Pesticide screening LC-QTOF, Agilent

National Food Institute, Technical University of Denmark Disposition National Food Institute –EURL –NRL –Personale og udstyr Kolibri Agilent LC-QTOF Bruker LC-QTOF Background – pesticide analysis –Number of pesticides –Quantitative analysis – LC-MS/MS –Pesticide screening Construction of Library Development of method

National Food Institute, Technical University of Denmark Pesticides Pesticide manual: 1436 pesticides –Metabolites –Isomers PCDL library of Agilent: 1664 pesticides including relevant metabolites Our library: 1716 pesticides including relevant metabolites – retention times of around 1/3 of these

National Food Institute, Technical University of Denmark Pesticides in our laboratory Number of pesticides introduced to the LC-QTOFQuantitative method: 311 PesticidesQualitative method: 680 pesticides

National Food Institute, Technical University of Denmark Disposition Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Disposition Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Disposition Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Disposition Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Disposition Screening analysis Library LC- QTOF analysis Validation

National Food Institute, Technical University of Denmark Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark PCD – CSV file

National Food Institute, Technical University of Denmark Formula – database - generator

National Food Institute, Technical University of Denmark PCDL - library

National Food Institute, Technical University of Denmark PCDL – library - spectra

National Food Institute, Technical University of Denmark PCDL – library - spectra

National Food Institute, Technical University of Denmark Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Pesticide screening Eluent A: 0.1% formic acid + 5 mM ammonia in water Eluent B: 0.1% formic acid in acetonitrile Analysis time: 20 min Full scan - m/z = In positive and negative mode

National Food Institute, Technical University of Denmark Overview on MS Analysis

National Food Institute, Technical University of Denmark No selection QTOF-analysis – MS scan No collision Used to determine retention time

National Food Institute, Technical University of Denmark Selection of single mass QTOF-analysis – Targeted MS/MS Collision 10, 20 and 40 V Obtaining compound spectra for Library Spectra of daughter ions from the single ion isolated in the quadropole

National Food Institute, Technical University of Denmark No selection QTOF-analysis – MS scan with collision Collision 10, 20 and 40 V Screening samples Spectra of daughter ions of all compounds eluting on a certain time point

National Food Institute, Technical University of Denmark Chromatogram - TIC

National Food Institute, Technical University of Denmark Spectra – MSscan incl. collision 0V 10V 20V 40V Ofurace, [M+H] +

National Food Institute, Technical University of Denmark Screening analysis Library LC- QTOF analysis

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search

National Food Institute, Technical University of Denmark Library Search - result

National Food Institute, Technical University of Denmark Screening analysis Library LC- QTOF analysis Validation

National Food Institute, Technical University of Denmark Qualitative versus Quantitative Screening methodConfirmatory method Qualitative (detectability)Quantitative (RSD) No full identification (selectivity)Unambiguous identification Result = + or –Result = value ± SD Automated MS-based methods using accurate mass instruments such ToFs Bioassays – now seldom used

National Food Institute, Technical University of Denmark Validation – screening method Requirement: Confidence in detection/identification of an analyte at a certain concentration has to be established Need to establish the lowest level of an analyte can 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 analysing unspiked (‘blank’) samples.

National Food Institute, Technical University of Denmark Validation – screening method Analyse 20 different samples for each commodity group spiked at the anticipated screening reporing level (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 should be acquired. For any new analyte further validation is required in order to be able to specify the Screening Detection Limit (SDL)

National Food Institute, Technical University of Denmark Considerations of validation setup How many different samples? –Can we make 2 samples of one apple? –Or do we need to purchase two apples? We have a wish to validate at 3 levels –This is many samples to clean up –Can we then spike after Clean up? No control of recovery!

National Food Institute, Technical University of Denmark Alternative validation plan 20 blank samples are cleaned up 20 samples spiked at 0.10 mg/kg are cleaned up a A b B, … t T 0.10 µg/mlBlank 0.10 µg/ml500 µl0 µl 0.05 µg/ml500 µl 0.01 µg/ml100 µl900 µl Blank0 µl500 µl The spiked samples are then diluted with the blank samples

National Food Institute, Technical University of Denmark