Trends
1, 2009
Residue analysis of dithiocarbamate
fungicides
Goranka Crnogorac, Wolfgang Schwack
We review the development of methods for the determination of dithiocar-
bamate fungicide residues in foodstuffs. We discuss all available methods,
from colorimetric carbon-disulfide methods to liquid chromatography
combined with tandem mass spectrometry. Our main focus is on the
relevance of analytical methods with respect to routine use for food control
and efficiency in meeting the requirements of current European legislation.
ª 2008 Elsevier Ltd. All rights reserved.
Keywords: Analysis; Determination; Dithiocarbamate fungicide; Carbon disulfide;
Colorimetry; LC-MS; LC-MS2; Liquid chromatography; Mass spectrometry; Residue
1. Introduction
Goranka Crnogorac,
Wolfgang Schwack*
Introduced 40–70 years ago, dithiocarba-
University of Hohenheim,
Institute of Food Chemistry, mate fungicides (DTCs) still represent an
Garbenstrasse 28, 70599 important class widely used in agriculture.
Stuttgart, Germany They are characterized by a broad spec-
trum of activity against various plant
pathogens, low acute mammal toxicity,
and low production costs. In combination
with modern systemic fungicides, they are
also used to manage resistances and to
broaden the spectrum of activity. It
therefore came as no surprise that the so-
called ‘‘maneb group’’ (zineb, maneb,
mancozeb, propineb, metiram) were some
of the most frequently detected pesticides
in the European Union, Norway, Iceland,
and Liechtenstein, and that this group also
had the highest frequency in exceeding
maximum residue limits (MRLs) [1].
Besides agriculture, DTCs are largely
used as slimicides in pulp and paper
manufacturing, wood preservatives or
vulcanization accelerators in the rubber
industry, which may also be a critical
source of non-residual contamination of
food and feed samples during handling.
Furthermore, DTCs are also used clini-
cally for the treatment of chronic alco-
holism and as anticancer and antitoxic
*
Corresponding author. drug agents [2–5].
Tel.: +49 711 4592 3978; DTCs can be categorized into three sub-
Fax: +49 711 4592 4096;
classes depending upon their carbon
E-mail:
wschwack@uni-hohenheim.de
skeleton [i.e. dimethyldithiocarbamates
40 0165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.10.008
Trends in Analytical Chemistry, Vol. 28, No.
(DMDs), ethylenebis(dithiocarbamates)
(EBDs), and propylenebis(dithiocarba-
mates) (PBDs)] (Table 1). They are mainly
complexed with transition metals (e.g.,
manganese or zinc). Thiram is the most
clearly-defined DMD (thiuramdisulfide),
whereas metiram refers to an unspecified
mixture of polythiuram disulfides and zinc
ammoniate bis(dithiocarbamate).
Concerning chemical structures, dazo-
met is exceptional, and is used as soil
sterilant (precursor of methyl isothiocya-
nate) as is metam, whereas DTCs are
typically applied to the aerial parts of
plants.
DTC analysis was first reviewed by Engst
and Schnaak, who covered the situation up
to 1973 [6]. In their reviews, Malik and
Faubel especially emphasized capillary
electrophoresis [7] and direct photometry
[8]. In addition to a brief discussion of
chromatographic and polarographic
methods, Rao et al. also reviewed colori-
metric methods of direct analysis [9].
Sharma et al. [10] reviewed degradation,
applications and analytical methods of
thiram.
Szolar [11] recently published a broader
survey of analytical methods for the
determination of DTCs. Coming from a
pharmaceutical company, he mainly fo-
cused on the mode of action in biological
systems and the analysis in body fluids.
Our aim is to provide a comprehensive
overview from the point of view of residue
analysts with special emphasis on
methods, that have had a practical impact
in routine residue analysis of DTCs in food
and feed.
Without sodium salts, EBDs and PBDs
forming polymeric chelates are almost
insoluble in both water and organic sol-
vents (Table 1). However, DMDs are
slightly soluble in water and in some polar
Trends in Analytical Chemistry, Vol. 28, No. 1, 2009 Trends

Table 1. Data on the most important dithiocarbamate fungicides (DTCs) [87–89]

Name Annex I1 Formula CAS No. Solubility Solubility in organic solvents

in water
(g/L, 20C)
Ziram + [(CH3)2N-CSS ]2Zn2+ 137-30-4 0.06 Chloroform, carbon disulfide
Ferbam – [(CH3)2N-CSS ]3Fe3+ 14484-64-1 0.13 Chloroform, acetone, acetonitrile
Asomate2 – [(CH3)2N-CSS ]3As3+ 3586-60-5 n.d.f.3 n.d.f
Thiram + (CH3)2N-CSS-SSC-N(CH3)2 137-26-8 0.018 Chloroform, dichloromethane,
acetone,
Metam + (CH3)2NH-CSS Na+ 144-54-7 722 Acetone, ethanol
Nabam – 142-59-6 200 p.i.4
CH2-NH-CSS¯
CH2-NH-CSS¯ 2 Na +

