QuEChERS Validation Method for acidic pesticides · CRL-SRM Community Reference Laboratory for...
Transcript of QuEChERS Validation Method for acidic pesticides · CRL-SRM Community Reference Laboratory for...
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Drazen Kostelac
Michelangelo Anastassiades
QuEChERS Validation Method for acidic pesticides
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Outline
Overview: acidic pesticides
Interlaboratory method validation experiment 2006
alkaline hydrolysis
Conclusion
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
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
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – pKa-values
4
1.9
2.3
2.7
3.1
3.8
4.8
9.9
3.1
3.8
4.2
4.7
5.2
6.5
7
9.5PropamocarbFenpropimorph
ImazalilCyromazine
ThiabendazoleCarbendazim
ProchlorazNitenpyram
DicambaClopyralid
2,4-DMCPAMCPPIoxynil2,4-DB
OPP
AcidspKa = pH above which
compounds lay in deprotonized form
BasespKa = pH below which
compounds lay in protonized form
pH-adjustment 1-2 units > or < pKa is recommended for quantitative recoveries
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – influence of pH on recovery
pH 6pH 5pH 4pH 3
MC
PB
2,4-
DB
Bro
mac
il
Flua
zifo
p
Bro
mox
ynil
Bent
azon
Ioxy
nil
Prop
yzam
id
2,4-
DP
2,4,
5-TP
Tric
lopy
r
Mec
opro
p
2,4,
5-T
Fluo
xypy
r
Nap
hthy
lace
tic a
cid
Imaz
aqui
n
2,4-
D
MC
PA
D
icam
ba
Imaz
etha
pyr
4-C
PA
Bena
zolin
Pic
lora
mC
lopy
ralid
Imaz
apyr
0
20
40
60
80
100
120
Recovery %
LC-MS/MS, ESI (-), No PSA Cleanup
max. pH 5.5
lower pKa general trend higher pKa
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – cleanup issue
Example: Extract of red currant
Losses of acidic components after cleanup
Acidic compounds interact with PSA
Citrate-BufferedSample pH 5.1
2,4
1,0 0,5
pH=3.5
pH=7.8 pH=8.3
0
2
4
6
8
10
12
Raw Extract PSA 25mg/mL
PSA 50mg/mL
mg/mL
0
1
2
3
4
5
6
7
8
9pH
mg co-extractives/mL ExtractpH
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – including into QuEChERS
QuEChERS has to be modified, to include acidic pesticides
Avoid special cleanup proceduresAvoid special cleanup proceduresSpecial conditions during extraction and Special conditions during extraction and chromatographychromatography
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Compound pKa-value Chemical Class acidic structure2,4,5-T 2,852,4,5-TP 2,842,4-D 2,732,4-DB 4,82,4-DP 3,674-CPA 3,56 Fenoxaprop-P 3,2Fluazifop 3,2Fluazifop-P-Butyl 3,1Haloxyfop 2,9MCPA 3,07MCPB 4,84MCPP 3,78Fluroxypyr 2,94Triclopyr 3,97Naphthoxyacetic acid, 2- naphthoxyacetic herbicideBromoxynil 3,86Ioxynil 3,96Clopyralid 2 pyridine herbicideDicamba 1,97 benzoic acidImazapyr 1,9Imazaquin 3,8Imazethapyr 2,1Quinmerac 4,32 quinolinecarboxylic acidPicloram 2,3 pyridine herbicideBentazone 3,3 unclassifiedFomesafen 2,83 amide herbicideImazosulfuron 4 pyrimidinylsulfonylurea herbicidesThifensulfuron-Methyl 4Metsulfuron-Methyl 3,3Metosulam 4,8 sulfonanilide herbicideCycloxydim 4,17 cyclohexene oxime herbicide special caseFludioxonil no acid
triazinylsulfonylurea herbicides
phenoxyalcanoic acid
pyridine herbicide
phenolic herbicide
imidazolinone herbicide
Acidic pesticides – pesticide list
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – pesticide list
CycloxidimThree mesomeric structuresThree mesomeric structuresDifficult chromatogramic behaviourDifficult chromatogramic behaviour
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides - findings
Pest
icid
e
CIT
RU
S &
PO
ME
FRU
IT
LEA
FY V
EGET
.
FRU
ITIN
G
VEG
ET.
CER
EALS
LEG
UM
ES
RO
OT
& T
UB
ER
ALL
CO
MM
OD
.
