2010 EurlFvSrmWorkshop SrmMethods

ADVANCES IN THE ANALYSIS OF
PESTICIDES TYPICALLY ANALYSED

BY SINGLE-RESIDUE METHODS
Michelangelo Anastassiades
Diana Strher Kolberg
Dorothea Mack
Irina Sigalova
Daniela Roux
EURL-SRM EU Reference Laboratory

for Pesticide Residues
EURL-FV/SRM WORKSHOP 2010
Almera/Spain, 27-28 October 2010 using Single Residue Methods
SRM-Phobia...
SRM-compounds
are not very popular...
SRM-Phobia...
SRMs often require...
tedious or unfamiliar sample preparation steps e.g.:

pH adjustments
Derivatization
Cleavage
Head-space sampling
separation/detection techniques different from those
typically used in MRMs and often not available in labs, e.g.:
Exotic columns for LC- or GC-separation
Exotic mobile phases (LC)
Exotic Instrumental-configurations
SRM-Phobia...
SRMs are thus often Costly
and Work-Intensive
Effort : Benefit-Ratio is less favourable than that of MRMs
As labs are typically

overloaded with work
..most try to avoid SRMs Covered

wherever possible Pesticides Effort
using them
only when there no other
EURL-FV/SRM way out
WORKSHOP ;-)
2010
Almera/Spain, 27-28 October 2010
using Single Residue Methods
SRM-Challenge...
If at all, labs employ SRMs very judiciously
targeting only specific pesticide/commodity-combinations
Examples:
Chlormequat (pears, cereals, mushrooms, carrots)
Glyphosate (cereals)
Maleic hydrazide (onions, potatoes)
( This targeted approach Improves the Effort to Benefit Ratio
However: Our knowledge about the residue

situation of SRM-Pesticides is still very limited
Question to be answered:
Which SRM-Pesticide/Commodity-Combinations
EURL-SRM
are worth-while monitoring ? EU Reference Laboratory
Lets take
a closer look
at the SRM pesticides

No makes
What matteraare
SRM-Pesticides the name
Divas they
compound
the remain Ugly
an SRM-compound?
among the Pesticides?
SAM
What is the best name?
SRM-Therapy
Single Single
WHYDOES
WHY DO
ALL
EVERYBODY
PEOPLE
Residue Analyte
HATE ME?
HATE ME?
Method Method
What
EURL-SRM
does Google
THE WORLDSsay?
UGLIEST EU Reference Laboratory
DOG METHOD for Pesticide Residues
What makes a compound an SRM-compound?
POSSIBLE SRM-
OBSERVATIONS / REASONS EXAMPLES
APPROACHES
SRM-Therapy
I. - Bad peak-shape - Organotins - Diff. Separation cond.
Poor Chr/phy - Poor detector response - Abamectin - Diff. Detection cond.
and/or Detection - Degradation (mostly GC) - Carbamates, dicofol - Deriv., APs, Temp. adj.
II. - Compound Degrades

- Hydrolysis - Captan, Folpet - Control pH, work fast,
Degradation -
- Oxidation - Ethiofencarb WHY
keepDO
WHY DOES temp. low
Losses - Reactions with matrix - Chlorothalonil
ALL
EVERYBODY
- Comp. interacts w. surfaces - Modify solvent (e.g. H+)
PEOPLE
- Organotins, Paraquat
- Compound too Polar HATE ME?
HATE ME?
- Quats, Glyphosate - One-phase extractions
III. - Compound Acidic/Basic
Adsorption / + unfavourable pH - Phenoxy acids - pH-adjustments
Partitioning
-Losses - Compound too Lipophilic
- Use non-polar solvents
+ high fat commodity - DDT, HCB
for total fat extraction
+ polar extraction solvent
IV. - Phosphine - Cryo-milling,
Evaporation - - Compound too Volatile - Ethylene oxide Special GC-conditions
Losses - CS2 (Headspace sampling)
- Legislative requirements: - Phenoxy-acids
V. - Cleavage steps to
EURL-SRM
Residues not in
Residue def. includes bound
residues or requires release of
- Dithiocarbamates,
Amitraz, Prochloraz,
release
EU bound residues
Reference or
Laboratory
Pesticide Residues
the form needed
EURL-FV/SRM WORKSHOP 2010 commonformoieties
common moiety
Vinclozolin, Diuron using Single Residue Methods
Classification SRM or MRM ?
Classification SRM/MRM varies
from lab to lab
over the course of time (as technology is progressing)
It depends on
Sample Preparation Technique Employed:
- Trend for methods better covering polar compounds, and being
LC/MS amenable (QuEChERS, ChemElut, modif. S19/Luke)
Instrumental Analysis Technique Available:

- Detection technology
Sensitivity/selectivity improves over time,
- Chromatographic separation technology

LC: UPLC, U-HPLC, new stationary phases e.g. HILIC
GC: fast GC, GCxGC
Many believe:
EURL-SRM In future virtually all pesticides will be MRM-amenable
EU Reference Laboratory
EURL-FV/SRM WORKSHOP (Dilute
2010
& Shoot or Quick & Dirty)?
I.
Poor Chromatography
and/or Detection

I. Poor Chromatography and/or Detection
Example: Organotins

Example: Organotins
5 QuEChERS-Recoveries 70 - 110 %
6 Very poor peak shapes using standard LC-MS/MS-conditions
Acidic conditions essential to obtain sharp peaks !!

H+ interrupts unwanted interactions of O-Tins with surfaces
Column:
Zorbax 3,5 m; Eclipse XDB-C18; 2,1x 50 mm
Mobile phase:
A: NH4formate (5 mmol) in water +1% formic acid
B: NH4formate (5 mmol) in methanol +1% formic acid

Example: Organotins Organotins at 0.001 mg/mL
ozinn 0.001 - Fenbutatinoxid 519.3 / 350.9 T1 (Standard) 519.3/350.9 amu - sample 3 of 103 from 070709sc.wiff
Area: 3.075e+003 counts Height: 6.70e+002 cps RT: 5.209 min
ozinn 0.001 - Fenbutatinoxid 517.3 / 195.0 T2 (Standard) 517.3/195.0 amu - sample 3 of 103 from 070709sc.wiff
1% FA in mobile phase
ozinn 0.001 - Fenbutatinoxid 519.3 / 197.0 (Standard) 519.3/197.0 amu - sample 3 of 103 from 070709sc.wiff
650 900 1300

600 1200
800
550 1100
500 700 1000
450 900
Fenbutatin oxide
600
In t e n s it y cp s
400 800
350 500 700
300 400 600
250 500
300
200 400
150 200 300
100 200
100
50 9.54
100 5.34
5.65 8.58 10.02 1.18
0 0 0
2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12
Time, min Time, min Time, min
Fenbutatinoxid 519.3 / 350.9 T1 Fenbutatinoxid 517.3 / 195.0 T2 Fenbutatinoxid 519.3 / 197.0

ozinn 0.001 - Cyhexatin 369.2 / 286.9 T1 (Standard) 369.2/286.9 amu - sample 3 of 103 from 070709sc.wiff ozinn 0.001 - Cyhexatin 367.2 / 202.9 T2 (Standard) 367.2/202.9 amu - sample 3 of 103 from 070709sc.wiff ozinn 0.001 - Cyhexatin 365.1 / 283.1 (Standard) 365.1/283.1 amu - sample 3 of 103 from 070709sc.wiff
Area: 6.717e+003 counts Height: 1.43e+003 cps RT: 4.697 min Area: 4.534e+003 counts Height: 9.59e+002 cps RT: 4.696 min Area: 3.128e+003 counts Height: 7.17e+002 cps RT: 4.697 min
1400 700
900
1200 800 600
700
Cyhexatin
1000 500
600
800 400
500
600 400 300
300
400 200
200
200 100
6.32 6.74 9.90 100 4.92 6.94
3.14 3.53 3.71 6.06 7.80 8.98 9.64 10.32
12.03 5.11 6.92 9.73 0.30 2.80 3.35 4.02 5.48 8.49 9.12 10.04
0.95 1.74 3.18 4.15 7.46 8.89 10.83
0 0 0
2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12
Cyhexatin 369.2 / 286.9 T1

ozinn 0.001 - Fentin 351.0 / 119.9 T1 (Standard) 351.0/119.9 amu - sample 3 of 103 from 070709sc.wiff
Cyhexatin 367.2 / 202.9
ozinn 0.001 - Fentin 349.0 / 195.0 T2 (Standard) 349.0/195.0 amu - sample 3 of 103 from 070709sc.wiff
T2 Cyhexatin 365.1 / 283.1
ozinn 0.001 - Fentin 347.0 / 115.9 (Standard) 347.0/115.9 amu - sample 3 of 103 from 070709sc.wiff
Area: 9.537e+003 counts Height: 1.40e+003 cps RT: 3.207 min Area: 6.179e+003 counts Height: 9.98e+002 cps RT: 3.224 min Area: 3.162e+003 counts Height: 4.89e+002 cps RT: 3.199 min
1400 1000
450
900
1200
400
800
Fentin
1000 350
700
300
600
800
500 250
600
400 200
300 150
400
200 100
200
EURL-SRM
100 8.60 50 4.94 10.43
7.29 0.96 4.33 5.06 5.56 6.46 7.91 8.81 10.46
11.01
4.95
0 0 0
2 4 6
Time, min
8 10 12 2 4 6
Time, min
8 10 12 2 4 6
Time, min
8 10
12
Fentin 351.0 / 119.9 T1 Fentin 349.0 / 195.0 T2 Fentin 347.0 / 115.9 for Pesticide Residues
I.
Poor Chromatography
and/or Detection
and
II.
Degradation - Losses
I./II. Poor Chromatography and Degradation losses
Example: Base-Labile Pesticides
Degradation at
High pH
High temperatures
FOLPET Phthalimide
e.g.:
During sample prep.
In final extract
CAPTAN In GC-inlet Tetrahydrophthalimide
Similar behaviour:
Dicofol, Captafol, Tolylfluanid, Dichlofluanid, Pyridate

Example: Base-Labile Pesticides
Reducing losses in extracts
Problem:
Degradation in extracts of original QuEChERS following PSA-cleanup
Rec. % Tolylfluanid stability in extract
100 Solution:
90 7 days
80 13 days Addition of acid
70 e.g.: Formic acid (5% in ACN)
60 pH 10 L per mL extract.
50
after
40
30 cleanup Brings pH to ~5
20 with PSA
10 QuEChERS-amenable!
0
pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 MeCN
Measured pH in extract
OK, but what about instrumental analysis?

