Analysis of Dithiocarbamates

Stewart Reynolds
Head of Analytical Services
Food Science Group
Central Science Laboratory, York, UK
SELAMAT Workshop, 5-7 July 2006, Bangkok
Introduction
What are dithiocarbamates use for
Chemical structures
Instability
Occurrence of residues
Method (no specific)
Method (More specific)
What are EBDCs used for?
Contact fungicides that are effective for
controlling many different fungal diseases
This means that they are extensively used on
a wide range of crops
What are EBDCs?
The ethylenebisdithiocarbamates are a group of
compounds that exist as strong complexes with
various metal ions, often in a polymeric form
This makes them difficult to analyse directly because
of their limited solubility in most organic solvents
Not amenable to multi-residue methods
Fungicides with the dithiocarbamate moiety
Dithiocarbamates
Dimethyldithiocarbamates Alkylenebis(dithiocarbamates)
Salts Ethylenebis(dithiocarbamates)
Thiram Ferbam (Fe) Maneb (Mn)
Ziram (Zn) Mancozeb (Mn/Zn)
Nabam (Na)
Zineb (Zn)
Propylenebis(dithiocarbamate)
Propineb (Zn)
Dimethyldithiocarbamates - thiram
N C S S C N
S
Ne
Ne
S
Ne
Ne
Dimethyldithiocarbamates
N C S
S
Ne
Ne
Ferbam: N = Fe, n= 3
Ziram: N = Zn, n = 2
N

Ethylenebisdithiocarbamates
S C N C C N C S N
S
Naneb: N = Nn
Zineb: N = Zn
Nancozeb: N = NnfZn

H H
H
H
H
H S
Ethylenebisdithiocarbamates
Na S C N C C N C S Na
S
Nabam
H H
H
H
H
H S
Propylenebisdithiocarbamate
S C N C C N C S Zn
S
Propineb

H
Ne
H
H
H
H S
Sub-sampling
Dithiocarbamate residues are extremely
heterogeneously dispersed over treated commodities
Homogenisation at room temperature can give up to
100% loss of residue
Duplicate analyses (CS
2
method) can give very poor
repeatability up a factor 10 difference
(Based on cutting segments from fruits and vegetables)
Cryogenic milling
Freeze sample then mill in the presence of dry ice
This reduces losses of dithiocarbamates spiked onto
frozen samples, but losses still significant (30-60%)
and are variable depending on the particualr
dithiocarbamate and the commodity
In general, slightly more loss of mancozeb compared
to thiram
Limited data set for apples, tomatoes and lettuce
MRL exceedances 2000 -2004
Where no suitable registration data MRLs
have been set at the LOD (0.05 mg/kg)
Mangoes from Puerto Rico
Avocado from Mexico
Edible-podded peas from Guatemala
Beans from Kenya
Lettuce & parsley from UK
Method of Analysis
Determined indirectly (and non-specifically)
by measuring the amount of carbon disulfide
(CS
2
) that is liberated by the action of Zn and
HCl
The liberated CS
2
is now almost exclusively
measured by GC-MS, or GC-FPD
Method of analysis
50 g of sample in a plastic coated Schott
bottle
Add 150 ml of tin(II) chloride/HCl soln
Add 25 ml iso-octane
Seal with screw cap fitted with 42 mm rubber
septa (screw cap has a small hole in it to
allow spiking)
Method of analysis
Use an organic (CS
2
free) sample of relevant
commodity for preparation of two blank samples and
a spike
Shake each bottle and put into a water bath at 80
o
C
+/- 5
o
C for 60 mins
Invert bottles ten times at 20, 40 & 60 mins
Place in cold water bath
Method of analysis
Transfer a portion of the iso-octane into a vial
Transfer as much as possible of the iso-
octane layer from each into a flask this is
used to make up the matrix-matched calibrant
solutions 0.025, 0.05, 0.10, 0.20 & 0.50
mg/kg CS
2

Gas Chromatography
Inject 1.0 l aliquots onto a 30 m x 0.53 mm
DB-1 column of 1.5 m film thickness
Inject 1-3 l aliquots onto 30 m x 0.32 mm CP
Sil5 CB column of 4 m film thickness
Example chromatograms of CS
2
Calibrant @ 0.05 mg/kg
Calibrant @ 0.025 mg/kg
min 0.6 0.8 1 1.2 1.4
150 pA
0
50
100
150
200
250
300
350
400
FPD1 B, (H:\PVMB\FPD-169\33674&\33674&41.D)


A
r
e
a
:

4
6
8
.
8
1
1

0
.
6
1
7

-


c
s
2
min 0.6 0.8 1 1.2 1.4
150 pA
0
50
100
150
200
250
300
350
400
FPD1 B, (H:\PVMB\FPD-169\33674&\33674&42.D)


