Revisiting the Advantages of the QuEChERS Approach to Sample Preparation Steven J. Lehotay
Revisiting the Advantages of the QuEChERS Approach to Sample Preparation Steven J. Lehotay Outline of the Presentation • Introduction to QuEChERS • QuEChERS for pesticides in foods • Other QuEChERS applications and options • Conclusions What is QuEChERS? www.quechers.com http://en.wikipedia.org/wiki/Quechers The Quechers method is a streamlined approach that makes it easier and less expensive for analytical chemists to examine pesticide residues in food. The name is a portmanteau word formed from "Quick, Easy, Cheap, Effective, Rugged, and Safe." Multiclass, Multiresidue Analytical Approach: extraction clean-up quantification identification Quick Easy QuEChERS method Cheap Effective Rugged Safe GC-MS(/MS) LC-MS/MS Slide Devised by Katerina Mastovska Sample Size and Comminution • Numerous studies have shown that the sample processing step is crucial (IUPAC, FAO/IAEA, CSL, FDA, USDA, EU studies, check samples). Conclusions from the Studies: • Use proven choppers of appropriate size • Chop frozen samples (dry ice or liquid N2) • 10-20 g subsample gives representative portion Minimizing Sample Size Minimizes Effort and Expense Minimizing Sample Size Minimizes Effort and Expense Freeze Sample (-20°C), Add dry ice, Homogenize to produce a flowable powder (-25 to -30°C) Improved homogeneity Slide adapted from Richard Fussell, FERA (UK) QuEChERS Approach Tomato Grape Spinach Strawberry 1) Shake sample with solvent and salts 2) Centrifuge for 1 min Spinach Tomato Grape Strawberry 4) Centrifuge for 1 min 5) Analyze Pesticides 3) Mix a portion with a sorbent The Birth of QuEChERS QuEChERS was born of two fathers at the USDAARS-ERRC, after 40 years of labor in the residue community, 8 years of labor of the fathers apart, and 2 years of their labor together. Birth announcement: 4th European Pesticide Residue Workshop Rome, Italy May 28-31, 2002 A rose by any other name would smell as sweet? QuEChERS without Rugged is: Fast Easy Cheap Effective Safe (FECES) Curtain plate after injection of 25 samples with extracts from raisins without cleanup Picture from Lutz Alder, BfR, Germany Traditional QuEChERS Blending Shaking Filtration Centrifugation Large solvent volumes Small solvent volumes Multiple partitioning steps Single partitioning Transfers of entire extract Take aliquots (use I.S.) Lot of glassware Single vessel Evaporation/Concentration Large volume injection Classical SPE Dispersive SPE QuEChERS Features and Impact • A single extract can be prepared in 10 min or a batch of 20 in an hr by a single analyst with ≈$1-3 of disposable materials per sample and generate <12 mL nonchlorinated solvent waste. • Consistently high recoveries (mostly 90-110% with RSDs < 10%) of a wide range of GC- and LC-amenable pesticides are achieved from many food matrices. • Countless labs have implemented QuEChERS successfully for up to 500 pesticides in food and increased efficiency (faster, less labor, lower cost, less waste, saves space, less labware, higher throughput). • QuEChERS concepts have spread to other applications. Recoveries in the