Trends Trends in Analytical Chemistry, Vol. 28, No.

1, 2009

Residue analysis of dithiocarbamate

fungicides
Goranka Crnogorac, Wolfgang Schwack

We review the development of methods for the determination of dithiocar- (DMDs), ethylenebis(dithiocarbamates)
bamate fungicide residues in foodstuffs. We discuss all available methods, (EBDs), and propylenebis(dithiocarba-
from colorimetric carbon-disulfide methods to liquid chromatography mates) (PBDs)] (Table 1). They are mainly
combined with tandem mass spectrometry. Our main focus is on the complexed with transition metals (e.g.,
relevance of analytical methods with respect to routine use for food control manganese or zinc). Thiram is the most
and efficiency in meeting the requirements of current European legislation. clearly-defined DMD (thiuramdisulfide),
ª 2008 Elsevier Ltd. All rights reserved. whereas metiram refers to an unspecified
mixture of polythiuram disulfides and zinc
Keywords: Analysis; Determination; Dithiocarbamate fungicide; Carbon disulfide;
ammoniate bis(dithiocarbamate).
Colorimetry; LC-MS; LC-MS2; Liquid chromatography; Mass spectrometry; Residue
Concerning chemical structures, dazo-
1. Introduction met is exceptional, and is used as soil
Goranka Crnogorac, sterilant (precursor of methyl isothiocya-
Wolfgang Schwack*
Introduced 40–70 years ago, dithiocarba- nate) as is metam, whereas DTCs are
University of Hohenheim, typically applied to the aerial parts of
Institute of Food Chemistry, mate fungicides (DTCs) still represent an
Garbenstrasse 28, 70599 important class widely used in agriculture. plants.
Stuttgart, Germany They are characterized by a broad spec- DTC analysis was first reviewed by Engst
trum of activity against various plant and Schnaak, who covered the situation up
pathogens, low acute mammal toxicity, to 1973 [6]. In their reviews, Malik and
and low production costs. In combination Faubel especially emphasized capillary
with modern systemic fungicides, they are electrophoresis [7] and direct photometry
also used to manage resistances and to [8]. In addition to a brief discussion of
broaden the spectrum of activity. It chromatographic and polarographic
therefore came as no surprise that the so- methods, Rao et al. also reviewed colori-
called ‘‘maneb group’’ (zineb, maneb, metric methods of direct analysis [9].
mancozeb, propineb, metiram) were some Sharma et al. [10] reviewed degradation,
of the most frequently detected pesticides applications and analytical methods of
in the European Union, Norway, Iceland, thiram.
and Liechtenstein, and that this group also Szolar [11] recently published a broader
had the highest frequency in exceeding survey of analytical methods for the
maximum residue limits (MRLs) [1]. determination of DTCs. Coming from a
Besides agriculture, DTCs are largely pharmaceutical company, he mainly fo-
used as slimicides in pulp and paper cused on the mode of action in biological
manufacturing, wood preservatives or systems and the analysis in body fluids.
vulcanization accelerators in the rubber Our aim is to provide a comprehensive
industry, which may also be a critical overview from the point of view of residue
source of non-residual contamination of analysts with special emphasis on
food and feed samples during handling. methods, that have had a practical impact
Furthermore, DTCs are also used clini- in routine residue analysis of DTCs in food
cally for the treatment of chronic alco- and feed.
holism and as anticancer and antitoxic Without sodium salts, EBDs and PBDs
*
Corresponding author. drug agents [2–5]. forming polymeric chelates are almost
Tel.: +49 711 4592 3978; DTCs can be categorized into three sub- insoluble in both water and organic sol-
Fax: +49 711 4592 4096;
classes depending upon their carbon vents (Table 1). However, DMDs are
E-mail: slightly soluble in water and in some polar
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. 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 Against this background, for decades, DTC-residue
and dazomet. analysis used hot-acid digestion, collecting evolving CS2,
Apart from problems associated with solubility, there which is determined by different techniques, as outlined
is another big problem in terms of low stability of DTCs below. Consequently, MRLs are specified worldwide as
in the presence of plant matrix. Especially when coming mg CS2/kg food.
into contact with acidic plant juices, DTCs rapidly de- However, specific MRLs are increasingly set by Euro-
grade, since free dithiocarbamic acids do not exist and pean legislation for certain fungicides to be determined
will decompose into carbon disulfide (CS2) and the as they are applied (i.e. thiram, ziram, and propineb)
respective amine, basically reversing the synthesis. [17–19], although validated methods of routine analysis
There is therefore no chance to homogenize plant sam- are not available at present. However, using state-of-the-
ples and to extract DTCs by organic solvent, as is stan- art techniques [e.g., mass spectrometry (MS)], some
dard procedure in pesticide-residue analyses [12–16]. promising approaches have been developed.

