Quantification of 3 4dichloroaniline, 3 5 ... - Büchi Labortechnik Gmbh

est
@buchi
www.buchi.com Information Bulletin Number 45�2007
Quantification of 3,4-dichloroaniline, 3,5-dichloroaniline
and 4-chloroaniline as residues of selected
pesticides
As alternative to the classical Hemmerling method a Distillation
Unit B-324 from Buchi was used offering an easier and more
simple sample handling.
en

est@buchi 45 / 2007 en
Authors: Berthold Kettner 2 , Nadin Epperlein 1,2 Wolfgang Bergmüller 2 , Barbara Kramhöller 2 and Karl Speer 1
Carmen Heiter 3 , Thomas Ziolko 3
1 TU Dresden, Institut für Lebensmittelchemie, Bergstraße 66, D-01062 Dresden
2 ILAU GmbH, Unterwöhrn 15, D-83543 Rott am Inn
3 : BÜCHI Labortechnik AG, Meierseggstrasse 40, 9230 Flawil, Switzerland
Quantification of 3,4�dichloroaniline, 3,5�dichloroaniline
and 4�chloroaniline as residues of selected pesticides
As alternative to the classical Hemmerling
method a Distillation Unit
B-324 from Buchi was used offering
an easier and more simple sample
handling.
Aniline derivative herbicides such as Diuron,
Linuron, Neburon, Propanil and Swep
in food samples are classically analysed
using the Hemmerling method. However,
this method is not easy to operate and
cannot be used with samples containing
starch. These samples cause strong
foaming even with the use of an antifoaming
agent. To counteract these problems
an alternative method was developed
by Epperlein et al. which can be
carried out with every kind of samples and
which is more user-friendly. The results
are comparable to those obtained with
the screening method by Hemmerling.
Both methods are applied to selected
pesticides and the results and handling
are compared.
Introduction
Pesticides are used worldwide in a very
high number of food cultures. In 2001,
pesticides for 27.1 Billion US dollars were
employed and nearly half of them were
herbicides. The residues of pesticides are
still detectable after several years. Responsible
management of pesticides and
good monitoring are therefore mandatory.
Due to their affiliation to different chemical
compound classes the determination
of pesticides is time-consuming and costintensive.
The investigated herbicides Diuron,
Linuron, Neburon, Buturon, Monuron,
Monolinuron and Diflubenzuron are
phenyl urea derivatives while Propanil is
an anilide and Swep is a carbamate.
Chlozolinate, Iprodione, Procymidone and
Vinclozolin are fungicides and belong to
the class of dicarboximides. Their enzymatic
degradation in plants, soil and
mammalian liver results in chloroaniline.
The same compound can be obtained in
the laboratory through alkaline hydrolysis
of the pesticides: 3,4-dichloroaniline
(3,4-DCA) (Fig. 1), 3,5-dichloroaniline
(3,5-DCA) (Fig. 2) and 4-chloroaniline (Fig. 3).
Cl
Cl
H O
N
Fig 1: 3,4-DCA.
Cl
3,5 Dichloroaniline
3,5-Dichloroaniline
Fig 2: 3,5-DCA.
Cl
Cl
Fig 3: 4-chloroaniline
N
CH 3
CH 3
Buturon
O
CH3 N N
H
OMe
CH N
3,4 Dichloroaniline
Methods
Classical method: Hemmerling
NH 2
Cl
The most-used method for the analysis
of aniline pesticides is the Hemmerling
method. The metabolites of the compounds
are analyzed with GC-MS after
alkaline hydrolysis. The equipment employed
is the Bleidner-apparatus (Fig. 4)
which is not really user-friendly and due
to the numerous glassware very delicate
to operate. Furthermore, this procedure
is not adequate for samples containing a
high amount of starch. In these samples
very strong foaming (Fig. 5) occurs during
hydrolysis, hindering a precise measurement.
Even the use of an anti-foaming
agent is not helpful.
