
with
respective
influence
the standard
a 0 , 1 ml g r a d u a t e d
reagent
of water
solutions
solutions
should
were
be tried,
1 ml
of water plus 2 ml of the reagent solution were used. no colour appeared at room temperature, ling water bath for different eventually
periodes
one drop of concentrated
If
heating on a boiof time were tried,
sulfuric acid was
added. The development studied
and stability
of the colour were
in a s p e c t r o p h o t o m e t e r . RESULTS A N D DISCUSSION
With all three reagents thionates and the sulfhydryl
the monoalkyl-aryl-phosphorocompounds
cysteine and 2-mercapto-ethanol) obeying Beer's
law (Fig.
lity the colourometry dardized
i).
gave fairly stable colours
To improve the reproducibi-
should be undertaken after a stan-
time, e.g. 3 minutes.
their absorbtion
(red. glutathione
The colours
obtained had
maxima between 425 nm and 435 nm.
For
the same compound the maxima could vary from experiment to experiment,
probably depending
solution.
366
on the exact pH of the
Water caused first-order
reaction
and therefore ce
in these
ether
has
colours
when w a t e r effect
or
incubation the
the
paration
compounds could
samples could
presence
B.
be r e a d .
of sulfhydryl
of blanks
it
is, in
absorbanEthyl-
w hen g r e a t
amounts
1 ml w a t e r ,
e.g.
liver
easily
buffer
or grain
be determined
When much p r o t e i n
colours,
however,
was
before
possibly
their inter-
due to
By c a r e f u l
possible
incubation
2),
B stable
compounds.
pre-
to determine
mixtures
by using
B.
W i t h 2 ml 0,4% DQC w / v colours
water.
developed
The b l a n k s
Propionic
acid
was t h e
had,
and the
only
presence
stabilizing
salt
the
Homogenate solutions
weak y e l l o w
a
reagent
had to be centrifuged
compounds directly
acetone,
and with
mixture,
2 ml o f r e a g e n t
after
was p r e s e n t .
in
by giving
in the
measure
Dissolved
absorbances
acid
to
colour
A or C (Fig.
was p r e s e n t .
present,
stable
reagent
was i m p o s s i b l e
reagents
the
also
by a d d i n g
reagent
of
obtained
homogenate,
the
- using
a stabilizing
solution,
the
it
fading
were therefore
of water
fered
rapid
of ether
ethanol,
formic
acid,
substances
367
1 ml
colour. and acetic
which gave stable T he o t h e r
solution
of
appreciable
combination
(butanol,
buffer
acid
in presence
however,
solvents
ethylacetate,
solutions,
slowly
of water.
agents
in propionic
solvents
colours tried
as
dimethylformamide, pyridine,
of different
strong pH) d i d
1.4
Abs43 banCe
1.2
in 3 m l /
1.0
/1
2/
~
///~4
-
0.8 0.6 0.4 0,2
30
i0
Fig. I.
50pg
Standard curves for some of the sulfhydryl compounds,
disulfides and mono alkyl-aryl-
phosphorothionates.
The absorbance is read
at the time of maximum colour which was about 3 min. for all but glutathione dimethoate
(35 min.) and
(15 min.).
i) 2-mercapto-ethanol,
2) Dimethoate (425 nm),
3) Cysteinhydrochloride methylparathion,
monohydrate,
4) Mono-
5) Monoethyl-bromophos,
6) Oxidized glutathione.
368
logA at
435
nm
0.0 -0
lb
1
-0.2 2b -0.3 -0.4
_
-0.5
_
-0.6
_
-0.7 -0.8
2a
-0.9
la
-1.0
Fig.
2.
The
fading
monomethyl ethanol
(2)
water,
b)
I
I
1
2
4
6 rain
of
the
colour
parathion in
a)
(1)
produced and
10 ~ g
2 ml reagent
2 ml reagent
369
B plus
by 50 pg 2-mercapto-
A plus
1 ml
1 ml water.
not however by themselves
inhibit colour development
or stability
B was u s e d .
buffers
when r e a g e n t
(e.g.
ment also
in case
solution. to
succinate)
This
B by d e c r e a s i n g
larger
B and water
reagent
A the
colour
and then
after
faded
acid.