Tolylfluanid, Dichlofluanid, Pyridate (amenable to LC-MS/MS)
EURL-SRM 5
Captan,
EURL-FV/SRM Folpet, Dicofol,
WORKSHOP 2010
Captafol (NOT amenable to LC-MS/MS) 4
Use of Analyte Protectants to improve GC-Analysis
Extract acidification also improves GC-behaviour of Folpet, Captan and Dicofol
But better results by addition of ANALYTE PROTECTANTS-MIX to extracts
Folpet, Captan work perfectly with APs 5
Dicofol still not fully satisfactory 4
Quantified against Errors due to Matrix Effects
solvent-based Overestimations
Calibration Std mix with and without Analyte Protectants
100 (no peak in solvent)
90
After ADDING APs
80 to Extract + Cal. Std
Absolute Error [%]
70
60
50
40
30
20
10
0
Carbofuran
Iprodione
Dichlorvos
Tolyfluanid
Carbaryl
Tetramethrin1
Dicofol
Diclofluanid
Captan
Parathion-e
TPP
Chlorothalonil
Trifloxystrobin
Dichlobenil
o-Phenylphenol
Parathion-m
HCH, gamma-
Folpet
Ditalimfos
Chlozolinate
Acrinathrin
Chinomethionat
Bupirimat
Methiocarb
Phenmedipham
Flubenzimine
Binapacryl
Bitertanol
Acephate
Procymidone
MEAN ALL
Vinclozolin
EURL-SRM Against Solvent w/o APs

EURL-FV/SRM WORKSHOP 2010 Against solvent with AP
Analyte Protectants Mixture
Ethylglycerol Shikimic acid

HO
O
HO O
HO
OH
OH
OH
Sorbitol -Gluconolactone
OH
OH OH
O O
HO
OH
OH OH HO OH
OH
Dicofol calibration curves with and w/o APs
Area
20000 Linear but not

18000 good enough
16000
14000
12000 R2 =0.9903 with APs
10000
8000
6000
4000
2000 w/o APs
0
0 0,1 0,2 0,3 0,4 0,5 0,6
g/mL
Dicofol Dicofol-with AP
No peak

Use of isotopically labelled ISTDs Better with APs
because w/o APs
often no peaks
Ratio Dicofol / DicofolD8 - with APs

1,30
1,15 1,05
1,00 0,91 0,93 0,94 0,95
0,85
0,70
0,55
0,40
0,25
0,10
-0,05
0,01 0,025 0,05 0,1 0,5
g/mL
Use of isotopically labelled ISTDs
4,000
Flchenverhltnis
3,000
2,000
y = 8,1676x + 0,8362 Dicofol
1,000
R2 = 0,9975 using IL-ISTD-ratio
0,000
0 0,1 0,2 0,3 0,4
g
Calculated via Standard
addition : 0.102 mg/kg
30000,000
25000,000
20000,000
Flche
15000,000 Dicofol
10000,000 y = 49538x + 10017
5000,000 R2 = 0,6532 using Area
0,000
0 0,1 0,2 0,3 0,4
g
EURL-SRM Calculated via Standard

addition : 0.201 mg/kg
Isotopically Labelled ISTDs
Folpet in Papaya (MRL=0.01 mg/kg) Captan in Blueberries (MRL=0.01 mg/kg)
Only via Area
Fl c h e p u r
Only via Area
Fl c h e p u r
140000
120000
0.019 mg/kg
Unacceptable R
Should be at
2 600000
500000 0.030 mg/kg

ISTDs
added to
100000
400000
80000
least 0.995 300000
60000
R2 =0.8318 R2 =0.9436
extract
40000 y = 3E+06x +45408 200000
y = 6E+06x +152616
2
20000 R = 0,8318 100000 R2 = 0,9436
0 0
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07
via ISTD (PCB 138)

FLV B S 13 8
via ISTD (PCB 138)
FLV B S 13 8
0,01
0,008 0.022 mg/kg

0,1
0,08 0.033 mg/kg

Violation
0,006 0,06
0,04
R2 =0.9585
0,004
y = 1,0722x +0,0252
0,002 R =0.7960
2
y = 0,1576x +0,003 0,02 R2 = 0,9585
2
R = 0,796 0
0
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07
via ISTD (Folpet D4)

FLV Fo l p e t D 4
via ISTD (Captan D6)
FLV C a p t a n D 6
0, 7 7
0, 6
0.018 mg/kg
6 0.030 mg/kg
0, 5 5

0, 4 4
0, 3 3 y = 71,164x +2,312
0, 2 R2 =0.9979
y = 12, 78x +0, 228
2 R2 =0.9979
R = 0,9979 2
R 2 = 0, 9979 1
0, 1
0 0
No violation
0 0, 005 0, 01 0, 015 0, 02 0, 025 0, 03 0, 035 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07

Impact of isotopically labelled ISTDs
EUPT-C3/SRM4 Chlormequat
z-scores (FFP 25%); (* NRL-SRM)
z-scores (FFP 25%); (* NRL-SRM)
4
1
z-score
-1
not Specified
-2 unacceptable: 1 labs 3%
questionable: 0 labs 0%
acceptable: 37 labs 97 %
-3
Median = 0.479 mg/kg
Using isotope labelled ISTD MRRL = 0.02 mg/kg
-4
*eupt 017
eupt 038
eupt 095
*eupt 003
*eupt 060
eupt 055
*eupt 086
*eupt 061
*eupt 084
eupt 054
*eupt 001
eupt 021
eupt 029
eupt 019
eupt 091
eupt 008
eupt 037
eupt 005
*eupt 007
*eupt 030
eupt 014
eupt 022
*eupt 046
eupt 026
eupt 042
*eupt 048
eupt 033
*eupt 040
eupt 018
eupt 044
eupt 053
*eupt 043
eupt 049
eupt 050
*eupt 063
eupt 117
eupt 034
*eupt 009
Impact of isotopically labelled ISTDs
EUPT-C3/SRM4
Impact of using isotopically labelled ISTD on Qn-RSDs
Qn 60
50 Median= 0.42
17 labs
40 Median= 0.48
Qn- 38 labs
RSD 30 Median= 0.48
21 labs
20 38,0
10
25,8
16,4
0
All Using isotop. labelled Other
Chlormequat

III. Partitioning Losses

Pesticide overview...
25
% of Pesticides
20
15 Less polar More polar

10
~4%
5
0
<0.01 0.01-0.1 0.1-1 1-10 10-100 100-1,000 1,000-10,000 10,000- >100,000
100,000
Solubility in Water in mg/L
30
% of Pesticides
25
20
15
More polar
10 ~4%
5
0
EURL-SRM
<-1 -1 to 1 0 to 1 1 to 2 2 to 3 3 to 4 4 to 5 5 to 6 >6
EURL-FV/SRM WORKSHOP 2010 LogKow of Pesticides
OUT OF SCOPE QuEChERS
Traditional MRMs
Amino acids Flavonoids/Anthocyanes Fatty Acids Phytosterols
-5 - -1 Polarity range covered by trad. MRMs
(pH dependent) 0-6 6-8.5 8.5-11.5
Sugars Monoterpenes
-5 - -2 2.5-5.5
Alkanoic acids (~40)
pH dependent Pyrethroids (~45)
Strepto 3.8 - 8.3
-mycin
-6.4 Basic Pesticides Carotenoids
OCs (~20)
pH dependent 3.5 - 7.0 11-18
Quats
-4.5 - -2.8 Ureas (~ 30)
Glyphosate 1.6 - 5.9 Chlorophyll
pH-dependent 17.2
OPs (~95 compounds)
-3.2
-0.9 - 5.7
Fosetyl-Al
pH-dependent Carbamates (~30) TGs
-2.7 20-24
-0.4 - 5.5
Maleic Hydr.
pH-dependent
EURL-SRM -1.9 LogP = EU
LogKow
Reference Laboratory
EURL-FV/SRM WORKSHOP 2010 for Pesticide Residues
-5 -4 -3
Almera/Spain, 27-28 -2 -1 20100
October 1 2 3 4 5 6 7 using Single Residue Methods
8 9 10 11
EURL-PROJECT
METHOD
FOR THE ANALYSIS OF
HIGHLY POLAR PESTICIDES

H2O sol. ADI/ARfD 91/414
Name Formulas LogKow pka Status
g /L mg/ kg bw
Fosetyl Al -2,7 (pH4) 111 (pH6) 4.7 3/- IN
2.82
Ethephon -1.89 (pH7) 800 (pH4) 0.03 / 0.03 IN
7.21
0.78
2.29
Glyphosate -3.2 (pH5) 900 (pH?) 0.3 / - IN
5.96
11
0.8
Glufosinate -3.77 (pH5) 500 (pH7) 2.9 0.02 / 0.02 IN
9.8
Maleic
-1.96 (pH7) 144 (pH7) 5.62 0.25 / - IN
Hydrazide
Chlormequat -3.47 886 - 0.05 / 0.05 IN
Mepiquat -2.82 500 - 0.3 / 0.3 IN
Paraquat -4.5 620 - 0.004 / 0.005 (OUT)
EURL-SRM
Diquat -4.6 700 -
0.002 / 0.01 Residues
for Pesticide IN
Recoveries with QuEChERS...
QuEChERS Recoveries from

Cuccumber
Name LogP
ORGANIC phase WATER phase
Maleic Hydrazide 30 % 74 % -1.96 (pH7)

Ethephon 2% 87 % -1.89 (pH7)
Daminozide 6% 87 % -1.5 (pH7)
Fosetyl Al <1% 83 % -2,7 (pH4)
Amitrol 5% 105 % -0.97 (pH7)
Method to be developed
should not involve liquid-liquid-partitioning

Goals of Project...
Develop an Attractive Method that labs can readily implement
Select relevant pesticides
Keep procedure simple and efficient
- Avoid troublesome steps (derivatisation, SPE-enrichm. etc.)
- Use isotopically labelled ISTDs!
Use instr. available or shortly available to most labs (LC-MS/MS)
Where possible, simultaneous sample prep. & chromatogr.
...LIMITATIONS!
EURL-SRM
Test real samples EU Reference Laboratory
to localize relevant Pesticide/Commodity Combinations
Overview LC-Conditions / ISTD-availability
Name LC-MS/MS LC-MS/MS Isotopically Labelled
ESI (+) ESI (-) ISTDs
Ethephon - ++ NEW (D4)
Glyphosate + ++ Existed
Glufosinate ++ ++ NEW (D3)
Chlormequat ++ - Existed
Daminozide ++ - Existed
HEPA - ++ Donation
ETU ++ - Existed
N-Acetylglufosinate - ++ NEW (D3)
Streptomycin ++ - dyhidrostreptomycin
Cyromazine ++ - Existed
MPPA - ++ NEW (D3)
AMPA - ++ Existed
Mepiquat ++ - Existed
Paraquat ++ - Existed
Diquat ++ - Existed
Amitrol ++ - ordered
Fosetyl Al + ++ NEW (D15)
Maleic Hydrazide + ++ NEW (D2)
Polar Pesticides
-
Short Introduction

Quats
Difenzoquat
Amenable
EURL-SRM to QuEChERS
EURL-FV/SRM WORKSHOP 2010 for Pesticide Residues
Chlormequat and Mepiquat
Uses:
- Growth regulators in many crops
(cereals, grapes, pears...)
-In cereals as stem shorteners / strengtheners
-Illegal use of chlormequat in carrots, pears
Mode of Action:
- Systemic
Toxicity to mammals:
- Comparably low

Paraquat and Diquat
Uses:
- Pre- and post-emergency herbicides
- Used on potatoes, canola for defoliation to facilitate mechanical harvest
- Used in banana, coffee, palm, citrus plantations to kill weeds
incl. for snake precaution
- Pre-season weed control for many cultivations
Mode of Action:
- Non-Systemic (contact-Herbicides)
- Non-selective (kill everything)
Dissipation:
- Quite stable, strongly bind on clay thus low residues expected on crops
Toxicity:
- Paraquat (high), Diquat (intermediate)
EURL-SRM
-EURL-FV/SRM
High AcuteWORKSHOP
Toxicity to2010
farmers upon inhalation (irreversible lung damage)
Maleic hydrazide

Maleic Hydrazide
Use:
Plant growth regulator, herbicide
- Inhibits sprouting (potatoes, onions, carrots)
- Induces dormancy in citrus fruits
- Used as herbicide in combination with 2,4-D
Mode of Action:
- Systemic, absorbed by leaves and roots,
inhibits cell division
Toxicity:
- Comparably low toxicity to mammals.