QuEChERS method 229 pesticides analyzed by GC/MS and LC/MS-MS 90% Percentage of Pesticides 80% 70% 60% 50% 40% 10 ng/g Lettuce 10 ng/g Orange 25 ng/g Orange 50 ng/g Lettuce 100 ng/g Lettuce 100 ng/g Orange 30% 20% 10% 0% <20 20-49 50-69 70-79 80-89 %Recovery 90-110 111-120 >120 Repeatabilities in the QuEChERS method 229 pesticides analyzed by GC/MS and LC/MS-MS 80% Percentage of Pesticides 70% 10 ng/g Lettuce 10 ng/g Orange 25 ng/g Orange 50 ng/g Lettuce 100 ng/g Lettuce 100 ng/g Orange 60% 50% 40% 30% 20% 10% 0% >0-5 >5-10 >10-15 >15-20 %RSD >20-30 >30 A QuEChERS Advantage QuEChERS “Waste” Mini-Luke Waste Same Number of Extractions and 15 g Sample Size (Nonhalogenated) (Halogenated) QuEChERS at General Mills vs. Traditional Approach Reduced cost per sample About 40-50% reduction in sample prep and operational costs ($72 savings per sample analyzed) Reduced and less hazardous waste 1:100 reduction; no chlorinated solvents Increased sample throughput About 3-fold increase Increased scope of pesticides - Successfully validated >180 pesticides Slide Devised by Katerina Mastovska Number of results reported in EU PT samples by method 1200 1000 800 2006: 2007: 2008: 2009: 2010: 2011: 2012: 129 labs x 16 pesticides = 2064 total 137 x 19 = 2603 132 x 18 = 2376 151 x 21 = 3171 153 x 18 = 2754 154 x 19 = 2926 167 x 18 = 3006 QuEChERS 600 EtOAc 400 200 Mini-Luke Specht 0 2006 MeOH MeCl2 2007 2008 2009 2010 2011 2012 Year Slide adapted from Paula Medina Pastor QuEChERS in the Literature 225 200 Citations to 1st Paper 175 QuEChERS & d-SPE Pubs 669 Total 659 Total * Number 150 125 100 According to ISI Web of KnowledgeSM 75 50 25 0 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Year *search conducted on Jan. 17, 2013 QuEChERS/d-SPE Applications Application Publications Pesticides ≈ 425 Veterinary drugs ≈ 50 Environmental (e.g. PAHs) ≈ 35 Mycotoxins ≈ 15 Natural Products (e.g. alkaloids) ≈ 15 Reviews ≈ 15 Other ≈ 15 (e.g. BPA, acrylamide, PFOS/PFOA, nerve agents, phthalates, dyes/ink, sildenafil, melamine, seafood toxins, Cu) Matrices Fruits/Vegetables, Grains, Processed Foods (e.g. baby food), Honey, Nuts, Animal Tissues (meat, liver, kidney), Fish/Seafood, Milk, (Vegetable) Oils, Soil/Sediment, Dried Fruits, Feeds, Eggs, Tea, Juices/Beverages, Plants, Wine/Beer, Mushrooms, Blood/Serum, Water, Cactus, Compost, Roots, Tobacco, Dietary Supplements, Chinese Medicines Vendors of QuEChERS Products b e k o lu t QuEChERS Update Steve and Angelo “Interviewed” by Ron Majors QuEChERS a Sample Preparation Technique that is “Catching On”: An Up-to-Date Interview with the Inventors, LC GC North America, July, 2010 The QuEChERS Revolution, LC GC Europe, Sept., 2010 Available from ChromatographyOnline.com What’s New with QuEChERS? • • • • • • • • • More vendors and formats (e.g. DPX) GCB or ChloroFiltr for chlorophyll reduction Type of MgSO4: can use 97% purity Shakers – e.g. Spex and Glas-Col Automation with robotic autosampler “Unified” method to undergo AOAC update d-SPE has a life of its own More applications (e.g. environmental, blood) Veterinary drug residue methods Limitations of QuEChERS? • • • • • • • • Too many modified