Water
inhibited The o n l y
to
give
after
colour
commercial
B plus
Beer's water
at
respective
The m e t h o d s a s thion-methyl, zinon, these
insecticides
with
With after
35
with
the sul-
( max ~ 412 n m ) . in all
cases.
insecticide
colour
colour
of all
1.8x104
absorbance described
parathion,
trichloronat
colour
found
was d i m e t h o a t e o
developed
t o a maximum
(~max ~ 4 2 5 ) .
g a v e no c o l o u r .
law were b e t w e e n
their
B.
of concentrated
l aw was o b e y e d
Th e m o l a r a b s o r b a n c e s Beer's
colour
thiophosphorus
and reproducible
acid
l a w was o b e y e d .
development
A or C a yellow
15 m i n .
Reagent
of one drop
l a w was o b e y e d
the
a stable
With reagent
gave a yellow
Beer's
mineral
developed
Beer's
develop-
of the
1 ml w a t e r ) .
slowly.
addition
furic
acidity
of reagent
was c o m p l e t e l y
of acid
colour
g a v e a w eak y e l l o w
(2 ml B p l u s
2onoethyl-trichloronat reagents
the
amounts
glutathione
reagent
minutes,
the
may b e o v e r c o m e b y a d d i n g
pH 2 o r by u s i n g Oxidized
may p r e v e n t
Presence
or
the
compounds obeying
and 2.2x104, maximum.
are
not suitable
demeton-methyl, fenitrothion.
gave a colour
370
measured
with
for
para-
bromophos~
However, reagent
dia-
some o f A or B
after sulfuric acid addition or by heating.
As these
colours did not follow Beer's law and gave bad reproducibility a hydrolytic step, giving the monoalkyl compounds could probably be included if a general analytical procedure
for thiophosphorus
Acknowledgments:
insecticides should be developed.
This work was carried out with financial
support from the Norwegian Research Council of Agriculture.
REFERENCES i.
MENN, J . J . , W.E. ERWIN and H.T. GORDON, J. A g r . Fd Chem. 5, 601 (1957)
2.
BRAITHWAITE, D.P., Nature 200, 1011 (1963)
3.
ROWLANDS, D.G., J. Sci. Fd Agric. i__66, 325 (1965)
4.
ROWLANDS, D.G., J. stored Prod. R e s . 2, 1 (1966)
5.
KAMIYA, S., Bunsekl Ka~aku 8, 596 (1959)
6.
CUZONI, M.T. and T. PIETRA LISSI, Farmaco (Pavia)
Ed. S c i . 7.
1_99, 981 ( 1 9 6 4 )
CUZONI, M.T. and r .
PIETRA L I S S I ,
37]
Ibid.
1__99, 771 ( 1 9 6 4 )
In studies of enzymatic formation and degradation of the monoalkyl derivatives of the thiophosphorus insecticides,
we had a great need for a simple and rapid
spectrophotometric method for monoalkyl-aryl-phosphorothionates.
The possible use of 2,6-dibromobenzoequinone-
4-chloroimide
(DQC) as a reagent for these compounds was
therefore investigated. I~C has been very successfully used as a chromatographic spray for detection of phosphorothionates Rowlands
(i-3).
(4) has used the reagent both for qualitative
and quantitative determinations
of organophosphorus
insecticides and their degradation products.
364 Bulletin of Environmental Contamination & Toxicology, Voh 2, No. 6, 1967, published by Springer-Verlag New York, Inc.
His
quantitative method involves extraction,
extensive puri-
fications and heating to obtain the colour reaction. The heating step is very critical and difficult to reproduce.
Kamiya
(5) has found that a great number of aroma-
tic and aliphatic thioketones which can enolize, coloured products with DQC in ethanol.
give
Cuzoni and Lissi
(6) have used DQC for qualitative and quantitative determination of aromatic and aliphatic disulfides.