Fosetyl Aluminium
Phoshorous acid (Phosphonic acid)
Fosetyl (Ethylphosphonic acid)
EURL-SRM
Current Residue Definition: EU Reference Laboratory
Fosetyl-Al (sum of fosetyl, phosphorous
acid and their salts, expressed
using Singleas fosetyl)
Residue Methods
Fosetyl-Aluminium
Use:
- Fungicide
- Control of diseases caused by fungi (e.g. Phythophthora)
- On a variety of crops (e.g. berries, fruiting vegetables, vine)
Note: Fosetyl-Al not the only source of phosphonic acid
- Phosphonic acid + salts used as plant strengtheners (incl. organic farming).
- Additional source: foliar P fertilizers.
Mode of Action:
Phosphonic acid degradant activates plants defence system
against fungal infections, e.g. peronospora in vine cultures
Toxicity:
EURL-SRM
Amitrol = 3AT

Amitrol
Use:
- Non-Selective Herbicide (unlike all other triazoles x Fungicides)
- Control annual weeds mostly in non-crop lands
- Also in food cultivations e.g. Kiwi (NZ), Soy, Cereals (pre-planting)
Mode of Action:
Toxicity:
- Proven endocrine disruption activity,
- Strongly suspected to induce thyroid cancer
Included in a biocide ban

EURL-SRM
approved by European Parliament in Jan. 2009
Ethephon/HEPA
Currently, no legal
relevance in food
but toxicological
concerns
HEPA
HO
Ethephon
Use:
- Plant growth regulator
- promotes pre-harvest ripening in apples, berries, citrus etc.
- facilitates harvesting by loosening the fruits
- shortens and strengthens the stem in cereals, maize etc.
- accelerates post-harvest ripening in mango, pine apples etc.
Mode of Action:
- Systemic, decomposes to ethylene,
affects growth and ripening
Toxicity:
Comparably low toxicity to mammals.
Daminozide

Daminozide
Use:
- Plant growth regulator
- Inhibition of internodial elongation. More compact plants.
- Formerly used on apples and peanuts
- Not used in Europe any more
Mode of Action:
- Systemic, absorbed by the leaves, translocated throughout plant
Toxicity:
Glyphosate, Glufosinate
Trimethylsulfonium
Trimesium
No legal Counter-ion of Glyphosate

relevance in food with proper MRL
3-MPPA = MPP NAG = N-acetyl-glufosinate
EURL-SRM
NAG is formed in transgenic crops as metabolite, next to check
Glyphosate and
Glufosinate
Use:
- Broad spectrum Herbicides
Against weeds, grasses,
As desiccants to dry off crops before harvest (potato, canola)
Transgenic crops resistant to glyphosate / glufosinate (Corn, Soy, Canola)
Combination with other herbicides (often double resistant GM-crops)
Glyphosate makes 60% of worldwide herbicide sales!!!

Mode of Action:
- Systemic
- Inhibit production of amino acids essential for plant growth
Toxicity:
EURL-SRM
-EURL-FV/SRM
Comparably low toxicity
WORKSHOP 2010 to mammals and insects. for Pesticide Residues
O H
H O P C H 2N H C H 2C O O H
+
OH
pKa=0.78
O H O H
H O P C H 2N H C H 2C O O H H O P C H 2N H C H 2C O O H
+
O- OH
pKa=2.29 Minor tautomer

O H
H O P C H 2N H C H 2C O O -
- +
O
pKa=5.96
-
O H
O P C H 2N H C H 2C O O -
Glyphosate,
O-
+
pH dependency
pKa=10.98
O
-O P C H -
EURL-SRM O -
2N H C H 2C O O Ref: Chamberlain et al. (1996). Pestic. Sci., 47.
Multiresidue Method for SRM Analytes
Weigh in 10g of frozen sample
(5 g for dry material)
Add ISTD-solution
Add 10 mL water (for dry material)
Add 10ml of Extraction solvent

(MeOH w. 1% Formic Acid)
Shake vigorously 1 min
Centrifuge 5min, 4000RPM
Filter Aliquot
(through 0,45m syringe filter)
Fill in vial
EURL-FV/SRM WORKSHOP
Perform 2010
LC-MS/MS analysis
Multiresidue Method for Paraquat Diquat
Weigh in 10g of frozen sample
(5 g for dry material)
Add ISTD-solution
Add 10 mL water (for dry material)
Add 10ml of Extraction solvent

MeOH + 0.1 M HCl in water (1:1)
Shake 1 min, heat 15 min at 80C and re-shake 1 min
Centrifuge 5min, 4000RPM
Filter Aliquot
Variation for
(through 0,45m syringe filter) Paraquat
Fill in vial + Diquat
Perform 2010
LC-MS/MS analysis
Polar Pesticides
Measurement
Validation
Analysis of real samples

Methods
Pesticide Name Isotopically labelled ISTDs ESI ESI
Amitrol Quats & Glyphosate Fosetyl neg pos
& Co. Co. & Co. /MH
Amitrol x Ordered
ETU x NEW D4 - ++
PTU x NEW D6 - ++
Chlormequat x x Existed D4 - ++
Mepiquat x x Existed D3 - ++
Daminozide x x Existed D6
Cyromazine x x NEW D6 + ++
Paraquat x Existed D4 - ++
Diquat x Existed D6 - ++
Glufosinate/MPPA/NAG x 3x NEW D3/D3/D3 ++ ++/++/-
G: 13C2 15N
Glyphosate/AMPA x Existed ++ (+)
AMPA: 13C 15N
Ethephon/HEPA x 2x NEW D4/D4 ++ -
N-Acetylglufosinate x NEW D3 ++ -
Fosetyl-Al Screening x NEW D15 (=3x5) ++ (+)
Maleic hydrazid x NEW D2 ++ (+)
COLUMN: ObeliscR (SIELC) 150x2mm 5;
Quats & Co. Eluent: A: 20 mmol NH4-formate in H2O (pH 3 w. FA); B: ACN (Gradient: 20%A to 80%A)
Amitrol & Co. Eluent: A: 50 mM NH4formate in H2O; B: ACN (Gradient: 3%A to 70%A)
Glyphosate& Co. COLUMN: AS11 (Dionex) 250x2mm;
EURL-SRM Eluent: A: H2O; B: 1mM citric acid adj. to pH11 w. DMA (Gradient: 0%B to 50%B)
EURL-FV/SRM WORKSHOPCOLUMN:
2010ObeliscR (SIELC) 150x2mm 5;
Fosetyl / MH
Eluent:2010
Almera/Spain, 27-28 October A: 50 mM NH4formate + 0,1% FA ; B: ACN (Gradient: 3%A to Single
using 70%A)Residue Methods
Quats & co.
Chlormequat
Mepiquat
Require stronger extraction
Diquat conditions than other compounds
Paraquat Generic conditions

OK for screening
Problems with
simultaneous analysis
of this pair Daminozide
Trimesium cation
Amitrol
LC-MS/MS Method: Quats & co.
Paraquat/Diquat and Amitrol could NOT be included in same LC-method
- Paraquat/Diquat require acidic eluent:

A: 20 mMol NH4formate in water (pH3 with FA)
B: ACN
Gradient: 20% A to 90% A
- Amitrol hates acidity

A: 50 mMol NH4formate in H2O;
B: ACN
Gradient: 3% A to 70% A
Chlormequat work well

Mepiquat under both
Daminozide conditions
EURL-SRM
Trimesium EU Reference Laboratory
LC-Column used...
Column used for Quats: ObeliscR by Sielc
R stands for RP-Charakter, but we have used it in NP-mode!
LiSC (Liquid Separation Cell)

Zwitterionic Ligands
Small pores act like living cells
Pore-openings act as a membrane
Different environment inside and outside the cells

LC-MS/MS Method: Quats-Co with ObeliscR
Trimesium Daminozide Chlormequat Mepiquat Diquat Paraquat
77/62 161/143 122/58 114/98 184/156 186/171
Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 10 of 38 from 080...
Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 10 of 38 from 080529_Validierun...
0.1 ppm on potato Karotte 1%As mit MeOH mit 0,1 ug/IS - Chlormequat 122.1 / 58.2 (Unknown) 122.1/58.2 amu - sample 9 of 38 from 080529_Validieru...
Karotte 1%As mit MeOH mit 0,1 ug/IS - Mepiquat 114.2 / 98.1 (Unknown) 114.2/98.1 amu - sample 9 of 38 from 080529_Validierung ...
Karotte 1%As mit MeOH mit 0,1 ug/IS - Diquat 184.2 / 156.1 (Unknown) 184.2/156.1 amu - sample 9 of 38 from 080529_Validierung Q...
Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Paraquat 186.2 / 171.1 (Unknown) 186.2/171.1 amu - sample 10 of 38 from 080529_Validieru...
4.02 1.90 2.77 3.60 6.59

3.2e4
1.5e5 1.00e4
9.5e4 5500
3.0e4 1.20e5 9500.00
9.0e4 1.4e5
2.8e4 9000.00
8.5e4 1.10e5 5000
1.3e5 8500.00
8.0e4 2.6e4
1.2e5 1.00e5 8000.00
4500
7.5e4 2.4e4 7500.00
7.0e4 1.1e5 9.00e4
2.2e4 4000 7000.00
6.5e4 1.0e5
8.00e4 6500.00
2.0e4 4.02
In te n s ity , c p s
I n t e n s i ty , c p s
I n t e n s it y , c p s
6.0e4
In te n s i t y , c p s
9.0e4 3500 6000.00
5.5e4 1.8e4 7.00e4
5500.00
8.0e4 3000
5.0e4 1.6e4 5000.00
6.00e4
4.5e4 7.0e4 4500.00
1.4e4 2500
4.0e4 5.00e4 4000.00
6.0e4
1.2e4
3.5e4 3.16 3500.00
5.0e4 4.00e4 2000
3.0e4 1.0e4 3000.00
2.5e4 4.0e4 3.00e4 1500
8000.0 2500.00
2.0e4 3.0e4 2000.00
6000.0 2.00e4
1.5e4 1000 1500.00
2.0e4
1.0e4 4000.0
1.00e4 1000.00
1.0e4 500
5000.0 2000.0 500.00
0.00
0.0 0.0 1 2 3 4 5 6 7 8 9 0.00
1 2 3 4 5 6 7 8 9 0.0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9 Time, min 1 2 3 4 5 6 7 8 9
Time, min Time, min
0.1 ppm on pineapple

Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 10 of 38 from 080... Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 10 of 38 from 080529_Validierun... Karotte 1%As mit MeOH mit 0,1 ug/IS - Chlormequat 122.1 / 58.2 (Unknown) 122.1/58.2 amu - sample 9 of 38 from 080529_Validieru... Karotte 1%As mit MeOH mit 0,1 ug/IS - Mepiquat 114.2 / 98.1 (Unknown) 114.2/98.1 amu - sample 9 of 38 from 080529_Validierung ... Karotte 1%As mit MeOH mit 0,1 ug/IS - Diquat 184.2 / 156.1 (Unknown) 184.2/156.1 amu - sample 9 of 38 from 080529_Validierung Q... Kartoffel 1%As mit MeOH mit 0,1 ug/IS - Paraquat 186.2 / 171.1 (Unknown) 186.2/171.1 amu - sample 10 of 38 from 080529_Validieru...
Area: 8.04e+005 counts Height: 1.01e+005 cps RT: 4.018 min Area: 3.96e+005 counts H eight: 3.20e+004 cps RT: 1.896 min Area: 1.95e+006 counts Height: 1.55e+005 cps RT: 2.771 min Area: 2.35e+006 counts Height: 1.28e+005 cps RT: 3.597 min Area: 7.99e+004 counts Height: 5.78e+003 cps RT: 6.738 min Area: 1.57e+005 counts Height: 1.02e+004 cps RT: 6.591 min
4.02 1.90 2.77 3.60 6.59