Ann
ex I
91/4
14
Fludioxonil 45 297 651 7 10 3283 PendingFluazifop 1 125 47 9 85 421 Out2,4-D 98 17 7 1 1 314 InHaloxyfop 1 44 5 2 6 146 OutBentazone 72 1 94 InMCPA 6 1 81 InClopyralid 30 1 49 InDicamba 8 4 31 Pending2,4-DP 14 2 25 InMCPP 2 2 24 Pending4-CPA 18 18 PendingNaphthoxyacetic acid, 2- 1 16 18 pendingBromoxynil 10 16 InTriclopyr 2 9Imazaquin 7 Pending2,4,5-T 1 6 InQuinmerac 1 3Fluroxypyr 1 2 InIoxynil 2 2 In2,4-DB 1 InCycloxydim 1 PendingMetosulam 1 1 Pending2,4,5-TP InFenoxaprop-P PendingFormesafenImazapyr OutImazethapyr OutImazosulfuron InMCPB OutMetsulfuron-MethylPicloram OutThifensulfuron-Methyl In
2004 - 2007N° of samples with positive findings
Relatively often found:
Fluazifop2,4-D
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Method Validation Experiment 2006 - facts
Method validation experimentModified QuEChERS without PSA-cleanup
August 2006:1919 Labs expressed interest from 99 Countries(Germany, Latvia, Spain, Czech Republic, Switzerland, Finland, Austria, The Netherlands and Great Britain)4 Matrices(Raisins, Cucumber, Lemon, Wheat Flour)32 Pesticides (LC-MS/MS, ESI pos and neg) with 2 Levels (0.1 and 0.01 mg/kg)
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Citrate-BufferedQuEChERS
Weigh 10 g of Frozen Sample
Shake
Shake and Centrifuge
Add ISTD-Solution
Analysis of acidic pesticides by LC-MS/MS
Add 10 mL Acetonitrile
Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer(pH 5-5.5)
optionally: Freeze-out of extracted
fat over night
QuEChERS – schematic method description
optionally: Add NaOH to adjust pH
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Interlab. Method Validation Experiment - evaluation
Collection of data and evaluationwww.crlwww.crl--pesticidespesticides--datapool.eudatapool.eu
ILMV Acids 2006
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
0
20
40
60
80
100
120
140
2,4,5-
T2,4
,5-TP
2,4-D
2,4-D
B2,4
-DP
4-CPA
Bentaz
one
Bromox
ynil
Clopyra
lidCyc
loxyd
imDica
mba
Fenox
aprop
-PFlua
zifop
Fluazif
op-P
-Buty
lFlud
ioxon
ilFlur
oxyp
yrFom
esafe
nHalo
xyfop
Imaz
apyr
Imaz
aquin
Imaz
ethap
yr
Imaz
osulf
uron
Ioxyn
ilMCPAMCPBMCPP
Metosu
lam
Metsulf
uron
-Meth
yl
Naphth
oxya
cetic
acid,
2-Picl
oram
Quinmer
ac
Thifen
sulfu
ron-M
ethyl
Triclop
yrm
ean
reco
very
in %
Spiking Level 0.1 mg/kg Spiking Level 0.01 mg/kg
ILMV 2006 Acids – Results Cucumber
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
ILMV 2006 Acids – Results Cucumber
CucumberHigh water contentHigh water content
Satisfying results for all pesticidesSatisfying results for all pesticidesSlightly problematic (mean recovery) for:Slightly problematic (mean recovery) for:
ClopyralidClopyralidCycloxydimCycloxydimPicloramPicloram
RSD > 20% for:RSD > 20% for:ImazapyrImazapyrCycloxydimCycloxydimPicloramPicloram
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
0
20
40
60
80
100
120
140
2,4,5-
T2,4
,5-TP
2,4-D
2,4-D
B2,4
-DP
4-CPA
Bentaz
one
Bromox
ynil
Clopyra
lidCyc
loxyd
imDica
mba
Fenox
aprop
-PFlua
zifop
Fluazif
op-P
-Buty
lFlud
ioxon
ilFlur
oxyp
yrFom
esafe
nHalo
xyfop
Imaz
apyr
Imaz
aquin
Imaz
ethap
yr
Imaz
osulf
