New Developments in QuEChERS methodology
New Developments in QuEChERS methodology
New Developments in QuEChERS methodology
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<strong>New</strong> <strong>Developments</strong> <strong>in</strong><br />
<strong>QuEChERS</strong> <strong>methodology</strong><br />
EPRW 2006, Corfu<br />
Slide 1<br />
Michelangelo Anastassiades<br />
Bünyam<strong>in</strong> Tasdelen<br />
Ellen Scherbaum<br />
Stuttgart Chemical and Veter<strong>in</strong>ary Control Laboratory
EPRW 2006, Corfu<br />
Slide 2<br />
Introduction, Orig<strong>in</strong>al <strong>QuEChERS</strong><br />
Modifications to broaden analyte spectrum<br />
Modified <strong>QuEChERS</strong><br />
Modifications to broaden matrix spectrum<br />
Results from method validation<br />
Results from EU-PT 6 + 7<br />
Summary<br />
Outl<strong>in</strong>e
<strong>QuEChERS</strong>:<br />
•Quick<br />
•Easy<br />
•Cheap<br />
•Effective<br />
•Rugged<br />
•Safe<br />
EPRW 2006, Corfu<br />
Slide 3<br />
Introduction<br />
Was first <strong>in</strong>troduced at the EPRW 2002 <strong>in</strong><br />
Rome and published <strong>in</strong> 2003 as<br />
“Fast and Easy Multiresidue Method<br />
Employ<strong>in</strong>g Acetonitrile Extraction/Partition<strong>in</strong>g<br />
and “Dispersive SPE” for the Determ<strong>in</strong>ation of<br />
Pesticide Residues <strong>in</strong> Produce“<br />
M. Anastassiades, S.J. Lehotay, D. Štajnbaher,<br />
F.J. Schenck, J. AOAC Int., 86 (2) 412-431, 2003
Orig<strong>in</strong>al <strong>QuEChERS</strong>-Method<br />
Weigh 10 g of Sample (50 mL Teflon-Tube)<br />
EPRW 2006, Corfu<br />
Slide 4<br />
Add 10 mL Acetonitrile<br />
Add ISTD-Solution<br />
Shake Vigorously 1 m<strong>in</strong><br />
Add 4 g MgSO 4 and 1 g NaCl<br />
GC-MSD and LC-MS<br />
Shake Vigorously 1 m<strong>in</strong><br />
Shake 30 s and Centrifuge<br />
Take Aliquot and Mix with MgSO 4 and PSA<br />
Shake Vigorously 30 s<br />
and Centrifuge<br />
Anastassiades et al.<br />
J. AOAC Int.<br />
86 (2) 412-431
EPRW 2006, Corfu<br />
Slide 5<br />
Analyte and Matrix Spectrum<br />
The orig<strong>in</strong>al <strong>QuEChERS</strong><br />
covers a broad analyte spectrum<br />
rang<strong>in</strong>g from the unpolar<br />
to the very polar end<br />
-2,6<br />
Propamocarb (pH4)<br />
-1,8<br />
Clopyralid (pH5)<br />
-0,9<br />
Acephat<br />
-0,4<br />
Oxamyl<br />
0,1<br />
Methomyl<br />
0,2<br />
2,4-D (pH 5)<br />
0,6<br />
Cymoxanil (pH5)<br />
0,8<br />
Acetamiprid<br />
1,0<br />
Kasugamyc<strong>in</strong><br />
1,2<br />
Aldicarb<br />
1,4<br />
Ofurace<br />
1,7<br />
Pirimicarb<br />
The orig<strong>in</strong>al method<br />
was developed for fruit and vegetables<br />
2,1<br />
Dichlorvos<br />
3,0<br />
Iprodion<br />
4,0<br />
Sp<strong>in</strong>osad<br />
5,0<br />
Tebufenpyrad<br />
6,0<br />
Fenpropathr<strong>in</strong><br />