3.2e4
1.5e5 1.00e4
9.5e4
3.0e4 1.20e5 5500 9500.00
9.0e4 1.4e5
9000.00
2.8e4
8.5e4 1.10e5 5000
1.3e5 8500.00
8.0e4 2.6e4
1.00e5 8000.00
7.5e4 1.2e5 4500
2.4e4 7500.00
7.0e4 1.1e5 9.00e4
2.2e4 7000.00
4000
6.5e4
1.0e5 6500.00
2.0e4 8.00e4
6.0e4 4.02 6000.00
In t e n s it y , c p s
3500
9.0e4
5.5e4 1.8e4 5500.00
7.00e4
5.0e4 8.0e4 3000 5000.00
1.6e4
4.5e4 6.00e4 4500.00
7.0e4
1.4e4 2500
4.0e4 4000.00
6.0e4 5.00e4
3.5e4 1.2e4 3500.00
3.16 2000
3.0e4 5.0e4 4.00e4 3000.00
1.0e4
2.5e4 2500.00
8000.0 4.0e4 1500
2.0e4 3.00e4 2000.00
6000.0 3.0e4
1.5e4 1000 1500.00
2.00e4
1.0e4 4000.0 2.0e4 1000.00
5000.0 1.00e4 500 500.00
2000.0 1.0e4
0.0 0.00
1 2 3 4 5 6 7 8 9 0.00 1 2 3 4 5 6 7 8 9
0.0 0.0 0
Time, min 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Time, min
Time, min Time, min Time, min Time, min
0.2 ppm on wheat flour

Mehl 1%As mit MeOH mit 0,1 ug/IS - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 12 of 38 from 080529... Mehl 1%As mit MeOH mit 0,1 ug/IS - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 12 of 38 from 080529_Validierung Qu... Mehl 1%As mit MeOH mit 0,1 ug/IS - Chlormequat 122.1 / 58.2 (Unknown) 122.1/58.2 amu - sample 12 of 38 from 080529_Validierung... Mehl 1%As mit MeOH mit 0,1 ug/IS - Mepiquat 114.2 / 98.1 (Unknown) 114.2/98.1 amu - sample 12 of 38 from 080529_Validierung Q... Mehl 1%As mit MeOH mit 0,1 ug/IS - Diquat 184.2 / 156.1 (Unknown) 184.2/156.1 amu - sample 12 of 38 from 080529_Validierung Qu... Mehl 1%As mit MeOH mit 0,1 ug/IS - Paraquat 186.2 / 171.1 (Unknown) 186.2/171.1 amu - sample 12 of 38 from 080529_Validierung ...
Area: 1.06e+006 counts Height: 1.03e+005 cps RT: 4.058 min Area: 3.62e+005 counts Height: 2.95e+004 cps RT: 1.898 min Area: 2.33e+006 counts Height: 2.21e+005 cps RT: 2.826 min Area: 1.73e+006 counts Height: 1.14e+005 cps RT: 3.538 min Area: 1.18e+005 counts Height: 1.04e+004 cps RT: 6.699 min Area: 8.48e+005 counts Height: 9.37e+004 cps RT: 6.543 min
4.06 1.90 2.83 3.54 6.70 6.54

2.2e5
1.00e5 1.10e5 1.00e4
2.8e4 9.0e4
9.50e4 9500.00
2.0e5 8.5e4
9.00e4 2.6e4 1.00e5 9000.00
8.0e4
8.50e4 8500.00
2.4e4 1.8e5 7.5e4
8.00e4 9.00e4 8000.00
7.50e4 2.2e4 7.0e4
1.6e5 7500.00
7.00e4 8.00e4 6.5e4
2.0e4 7000.00
6.50e4 1.4e5 6500.00 6.0e4
7.00e4
1.8e4
I n te n s i t y , c p s
In t e n s i t y , c p s
6.00e4 6000.00 5.5e4

5.50e4 1.6e4 1.2e5 5500.00 5.0e4
6.00e4
5.00e4 5000.00 4.5e4
1.4e4
4.50e4 1.0e5
5.00e4 4500.00 4.0e4
4.00e4 1.2e4 4000.00
8.0e4 3.5e4
3.50e4 4.00e4 3500.00
1.0e4 3.0e4
3.00e4 3000.00
8000.0 6.0e4 2.5e4
2.50e4 3.00e4 2500.00
2.00e4 2.0e4
6000.0 4.0e4 2000.00
1.50e4 2.00e4 1.5e4
1500.00
4000.0
1.00e4 1000.00 1.0e4
2.0e4
1.00e4
5000.00 2000.0 500.00 5000.0
0.00 0.0 0.00 0.0
1 2 3 4 5 6 7 8 9 0.0 1 2 3 4 5 6 7 8 9 0.00 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Time, min 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Time, min Time, min

Peak most probably
LC-MS/MS of Diquat from [M-H+]+ formed in
non-acidified extracts
Importance of Extract Composition and eluting as such
Mixneutral 0,1ug/mL- Diquat 92.1/ 92.1(Unknown) 92.1/92.1amu- sample2of 38from080529_ValidierungQuats.wiff Mix neutral 0,1 ug/mL- Diquat 183.2/ 157.1 (Unknown) 183.2/157.1amu - sample2 of 38from080529_Validierung Quats.wiff Mix neutral 0,1 ug/mL- Diquat184.2 / 128.0 (Unknown) 184.2/128.0 amu - sample 2 of 38 from080529_Validierung Quats.wiff
Area: 2.22e+005counts Height: 1.03e+004cps RT: 7.979min Area: 5.85e+006counts Height: 4.91e+005 cps RT: 2.939min Area: 1.12e+005 counts Height: 5.86e+003 cps RT: 7.942 min
7.98 2.94
[M]2+
1.00e4
9500.00
9000.00
4.5e5
4.0e5
105 Most
5500
5000
8500.00
8000.00
7500.00 3.5e5
abundant 4500
7000.00
6500.00 3.0e5
MRM 4000
Std in
I n te n s i ty , c p s
3500
6000.00
5500.00 2.5e5 3000
5000.00
Very
MeOH
4500.00 2500
2.0e5
4000.00
3500.00 2000
small
1.5e5
3000.00
2500.00 1500
1.0e5
2000.00
1000
1500.00
5.0e4
1000.00
500
500.00
0.00 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time, min Time, min
Time, min
Mixsauer 0,1 ug/mLverdin1%Asmit MeOH- Diquat 92.1/ 92.1(Unknown) 92.1/92.1 amu- sample4of 38 from080529_Validierung... Mixsauer 0,1ug/mLverdin1%Asmit MeOH- Diquat 183.2/ 157.1(Unknown) 183.2/157.1amu- sample 4of 38from080529_Validie... Mix sauer 0,1 ug/mLverd in1%As mit MeOH- Diquat184.2 / 128.0 (Unknown) 184.2/128.0 amu- sample 4 of 38 from080529_Validier...
Area: 2.28e+005counts Height: 1.42e+004cps RT: 7.822min Area: 4.21e+004counts Height: 3.04e+003cps RT: 7.797min Area: 2.83e+005 counts Height: 1.76e+004 cps RT: 7.796 min
7.82 7.80
3000
1.4e4 1.7e4
2800
1.3e4 1.6e4
103
2600 1.5e4
1.2e4
1.1e4
2400
2200
small 1.4e4
1.3e4
Std in
1.0e4
1.2e4
2000
MeOH + 1% FA
9000.0 1.1e4
In t e n s ity , c p s
1800
1.0e4
8000.0
1600
9000.0
7000.0
1400 8000.0
6000.0
1200 7000.0
5000.0 6000.0
1000
4000.0 5000.0
800
4000.0
3000.0 600
3000.0
2000.0 400 2000.0
1000.0 200 1000.0
0.0 0 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14
m/z 92/92 m/z 183/157 m/z 184/128

M2+ [M-H+]+ M+
EURL-SRM Deprotonated D. Reduced D. EU Reference Laboratory
EURL-FV/SRM WORKSHOP 2010 (Radical)
Recoveries - Quats & Co.-method
Matrix: Wheat flour 0.1 mg/kg
Name Matrix mached cal. matrix mached cal.) Matrix Effects
(with ISTD) (w/o ISTD) cal. in solv.= 100%
Rec. RSD Rec. RSD Compound ISTD
Chlormequat 106 5.5 106 5.5 82 80

Mepiquat 106 1.9 108 3.1 77 70
Diquat 103 6.2 69 4.4 72 60
Paraquat 114 6.5 75 9.4 37 34
Daminozide 101 2.8 113 4.5 95 99
Trimesium no ISTD no ISTD 104 1.9 85 -
Recoveries using the generic extraction method
Column: ObeliscR (SIELC) 150x2mm 5;

Eluent: A: 20 mMol NH4formate at pH 3.0; B: acetonitrile (gradient: 20% A to 80% A)

Recoveries - Amitrol & Co.-method
Matrix: Cucumber 0.01 mg/kg
Name Matrix-Mached cal. Solvent-Based Cal. Matrix Effects
(with ISTD) (with ISTD) cal. in solv.= 100%
Rec. RSD Rec. RSD Compound
Amitrol 107 4.6 90 3.6 93

(no ISTD) (no ISTD)
Mepiquat 106 5.7 105 5.3 93
Chlormequat 107 1.6 103 1.6 100
ETU 95 2.2 93 2.8 96
PTU 99 3.1 91 3.7 93
(no ISTD) (no ISTD)
Cyromazine 97 4.3 106 3.2 94
Daminozide 107 3.5 86 3.1 80

Recoveries
Matrix: Orange 0.01 mg/kg
Name Matrix-Mached cal. Solvent-Based Cal. Matrix Effects
(with ISTD) (with ISTD) cal. in solv.= 100%
Rec. RSD Rec. RSD Compound
Amitrol 109 6.0 56 5.5 46

(no ISTD) (no ISTD)
Mepiquat 107 8.2 101 9.0 38
Chlormequat 102 3.7 101 3.5 42
Daminozide 120 7.7 72 5.9 50
Column: ObeliscR (SIELC) 150x2mm 5;

Eluent: A: 50 mMol NH4formate; B: acetonitrile

Recoveries - Amitrol & Co.-method
Matrix: Cucumber 0.01 mg/kg
Amitrol 85 /43 Mepiquat 114/98 Chlormequat 122/58 Daminozide 161 /143
Cyromazine 167/68 ETU 103/44 PTU 117/100

Glyphosate & Co.
Glyphosate
Glufosinate
AMPA
3-MPPA
Ethephon
HEPA
LC-Column used
Anion-Exchange Column
Active group: e.g. quarternary amine (permanently cationic)
-N(CH3)3+ OH-
Column used for Glyphosate & Co.:

IonPac AS11 by Dionex
Quarternary Ammonium bound on a DVB/Ethyl-DVB co-polymer

problems with RT-shift / peak shape
LC-MS/MS can occur depending on matrix!
(ISTD fortunately matches for this)
Glyphosate AMPA Glufosinate MPPA Ethephon