versions Cereals require a separate protocol Still problems with captan, folpet, captafol Spices, tea, and oils give problems Works best with modern MS systems Need PTV or solvent exchange for low LOD Matrix effects in complicated matrices Even simpler sample prep possible QuEChERS Methods 2003 Anastassiades et al. 2005 Lehotay et al. 2007 Anastassiades et al. Original AOAC 2007.01 CEN 15662 10-15 g sub sample 10-15 g sub sample 10-15 g sub sample 10-15 mL 1% HOAc in MeCN 10-15 mL MeCN shake shake centrifuge shake & centrifuge 0.4 g/mL anh.MgSO4 0.1 g/mL NaCl 0.1g/mL Na3Cit2H2O 0.05 g/mL Na2Cit1.5H2O 0.4 g/mL anh.MgSO4 0.1 g/mL NaOAc shake shake shake 0.4 g/mL anh.MgSO4 0.1 g/mL NaCl 150 mg/mL anh.MgSO4 25 mg/mL PSA 10-15 mL MeCN Option: + 50 mg C18 & 7.5 mg GCB centrifuge 150 mg/mL anh.MgSO4 50 mg/mL PSA shake Option: + 50 mg C18 & 2.5-7.5 mg GCB shake & centrifuge centrifuge 150 mg/mL anh.MgSO4 25 mg/mL PSA shake & centrifuge Option: Scale-Up & Conc. in Toluene Slide Devised by Urairat Koesukwiwat Comparison of QuEChERS Methods Dimethoate 120% Recovery 100% 80% 60% Original QuEChERS 40% CEN (Citrate buffer) 20% 0% Apple/Berry Peas Limes AOAC (Acetate buffer) No significant difference in recoveries for the majority of pesticides. Comparison of QuEChERS Methods Tolylfluanid F 120% Cl S 100% Cl O N Recovery S N 80% O 60% Original QuEChERS 40% CEN (Citrate buffer) 20% 0% Apple/Berry Peas Limes AOAC (Acetate buffer) Base-sensitive pesticides remain problematic in matrices with a higher pH. Comparison of QuEChERS Methods Pymetrozine 120% N Recovery 100% 80% 60% N NH N O N Original QuEChERS 40% 20% 0% CEN (Citrate buffer) AOAC (Acetate buffer) Apple/Berry Peas Limes For basic pesticides in low-pH matrices, the AOAC method with acetate buffer gives higher and more consistent recoveries. Unified QuEChERS Method per 1 g sample, 1 mL of MeCN w/ 1% HOAc for fruits and vegetables (10-15 g) add internal standard per g sample, add 0.4 g anh. MgSO4 + 0.1 g anh. NaOAc shake or blend centrifuge per mL of the upper layer: 150 mg MgSO4 + 50 mg PSA + 50 mg C18 + 7.5 mg GCB mix and centrifuge QuEChERS for Grains, Nuts, Doughs 2.5 - 5 g sample + 10 mL water* + 10 mL MeCN + internal standards shake for 1 h add 4 g MgSO4 + 1 g NaCl shake vigorously for 1 min centrifuge for 1 min *15 mL water for 5 g of rice 1 mL of the upper layer + 150 mg PSA + 50 mg C18 + 150 mg MgSO4 mix for 30 s centrifuge for 1 min Slide adapted from Kate Mastovska Buffered vs. Non-Buffered Approach 9.00E+04 GC-TOF MS Endrin 8.00E+04 7.00E+04 Dieldrin 6.00E+04 5.00E+04 4.00E+04 3.00E+04 Slide by Kate Mastovska 2.00E+04 1.00E+04 0.00E+00 1020 1040 1060 1080 1100 Time (s) 1120 1140 1160 Is a Freeze-Out Step Worthwhile? Effect of Freezing Out Lipids from QuEChERS Flaxseed Extracts Co-Extractives by Weight 2.5% Before d-SPE After d-SPE 2.0% 1.5% 1.0% 0.5% 0.0% 22 4 -10 -20 "Freeze-Out" Temperature (deg C) -85 (-45) Pesticide Recoveries vs. Fat Content 120% Pesticide Recovery 100% 80% 60% chlordane 40% 20% DDE HCB lettuce/orange avocado 0% 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 Pesticide Solubility in Water (mg/L) Pesticide Recoveries vs. Fat Content 