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Cryogenic Milling: An Enabling Technology for High Throughput Residue Sample Preparation. Leah S. Riter and Chad E. Wujcik

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Cryogenic Milling: An Enabling Technology for High Throughput Residue Sample Preparation Leah S. Riter and Chad E. Wujcik Serial Sample Preparation Creates Bottlenecks thru the entire workflow Process
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Cryogenic Milling: An Enabling Technology for High Throughput Residue Sample Preparation Leah S. Riter and Chad E. Wujcik Serial Sample Preparation Creates Bottlenecks thru the entire workflow Process bottlenecks limit the capacity of an workflow - Identification and elimination of bottlenecks (or rate limiting steps) can improve throughput - Increasing the capacity of non-rate limiting steps may not improve workflow performance Tube Based Sample Preparation is Serial in Nature Tubes are Standard due to Limitations in Comminution Methods Limited comminution of VCM based methods determine that large (g quantity) samples are needed to represent the field R.J. Fussell et.al., J. Agric. Food Chem., 2007, 55 (4), p 1062. Reducing Particle Size Enables Parallel Processing Diameter cubed is proportional to required mass of analytical portion 10 λ d M = S E 2 3 M = Mass of sample (grams) λ = density (g/cm 3 ) d = diameter of largest particles (cm) E = tolerable fundamental error (%) Scale Down Serial Processing Parallel Processing Correct Sampling Using the Theories of Pierre Gy, AAFCO Guidelines for Preparing Laboratory Samples, Third Edition. Particle Size Determines the Size of Analytical Test Portion, slide 1 7% brown particles Acceptable accuracy only with replication (N=6) Even with replicates, precision stays poor N Individual Results Avg. (Accuracy) %RSD (286%) N/A 3 20, 10, (190%) 43% 6 20, 10, 10, 0, 10, (119%) 111% Particle Size Determines the Size of Analytical Test Portion, slide 2 Diameter 5x smaller Theoretically 125x less mass for analytical test portion Good accuracy (N=1-6) Precision is good Precision can be used to characterize suitability N Individual Results Avg. (Accuracy) %RSD (103%) N/A 3 7.2, 6.4, (103%) 11% 6 7.2, 6.4, 8.0, 7.6, 8.0, (106%) 8% Parallel Sample Preparation is Enabled by a Two-Step Milling Process Dry Ice VCM LN 2 Freezermill 8 The Analytical Test Portion Must Accurately Represent the Field Field VCM Field Sample (kg) Freezermill Subsample (g) Analytical Test Portion (mg) Sample Collection Methods are Critical for All Residue Studies Evaluation of sample collection methods is outside of the scope of this study. Field VCM Field Sample (kg) Freezermill Subsample (g) Analytical Test Portion (mg) Lehotay and Cook recent perspective in J Ag Food Chem. covered this topic Current practices have acceptable precision ( 25%) when common regulatory guidelines are followed. Lehotay, S. J., & Cook, J. M. (2015). Sampling and Sample Processing in Pesticide Residue Analysis. Journal of agricultural and food chemistry. Examination of Fortified Residues Characterizes the Freezermill Step Fortification at the step before Freezermill enabled optimization of freezermill procedures. Precision used to characterize suitability of method for mg analytical test portions. Accuracy can further assess suitability of method. Field VCM Field Sample (kg) Freezermill Subsample (g) Analytical Test Portion (mg) 75 mg subsamples accurately & precisely represent a 10 g fortified sample Three replicate 10 g sub-samples of intact undelinted cotton seed in separate mills Fortified to 3 ppm of glyphosate and AMPA using 100 µl of a fortification solution, in the freezer/mill vessel prior to milling. - Six replicates 75 mg analytical test portions analyzed by LC-MS/MS Recovery from Undelinted System Cotton Seed (%RSD) Glyphosate AMPA Mill #1 99.2% (6%) 92% (5%) Mill #2 97.7% (3.1%) 94.6% (4.1%) Mill #3 97.1% (6%) 94.6% (6.3%) Overall 98% (5%) 93.7% (5.1%) 12 Examination of Field