1. Extracting Phenoxyacid pesticides from soil and vegetation
Where we have been, where we are, and where we will be

2. Overview Pesticide residue overview Where we have been
Previous method
Where we are now
Current method
Advantages of current method
Where we will be
Future plans for method

3. Pesticide Residue FERN FSIS CAP lab – chemistry Mycotoxins
“Seasonal” work – regulatory samples only
Usually ~May – August/Sept
Soil, vegetation (grass, tree leaves, soybean plants, etc.), water, swabs, clothing, milk, etc.
FERN FSIS CAP lab – chemistry
LC/MS
GC/MS
Metals
Mycotoxins
Aflatoxins, fumonisins, deoxynivalenol, zearalenone
Analyzed by LC/MS

4. Extractions Major methods - covers vast majority of samples analyzed
Glyphosate – vegetation – Pickering method
Soil glyphosate – LC/MS
QuEChERS – vegetation, soil, bees
GC/MS and LC/MS
Wide variety of compounds
Swabs – acetone extraction
Phenoxyacids – modified QuEChERS

5. Previous method PAM I, 222.13 A 7/69 version
Extracted with acidified organic mixture
Could be split into two fractions
Acidic fraction – phenoxyacids
Basic/neutral fraction
The acidic fraction was derivatized with diazomethane - methyl ester; made compound amenable to GC/MS
CH2=N+=N-

6. Previous method Was a reliable method Disadvantages Decent recoveries
Extraction time
Solvent use
Glassware – clean up
Diazomethane - toxic

7. QuEChERS Quick, Easy, Cheap, Efficient, Rugged, Safe
Developed by Lehotay and Anastassiades in 2003
Initially, for produce (fruits and vegetables), which have a fairly high moisture content
Has expanded to many different matrix types
Can easily be adapted to suit the needs of the extraction

8. Current method Modified QuEChERS Sample weights Acidified acetonitrile
3g vegetation; 5g soil
Lower moisture level; “fluffy” matrix
Acidified acetonitrile
1% formic acid
Keep analytes protonated
C18 dSPE
Analytes would absorb onto PSA

9. Testing of method conditions
Is base hydrolysis necessary?
Which QuEChERS salts?
No cleanup or C18 cleanup?
Plain acetonitrile or acidified acetonitrile?
For soil and vegetation
Several different phenoxyacid herbicides

10. Screening Results – Vegetation example
Compound
Original QuEChERS
acetonitrile
1% formic in ACN
5N NaOH/H2SO4
weaker base/acid
no hydrolysis
C18
none
None
2,4-D
100.30%
106.80%
110.00%
105.10%
105.70%
62.98%
114.30%
95.55%
107.60%
97.11%
105.40%
96.86%
dicamba
62.54%
65.67%
67.315
68.59%
70.82%
67.49%
51.43%
60.70%
69.25%
61.43%
67.87%
65.20%
triclopyr
109.70%
113.50%
115.70%
112.80%
59.92%
115.50%
120.00%
104.80%
103.00%
121.50%
114.60%
107.20%
MCPA
101.40%
99.84%
103.10%
101.70%
106.50%
103.90%
91.32%
91.25%
103%
99.19%
90.30%
MCPP
107.50%
102.10%
113.90%
107.90%
110.10%
106.40%
110.30%
93.46%
104.60%
94.26%
98.46%
94.14%
clopyralid
42.24%
10.31%
30.32%
8.36%
14.13%
8.03%
95.23%
72.01%
93.13%
80.46%
95.12%
84.08%
picloram
5.59%
5.61%
6.46%
1.68% 0%
8.72%
87.46%
78.75%
95.54%
87.39%
98.79%
81.70%
diflufenzopyr
95.50%
105.50%
104.10%
85.68%
111.20%
78.45%
69.21%
70.04%
65.29%
73.28%
67.53%
chloramben
77.11%
77.54%
86.35%
85.06%
83.92%
83.48%
82.51%
72.16%
87.44%
79.48%
81.31%
78.38%
acifluorfen
56.42%
109.00%
79.97%
42.98%
97.99%
107.40%
93.40%
92.95%
79.85%
97.57%
90.43%
aminopyralid
5.01%
33.85%
17.36%
34.23%
21.97%
28.21%
23.20%
5-OH dicamba
23.15%
28.51%
34.74%
35.12%
35.72%
39.44%
84.54%
71.04%
83.96%
78.09%
78.36%
77.70%
DCSA
8.48%
65.40%
44.17%
54.65%
13.79%
52.01%
64.41%
65.83%
26.53%
55.13%
29.49%
53.59%
6-CPA
92.18%
104.90%
94.34%
102.90%
92.84%
100.70%
79.50%
95.14%
90.715
98.22%
88.22%
quinclorac
83.24%
76.46%
57.86%
20.65%
49.41%
29.25%
54.45%
68.92%
85.19%
75.11%
80.56%
73.88%