7,0<br />
Etofenprox
EPRW 2006, Corfu<br />
Slide 6<br />
Broaden Analyte Spectrum<br />
Some pesticides are sensitive to pH<br />
Some get ionized at certa<strong>in</strong> pH-values<br />
Acids: HX � H + + X -<br />
Bases: B + H + � BH +<br />
� Ionic form prefers to stay <strong>in</strong> the waterphase<br />
pH-Range of agricultural samples: ~2.5 – 7
Ionizable Compounds - Basic Pesticides<br />
Matrix: apple; fortif. level: 0.1 mg/kg; analysis: LC/MS; ESI (+)<br />
Propamocarb<br />
Thiabendazole<br />
Carbendazim<br />
Fenpropimorph<br />
Spiroxam<strong>in</strong>e<br />
EPRW 2006, Corfu<br />
Slide 7<br />
Imazalil<br />
Prochloraz<br />
pKa of corresp. acid 9.5<br />
pKa of corresp. acid 6.5<br />
pKa of corresp. acid 3.8<br />
pKa of corresp. acid 4.7<br />
pKa of corresp. acid 4.2<br />
pKa of corresp. acid 6.9<br />
pKa of corresp. acid 7.0<br />
0 20 40 60 80 100 120<br />
Recovery %<br />
pH 2.9
Ionizable Compounds - Acidic Pesticides<br />
LC-MS/MS, ESI (-), No PSA Cleanup<br />
EPRW 2006, Corfu<br />
Slide 8<br />
Recovery %<br />
Imazapyr<br />
Clopyralid<br />
Picloram<br />
Benazol<strong>in</strong><br />
4-CPA<br />
Imazethapyr<br />
Dicamba<br />
MCPA<br />
2,4-D<br />
pH5.5<br />
Imazaqu<strong>in</strong><br />
Naphthylacetic acid<br />
Fluoxypyr<br />
2,4,5-T<br />
Mecoprop<br />
Triclopyr<br />
2,4,5-TP<br />
Lower pKa values General Trend Higher pKa values<br />
2,4-DP<br />
Propyzamid<br />
Ioxynil<br />
Bentazon<br />
Bromoxynil<br />
Fluazifop<br />
Bromacil<br />
2,4-DB<br />
MCPB<br />
pH 6<br />
pH 5<br />
pH 4<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
pH 3
pH-Labile Compounds<br />
EPRW 2006, Corfu<br />
Slide 9<br />
Some Pesticides are sensitive to pH<br />
and degrade at high or low pH-values<br />
• In the sample (dur<strong>in</strong>g process<strong>in</strong>g and storage)<br />
� Keep low temperatures<br />
• Dur<strong>in</strong>g sample preparation<br />
� Work fast, adjust pH<br />
• In the extract dur<strong>in</strong>g storage (1 week is common)<br />
� Keep low temperatures, adjust pH,<br />
after dispersive SPE with PSA, pH may reach values exceed<strong>in</strong>g 8
Optimal pH for <strong>QuEChERS</strong> ?<br />
Goals:<br />
• Still good extraction for the most strong acids<br />
• Still good protection for base-sensitives<br />
EPRW 2006, Corfu<br />
Slide 10<br />
tolylfluanid, dichlofluanid, captan, folpet,<br />
dicofol, pyridate…<br />
•Still good protection for acid-sensitives<br />
sulfonylureas, pymetroz<strong>in</strong>e, carbosulfan,<br />
dioxacarb...<br />
Extraction<br />
Extract<br />
storage
Optimal pH for <strong>QuEChERS</strong> ?<br />
pH adjustment before extraction (Buffer<strong>in</strong>g)<br />
1) Buffer<strong>in</strong>g to pH of ~6, Lehotay et al. with acetate buffer<br />
� good recovery for pymetroz<strong>in</strong>e<br />
� Low recoveries for some acidic pesticides<br />