Incurred Residues Characterizes Overall Suitability Field incurred residues are present for entire laboratory workflow. Evaluation of precision can characterize comminution process. Accuracy determination difficult (or indirect). Field VCM Field Sample (kg) Freezermill Subsample (g) Analytical Test Portion (mg) Excellent Precision of Endogenous Residues Shows Method Suitability Residue field trial samples with endogenous glyphosate and AMPA Eighteen replicate 10 g sub-samples of each matrix (six per mill) - Six 75 mg analytical test portions analyzed by LC-MS/MS Intra-mill (inter-batch) variability generally RSD 7% Inter-mill variability ranged from % RSD. Corn Forage Alfalfa Hay Canola Seed System Glyphosate AMPA Glyphosate AMPA Glyphosate AMPA % RSD % RSD % RSD % RSD % RSD % RSD Mill # Mill # Mill # Overall Results Comparable to Previous Method for Endogenous Residues Residue field trial samples with endogenous glyphosate and AMPA Previously analyzed by OPA based glyphosate method Six replicate 10 g sub-samples of each matrix - Six 75 mg analytical test portions analyzed by LC-MS/MS - Excellent precision ( %) - Results from two methods generally agree Glyphosate AMPA Matrix % of OPA method % RSD % of OPA method % RSD Corn Forage Corn Seed Cotton Seed Cotton Gin Byproducts Canola Seed Alfalfa Hay Adding Internal Standard upfront Corrects for Inefficiencies in Workflow 12 analytical portions per matrix IS added either before or after extraction (6 each) Calculated recoveries increased from 2-11% by adding the internal standard upfront Small sample sizes enable IS to be added earlier in process Glyphosate AMPA Recovery Recovery Matrix Difference %RSD Difference %RSD Corn Grain +11% 1.8% +7% (2.1%) 2.1% Corn Forage +8% 6.0% +5% (4.9%) 4.9% Canola Seed +2% 1.6% +4% (2.1%) 2.1% 16 Validated Small Scale Glyphosate Residue Method Net sample throughput 60 samples/day 0.05 ppm LLMV in RACs MS: AB Sciex API 5000 (ESI negative) Column: Bio-Rad Cation-H Guard Column 30 mm x 4.6 mm Injection Volume: 5 µl; Flow Rate: 500 µl/minute Glyphosate LC: Isocratic at 80 % A (0.1% FA) 20% B (MeCN) Valve Switch: From waste to MS at 1.2 minutes AMPA Time, min Small Scale Glyphosate Residue Method Validated for Many Matrices Matrices Validated - corn (grain, forage, and stover) - cotton (undelinted seed, and meal) - canola (seed) - soy (seed, meal, forage, and hay) - alfalfa (forage and hay) - sugar beet (root and tops) - onion bulbs 6 reps at 0.05 & 0.5 mg/kg glyphosate and AMPA Recovery % %RSD % 18 Small Scale Glyphosate Residue Method Validated for Many Matrices Recovery Range (%RSD) Matrix Glyphosate AMPA 0.05 ppm 0.5 ppm Fortification 0.05 ppm Fortification 0.5 ppm Fortification Fortification Corn Grain % (5.1%) % (2%) % (4.5%) % (2.2%) Corn Stover % (4.4%) % (1.2%) % (2.8%) % (1.9%) Corn Forage % (6.4%) % (3.3%) % (3.6%) % (1.7%) Soybean Seed % (3.7%) % (2.3%) % (4.7%) % (0.9%) Cotton Seed % (1.8%) % (3.6%) % (7.2%) % (4.2%) Soy Forage % (1.6%) % (2.6%) % (6.2%) % (2.2%) Soybean Hay % (1.6%) % (1.9%) % (3.1%) % (3%) Canola Seed % (1.2%) % (1.4%) % (7.2%) % (1.6%) Sugar Beet Root % (3.4%) % (1%) % (1.9%) % (1.4%) Alfalfa Forage % (11.8%) % (2.6%) % (11.5%) % (4.9%) Onion % (3.3%) % (1.5%) % (3.7%) % (0.8%) Sugar Beet Tops % (4.1%) % (2.2%) % (3.7%) % (1.9%) Alfalfa Hay % (4.2%) % (4%) % (2.9%) % (4%) Soybean Meal % (0.8%) % (0.9%) % (5.9%) % (4.7%) Cotton Seed Meal % (1.9%) % (2.8%) % (1.2%) % (0.8%) Two-Step Milling Enables Accurate Representation of Field Two-step milling process enables accurate and precise representation of field collected samples with mg analytical test portions Performed by VCM entire field sample, then subsample for further processing Field VCM Field Sample (kg) Freezermill Subsample (g) Analytical Test Portion (mg) Dry Ice VCM LN 2 Freezermill Acknowledgements ESTC Leadership Susan Martino- Catt Monte Marshall Chad Wujcik RAC Team Wei Huang Pamela Jensen Krystal Easter Albert White Thank You! 22
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