168/63 110/63 180/63 150/63 143/107
Spargel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glyphosat 167.9 / 62.9 (Unknown) 167.9/62.9 amu - sample 25 of 34 fromValidierun... Spargel mit 0.05 ug/IS bw. mLin MeOH+1%AS - AMPA 110.0 / 62.8(Unknown) 110.0/62.8 amu - sample 25of 34fromValidierung Et... Spargel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Glufosinate 180.0 / 62.9 (Unknown) 180.0/62.9 amu- sample 25 of 34 fromValidieru... Spargel mit 0.05 ug/IS bw. mL in MeOH+1%AS - MPPA 150.9 / 62.8(Unknown) 150.9/62.8 amu - sample 25 of 34 fromValidierung Et... Spargel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9amu - sample 25of 35 fromValidieru...
Area: 2.12e+004counts Height: 3.43e+003 cps RT: 9.792min Area: 3.99e+004 counts Height: 3.78e+003 cps RT: 7.341min Area: 4.42e+004 counts Height: 7.09e+003 cps RT: 7.269 min Area: 1.27e+005counts Height: 2.01e+004 cps RT: 7.836 min Area: 4.77e+004 counts Height: 6.63e+003 cps RT: 9.096 min
9.79 7.34 7.27 7.84 9.10

3400 2.0e4
3600 6500
3200 6500 1.9e4
3400
0,1 ppm
1.8e4 6000
3000
3200 6000
1.7e4
2800 5500
3000 1.6e4
5500
2600 2800 1.5e4 5000
2400 5000
2600 1.4e4
4500
2200 2400 4500 1.3e4
1.2e4 4000
2000 2200
4000
2000 1.1e4
asparagus
1800 3500
3500 1.0e4
1600 1800
9000.0 3000
1600 3000
1400
8000.0
1400 2500
1200 2500 7000.0
1200
1000 6000.0 2000
1000 2000
800 5000.0
800 7.83 1500
1500 4000.0
600
600
1000 3000.0 1000
400 400
2000.0
200 200 500 500
1000.0
0 0 0.0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
Time, min Time, min
Kartoffel mit 0.05 ug/ISbw. mL in MeOH+1%AS - Glyphosat 167.9 / 62.9(Unknown) 167.9/62.9amu - sample 27of 34 fromValidieru... Kartoffel mit 0.05 ug/IS bw. mL in MeOH+1%AS - AMPA 110.0 / 62.8(Unknown) 110.0/62.8 amu - sample 27 of 34 fromValidierung E... Kartoffel mit 0.05 ug/ISbw. mLin MeOH+1%AS- Glufosinate 180.0 / 62.9 (Unknown) 180.0/62.9amu - sample 27 of 34 fromValidier... Kartoffel mit 0.05 ug/IS bw. mL in MeOH+1%AS - MPPA 150.9 / 62.8 (Unknown) 150.9/62.8 amu - sample 27 of 34 from Validierung E... Kartoffel mit 0.05 ug/IS bw. mL in MeOH+1%AS - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 27 of 35fromValidier...
Area: 2.06e+004 counts Height: 3.38e+003 cps RT: 9.627 min Area: 4.11e+004 counts Height: 5.63e+003 cps RT: 7.559 min Area: 4.74e+004counts Height: 7.01e+003 cps RT: 7.535 min Area: 2.00e+005 counts Height: 2.62e+004 cps RT: 7.723 min Area: 4.78e+004 counts Height: 7.27e+003 cps RT: 9.035 min
9.63 7.56 7.53 7.72 9.04

7000 2.6e4
5500 7000
0,1 ppm
3200
6500 2.4e4
6500
3000 5000
6000 2.2e4
2800 6000
4500
2600 5500 2.0e4 5500
2400 4000 5000

1.8e4 5000
2200
3500 4500 4500
1.6e4
2000
potato
4000 4000
1800 3000 1.4e4
1600
3500 3500
2500 1.2e4
1400 3000 3000
1.0e4
1200 2000 2500 2500
1000 8000.0
1500 2000 2000
800 6000.0
1500 1500
600 1000
4000.0
1000 1000
400
500
2000.0 500
200 8.15 500
8.15
0 0 0.0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
Time, min Time, min
1 2 3 4 5 6 7 8 9 10 11
Time, min Time, min
Time, min
Erdbeere mit 0.05ug/ISbw. mL inMeOH+1%AS- Glyphosat 167.9 / 62.9(Unknown) 167.9/62.9 amu- sample28of 34 fromValidieru... Erdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - AMPA 110.0 / 62.8 (Unknown) 110.0/62.8amu - sample 28of 34 fromValidierung E... Erdbeere mit 0.05 ug/ISbw. mL in MeOH+1%AS- Glufosinate 180.0 / 62.9 (Unknown) 180.0/62.9 amu - sample 28 of 34 fromValidier... Erdbeere mit 0.05 ug/IS bw. mL in MeOH+1%AS - MPPA 150.9 / 62.8(Unknown) 150.9/62.8 amu - sample 28of 34 fromValidierung E... Erdbeere mit 0.05 ug/ISbw. mL inMeOH+1%AS- Ethephon142.9/ 106.9 (Unknown) 142.9/106.9 amu- sample 28 of 35 fromValidier...
Area: 2.37e+004counts Height: 3.90e+003cps RT: 9.601min Area: 8.87e+004 counts Height: 9.32e+003 cps RT: 7.086 min Area: 8.30e+004counts Height: 1.25e+004 cps RT: 7.032 min Area: 1.25e+005 counts Height: 1.96e+004cps RT: 7.386 min Area: 5.16e+004counts Height: 7.93e+003 cps RT: 9.060 min
9.60 7.09 7.03 7.39 9.06

9000 1.9e4
3800
1.20e4 7500
8500 1.8e4
3600
8000 1.7e4 7000
3400 1.10e4
0,1 ppm
7500 1.6e4 6500
3200
1.00e4
7000 1.5e4
3000 6000
6500 1.4e4
2800 9000.00
5500
1.3e4
2600 6000
8000.00 5000
2400 5500
1.2e4
In t e n s i ty , c p s
2200 1.1e4 4500

5000 7000.00
2000 1.0e4 4000
4500
strawberry
6000.00 9000.0
1800 4000 3500
1600 8000.0
3500 5000.00
3000
1400 7000.0
3000
1200 4000.00 6000.0 2500
2500
1000 5000.0 2000
2000 3000.00
800 4000.0
1500
1500
600 2000.00 3000.0
1000 1000
400 2000.0
500
1000.00
7.76 500 1.58
200 1000.0
0
0 1 2 3 4 5 6 7 8 9 10 11 0.00 0.0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
Time, min

Note RT shift, but ISTD
HEPAinrealsamples behaves the same
Oats
0.034mg/kg
Figs
0.12mg/kg
Pineapple
0.009mg/kg
HEPAD4(IS) HEPA HEPA HEPA

129/78.8 125/94.8 125/78.9T 125/62.8
Chromatographic experiments - Ethephon
Ethephon 0,1 ug/mL MeOH 27.09. - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 69 of 112 from 0709...
Area: 1.03e+005 counts Height: 1.29e+004 cps RT: 9.658 min Ethephon 21.09. 0,1 ug/mL in MeOH - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 4 of 75 from 0709...
1.20e4 1.10e4
Ethephon
1.10e4 1.00e4
1.00e4 9000.00
9000.00
8000.00
8000.00
7000.00
7000.00
6000.00
5000.00
0,1 g/mL 6000.00
5000.00
11.32
11.51
4000.00 4000.00
3000.00
2000.00
In SOLVENT 3000.00
2000.00 12.29
1000.00
13.12
1000.00 15.21 15.71
0.00
2 4 6 8 10 12 14
Time, min 0.00
2 4 6 8 10 12 14 16 18
Time, min
Kalib Apfel 0,05 ug/mL Ethephon - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 12 of 93 from 07092... Kalib Apfel 0,05 ug/mL Ethephon - Ethephon 142.9 / 106.9 (Unknown) 142.9/106.9 amu - sample 7 of 75 from 070921...
Area: 2.71e+004 counts Height: 3.62e+003 cps RT: 10.25 min Area: 1.16e+004 counts Height: 7.16e+002 cps RT: 10.93 min
3500
Ethephon
8000
3000 nearly no 7000

extreme
2500
suppression 6000 7.90
suppression
2000 0,05 g/mL 5000
4000
1500
in APPLE
7.65
3000 8.86
1000
1.64
2000
500
1000 10.93
9
0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 16 18
Time, min
Time, min
Dionex AS11 250x2mm Atlantis HILIC 100x2mm

A: water A: 100 mMol NH4formate in water
B: 1 mMol citric acid in water, adjusted
B: acetonitrile
to pH 11 with Dimethylamin
Recoveries - Glyphosate & Co.- Method
Matrix: Wheat flour 0.1 mg/kg
Name Matrix mached cal.
Matrix mached cal. Matrix Effects
(with ISTD) (w/o ISTD) cal. in solv.= 100%
Glyphosate 118 5.8 83 5.9 277 289
Glufosinate no ISTD no ISTD 85 4.6 194 -
AMPA 119 5.7 83 7.7 247 236
3-MPPA no ISTD no ISTD 85 1.3 161 -
Ethephon 109 5.9 91 7.1 143 141

HEPA-Recoveries
Matrix: Orange 0.02 mg/kg
Name Matrix mached cal.
(with ISTD)
Rec. RSD (n=5)
HEPA 119 3.5
Isotope-labelled ISTD provided by Bayer CropScience

Fosetyl, MH - Method
Fosetyl-Aluminium
Maleic Hydrazide

LC-Column used...
Column used for Fosetyl andMH: ObeliscR by Sielc
R stands for RP-Charakter, but we have used it in NP-mode!
LiSC (Liquid Separation Cell)

Zwitterionic Ligands
Small pores act like living cells
Pore-openings act as a membrane
Different environment inside and outside the cells

LC-MS/MS Maleic Hydrazide
Onion with 0.06 mg/kg maleic hydrazide
Kalib 0,03 ug/IS auf Zwiebel 1%AS in MeOH - Maleic hydrazid 111.0 / 81.8 T (Unknown) 111.0/81.8 am u - sample 28 of 38 from Vali... Kalib 0,03 ug/IS auf Zwiebel 1%AS in MeOH - Maleic hydrazid 111.0 / 41.9 (Unknown) 111.0/41.9 am u - sample 28 of 38 from Validie...
7.78 2.99
6000
5000
5500
4500 2.82
5000
4000
4500
4000 3500
3500 3000
3000
2500
1.88
2500
2000 7.78 12.28
2000
1500
1500
1000 6.70
1000 6.37
500
500
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time, min Time, min
Maleic hydrazid 111/82 Maleic hydrazid 111/42

Kalib 0,03 ug/IS auf Zwiebel 1% AS in MeOH - Maleic hydrazid D2 113/84(IS) (Unknown) 113.0/83.8 amu - sample 28 of 38 from Vali... Kalib 0,03 ug/IS auf Zwiebel 1%AS in MeOH - 113.0 / 41.9 (Unknown) 113.0/41.9 am u - sample 28 of 38 from Validierung_Malein_Fo...
7.78 7.78
2.2e5
2.1e5
6.0e4
2.0e5
1.9e5
5.5e4
1.8e5
1.7e5 5.0e4
1.6e5
1.5e5 4.5e4
1.4e5
4.0e4
1.3e5
13.00
1.2e5 3.5e4
1.1e5
1.0e5 3.0e4
9.0e4
2.5e4
8.0e4
7.0e4
2.0e4
6.0e4
5.0e4 1.5e4
4.0e4
3.0e4 6.67 1.0e4
2.0e4
5000.0
1.0e4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time, min
Time, min
ISTD
ISTDMaleic
Maleichydrazid D2 111/84
H. D2 113/84 ISTD
ISTDMaleic
Maleichydrazid D2 111/42
H. D2 113/42

Longer RT, less
suppression with
LC-MS/MS of Fosetyl neutral extracts
Grapes 0,5 g/mL with 0,03 mg/kg

