This document summarizes the development and validation of an UHPLC-ESI-MS/MS method for the determination of ethylene- and propylenethiourea (ETU and PTU), metabolites of carbamate fungicides, in human urine. The method involves liquid-liquid extraction of urine samples followed by UHPLC separation and detection using MS/MS. Validation studies demonstrated the method has good accuracy, precision, sensitivity, selectivity and matrix effects. The validated method will be applied to establish ETU and PTU reference values in populations exposed to carbamate fungicides through agricultural work and diet.

1. Biological Monitoring of Exposure to Carbamate
Fungicides: determination of ethylene- and
propylenethiourea by UHPLC-ESI-MS/MS
Cristina Sottani
Lowry Centre, Manchester
9th - 11th September 2013

2. Plant Protection Products (PPPs)
The most widely used fungicides in agriculture are the alkali and metal salts of
the alkelenebis-(dithiocarbamate) acids. The alkelenebis-(dithiocarbamates)
(DTCs) can be divided into 3 subgroups:
dimethyl dithiocarbamates
(ferbam, thiram and ziram);
ethylene bisdithiocarbamates (EBDCs)
(mancozeb, maneb, metiram, nabam and zineb);
propylene bisdithiocarbamates (propineb).
15/10/2013
Cristina Sottani,
Fondazione Salvatore Maugeri
2

3. Use and Composition of PPPs in Europe and in Italy
Total amount (220.000 tons ) of PPPs in the EU-25, 2007
Quantity of the PPP in the Top 5 Member States
64% of fungicides
Total amount (50.000 tons) and Composition
of Plant Protection Products in Italy
Tonnes of
AS
80000
83% of fungicides
60000
40000
20000
0
1992 1993 1994 1995 1996
1997
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Fungicides
Herbicides
Insecticides
Others
Cristina Sottani,
Fondazione Salvatore Maugeri
1998
1999
2000
2001
2002
2003
3

4. Proposed animal metabolism of DTCs
(M01)
Propineb
Propineb
Pro
(M01)
R
Mancozeb
R=H
Ethylenethiourea (ETU)
R=CH3 Propylenethiourea (PTU)
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Cristina Sottani,
Fondazione Salvatore Maugeri
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5. Analytical methods to measure ETU in human urine
Method
Sample Preparation
Analytical System
Detection Limit
Aprea et al., 1993
LLE with dichloromethane
HPLC/DAD
LOQ=0.5 mg/g
(creatinine)
Debbart & Moore, 2002
LLE with dichloromethane
HPLC/UV
LOD=0.5 mg/L
Sottani et al., 2003
LLE with dichloromethane
HPLC/ESI‐MS/MS
LOD=0.5 mg/L
GC/MS
LOD=0.6 mg/L
Fustinoni et al., 2005
LLE with dichloromethane,
BSTFA derivatization
El Balkhi et al., 2005
SPE with dichloromethane
HPLC/DAD
LOQ=1.0 mg/L
Montesano et al., 2007
Lyophilization , extraction with
dichloromethane
HPLC/APCI‐MS/MS
LOD=0.16 mg/L
Lindh et al., 2008
Single Step Extraction/PFBr
derivatization
LC/ESI‐MS/MS
LOD=0.05 mg/L
Jones et al., 2010
LLE with dichloromethane
LC/APCI‐MS
LOD=0.25 mg/L
Jayatilaka & Montesano
et al., 2011
Lyophilization,96 well‐plate
automated extraction with
dichloromethane
HPLC/APCI‐MS/MS
LOD= 0.01 mg/L
Ekman & Lindh et al., 2013
Incomplete hydrolysis/PFBr
derivatization, 96 well‐plate
automated extraction with
dichloromethane
LC/APCI‐MS/MS
LOQ=0.5 mg/L
5

6. ETU and PTU detection in human urine
UHPLC system:
1290 LC infinity
Column – method #1
Pinnacle DB PFPP 1.9 mm 100 x 2.1 mm
Column – method #2
ZORBAX SB-Aq 2.1 x 100 1.8
Mobile Phase
A= 0.01% (v/v) formic acid in water; B= MeOH
Flow rate
0.4 ml/min
Gradient
Isocratic condition
Temperature
20°C
Injection
2 ml with needle wash
Detection
MS/MS
Ionization
Electrospary, positive ionization
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7. MS/MS Fragmentation Pattern of ETU and D4-IS
+
+
H
N
S
NH
102 MW
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+H+
H2
N
S
HN
2
i
NH
m/z 103
S
NH
m/z 103
i
H2N+
H3C
m/z 44
7