120% Recovery 100% penconazole chlorpyrifos-methyl lindane heptachlor epoxide permethrin chlordane DDE hexachlorobenzene 80% 60% 40% 20% orange lettuce milk 0% 0% eggs 5% 10% Fat content avocado 15% H C Q ui M B nt ire o p, ze x p n ci o '-D e s- ,p D Ch '- E D l o H rd DT ep a C C hl tac ne hl or h or py lo py r r L i B r if in fo ro o d s m s-m an E n C op e e do yp r op thy su er yl l lfa m e ate n th S r Tr ulf in O ifl at xy u r e Pi rim P flu ali n ip erm or fe ho e n s- thr m i F e et n Vi nt hy C ncl hio l ou oz n m ol ap i n ho s Recovery 6 Data Sets from 3 Types of Flaxseeds QuEChERS of Milled Flaxseeds 100% Why not correct for recoveries? 80% 60% 40% 20% 0% n = 26 “Just-Enough” Selectivity in Extraction Volatility dichlorvos methiocarb sulfone deltamethrin pKa Polarity pymetrozine Objective: consistent recoveries at low LOQ Factors in Extraction Sample Type Extraction Solvent(s) Sample:Solvent Ratio Type(s) of Salt Added pH Temperature Comminution Water Content Extraction Method Amt. of Salt(s) Added Time of Extraction Pressure All Adjustable Parameters were Isolated and Measured using Quantifiable Factors (Recoveries, NMR, Gravimetry, GC/MS, Color) Extraction/Cleanup for Acrylamide in Foods FEP tube discard ...... removal of fat hexane layer MeCN layer matrix take 1 mL aliquot for dispersive SPE clean-up water layer excessive salts Slide by Katerina Mastovska Effect of NaCl Addition NaCl modifies extraction selectivity (amount of polar co-extractives): LC-MS/MS analysis (m/z 72 55) of potato chips extract no cleanup dispersive PSA cleanup Intensity (cps) Intensity (cps) 6.0e5 Acrylamide 7.0e4 Interference 6.0e4 5.0e5 Amount of NaCl: 0g 0.5 g 1g Acrylamide 1.5 g 2g 4.0e5 3.0e5 2.0e5 1.0e5 0.0 5.0e4 4.0e4 Interference 3.0e4 2.0e4 1.0e4 0.0 4.6 5.0 5.4 5.8 6.2 6.6 7.0 7.4 7.8 4.6 5.0 Time (min) 5.4 5.8 6.2 6.6 Time (min) Slide by Katerina Mastovska 7.0 7.4 7.8 Optimized Method for Acrylamide Analysis 1 g sample in a 50 mL FEP tube add d3-acrylamide at 500 ng/g e ra t x c n o ti add 5 mL hexane, vortex add 10 mL water + 10 mL MeCN + 4 g MgSO4 + 0.5 g NaCl shake vigorously for 1 min centrifuge for 5 min at 3450 rcf discard the hexane layer e cl a p u n- 1 mL of the upper layer + 50 mg PSA + 150 mg MgSO4 mix for 30 s centrifuge for 1 min at 3450 rcf Slide adapted from Katerina Mastovska Cleanup Solid-Phase Extraction (SPE) is a common efficient and effective approach to cleanup of extracts in residue analysis. Slide Devised by Katerina Mastovska Dispersive-SPE • Why use an SPE apparatus for “chemical filtration?” • Dispersive-SPE involves the mixing of the sorbent with the extract in a mini-centrifuge tube to retain matrix interferants, but not analytes. Pictures by Michelangelo Anastassiades Advantages of Dispersive-SPE • No SPE Apparatus, No SPE Cartridges, No Vacuum, No pretreatment, No Channeling, No Drying Out, No Collection Tube, No Flow Control, No Elution Solvent, No Dilution of Extract, No Solvent Evaporation, No Knowledge or Skill Needed • Less Sorbent, Less Time, and Less Cost • Fast, Easy Procedure (no need for automation) Disadvantages: less cleanup than SPE; half of extract is