Pesticide screening

1. Pesticide screening LC-QTOF, Agilent

2. Disposition • National Food Institute • EURL • NRL • Personale og udstyr • Kolibri • Agilent LC-QTOF • Bruker LC-QTOF • Background – pesticideanalysis • Number of pesticides • Quantitativeanalysis – LC-MS/MS • Pesticide screening • Construction of Library • Development of method

3. Pesticides • Pesticide manual: 1436 pesticides • Metabolites • Isomers • PCDL library of Agilent: 1664 pesticidesincluding relevant metabolites • Ourlibrary: 1716 pesticidesincluding relevant metabolites – retention times of around 1/3 of these

4. Pesticides in ourlaboratory

5. Disposition

6. Disposition

7. Disposition

8. Disposition

9. Disposition Validation

11. PCD – CSV file

12. Formula – database - generator

13. PCDL - library

14. PCDL – library - spectra

15. PCDL – library - spectra

17. Pesticide screening QuEChERS Q-TOF Full scan - m/z = 50-1000 In positive and negative mode UPLC-method Eluent A: 0.1% formicacid + 5 mMammonia in water Eluent B: 0.1% formicacid in acetonitrile Analysis time: 20 min

18. Overview on MS Analysis

19. QTOF-analysis – MS scan • Used to determine retention time No selection No collision

20. QTOF-analysis – Targeted MS/MS • Obtainingcompoundspectra for Library • Spectra of daughter ions from the single ion isolated in the quadropole Selection of single mass Collision 10, 20 and 40 V

21. QTOF-analysis – MS scan with collision • Screening samples • Spectra of daughter ions of all compoundseluting on a certain time point No selection Collision 10, 20 and 40 V

22. Chromatogram - TIC

23. Spectra – MSscanincl. collision Ofurace, [M+H]+ 0V 10V 20V 40V

25. Library Search

26. Library Search

27. Library Search

28. Library Search

29. Library Search

30. Library Search

31. Library Search

32. Library Search

33. Library Search - result

34. Validation

35. Qualitative versus Quantitative • Automated MS-based methods using accurate mass instruments suchToFs • Bioassays– nowseldomused

36. Validation – screening method Requirement: Confidencein detection/identification of an analyte at a certain concentration has to be established • Need to establish the lowest level of an analytecan be detected in 95% of samples (false negative rate of 5% is acceptable) • No requirements with regard to linearity or recovery • Exclude false positive results by analysingunspiked(‘blank’) samples.

37. Validation – screening method • Analyse 20 different samples for eachcommoditygroupspiked at the anticipated screening reporinglevel (SRL) • Samples should cover multiple matrices from the commodity with a minimum of 2 samples per matrix group. • Once applied routinely, on-going QC data shouldbeacquired. • For any new analyte further validation is required in order to be able to specify the Screening Detection Limit (SDL)

38. Considerations of validationsetup • How manydifferent samples? • Can wemake 2 samples of oneapple? • Or do weneed to purchasetwoapples? • We have a wish to validate at 3 levels • This is many samples to clean up • Can wethenspikeafterClean up? • No control of recovery!

39. Alternative validation plan 20 blank samples arecleaned up a b t , … 20 samples spiked at 0.10 mg/kg arecleaned up A B , … T The spiked samples arethendiluted with the blank samples

40. Thankyou for your attention