Options for Fast, Reliable Pesticide Residues Analysis in Food by Triple Quadrupole GC-MS/MS

Richard Fussell1

Vertical Marketing Manager, Food and Beverage Michael T. Hetmanski2, Cristian Cojocariu3, Victoria Bailey-Horne2, Dominic Roberts3 and Paul Silcock3 1Thermo Fisher Scientific, Hemel Hempstead, UK, 2 Food and Environment Research Agency (FERA), York, UK , 3Thermo Fisher Scientific, Runcorn, UK

PP71668-EN 0515S

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Outline

• The analytical challenge

• User requirements for GC-MS/MS analysis of pesticides • Collaboration between Fera and Thermo Fisher Scientific;

method optimisation, results and learning experiences

• Latest GC-MS/MS developments • Summary

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• Wide range of matrices • Food • Environment

• Wide analytical scope • Low limits of detection • High sample throughput • Fast turnaround • Low cost of analysis

Analytical Challenges

RT: 4.57 - 37.12

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14.969.50 23.13 29.6117.388.89

12.207.315.77 19.1315.57 15.848.20

26.4712.87 17.9513.77 23.58

28.7125.3620.625.09 21.7023.8319.25

27.7724.12

35.78

34.8134.3932.9230.11 31.00

NL:3.94E8TIC MS 2july2104_011

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Requirements for GC-MS/MS

• Selectivity

• High sensitivity for 2nd transition to enable identification of pesticides at low target concentrations

• Robustness in operation 24/7 and ease of use

• Reproducibility

• Software: Effective and ease of use

• Cost, maintenance and service

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1. Sample Prep: March 24th 2. LC-MS Analysis: April 29th

3. GC-MS Analysis: June 17th 4. Data Processing/Analysis: July 15th

Typical Pesticides Workflow Register at www.chromatographyonline.com/LCGCwebseminars

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Pesticide Analysis: Triple Quad GC-MS

Highly Selective Reaction Monitoring (SRM) Improved detection limits

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Selectivity: SIM and SRM

DDE-p,p’, 0.05 mg/kg in green tea, 1.0 uL splitless injection

Ion ratio = 2.7 Ion ratio = 3.5

Thermo Fisher Scientific and Fera GC-MS/MS Collaboration (1)

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Analysis of QuEChERS Acetonitrile by GC?

• High expansion coefficient-limited injection volume

• Offline solvent exchange - requires care to avoid losses of volatile or unstable pesticides

• LVI with higher capacity liners or liner packing materials, but potential for adsorption of pesticides?

• Will repeated injection of acetonitrile extracts lead to a decrease in column efficiency?

• How quickly will matrix extracts contaminate the GC-MS

system?

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Evaluation of high sensitivity GC-MS/MS

• Thermo Scientific™ TSQ Quantum™ GC-MS/MS system

• Thermo Scientific™ Trace™ Ultra GC Chromatograph • Thermo Scientific™ TriPlus RSH ™ Autosampler • Column: TR-Pesticide (II), 30 m x 0.25 mm i.d., 0.25 µm film

• ‘test matrix’- fruit preserve

• 96 pesticides/metabolites:192 transitions

Evaluate direct analysis, of QuEChERS acetonitrile extracts

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SRM chromatograms – 1µL splitless

Splitless injection (1 µL) of acetonitrile extracts (d-PSA clean-up) of fruit preserve spiked at 0.01 mg/kg (= 10 pg on-column)

184.95>92.88 219.95>1

Dichlorvos

6.44

Pirimiphos- methyl

290.09>233.07 305.10>290.09

21.05

148.97>69.98 148.97>104.98

Captan (0.02 mg/kg)

24.9

226.03>206.03 163.02>206.03

Cyfluthrin (0.02 mg/kg)

47.03

180.99>151.99 252.99>93.00

Deltamethrin 51.6

Courtesy of Fera, UK

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Summary of validation data (1 µL splitless)

• QuEChERS extraction (citrate buffer/d-PSA) of fruit preserve

• Except chlorothalonil, recovery and % RSD data for the other 95 compounds met EU DG SANCO validation criteria

Results courtesy of Fera, UK

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Robustness (1 year) of instrument performance

• TSQ Quantum XLS • Analysed ~ 200 batches of samples

• ~ 6000 matrix injections (PTV-backflush)

• Kidney, liver, fish, meat, crustaceans, milk

• Response does vary but is always sufficient

• Analytical capillary column not replaced

• 2 breakdowns requiring service engineer

Information courtesy of Fera, UK

Themo Fisher Scientific and Fera GC-MS/MS Collaboration (2)

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Thermo Scientific TSQ 8000 GC-MS/MS