lost; “chemical filtration” applications only Comparison of Sorbents in Dispersive-SPE Mixture of Apple, Orange, Grape, Strawberry, Spinach, Carrot, Lettuce, and Tomato Dispersive-SPE clean-up: Mixed Extract None C18 Nexus NH2 SAX PSA AL-N GCB Evaporated Mixed Extracts Before and After Dispersive-SPE No Cleanup PSA PSA + GCB 2.2 mg residue 12.5 mg residue 4.2 mg residue The Problem with GCB %Recoveries of pesticides when GCB used for cleanup Pesticide o-Phenylphenol Chlorothalonil Vinclozolin Cyprodinil Thiabendazole Coumaphos Orange 92 30 94 48 44 41 Tomato 93 4 92 28 27 7 Strawberry 84 24 88 28 17 18 GCB Strongly Retains Planar Analytes Spiked spinach QuEChERS extracts (at 625 ng/mL) w/o GCB clean-up (5 μL PTV injections) Not OK after 100 injections Spiked spinach QuEChERS extracts (at 625 ng/mL) w/o GCB clean-up (5 μL PTV injections) OK after 100 injections Comparison of 16B (ChloroFiltr) and GCB HCB in Lettuces (QuEChERS w/MeCN) 16B - chlorophyll removal GCB - chlorophyll removal 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 16B - recovery GCB - recovery 5-10 mg GCB is like 50 mg 16B 0 10 20 30 40 50 Amount of sorbent (mg per mL extract) Comparison of GCB-Type Sorbents Chlorophyll %Removed Measured by Absorbance @ 680 nm Recovery of Hexachlorobenzene (HCB) at 150 ng/g in Kale Sorbent (7.5 mg/mL) ENVI-Carb Hypercarb CarbonX CarboPrep-90 CarboPrep-200 GCB “A” GCB “B” GCB “C” ChloroFiltr* %Removed 89 ± 5 96 ± 9 61 ± 1 92 ± 2 94 ± 2 93 ± 7 95 ± 6 93 ± 5 90 ± 4 * 50 mg/mL extract in d-SPE HCB %Recovery, n=4 71 ± 6 73 ± 12 89 ± 12 63 ± 0 68 ± 10 65 ± 17 42 ± 3 47 ± 3 78 ± 4 Use of MeCN/EtOAc with GCB Hexachlorobenzene 100% EtOAc MeCN Recovery 80% 60% 40% 20% 0% 2.5 5.0 7.5 10 25 50 Amount of GCB sorbent (mg per mL extract) Our MMM for Veterinary Drugs 2 g tissue in a 50 mL tube, add IS mix add 10 mL of 4/1 (v/v) MeCN/water vortex briefly, shake for 5 min centrifuge for 5 min >3500 rcf supernatant + 500 mg C18 + 10 mL hexane sat’d w/MeCN; mix for 30 s, centrifuge for 5 min > 3500 rcf; aspirate hexane to waste evaporate 5 mLs extract and dilute with water to a final volume of 1 mL add atrazine filter extract with the syringeless PVDF filter vial UHPLC-MS/MS analysis Effectiveness of Clean-Up in Removing Co-Extractives Comparison of Sorbents 60% % Co-Extractives Removed Cleanup of Beef Muscle Tissue 50% 40% 30% 20% 10% 0% Z-Sep Z-Sep + hexane Hybrid + hexane Average Recoveries and Reproducibilities 80% 70% For VD Group 2 – end-capped C18 was found to be more effective for quantitative results 60% 40% 70 – 120% 30% 25% RSD 50% 20% 10% 87 analytes 0% 0% 25% 50% 75% 100% Recovery 125% 150% 175% Method Logistics 1 chemist was able to process 60 pre-homogenized samples in an 8-hr day for an overnight sequence (longest step was 1 hr to evaporate MeCN) No glassware to be cleaned afterwards Cost of materials ≈ $3/sample (using bulk C18) Waste = 10 mL hexane and 5 mL MeCN (and two 50 mL and one 15 mL PP tubes) Conclusions • This presentation has barely scratched the surface to discuss what is possible with the “QuEChERS” template. • QuEChERS doesn’t work for everything, but it is easy, fast, and cheap to try, and