uron
Ioxyn
ilMCPAMCPBMCPP
Metosu
lam
Metsulf
uron
-Meth
yl
Naphth
oxya
cetic
acid,
2-Picl
oram
Quinmer
ac
Thifen
sulfu
ron-M
ethyl
Triclop
yrm
ean
reco
very
in %
Spiking Level 0.1 mg/kg Spiking Level 0.01 mg/kg
ILMV 2006 Acids – Results Lemon
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
ILMV 2006 Acids – Results Lemon
LemonAcidic matrixAcidic matrix
Satisfying results for most pesticidesSatisfying results for most pesticidesSlightly problematic (mean recovery) for:Slightly problematic (mean recovery) for:
ClopyralidClopyralidPicloramPicloram
RSD > 20% for:RSD > 20% for:Imazapyr (only 0.01 mg/kg)Imazapyr (only 0.01 mg/kg)PicloramPicloramThifensulfuronThifensulfuron--MethylMethyl (only 0.1 mg/kg)(only 0.1 mg/kg)
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
0
20
40
60
80
100
120
140
2,4,5-
T2,4
,5-TP
2,4-D
2,4-D
B2,4
-DP
4-CPA
Bentaz
one
Bromox
ynil
Clopyra
lidCyc
loxyd
imDica
mba
Fenox
aprop
-PFlua
zifop
Fluazif
op-P
-Buty
lFlud
ioxon
ilFlur
oxyp
yrFom
esafe
nHalo
xyfop
Imaz
apyr
Imaz
aquin
Imaz
ethap
yr
Imaz
osulf
uron
Ioxyn
ilMCPAMCPBMCPP
Metosu
lam
Metsulf
uron
-Meth
yl
Naphth
oxya
cetic
acid,
2-Picl
oram
Quinmer
ac
Thifen
sulfu
ron-M
ethyl
Triclop
yrm
ean
reco
very
in %
Spiking Level 0.1 mg/kg Spiking Level 0.01 mg/kg
ILMV 2006 Acids – Results Raisins
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
ILMV 2006 Acids – Results Raisins
RaisinsDry and sugar containing matrixDry and sugar containing matrix
Satisfying results for most pesticidesSatisfying results for most pesticidesProblematic (mean recovery) for:Problematic (mean recovery) for:
ClopyralidClopyralidPicloramPicloram
RSD > 20% for:RSD > 20% for:ImazosulfuronImazosulfuronClopyralidClopyralidPicloramPicloramThifensulfuronThifensulfuron--MethylMethyl (only 0.1 mg/kg)(only 0.1 mg/kg)
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
0
20
40
60
80
100
120
140
2,4,5-
T2,4
,5-TP
2,4-D
2,4-D
B2,4
-DP
4-CPA
Bentaz
one
Bromox
ynil
Clopyra
lidCyc
loxyd
imDica
mba
Fenox
aprop
-PFlua
zifop
Fluazif
op-P
-Buty
lFlud
ioxon
ilFlur
oxyp
yrFom
esafe
nHalo
xyfop
Imaz
apyr
Imaz
aquin
Imaz
ethap
yr
Imaz
osulf
uron
Ioxyn
ilMCPAMCPBMCPP
Metosu
lam
Metsulf
uron
-Meth
yl
Naphth
oxya
cetic
acid,
2-Picl
oram
Quinmer
ac
Thifen
sulfu
ron-M
ethyl
Triclop
yrm
ean
reco
very
in %
Spiking Level 0.1 mg/kg Spiking Level 0.01 mg/kg
ILMV 2006 Acids – Results Wheat Flour
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
ILMV 2006 Acids – Results Wheat Flour
Wheat FlourFat containing matrixFat containing matrix
Satisfying results for most pesticidesSatisfying results for most pesticidesproblematic (mean recovery) for:problematic (mean recovery) for:
ClopyralidClopyralidPicloramPicloram
RSD > 20% for:RSD > 20% for:2,42,4--DB (only 0.01 mg/kg)DB (only 0.01 mg/kg)ClopyralidClopyralidDicamba Dicamba PicloramPicloram
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
QuEChERS – alkaline hydrolysis
Acids are often covalently bound to matrix components Acids are often covalently bound to matrix components and thus their concentration underestimated!and thus their concentration underestimated!