� PSA cleanup is much less efficient<br />
2) Buffer<strong>in</strong>g to pH of 5 to 5.5, citrate buffer,<br />
EPRW 2006, Corfu<br />
Slide 11<br />
• 4 g Magnesium sulphate anhydrous,<br />
• 1 g Sodium chloride,<br />
• 1 g Trisodium citrate dihydrate and<br />
• 0.5 g Disodium hydrogencitrate sesquihydrate
EPRW 2006, Corfu<br />
Slide 12<br />
Portion<strong>in</strong>g of sorbents and salts<br />
Rapid and Easy
Optimal pH for <strong>QuEChERS</strong> ?<br />
pH adjustment after cleanup<br />
Use of formic acid <strong>in</strong> acetonitrile to achieve a “pH” of 5<br />
Base-labile compounds are stabilized:<br />
tolylfluanid,<br />
dichlofluanid,<br />
captan,<br />
Rec. %<br />
100<br />
90<br />
80<br />
70<br />
Tolylfluanid extract stability<br />
7 days<br />
13 days<br />
folpet,<br />
60<br />
50<br />
dicofol,<br />
40<br />
30<br />
pyridate<br />
20<br />
5% formic acid: 10 µL/mL<br />
EPRW 2006, Corfu<br />
Slide 13<br />
10<br />
0<br />
pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 MeCN<br />
Measured pH <strong>in</strong> extract
Sulfonylureas,<br />
Carbosulfan<br />
acid labile...<br />
Rec. <strong>in</strong> %<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
EPRW 2006, Corfu<br />
Slide 14<br />
7 days<br />
13 days<br />
Primisulfuron-Methyl<br />
pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 MeCN<br />
Measured pH of extract<br />
If these<br />
compounds are<br />
<strong>in</strong>cluded <strong>in</strong> the<br />
target spectrum<br />
use an aliquot of<br />
the f<strong>in</strong>al extract<br />
before acidify<strong>in</strong>g
Alox N Supelco, C18<br />
C8-Varian, C8-Am<strong>in</strong>o 50mg<br />
C8/SAX, ENVI-18<br />
Polymeric Strata x, PSA Varian<br />
Si-PFP, Strata NH2<br />
Strata polym. w. Anion, Waters Wax cc6<br />
Si-Triam<strong>in</strong>e, Si-Diam<strong>in</strong>e<br />
H2O Phobic WA-DVB Speedisk, Si-Am<strong>in</strong>e<br />
NH2 Am<strong>in</strong>o Speedisk, Si-Dimethylam<strong>in</strong>e<br />
C8-Am<strong>in</strong>o25, Strata Screen-A<br />
Diam<strong>in</strong>o MN, UP60-50NH2 <strong>in</strong>terchrom<br />
1,2,Am<strong>in</strong>o Bakerbond, Am<strong>in</strong>o SPE J.T.Baker<br />
Alox Aldrich, Am<strong>in</strong>opropyl modif. MN<br />
Alum<strong>in</strong>a-B Supelclean, Alox Aldrich acidic<br />
DEA Varian, Alox N MN<br />
Alum<strong>in</strong>a A Supelclean, Dimethylam<strong>in</strong>o modif. MN<br />
LiChrolut Sax, PA Chromabond<br />
DPA-6S, PF-AEV <strong>in</strong>terchrom<br />
DSC-MCAX, Cation mixed-mode polym. Strata<br />
PCA MN, PH Varian ...<br />
EPRW 2006, Corfu<br />
Slide 15<br />
Improved cleanup<br />
For samples, with a high content<br />
of carot<strong>in</strong>oides<br />
or chlorophyll<br />
cleanup with PSA<br />
is not satisfy<strong>in</strong>g.....<br />
We tested more than 50 SPE sorbents!
