e-seminar

January 29, 2008

New Sample Preparation and Data

Analysis for Analyzing Hundreds of Pesticides

in Foods

E. Blanke, S. Bracht, P. FürstChemical and Veterinary ControlLaboratoryD – 48147 Münster, Germany

Chin-Kai (Kai) Meng, Ph.D. Agilent TechnologiesWilmington, DE 19808, USA

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January 29, 2008

Greetings from Münster, Germany

Sabine Bracht Peter Fürst Eva Blanke

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January 29, 2008

Chemical and Veterinary Control LaboratoryOverview

Governmental institution of the federal state, North Rhine-Westphalia Central official laboratory for inspecting:

Food of animal and plant origin, wine, tobacco

Commodities, materials with food contact

Cosmetics

Toys

Diagnosis of animal diseases and epizootics

>160 personnels (32 scientists)

Accreditated under ISO/DIN/EN 17025

Around 700 validated methods

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January 29, 2008

Samples Analyzed Yearly

• 8,500 food samples (including cosmetics)

• 25,000 meat samples (3 plate inhibition screening tests for antibiotics)

• 250,000 blood samples from food producing animals (for animal diseases)

• 30,000 cerebric samples for BSE

• 1,500 feed samples

• 400 samples for radioactivity

• 800 samples (food/feed) for pesticides

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January 29, 2008

Sample Prep Method Used Previously

DFG S 19

• sample weight: 100 g• extraction with acetone/water• liquid – liquid partitioning with cyclohexane/ethyl acetate• Gel permeation chromatography (GPC)

Clean up with silica gel GC/MS; GC/NPD

GC-ECD

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January 29, 2008

Sample Prep Method Used Previously

DFG S 19

☺ multi-residue method(more than 300 pesticides)

☺ sensitive (4 g sample/mL)

☺ applicable for multitude of matrices

labor- and time- consuming

high solvent consumption

several GC runs (>10) required for each sample

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January 29, 2008

extraction

clean-up QuEChERS method

QuickEasyCheapEffectiveRuggedSafe

GC-MSLC-MS/MS

QuEChERS - Multi-class, Multi-residue Approach

A Low Cost, Highly Effective Sample Preparation Technique for Pesticide Residue Analysis

M. Anastassiades et al., 2003, J. AOAC Int, 86:412–431.

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January 29, 2008

QuEChERS OverviewSamples are milled in frozen state (dry ice is added) to get the best

recovery. Extraction is done in acetonitrile buffered at pH 5 – 5.5. After centrifuged, the organic phase is cleaned-up by dispersive SPE using PSA (and GCB as necessary). Additional MgSO4 is added to remove any residual water. The PSA treated extract is acidified with formic acid to improve the stability of base-sensitive pesticides. The extract is ready for GC and LC analysis.

For samples with low water content (<80%), water is added before the initial extraction to get a total of ca. 10 mL water.

Quality control is performed by adding ISTD to the acetonitrileextraction step.

PSA: primary secondary amine GCB: graphitized carbon black

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January 29, 2008

Sample Requirement

Sample size: 1 – 2 kg (representative sample)

Cutting sample into suitable size (2 cm x 2 cm) for milling

Put cut samples in a polyethylene bag, seal the bag and freeze (@ -18 oC) overnight prior to cryogenic milling

Store flat in freezer

Seal the bag

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January 29, 2008

Sample Preparation (Strawberry) - cryogenic milling

Initial milling without dry ice

Add dry ice, continue milling

Stir while milling

Gradually increase milling speed

Frozen sample

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January 29, 2008

Pictorial Representation of the QuEChERS Steps

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January 29, 2008

Pictorial Representation of the QuEChERS Steps

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January 29, 2008

QuEChERS Uses Less Glassware

QuEChERS

Courtesy of Dr. Jon Wong, FDA, College Park, Maryland

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January 29, 2008

A batch of A batch of 66--12 extracts12 extracts can be prepared can be prepared inin 3030--40 min40 min by a by a single analystsingle analyst with with ≈≈$1$1--33 of disposable materials per sample and of disposable materials per sample and generate generate <12 mL solvent waste<12 mL solvent waste and only a and only a rere--usable itemusable item (FEP tube) for cleaning.(FEP tube) for cleaning.