The
coloured products were found to be quinone-sulfeneimines (7). In this paper the use of DQC for the determination of monoalkyl-aryl-phosphorothionates interesting sulfhydryl compounds
and some biological
is reported.
EXPERIMENTAL Reagents:
0,4% W/V of DQC (Fluka) in: A) acetic acid B) acetic acid - ethyl ether
(i:i) and
C) absolute ethanol adjusted to pH i~5 with 1 N
HCl
The reagents are stable for some days if kept in dark bottles
in refrigerator.
Standard solutions:
0,1% w/v of the compounds
in acetone
or water according to their solubilities. The compounds tried were: Parathion-methyl, demeton-methyl, ronat,
dimethoate,
fenitrothion,
bromophos
bromophos,
diazinon,
monomethyl-parathion,
trichlo-
monomethyl-
(sodium salt), monoethyl-bromophos, 365
parathion,
monoethyl-
trichloronat thionate),
(2,
4,
5-trichlorophenyl-ethyl-phosphono-
monomethyl-fenitrothion,
glutathione
oxidized,
glutathione
reduced,
cysteinhydrochloridemonohydrate,
2-mercapto-ethanol. Procedure:
O, 10,
were transferred pipette. added.
30 a n d 5 0 ~ 1
to test
3 ml o f t h e When t h e
of
tubes
with
respective
influence
the standard
a 0 , 1 ml g r a d u a t e d
reagent
of water
solutions
solutions
should
were
be tried,
1 ml
of water plus 2 ml of the reagent solution were used. no colour appeared at room temperature, ling water bath for different eventually
periodes
one drop of concentrated
If
heating on a boiof time were tried,
sulfuric acid was
added. The development studied
and stability
of the colour were
in a s p e c t r o p h o t o m e t e r . RESULTS A N D DISCUSSION
With all three reagents thionates and the sulfhydryl
the monoalkyl-aryl-phosphorocompounds
cysteine and 2-mercapto-ethanol) obeying Beer's
law (Fig.
lity the colourometry dardized
i).
gave fairly stable colours
To improve the reproducibi-
should be undertaken after a stan-
time, e.g. 3 minutes.
their absorbtion
(red. glutathione
The colours
obtained had
maxima between 425 nm and 435 nm.
For
the same compound the maxima could vary from experiment to experiment,
probably depending
solution.
366
on the exact pH of the
Water caused first-order
reaction
and therefore ce
in these
ether
has
colours
when w a t e r effect
or
incubation the
the
paration
compounds could
samples could
presence
B.
be r e a d .
of sulfhydryl
of blanks
it
is, in
absorbanEthyl-
w hen g r e a t
amounts
1 ml w a t e r ,
e.g.
liver
easily
buffer
or grain
be determined
When much p r o t e i n
colours,
however,
was
before
possibly
their inter-
due to
By c a r e f u l
possible
incubation
2),
B stable
compounds.
pre-
to determine
mixtures
by using
B.
W i t h 2 ml 0,4% DQC w / v colours
water.
developed
The b l a n k s
Propionic
acid
was t h e
had,
and the
only
presence
stabilizing
salt
the
Homogenate solutions
weak y e l l o w
a
reagent
had to be centrifuged
compounds directly
acetone,
and with
mixture,
2 ml o f r e a g e n t
after
was p r e s e n t .
in
by giving
in the
measure
Dissolved
absorbances
acid
to
colour
A or C (Fig.
was p r e s e n t .
present,
stable
reagent
was i m p o s s i b l e
reagents
the
also
by a d d i n g
reagent
of
obtained
homogenate,
the
- using
a stabilizing
solution,
the
it
fading
were therefore
of water
fered
rapid
of ether
ethanol,
formic
acid,
substances
367
1 ml
colour. and acetic
which gave stable T he o t h e r
solution
of
appreciable
combination
(butanol,
buffer
acid
in presence
however,
solvents
ethylacetate,
solutions,
slowly
of water.
agents
in propionic
solvents
colours tried
as
dimethylformamide, pyridine,
of different
strong pH) d i d
1.4
Abs43 banCe
1.2
in 3 m l /
1.0
/1
2/
~
///~4
-
0.8 0.6 0.4 0,2
30
i0
Fig. I.