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

2. Carbon-disulfide methods The first modifications were introduced by Cullen [27],

who recognized that technical-grade triethanolamine
2.1. Spectrophotometry contained high percentages of diethanolamine, which
With respect to the non-extractability of DTCs from also entered into the color reaction forming a yellow
homogenized plant samples, the development of residue- copper chelate (kmax 435 nm). He therefore simplified the
analysis methods used CS2 as collective marker, follow- reagent, just using diethanolamine and copper acetate
ing release from DTCs upon acidic hydrolysis. dissolved in ethanol. But he pointed out that two chelate
Following a method used in the rubber industry for the complexes (1:1 and 1:2) are formed with absorption
determination of vulcanization accelerators, first at- maxima of 380 nm and 435 nm, depending upon the
tempts used iodometric microtitration after absorption of copper/CS2 ratio. Two differently concentrated color re-
evolved CS2 in methanolic potassium-hydroxide solution agents of 0.016 g/L and 0.048 g/L copper acetate were
[20,21]. But the same authors also presented photo- therefore suggested for the determination of 200–1000
metric CS2 determination using a diethylamine/copper- lg and <200 lg CS2, respectively. Cullen also substi-
acetate reagent dissolved in a mixture of triethanolamine tuted the lead-acetate scrubbing solution, formerly used,
and ethanol, when yellow copper diethyldithiocarba- with zinc acetate, which facilitated cleaning the trap.
mate is formed and measured at 430 nm. This proce- Concerning erratic recovery results for some DTCs and
dure, based on results of Viles [22] and Dickinson [23], DTCs in presence of certain crops (tobacco, cottonseed,
was simultaneously published by Lowen [24]. Applying apricots, cereals), especially those having a relatively
the method to standards, recoveries were satisfactory, high organic sulfur content, which interfere with CS2
but both groups reported the great instability of DTCs in determination, Keppel evaluated the methodology with
the presence of plant pulps and in trials of surface special emphasis on scrubbing solutions for trapping
extraction (ÔstrippingÕ) of plant samples followed by the hydrogen sulfide from the CS2-gas stream and on acidic
hydrolysis of the extract. Nevertheless, the method of hydrolysis conditions [28]. He found that an aqueous
digestion/distillation was accepted for several years potassium-hydroxide solution (6.5%) instead of lead
[25,26] (see Fig. 1 for the general procedure). 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
Figure 1. Digestion/distillation apparatus for the determination of
formation of side products (COS, H2S) is favored at the
CS2 from residues of dithiocarbamate fungicides. The sample is
weighed into the flask (a), covered by dilute acid and heated under expense of CS2 [32].
reflux (b). Released CS2 is pushed through the entire system by a To determine CS2 and COS separately, different amines
stream of nitrogen (e) or by applying slight vacuum (f), passing can be used as absorbing reagents [33], so, as recom-
one or two scrubbing solutions for clean up (c) and finally the mended by DFG method S 15 [30], it is important to
reagent solution for CS2 absorption (d). A second absorption tube
bring the digestion/distillation flask to boiling tempera-
may be used to control break-through due to saturation.
tures quickly, so a high-power heating mantle is