CH2-NH-CSS¯
Zineb – 12122-67-7 p.i. p.i
CH2-NH-CSS¯ Zn 2+

CH2-NH-CSS¯
Maneb + 12427-38-2 p.i. p.i.
CH2-NH-CSS¯ Mn 2+

CH2-NH-CSS¯
Mancozeb + 8018-01-7 p.i. p.i.
CH2-NH-CSS¯ Mn 2+/ Zn2+

CH2-NH-CSS¯
Mancopper – 53988-93-5 p.i. p.i.
CH2-NH-CSS¯ Mn 2+/ Cu2+

CH2 NH CSS CH2 NH CSS

Metiram + 9006-42-2 p.i. p.i.
CH2 NH CSS Zn(NH3) CH2 NH CSS
3 x

CH2 NH CSS Zn SSC N(CH3)2

Polycarbamate5 – 64440-88-6 n.d.f. n.d.f.
CH2 NH CSS Zn SSC N(CH3)2
(Bis-Dithane)
CH3
Propineb + CH2-NH-CSS¯ 12071-83-9 p.i. p.i.
CH2-NH-CSS¯ Zn 2+

S S
Dazomet – 533-74-4 3 Chloroform, acetone, cyclohexane
N N
H3C CH3

1
Annex I, Council Directive 91/414/EEC [86]: included (+), out ( ), pending (p).
2
Used in China.
3
n.d.f., No data found.
4
Practically insoluble.
5
Used in Japan as both plant protectant and antifouling agent in coatings.

organic solvents, especially neutral compounds thiram
and dazomet.
Apart from problems associated with solubility, there
is another big problem in terms of low stability of DTCs
in the presence of plant matrix. Especially when coming
into contact with acidic plant juices, DTCs rapidly de-
grade, since free dithiocarbamic acids do not exist and
will decompose into carbon disulfide (CS2) and the
respective amine, basically reversing the synthesis.
There is therefore no chance to homogenize plant sam-
ples and to extract DTCs by organic solvent, as is stan-
dard procedure in pesticide-residue analyses [12–16].
Against this background, for decades, DTC-residue
analysis used hot-acid digestion, collecting evolving CS2,
which is determined by different techniques, as outlined
below. Consequently, MRLs are specified worldwide as
mg CS2/kg food.
However, specific MRLs are increasingly set by Euro-
pean legislation for certain fungicides to be determined
as they are applied (i.e. thiram, ziram, and propineb)
[17–19], although validated methods of routine analysis
are not available at present. However, using state-of-the-
art techniques [e.g., mass spectrometry (MS)], some
promising approaches have been developed.

