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Michelangelo Anastassiades

QuEChERS Theory – Part 2. Michelangelo Anastassiades. Michelangelo Anastassiades. QuEChERS with Citrate Buffer. Shake. Shake & Centrifuge. Shake & Centrifuge. Official Method in Germany (§64 LFGB) + in Europe (EN 15662). Weigh 10 g of Frozen Sample. Add 10 mL Acetonitrile.

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Michelangelo Anastassiades

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  1. QuEChERS Theory – Part 2 Michelangelo Anastassiades Michelangelo Anastassiades

  2. QuEChERS with Citrate Buffer Shake Shake & Centrifuge Shake & Centrifuge Official Methodin Germany (§64 LFGB) + in Europe (EN 15662) Weigh 10 g of Frozen Sample Add 10 mL Acetonitrile Add ISTD-Solution Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer (pH 5-5.5) Optionally: Acidic Pest. by LC-MS/MS Mix an Aliquot w. MgSO4 & Sorbents, freeze-out Optionally: SUs by LC-MS/MS Acidify extract to pH ~5 to protect base-sensitive pesticides Multiresidue Analysis by GC-MS, LC-MS ... Optionally: Add other “Analyte Protectants”

  3. Improving Selectivity • At Extraction/partitioning Step • pH • Salts • At Cleanup Step • Lipids, Sugars • Chlorophyll, Carotenoids Sigma / Aldrich / Supelco Seminar 17. November 2006, Fellbach

  4. Role of pH in the Selectivity of Extraction/Partitioning The higher the pH the less co-extractives… Sigma / Aldrich / Supelco Seminar 17. November 2006, Fellbach

  5. Role of pH in the Selectivity of the Extraction/Partitioning Step, red currant Red Currant (a different one) - Buffering to pH ~5 reduces acidic components in the extract - Acetate buffer negatively affects PSA cleanup efficiency - Citrate buffer does not show this effect, however, pH rises very high after PSA-cleanup and needs to be lowered

  6. Selectivity of Cleanup More than 50 SPE Sorbents and freezing-out tested! The sorbents mainly removed the following: • Amino-Sorbents, Alumina: • Acids (including fatty acids) • Sugars • Pigments (Anthocyanes, some Chlorophyll) Risk: Losses of acidic pesticides • Carbon-based Sorbents: • Carotinoids, Chlorophyll, Sterols Risk: Losses of planar pesticides • Reversed-Phase Sorbents: • Lipids and Waxes No losses observed • Freeze-out: • Lipids and Waxes • Sugars No losses observed Sigma / Aldrich / Supelco Seminar 17. November 2006, Fellbach

  7. Use of Carbon Sorbents PSA  PSA not satisfying when high contents ofcarotinoidsorchlorophyll • Carbon Sorbents more Effective Many tested, GCB (Graphitized Carbon Black)was best in handling - Used in combination with PSA at small amounts • - Cleanup time (shaking) extended from 30 s to 2 min  Small GCB amounts are difficult to handle … SOLUTION: Pre-mixtures of GCB/MgSO4 (powder) facilitate weighing 7

  8. Handling of GCB: Planar pesticides have a high affinity towards GCB e.g. hexachlorobenzene, chlorothalonil, thiabendazole But chlorophyll has higher affinity than all pesticides • Final extract should remain slightly coloured!!    Anthracene may be used as surrogate QC standard. Recoveries > 70% will indicate that no unacceptable losses of pesticides have occurred. 8

  9. Removal of co-extracted lipids by C18 and freezing out 5 OIL EXTRACTS 4,20 4,03 4 3 Extractives [mg/ml] 2 1,48 1,33 1,25 1,23 1,15 1,10 1 0 C18 PSA PSA/C18 Raw extract Freezing out C18/freezing out PSA/freezing out PSA/C18/freezing out Sigma / Aldrich / Supelco Seminar 17. November 2006, Fellbach

  10. Removal of co-extractives from Whole-Wheat flour

  11. Removal of co-extractives with D-SPE and Freeze-out D-SPE/freeze-out combination gives best results but more work intensive …

  12. Comparisson Freeze-out vs. GPC 1) QuEChERS method with Freezing-Out step - 2 g oil extracted with 10 mL ACN (0.2 g sample / mL extract) - cleanup by freeze out step:Impact: - From 4.6 mg lipids/mL raw extract to 0.9 mg/mL after cleanup - 99.55 % of the original 2 g oil was removed (0.45 % left) 2) CH:EA extracts by GPC- 0.5 g oil dissolved in 4 mL EtAC/CH 1:1 - Passed through Bio-beads SX3 column in two GPC runs - fractionation-settings were as typically used for pesticides Impact: - 0.5 g oil resulted in 5.5 mg oil residue in total - 98.89 % of the original 0.5 g oil was removed in 2 GPC runs (1.1 % left) Besides the less efficient lipid removal other GPC-problems include:- adsorptive losses of certain basic pesticides- cut-off of pyrethroids that coelute with the TG-tailing- High solvent and Time consumption