Example 2,4-D
Selective systemic herbicideSelective systemic herbicideControl of broad leaved weedControl of broad leaved weedPlant growth regulator used to prevent premature fruit dropPlant growth regulator used to prevent premature fruit dropFormulations include Formulations include freefree acidacid, , saltssalts, , estersestersMay form conjugatesMay form conjugatesMethod designed to convert all possible residues to free acidMethod designed to convert all possible residues to free acid
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Citrate-Buffered QuEChERS
Weigh 10 g of Frozen Sample
Shake
Shake and Centrifuge
Add ISTD-Solution
Analysis of acidic pesticides by LC-MS/MS
Add 10 mL Acetonitrile
Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer(pH 5-5.5)
optionally: Freeze-out of extracted
fat over night
QuEChERS – schematic description with AH
optionally: Add NaOH to adjust pH
alkaline hydrolysis: Add NaOH and store for
30 min at RT,then neutralize
Shake
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Alkaline Hydrolysis – Level of 2,4-D in different citrus samples
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
sample 01 sample 02 sample 03 sample 04 sample 05 sample 06 sample 07 sample 08 sample 09 sample 10
mg/
kg
no alkaline hydrolysis with alkaline hydrolysis
factor: 1.8 – 5.8x 1.8
x 1.8
x 2.2
x 2.6
x 2.5
x 5.8
x 3.4
x 5.0
x 3.0
x 3.3
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Alkaline Hydrolysis – Level of MCPA in berries
0
50
100
150
200
250
300
350
no alkaline hydrolysis with alkaline hydrolysis
Rel
ativ
e Res
pons
e of
MCPA
[%
]
Goosebeery Strawberry
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – alk. hydrolysis legally required
NameAnnex I91/414
Chem. group EU-Harmonized MRL? Free Acid ESTERS SALTS CONJUGATES NOTES
Dichlorprop (2,4-DP) In COOH since 31.12.1993 YES incl. Dichlorprop-PMecoprop (MCPP) In COOH since 04.12.2006 YES incl. Mecoprop-P2,4-DB In COOH since 04.06.2005 YES2,4-D In COOH since 01.07.2003 YES YES YESFluroxypyr In COOH since 01.03.2002 YES YESClopyralid In COOH PendingMCPA In COOH Pending (YES) (YES) (YES) (YES)MCPB In COOH PendingMesosulfuron In COOH PendingTriclopyr In COOH PendingQuinmerac Pending COOH NoPicloram Pending COOH NoImazaquin Pending COOH NoHaloxyfop-R Pending COOH NoFenoxaprop-P Pending COOH NoDicamba Pending COOH No2-Naphthyloxyacetic acid Notified COOH NoBromoxynil In OH after 21.04.2007 YES YES YESIoxynil In OH after 21.04.2007 YES YES YES
Current residue definitions
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007Sigma / Aldrich / Supelco Seminar
17. November 2006, Fellbach
Conclusion
Modified QuEChERS method without PSA cleanup is Modified QuEChERS method without PSA cleanup is suitable for analysis of most acidic pesticidessuitable for analysis of most acidic pesticidesProblematic in all matrices are:Problematic in all matrices are:
ClopyralidPicloram
Some other pesticides have RSD (> 20%)Some other pesticides have RSD (> 20%)Alkaline hydrolysis is strongly recommended, in order not Alkaline hydrolysis is strongly recommended, in order not to underestimate the level of acidic pesticidesto underestimate the level of acidic pesticidesEU should reconsider the existing residue definitions of EU should reconsider the existing residue definitions of harmonized MRLs to include conjugates where harmonized MRLs to include conjugates where appropriateappropriate
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acidic pesticides – influence of pH on recovery
pH 6pH 5pH 4pH 3
MC
PB
2,4-
DB
Bro
mac
il
Flua
zifo
p
Bro
mox
ynil
Bent
azon
Ioxy
nil
Prop
yzam
id
2,4-
DP
2,4,
5-TP
Tric
lopy
r
Mec
opro
p
2,4,
5-T
Fluo
xypy
r
Nap
hthy
lace
tic a
cid
Imaz
aqui
n
2,4-
D
MC
PA
D
icam
ba
Imaz
etha
pyr
4-C
PA
Bena
zolin
Pic
lora
mC
lopy
ralid
Imaz
apyr
0
20
40
60
80
100
120
Recovery %
LC-MS/MS, ESI (-), No PSA Cleanup
max. pH 5.5
lower pKa general trend higher pKa
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007Sigma / Aldrich / Supelco Seminar
17. November 2006, Fellbach
....the modified QuEChERS method including all presented modifications and a lot of background information is available via the internet.
www.quechers.com
www.QuEChERS.com
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsSigma / Aldrich / Supelco Seminar 17. November 2006, Fellbach
Thank you very much for your Attention!
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acids validation - Matrices
CucumberHigh water contentHigh water content
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acids validation - Matrices
LemonAcidic matrixAcidic matrix
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acids validation - Matrices
RaisinsSugar containing Sugar containing matrixmatrix
CRL-SRM Community Reference Laboratoryfor Pesticide Residues
using Single Residue MethodsCRL Workshop Valencia Spain26. – 29. September 2007
Acids validation - Matrices
Wheat FlourFat containing Fat containing matrixmatrix