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Trends
2. Carbon-disulfide methods
2.1. Spectrophotometry
With respect to the non-extractability of DTCs from
homogenized plant samples, the development of residue-
analysis methods used CS2 as collective marker, follow-
ing release from DTCs upon acidic hydrolysis.
Following a method used in the rubber industry for the
determination of vulcanization accelerators, first at-
tempts used iodometric microtitration after absorption of
evolved CS2 in methanolic potassium-hydroxide solution
[20,21]. But the same authors also presented photo-
metric CS2 determination using a diethylamine/copper-
acetate reagent dissolved in a mixture of triethanolamine
and ethanol, when yellow copper diethyldithiocarba-
mate is formed and measured at 430 nm. This proce-
dure, based on results of Viles [22] and Dickinson [23],
was simultaneously published by Lowen [24]. Applying
the method to standards, recoveries were satisfactory,
but both groups reported the great instability of DTCs in
the presence of plant pulps and in trials of surface
extraction (ÔstrippingÕ) of plant samples followed by the
hydrolysis of the extract. Nevertheless, the method of
digestion/distillation was accepted for several years
[25,26] (see Fig. 1 for the general procedure).
Figure 1. Digestion/distillation apparatus for the determination of
CS2 from residues of dithiocarbamate fungicides. The sample is
weighed into the flask (a), covered by dilute acid and heated under
reflux (b). Released CS2 is pushed through the entire system by a
stream of nitrogen (e) or by applying slight vacuum (f), passing
one or two scrubbing solutions for clean up (c) and finally the
reagent solution for CS2 absorption (d). A second absorption tube
may be used to control break-through due to saturation.
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Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
The first modifications were introduced by Cullen [27],
who recognized that technical-grade triethanolamine
contained high percentages of diethanolamine, which
also entered into the color reaction forming a yellow
copper chelate (kmax 435 nm). He therefore simplified the
reagent, just using diethanolamine and copper acetate
dissolved in ethanol. But he pointed out that two chelate
complexes (1:1 and 1:2) are formed with absorption
maxima of 380 nm and 435 nm, depending upon the
copper/CS2 ratio. Two differently concentrated color re-
agents of 0.016 g/L and 0.048 g/L copper acetate were
therefore suggested for the determination of 200–1000
lg and <200 lg CS2, respectively. Cullen also substi-
tuted the lead-acetate scrubbing solution, formerly used,
with zinc acetate, which facilitated cleaning the trap.
Concerning erratic recovery results for some DTCs and
DTCs in presence of certain crops (tobacco, cottonseed,
apricots, cereals), especially those having a relatively
high organic sulfur content, which interfere with CS2
determination, Keppel evaluated the methodology with
special emphasis on scrubbing solutions for trapping
hydrogen sulfide from the CS2-gas stream and on acidic
hydrolysis conditions [28]. He found that an aqueous
potassium-hydroxide solution (6.5%) instead of lead
acetate or zinc acetate was effective for removing
hydrogen sulfide and other unspecified interferences
from the CS2-gas stream.
While in former studies diluted sulfuric acid was used
for DTC hydrolysis, Keppel introduced hydrochloric acid
(1.6 M) and added tin(II) chloride as reducing agent,
which significantly improved recoveries of different DTCs
from different crop samples. A first collaborative study
found that KeppelÕs method worked well, but disclosed
some variables to be respected and to be investigated [29].
In following years, the method was adopted by official
methods in many countries (e.g., the ÔDFG method S 15Õ
introduced in 1979 by the German Pesticides Commis-
sion [30] or a European norm method [31]). A limit of
detection (LOD) was calculated to be 0.1 mg/kg, which
can be reached using a 500-g sample. During a collab-
orative study, mean recoveries in the range 74–103%
were achieved for different fungicides spiked to different
crops [30].
Concerning special variables during the analysis,
heating rate has to be especially respected. Clarke et al.
reported that EBD can decompose yielding only one mole
CS2 (instead of two moles) at temperatures below boiling
[21]. For thiram as an example of thiuram disulfides, it
was shown that, at lower temperatures (<80C), the
formation of side products (COS, H2S) is favored at the
expense of CS2 [32].
To determine CS2 and COS separately, different amines
can be used as absorbing reagents [33], so, as recom-
mended by DFG method S 15 [30], it is important to
bring the digestion/distillation flask to boiling tempera-
tures quickly, so a high-power heating mantle is
Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
essential. It is worth noting that leakages at multiple ball
joints account for CS2 losses, which need to be especially
considered, when nitrogen is used as carrier gas, by
setting the system under slight positive pressure. In this
respect, use of air is advantageous, while the system
outlet is connected to a vacuum pump regulating the
flow (Fig. 1).
The copper reagent meets its limit when residues
below 0.1 mg/kg are to be determined, as is essential for
dietetic food, including baby food, as well as for control
of ÔorganicÕ food. DFG method S 15 needs to use a
methanolic potassium hydroxide reagent, if it is to
determine <50 lg CS2, and needs to measure the
absorption of the xanthogenate formed at 302 nm using
a 2-cm cuvette. This alternative, intensively studied by
Schwack and Nyanzi [34], was validated by the German
Pesticide Commission [35] and finally adopted by
European norm methods [36]. While the official methods
use a three-wavelength measurement for background
correction, second-derivative spectroscopy has been
recommended to overcome background signals, to im-
prove signal-to-noise ratios and to result in LODs clearly
below 0.01 mg CS2/kg [34,37].
During these studies, different pairs of scrubbing
reagents were also tested [37]. The combination of
sodium-hydroxide solution (10%) and concentrated
sulfuric acid, filling the first and second scrubbing units,
was found to be as effective as the combination of lead-
acetate solution and sulfuric acid, but it was clearly more
effective to use lead-acetate and sodium-hydroxide
scrubbing reagents [30,31]. Sulfuric acid was found to
reduce background interferences significantly. Under
both ecological and economical aspects, the proposed
scrubbing reagents were beneficial and were adopted by
official methods [36].
So far, the digestion/distillation apparatus has com-