  13. Overview of cleanup options

  14. Pesticides and Co-extractives... Fatty Acids 6-8.5 D-SPE (PSA) D-SPE (PSA) LLP/ D-SPE Amino acids -5 - -1(pH dependent) Flavonoids/Anthocyanes 0 - 6 Polarity range covered by trad. MRMs Phytosterols 8.5-11.5 Sugars -5 - -2 Monoterpenes 2.5-5.5 Vit. E 11.5 LLP/ D-SPE (PSA)(Freeze-out) Pyrethroids (~45) 3.8 - 8.3 Acidic Pesticides (~40) pH dependent Carotenoids 11-18 D-SPE (GCB) OCs (~20) 3.5 - 7.0 Strepto-mycin -7.5 Ureas (~ 30) 1.6 - 5.9 D-SPE (GCB) Chlorophyll 17.2 OPs (~95) -0.9 - 5.7 Glyphosate -4 TGs 20-24 Carbamates (~30) -0.4 - 5.5 LLP/ D- SPE (C18, freezing) PBDEs 6.2 - 9.5 PAHs 3.3 – 6.8 Basic Pesticides pH dependent Quats -4.5 - -2.8 PCBs 5 – 8.5 Phthalates 2.5 - 6 LogKow 11 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

  15. Broadening of commodity scope Dry commodities (cereals, dried fruits) Fatty Commodities

  16. Broadening of Commodity Spectrum – Dry Commodities E.g. cereals, dried fruits • Water-Addition prior to extraction • to weaken interactions of pesticides with matrix and to ensure adequate partitioning. Sample amount is reduced and water is brought to 10 mL Co-extracted fat removed by freezing out or C18, if necessary....

  17. Dry Commodities

  18. Removal of co-extractives from honey extracts (0.5 g/mL) Raw-extract

  19. Broadening of Commodity Spectrum – Fatty commodities Commodities with a high lipid load, such as avocados or plant oils can be employed.

  20. QuEChERS- Method for oil Shake & Centrifuge Note: non-polar pesticides , e.g. HCB, DDT, DDEgive recoveries <70% Recovery correction is possible since partitioning is highly defined Weigh 2 g of Sample Add 10 mLAcetonitrile Shake & Centrifuge Take aliquot Add ISTD-Solution Perform cleanup Freeze-out followed by D-SPE with PSA or D-SPE with PSA/ODS Shake & Centrifuge Multiresidue Analysis by GC-MS, LC-MS ...

  21. Recoveries of pesticides in high fat samples Recoveries % 2g g Oil / 10 mL ACN Values in absense of water PCB 138 or 153 may be used as surrogate QC standards Rec. > 70% will indicate that no unacceptable pesticide losses occurred • The tolerable lipid-amount depends on the selection of pesticides to be covered • e.g. for HCB 0.4 g lipids are still OK (>70% rec.), for DDE 1 g, for Endosulfane 5 g • (NOTE: In presence of water (ternary system) values are different, less lipid is tolerable) • Compromise for Oil samples: 2 g oil + 10 mL ACN • HCB and DDE give recoveries <70%… • but equilibrium is defined and recovery-correction is justified

  22. Impact of lipid amount on pesticide recoveries in presence and absence of water

  23. Removal of co-extractives from Olives extracts (0.5 g/mL) Raw-extract

  24. Removal of co-extractives from Egg extracts (0.5 g/mL)

  25. Grouping of plant products for QuEChERS sample preparation.

  26. Cleanup of Tea extracts - use of CaCl2 in d-SPE CaCl2removes more water from the extract than MgSO4. Thus interctions with the sorbent (H-binding, ionic) become stronger  Better Cleanup results Methamidophos recov. normalized Recoveries of Methamidophos drop when more CaCl2 is used If polar pesticides are not within the scope CaCl2 offers a good possibility to remove the majority of the co-extractives Compromize 50 mg CaCl2 and 50 mg PSA per mL tea-extract Methamidophos recovery still at 80% (not shown here)

  27. MODIFICATION OF SAMPLE PREPARARTIONPROCEDURERelease of covalently-bound acidic pesticidesby alkaline hydrolysis

  28. Acidic Pesticides - Bound Residues

  29. Citrate-Buffered Sample pH 5.1 12 9 pH=8.3 pH pH=7.8 mg/mL 8 10 7 8 6 5 6 4 pH=3.5 4 3 2,4 2 2 1,0 1 0,5 0 0 Raw Extract PSA 25 PSA 50 mg/mL mg/mL mg co-extractives/mL Extract pH Acidic pesticides – cleanup issue Example: Extract of red currant • Losses of acidic components after cleanup • Acidic compounds interact with PSA. Thus skip PSA cleanup

  30. QuEChERS – alkaline hydrolysis Example 2,4-D • Selective systemic herbicide • Control of broad leaved weed • Plant growth regulator used to prevent premature fruit drop • Formulations include freeacid, salts, esters • May form conjugates • Method designed to convert all possible residues to free acid • Acids are often covalently bound to matrix components and thus their concentration underestimated!