EPRW 2006, Corfu<br />
Slide 16<br />
Improved cleanup<br />
GCB, Graphitized Carbon Black, was best <strong>in</strong> handl<strong>in</strong>g and effect<br />
Some pesticides have a high aff<strong>in</strong>ity towards GCB<br />
e.g. hexachlorobenzene, chlorothalonil, thiabendazole<br />
Anthracene may be used as<br />
surrogate QC standard.<br />
Recoveries > 70% will <strong>in</strong>dicate that no<br />
unacceptable losses of pesticides<br />
have occurred.<br />
Lettuce Extract<br />
PSA GCB
EPRW 2006, Corfu<br />
Slide 17<br />
Improved cleanup<br />
The cleanup time (shak<strong>in</strong>g) is<br />
extended from 30 s to 2 m<strong>in</strong>,<br />
Pre-mixtures of MgSO 4 and GCB<br />
facilitates weigh<strong>in</strong>g<br />
Dispersive SPE is performed us<strong>in</strong>g<br />
a comb<strong>in</strong>ation of PSA and GCB<br />
F<strong>in</strong>al extract should rema<strong>in</strong> slightly coloured<br />
PSA GCB
EPRW 2006, Corfu<br />
Slide 18<br />
Weigh 10 g of Frozen Sample<br />
Add 10 mL Acetonitrile<br />
Add ISTD-Solution<br />
Shake<br />
Add 4 g MgSO 4 / 1 g NaCl /<br />
Buffered to pH 5-5.5 with Citrate Buffer<br />
Shake & Centrifuge<br />
Mix an Aliquot with MgSO4 & PSA/(GCB)<br />
Shake & Centrifuge<br />
Acidify extract to pH ~5<br />
to protect base-sensitive pesticides<br />
Optionally: Add other “Analyte Protectants”<br />
Optionally:<br />
Acidic Pesticides LC-MS<br />
Optionally:<br />
SUs LC-MS<br />
<strong>QuEChERS</strong><br />
Multiresidue Analysis<br />
by GC-MS, LC-MS ...
EPRW 2006, Corfu<br />
Slide 19<br />
Broaden matrix spectrum<br />
Dry commodities (cereals, dried fruits) Fatty Commodities
EPRW 2006, Corfu<br />
Slide 20<br />
Broaden matrix spectrum –<br />
E.g. cereals, dried fruits<br />
Dry Commodities<br />
require the addition of water prior to extraction<br />
to weaken <strong>in</strong>teractions of pesticides with the<br />
matrix and to ensure adequate partition<strong>in</strong>g.<br />
Weigh 5 g of sample add 10 mL water....<br />
Remove co-extracted fat by freez<strong>in</strong>g out or C18, if<br />
necessary....
Sample type<br />
Cereals<br />
Dried fruits<br />
Fruit/Vegetables<br />
(water >80 %)<br />
Fruit/Vegetables<br />
(water 30-80 %)<br />
Honey<br />
Spices<br />
EPRW 2006, Corfu<br />
Slide 21<br />
Dry Commodities<br />
Weigh<br />
5 g<br />
5 g<br />
10 g<br />
10 g<br />
5 g<br />
2 g<br />
Water<br />
10 g<br />
7.5 g<br />
-<br />
X g<br />
10 g<br />
10 g<br />
Annotation<br />
Add water to<br />
comm<strong>in</strong>ute, weigh<br />
12.5 g of homogenate<br />
X = 10 g – water<br />
amount <strong>in</strong> 10 g sample
EPRW 2006, Corfu<br />
Slide 22<br />
Broaden matrix spectrum –<br />
Fatty commodities<br />
Commodities with a high lipid load, such as<br />
avocados or plant oils can be employed.<br />
Problems:<br />
�Highly non-polar pesticides<br />
may give recoveries < 70% (e.g. HCB and DDT)<br />