Consistently high recoveries (mostly 90Consistently high recoveries (mostly 90--110% with RSDs < 5%) of a wide range of 110% with RSDs < 5%) of a wide range of GCGC-- and LCand LC--amenable pesticides are amenable pesticides are achieved from many matrices. achieved from many matrices.

QuEChERS Advantages

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January 29, 2008

229 pesticides analyzed by GC-MS and LC-MS-MS

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

<20 20-49 50-69 70-79 80-89 90-110 111-120 >120

%Recovery

Perc

enta

ge o

f Pes

ticid

es

10 ng/g Lettuce10 ng/g Orange25 ng/g Orange50 ng/g Lettuce100 ng/g Lettuce100 ng/g Orange

Recoveries in the QuEChERS method

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January 29, 2008

Dispersive SPE Comparable to Cartridge SPE

Courtesy of Dr. Frank Schenck, FDA, Atlanta Georgia

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January 29, 2008

inter-laboratory validation of the “buffered” QuEChERS method

13 labs in 7 countries (USA, Canada, Denmark, Germany, The Netherlands, Spain, and UK)

21 fortified samples (at 10 - 1,000 ng/g at 3 paired levels) with 20 diverse pesticides plus 6 incurred pesticides in 3 commodities (grape, lettuce, and orange)

analysis by (LVI-)GC/MS and LC/MS-MS

QuEChERS Collaborative Study

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January 29, 2008

JAOAC Int, Vol 88, No 2, pp595-614 (2005).By S. Lehotay et al

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January 29, 2008

New Pesticide Analysis Method

QuEChERS☺ Multi-residue method

☺ fast sample preparation

☺ low solvent consumption

low final sample concentration (1 g/mL)

normally several analytical runs (in SIM) necessary

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January 29, 2008

Sample Analysis Objective

Multi-residue and data analysis for 927pesticides with one GC/MS injection

• injection of 10 µL

• simultaneous SIM/Scan data acquisition

• data analysis with spectra deconvolution (AMDIS)

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January 29, 2008

System Configuration

• 7890GC / 5975 inert MSD, Retention Time locked

• PTV injection port

• 30 m x 0.25 mm x 0.25 µm HP-5MS column

• Synchronous SIM/Scan acquisition

• Deconvolution Reporting Software, DRS (G1716AA)

• RT locked DRS Pesticide Library (927 compounds)

(G1672AA)

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January 29, 2008

What is Deconvolution?“…in the broad sense of extracting one signal from a complex mixture...” (From AMDIS)

There are four deconvoluted components under the white TIC.

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January 29, 2008

AMDIS

Automatic Mass spectral Deconvolution andIdentification System

• Developed by the National Institute of Standards and Technology (NIST)

• Developed to detect chemicals in violationof Chemical Weapons Convention (mustminimize false positives and false negativesin reporting)

• Used to identify target compounds incomplex matrices

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50

170 280

31075

185

160

How Does Deconvolution Work?

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50

170 280

50170280

31075

75

185

185

310

160

160

Eliminate Ions Don’t Fit the Criteria

Extracted Ion Chromatograms

(EIC)

Ion grouping criteria:

1. Same RetTime at apex

2. Same peak width

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50

170 280

50170280

Spectrum is Deconvoluted/Cleaned

Related ions are grouped together as

a component.

A component in AMDIS

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January 29, 2008

10 15 20 25 30 35 40

Ion 41

Ion 42

Ion 43

Ion 154

Ion 98

Ion 55

Ion 56

Ion 57

Ion 99

Ion 116

Ion 131

Ion 248

Deconvolve EICs (after de-skewing) of a Pear Extract

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January 29, 2008

Extract All “Components” from EICs

11.20 11.40 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40

No,

Ions not related

Yes,

Ions are related

Ion grouping criteria:

1. Same RetTime at apex

2. Same peak width

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January 29, 2008

AMDIS Deconvolution Pulls Out Individual Components and their Spectra

TIC & Spectrum

TIC Sum of ions in Component 1

Sum of ions in Component 3

Sum of ions in Component 2

Deconvolution

Deconvoluted peaks and spectra

Search “cleaned” components against a library for hits.

