Pesticide screening
LC-QTOF, Agilent
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
LCQTOF
analysis
Library
Screening
analysis
National Food Institute, Technical University of Denmark
Disposition
LCQTOF
analysis
Library
Screening
analysis
National Food Institute, Technical University of Denmark
Disposition
LCQTOF
analysis
Library
Screening
analysis
National Food Institute, Technical University of Denmark
Disposition
LCQTOF
analysis
Library
Screening
analysis
National Food Institute, Technical University of Denmark
Disposition
LCQTOF
analysis
Library
Screening
analysis
Validation
National Food Institute, Technical University of Denmark
LCQTOF
analysis
Library
Screening
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
LCQTOF
analysis
Library
Screening
analysis
National Food Institute, Technical University of Denmark
Pesticide screening
Full scan - m/z = 50-1000
In positive and negative mode
Eluent A: 0.1% formic acid +
5 mM ammonia in water
Eluent B: 0.1% formic acid in
acetonitrile
Analysis time: 20 min
National Food Institute, Technical University of Denmark
Overview on MS Analysis
National Food Institute, Technical University of Denmark
QTOF-analysis – MS scan
• Used to determine retention time
No selection
No collision
National Food Institute, Technical University of Denmark
QTOF-analysis – Targeted MS/MS
• Obtaining compound spectra for Library
• Spectra of daughter ions from the single
ion isolated in the quadropole
Selection of
single mass
Collision
10, 20 and 40 V
National Food Institute, Technical University of Denmark
QTOF-analysis – MS scan with collision
• Screening samples
• Spectra of daughter ions of all compounds
eluting on a certain time point
No selection
Collision
10, 20 and 40 V
National Food Institute, Technical University of Denmark
Chromatogram - TIC
National Food Institute, Technical University of Denmark
Spectra – MSscan incl. collision
Ofurace, [M+H]+
0V
10V
20V
40V
National Food Institute, Technical University of Denmark
LCQTOF
analysis
Library
Screening
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
LCQTOF
analysis
Library
Screening
analysis
Validation
National Food Institute, Technical University of Denmark
Qualitative versus Quantitative
Screening method
Confirmatory 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
a
b
,… t
20 samples spiked at 0.10 mg/kg are cleaned up
A
B
,…
T
The spiked samples are then diluted with the blank samples
0.10 µg/ml
0.10 µg/ml
500 µl
Blank
0 µl
0.05 µg/ml
500 µl
500 µl
0.01 µg/ml
100 µl
900 µl
Blank
0 µl
500 µl
National Food Institute, Technical University of Denmark
National Food Institute, Technical University of Denmark