The new Trace 1300 Series offers the most versatile GC platform in the market, with unique “instant connect” modularity for ground-breaking ease of use and performance, setting a new era in GC technology

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(No I/S)

Repeatability using the baffled liner

• [10ul (1/10 diln with EtAc] speed controlled solvent vent] • PTV Baffle Liner deactivated (Siltek),

Results courtesy of Fera, UK

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Repeatability Using Asymmetric Liners

• QuEChERS extracts of baby food (no IS) • High number of injector parameters to optimise

Results courtesy of Fera, UK

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Validation Results

Dichlorvos (m/z 185-93, 186.9-93)

Cyfluthrin (m/z 226.0-206.1, 163.0-127)

• Excellent results obtained • Cycle time approximately 40 minutes

(0.01 mg/kg, 10 pg on column) (0.001 mg/kg 2.5 pg on column)

Results courtesy of Fera, UK

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QuEChERS- GC-MS/MS Workflow Validation

Matrix Recovery (%) %RSD Average No out-with

criteria Average No outwith

criteria Apple 95 3 4.7 0 Grapes 100 4 4.1 2 lettuce 104 10 4,0 0 Oats 96 16 5.9 0

• According to SANCO/12571/2013 criteria • Recovery 70-120 % , RSDr (<20%)

• Splitless (1 µL) with cyclo-liner

Preliminary Data

Results courtesy of Fera, UK

Thermo Scientific™ TSQ ™ 8000 Evo GC-MS/MS

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Increasing Laboratory Productivity

• Decrease analysis time by shortening the GC run times. • More samples in less time.

• Increase the number of pesticides in a run. • More SRMs to accommodate within an analytical run.

• Improve selectivity for various matrices.

• Increased number of SRMs per compound.

• See beyond the targets. • Full Scan and SRM data acquisition in the same experiment.

Expect More Performance

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Fast GC-MS Pesticide Residue Analysis

Challenges:

• Complexity of elution when using fast GC

• Large number of compounds (SRMs) in short time

• Many SRM transitions can result in sensitivity loss

Solution:

• High speed analyzer • Fast collision cell • Short SRM dwell times with very

short inter-scan delays

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TSQ 8000 Evo GC-MS/MS

Expect More Capacity

• Analytical instrumentation:

TSQ 8000 Evo GC-MS/MS Pesticide Analyzer TRACE 1310 GC Gas Chromatograph TriPlus RSH Autosampler (liquid injection set-up)

• EvoCell

• Rapid, innovative collision cell technology • Increased method capacity • More compounds • More SRM transitions • Up to 4x more transitions whilst maintaining method sensitivity low analyte concentrations

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Pesticide MRM Database

The problem:

• Growing list of target compounds require continuous adjustment to an existing SRM database.

• Some SRM transitions are not suitable for all matrices. Addition of new SRM transitions can be time consuming.

The solution:

• Automated SRM development with AutoSRM.

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AutoSRM: Fast, Simple Route to Optimized SRM

1) Precursor ion selection

2) Product ion selection

3) Collision energy optimization

AutoSRM automates the development of SRM methodology

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Highlights of AutoSRM

• Automates the following: • Creation of full scan, product ion scan, and SRM methods • Creation of sample sequences • Creation of data layouts for analyzing results • Selection of precursor, product, and collision energies

End result showing optimized transition

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Timed-SRM: Using Dwell Times Efficiently

Classical segmented SRM

TSQ 8000 EVO timed SRM

Classical segmented SRM:

• Complex to set up • Wasted dwell time • Reduced sensitivity • Reduced tolerance to RT shifts

TSQ 8000 Evo timed-SRM:

• Automated set-up • Full optimized dwell time • Optimal sensitivity • Increased resistance to RT shifts

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Increasing Laboratory Productivity

• Decrease the analysis time by shortening the GC run times. • More samples in less time. • More SRMIncrease the number of pesticides in a run. • s to accommodate within an analytical run.

• Improved selectivity for various matrices

• Increase the number of SRMs per compound.

• Seeing beyond the targets • Full Scan and SRM data acquisition in the same experiment.