little is lost if it fails. • Even so, the QuEChERS template is so flexible that conditions can be found to work eventually. Acknowledgments • Michelangelo Anastassiades • Katerina Mastovska • Lucía Geis-Asteggiante • Alan Lightfield • Urairat Koesukwiwat • Brian Kinsella • Yelena Sapozhnikova + + Tanks Berry Mulch! ! Recoveries of Tylosin (100-400 ng/g) 100% 90% 80% Reproducibility = 21% RSD 70% 60% 50% Marilyn Christine n = 150 20% Alan 30% Lucia 40% Steve Recovery n = 30 each day (10 reps x 3 levels) Avg. Repeatability = 12% RSD Day 1 Day 2 Day 3 Day 4 Day 5 Average 10% 0% Average Recoveries of the 62 Drugs Matrix-matched stds w/o internal stds n = 150 per drug (10 reps x 3 levels x 5 days) 20 different kidneys No. of Drug Analytes 60 50 40 30 20 10 0 70-100 50-69 %Recovery <50 Precision in the Analysis of the 62 Drugs No. of Drug Analytes 60 Reproducibility 50 Repeatability n = 150 per drug (10 reps x 3 levels x 5 days) 20 different kidneys 40 30 20 10 0 ≤15 16-20 %RSD 21-25 >25 Using matrix-matched stds w/o normalization to internal stds ME Profiles Before and After Clean-Up Z-Sep + hexane shows enhancement Suppression is not reduced the front of the run Effect of Clean-Up on Recoveries Effect of Clean-Up on Repeatabilities Dilution Effects on ME Profiles Extracts obtained after C18 + hexane clean-up were diluted by a dilution factor (DF) of 2 and 4. Numbers of false negatives at lowest spiking level increased vs. DF Comparison of Extraction Solvents EtOAc Acetone MeCN High Recoveries Avoid Lipids Avoid Proteins Avoid Sugars Water Removal Ease GC Compatible RP-LC/SPE Compatible GPC Compatible Ease of Evaporation Low Cost Low Toxicity Comparison of Extraction Solvents Co-Extracted Matrix (mg/g) 3.5 2.8 2.1 1.4 0.7 0.0 Ac t E QuEChERS Method of Mixed Produce CN e M ac : CN e M e n o et ac e n o et Extraction Solvent w/o PSA SPE Cleanup w/PSA SPE Cleanup Problematic Pesticide/ Solvent Combinations Pesticide(s) Solvent(s) Dicofol acetone, MeCN Chlorothalonil acetone, MeCN Pesticides with a thioether group (fenthion, phorate, disulfoton etc.) EtAc, acetone Pyrethroids (deltamethrin, cyhalothrin-lambda) acetone, MeCN N-trihalomethylthio pesticides (captan, folpet, dichlofluanid) potentially MeCN Slide by Katerina Mastovska Effect of pH and Extraction Solvents %Recoveries in pH-adjusted apple juice Thiabendazole Imazalil EtOAc MeCN EtOAc MeCN pH 2.5 57 92 58 101 pH 3 54 90 51 92 pH 4 85 90 73 94 pH 5 96 84 84 86 pH 6 104 90 94 90 pH 7 104 94 94 89 Addition of 0.1% acetic acid to extracts improves stability of folpet, dichlofluanid, captan, captafol and other pesticides or ot h en to alo do ly su lflu nil lfa an n ch su id lo ph lf rp a yr osa te ifo lo s- ne m et hy co fo l um lpe t pr oc aph ym os he ido pt ne ac h di lor ch azi lo no rp n y cy rifo pr s od at inil di raz m in et e m ho a e ci vin te sch ph lo os rd et an ho e di pr ch op pe lorv rm os et hr in ch l Recovery Comparison of MeCN and EtOAc Acetate-Buffered QuEChERS (GC-MS) 140% 120% MeCN EtOAc 100% 80% 60% 40% 20% 0% Extraction of Produce, n = 36 The Extraction/Partitioning Step • MeCN forms a strong partition with water by adding salt(s) (also