Atlantis HILIC - late RT Dionex-AS11 - early RT
32744 Traube 0,5 g/mL - Fosetyl-Al 109.0 / 81.0 (Unknown) 109.0/81.0 amu - sample 55 of 86 from 071019Mc_Malein.... 32744 Traube 0,5 g/mL - Fosetyl-Al 109.0 / 63.0 (Unknown) 109.0/63.0 amu - sample 55 of 86 from 071019Mc_Malein....
Area: 2.81e+004 counts Height: 5.69e+003 cps RT: 9.708 min Area: 8.38e+003 counts Height: 1.60e+003 cps RT: 9.704 min 32744 Traube 0,5 g/mL - Fosetyl-Al 109.0 / 81.0 (Unknown) 109.0/81.0 amu - sample 37 of 67 from 071017Mc_Eth 32744 Traube 0,5 g/mL - Fosetyl-Al 109.0 / 63.0 (Unknown) 109.0/63.0 amu - sample 37 of 67 from 071017Mc_Ethep
1600
9000 1000
4000
8000 1400 900
3.14
3500
7000 800
1200
2.87 3000
9.71 700
6000
1000
2500 600
5000
800 500
3.63 2000
4000
4.23 1.38 2.45 400
600 1500 2.87 4.01
3000 4.68
4.80
4.96 300
2.27 400 1000
2000 6.71 5.56 200
7.38 10.94
6.48 7.04 7.86 8.52 9.07
200 500 6.44 9.92
1000 10.92 11.31 12.52 100
9.27
0 0 0 0
2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 2 4 6 8 10
Fosetyl-Al 109.0 / 81.0 Fosetyl-Al 109.0 / 63.0 Fosetyl-Al 109.0 / 81.0 Fosetyl-Al 109.0 / 63.0
Moderate matrix effects Strong matrix effects

(20% suppression) (80% suppression!)
Glyphosate & Co.-Method

HILIC also works not optimal but suitable for
well for Fosetyl Fosetyl SCREENING

LC-MS/MS of Phosphorous acid
w. Glyphosate+co method
0.1 ppm
81/63
Problem: no isotopically labelled ISTD
Phoshorous acid (Phosphonic acid)
81/35
Shifts of H between O and P

Exchange of OH in aqueous media

Recoveries - Fosetyl & MH- method
Fosetyl: Strawberry 0.1 mg/kg
Maleic Hydrazide: Onion 0.1 mg/kg
Compound
Matrix mached cal. Matrix mached cal. Matrix Effects
(with ISTD) (No ISTD) cal. in solv.= 100%
Fosetyl-Al 98 4.6 96 4.3 54 44
Maleic 105 3.0 112 1.9 62 72

Hydrazide

LODs
Limits of detection in fruits, vegetables and cereals
Method
Pesticide Cucumber Orange Barley
M1 Amitrol 0.01 0.01 0.02

M1 ETU 0.01 0.02 0.02
M1 PTU 0.01 0.02 0.02
M1/M2 Chlormequat 0.005/0.005 0.005/0.005 0.01/0.01
M1/M2 Mepiquat 0.005/0.01 0.005/0.01 0.001/0.02
M1/M2 Cyromazine 0.01/0.01 0.01/0.01 0.02/0.02
M1/M2 Daminozide 0.01/0.02 0.01/0.02 0.02/0.04
M1/M2 Trimethylsulfonium-Cation 0.01/0.005 0.01/0.005 0.02/0.01
M1/M2 Morpholine 0.01 0.01 n.a.
M2 N,N-Dimethylhydrazine 0.005 0.005 0.01
M2 Diquat 0.005 0.005 0.005
M2 Paraquat 0.005 0.005 0.005
M3 Ethephon 0.01 0.01 0.02
M3 HEPA 0.01 0.01 0.02
M3 Glyphosate 0.01 0.02 0.02
M3 AMPA 0.01 0.02 0.02
M3 Glufosinate 0.01 0.02 0.02
M3 MPPA 0.01 0.02 0.02
M3 N-AG 0.02 0.02 0.02
M3/M4 Fosetyl 0.005/? 0.005/? 0.005/?
M4 Maleic Hydrazide 0.01 0.01 0.02
M5 Streptomycin 0.01 n.a. n.a.
M5 Kasugamycin 0.01 n.a. n.a.

Use of ISTDs
Approx. cost of ISTDs
Name of ISTD -cent per portion
(exemplary)
If added to If added to
sample (2g) 1 mL sample extract aliquot (0.1g)
Chlormequat D4 6c 0.3 c
Mepiquat D3 136 c 7c
Diquat D4 8c 2c
Paraquat D6 146 c 7c
Glyphosate 1,2-13C2 15N 660 c 33 c
Ethephon D4 13 c 0.6 c
Fosetyl-Al D15 8c 0.4 c
Maleic hydrazide D2 12 c 0.6 c
Notes:
If detections are rather seldom ISTD can be added in a 2nd analysis in case of positives
If RTs are shifting, adding ISTD is highly recommended, at least to the 1 mL aliquot (vial)
- RT of ISTD gives additional certainty to identification

Residue findings
of polar pesticides in real
samples

Chlormequat and Mepiquat -
Residues
Data from CVUA Stuttgart 2005-2008
Commodities Chlormequat Mepiquat
Mushrooms* +++ +++

(champignons, oyster) (also in organic*) (also in organic*)
Pears ++ ++
(decreasing) (decreasing)
Cereals +++ +++
(also in organic**) (also in organic**)
Fruiting vegetables
+ +
(tomato, sweet pepper)
* From the cereals used as a substrate for fungi growth

** In organic often due to cross-contamination in mills
Glyphosate / Glufosinate - Residues
Glyphosate:
Analyzed: 261 samples (28 different commodities) from 16 countries
Positive: 5 samples (asparagus, shallots, oats, bran) from 3 countries
MRL-violations: none
Samples analyzed with residues Min (mg/kg) Max (mg/kg)

Asparagus 37 2 0,01 0,04
Onions, shallots 4 1 0,02
Cereals 24 2 0,52 0,61
Glufosinate: 1x in potatoes
AMPA, 3-MPPA: No detections
Ethephon - Residues
Analyzed: 1162 samples (79 different commodities) from 45 countries
Positive: 74 samples (12 commodities) from 18 countries, MRL-violations: 11
No.
C o u n tr y of No. of E th e p h o n O r g a n ic
M a tr ix >M RL
o f o r ig in s a m - fin d in g s m g /k g g ro w th
p le s
P in e a p p le A f r ic a 1 1 0 .5 4
B r a z il 1 1 0 .0 3
C o s t a R ic a 16 10 0 .0 4 to 1 .0
G hana 5 1 0 .2 9
H o n d u ra s 1 1 0 .3 0
K a m e ru n 4 3 0 .0 3 1 to 1 .2 5 2 x y e s
U nknow n 1 1 0 .1 7
Panam a 2 2 0 .0 1 6 to 0 .4
P in e a p p le J u ic e U n k n o w n 10 7 0 .0 0 5 to 0 .2 2 4 x y e s
A p p le G e rm a n y 75 6 0 .0 0 5 to 0 .1
Pear I t a ly 11 1 0 .0 1
D u r ia n T h a ila n d 2 2 0 .3 1 to 1 0 .1 2
F ig g s B r a z il 3 3 0 .2 2 to 0 .5 5 3

Ethephon Residues cont.
No. >MRL
of
Country sam- No. of Ethephon Organic
Matrix of origin ples findings mg/kg growth
Sweet Pepper Spain 28 4 0.049 to 1.2
Kaki Italy 3 1 0.12 1
Spain 27 4 0.024 to 0.61 1
Pomelo China 5 3 0.032 to 0.24 2
Cherry Germany 12 1 0.022
Grapes Argentina 5 1 0.063
Italy 48 7 0.28 to 0.78
Namibia 2 1 1.43 1
Spain 2 1 0.016
South Africa 25 6 0.004 to 0.4 1 x yes
Tomato Belgium 7 2 0.12 and 1.3 1
The Netherlands 11 4 0.18 to 0.68
Total
(positiv matrices) 307 74 7 11

Ethephon Residues newer data (1890 samples)
47
Pine apple s 1
1
18
Grape s 1
16
Tomatoe s 2
4
16
Swe e t pe ppe rs 1
2
13
Figs 11
10
Apple s 6 ARfD
8
Pine apple juice s exceedances !!
6
Pe rsimmons 2
4
Pome los 2
4
Orange s 1
3
2
Che rrie s
2
Durians 2
1
Plums
1 with residues
Pe ache s
1
M angoe s 1 residues > MRL
1 ARfD exceedance
Grape fruits
1
Pe ars
EURL-SRM numbe r of sample s
Ethephon and HEPA in real samples
Sample Ethephon mg/kg HEPA mg/kg
Pomelo 0.24 0.032
Figs 0.22 0.071
Grapes 1.43 0.220
Pomelo 0.23 0.063
Figs 0.55 0.120
Figs 0.52 0.096
Figs 0.81 0.122
Figs 0.13 0.038
Figs 0.42 0.093
Orange 0.55 0.040
Pineapple 1.2 0.0092
Pineapple 0.65 0.024
From straw
Oat - used to grow 0.034
Champignons - on mushrooms 0.17
Champignons - 0.23
EURL-SRM
Champignons - 0.33
Maleic Hydrazide - Residues
Analyzed: 177 samples (15 commodities) from 13 countries
Positive: 15 samples (3 commodities garlic, onion and shallots) from 4 countries.
MRL-violations: 1 MRL-violation in shallots from France with 17.2 mg/kg.
1 organic onion sample from Italy with 1.7 mg/kg.
Minimum Maximum
No. of
Commodity Country of Origin Value Value Remarks
findings
(mg/kg) (mg/kg)
Garlic France 1 14,3
France 4 0.27 15
Germany 1 1.4 Illegal use
Onions Italy 1 1.7 1 organic
The Netherlands 2 4.1 8.4
Unknown 1 0,2
Shallots France 5 6.6 17.2 1 > MRL

Fosetyl Aluminium Residue Findings
Analyzed: 1241 samples (85 commodities) from 44 countries
Positive: 37 samples (in 9 dif. commodities) from 9 countries
MRL-violations: None
No. of
Country No. of Fosetyl Organic
Matrix fin-
of origin samples mg/kg growth
dings
Lettuce, ice Spain 4 3 0.017 to 0.085
Lettuce, cabbage Germany 20 1 0.009
Lettuce, lollo France 2 1 0.008
Strawberry Germany 29 13 0.019 to 0.067
Greece 2 1 0.047
Spain 59 2 0.82 to 1.1
Cucumber Germany 12 2 0.68 to 2.6
Italy 4 3 0.008 to 0.3 3 x yes
Maroc 2 1 0.045 1 x yes
Spain 3 2 0.032 and 0.43 2 x yes
Litchi Thailand 1 1 0.027
Maracuja Columbia 7 1 0.006
Melon Italy 2 1 0.024
Rucola Germany 6 1 0.01
Italy 2 1 0.055
Tomato Italy 9 1 0.020
Spain 33 1 0.014
EURL-SRM
Grape
The Nether-
lands
1
1 0.040 EU Reference Laboratory
for Pesticide
EURL-FV/SRM
Total WORKSHOP 2010 198 37 6 Residues
Intermediate
Conclusion