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Decrease the Analysis Time

RT: 4.57 - 37.12

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14.969.50 23.13 29.6117.388.89

12.207.315.77 19.1315.57 15.848.20

26.4712.87 17.9513.77 23.58

28.7125.3620.625.09 21.7023.8319.25

27.7724.12

35.78

34.8134.3932.9230.11 31.00

NL:3.94E8TIC MS 2july2104_011

Full scan 144 pesticides in baby food @ 0.2 mg/kg TG-5 SILMS, 30m x 0.25 mm x 0.25 µm GC run time: ~37 min

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Fast GC: 144 Pesticides in Baby Food

As more compounds are added to a single run, more complexity arises with the management of acquisition windows and compound co-elution. RT: 3.68 - 10.75

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8.68 8.819.31

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8.258.007.78 8.49 9.61

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9.687.00 7.31

6.56 8.236.83

7.096.08

7.46 9.958.045.956.36

5.25 6.28 10.359.58

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4.90 5.43 10.055.894.10 5.68

4.46 10.574.44 5.113.74 3.99

NL:1.28E9TIC MS 2july2104_057_140704112840SRM acquisition of 144 pesticides in baby food @ 0.2 mg/kg

GC column: TG-5 SILMS, 20m x 0.18 mm x 0.18 µm, faster temperature ramp GC run time: <11 min

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Increase Laboratory Productivity

• Decrease the analysis time by shortening the GC run times. • More samples in less time.

• Increase the number of pesticides in a run. • More SRMs to accommodate within an analytical run.

• Improved selectivity for various matrices

• Increase the number of SRMs per compound.

• Seeing beyond the targets • Full Scan and SRM data acquisition in the same experiment.

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More SRM Transitions/Compound for More Confidence

Tecnazene in baby food at 0.01 mg/kg level

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RT: 14.95 - 16.71

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RT: 15.79AA: 4093580SN: INF

RT: 15.79AA: 1901556SN: INF

NL: 2.60E6TIC F: + c EI SRM ms2 246.000@cid28.00 [176.095-176.105] MS Genesis 19May2014_03

NL: 1.21E6TIC F: + c EI SRM ms2 317.800@cid20.00 [245.995-246.005] MS Genesis 19May2014_03

RT: 14.84 - 16.62

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RT: 15.79AA: 3837740

RT: 15.79AA: 1690756

NL: 2.35E6TIC F: + c EI SRM ms2 246.000@cid28.00 [176.095-176.105] MS Genesis 19May2014_05

NL: 1.03E6TIC F: + c EI SRM ms2 317.800@cid20.00 [245.995-246.005] MS Genesis 19May2014_05

More Speed Maintaining the Sensitivity

DDE-p,p’ in green tea, 1917 SRMs Inj. 0.01 mg/kg on column

DDE-p,p’ in green tea, 44 SRMs Inj. 0.01 mg/kg on column

0.7 ms 27 ms

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Linearity: Dichlorvos

• Dichlorvos peak area response over 0.5–10 ppb (mg/kg), matrix-matched standard (baby food).

• Chromatograms (quantification and confirmation ions) at 10 ppb level.

8 SRMs/compound

2 SRMs/compound R2= 0.998

R2= 0.997

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Examples of Peak Area Repeatability: Fast GC method

Data analysis method maintenance

GC Performance Maintenance

MS Performance Maintenance

Acquisition Method Maintenance

Routine results

%RSD pesticides (0.001 mg/kg on column) baby food matrix ~144 compounds in <10 minutes

Compound % RSD (n = 10) BHC, Alpha 7.0 BHC, Beta 8.8 BHC, gamma 9.2 Chlorobenzilate 12.5 Chlorothalonil 12.6 Clomazone 6.3 Cyfluthrin peaks 1-4 9.3 DDE p, p 8.2 Dichlobenil 5.3 Dichlorvos 8.1 EPTC 5.3 Etridiazole (Terrazole) 4.6 Hexachlorobenzene 8.9 Methacrifos 7.6 Propachlor 11.0 Propham 11.5 Simazine 9.1 Tecnazene 5.6 Tefluthrin 7.0 Triallate 11.0

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Comprehensive Detection of Pesticides in a Sample

• Targeted screening and quantification of a number of pesticides is important.

• Increased interest in screening the samples for additional compounds beyond a target list.

• Less common or new pesticides or unknown

metabolic/transformation products could be detected in addition to targeted compounds.

• This requires fast analytical instrumentation able to acquire both full scan and SRM/SIM data simultaneously.

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Summing up

• Compound database management: • Auto-SRM automates SRM transitions development.

• Using the available dwell time wisely: • Timed-SRM ensures minimal loss of time spent to acquire data.

• Enhancing the number of compounds and increasing the number of SRM transitions per compound:

• EvoCell fast collision cell technology.

• Comprehensive detection of target pesticides and of additional contaminants potentially hazardous to human health:

• Simultaneous full scan and SRM/SIM data acquisition.

• A full validation of this fast method is ongoing and will be available soon.

Fast, easy, and robust pesticide analysis can be achieved using:

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Thermo Scientific Food and Beverage Community

• View application notes, on-demand webinars, product information, and many more resources on our Pesticides and Food Communities Libraries:

www.thermoscientific.com/pesticides www.thermoscientific.com/foodandbeverage

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Thank You for Listening

Questions?

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