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Pesticide screening LC-QTOF, Agilent
Pesticide screeningLC-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-QTOF
Quantitative method: 311 Pesticides
Qualitative method: 680 pesticides
National Food Institute, Technical University of Denmark
Disposition
Screening analysis
LibraryLC-
QTOF analysis
National Food Institute, Technical University of Denmark
Disposition
Screening analysis
LibraryLC-
QTOF analysis
National Food Institute, Technical University of Denmark
Disposition
Screening analysis
LibraryLC-
QTOF analysis
National Food Institute, Technical University of Denmark
Disposition
Screening analysis
LibraryLC-
QTOF analysis
National Food Institute, Technical University of Denmark
Disposition
Screening analysis
LibraryLC-
QTOF analysis
Validation
National Food Institute, Technical University of Denmark
Screening analysis
LibraryLC-
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
QuEChERS
UPLC-methodEluent A: 0.1% formic acid + 5 mM ammonia in waterEluent B: 0.1% formic acid in acetonitrileAnalysis time: 20 min
Q-TOFFull scan - m/z = 50-1000In 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
Collision10, 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
Collision10, 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
LibraryLC-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
LibraryLC-QTOF analysis
Validation
National Food Institute, Technical University of Denmark
Qualitative versus QuantitativeScreening method Confirmatory methodQualitative (detectability) Quantitative (RSD)No full identification (selectivity) Unambiguous identificationResult = + 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 methodRequirement: 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/ml Blank0.10 µg/ml 500 µl 0 µl0.05 µg/ml 500 µl 500 µl0.01 µg/ml 100 µl 900 µlBlank 0 µl 500 µl
The spiked samples are then diluted with the blank samples
National Food Institute, Technical University of Denmark
Thank you for your attention