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

essential. It is worth noting that leakages at multiple ball design, scrubbing efficiency was shown to be superior
joints account for CS2 losses, which need to be especially over traditional apparatus, and that is advantageous
considered, when nitrogen is used as carrier gas, by with respect to sensitivity of the xanthogenate method,
setting the system under slight positive pressure. In this especially for samples prone to background interferences
respect, use of air is advantageous, while the system (e.g., peppermint tea).
outlet is connected to a vacuum pump regulating the CS2 methods generally face a big problem when crops
flow (Fig. 1). of phytogenic CS2 sources are to be analyzed, especially
The copper reagent meets its limit when residues members of the Brassica or Carica families (e.g., broccoli,
below 0.1 mg/kg are to be determined, as is essential for cabbage, cauliflower or papaya) as these are known to
dietetic food, including baby food, as well as for control produce glucosinolates and mustard oils, which are
of ÔorganicÕ food. DFG method S 15 needs to use a responsible for a false-positive CS2 formation during hot-
methanolic potassium hydroxide reagent, if it is to acid hydrolysis [40].
determine <50 lg CS2, and needs to measure the
absorption of the xanthogenate formed at 302 nm using 2.2. Gas chromatography
a 2-cm cuvette. This alternative, intensively studied by To confirm spectrophotometric results and to replace
Schwack and Nyanzi [34], was validated by the German spectrophotometry, evolved CS2 can be determined by
Pesticide Commission [35] and finally adopted by gas chromatography (GC) and electron-capture or flame-
European norm methods [36]. While the official methods photometric detection (GC-ECD or GC-FPD). Hot-acid
use a three-wavelength measurement for background digestion is therefore performed in a septum-closed ves-
correction, second-derivative spectroscopy has been sel, and CS2 is sampled from the headspace (HS) or
recommended to overcome background signals, to im- simultaneously extracted into an organic solvent (e.g.,
prove signal-to-noise ratios and to result in LODs clearly hexane or isooctane), from which a sample is taken for
below 0.01 mg CS2/kg [34,37]. GC.
During these studies, different pairs of scrubbing McLeod and McCully were the first to report an HS-GC
reagents were also tested [37]. The combination of method using both 10 M sulfuric acid and 4 M hydro-
sodium-hydroxide solution (10%) and concentrated chloric acid/tin(II) chloride for hydrolysis, but they found
sulfuric acid, filling the first and second scrubbing units, unsatisfactory recoveries for EBDs [41]. As they per-
was found to be as effective as the combination of lead- formed the hydrolysis at only 60C, the low recoveries
acetate solution and sulfuric acid, but it was clearly more agreed with the findings of Clarke et al. [21] mentioned
effective to use lead-acetate and sodium-hydroxide above.
scrubbing reagents [30,31]. Sulfuric acid was found to Following the method of McLeod and McCully, but
reduce background interferences significantly. Under heating the flask at 80C overnight, Van Haver and
both ecological and economical aspects, the proposed Gordts found good correlation between KeppelÕs method
scrubbing reagents were beneficial and were adopted by and HS-GC, both applied to 95 salad samples [42].
official methods [36]. The British Panel on Determination of Dithiocarba-
So far, the digestion/distillation apparatus has com- mate Fungicides recommended 1.5% tin(II) chloride/5 M
prised an assembly, horizontally mounted in series hydrochloric-acid solution and a water bath of 80C for
(Fig. 1). Besides cumbersome assembling/dismantling hydrolysis, under which conditions recoveries were sat-
and cleaning, it was prone to be set up under tension, isfactory [43]. Hill and Edmunds briefly and critically
susceptible to breakages, and vulnerable to gas leakages reviewed the state of the art of sample preparation and
at the connections. HS sampling [44].
A ground-breaking new design was published by Ahmad et al. [45] published further improvements in
Caldas et al. [38], whereby scrubbing and absorption the HS-GC technique. They used thiophene as internal
units were arranged vertically above the reflux con- standard – already introduced by Blaicher et al. [46] – to
denser. Such a vertical design is easier to assemble, less compensate for injection-volume errors, reduced sample
prone to gas leakages, and especially more space-saving size to 20 g (finely cut) and used a smaller sample bottle
than the traditional horizontal system. The construction (140 mL). They reported high recoveries and an LOD of
of the scrubbing unit containing sodium-hydroxide 0.001 mg/kg.
solution was solved by using an anti-reflux retention While these attempts – also adopted by a European
valve. norm [47] – employed relatively large HS vessels,
Later, Schwack et al. presented a vertical digestion/ unsuitable for an autosampler, Friedrichs et al. aimed to
distillation system, using two Ôsolid-phase extractionÕ use standard 20-mL HS vials, thus reducing sample
(SPE) sintered glass-bottomed scrubbing traps arranged weight to 5 g (coarsely sliced) [48]. The closed vial was
in a vertical set-up and filled with boiling chips wetted first pretreated in an ultrasonic bath for 15 min and then
with sodium hydroxide solution (50%) and concentrated placed in the autosampler; the hydrolysis conditions
sulfuric acid, respectively [39]. Besides the vertical (6 M hydrochloric acid/tin(II) chloride) were 80C for