F
F
O
H
O
Diflubenzuron
N
H
O
H C 3
N N
H C H
3
Monolinuron Monuron
Cl
Cl
Cl
O
N
O
O
Vinclozolin
H O
N
N
O
N
O
CH2 C
H
CH3 N
3,4 Dichloroaniline
4-Chloroaniline
O
N
H
CH 3
CH 3
Cl
NH 2
H O O
3C
H3C C O
H2 N
O
O
Iprodione Chlozolinate
CH 3
CH 3
Buturon
O
CH3 N N
H
OMe
Cl
C
H 3
C
H 3
CH N
NH 2
Cl
4-Chloroaniline
4-Chloroaniline
F
F
O
H
O
Cl
O
N
Procymidone
O
Diflubenzuron
N
H
O
H C 3
N N
H C H
3
Monolinuron Monuron
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Fig 4: Bleidner-apparatus
Fig 5: Foaming of a rice sample in a Bleidner-apparatus
Alternative method: Epperlein
An alternative method was developed by
Epperlein in order to counteract the foaming
problem. The procedure is divided into
two steps. In the first step, an alkaline hydrolysis
of the sample is carried out with
a simultaneous steam distillation using a
Kjeldahl distillation unit (Fig. 6).
In the second step, an alkaline steam distillation
is performed at the same time as
an extraction using the Clevenger-equipment
(Fig. 7).
This method has several advantages in
comparison with the classical Hemmerling
procedure. The sample matrix is not
a problem anymore, and the Clevengerapparatus
must not be constantly supervised
during the extraction. The resulting
solution can be directly injected into a GC
instrument without any further cleaning or
concentrating step. The Clevenger-appa-

atus is more robust and less cost-intensive
than the Bleidner-apparatus used in
the Hemmerling method. All these advantages
together make this method a more
user-friendly and a less time-consuming
analysis procedure.
Fig 6: B-324 Distillation Unit Fig 7: Clevengerequipment
Analysis
After fine grinding, 10 g of the sample are
weighed into a Kjeldahl glass. Prior to the
distillation with the Buchi Kjeldahl unit,
40 ml of 10 M potassium hydroxide are
added. The distillation is carried out for
9 min (see parameters in Table 1). The
distillate is collected in a 500-ml wideneck
flask containing 10 ml of 0.1 M hydrochloric
acid. At the end of the distillation
the pH value of the distillate should
be still in the acidic range. Between the
samples, the Buchi Kjeldahl unit should
be rinsed thoroughly with water.
Another 40 ml of 10 M potassium hydroxide
as well as four boiling stones are added
to the distillate. The round flask is
connected to the Clevenger-equipment.
The interconnection of the apparatus is
first filled with 5 ml water and then 5 ml
isooctane are added. After the 3 h extraction,
isooctane is transferred into a 10-ml
test tube and dried with 2 g sodium sulphate.
The organic solution is directly injected
in the GC-MS instrument (see parameters
in Table 2).
Distillation (Kjeldahl Distillation Unit B-324)
Water 0 mL
Sodium hydroxide 0 mL
Boric acid 0 mL
Delay 10 s
Distillation 9 min
Steam 75%
Aspiration OFF
Table 1: Parameters for distillation
GC-MS conditions
Capillary gaschromatograph HP 6890 split/splitless injector with autosampler HP 6890
Injector: 210°C
Carrier gas: Helium (Air liquide Germany GmbH)
Injection volume: 2 µl pulsed splitless, purge on: 1.5 min
Oven:
Table 2: Parameters for GC-MS analysis
Results and Validation
The pesticides were analysed at different concentrations and calculated against
3,4-DCA, 3,5-DCA or 4-chloroaniline. The recoveries are shown in Table 3 and 4.
Calculate
against
3,4-DCA
3,5-DCA
4-Chloroaniline
60°C 1 min, 40°C/min to 110°C, 6°C/min to 220°C, 20°C/min
to 280°C 10 min
Column: DB-35ms, 30 m x 0.32 mm x 0.25 mm
Flow: Constant, 1.2 ml/min
Transfer line: 300°C
Mass spectrometer : HP 5972
Mode : EI, positiv
Ionisation energy: 70 eV
Pesticides Recoveries in %
0.005 mg/kg 0.01 mg/kg 0.05 mg/kg 0.1 mg/kg 0.3 mg/kg 0.5 mg/kg
Diuron - - 68.7 70.1 - -
Linuron - 83.6 89.8 92.5 - -
Neburon - 93.4 94.1 96.4 - -
Propanil - 94.9 92.0 91.2 - -
Swep - 73.7 75.8 75.3 - -
Chlozolinate - 84.8 78.4 83.4 93.9 -
Iprodione - 39.5 31.6 37.4 35.5 -
Procymidone - 100.2 96.6 99.6 102.0 -
Vinclozolin - 103.6 101.1 107.2 108.2 -
Buturon 16.2 7.3 4.0 3.4 3.3 3.2
Diflubenzuron 83.1 85.0 93.7 83.5 84.5 89.5
Monolinuron 111.9 111.4 133.2 111.9 128.4 129.2
Monuron 98.1 101.6 114.9 118.0 123.2 132.7
Table 3: Recovery of each pesticide (method: Epperlein et al. 3,4 )
The recoveries for the metabolite 3,4-DCA are between 65 and 100% and for the metabolites
3,5-DCA and 4-chloroaniline between 83.4 and 133.2%. The lower recoveries
for Buturon (3.3 – 16.2%) and for Iprodione (31.6 – 39.5%) are comparable with the
recoveries of the screening method by Hemmerling (Table 4). Both methods are not
suitable for the determination of Buturon and Iprodione.