sugars). • No dilution of the extract with nonpolar cosolvents as needed with acetone. • Amount and type of salts controls MeCN:water partitioning to affect polarity range. • MeCN:water extracts polars, MeCN after partitioning can extract nonpolars. • MeCN is immiscible with nonpolar solvents to give the possibility to remove lipid co-extractives. MgSO4 Removes Water from MeCN Better than Other Dessicants Dessicant NaCl Na2SO4 Mol. Sieves MgSO4 *NaCl Amt. Added 2 g per 27 mL 0.8 g per 4 mL 0.8 g per 4 mL 0.8 g per 4 mL Initial %H2O 27 6.0 6.0 6.0 Final %H2O 6.0* 3.9 3.2 0.6 was used to form 2 layers in a MeCN:H2O solution; 6% H20 remained in upper layer Solubility of MgSO4 in Different Solvents eC N/ wa te eC N/ wa t 55 /45 M M /2 98 r er eC N M c Et A ne to Ac e eO H M ne 16 14 12 10 8 6 4 2 0 To l ue solubility (mg/mL) Solubility of MgSO4 in different solvents Use of Different Types of MgSO4 Matrix Lime Broccoli Type of MgSO4 Temp (°C) % Matrix Removed anh. MgSO4 ≥99% purity 45 57 anh. MgSO4 ≥97% purity 39 55 anh. MgSO4 ≥99% purity 48 62 anh. MgSO4 ≥97% purity 39 67 Effect of Extraction Temperature Broccoli No ice Broccoli Ice Lime No ice Lime Ice 120% Recovery 100% 80% 60% 40% 20% 0% Phthalimide Tetrahydrophthalimide Chlorothalonil Captan Folpet Use of Ice to Lower Extraction Temp. may Help for Base-Sensitive Analytes in Broccoli (non-acidic), but it has No Effect in Lime (acidic) How Salting Out Effects Recoveries Results in Different Commodities MgSO4 NaCl Water (g) (g) (mg/mL) 0 1 124 0 2 83 0 3 61 2 0 195 4 0 138 6 0 128 1 2 76 3 2 63 5 2 62 Methamid. (%recov.) 41 43 53 79 98 94 51 79 88 Acephate Omethoate (%recov.) (%recov.) 44 44 43 45 53 64 97 96 102 103 99 106 57 67 88 92 85 88 Higher Recoveries were Achieved with MgSO4 than NaCl in Combination or Alone... How Salting Out Effects Selectivity 5000000 4500000 2 g NaCl 4000000 3500000 1 g NaCl 3000000 0.5 g NaCl 2500000 2000000 1500000 1000000 500000 fructose degradant (HMF) Apple extracts 5 g MgSO4 used in all cases 0 g NaCl 0 6.00 8.0010.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.00 … but Time-->Combination of MgSO4 with NaCl Improved Selectivity by Avoiding Sugar Co-Extractives Evaluation of Incurred Samples • FSIS provided 10 kidneys found in their monitoring program to contain drug residues. • We analyzed the samples in blind fashion (unknown drugs and unknown levels). • These were each analyzed in duplicate using the different features of 3 multiclass, multiresidue methods (MMMs) to compare and assess their performances on real samples. Analysis of Incurred Kidney (2 g) Pirlimycin in Incurred Kidney (no IS) Concentration (ng/g) 250 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid 200 150 100 50 0 1 2 3 4 5 6 Kidney Sample 7 8 9 10 Analysis of Incurred Kidney (2 g) Sulfamethazine in Incurred Kidney (no IS) 180 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Concentration (ng/g) 160 140 120 100 80 60 40 20 0 1 2 3 4 5 6 Kidney Sample 7 8 9 10 Analysis of Incurred Kidney (2 g) Beta/Dexamethasone in Incurred Kidney (no IS) 450 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Concentration (ng/g) 400 350 300 250 200 150 