Acidic pesticdes

using QuEChERS
method

Acidic pesticides
pH-issue: Ionization of pesticides at low or high pH-values
1

Acids: HX 1 H+ + X-

Bases: B + H+ 1 BH+

3 Ionic form prefers to stay in the water phase

pH-range of agricultural
samples: ~2.5 7

MCPB

123456457895
837 123456 789


pH

pH

pH

pH

lower pKa 4 general trend 5 higher pKa

2,4-DB

Bromacil

Fluazifop

Bromoxynil

Bentazon

Ioxynil

Propyzamid

2,4-DP

2,4,5-TP

Triclopyr

Mecoprop

2,4,5-T

Fluoxypyr

Naphthylacetic acid

Imazaquin

2,4-D

MCPA

Dicamba

Imazethapyr

4-CPA

Imazapyr
Clopyralid
Picloram
Benazolin

Acidic pesticides
influence of pH on recovery
120

Recovery
%
100

80

max. pH 5.5
60

40

20

Acidic pesticides cleanup issue

Example:
Extract of red currant

12

Citrate-Buffered
Sample pH 5.1
pH=7.8

mg/mL

7Losses of acidic
components after
cleanup

pH=8.3

pH

10
7
8

6
5

6
4

pH=3.5
4

7Acidic compounds
interact with PSA

2,4
2

1,0

0,5

0
Raw Extract PSA 25 PSA 50
mg/mL
mg/mL
mg co-extractives/mL Extract
pH

1
0

QuEChERS has to be modified,

to include acidic pesticides
7 Avoid

special cleanup with PSA

7 Special conditions during chromatography

Acidic pesticides pesticide list

Compound
2,4,5-T
2,4,5-TP
2,4-D
2,4-DB
2,4-DP
4-CPA
Fenoxaprop-P
Fluazifop
Fluazifop-P-Butyl
Haloxyfop
MCPA
MCPB
MCPP
Fluroxypyr
Triclopyr
Naphthoxyacetic acid, 2Bromoxynil
Ioxynil
Clopyralid
Dicamba
Imazapyr
Imazaquin
Imazethapyr
Quinmerac
Picloram
Bentazone
Fomesafen
Imazosulfuron
Thifensulfuron-Methyl
Metsulfuron-Methyl
Metosulam
Cycloxydim
Fludioxonil

pKa-value
2,85
2,84
2,73
4,8
3,67
3,56
3,2
3,2
3,1
2,9
3,07
4,84
3,78
2,94
3,97

Chemical Class

acidic structure

phenoxyalcanoic acid

pyridine herbicide
naphthoxyacetic herbicide

3,86
3,96
2
1,97
1,9
3,8
2,1
4,32
2,3
3,3
2,83
4
4
3,3
4,8
4,17

phenolic herbicide
pyridine herbicide
benzoic acid
imidazolinone herbicide
quinolinecarboxylic acid
pyridine herbicide
unclassified
amide herbicide
pyrimidinylsulfonylurea herbicides
triazinylsulfonylurea herbicides
sulfonanilide herbicide
cyclohexene oxime herbicide

special case
no acid

Acidic pesticides pesticide list

Cycloxydim
7
7

Three mesomeric structures

Difficult chromatogramic behaviour

Acidic pesticides - findings

Nof samples with positive findings

14
2

651
47
7
5

7
9
1
2
1

10
85
1
6

3283
421
314
146
94
81
49
31
25
24
18
18
16
9
7
6
3
2
2
1
1
1

1
1
4

18
16

1
10
2
1
1

1
2

Annex I 91/414

LEGUMES

CEREALS

FRUITING
VEGET.

LEAFY VEGET.
297
125
17
44
72
6
30
8
2
2

ALL COMMOD.