8. MS/MS Fragmentation Pattern of PTU
S
HN
S
NH
+H+
PTU
S
H2N+ NH
i
H2N+ NH
H 3C
H3C
H 3C
CH3 H
H3C
m/z 58
m/z 60
m/z 117
+
HN
CH3 H
H 3C
m/z 117
116 MW
+
H2N
i
+
Hydrocarbon fragment
m/z 41
S
PTU
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NH
H
N
116 MW
+H+
H2N
S
H
N
m/z 117
i
S +H2N
HN
m/z 117
i
H 2N
+
HN
H
H3C
m/z 60
+
H
H3C
m/z 58
8

9. Calibration curves
Level
1
0.5
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Cristina Sottani,
Fondazione Salvatore Maugeri
4.0
5.0
8.0
QC3
10.0
7
PTU
2.0
6
4
1.0
5
10
0.5
QC2
0.5
0.4
4
4
2
3
ETU
0.2
QC1
10
μg/L
20.0
9

10. SRM LC-ESI-MS/MS profiles at QC2 level for ETU and PTU
ETU (Rt 1.8 min)
ISTD (Rt 1.8 min)
PTU (Rt 2.5 min)
Strong matrix effect leading to a signal
suppression was observed from the most
analyzed matrices.
SRM profile of standard samples (set A)
ISTD (Rt 1.8 min)
ISTD (Rt 1.8 min)
ETU (Rt 1.8 min)
ETU (Rt 1.8 min)
PTU (Rt 2.5 min)
PTU (Rt 2.5 min)
SRM profile of a blank urine extracted
sample spiked after extraction (set B)
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SRM profile of a blank urine sample
spiked before extraction (set C)
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Fondazione Salvatore Maugeri
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11. Matrix Effect
Sample
Level
RT
Area
Area
Matrix Effect
%
set A
Name
set B
set B/set A*100
Accuracy
0.2
1
1.8
2808
1669
59
99
0.4
2
1.8
5545
3384
61
96
0.5
QC1
1.8
8004
3929
49
92
1
3
1.8
14146
8344
59
101
2
4
1.8
29538
14847
50
100
4
QC2
1.8
58866
30631
52
100
5
5
1.8
63762
39722
62
105
8
6
1.8
114708
61170
53
98
10
QC3
1.8
140210
77539
55
102
20
7
1.8
307868
153814
50
99
Area
Area
Matrix Effect
%
set A
set B
set B/set A*100
Sample
Name
Level
RT
Accuracy
0.2
1
2.5
6692
3318
50
94
0.4
2
2.5
11925
6926
58
101
0.5
QC1
2.5
16728
8099
48
98
1
3
2.5
28836
15862
55
103
2
4
2.5
59290
27671
47
101
4
QC2
2.5
110481
56883
51
101
5
5
2.5
151978
72009
47
104
8
6
2.5
220357
113349
51
99
10
QC3
2.5
261265
141931
54
102
20
7
2.5
463100
280953
61
100
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12. Sample
ISTD Resp. Ratio
ISTD Resp. Ratio
Matrix Effect
%
ISTD Resp. Ratio
Recovery
%
Name
RT
set A
set B
set B/set A*100
set C
set C/set BC*100
0.2
1.8
0.010
0.0100
1.8
0.018
0.0202
99
115
89
104
101
96
84
99
100
88
0.0081
0.4
81
84
97
96
96
98
96
91
80
75
0.0170
0.5
1.8
0.030
0.0263
1
1.8
0.052
0.0548
0.0256
2
1.8
0.108
0.1091
4
1.8
0.252
0.2405
5
1.8
0.347
0.2908
8
1.8
0.433
0.4293
10
1.8
0.560
0.5597
20
1.8
1.362
1.1955
ISTD Resp. Ratio
ISTD Resp. Ratio
Matrix Effect
%
ISTD Resp. Ratio
Recovery
%
0.0527
0.1046
0.2348
0.2788
0.3886
0.4469
0.8954
Sample
Name
RT
set A
set B
set B/set A*100
set C
set C/set B*100
0.2
2.5
0.0241
0.0199
2.5
0.0422
0.0414
0.5
2.5
0.0660
0.0501
1
2.5
0.1070
0.1041
2
2.5
0.2176
0.2033
4
2.5
0.4964
0.4048
5
2.5
0.5659
0.5228
8
2.5
0.8312
0.7956
10
2.5
1.0432
1.0245
20
1.8
2.1698
2.0011
83
98
76
97
93
82
92
96
98
92
0.0159
0.4
80
93
92
108
105
90
98
92
89
94
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0.0384
0.0459
0.1120
0.2141
0.3640
0.5134
0.7312
0.9135
1.8860
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13. Efficiency of the chromatographic conditions and sample preparation
ISTD (Rt 1.8 min)
ETU (Rt 1.8 min)
PTU (Rt 2.5 min)
SRM profile of a blank urine sample SPE spiked and
extracted (set C)