best@buchi 45 / 2007 en

est@buchi 45 / 2007 en
Pesticide Recoveries in %
According to Epperlein et al. 4 According to Hemmerling 2
0.05 mg/kg 0.1 mg/kg 0.05 mg/kg 0.1 mg/kg
Buturon 4.0 3.4 5.8 5.0
Iprodion 31.6 37.4 37.4 38.6
Table 4: Recoveries of Buturon and Iprodione
The limit of quantification (LOQ), the limit of detection (LOD) and the lowest legally allowed
maximum residue level for some selected pesticides are shown in Table 5.
A comparison of both methods with different native rice samples showed the same
results (Table 6). The alternative method has a good reproducibility and is therefore
more appropriate for the comparison among different laboratories.
Conclusion
The alternative method by Epperlein at al. can be used as a routine analysis procedure.
Compared to the classical Hemmerling method, this new method is less timeconsuming,
easier to perform and adequate for all types of samples.
The quantification of the 3,4-DCA, 3,5-DCA and 4-chloroaniline with this new method
has shown excellent results in terms of recovery, limit of detection and handling. The
aniline derivative pesticides can be easily analyzed with this method.
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lowest legally allowed
maximum residue level 1 LOQ * LOD *
Diuron 0.05 0.03 0.010
Linuron 0.05 0.01 0.003
Neburon 0.05 0.01 0.003
Propanil 0.05 0.01 0.003
Swep 0.01 0.01 0.003
Chlozolinate 0.05 0.01 0.003
Procymidone 0.02 0.01 0.003
Vinclozolin 0.05 0.01 0.003
Diflubenzuron 0.05 0.01 0.003
Monolinuron 0.05 0.01 0.003
Monuron 0.05 0.01 0.003
*calculated with “Kalibo” according to DIN 32645
Table 5: Limit of quantification and limit of detection (method: Epperlein et al.)
Rice
TU DresdenMethod according
to Hemmerling 2
ILAU GmbHMethod according to
Epperlein et al. 3
A 0.02
Results in mg/kg
0.02 0.03
B 0.02 0.02 0.02
C 0.01 0.03 0.02
D 0.06 0.07 0.08
Table 6: Interlaboratory Recovery Test” with native rice samples
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Literature
[1] Verordnung über Höchstmengen an
Rückständen von Pflanzenschutz-
und Schädlingsbekämpfungsmitteln,
Düngemitteln und sonstigen Mitteln
in oder auf Lebensmitteln (Rückstands-Höchstmengenverordnung
–
RHmV) in der Neufassung vom
21.10.1999, 18. Verordnung zur Änderung
der Rückstands-Höchstmengenverordnung
vom 20.04.2007
[2] Hemmerling, C: Screeningmethode
zur schnellen Untersuchung von Lebensmitteln
auf Rückstände von Phenylharnstoffherbiziden
und weiteren
PSM-Wirkstoffen durch alkalische
Hydrolyse und GC-MS-Bestimmung,
Deutsche Lebensmittel-Rundschau
95, 350-361 (1999)
[3] Epperlein et al. : Die Quantifizierung
von Rückständen ausgewählter Herbizide
(Diuron, Linuron, Neburon,
Propanil und Swep) in Lebensmitteln
nach alkalischer Hydrolyse mit Hilfe
der GC/MS, Deutsche Lebensmittelrundschau
102, 53-57 (2006)
[4] Kettner et al. : Die Bestimmung von
3,5-Dichloranilin und 4-Chloranilin
mittels GC/MS-Analytik nach alkalischer
Hydrolyse ausgewählter Pflanzenschutzmittel,
Deutsche Lebensmittel-Rundschau
103, 4-7 (2007)
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