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

60 min. But, it was pointed out that the whole procedure dried grape leaves, the best results were obtained with
requires a certain routine to achieve reproducible results. the xanthogenate method (median 3.44 mg/kg, RSD
Recoveries obtained were in the range 70–90% (below 36%), while the results obtained by different GC methods
0.9 mg/kg). Despite low sample weights, an LOD of were significantly lower and accompanied by larger
0.004 mg/kg was reported by using an improved FPD. latitude (median 1.88 mg/kg, RSD 73%) [58]. The same
The method is also used (e.g., by Zurich Cantonale trend was found during a former proficiency test [59].
Laboratory [49] and in the baby-food industry) where While sub-sample variability (keeping in mind that res-
juices and pulps packed into glass jars are already idues normally are not uniformly distributed) also holds
homogenized. true for digestion/distillation methods, a major problem
However, for hot-acid digestion inside a HS vessel, it is may result from weighed samples (down to 5 g) for HS-
not only CS2 that is sampled and injected into GC, but GC methods, which are ÔfinelyÕ cut to facilitate Ôhomog-
also a couple of volatile (also sulfur-containing) com- enizationÕ and to pass the neck of the bottle, but are also
pounds that possibly interfere with CS2. Also, large responsible for decomposition and losses of DTCs. Cryo-
amounts of hydrogen chloride are present in HS, which genic processing (comminution under dry ice at
must be taken into account in terms of stability of both < 20C) of 1–5 kg samples has therefore been applied
the column and the instrument, when, more than ever, and shown to improve precision and to guarantee sta-
a mass spectrometer may be used for selective detection bility of residues during storage at 20C [60]. However,
[50,51]. To overcome that problem, simultaneous in contrast to incurred residues, cryo-milling could not
extraction of CS2 by hydrocarbon solvents during sample prevent losses of DTCs spiked onto sample surfaces.
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 3. Liquid chromatography methods for entire
the British Panel [43], but they considered the HS pro- dithiocarbamates
cedure to be more satisfactory.
The procedure was re-evaluated and introduced as 3.1. Liquid chromatography with photometry
routine by the Central Science Laboratory, using tri- In addition to the CS2 digestion/distillation methods de-
methylpentane as extraction solvent, thus replacing the scribed above, several techniques for direct determina-
HS procedure [52]. Briefly, a 50-g sample was incubated tion of ÔintactÕ DTCs have been published, where ÔintactÕ
in the presence of hydrochloric acid (5.6 M, 150 mL) and refers to DTC anions being accessible to analysis after
trimethylpentane (25 mL) at 80C for 1 h, whilst being alkaline treatment in presence of complexing agents
shaken periodically. After cooling the reaction mixture, a [e.g., ethylenediaminetetraacetic acid (EDTA)].
sub-sample of organic layer (5 lL) was injected into DTCs are non-systemic fungicides, so their residues
GC-FPD or GC-MS. For both GC detectors, an LOD of remain on only the surfaces of fruits and vegetables [61].
0.01 mg CS2/kg was reported; data on recoveries (92– Pflugmacher and Ebing used this fact for the first time
100%) were presented for thiram only, spiked to pears and published a gel-permeation chromatography (GPC)
and lettuce at 0.1 mg/kg. The same procedure was method for the determination of EBDs (maneb, zineb,
introduced by the Dutch Food Inspection Service using mancozeb) and PBD (propineb) on fruits and vegetables
isooctane for CS2 extraction [53]. Method validation for after sample-surface extraction with aqueous EDTA
different crops resulted in recoveries of 55–100%, and an solution and separation on a Sephadex LH-20 column
LOD 0.02 mg/kg (GC-ECD) [54]. Identical results were with 0.1 M aqueous EDTA as eluent [62]. The mean
reported using GC-FPD or GC-MS [55,56]. recoveries were 78–106% after spiking of samples in the
HS-GC-ECD can also be used for confirmation of a range 0.25–2.0 mg/kg. For different crops, an LOQ of
spectrophotometric result. For example, a 4-mL aliquot 0.05 mg/kg was reported except for samples of lettuce
of xanthogenate reagent solution was transferred to an with a higher LOQ of 0.5 mg/kg. This method was
HS vial, internal standard (ethyl bromide) and sufficient adopted as an official method (i.e. DFG method S 21,
0.35 M sulfuric acid (6 mL) were added, and the sample introduced in 1987 by German Research Foundation for
analyzed for liberated CS2 [57]. Following this proce- the analysis of ethylene and propylene(bisdithiocarba-
dure, LODs of about 0.1 mg CS2/L reagent solution were mate) fungicides) [63].
obtained. Since the digestion/distillation can be viewed Gustafsson and Fahlgren determined thiram, ziram,
as a clean-up process, interferences by volatiles from the and zineb by high-performance liquid chromatography
matrix were strongly reduced, and, using sulfuric acid, (HPLC-UV) on a reversed-phase column (Nucleosil RP-
the GC system was protected against damage from 18) after two separate extraction techniques. Thiram
vapors of hydrochloric acid usually used. was extracted by chloroform and directly injected into
For residue analyses in general, nice recoveries do not HPLC. Zineb and ziram were extracted from the surface
demonstrate that one can correctly analyze real samples. of the crops with aqueous alkaline EDTA/cysteine solu-
During a proficiency test with incurred DTC residues in tion; subsequently, the DTC anions were extracted into