�Co-extracted lipids have to be removed<br />
prior to GC-analysis
Removal of co-extracted lipids<br />
Extractives [mg/ml]<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
EPRW 2006, Corfu<br />
Slide 23<br />
4,20<br />
raw extract<br />
4,03<br />
PSA<br />
1,48<br />
freez<strong>in</strong>g out<br />
1,33 1,25 1,23 1,15 1,10<br />
C18<br />
PSA/freez<strong>in</strong>g out<br />
C18/freez<strong>in</strong>g out<br />
PSA/C18<br />
PSA/C18/freez<strong>in</strong>g out
100<br />
75<br />
50<br />
25<br />
0<br />
EPRW 2006, Corfu<br />
Slide 24<br />
BS138<br />
Recoveries for oil matrix<br />
Hexachlorobenzene<br />
p,p' -DDE<br />
Dieldr<strong>in</strong><br />
Endosulfan<br />
HCH, gamma-<br />
Chlorpyriphos-methyl<br />
Carbaryl<br />
Chlorpyriphos<br />
Fenthion<br />
Pirimiphos-methyl<br />
Diaz<strong>in</strong>on<br />
TPP<br />
Triflural<strong>in</strong><br />
Deltamethr<strong>in</strong><br />
Malathion<br />
Cypermethr<strong>in</strong><br />
1g<br />
2g<br />
3g
100<br />
75<br />
50<br />
25<br />
0<br />
EPRW 2006, Corfu<br />
Slide 25<br />
BS138<br />
Recoveries for oil matrix<br />
PCB 138 or 153 may be used as<br />
surrogate QC standards<br />
Hexachlorobenzene<br />
p,p' -DDE<br />
Dieldr<strong>in</strong><br />
Endosulfan<br />
HCH, gamma-<br />
Chlorpyriphos-methyl<br />
Carbaryl<br />
Chlorpyriphos<br />
Recoveries > 70% will <strong>in</strong>dicate that no unacceptable<br />
losses of pesticides have occurred.<br />
Fenthion<br />
Pirimiphos-methyl<br />
Diaz<strong>in</strong>on<br />
TPP<br />
Triflural<strong>in</strong><br />
Deltamethr<strong>in</strong><br />
Malathion<br />
Cypermethr<strong>in</strong><br />
1g<br />
2g<br />
3g
EPRW 2006, Corfu<br />
Slide 26<br />
Interlaboratory Validation<br />
3 Validation studies were performed <strong>in</strong> 2005 and 2006 <strong>in</strong><br />
Germany<br />
Thanks to:<br />
Dr. Lutz Alder Berl<strong>in</strong>, PeterBaumann Dortmund, Dr. Sab<strong>in</strong>e<br />
Bracht Münster, Dr. Franz Haslbeck Pfaffenhofen, Dr.<br />
Matthias He<strong>in</strong>zler Kassel, Dr. Günther Kempe Chemnitz,<br />
Labor Specht Hamburg, Dr. Jürgen Lip<strong>in</strong>ski Berl<strong>in</strong>, Dr. Peter<br />
Seulen Kiel, Dr. Magnus Jezussek Erlangen, Dr. Iris Suckrau<br />
Oldenburg, Dr. Harald Kubel Potsdam, Anja Bostel Stuttgart,<br />
Dr. Norbert Reichert Taunusste<strong>in</strong>, Dr. Graf Rostock
GC-MS and LC-MS/MS Inter-Laboratory Validation Study (GDCh)<br />
EPRW 2006, Corfu<br />
Slide 27<br />
GC<br />
LC (+)<br />
LC (-)<br />
Mean recovery, all laboratories, all analytes<br />
101% 100% 105% 102% 100% 100%<br />
Apple Lettuce Orange
GC-MS and LC-MS/MS Inter-Laboratory Validation Study (GDCh)<br />
EPRW 2006, Corfu<br />
Slide 28<br />
GC<br />
LC (+)<br />
LC (-)<br />
Mean RSD %, all laboratories, all analytes<br />
3% 4% 8% 9% 5% 3%<br />
Apple Lettuce Orange
