Pesticide screening LC-QTOF, Agilent. National Food Institute, Technical University of Denmark Disposition National Food Institute –EURL –NRL –Personale.

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


Disposition

Screening analysis

LibraryLC-

QTOF analysis


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


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


Library Search


Library Search


Library Search


Library Search


Library Search


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 Disposition National Food Institute –EURL –NRL –Personale.

Documents

ms analysis slide

agilent slide

quadropole slide

pesticide screening

retention time slide

acetonitrile analysis

negative mode slide

certain time point slide