  31. Acidic pesticides pH-issue: Ionization of pesticides at low or high pH-values • Acids: HX  H+ + X- • Bases: B+H+  BH+ •  Ionic form prefers to stay in the water phase pH-range of agricultural samples: ~2.5 – 7

  32. LC-MS/MS, ESI (-), No PSA Cleanup 120 100 Recovery % 80 60 40 20 4-CPA Imazapyr Picloram Clopyralid 2,4-D MCPA Benazolin Dicamba 0 2,4,5-T Imazethapyr Imazaquin Fluoxypyr Triclopyr 2,4-DP Mecoprop 2,4,5-TP Ioxynil Bentazon Propyzamid 2,4-DB Fluazifop pH 3 pH 4 Bromoxynil Naphthylacetic acid MCPB pH 5 Bromacil pH 6 Acidic pesticides – influence of pH on recovery max. pH 5.5 lower pKa  general trend higher pKa

  33. Shake Shake and Centrifuge QuEChERS – schematic description with AH Citrate-Buffered QuEChERS Weigh 10 g of Frozen Sample alkaline hydrolysis: Add NaOH and store for 30 min at RT, then neutralize w. H2SO4 Shake Add 10 mLAcetonitrile Add ISTD-Solution Add 4 g MgSO4 / 1 g NaCl/ Citrate Buffer (pH 5-5.5) optionally: Freeze-out of extracted fat over night Analysis of acidic pesticides by LC-MS/MS

  34. Alkaline cleavage or the release of phenoxy-acids Wheat flour with incurred residues

  35. Alkaline Hydrolysis – Level of MCPA in berries

  36. Alkaline Hydrolysis – Level of 2,4-D in different citrus samples x 2.5 x 2.2 factor: 1.8 – 5.8 x 1.8 x 3.4 x 3.3 x 2.6 x 5.0 x 1.8 x 3.0 x 5.8

  37. Example MCPA in Cereals (from EUPT in 2007) Median MCPA (with AH) Mean Factor x 7.1 Median MCPA

  38. Nicotine in Mushrooms

  39. Nicotine in Mushrooms - Background Information: • In 2008 high levels of Nicotine detected in mushrooms at CVUA Sigmaringen • CVUA Stuttgart consulted for confirmation (LC-TOF, -MS/MS  GC-MS ) • Since then many findings by various labs in mushrooms mainly from China • Most affected dried Porcini (Boletus edulis) but also Truffles and Chanterelles • Porcini are reported to be not cultivable • China (Yunnan Region) largest producer (80% of EU-imports from CH) • Chinese authorities say : tobacco is also widely cultivated in Yunnan region • Nicotine is a naturally occurring alkaloid in tobacco (Nicotiana tabacum) where it occurs at concentrations ranging from 2% to 8% • Cross-contamination in drying/packing sites may be an issue • Intentional use of nicotine as pesticides is also speculated

  40. Nicotine in Mushrooms - Risk assessment • EFSAARfD : 0.0008 mg/kg body weight; ADI: 0.0008 mg/kg bw per day • 99% of samples contained Nicotine (conc. often above 1 ppm) • Safe concentration in fresh mushrooms 0.036 mg/kg (=highest level not exceeding ARfD for Italian consumer) • Proposal: Dried ceps with a nicotine level higher than 2,3 mg/kg should be withdrawn from the market and safely disposed of. • Monitoring program for European wild mushrooms initiated

  41. NICOTINE PROPERTIES: • Basic: pKa1 = 3.1; pKa2 = 8.2 (i.e. predominantly protonated at pH<8.2 and double protonated at pH<3.1) • Polar:logP = 0.93 (25 °C/unionised) , the lower the pH the lower the logP • Volatile:Pvap = 5.6 Pa (25 °C). Evaporation losses reduced at low pH (ionized) (K. Chamberlain et al., Pestic. Sci., 47, 265 (1996)

  42. Nicotine by QuEChERS – Optimisation of pH at extraction/partitioning Recovery of Nicotine from fresh mushrooms at different pH(spiked residues) Natural pH LC-QToF, ISTD TPP Using Nicotine D3 as ISTD losses were compensated (rec. 93 - 120 %)

  43. QuEChERS, variation of pH Extraction of incurred residues from Dried Porcini

  44. Extraction of incurred residues from Dried Porcini QuEChERS pH 10, Variation of Temperature and Time

  45. QuEChERS modification for Nicotine 10 g Fresh mushrooms or 2 g dried sample + 10 mL water Bring pH to 10-11 by addition of NaOH 5N Add 4 g MgSO4+ 1g NaCl Shake 1 min by hand Centrifugue 5 min. at 3500 r.p.m. Take alicuot Add 150 mg MgSO4 anh. + 50 mg PSA per mL extract Shake for 30s Validation (n=5) using Champignon 10 g Mean Recovery:106% RSD: 4,1% Centrifugue 5 min. at 3500 r.p.m. Take aliquot and acidify w. 5% Formic Acid to pH~5 Analysis via GC or LC

  46. THANK YOU FOR YOUR ATTENTION!!

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