prised an assembly, horizontally mounted in series
(Fig. 1). Besides cumbersome assembling/dismantling
and cleaning, it was prone to be set up under tension,
susceptible to breakages, and vulnerable to gas leakages
at the connections.
A ground-breaking new design was published by
Caldas et al. [38], whereby scrubbing and absorption
units were arranged vertically above the reflux con-
denser. Such a vertical design is easier to assemble, less
prone to gas leakages, and especially more space-saving
than the traditional horizontal system. The construction
of the scrubbing unit containing sodium-hydroxide
solution was solved by using an anti-reflux retention
valve.
Later, Schwack et al. presented a vertical digestion/
distillation system, using two Ôsolid-phase extractionÕ
(SPE) sintered glass-bottomed scrubbing traps arranged
in a vertical set-up and filled with boiling chips wetted
with sodium hydroxide solution (50%) and concentrated
sulfuric acid, respectively [39]. Besides the vertical
Trends
design, scrubbing efficiency was shown to be superior
over traditional apparatus, and that is advantageous
with respect to sensitivity of the xanthogenate method,
especially for samples prone to background interferences
(e.g., peppermint tea).
CS2 methods generally face a big problem when crops
of phytogenic CS2 sources are to be analyzed, especially
members of the Brassica or Carica families (e.g., broccoli,
cabbage, cauliflower or papaya) as these are known to
produce glucosinolates and mustard oils, which are
responsible for a false-positive CS2 formation during hot-
acid hydrolysis [40].
2.2. Gas chromatography
To confirm spectrophotometric results and to replace
spectrophotometry, evolved CS2 can be determined by
gas chromatography (GC) and electron-capture or flame-
photometric detection (GC-ECD or GC-FPD). Hot-acid
digestion is therefore performed in a septum-closed ves-
sel, and CS2 is sampled from the headspace (HS) or
simultaneously extracted into an organic solvent (e.g.,
hexane or isooctane), from which a sample is taken for
GC.
McLeod and McCully were the first to report an HS-GC
method using both 10 M sulfuric acid and 4 M hydro-
chloric acid/tin(II) chloride for hydrolysis, but they found
unsatisfactory recoveries for EBDs [41]. As they per-
formed the hydrolysis at only 60C, the low recoveries
agreed with the findings of Clarke et al. [21] mentioned
above.
Following the method of McLeod and McCully, but
heating the flask at 80C overnight, Van Haver and
Gordts found good correlation between KeppelÕs method
and HS-GC, both applied to 95 salad samples [42].
The British Panel on Determination of Dithiocarba-
mate Fungicides recommended 1.5% tin(II) chloride/5 M
hydrochloric-acid solution and a water bath of 80C for
hydrolysis, under which conditions recoveries were sat-
isfactory [43]. Hill and Edmunds briefly and critically
reviewed the state of the art of sample preparation and
HS sampling [44].
Ahmad et al. [45] published further improvements in
the HS-GC technique. They used thiophene as internal
standard – already introduced by Blaicher et al. [46] – to
compensate for injection-volume errors, reduced sample
size to 20 g (finely cut) and used a smaller sample bottle
(140 mL). They reported high recoveries and an LOD of
0.001 mg/kg.
While these attempts – also adopted by a European
norm [47] – employed relatively large HS vessels,
unsuitable for an autosampler, Friedrichs et al. aimed to
use standard 20-mL HS vials, thus reducing sample
weight to 5 g (coarsely sliced) [48]. The closed vial was
first pretreated in an ultrasonic bath for 15 min and then
placed in the autosampler; the hydrolysis conditions
(6 M hydrochloric acid/tin(II) chloride) were 80C for
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Trends
60 min. But, it was pointed out that the whole procedure
requires a certain routine to achieve reproducible results.
Recoveries obtained were in the range 70–90% (below
0.9 mg/kg). Despite low sample weights, an LOD of
0.004 mg/kg was reported by using an improved FPD.
The method is also used (e.g., by Zurich Cantonale
Laboratory [49] and in the baby-food industry) where
juices and pulps packed into glass jars are already
homogenized.
However, for hot-acid digestion inside a HS vessel, it is
not only CS2 that is sampled and injected into GC, but
also a couple of volatile (also sulfur-containing) com-
pounds that possibly interfere with CS2. Also, large
amounts of hydrogen chloride are present in HS, which
must be taken into account in terms of stability of both
the column and the instrument, when, more than ever,
a mass spectrometer may be used for selective detection
[50,51]. To overcome that problem, simultaneous
extraction of CS2 by hydrocarbon solvents during sample
hydrolysis inside a septum-closed bottle has been pre-
sented as an alternative. This type of Ôsolvent-layerÕ
procedure had already been checked and discussed by
the British Panel [43], but they considered the HS pro-
cedure to be more satisfactory.
The procedure was re-evaluated and introduced as
routine by the Central Science Laboratory, using tri-
methylpentane as extraction solvent, thus replacing the
HS procedure [52]. Briefly, a 50-g sample was incubated
in the presence of hydrochloric acid (5.6 M, 150 mL) and
trimethylpentane (25 mL) at 80C for 1 h, whilst being
shaken periodically. After cooling the reaction mixture, a
sub-sample of organic layer (5 lL) was injected into
GC-FPD or GC-MS. For both GC detectors, an LOD of
0.01 mg CS2/kg was reported; data on recoveries (92–
100%) were presented for thiram only, spiked to pears
and lettuce at 0.1 mg/kg. The same procedure was
introduced by the Dutch Food Inspection Service using
isooctane for CS2 extraction [53]. Method validation for
different crops resulted in recoveries of 55–100%, and an
LOD 0.02 mg/kg (GC-ECD) [54]. Identical results were
reported using GC-FPD or GC-MS [55,56].
HS-GC-ECD can also be used for confirmation of a
spectrophotometric result. For example, a 4-mL aliquot
of xanthogenate reagent solution was transferred to an
HS vial, internal standard (ethyl bromide) and sufficient
0.35 M sulfuric acid (6 mL) were added, and the sample
analyzed for liberated CS2 [57]. Following this proce-
dure, LODs of about 0.1 mg CS2/L reagent solution were
obtained. Since the digestion/distillation can be viewed
as a clean-up process, interferences by volatiles from the
matrix were strongly reduced, and, using sulfuric acid,
the GC system was protected against damage from
vapors of hydrochloric acid usually used.
For residue analyses in general, nice recoveries do not
demonstrate that one can correctly analyze real samples.
During a proficiency test with incurred DTC residues in
