Pesticide screening LC-QTOF, Agilent. National Food Institute, Technical University of Denmark...

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


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


Pesticides in our laboratory

Number of pesticides introduced to the LC-QTOF

Quantitative method: 311 Pesticides

Qualitative method: 680 pesticides


Disposition

Screening analysis

LibraryLC-

QTOF analysis


Disposition

Screening analysis

LibraryLC-

QTOF analysis

Validation


Screening analysis

LibraryLC-

QTOF analysis


PCD – CSV file


Formula – database - generator


PCDL - library


PCDL – library - spectra


Screening analysis

LibraryLC-QTOF analysis


Pesticide screening

QuEChERS

UPLC-methodEluent A: 0.1% formic acid + 5 mM ammonia in water

Eluent B: 0.1% formic acid in acetonitrile

Analysis time: 20 min

Q-TOFFull scan - m/z = 50-1000

In positive and negative mode


Overview on MS Analysis


No selection

QTOF-analysis – MS scan

No collision

• Used to determine retention time


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


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


Chromatogram - TIC


Spectra – MSscan incl. collision

0V

10V

20V

40V

Ofurace, [M+H]+


Screening analysis



Library Search


Library Search - result


Screening analysis


Validation


Qualitative versus QuantitativeScreening 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


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.


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)


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!


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 Blank

0.10 µg/ml 500 µl 0 µl

0.05 µg/ml 500 µl 500 µl

0.01 µg/ml 100 µl 900 µl

Blank 0 µl 500 µl

The spiked samples are then diluted with the blank samples


Thank you for

your attention

Pesticide screening LC-QTOF, Agilent. National Food Institute, Technical University of Denmark...

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