LC-MS/MS Inter-Laboratory Validation Study (BLAPS-Work<strong>in</strong>g Group)<br />
EPRW 2006, Corfu<br />
Slide 29<br />
Mean recovery, all laboratories, all analytes<br />
97% 97% 98% 98% 96% 98% 95% 97%<br />
Cucumber Lemon Wheat Rais<strong>in</strong>s<br />
Ethiofencarb<br />
was oxidized<br />
<strong>in</strong> cucumber<br />
SUs degraded<br />
<strong>in</strong> acidified extract
LC-MS/MS Inter-Laboratory Validation Study (BLAPS-Work<strong>in</strong>g Group II)<br />
Acids, lost <strong>in</strong><br />
PSA cleanup<br />
EPRW 2006, Corfu<br />
Slide 30<br />
Mean recovery, all laboratories, all analytes<br />
101% 98% 99% 94% 100% 101% 100% 97%<br />
Cucumber Lemon Wheat Rais<strong>in</strong>s<br />
Oxidation<br />
Degraded <strong>in</strong> the standard<br />
solution provided
EU - Proficiency Test 6 and 7<br />
EU-PT 6, Tomates (mg/kg)<br />
Pesticide<br />
Acr<strong>in</strong>athr<strong>in</strong><br />
Azoxystrob<strong>in</strong><br />
Bromopropylate<br />
Chlorothalonil<br />
Diaz<strong>in</strong>on<br />
Dimethoate<br />
Endosulfan<br />
Imazalil<br />
Imidacloprid<br />
Oxydemeton-me<br />
Procymidone<br />
Thiabendazole<br />
EPRW 2006, Corfu<br />
Slide 31<br />
CVUA S<br />
<strong>QuEChERS</strong><br />
0.2<br />
0.23<br />
0.46<br />
2.2<br />
4.4<br />
0.13<br />
0.31<br />
0.16<br />
0.23<br />
0.24<br />
0.41<br />
0.33<br />
No. 4<br />
Median<br />
(n=130)<br />
0.284<br />
0.225<br />
0.490<br />
1.626<br />
3.955<br />
0.132<br />
0.344<br />
0.167<br />
0.232<br />
0.199<br />
0.412<br />
0.330<br />
No. 1<br />
EU-PT 7, Grapes (mg/kg)<br />
Pesticide<br />
Acetamiprid<br />
Carbaryl<br />
Cyprod<strong>in</strong>il<br />
Diaz<strong>in</strong>on<br />
Dimethoate<br />
Fenhexamid<br />
Fludioxonil<br />
Imidacloprid<br />
Iprodione<br />
Kresoxim-me<br />
Methomyl<br />
Monocrotophos<br />
Procymidone<br />
Pyrimethanil<br />
Tetraconazole<br />
Thiabendazole<br />
CVUA S<br />
<strong>QuEChERS</strong><br />
0.324<br />
1.68<br />
0.472<br />
0.318<br />
0.144<br />
0.772<br />
0.242<br />
0.598<br />
0.538<br />
0.502<br />
0.153<br />
0.761<br />
1.98<br />
0.148<br />
0.076<br />
0.745<br />
Median<br />
(n=128)<br />
0.312<br />
1.62<br />
0.437<br />
0.287<br />
0.139<br />
0.706<br />
0.238<br />
0.549<br />
0.524<br />
0.453<br />
0.167<br />
0.643<br />
1.90<br />
0.149<br />
0.064<br />
0.656
www.<strong>QuEChERS</strong>.com<br />
EPRW 2006, Corfu<br />
Slide 32<br />
....the modified <strong>QuEChERS</strong> method<br />
<strong>in</strong>clud<strong>in</strong>g all presented modifications<br />
and a lot of background <strong>in</strong>formation is<br />
available via the <strong>in</strong>ternet, as well as the<br />
validation data.<br />
www.quechers.com
Thank you for your attention!<br />
....and the CVUA-Team for their dedicated work and their support!<br />
EPRW 2006, Corfu<br />
Slide 33
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