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January 29, 2008

Carbaryl

PeachScan at 5.615 min

Deconvoluted/extracted spectrum

Library spectrum

The power of deconvolution is appreciated while comparing the top two spectra (raw scan and the compound spectrum hidden in the raw scan).

A component in the scan above.

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January 29, 2008

Fenbuconazole

PeachScan at 10.776 min

Deconvoluted/extracted spectrum

Library spectrum

A component in the scan above.

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January 29, 2008

Endosulfan, alpha

Scan at 7.538 min

Deconvoluted/extracted spectrum

Library spectrum

Peach

A component in the scan above.

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January 29, 2008

Fast GC and 3-Way Splitter System(Sample prepared using QuEChERS protocol at US FDA)

• Agilent application simultaneously collected GC and MS signals (RT locked) using a 3-way splitter. The location (RT) of the GC peaks from µECD and FPD (P) helped to confirm the targets found by AMDIS (deconvolution).

• Each GC run is at the 3x speed using a 240V oven. With deconvolution, less peak resolution is required. Each run is finished in less than 15 minutes.

FDA uses 4 injections (FPD + SIM, ELCD + SIM), 50 minutes each, for multi-residue analysis

Application Note 5989-7670EN: Replacing Multiple 50-Minute GC and GC-MS/SIM Analyses with One 15-Minute Full-Scan GC-MS Analysis for Non-targeted Pesticides Screening and >10x Productivity Gain

DRS Report - Peach

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

5000000

1e+07

1.5e+07

2e+07

2.5e+07

3e+07

3.5e+071.838

2.703

3.519

3.932

4.696

5.640 6.6807.093

7.556

9.081

9.538

10.559 12.382

Endosulfan (alpha)

Tetrachloro-m-xylene

Captan

Phosmet

Carbaryl

µECD

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

1000000

2000000

3000000

4000000

50000003.641

4.773

9.358

9.523

9.826

9.91011.066

Tributyl phosphate Phosmet

FPD (P)

TIC

Peach

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00200000

600000

1000000

1400000

1800000

2200000

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January 29, 2008

DRS Report from a SIM data file – for Additional Confirmation (4 ions for each target compound)

Full-scan Deconvolution vs. FPD/ELCD/SIM

21 ± 614 ± 2Endosulfan sulfateEndosulfan sulfate47 ± 534 ± 4Endosulfan (beta)Endosulfan (beta)26 ± 416 ± 2Endosulfan (alpha)Endosulfan (alpha)

153 ± 47205 ± 10ChlorothalonilChlorothalonilTomatoFenbuconazolePyridaben

230 ± 23320 ± 37PhosmetPhosmetPropiconazole I and IIEndosulfan (alpha)CaptanCarbarylPeachAzoxystrobinChlorthal-dimethyl

25 ± 2 ppb25 ± 3 ppbDiazinonDiazinonGinsengGC-MS/SIMGC-FPDFDA (FPD,ELCD,SIM)Agilent DRS (full scan/TID)

1 15-min injection (splitter) found these

4 50-min injections found these

FDA quant results

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January 29, 2008

Can We Quant on Deconvoluted Ions?

• YES!

• MSD ChemStation Rev. E.02

• DRS A.04

Software Demo

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January 29, 2008

Summary

QuEChERS - Analyses of real samples had same results as earlier (labor- and time- consuming) sample prep protocol

GC/MS in synchronous SIM/Scan mode combined with GC detectors and deconvolution reporting software (DRS) enables pesticide residue analysis at 10 µg/kg (ppb) and below in various food commodities from one injection

Quantitiation on the deconvoluted (cleaned) ions provides more accurate results

Data analysis time was about 15 min for each sample (screen and semi-quant 927 pesticides)

QuEChERS+DRS is a fast and comprehensive multi-residuepesticide analysis methodology.

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January 29, 2008

Questions?

chin_meng@agilent.com

Application Note 5989-7670EN: Replacing Multiple 50-Minute GC and GC-MS/SIM Analyses with One 15-Minute Full-Scan GC-MS Analysis for Non-targeted Pesticides Screening and >10x Productivity Gain

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January 29, 2008

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