11. Acetate Buffered example
Compound
Acetate buffered
1% acetic in acetonitrile
1% formic in ACN
5N NaOH/H2SO4
weaker base/acid
no hydrolysis
C18 cleanup
no cleanup
2,4-D
54.73%
70.05%
62.02%
69.72%
58.29%
70.46%
66.23%
69.00%
60.28%
73.71%
61.08%
68.46%
dicamba
34.47%
55.17%
39.43%
52.16%
40.78%
47.74%
39.25%
50.19%
42.41%
48.05%
42.24%
46.63%
triclopyr
50.40%
70.75%
54.37%
47.78%
42.215
69.79%
74.58%
81.96%
68.23%
80.77%
66.18%
81.72%
MCPA
63.11%
76.62%
69.80%
71.06%
69.45%
66.03%
78.10%
81.09%
75.85%
70.72%
74.14%
69.87%
MCPP
77.62%
81.74%
83.65%
78.11%
79.30%
79.83%
84.83%
76.40%
78.07%
77.54%
79.80%
75.58%
clopyralid -
65.26%
56.99%
67.64%
51.08%
67.85%
57.37%
picloram
diflufenzopyr
47.44%
97.45%
39.91%
49.43%
17.485
85.24%
91.45%
126.60%
67.86%
123.50%
62.88%
109.80%
chloramben
36.58%
46.73%
43.01%
48.41%
42.56%
48.33%
52.94%
53.93%
47.89%
56.32%
48.00%
51.61%
acifluorfen 0%
2.92%
4.79%
18.41%
12.64%
10.73%
aminopyralid
5-OH dicamba
22.14%
33.31%
26.87%
34.73%
25.49%
36.29%
30.95%
41.74%
41.88%
28.92%
38.30%
DCSA
6-CPA
25.41%
30.42%
26.71%
27.25%
24.45%
27.14%
34.17%
33.11%
34.78%
29.86%
33.59%
31.32%
quinclorac
29.94%
44.25%
33.85%
41.35%
33.92%
42.72%
42.19%
55.92%
44.20%
43.30%
51.56%

12. Screening observations
No difference between hydrolysis and no hydrolysis
C18 cleanup gave better recoveries
Except for dicamba
Original salts and European buffered salts gave best recoveries
Acetate buffered – poor results
Acidified acetonitrile
Significantly better recoveries

13. “Final” method Weigh sample – 3g vegetation, 5g soil
Add 15 mL 1% formic acid in ACN
Add water – to get to 15g
Add QuEChERS salts - original
Cleanup – dSPE (C18) – 1 mL ACN extract
Shake for 30 minutes
Shake on GenoGrinder
Dilute, filter, analyze

14. Comparison of cleanup steps
QuEChERS extract
After C18
After dilution
After filtration

15. Results – regulatory work
For the most part, good recoveries
Aminopyralid
~30% recoveries consistently
Aminocyclopyrachlor
Very poor recoveries
More hydrophilic phenoxyacid compounds
Clopyralid and picloram

16. Results – validation work
Validated for several compounds on two LC/MS instruments
Varian 325-MS
Sciex 4500 Qtrap
Soil validation
Easy; no matrix issues
Vegetation validation
Some matrix interference; some co-eluting peaks (MCPP-p)
Continuing validation

17. How this method has helped us
Old method
New method
Extraction time – 3 days, at best
Solvent use - ~4 liters of ether per set of samples
Glassware – separatory funnels and florisil columns
Safety – use of diazomethane and chlorinated solvents
Derivatization - with diazomethane; for GC/MS analysis
Extraction time – half a day, at worst
Solvent use – mL of acetonitrile
Glassware – none for extraction itself
Safe – no diazomethane or chlorinated solvents
Derivatization - none

18. Dicamba 2017 – reformulated to lower volatility 218 samples
Degrades readily, need to look for metabolites; 5-OH dicamba and DCSA
Needed to look at lower levels
2018
EPA – approved the use of reformulated dicamba
Expect similar numbers of samples

19. Dicamba “Regular” extraction “Dicamba” extraction 3g vegetation
15 mL acidic ACN
10 mL acidic ACN
Calibration range – 1 to 100 ppb
Calibration range – 0.1 to 10 ppb
Normally 10 μL injection
50 μL injection

20. Future of method Finish adding phenoxyacid herbicides
Adding imidazolinones (“imis”)
Adding “normal” QuEChERS compounds
Oftentimes, have other compounds to analyze besides phenoxyacid compounds
Speeding up analysis

21. Acknowledgements Residue Group Travis Knight and Melanie Banta EPA
Varuni Subramaniam
Travis Knight and Melanie Banta
EPA

22. Lab overview Pesticide Residue – 5 chemists
Pesticide Formulation – 1 chemist
Feed and Fertilizer (Vitamin) – 3 analysts
Meat and Poultry – 2 microbiologists
Dairy – 2 microbiologists
Administrative
Travis Knight – Ag Lab Bureau chief
Melanie Banta – Chemistry supervisor
Bill Gamble – the ISO guy

23. Current method – How method came together
Office of the Indiana State Chemist – Ping Wan
Used their method initially
Plain acetonitrile
Base hydrolysis/acid neutralization
C18 cleanup
As mentioned in previous slide
Indiana does not use any cleanup
Acidified acetonitrile
Observation by one of our chemists
NACRW – a talk by Anastassiades and poster by Sack

24. How this method has helped us
Sample and analyte load
Slight increase in sample load over the years
More active ingredients to analyze per sample
More actives are analyzed on LC/MS