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Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
dried grape leaves, the best results were obtained with
the xanthogenate method (median 3.44 mg/kg, RSD
36%), while the results obtained by different GC methods
were significantly lower and accompanied by larger
latitude (median 1.88 mg/kg, RSD 73%) [58]. The same
trend was found during a former proficiency test [59].
While sub-sample variability (keeping in mind that res-
idues normally are not uniformly distributed) also holds
true for digestion/distillation methods, a major problem
may result from weighed samples (down to 5 g) for HS-
GC methods, which are ÔfinelyÕ cut to facilitate Ôhomog-
enizationÕ and to pass the neck of the bottle, but are also
responsible for decomposition and losses of DTCs. Cryo-
genic processing (comminution under dry ice at
< 20C) of 1–5 kg samples has therefore been applied
and shown to improve precision and to guarantee sta-
bility of residues during storage at 20C [60]. However,
in contrast to incurred residues, cryo-milling could not
prevent losses of DTCs spiked onto sample surfaces.
3. Liquid chromatography methods for entire
dithiocarbamates
3.1. Liquid chromatography with photometry
In addition to the CS2 digestion/distillation methods de-
scribed above, several techniques for direct determina-
tion of ÔintactÕ DTCs have been published, where ÔintactÕ
refers to DTC anions being accessible to analysis after
alkaline treatment in presence of complexing agents
[e.g., ethylenediaminetetraacetic acid (EDTA)].
DTCs are non-systemic fungicides, so their residues
remain on only the surfaces of fruits and vegetables [61].
Pflugmacher and Ebing used this fact for the first time
and published a gel-permeation chromatography (GPC)
method for the determination of EBDs (maneb, zineb,
mancozeb) and PBD (propineb) on fruits and vegetables
after sample-surface extraction with aqueous EDTA
solution and separation on a Sephadex LH-20 column
with 0.1 M aqueous EDTA as eluent [62]. The mean
recoveries were 78–106% after spiking of samples in the
range 0.25–2.0 mg/kg. For different crops, an LOQ of
0.05 mg/kg was reported except for samples of lettuce
with a higher LOQ of 0.5 mg/kg. This method was
adopted as an official method (i.e. DFG method S 21,
introduced in 1987 by German Research Foundation for
the analysis of ethylene and propylene(bisdithiocarba-
mate) fungicides) [63].
Gustafsson and Fahlgren determined thiram, ziram,
and zineb by high-performance liquid chromatography
(HPLC-UV) on a reversed-phase column (Nucleosil RP-
18) after two separate extraction techniques. Thiram
was extracted by chloroform and directly injected into
HPLC. Zineb and ziram were extracted from the surface
of the crops with aqueous alkaline EDTA/cysteine solu-
tion; subsequently, the DTC anions were extracted into
Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
an organic solvent as ion pairs with tetrabutylammo-
nium hydrogen sulfate and derivatized with methyl
iodide in one step [64]. The reported average recoveries
for different crops and water spiked at 0.5 mg/kg were
59–85%; LODs of 0.01 mg/kg, 0.02 mg/kg, and 0.01
mg/kg were reported for thiram, zineb, and ziram,
respectively.
Many years later, Lo et al. used the same method, but
for the analysis of EBDs (zineb, maneb, mancozeb) and
PBD (propineb) in formulated fungicides only. EBD fun-
gicides were distinguished by atomic absorption spec-
trophotometry (AAS) [65].
Bardarov et al. were the first to apply ion-exchange LC
for determination of propineb (technical product) in air
after aspiration through a filter and surface extraction of
the filter with an aqueous alkaline EDTA solution, but
they did not validate the method and apply it to food
samples [66].
Ion-pair LC methods applying tetraalkylammonium
salts [i.e. cetyltrimethylammonium bromide (CTAB),
tetrabutyl ammonium chloride (TBAC) or tetrabutyl
ammonium hydrogen sulfate (TBAHS)] have often been
reported in literature.
Irth et al. determined zineb and maneb in soil after
extraction with an aqueous alkaline EDTA solution and
selective pre-concentration as ion-pairs on a C18-pre-
column, which was loaded on-line with CTAB [67]. In a
separate run, thiram was determined in samples of soil,
apple, and lettuce after extraction with dichloromethane
using a reversed-phase column and post-column com-
plexation with copper(II). For soil samples, spiked at
1 mg/kg, the reported recoveries for zineb, maneb, and
thiram were high (>98%) and LODs were 0.1 mg/kg.
LODs for thiram were generally rather low (0.005–0.01
mg/kg) apart from those for lettuce (0.05–0.1 mg/kg).
Dhoot et al. separated metam-sodium and its metab-
olite, methyl isothiocyanate, in tap water and raw sur-
face water on a strong anion-exchange column with
methanol/buffered water containing 10 mM CTAB as
eluent [68] and reported recoveries of 108–114%.
Determination of ziram residues on spinach by ion-
pair LC using aqueous solution containing 1 mM EDTA
and 10 mM TBAC as eluent was published by Atienza
et al. [69]. The authors tested two different extraction
procedures for lyophilized samples:
a) with methanol/aqueous EDTA solution and subse-
quent liquid-liquid partition with hexane as clean
up; and,
b) with supercritical carbon dioxide containing metha-
nol as organic modifier.
Recoveries at high fortification levels obtained by
extraction procedure b) were generally lower than those
by a), especially when sample weights >0.25 g were
used. At a fortification level of 0.7 mg/kg, recoveries of
96% and 20% were determined using method b) for
0.25-g and 1.0-g samples, respectively. Additionally,
Trends
recoveries decreased with increasing fortification levels
for method b). However, extraction method a) was not
affected by sample amount or fortification level. LODs of
approximately 0.05 mg/kg were reported.
Applying ion-pair LC with chemiluminescence detec-
tion, Nakazawa et al. determined mancozeb and propi-
neb in cucumbers and apples [70]. They extracted
samples cut into small pieces with aqueous buffer com-
prising 10 mM TBAHS, EDTA, disodium hydrogen
phosphate, and 0.1% tri(2-carboxyethyl)phosphine
(TCEP) as stabilizer for DTCs. Method validation resulted
in recoveries of 88–109% for cucumbers and apples
spiked in the range 1–10 mg/kg; LODs were 0.09 lg/kg
and 0.4 lg/kg for mancozeb and propineb, respectively.
Van Lishaut and Schwack were the first to determine
all three DTC sub-classes [i.e. DMD (ziram, ferbam), EBD
(maneb, zineb, mancozeb), and PBD (propineb)] by a
rapid ion-pair LC method with UV and electrochemical
detection [71]. The surface extraction was performed
applying an aqueous extraction buffer (pH 11.0) con-
taining TBAHS, EDTA, and disodium hydrogen phos-