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

an organic solvent as ion pairs with tetrabutylammo- recoveries decreased with increasing fortification levels
nium hydrogen sulfate and derivatized with methyl for method b). However, extraction method a) was not
iodide in one step [64]. The reported average recoveries affected by sample amount or fortification level. LODs of
for different crops and water spiked at 0.5 mg/kg were approximately 0.05 mg/kg were reported.
59–85%; LODs of 0.01 mg/kg, 0.02 mg/kg, and 0.01 Applying ion-pair LC with chemiluminescence detec-
mg/kg were reported for thiram, zineb, and ziram, tion, Nakazawa et al. determined mancozeb and propi-
respectively. neb in cucumbers and apples [70]. They extracted
Many years later, Lo et al. used the same method, but samples cut into small pieces with aqueous buffer com-
for the analysis of EBDs (zineb, maneb, mancozeb) and prising 10 mM TBAHS, EDTA, disodium hydrogen
PBD (propineb) in formulated fungicides only. EBD fun- phosphate, and 0.1% tri(2-carboxyethyl)phosphine
gicides were distinguished by atomic absorption spec- (TCEP) as stabilizer for DTCs. Method validation resulted
trophotometry (AAS) [65]. in recoveries of 88–109% for cucumbers and apples
Bardarov et al. were the first to apply ion-exchange LC spiked in the range 1–10 mg/kg; LODs were 0.09 lg/kg
for determination of propineb (technical product) in air and 0.4 lg/kg for mancozeb and propineb, respectively.
after aspiration through a filter and surface extraction of Van Lishaut and Schwack were the first to determine
the filter with an aqueous alkaline EDTA solution, but all three DTC sub-classes [i.e. DMD (ziram, ferbam), EBD
they did not validate the method and apply it to food (maneb, zineb, mancozeb), and PBD (propineb)] by a
samples [66]. rapid ion-pair LC method with UV and electrochemical
Ion-pair LC methods applying tetraalkylammonium detection [71]. The surface extraction was performed
salts [i.e. cetyltrimethylammonium bromide (CTAB), applying an aqueous extraction buffer (pH 11.0) con-
tetrabutyl ammonium chloride (TBAC) or tetrabutyl taining TBAHS, EDTA, and disodium hydrogen phos-
ammonium hydrogen sulfate (TBAHS)] have often been phate. For stabilization of DTCs, sodium hydrogen sulfite
reported in literature. (1 g/L) was added to the extraction buffer before use. The
Irth et al. determined zineb and maneb in soil after DTCs were separated on a Supelcogel ODP-50 (C18-
extraction with an aqueous alkaline EDTA solution and polyvinylalcohol) column using methanol/extraction
selective pre-concentration as ion-pairs on a C18-pre- buffer as eluent. The method validation for different
column, which was loaded on-line with CTAB [67]. In a fruits and vegetables resulted in recoveries of 72–111%
separate run, thiram was determined in samples of soil, at different fortification levels and LODs of 4–8 lg CS2/L
apple, and lettuce after extraction with dichloromethane using electrochemical detection.
using a reversed-phase column and post-column com- Three years later, Perz and Schwack reported an im-
plexation with copper(II). For soil samples, spiked at proved method using a new alkaline-stable reversed-
1 mg/kg, the reported recoveries for zineb, maneb, and phase column (XTerra RP18) and increased the stability
thiram were high (>98%) and LODs were 0.1 mg/kg. of DTCs in alkaline-sample extracts by adding cysteine
LODs for thiram were generally rather low (0.005–0.01 [72]. Fig. 2 shows as an example the chromatogram of
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 Figure 2. Separation of dithiocarbamates (DTCs) by ion-pair liquid
chromatography with ultraviolet detection on the stable alkaline
96% and 20% were determined using method b) for
XTerra RP18 column [72].
0.25-g and 1.0-g samples, respectively. Additionally,

