New Sample Preparation and Data Analysis for Analyzing Hundreds of Pesticides in Foods

E. Blanke, S. Bracht, P. Frst Chemical and Veterinary Control Laboratory D 48147 Mnster, Germany Chin-Kai (Kai) Meng, Ph.D. Agilent Technologies Wilmington, DE 19808, USA

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Greetings from Mnster, Germany

Sabine Bracht

Peter Frst

Eva Blanke
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Chemical and Veterinary Control Laboratory Overview

Governmental institution of the federal state, North RhineWestphalia 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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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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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-ECD

GC/MS; GC/NPD

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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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QuEChERS - Multi-class, Multi-residue Approach

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

extraction clean-up

QuEChERS method

GC-MS LC-MS/MS

Quick Easy Cheap Effective Rugged Safe

M. Anastassiades et al., 2003, J. AOAC Int, 86:412431.

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QuEChERS Overview
Samples 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 acetonitrile extraction step.
PSA: primary secondary amine GCB: graphitized carbon black
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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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Sample Preparation (Strawberry) - cryogenic milling

Initial milling without dry ice

Frozen sample Add dry ice, continue milling Stir while milling

Gradually increase milling speed

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Pictorial Representation of the QuEChERS Steps

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Pictorial Representation of the QuEChERS Steps

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QuEChERS Uses Less Glassware

QuEChERS

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

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QuEChERS Advantages A batch of 6-12 extracts can be prepared in 30-40 min by a single analyst with $13 of disposable materials per sample and generate <12 mL solvent waste and only a re-usable item (FEP tube) for cleaning. Consistently high recoveries (mostly 90110% with RSDs < 5%) of a wide range of GC- and LC-amenable pesticides are achieved from many matrices.

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Recoveries in the QuEChERS method 229 pesticides analyzed by GC-MS and LC-MS-MS
90% 80%

Percentage of Pesticides

70% 60% 50% 40% 30% 20% 10% 0%

10 ng/g Lettuce 10 ng/g Orange 25 ng/g Orange 50 ng/g Lettuce 100 ng/g Lettuce 100 ng/g Orange

<20

20-49

50-69

70-79

80-89

90-110

111-120

>120

%Recovery
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Dispersive SPE Comparable to Cartridge SPE

Courtesy of Dr. Frank Schenck, FDA, Atlanta Georgia

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QuEChERS Collaborative Study

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

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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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Sample Analysis Objective

Multi-residue and data analysis for 927 pesticides with one GC/MS injection
injection of 10 L simultaneous SIM/Scan data acquisition data analysis with spectra deconvolution (AMDIS)

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System Configuration
7890GC / 5975 inert MSD, Retention Time locked PTV injection port 30 m x 0.25 mm x 0.25 m HP-5MS column Synchronous SIM/Scan acquisition Deconvolution Reporting Software, DRS (G1716AA) RT locked DRS Pesticide Library (927 compounds) (G1672AA)

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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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AMDIS

Automatic Mass spectral Deconvolution and Identification System Developed by the National Institute of Standards and Technology (NIST) Developed to detect chemicals in violation of Chemical Weapons Convention (must minimize false positives and false negatives in reporting) Used to identify target compounds in complex matrices
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How Does Deconvolution Work?

170 50 75 160 185

280

310

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Eliminate Ions Dont Fit the Criteria

170 50 75 160 185

280

310

Ion grouping criteria: 1. Same RetTime at apex 2. Same peak width 160 50 Extracted Ion 170 Chromatograms 280 (EIC) 185 75 310

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Spectrum is Deconvoluted/Cleaned
170 50

280

A component in AMDIS

50 170

Related ions are grouped together as 280 a component.

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

Ion 41 Ion 42 Ion 43

Ion 55 Ion 56 Ion 57

Ion 98 Ion 99 Ion 116 Ion 131 Ion 154 Ion 248 10 15 20 25 30 35 40
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Extract All Components from EICs

Ion grouping criteria: 1. Same RetTime at apex 2. Same peak width
Yes, Ions are related No, Ions not related

11.20 11.40 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40
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AMDIS Deconvolution Pulls Out Individual Components and their Spectra

TIC & Spectrum TIC Deconvoluted peaks and spectra
Sum of ions in Component 1

Search cleaned components against a library for hits.

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Deconvolution

Sum of ions in Component 2

Sum of ions in Component 3

Scan at 5.615 min

Peach

Deconvoluted/extracted spectrum

A component in the scan above.

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

Library spectrum

Carbaryl

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Scan at 10.776 min

Peach

Deconvoluted/extracted spectrum

A component in the scan above.

Library spectrum

Fenbuconazole

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Scan at 7.538 min

Peach

Deconvoluted/extracted spectrum

A component in the scan above.

Library spectrum

Endosulfan, alpha

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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 ECD 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
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DRS Report - Peach

2200000 1800000 1400000 1000000 600000 200000 2.00 1.838 3.5e+07 3e+07 2.5e+07 2e+07 1.5e+07 1e+07 5000000 2.00 5000000 4000000 3000000 2000000 9.358 1000000 4.773 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 9.910 9.826 10.00 3.00 2.703 3.932 4.00 3.641 4.696 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Peach
TIC

11.00

12.00

13.00

Tetrachloro-m-xylene
3.519

Endosulfan (alpha) 7.556 Phosmet Captan Carbaryl

5.640 5.00 6.00 7.093 6.680 7.00 8.00 9.081 9.00 10.559 10.00 9.523 11.00 9.538

ECD

12.382 12.00 13.00

Tributyl phosphate

Phosmet

FPD (P)

11.066 11.00 12.00 13.00

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

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Full-scan Deconvolution vs. FPD/ELCD/SIM

Agilent DRS (full scan/TID) FDA (FPD,ELCD,SIM) Ginseng Diazinon Chlorthal-dimethyl Azoxystrobin Peach Carbaryl Captan Endosulfan (alpha) Propiconazole I and II Phosmet Pyridaben Fenbuconazole Tomato Chlorothalonil Endosulfan (alpha) Endosulfan (beta) Endosulfan sulfate 1 15-min injection (splitter) found these Chlorothalonil Endosulfan (alpha) Endosulfan (beta) Endosulfan sulfate 4 50-min injections found these 205 10 16 2 34 4 14 2 153 47 26 4 47 5 21 6 Phosmet 320 37 230 23 Diazinon GC-FPD 25 3 ppb GC-MS/SIM 25 2 ppb

FDA quant results

Can We Quant on Deconvoluted Ions?

YES! MSD ChemStation Rev. E.02 DRS A.04

Software Demo

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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 g/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-residue pesticide analysis methodology.
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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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