45
1
98
1

ROOT & TUBER

Fludioxonil
Fluazifop
2,4-D
Haloxyfop
Bentazone
MCPA
Clopyralid
Dicamba
2,4-DP
MCPP
4-CPA
Naphthoxyacetic acid, 2Bromoxynil
Triclopyr
Imazaquin
2,4,5-T
Quinmerac
Fluroxypyr
Ioxynil
2,4-DB
Cycloxydim
Metosulam
2,4,5-TP
Fenoxaprop-P
Formesafen
Imazapyr
Imazethapyr
Imazosulfuron
MCPB
Metsulfuron-Methyl
Picloram
Thifensulfuron-Methyl

CITRUS & POME

FRUIT

Pesticide

2004 - 2007

Pending
Out
In
Out
In
In
In
Pending
In
Pending
Pending
pending
In
Pending
In
In
In
In
Pending
Pending
In
Pending
Out
Out
In
Out
Out
In

Relatively often found:

7 Fluazifop
7 2,4-D

Interlaboratory Method Validation Study

Modified QuEChERS (without PSA-cleanup)
August 2006:
7 19 Labs expressed interest from 9 Countries
(Germany, Latvia, Spain, Czech Republic, Switzerland,
Finland, Austria, The Netherlands and Great Britain)
7 4 Commodities
(Raisins, Cucumber, Lemon, Wheat Flour)
7 32 Pesticides (LC-MS/MS, ESI pos and neg) with
2 Levels (0.1 and 0.01 mg/kg)

M
C
M
M
PP
et
N
e
su
to
ap
su
lfu
ht
la
ho
ro
m
n
xy
ac -Me
et
th
y
ic
ac l
id
,2
Pi
Th
cl
or
ife
Q
ns
ui am
nm
ul
fu
er
ro
ac
nM
et
hy
l
Tr
ic
lo
py
r

Spiking Level 0.1 mg/kg

M
C
PB

2,
4D
2,
4D
B
2,
4D
P
4C
PA
Be
nt
az
on
Br
om e
ox
yn
C
il
lo
py
ra
C
l id
yc
lo
xy
di
m
D
Fe ica
m
no
xa ba
pr
op
-P
Fl
ua Flu
az
zi
i
fo
p- fop
PBu
ty
Fl
l
ud
io
xo
Fl
ni
ur
l
ox
y
Fo
py
r
m
es
af
en
H
al
ox
yf
Im o p
az
ap
Im
yr
az
Im aqu
az
in
Im eth
a
az
py
os
r
ul
fu
ro
n
Io
xy
ni
l
M
C
PA

2,
4,
5T
2,
4,
5TP

mean recovery in %

ILMV 2006 Acids

Results (Example Cucumber)

140
Spiking Level 0.01 mg/kg

120

100

80

60

40

20

QuEChERS alkaline hydrolysis

Example 2,4-D
7
7
7
7
7
7

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!

QuEChERS schematic description with AH

Citrate-Buffered QuEChERS
Weigh 10 g of Frozen Sample
Shake

Add 10 mL Acetonitrile

alkaline hydrolysis:
Add NaOH and store for
30 min at RT,
then neutralize

Add ISTD-Solution
Shake

Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer

(pH 5-5.5)
Shake and Centrifuge

Analysis of acidic pesticides by LC-MS/MS

optionally:
Add NaOH to adjust pH
optionally:
Freeze-out of extracted
fat over night

 8888883 
!8"8#8
Alkaline Hydrolysis for the release of phenoxy-acid pesticides
" 8 "

wheat sample

At 70 C

At Room Temperature

1200

pH 12
30 min

1000
800

pH 10
30 min

pH 9
30 min

715

619

606

pH 10
60 min

600

336

400

275
135

200

100

dr
ol

in

w/
o
ER
S
Q
uE
Ch

pH

9/
3

hy

m
in
10
/

pH

pH

10
/

60

30

m
in

in
30
m
12
/
pH

30
10
/
pH

12
/

30

m
in

m
in

pH

Relativ concentration

1400

Alkaline Hydrolysis
Level of MCPA in berries

(%$
./

5#/

)8)8842* 8+,-

($$
'%$
'$$
&%$
&$$
%$
$
88 

#88 

Alkaline Hydrolysis
Level of 2,4-D in different citrus samples
12648



88 

#88 

12646




12345

08&1283 %12
1294




12949
12647



1284

12345

1294

12845






123456789

12345678

12345678

12345678

12345678

12345678

12345678

12345678

12345678

123456798