1. Evaluation of Zirconia Adsorbents in QuEChERS Cleanup for Hazelnut, Avocado and Soybeans Hans Ragnar Norli, Gunvor Viki Seneset, Nina Svendsen, Agnethe Christiansen and Sven Roar Odenmarck Bioforsk Plant Health and Plant Protection Høgskoleveien 7 N-1430 Ås, Norway

2. Primarily establish a QuECheRS cleanup method for analysis of nuts including other fatty matrices Develop a simpler and faster in house cleanup method for fatty matrices without a freeze out step and CaCl 2 treatment Investigate the new zirconia materials from Supelco Background

3. Outline Matrices: – Hazelnut (60-70% fat) – Avocado (10-15% fat) – Soybean (≈ 40 % fat) Pesticides: – Total: 333 – 122: GC-MS/MS – 211: LC-MS/MS Cleanup: – In house – Z-sep – Z-sep+ Matrices: – Hazelnut (60-70% fat) – Avocado (10-15% fat) – Soybean (≈ 40 % fat) Pesticides: – Total: 333 – 122: GC-MS/MS – 211: LC-MS/MS Cleanup: – In house – Z-sep – Z-sep+

4. Instruments Agilent 7890 A GC, 7000B GC/MS-MS (2012)  Multimode inlet (Cooled by Pressurized Air)  Backflush at column midpoint  Rt-lock (ditalimfos) Agilent 7890 A GC, 7000B GC/MS-MS (2012)  Multimode inlet (Cooled by Pressurized Air)  Backflush at column midpoint  Rt-lock (ditalimfos) Agilent 1200 HPLC, 6410 Triple Quad LC/MS (2009)  ESI+  Dynamic MRM  Programmed injection (sample dilution) Agilent 1200 HPLC, 6410 Triple Quad LC/MS (2009)  ESI+  Dynamic MRM  Programmed injection (sample dilution)

5. QuEChERS Sample Preparation In house method Avocado Hazelnut Soybean With Z-sep Avocado Hazelnut Soybean With Z-sep+ Avocado Hazelnut Soybean Amount (g)555 Water (mL)10 (0 avocado) Spiking mg/kg0.1 ACN (mL)10 Phase separation Supel QuE Citrate (EN) tube Cleanup 1Freezing out -20°C 1.5 h*Supel QuE Z- sep(EN) tube Supel QuE Z-sep+ (EN) tube Cleanup 20.5 g CaCl 2 Cleanup 3 Only for GC (Omitted for LC) Supel QuE PSA/C18 (EN) tube *Hans Ragnar Norli, Agnethe Christiansen, Ermias Deribe Journal of Chromatography A, 1218 (2011) 7234– 7241

6. Removal of lipids with freezing Hans Ragnar Norli, Agnethe Christiansen, Ermias Deribe Journal of Chromatography A, 1218 (2011) 7234– 7241

7. Gravimetric Studies

8. Visual Inspection of Residues No cleanupIn houseZ-sepZ-sep+ Percent residue removed Avocado0895927 Hazelnut0895761 Soybean0945751 No CleanupIn-HouseZ-sepZ-sep+ Hazelnut

9. Chromatograms (LC-MS/MS) Hazelnut extracts Red= Z-sep Green= Z-sep+ Black= In-House Full Scan: m/z 100-1000

10. Recovery and Precision HazelnutAvocadoSoybeans Recovery % In- House Z-sepZ-sep+In-HouseZ-sepZ-sep+In- House Z-sepZ-sep+ <60861076138511 60-69425214101 70-120848976878968779063 121-14011412210 212 >1403342253312 Precision% < =209398 919699949663 > 206229415437 Percent of pesticides of 333 within different recovery categories