phate. For stabilization of DTCs, sodium hydrogen sulfite
(1 g/L) was added to the extraction buffer before use. The
DTCs were separated on a Supelcogel ODP-50 (C18-
polyvinylalcohol) column using methanol/extraction
buffer as eluent. The method validation for different
fruits and vegetables resulted in recoveries of 72–111%
at different fortification levels and LODs of 4–8 lg CS2/L
using electrochemical detection.
Three years later, Perz and Schwack reported an im-
proved method using a new alkaline-stable reversed-
phase column (XTerra RP18) and increased the stability
of DTCs in alkaline-sample extracts by adding cysteine
[72]. Fig. 2 shows as an example the chromatogram of
Figure 2. Separation of dithiocarbamates (DTCs) by ion-pair liquid
chromatography with ultraviolet detection on the stable alkaline
XTerra RP18 column [72].
http://www.elsevier.com/locate/trac 45
Trends
DTC standards. Recoveries of 93–120% were determined
for different fruits and vegetables. Thiram could also be
determined using the same chromatography, but after
separate extraction by a buffer containing sulfite, which
guaranteed thiram transformation into a DMD-sulfite
adduct. Thiram was recovered from cucumbers spiked at
0.05–0.5 mg CS2/kg in the range 78–91%.
Aulakh et al. published an HPLC-UV method for the
determination of thiram and thiourea in wheat grains
and in a commercial fungicide (Thiram 75 WP) [73].
After extraction of the crushed sample with chloroform,
filtration, and evaporation of extracts, the residues were
dissolved in acetonitrile and separated on a C18 column
with acetonitrile/water as eluent. LODs of 5.2 lg/L and
4.9 lg/L were determined for thiram and thiourea,
respectively. From wheat grain spiked with thiram in the
range 0.2–0.8 mg/kg, recoveries were 90–99%.
Garcinuno et al. determined maneb and its main
metabolites [ethylenethiourea (ETU), ethylenebis(iso-
thiocyanate) sulfide (EBIS) and ethyleneurea (EU)] in
tomatoes, which were chopped and extracted using a
mixture of acetonitrile, dichloromethane, and chloro-
form [74]. Analysis was performed on a reversed-phase
column with acetonitrile/methanol/100 mM aqueous
sodium dodecylsulfate (SDS) as eluent, but EU and
maneb coeluted.
Özhan and Alpertunga also published a method for
determination of maneb and its main degradation
product (ETU) in different fruit juices using two reversed-
phase columns (Luna CN and C18) with different
retention mechanisms [75]. The authors tested two dif-
ferent extraction procedures using dichloromethane: a)
SPE; and, b) liquid-liquid extraction (LLE), whereby SPE
was favored. For maneb in tomato juice, recoveries of
90–101% and an LOQ of 0.1 mg/L were reported. On
studying different samples of juice, maneb was found in
one tomato juice at a concentration of 0.45 mg/L.
3.2. Liquid chromatography with mass spectrometry
Compared to LC-UV, LC-MS is quite rarely reported in the
literature (Table 2). Concerning demands, only LC-MS
methods fulfill the requirements for both selectivity and
sensitivity.
Based on sample preparation and derivatization with
methyl iodide introduced by Gustafsson and Fahlgren
[64] and clean up on Sep-PAK C18 cartridges, Hanada
et al. determined EBDs (zineb, maneb, and mancozeb) in
environmental water samples by LC-electrospray ioni-
zation (ESI)-MS in negative-ion mode [76]. The average
recovery at sub-ppb level was 79% with a coefficient of
variation of 29%.
Several LC-atmospheric pressure chemical ionization
(APCI)-MS methods in positive-ion mode have been re-
ported.
Moriwaki et al. presented an LC-APCI-MS method for
the determination of ziram, but only standards dissolved
46 http://www.elsevier.com/locate/trac
Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
in chloroform (2–20 mg/L) were separated on a GPC
column [77].
Blasco et al. also published an LC-APCI-MS method,
which referred to the determination of neutral DTCs only
(i.e. thiram, dazomet and disulfiram, including ETU and
PTU) in different plants [78]. After cutting and homo-
genizing the samples, extraction and clean up was per-
formed by matrix solid-phase dispersion and column
elution with dichloromethane/methanol. With the help
of matrix-matched standards, recoveries of 60–101%
(standard deviation 4–21%) were obtained for thiram,
spiked at 2.5 mg/kg in different matrices. However,
thiram could not be recovered from high acidic fruits
(e.g., oranges and lemons). Surprisingly, disulfiram
could not be recovered from nuts.
Crnogorac and Schwack published an LC-MS method
for all three DTC sub-classes (i.e. DMD, EBD, and PBD)
using a new alkaline-stable polymeric HILIC (hydrophilic
interaction LC) phase and a simple mobile phase com-
prising acetonitrile and aqueous ammonia (10 mM)
[79]. For the surface extraction of whole samples, they
used a sodium hydrogen carbonate buffer (pH 12) con-
taining D,L-penicillamine to stabilize DTCs. They used
deuterated DMD and EBD as internal standards, allowing
isotope-dilution assay and compensating for losses from
extracts by degradation. They operated the single-
quadrupole MS in negative ESI mode. LODs (expressed as
CS2) of 16 lg/kg, 24 lg/kg, and 20 lg/kg were deter-
mined for DMD, EBD, and PBD, respectively. Recovery
studies with grapes, cucumbers, tomatoes, and rucola
revealed recovery rates in the range 90–100% at dif-
ferent spiking levels of ziram, dithane, and antracol.
Concerning both selectivity and sensitivity, LC-tandem
MS (LC-MS2) is preferred over LC-MS, if low LODs are
required, especially for crops to be processed into baby
food (diet regulations) [80].
Hayama et al. determined fungicide polycarbamate in
tap water and river water by LC-APCI-MS2 after adding
an alkaline EDTA/cysteine solution to the water sample,
derivatization with dimethyl sulfate and subsequent
clean up using SPE [81]. Mean recoveries of polycarba-
mate from distilled water in the spiking range 0.25–5.0
lg/L were 63–74% in the form of DMD-methyl and EBD-
dimethyl, respectively. The reported LODs in water
samples were amazingly low (i.e. 0.061 lg/L and 0.032
lg/L for DMDs and EBDs, respectively).
Brewin et al. published an LC-MS2 method for the
determination of EBDs (mancozeb, maneb, zineb, nabam,
metiram) in different fruits and vegetables after sample
extraction with an aqueous EDTA/cysteine solution,
derivatization with methyl iodide and subsequent SPE
clean up [82]. They obtained an LOQ for EBDs in apples,
grapes, and tomatoes of 0.01 mg/kg and recoveries of
79–104% from lettuce, wheat and onions spiked at 0.01
mg/kg. The authors used this method within their rou-
tine analyses of wheat samples for registration studies
 Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
Table 2. LC-MS and LC-MS2 methods for determination of dithiocarbamate fungicide (DTC) residues