50pg
Standard curves for some of the sulfhydryl compounds,
disulfides and mono alkyl-aryl-
phosphorothionates.
The absorbance is read
at the time of maximum colour which was about 3 min. for all but glutathione dimethoate
(35 min.) and
(15 min.).
i) 2-mercapto-ethanol,
2) Dimethoate (425 nm),
3) Cysteinhydrochloride methylparathion,
monohydrate,
4) Mono-
5) Monoethyl-bromophos,
6) Oxidized glutathione.
368
logA at
435
nm
0.0 -0
lb
1
-0.2 2b -0.3 -0.4
_
-0.5
_
-0.6
_
-0.7 -0.8
2a
-0.9
la
-1.0
Fig.
2.
The
fading
monomethyl ethanol
(2)
water,
b)
I
I
1
2
4
6 rain
of
the
colour
parathion in
a)
(1)
produced and
10 ~ g
2 ml reagent
2 ml reagent
369
B plus
by 50 pg 2-mercapto-
A plus
1 ml
1 ml water.
not however by themselves
inhibit colour development
or stability
B was u s e d .
buffers
when r e a g e n t
(e.g.
ment also
in case
solution. to
succinate)
This
B by d e c r e a s i n g
larger
B and water
reagent
A the
colour
and then
after
faded
acid.
Water
inhibited The o n l y
to
give
after
colour
commercial
B plus
Beer's water
at
respective
The m e t h o d s a s thion-methyl, zinon, these
insecticides
with
With after
35
with
the sul-
( max ~ 412 n m ) . in all
cases.
insecticide
colour
colour
of all
1.8x104
absorbance described
parathion,
trichloronat
colour
found
was d i m e t h o a t e o
developed
t o a maximum
(~max ~ 4 2 5 ) .
g a v e no c o l o u r .
law were b e t w e e n
their
B.
of concentrated
l aw was o b e y e d
Th e m o l a r a b s o r b a n c e s Beer's
colour
thiophosphorus
and reproducible
acid
l a w was o b e y e d .
development
A or C a yellow
15 m i n .
Reagent
of one drop
l a w was o b e y e d
the
a stable
With reagent
gave a yellow
Beer's
mineral
developed
Beer's
develop-
of the
1 ml w a t e r ) .
slowly.
addition
furic
acidity
of reagent
was c o m p l e t e l y
of acid
colour
g a v e a w eak y e l l o w
(2 ml B p l u s
2onoethyl-trichloronat reagents
the
amounts
glutathione
reagent
minutes,
the
may b e o v e r c o m e b y a d d i n g
pH 2 o r by u s i n g Oxidized
may p r e v e n t
Presence
or
the
compounds obeying
and 2.2x104, maximum.
are
not suitable
demeton-methyl, fenitrothion.
gave a colour
370
measured
with
for
para-
bromophos~
However, reagent
dia-
some o f A or B
after sulfuric acid addition or by heating.
As these
colours did not follow Beer's law and gave bad reproducibility a hydrolytic step, giving the monoalkyl compounds could probably be included if a general analytical procedure
for thiophosphorus
Acknowledgments:
insecticides should be developed.
This work was carried out with financial
support from the Norwegian Research Council of Agriculture.
REFERENCES i.
MENN, J . J . , W.E. ERWIN and H.T. GORDON, J. A g r . Fd Chem. 5, 601 (1957)
2.
BRAITHWAITE, D.P., Nature 200, 1011 (1963)
3.
ROWLANDS, D.G., J. Sci. Fd Agric. i__66, 325 (1965)
4.
ROWLANDS, D.G., J. stored Prod. R e s . 2, 1 (1966)
5.
KAMIYA, S., Bunsekl Ka~aku 8, 596 (1959)
6.
CUZONI, M.T. and T. PIETRA LISSI, Farmaco (Pavia)
Ed. S c i . 7.
1_99, 981 ( 1 9 6 4 )
CUZONI, M.T. and r .
PIETRA L I S S I ,
37]
Ibid.
1__99, 771 ( 1 9 6 4 )