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

DTC standards. Recoveries of 93–120% were determined in chloroform (2–20 mg/L) were separated on a GPC
for different fruits and vegetables. Thiram could also be column [77].
determined using the same chromatography, but after Blasco et al. also published an LC-APCI-MS method,
separate extraction by a buffer containing sulfite, which which referred to the determination of neutral DTCs only
guaranteed thiram transformation into a DMD-sulfite (i.e. thiram, dazomet and disulfiram, including ETU and
adduct. Thiram was recovered from cucumbers spiked at PTU) in different plants [78]. After cutting and homo-
0.05–0.5 mg CS2/kg in the range 78–91%. genizing the samples, extraction and clean up was per-
Aulakh et al. published an HPLC-UV method for the formed by matrix solid-phase dispersion and column
determination of thiram and thiourea in wheat grains elution with dichloromethane/methanol. With the help
and in a commercial fungicide (Thiram 75 WP) [73]. of matrix-matched standards, recoveries of 60–101%
After extraction of the crushed sample with chloroform, (standard deviation 4–21%) were obtained for thiram,
filtration, and evaporation of extracts, the residues were spiked at 2.5 mg/kg in different matrices. However,
dissolved in acetonitrile and separated on a C18 column thiram could not be recovered from high acidic fruits
with acetonitrile/water as eluent. LODs of 5.2 lg/L and (e.g., oranges and lemons). Surprisingly, disulfiram
4.9 lg/L were determined for thiram and thiourea, could not be recovered from nuts.
respectively. From wheat grain spiked with thiram in the Crnogorac and Schwack published an LC-MS method
range 0.2–0.8 mg/kg, recoveries were 90–99%. for all three DTC sub-classes (i.e. DMD, EBD, and PBD)
Garcinuno et al. determined maneb and its main using a new alkaline-stable polymeric HILIC (hydrophilic
metabolites [ethylenethiourea (ETU), ethylenebis(iso- interaction LC) phase and a simple mobile phase com-
thiocyanate) sulfide (EBIS) and ethyleneurea (EU)] in prising acetonitrile and aqueous ammonia (10 mM)
tomatoes, which were chopped and extracted using a [79]. For the surface extraction of whole samples, they
mixture of acetonitrile, dichloromethane, and chloro- used a sodium hydrogen carbonate buffer (pH 12) con-
form [74]. Analysis was performed on a reversed-phase taining D,L-penicillamine to stabilize DTCs. They used
column with acetonitrile/methanol/100 mM aqueous deuterated DMD and EBD as internal standards, allowing
sodium dodecylsulfate (SDS) as eluent, but EU and isotope-dilution assay and compensating for losses from
maneb coeluted. extracts by degradation. They operated the single-
Özhan and Alpertunga also published a method for quadrupole MS in negative ESI mode. LODs (expressed as
determination of maneb and its main degradation CS2) of 16 lg/kg, 24 lg/kg, and 20 lg/kg were deter-