Ethephon
Fosetyl-Al
Glyphosate
Chlormequat
Maleic hydrazide

Do SRM!
Dont do SRM
Do SRM!
Dont do SRM
Do SRM!
Dont do SRM
Do SRM!
Dont do SRM

III.
Partitioning Losses
(due to unfavorable pH)

Nicotine in Mushrooms

Nicotine in Mushrooms - Background Information:
In 2008 high levels of Nicotine detected in mushrooms at CVUA Sigmaringen
CVUA Stuttgart consulted for confirmation (LC-TOF, -MS/MS 3 GC-MS 3)
Since then many findings by various labs in mushrooms mainly from China
Most affected dried Porcini (Boletus edulis) but also Truffles and Chanterelles
Porcini are reported to be not cultivable
China (Yunnan Region) largest producer (80% of EU-imports from CN)
Chinese authorities say : tobacco is also widely cultivated in Yunnan region
Nicotine is a naturally occurring alkaloid in tobacco (Nicotiana tabacum)
where it occurs at concentrations ranging from 2% to 8%
Cross-contamination in drying/packing sites may be an issue

Intentional use of nicotine as pesticides is also speculated

Nicotine in Mushrooms
Risk assessment/management
EFSA
ARfD : 0.0008 mg/kg body weight;
ADI: 0.0008 mg/kg bw per day
99% of dried mushroom samples contained Nicotine

(conc. often above 1 ppm)
Provisional MRL-Proposal:
Dried ceps with a nicotine > 2,3 mg/kg should be withdrawn from
the market and safely disposed of.
Monitoring program for European wild mushrooms initiated
NICOTINE PROPERTIES:
(K. Chamberlain et al., Pestic. Sci., 47, 265 (1996)
Basic:
pKa1 = 3.1; pKa2 = 8.2
(i.e. predominantly protonated at pH<8.2 and double protonated at pH<3.1)
Polar:
logP = 0.93 (25 C/unionised) , the lower the pH the lower the logP
Volatile:
Pvap = 5.6 Pa (25 C). Evaporation losses reduced at low pH (ionized)
N CH3
N
H
Nicotine by QuEChERS
Optimisation of pH at extraction/partitioning-step
Recoveryfor
Recovery of Nicotine
Nicotine from fresh
with mushrooms
QuEChERS at different pH
- pH-dependant
100
(spiked residues)
90
80
Natural pH
Recovery (%)
70
60
50
40
30
20
10
LC-QToF, ISTD TPP
0
6 7 8 9 10 11
pH-Value before extraction
Using Nicotine D3 as ISTD losses were compensated irrespective of pH

(Rec. 93 - 120 %)

Extraction of incurred residues from Dried Porcini
QuEChERS - impact of pH
E x tra c tio n ra te s o f N ic o tin e fro m P o rc in i u s in g Q u E C h E R S
(in c u rre d re s id u e s )
a re a /a re a IS T D
2,5
1,5
0,5
0
natural 8 9 10 11
p H -V a lu e
Extraction of incurred residues from Dried Porcini
QuEChERS pH 10, Variation of Temperature and Time

E xtractio n rates o f N ico tin e fro m P o rcin i u sin g Qu E C h E R S
(in cu rred resid u es)
area/area IS TD
2,0
1,5
1,0
0,5
0,0
1 m in (RT) 10 m in (RT) 30 m in (RT) 1 m in Ultra- 20 m in (60C) 20 m in (90C)
Turrax (RT)
e x tra ction tim e

QuEChERS modification for Nicotine
10 g Fresh mushrooms or
2 g dried sample + 10 mL water
Bring pH to 10-11
by addition of NaOH 5N
Add 4 g MgSO4+ 1g NaCl
Shake 1 min by hand

Centrifugue 5 min.
at 3500 r.p.m.
Take alicuot
Add 150 mg MgSO4 anh.
+ 50 mg PSA per mL extract
Shake for 30s
Centrifugue 5 min. Validation (n=5)
at 3500 r.p.m. using Champignon 10 g
Mean Recovery: 106%
Take aliquot and acidify
w. 5% Formic Acid to pH~5 RSD: 4,1%
EURL-SRM Analysis via GC or LC

Ad-hoc PT on Nicotine in dried mushrooms
in collaboration with COOP-Switzerland
2 Boletus (CN, BiH) + 1 Mu-Err (CN) w. incurred Nicotine (~1.0/0.7/2.5 mg/kg)
z-scores
Preliminary Data
4,0
38 Acceptable (= 90%)
3,0 Possibly systematically 2 Questionable (= 5%)
underestimating 2 Unacceptable (= 5%)
2,0
1,0
z-score
0,0
-1,0
-2,0
= Methanol employed for extraction
-3,0
= Petrol ether employed for extraction
ND All other labs variations of QuEChERS
-4,0
5 8 11 14 9 4 10 7 3 12 6 13 2 1
z-scores for Boletus mushroom (Bosnia and Herzegovina) Lab-Code
z-score for Boletus mushroom (China)
EURL-SRM z-score for Mu-Err mushroom (China)

V. Pesticides requiring
inclusion of
metabolites or
reaction products

Acidic Pesticides
-
Bound Residues

Acidic pesticides cleanup issue
Example: Extract of red currant Citrate-Buffered

12
Sample pH 5.1 9
pH=8.3 pH
mg/mL pH=7.8
8
Losses of acidic components 10
7
after cleanup 8 6
5
6
Acidic compounds interact with pH=3.5
4
PSA. Thus skip PSA cleanup 4 3

2,4 2
2
1,0 1
0,5
0 0
Raw Extract PSA 25 PSA 50
mg/mL mg/mL
mg co-extractives/mL Extract
pH

QuEChERS alkaline hydrolysis
Example 2,4-D
Selective systemic herbicide

Control of broad leaved weed
Plant growth regulator used to prevent premature fruit drop
Formulations include free acid, salts, esters
May form conjugates
Method designed to convert all possible residues to free acid
Acids are often covalently bound to matrix components

and thus their concentration underestimated!

Acidic pesticides
pH-issue: Ionization of pesticides at low or high pH-values

Acids: HX ' H+ + X-
Bases: B + H+ ' BH+

Ionic form prefers to stay in the water phase
pH-range of agricultural
samples: ~2.5 7

Acidic pesticides influence of pH on recovery
LC-MS/MS, ESI (-), No PSA Cleanup 120
100
Recovery
%
80
60
max. pH 5.5
40
20
Clopyralid
Picloram
Imazapyr
Benazolin
Imazethapyr
4-CPA
Naphthylacetic acid
Dicamba
Imazaquin
MCPA
2,4-D
Fluoxypyr
2,4,5-T
Mecoprop
Triclopyr
2,4,5-TP
Propyzamid
2,4-DP
Bentazon
Bromoxynil
Ioxynil
0
Fluazifop
Bromacil
2,4-DB
MCPB
pH
pH
pH
pH
3
4
5
6
lower pKa general trend higher pKa

QuEChERS schematic description with AH
Citrate-Buffered QuEChERS
Weigh 10 g of Frozen Sample

alkaline hydrolysis:
Shake
Add NaOH and store for
Add 10 mL Acetonitrile 30 min at RT,
then neutralize w. H2SO4
Add ISTD-Solution
Shake
Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer
(pH 5-5.5)
Shake and Centrifuge optionally:
Freeze-out of extracted
Analysis of acidic pesticides by LC-MS/MS fat over night

Alkaline cleavage or the release of phenoxy-acids
Alkaline Hydrolysis for the release of phenoxy-acid pesticides

wheat sample
1400 At 70 C At Room Temperature

1200
Wheat flour
Relativ concentration
pH 12
30 min
pH 10
60 min
pH 10
30 min
pH 9
30 min
with incurred
1000
residues
800 715
606 619
600
336
400 275
200 135 100
0
in
in
in
in
in
in
.
ol
m
m
m
dr
30
0
30
30
60
30
hy
3
/
9/
12
/
/
12
10
10
10
o
pH
w/
pH
pH
pH
pH
pH
RS
hE
EC
Qu
Alkaline Hydrolysis Level of MCPA in berries
350
Goosebeery Strawberry
300
Relative Response of MCPA [%]
250
200
150
100
50
0
no alkaline hydrolysis with alkaline hydrolysis

Alkaline Hydrolysis Level of 2,4-D in different citrus samples
0,7
no alkaline hydrolysis with alkaline hydrolysis

x 2.5
0,6
x 2.2
0,5
0,4
mg/kg
x 1.8 factor: 1.8 5.8

x 3.4
0,3
x 3.3
x 2.6
0,2
x 5.0
x 1.8
x 3.0
x 5.8
0,1
0
sample 01 sample 02 sample 03 sample 04 sample 05 sample 06 sample 07 sample 08 sample 09 sample 10

Matrix: Cucumber Experiments performed in advance to QuEChERS
Redidue Definition
Method: QuEChERS I II III IV V VI VII VIII
AH AH AH EH EH EH EH
(1 mg) (2 mg) (5 mg) (2 mg)
Acid Ester Acid Esters Conjug. Residue Definition VII + II
RT 80C RT RT RT RT RT
30 min 30 min 16 h 3h 3h 3h 16 h
2-butyl 63 33 19 0 0 0 0 0
butoxyethyl 7 0 0 0 0 0 0 0
ethylhexyl 103 47 26 7 4 2 0 0
2,4,5-T isooctyl x 2,4,5-T (F) 104 48 26 8 5 3 0 0
isopropyl 15 0 0 0 0 0 0 0
methyl 1 0 1 2 2 2 1 0
octyl 38 10 3 3 2 1 1 0
methyl 0 0 0 0 0 0 0 0
butyl 0 0 0 0
ethyl 2,4-D (sum of 2,4-D and its esters exp. as 0 0 0 0
2,4-D isobutyl
x x
2,4-D) 0 0 0 0
isooctyl 9 2 3 0
isopropyl 0 0 0 0
2,4-DP methyl x Dichlorprop, incl. Dichlorprop-p
0 0 0 0 0 0 0 0
Carfentrazone-ethyl (determined as
Carfentrazone ethyl x x carfentrazone and expr. as carfentrazone-
ethyl)
0 0 0 0 0 0 0 0
Chlorthal dimethyl x Chlorthal-dimethyl 100 61 78 93 89 78 57 24
Cinidon-ethyl (sum of cinidon ethyl and its
Cinidon ethyl x
E-isomer)
10 0 0 2 1 0 0 0
Clodinafop and its S-isomers, expr. as
Clodinafop propargyl x
clodinafop (F) 2 0 0 0 0 0 0 0
Cyhalofop-butyl (sum of cyhalofop butyl
Cyhalofop butyl x x
and its free acids) 79 2 0 0 0 0 0 0
Dicamba methyl x Dicamba 32 5 1 0
Dichlorprop 2-ethylhexyl x Dichlorprop, incl. Dichlorprop-p 70 58 41 8 3 1 1 0
Diclofop (sum diclofop-methyl and diclofop
Diclofop methyl x x
acid expr. as diclofop-methyl) 60 1 0 1 1 1 0 0
Diethatyl ethyl 8 0 0 12 2 0 0 0
Dinoseb acetate x Dinoseb 8 0 0 0 0 0 0 0
isooctyl 64 11 11 0
Fenoprop methyl 0 0 0 0
Fenoxaprop P-ethyl 37 0 0 1 0 0 0 0
Flamprop isopropyl 103 33 20 97 92 76 53 15
butyl 77 3 1 1 0 0 0 0
Fluazifop methyl 1 0 0 0 0 0 0 0
Flumiclorac pentyl 13 0 0 0 0 0 0 0
EURL-SRM
remaining esters 5%
remaining esters 5-20%
AH = alkaline hydrolysis, pH 12, re-neutralized with H2SO4
EH = incubation of buffered samples with esterase from porcine liver (buffering at pH 6-9 with Pesticide
forphosphate Residues
buffer
remaining esters > 20% 2010
Matrix: Cucumber Experiments performed in advance to QuEChERS
Redidue Definition
Method: QuEChERS I II III IV V VI VII VIII
AH AH AH EH EH EH EH
(1 mg) (2 mg) (5 mg) (2 mg)
Acid Ester Acid Esters Conjug. Residue Definition VII + II
RT 80C RT RT RT RT RT
30 min 30 min 16 h 3h 3h 3h 16 h
ethoxyethyl Haloxyfop incl. haloxyfop-R (Haloxyfop-R 9 0 0 0 0 0 0 0
methyl ester, haloxyfop-R and conjugates
Haloxyfop x x x
methyl of haloxyfop-R expr. as haloxyfop-R) (F)
(R) 6 0 0 1 1 1 0 0
Ioxynil octanoat (x) x Ioxynil, incl. its esters expr. as ioxynil (F)
70 16 0 3 2 1 0 0
1-butyl 1 0 0 0
butoxyethyl MCPA and MCPB (MCPA, MCPB incl. their 0 0 0 0
MCPA ethyl x x x salts, esters and conjugates expr. as 0 0 0 0
ethylhexyl MCPA) (F) (R) 21 3 3 0
thioethyl 3 0 0 0 0 0 0 0
MCPA and MCPB (MCPA, MCPB incl. their
MCPB ethyl x x x salts, esters and conjugates expr. as
MCPA) (F) (R) 3 1 1 1
methyl 0 0 0 0 0 0 0 0
Mecoprop (sum of mecoprop-p and
Mecoprop 1-octylester x 103 85 54 8 5 3 2 1
mecoprop expressed as mecoprop)
2,4,4-trimethylpentyl 104 83 44 7 4 3 0 0
Mefenpyr diethyl 2 0 0 0 0 0 0 0
Nitrothal di-isopropyl 19 0 0 2 1 0 0 0
Picloram isooctyl x Picloram 14 1 1 0
Triclopyr 2-butoxyethyl x Triclopyr 0 0 0 0
Trinexapac ethyl x Trinexapac 8 0 0 82 64 29 10 0
remaining esters 5% AH = alkaline hydrolysis, pH 12, re-neutralized with H2SO4