11. Pesticides giving recoveries < 60 % in all three matrices after In house and Z-sep+ cleanup PesticideRecovery range (%)PesticideRecovery range (%) In-HouseZ-sep+PesticideIn-HouseZ-sep+ Acephate52-56Propamocarb50-54 4-Bromophenylurea52-55Prothiconazole- desthio 11-27 Aldicarb-sulfoxide42-51Tebuconazole29-49 Chinomethionate5-10Thiabendazole40-46 Diclofluanide13-33Tolylfluanid12-36 Diniconazole21-41Trihclorfon29-52 Fenamiphos47-54Tricyclazole46-56 Fenamiphos-sulfoxideTriticonazole21-40 Fenhexamid35-56 Fluazinam20-40 Hexaconazole14-32 Metconazole21-40 Omethoate43-49 Pinoxaden28-33 Prochloraz56-58

12. Combination of matrix and pesticide with recoveries < 60 % in all cleanup methods PesticideMatrixRecovery range Fenbutadin oxideHazelnut35-56 CarboxinAvocado16-47 BenfuracarbHazelnut0-31 DiafenthiuronAvocado13-27 DiafenthiuronSoybean23-37 Recoveries < 60 % for all 9 combinations of matrices and cleanup methods PesticideRecovery range Pymetrozine5-59 Spirotetramat-enol 0-12 Cyromazine20-55

13. Matrix Effects Normally related to quantification of pesticides using calibration standards in pure solvent Analyte signal interferences; either suppression or enhancement caused by co- eluting sample components Quantification is either over- or under- estimated

14. The nature of Matrix effects* In LC-MS/MS with electrospray signal suppression is most predominant – Caused by competition between matrix components and analytes to obtain available protons Signal enhancement may be caused by co - elution of isobaric ions *Journal of Chromatography A, 1304(2013)109-120

15. The nature of Matrix effects # In GC-MS/MS signal enhancement is caused by matrix components blocking active sites: – more analyte molecules can reach the detector Signal suppression can be observed when non-volatile compounds accumulate in the GC-system creating new active sites # Talanta 140(2015)81-87

16. Matrix effects TypeSupression or Enhancement Soft0-20 % Medium20 -50 % Strong> 50 % Medium or strong matrix effects are overcome by matrix matched calibration standards, analyte protectants (GC) or dilution of sample

17. Slope Solvent > Slope matrix OK ! ! Slope Solvent Slope Matrix Too Low! Response Concentration

18. Slope Solvent < Slope matrix Too High ! Slope Matrix Slope Solvent OK ! Response Concentration

19. Percent of pesticides in hazelnut within different matrix effect categories Soft 0-20 (%)Medium 20-50 (%)Strong > 50 (%) LCGCLCGCLCGC In house9475514111 Z-Sep947831735 Z-sep+968641004

20. Distribution of matrix effects GC- MS/MS (Hazelnut)

21. Distribution of matrix effects LC- MS/MS (Hazelnut)

22. Distribution of matrix effects > 50 for Hazelnut PesticideInstrumentIn-HouseZ-sepZ-sep+Comments 4-ChloroanilineGC-76-78Metabolite of diflubenzurone PhtalimideGC69Degradation product of folpet EPNGC59 Parathion methylGC59 Dicofol, p,p'-GC63134 Deltamethrin isomer # 1GC177-52-79 Always difficult on GC (Move to LC) Deltamethrin isomer # 2GC65 Always difficult on GC (Move to LC) DichlofluanidGC71 FluazinamGC7113855 ParaoxonGC72 TolylfluanidGC76 Endosulfan sulfateGC103 ChlorothalonilGC146 Paraoxon-methylGC833 FolpetGC2992-61 Phenyl phenol -ortoGC91 PropyzamideGC-58 AmitrazLC-89-85-86Degrades! Bromukonazole isomer #2LC772 3-OH-CarbofuraneLC817Unstable CyanazineLC819 6Cl-4OH-PhenylpyridazineLC-657Metabolite of pyridate Atrazine desisopropylLC783 FlonicamidLC844 Phosmet oxonLC789

23. Thanks to: Gunvor: Sample prepartation and photograhps Nina: LC-MS/MS results Agnethe: Experimental design and discussion of results Sven: LC-MS/MS-Chromatograms