product (ETU) in different fruit juices using two reversed- mined for DMD, EBD, and PBD, respectively. Recovery
phase columns (Luna CN and C18) with different studies with grapes, cucumbers, tomatoes, and rucola
retention mechanisms [75]. The authors tested two dif- revealed recovery rates in the range 90–100% at dif-
ferent extraction procedures using dichloromethane: a) ferent spiking levels of ziram, dithane, and antracol.
SPE; and, b) liquid-liquid extraction (LLE), whereby SPE Concerning both selectivity and sensitivity, LC-tandem
was favored. For maneb in tomato juice, recoveries of MS (LC-MS2) is preferred over LC-MS, if low LODs are
90–101% and an LOQ of 0.1 mg/L were reported. On required, especially for crops to be processed into baby
studying different samples of juice, maneb was found in food (diet regulations) [80].
one tomato juice at a concentration of 0.45 mg/L. Hayama et al. determined fungicide polycarbamate in
tap water and river water by LC-APCI-MS2 after adding
3.2. Liquid chromatography with mass spectrometry an alkaline EDTA/cysteine solution to the water sample,
Compared to LC-UV, LC-MS is quite rarely reported in the derivatization with dimethyl sulfate and subsequent
literature (Table 2). Concerning demands, only LC-MS clean up using SPE [81]. Mean recoveries of polycarba-
methods fulfill the requirements for both selectivity and mate from distilled water in the spiking range 0.25–5.0
sensitivity. lg/L were 63–74% in the form of DMD-methyl and EBD-
Based on sample preparation and derivatization with dimethyl, respectively. The reported LODs in water
methyl iodide introduced by Gustafsson and Fahlgren samples were amazingly low (i.e. 0.061 lg/L and 0.032
[64] and clean up on Sep-PAK C18 cartridges, Hanada lg/L for DMDs and EBDs, respectively).
et al. determined EBDs (zineb, maneb, and mancozeb) in Brewin et al. published an LC-MS2 method for the
environmental water samples by LC-electrospray ioni- determination of EBDs (mancozeb, maneb, zineb, nabam,
zation (ESI)-MS in negative-ion mode [76]. The average metiram) in different fruits and vegetables after sample
recovery at sub-ppb level was 79% with a coefficient of extraction with an aqueous EDTA/cysteine solution,
variation of 29%. derivatization with methyl iodide and subsequent SPE
Several LC-atmospheric pressure chemical ionization clean up [82]. They obtained an LOQ for EBDs in apples,
(APCI)-MS methods in positive-ion mode have been re- grapes, and tomatoes of 0.01 mg/kg and recoveries of
ported. 79–104% from lettuce, wheat and onions spiked at 0.01
Moriwaki et al. presented an LC-APCI-MS method for mg/kg. The authors used this method within their rou-
the determination of ziram, but only standards dissolved tine analyses of wheat samples for registration studies

46 http://www.elsevier.com/locate/trac
 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 Trends

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