remaining esters 5-20%
EH = incubation of buffered samples with esterase from porcine liver (buffering at pH 6-9 with phosphate buffer
remaining esters > 20%

Amitraz

Amitraz
Use:
- Acaricide and Insecticide
mite control in many different crops
pome fruit, citrus, stone fruit, strawberries, cucurbits,
solanaceous etc.
To control varroa mite in Honey-production
Mode of Action:
- Non-systemic
- Interacts with the nervous system of mites and ticks
Toxicity:
Relatively high toxicity to mammals
Amitraz
Recoveries with QuEChERS quite good
Better stability of Amitraz in extracts at high pH
PSA cleanup (w/o acidification) increases pH and thus stability
Amitraz
100,0
90,0 Measurement
80,0
of acidified extract
70,0
60,0
0 Tage should be performed
1 Tag
50,0 2 Tage within one day.
4 Tage
40,0
9 Tage
30,0
20,0
10,0
Better dont acidify
0,0 QuEChERS extract !
pH3 pH4 pH5 pH6 pH7 pH8 pH9 pH10

Amitraz
Degradation of amitraz and formation of DMPF (amidine)
during storage of QuEChERS extracts at diff. pH
Residual Amitraz in % DMPF formation expressed in amitraz e quivale nts
100
120
90
100 80
70
80
60
60 50
40
40
30
20 20
10
0
0
3
3
0d
0d
4
1d
1d
5
2d
2d
6
3d
3d
7
4d
Ext ract 8 pH pH
4d
Extract 8
9d
9
Storag e time 9
9d
10 Storage time
17d
10
17d
Amitraz
Residue Definition:
Amitraz including all metabolites containing
2,4-dimethylaniline expressed as Amitraz
Traditional methods involve:

Conversion of Amitraz and degrad. products to 2,4-dimethylanilin
Different approaches:
- Alkaline hydrolysis,
- Acidic hydrolysis,
- Combination of both

Degradation paths of Amitraz
CH3 CH3 CH3 H3C CH3
N N N 1:1 N N
H
H3C Amitraz CH3 H3C
(minor residue) 1:1 Amidine

(main residue)
H+ 1:2 1:2
In presence of
matrix non- OH- OH-
In presence of
quantitative 1:1 matrix non-
quantitative
CH3
H+ CH3 O
NH2 NH
1:1 H
H3C
H3C
EURL-SRM Anilineor DMA Amid
EURL-FV/SRM
(very WORKSHOP
minor/ 2010
n.d.)
Almera/Spain, 27-28 October 2010 (minor
using residue)
Single Residue Methods
Amitraz
Acidic hydrolyses were quantitative:
Amitraz @ Aniline
Amid @Aniline
Amidin remains stable
Aniline remains stable
Alkaline hydrolyses were not quantitative
(especially in presence of matrix)
Amitraz @Amid
Amid @Aniline
Amidin @Aniline
Aniline remains stable (closed vessel to avoid losses!)
Amitraz
Amitraz and its 3 main metabolites well detectable by LC-MS/MS
So, total DMA can be calculated (virtual hydrolysis)
Virtual vs. chemical Hydrolysis:

Virtual hydr.: only covers the detected compounds.
Chemical hydr.: covers also other DMA-containing metab./conj.
Question: Are there any significant levels of other
metabolites/conjugates releasing DMA upon hydrolysis?
Observation: Alkaline hydrolysis did not increase determined
residue level in real samples
In any case:
Acidic hydrolysis does not transform amidine to aniline
EURL-SRM
Risk of losses alkaline hydrolysis is very high. EU Reference Laboratory
Amitraz - Calculating the Total Residue
Important: Mind stechiometries of degradation pathways
- Amidin @ Amitraz 1:1
- Amid @ Amitraz 2:1 or 1:1 (depending of pathway)
- DMA @ Amitraz 2:1
CASE 1: Amidin is the only metabolite detected (as in pears)

& 1:1 stechiometry (conversion factor based on MW-difference)
CASE 2: Amidin + at least one of other metabolites present

1) Expression of all metabolites as DMA (1:1 stechiometry),
and calculation of Amitraz from DMA (2:1 stechiometry)
2) Expression of Amidin as Amitraz (1:1),
Amid + DMA are ignored as they can be formed as by-products during
the degradation of Amitraz to Amidin.
EURL-FV/SRM
Take the WORKSHOP
highest of 2010
both
Amitraz-Findings in pears (2008): CVUA Stuttgart*
Amitraz Metabolite: Amitraz MRL- ARfD-
Origin (parent) N-2,4-Dimethylphenyl-N-methylformamidine (sum) Exceedance Exceedance [%]
Turkey ND 1,6 2,9 Yes 2647
Turkey 0,001 1,2 2,2 Yes 2008
Turkey <0,001 1,21 2,2 Yes 2008
Turkey 0,003 0,96 1,7 Yes 1552
Turkey ND 0,84 1,52 Yes 1387
Turkey ND Amitraz parent 0,81 1,5 Yes 1369
Turkey ND not detected or at 0,69 1,2 Yes 1095
Turkey ND very low levels in 0,59 1,1 Yes 1004
Turkey ND 2008 0,59 1,1 Yes 1004
Turkey ND Residue situation 0,48 0,87 Yes 794
is clearly
Turkey ND underestimated 0,28 0,51 Yes 465
Turkey ND if only parent is 0,27 0,49 Yes 447
Spain ND 0,013 0,024 No 22
targeted
Italy ND 0,32 0,58 Yes 529
Unknown ND 0,9 1,6 Yes 1460
Risk Assessment: German Child 2-5 y. old, 97.5 percentile (232 g/portion);
EURL-SRM
Variability Factor: 7 (JMPR model); ARfD 0.01 (EFSA) EU Reference Laboratory
* www.cvuas.de; Amitraz-article: http://www.cvuas.de/pub/beitrag.asp?subid=1&Thema_ID=5&ID=881&Pdf=No
Amitraz-Findings in Pears (2008): CVUA Stuttgart
Turkish Pear-samples: 18 analyzed; 18 >ARfD

Other countries:
Italy (11/1 >ARfD),
Argentina (10 samples/0 findings),
Germany (7/0),
Spain (6/1 <ARfD),
South Africa (5/0),
China (4/0)
Chile (1/0),
France (1/0),

Amitraz-Findings in Pears (2009): CVUA Stuttgart
8 Turkish Pear-samples analyzed, 8 >ARfD

Total Amitraz parent % ARfD
Amitraz
15.7 0.35 14327 (~140 x)
10.4 0.04 9491 (~100 x)
9.8 1.3 8943
6.0 0.02 5475
5.9 0.06 5384
4.3 0.007 3924
1.8 nd 1643
0.2 nd 183
Turkey has withdrawn amitraz registration.
Farmers have 2-year period of grace to get rid of stock.
This had obviously a negative effect in terms of agricultural practice
SRM
Thank you very much

EURL-SRM
for your attention!

2010 EurlFvSrmWorkshop SrmMethods

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ADVANCES IN THE ANALYSIS OF

PESTICIDES TYPICALLY ANALYSED

EURL-SRM EU Reference Laboratory

SRMs often require...

tedious or unfamiliar sample preparation steps e.g.:

Effort : Benefit-Ratio is less favourable than that of MRMs

As labs are typically

..most try to avoid SRMs Covered

( This targeted approach Improves the Effort to Benefit Ratio

However: Our knowledge about the residue

EURL-SRM EU Reference Laboratory

II. - Compound Degrades

Instrumental Analysis Technique Available:

- Chromatographic separation technology

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

Acidic conditions essential to obtain sharp peaks !!

EURL-SRM EU Reference Laboratory

650 900 1300

Fenbutatinoxid 519.3 / 350.9 T1 Fenbutatinoxid 517.3 / 195.0 T2 Fenbutatinoxid 519.3 / 197.0

1200 800 600

600 400 300

Cyhexatin 369.2 / 286.9 T1

EURL-SRM EU Reference Laboratory

OK, but what about instrumental analysis?

EURL-SRM Against Solvent w/o APs

Ethylglycerol Shikimic acid

20000 Linear but not

EURL-SRM EU Reference Laboratory

Ratio Dicofol / DicofolD8 - with APs

EURL-SRM Calculated via Standard

500000 0.030 mg/kg

via ISTD (PCB 138)

0,008 0.022 mg/kg

0,08 0.033 mg/kg

via ISTD (Folpet D4)

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

15 Less polar More polar

EURL-SRM EU Reference Laboratory

Fosetyl Al -2,7 (pH4) 111 (pH6) 4.7 3/- IN

Chlormequat -3.47 886 - 0.05 / 0.05 IN

Mepiquat -2.82 500 - 0.3 / 0.3 IN

Paraquat -4.5 620 - 0.004 / 0.005 (OUT)

QuEChERS Recoveries from

Maleic Hydrazide 30 % 74 % -1.96 (pH7)

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

EURL-SRM EU Reference Laboratory

Fosetyl (Ethylphosphonic acid)

EURL-SRM EU Reference Laboratory

Included in a biocide ban

EURL-SRM EU Reference Laboratory

No legal Counter-ion of Glyphosate

3-MPPA = MPP NAG = N-acetyl-glufosinate

Glyphosate makes 60% of worldwide herbicide sales!!!

pKa=2.29 Minor tautomer

Add 10 mL water (for dry material)

Add 10ml of Extraction solvent

Add 10 mL water (for dry material)