A
r
e
a
:

1
5
8
.
1
5

0
.
6
1
6

-


c
s
2
Example chromatograms of CS
2
Blank (Apple)
Spike @ 0.1 mg/kg Thiram (equivalent to 0.062 mg/kg CS
2
)
min 0.6 0.8 1 1.2 1.4
150 pA
0
50
100
150
200
250
300
350
400
FPD1 B, (H:\PVMB\FPD-169\33674&\33674&43.D)
min 0.6 0.8 1 1.2 1.4
150 pA
0
50
100
150
200
250
300
350
400
FPD1 B, (H:\PVMB\FPD-169\33674&\33674&60.D)

0
.
6
1
6

-


c
s
2
GC-MS
Monitor for ions
m/z = 76 & m/z = 78 (S-34 isotope)
m/z = 78 around 10% abundance of m/z = 76
GC-MS SIM of CS
2
Calibrant @ 0.05 mg/kg
R T : 1 .5 0 - 3 .0 0
1 .6 1 .8 2 .0 2 .2 2 .4 2 .6 2 .8 3 .0
T i m e ( m i n )
0
2 0
4 0
6 0
8 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
R
e
l
a
t
i
v
e

A
b
u
n
d
a
n
c
e
0
2 0
4 0
6 0
8 0
1 0 0
2 .3 3
2 .5 7
2 .5 6 2 .8 3
2 .6 1 2 .7 6
2 .8 6
1 .8 5 1 .7 7 1 .5 6 1 .6 0
2 .0 8 2 .2 4 1 .9 2 2 .1 8
1 .5 6
1 .6 2
1 .8 9 2 .3 5
1 .7 0 2 .7 6
2 .7 3
2 .0 6 1 .7 6
2 .7 7 2 .3 2
1 .9 5
2 .6 3 2 .4 2
2 .5 8
2 .8 1 2 .1 9
2 .0 8
2 .8 8
2 .5 6 2 .2 9
1 .8 7
2 .7 7
1 .6 4
1 .7 4
2 .0 7 2 .1 4 2 .3 4 2 .5 8 2 .8 8 2 .2 0 2 .3 8 2 .5 3 2 .6 7
N L :
4 .2 0 E 4
m /z=
7 5 .5 0 -
7 6 .5 0 MS
2 7 4 6 2 2 1
N L :
8 .4 2 E 3
m /z=
7 7 .5 0 -
7 8 .5 0 MS
2 7 4 6 2 2 1
N L :
4 .1 0 E 6
T IC MS
2 7 4 6 2 2 1
Problems associated with this approach
Cannot tell which dithiocarbamate is present
CS
2
can be can be produced by natural
precursors in in certain commodities, eg
cruciferacae
Easy to contaminate the sample if in contact
with rubber or latex
Difficult to obtain representative sub-samples
as dithiocarbamates unstable during normal
sample processing - homogenisation
Problems with the CS
2
approach
Cannot distinguish between different
dithiocarbamates
Thiram has different EU MRLs from the
ethylenebisdithiocarbamates
Non-specific method (prone to false +ves)
Some crops contain natural precursors that
produce CS
2
when treated with Sn/HCl
CS
2
produced from accelerators used in
rubbers (e.g. latex gloves)
Analysis of ethylenebisdithiocarbamates by
LC-MS/MS
Method is based on the direct quantification of
[-SCSNHCH
2
CH
2
NHCSS-] chain after derivatisation
Extract with EDTA, cysteine and iodomethane
The EDTA breaks the polymeric chain and the
iodomethane methylates the EBDC to form:
CH
3
SCSNHCH
2
CH
2
NHCSSCH
3
- a stable derivative
Analysis of EBDCs
Centrifuge and pour off the supernatant
Clean-up on a C
18
based SPE cartridge
Quantification by LC-MS/MS using +ve
electrospray
LC-MS/MS
Obtain the protonated molecular ion of
m/z 241
Fragmentation of m/z 241 produces a daughter ion of
m/z 134
Method has been validated down to 0.01 mg/kg for
maneb, mancozeb, metiram, nabam & zineb on
apples, grapes and tomatoes
Method validation data
Individual recovery values fell between 70 -110%
Mean recovery values fell between 74 - 100%
CVs of <14% for all 5 EBDCs in all 3 commodities
The method is specific for EBDCs as
dimethyldithiocarbamates and propineb will form
different derivatives
Poster at EPRW 2006
Stephen Brewin et al
Huntingdon Life Sciences,
Eye
Suffolk IP23 7PX
UK
www.huntingdon.com
Conclusions
If using the methods based on CS
2
then need
to take extra care:
Ensure you are measuring CS
2
Could commodity be producing CS
2
naturally
Could the sample have been contaminated
Use the LC-MS/MS based more direct
method if possible