Author Method DTCs Matrix LOD/LOQ/Recoveries Comments Year [reference]

Moriwaki ziram no matrix, LOD: 2001 [77]
et al. LC-APCI-MS (positive-ion mode) just ziram 2 mg/L Calibration graph in the high
Chromatography: standard range of 2–20 mg/L
 Tosoh TSK-GEL-2500 GPC-column High LOD
 eluent: methanol

Hanada et al. environ- 2002 [76]

EBDs:
LC-ESI-MS (negative-ion mode) mental LOD: Extraction with chloroform
Chromatography:  mancozeb mancozeb 0.043 lg/L Derivatization with methyl
water
 Inertsil ODS-80A column (RP-18)  maneb Recoveries at sub-ppb: iodide (carcinogen)
 eluent: acetonitrile/water (gradient)  zineb 79% (RSD 29%) High RSD (29%)

Sample preparation:
 Gustafsson and Fahlgren method [64]
Blasco et al. 2004 [78]
LC-APCI-MS (positive-ion mode) Neutral DTCs: avocados LOQs: Unsatisfactory for fruits with
Chromatography:  dazomet cherries dazomet 0.5 mg/kg high acid content (orange and
 disulfiram lemons disulfiram 2.5 mg/kg lemon), thiram/disulfiram not
 C8-column
 thiram nuts thiram 2.5 mg/kg recovered
 eluent: methanol/water (gradient) oat ETU 0.25 mg/kg Unsatisfactory for nuts, disul-
Sample preparation: Metabolites: oranges
PTU 0.25 mg/kg firam not recovered
1. chopping and homogenization  ETU peaches
Recoveries at LOQ and High RSD: 4–21%
 PTU rice 10xLOQ: 33–109%
2. matrix solid-phase dispersion with
tomatoes
Carbograph, elution with dichloro-
methane/methanol (80:20, v/v)
Hayama et al. polycarbamate Only water samples 2007 [81]
LC-APCI-MS2 (positive-ion mode) tap water LOQs:
Chromatography: river water DMD 0.2 lg/L
 Luna C18-column EBD 0.11 lg/L
 eluent: methanol/water (3:2, v/v) Recoveries from distilled
water at 0.25–5.0 lg/L:
Sample preparation: DMD 63%
1. treatment with alkaline EDTA/cysteine EBD: 74%
2. derivatization with dimethyl sulfate
3. solid-phase extraction with Oasis HLB
http://www.elsevier.com/locate/trac

cartridges
Crnogorac 2007 [79]
LC-ESI-MS (negative-ion mode) DMDs tomatoes LOQs for tomatoes: Multi-analyte method
and Schwack
Chromatography: EBDs grapes DMDs 0.04 mg/kg No derivatization
PBDs
 ZIQ-pHILIC column cucumbers EBDs 0.05 mg/kg No clean up
 eluent: acetonitrile/10 mM ammonia (gradient) rucola PBDs 0.04 mg/kg
Recoveries at 0.01–0.9 mg/kg:
Sample preparation: 90–100% (RSD 2%)
extraction with alkaline buffer (sodium
hydrogen carbonate/DL-penicillamine)
(continued on next page)

