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

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

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

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

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-

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

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

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

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

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%

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%

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

“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

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

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

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

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

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

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

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

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

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

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

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