100 50 Beta/Dexamethasone was missed by FSIS 0 1 2 3 4 5 6 7 Kidney Sample 8 9 10 Analysis of Incurred Kidney (2 g) Flunixin in Incurred Kidney (no IS) Concentration (ng/g) 600 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid 500 400 300 200 100 0 1 2 3 4 5 6 7 Kidney Sample 8 9 10 Analysis of Incurred Kidney (2 g) Flunixin vs. Flunixin-d3 IS in Incurred Kidney 700 Concentration (ng/g) 5 min shake; w/o acid 600 5 min shake; w/ acid 500 30 min shake; w/o acid 30 min shake w/ acid 400 60 min heat; w/o acid 60 min heat; w/ acid 300 200 100 0 1 2 3 4 5 6 7 Kidney Sample 8 9 10 Analysis of Incurred Kidney (2 g) Penicillin G vs. PenG-d7 IS in Incurred Kidney Concentration (ng/g) 60 5 min shake; w/o acid 30 min shake; w/o acid 60 min heat; w/o acid 50 40 5 min shake; w/ acid 30 min shake w/ acid 60 min heat; w/ acid No need for long shake nor heat, so fast and cool is fine! 30 Spikes were ok, but incurred penicillins can’t tolerate acids 20 10 0 1 2 3 4 5 6 7 Kidney Sample 8 9 10 Comparison of Cleanup 45% 40% % Removed 35% 30% 25% 20% 15% 10% 5% 0% Cleanup of Beef Kidney Tissue <4% of matrix co-extracted initially “QuEChERS” w/o Partitioning 2.5 g sample + 15 mL MeCN/H2O (75/25) with 1% formic acid Vortex 30 min Centrifuge at 3000 rcf 5 min Based on Mol et. al. (2008) Anal. Chem. 80, 9450-9459. Take a 2 mL aliquot Partition with 2 mL of hexane saturated in MeCN Take a 0.5 mL aliquot Dilute with 0.5 mL of H20 Filter using 0.45 μm PVDF filter vials Inject in LC-MS/MS Slide adapted from Lucía Geis-Asteggiante Mycrocystins (MCs) and Nodularin D-Glu Mdha D-Ala Adda L-Leu MC-LR Nodularin D-MeAsp L-Arg Two variable L-amino acids Slide adapted from Lucía Geis-Asteggiante MCs Recoveries and Precision: 4 levels (10, 25, 50, and 100 ng/g), 5 replicates each on 5 different days (n=42) Slide adapted from Lucía Geis-Asteggiante QuEChERS for Anthelmintics Place 10 g tissue / 10 ml milk into tube Add 10 ml MeCN + 4 g MgSO4 + 1 g NaCl Add internal standard (Cyprodinil + 2,4D) Shake for 1 min Centrifuge for 5 min Place 1 ml supernatant into 2 ml centrifuge tube containing 150 mg MgSO4 and 50 mg sorbent (C18) Mix for 1 min Centrifuge for 1 min Place 0.5 ml into auto sampler vial Add QC spike (TPP) Inject onto LC-MS/MS (ESI+ and ESI-) Slide adapted from Brian Kinsella Recoveries of 39 Anthelmintics 120 110 100 90 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Recovery 80 Slide adapted from Brian Kinsella Anthelmintics ESI + Triphenyl Phosphate (IS) Cyprodinil (QC spike) Abamectin Doramectin Emamectin Eprinomectin Moxidectin Ivermectin Selamectin Dichlorvos Coumaphos Coumaphos-Oxon Haloxon Morantel Levamisole ESI – Albendazole Albendazole-Sulfoxide Albendazole-Sulfone Albendazole-Amino-Sulfone Cambendazole Flubendazole Amino-Flubendazole Hydroxy-Flubendazole Fenbendazole Fenbendazole-Sulfone Oxfendazole Mebendazole Amino-Mebendazole Hydroxy-Mebendazole Oxibendazole Thiabendazole 5-Hydroxy-Thiabendazole Triclabendazole Triclabendazole-Sulfoxide 2,4 Dichlorophenoxyace tic acid (IS) Bithionol Clorsulon Closantel Niclosamide Nitroxynil Oxyclozanide Rafoxanide Triclabendazole Slide adapted from Brian Kinsella
© Copyright 2024