Trends
47
Trends Trends in Analytical Chemistry, Vol. 28, No. 1, 2009

Year [reference]
2008 [82]

2008 [83]
iodide
No multi-analyte method

Multi-analyte method
methyl

No derivatization
(carcinogen)

No clean up
Comments

Use of

97–101%
LOQs for apples, grapes, and

lettuce,

Recoveries from tomatoes at

wheat, and onions at 0.01 mg/
LOD/LOQ/Recoveries

tomatoes: 0.01 mg/kg

LOQs for tomatoes:

from

mg/kg:
DMDs 8 lg/kg
EBDs 3 lg/kg
PBDs 5 lg/kg
kg: 79–104%
Recoveries

(RSD 3%)

Figure 3. LC-MS2 (MRM) chromatograms of (a) DTC standards and

0.05–1

(b) deuterium-labeled DTC standards (each 100 lg/L), separated on

a ZIC-pHILIC column.

and found good correlations between LC-MS and the CS2

cocktail tomatoes
cherry tomatoes

cooking method.
Employing an LC-MS2 system, Crnogorac et al. could
cucumbers

tamarillos
tomatoes

tomatoes

improve on their previously published HILIC (LC-MS)

broccoli
Matrix

papaya
lettuce

onions
grapes

grapes
apples

apples
wheat

pears

method (Fig. 3). This work resulted in LODs reduced by a

factor of about 10 (i.e. approximately 1–5 lg/kg) [83].
After studying several fruits and vegetables from the
 mancozeb

 metiram
 nabam
 maneb

local market and different countries of origin, the

 zineb

authors concluded that the results obtained by both LC-

DMDs
DTCs

EBDs

EBDs
PBDs

MS and LC-MS2 were in good agreement each other and

with the results obtained by the CS2 digestion/distillation
method [36], except for false-positive CS2 findings in
 eluent: acetonitrile/10 mM ammonia (gradient)

samples of rucola and broccoli. CS2 found by the diges-

tion/distillation method clearly indicated the phytogenic
2. methylation with methyl iodide
1. extraction with EDTA/cysteine

sources of CS2 (mentioned above in Section 2).

extraction with alkaline buffer (sodium
hydrogen carbonate/DL-penicillamine)
3. clean up with C18 cartridges

LC-ESI-MS2 (negative-ion mode)

LC-ESI-MS2 (positive-ion mode)

4. Summary
 column: no information
 eluent: no information

 ZIQ-pHILIC column

Over the long period of usage of DTC fungicides, method

Sample preparation:

Sample preparation:

development of residue analysis has been continually

Chromatography:

Chromatography:

improved in terms of selectivity and sensitivity. Whereas

digestion/distillation methods combined with spectro-
photometry have the longest history and are still prac-
Method

tically quite important, activities were especially initiated

Table 2. (continued)

to introduce more rapid methods to circumvent distilla-

tion and result in HS-GC techniques, that are also having
Brewin et al.

a practical impact. LC-MS and LC-MS2 methods for the

Crnogorac

direct determination of DTC anions avoid derivatization

Author

et al.

by alkylating agents, so they open new avenues for ra-

pid, sensitive analysis.

48 http://www.elsevier.com/locate/trac
Trends in Analytical Chemistry, Vol. 28, No. 1, 2009
Very recently, new rules for EU-harmonized MRLs for
pesticides in food were published [84,85]. MRLs of DTCs
(including maneb, mancozeb, metiram, propineb,
thiram, and ziram) are generally expressed as mg CS2/kg
food. However, as was pointed out: ‘‘the MRLs expressed
as CS2 can arise from different dithiocarbamates and
therefore they do not reflect a single Good Agricultural
Practice (GAP). It is therefore not appropriate to use
these MRLs to check compliance with a GAP.’’ Besides
CS2, specific MRLs are fixed for thiram, ziram and pro-
pineb, whereas it was commented that: ‘‘there are single
residue methods available, and that these methods
should be implemented on a case by case basis when the
specific quantification of propineb, ziram and/or thiram
is required’’. But, no reference was given.
In addition, some DTCs were not included in Annex I
[86], so they are not allowed to be used and a general
MRL of 0.01 mg/kg applies, unless otherwise specified.
In our view, there is at present no chance to control
unauthorized fields of application as well as to control
residues of distinct DTC compounds, so residue analysis
of DTC fungicides faces quite a challenge to fulfill the
requirements already regulated by European legislation,
while still lacking potential analytical or validated
methods.
However, with the development of new column
materials to be used effectively for LC separation of DTCs
and detection by MS, we have clearly set out opportu-
nities within the field of DTC-residue analysis. They
promise very sensitive analyses while avoiding false-po-
sitive results, as reported for CS2 methods, and method
validation is in progress.
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