ISTD (Rt 1.8 min)
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SRM profile of a blank urine sample SPE extracted
(set C)
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Fondazione Salvatore Maugeri
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14. 1.600
yA = 0.0662x ‐ 0.0212
R² = 0.988
1.400
1.200
Set A
Set B
yB = 0.0591x ‐ 0.0084
R² = 0.998
1.000
Set C
yC = 0.0446x + 0.0175
R² = 0.99338
0.800
0.600
ETU
0.400
0.200
0.000
‐0.200
0
2.5000
5
10
15
20
yA = 0.1072x + 0.0084
R² = 0.99824
2.0000
Set A
Set B
Set C
yB = 0.1002x + 0.0046
R² = 0.99976
1.5000
25
PTU
yC = 0.0933x + 0.0065
R² = 0.9987
1.0000
0.5000
0.0000
0
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5
10
15
20
25
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Fondazione Salvatore Maugeri
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15. Method Development
Coefficient of Regressions of repeated calibration curves
Limit of detection
✔
✔
Limit of quantification:
✔
CVs Intra-Run ; Inter-Run
✔
Matrix Effect Assessment
✔
Recovery
✔
Reproducibility : ✔
Accuracy and Precision
CVs Intra-Run ; Inter-Run
✔
✔
Stability Studies
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Fondazione Salvatore Maugeri
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16. Discussion
•
The world‐wide consumption of dithiocarbamates is between 25 000
and 35 000 metric tons per year.
•
Residues of DTCs and ETU are found in and/or on crops treated with
dithiocarbamates. The residue levels change during storage, processing,
and cooking due to environmental factors. During these processes, the
parent compounds may be converted to ETU and/or PTU.
•
Human exposure to dithiocarbamates was calculated for the population
of the USA on the basis of estimated consumption of dietary residues of
ETU in treated crops.
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY (ICPS)
ENVIRONMENTAL HEALTH CRITERIA; DITHIOCARBAMATE PESTICIDES, ETHYLENETHIOUREA AND
PROPYLENETHIOUREA Published under the joint sponsorship of the United Nations Environment
Programme, the International Labour Organisation, and the World Health Organization. Geneva 1988.
•
Science has recently published a paper intended to create greater
awareness in matter of Pesticides for Food Security and Safety.
Verger PJP,WHO, Boobis A.R. Imperial College UK, Science, 341, 2013.
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17. Conclusions
①The aim of our study is to validate an analytical method with a
reproducibility (accuracy and precision) and sensitivity
sufficient enough to measure human exposure to metabolites
of dithiocarbamates (ETU and PTU).
②The establishment of the reference values of ETU and PTU is a
growing need to study the quality of the environment in
countries where grape production is predominant such as Italy
and other Southern European lands.
③ETU and PTU reference values will be available and reported in
next studies. Data will be suitable to give us the basis for the
risk assessment of the impact of fungicides on the human
health.
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Fondazione Salvatore Maugeri
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18. Thanks for your attention !!!
15/10/2013
Cristina Sottani,
Fondazione Salvatore Maugeri
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