14.4 sottani

Biological Monitoring of Exposure to CarbamateFungicides: determination of ethylene- andpropylenethiourea by UHPLC-ESI-MS/MS

Cristina Sottani

Lowry Centre, Manchester9th - 11th September 2013

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).

Cristina Sottani, Fondazione Salvatore Maugeri 15/10/2013 2

0

20000

40000

60000

80000

1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003

To

nn

es

of

A

S

Total amount (50.000 tons) and Composition of Plant Protection Products in Italy

FungicidesHerbicidesInsecticidesOthers

83% of fungicides

Total amount (220.000 tons ) of PPPs in the EU-25, 2007Quantity of the PPP in the Top 5 Member States

64% of fungicides

Use and Composition of PPPs in Europe and in Italy

Cristina Sottani, Fondazione Salvatore Maugeri

15/10/2013 3

Proposed animal metabolism of DTCs

Propineb

R=H Ethylenethiourea (ETU) R=CH3 Propylenethiourea (PTU)

Pro R

(M01) (M01)

Propineb Mancozeb


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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

Fustinoni et al., 2005 LLE with dichloromethane, BSTFA derivatization GC/MS LOD=0.6 mg/L

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/PFBrderivatization 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 & Montesanoet al., 2011

Lyophilization,96 well‐plate automated extraction with

dichloromethaneHPLC/APCI‐MS/MS LOD= 0.01 mg/L

Ekman & Lindh et al., 2013

Incomplete hydrolysis/PFBrderivatization, 96 well‐plate automated extraction with

dichloromethane

LC/APCI‐MS/MS LOQ=0.5 mg/L

5

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 6

++

iNH

NHS

N2H

NHS NHS

N2Hi+H+

102 MW m/z 103 m/z 103 m/z 44

H2N+

H3C

MS/MS Fragmentation Pattern of ETU and D4-IS

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116 MW

116 MW

NH

NH

S

NH

S

m/z 117

NH

S N2+H

m/z 117

i

m/z 60 m/z 58

H2N

HN NH

H3C

S

+H+

+H+H2N+ NH

H3C

S

iH2N+ NH

H3C

S

H2N

H3C

CH3

HN

H3C

CH3

m/z 117 m/z 117 m/z 60 m/z 58

i

m/z 41

+

i

PTU

PTUHN

H3C

+

N2H

H3C

H

+H

+

H

+H

Hydrocarbon fragment

MS/MS Fragmentation Pattern of PTU

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ETU

PTU

Level μg/L

1 0.2

2 0.4

QC1 0.5

3 1.0

4 2.0

QC2 4.0

5 5.0

6 8.0

QC3 10.0

7 20.0

0.5

4

10

0.5

4

10

Calibration curves


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SRM profile of a blank urine extracted sample spiked after extraction (set B)

SRM profile of a blank urine samplespiked before extraction (set C)

PTU (Rt 2.5 min)

ETU (Rt 1.8 min) ISTD (Rt 1.8 min)

SRM profile of standard samples (set A)

Strong matrix effect leading to a signal suppression was observed from the most analyzed matrices.

SRM LC-ESI-MS/MS profiles at QC2 level for ETU and PTU


PTU (Rt 2.5 min)ETU (Rt 1.8 min)

ISTD (Rt 1.8 min)

PTU (Rt 2.5 min)

ETU (Rt 1.8 min)

ISTD (Rt 1.8 min)

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Matrix Effect

Sample

Name Level RT Area AreaMatrix Effect

%Accuracy

set A set B set B/set A*100

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

Sample

Name Level RT Area AreaMatrix Effect

%Accuracy

set A set B set B/set A*100

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 11

Sample

ISTD Resp. Ratio ISTD Resp. RatioMatrix 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 99 0.0081 810.4 1.8 0.018 0.0202 115 0.0170 840.5 1.8 0.030 0.0263 89 0.0256 971 1.8 0.052 0.0548 104 0.0527 962 1.8 0.108 0.1091 101 0.1046 964 1.8 0.252 0.2405 96 0.2348 985 1.8 0.347 0.2908 84 0.2788 968 1.8 0.433 0.4293 99 0.3886 91

10 1.8 0.560 0.5597 100 0.4469 8020 1.8 1.362 1.1955 88 0.8954 75

Sample

ISTD Resp. Ratio ISTD Resp. RatioMatrix Effect

%ISTD Resp. Ratio

Recovery %

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 83 0.0159 800.4 2.5 0.0422 0.0414 98 0.0384 930.5 2.5 0.0660 0.0501 76 0.0459 921 2.5 0.1070 0.1041 97 0.1120 1082 2.5 0.2176 0.2033 93 0.2141 1054 2.5 0.4964 0.4048 82 0.3640 905 2.5 0.5659 0.5228 92 0.5134 988 2.5 0.8312 0.7956 96 0.7312 92

10 2.5 1.0432 1.0245 98 0.9135 8920 1.8 2.1698 2.0011 92 1.8860 9415/10/2013 12

PTU (Rt 2.5 min)

ETU (Rt 1.8 min)

ISTD (Rt 1.8 min)

ISTD (Rt 1.8 min)

SRM profile of a blank urine sample SPE extracted (set C)

SRM profile of a blank urine sample SPE spiked and extracted (set C)

Efficiency of the chromatographic conditions and sample preparation

Cristina Sottani, Fondazione Salvatore Maugeri 15/10/2013 13


yA = 0.0662x ‐ 0.0212R² = 0.988

yB = 0.0591x ‐ 0.0084R² = 0.998

yC = 0.0446x + 0.0175R² = 0.99338

‐0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 5 10 15 20 25

yA = 0.1072x + 0.0084R² = 0.99824

yB = 0.1002x + 0.0046R² = 0.99976

yC = 0.0933x + 0.0065R² = 0.9987

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

0 5 10 15 20 25

Set A Set B

Set C

Set A Set BSet C

ETU

PTU

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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

Method Development

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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 withdithiocarbamates. The residue levels change during storage, processing,and cooking due to environmental factors. During these processes, theparent compounds may be converted to ETU and/or PTU.

• Human exposure to dithiocarbamates was calculated for the populationof the USA on the basis of estimated consumption of dietary residues ofETU in treated crops.INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY (ICPS)ENVIRONMENTAL HEALTH CRITERIA; DITHIOCARBAMATE PESTICIDES, ETHYLENETHIOUREA ANDPROPYLENETHIOUREA Published under the joint sponsorship of the United Nations EnvironmentProgramme, the International Labour Organisation, and the World Health Organization. Geneva 1988.

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• Science has recently published a paper intended to create greaterawareness in matter of Pesticides for Food Security and Safety.

Verger PJP,WHO, Boobis A.R. Imperial College UK, Science, 341, 2013.

Conclusions


①The aim of our study is to validate an analytical method with areproducibility (accuracy and precision) and sensitivitysufficient enough to measure human exposure to metabolitesof dithiocarbamates (ETU and PTU).

②The establishment of the reference values of ETU and PTU is agrowing need to study the quality of the environment incountries where grape production is predominant such as Italyand other Southern European lands.

③ETU and PTU reference values will be available and reported innext studies. Data will be suitable to give us the basis for therisk assessment of the impact of fungicides on the humanhealth.

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Thanks for your attention !!!


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14.4 sottani

Technology

minptu rt

minetu rt

ptuetu rt

ministd rt

aistd rt

sample preparationistd

anameset bset bset

sample istd