Maximizing Efficiency in Analysis through New GC-MS Approaches

Richard Fussell

Vertical Marketing Manager, Food and Beverage, Thermo Fisher Scientific, Hemel Hempstead, UK Dominic Roberts Senior Applications Scientist, GC-MS, Thermo Fisher Scientific, Runcorn, UK PO71686-EN 0615S

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Overview •  The analytical challenge

• User requirements for GC-MS/MS analysis of pesticides • Critical aspects of the method & improving efficiency

•  Injector •  Column configuration •  Instrumental parameters

•  Latest GC-MS/MS developments including GC-Orbitrap for pesticide screening

• Summary

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Typical Pesticides Workflow Register for future webinars and to view recordings of past webinars at www.chromatographyonline.com/LCGCwebseminars

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

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

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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 for GC Pesticides Analysis

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

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NL:3.94E8TIC MS 2july2104_011

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Pesticide Analysis: GC-MS

•  Many compounds not amenable to LC separation •  Low polarity–poor atmospheric pressure ionization

•  GC offers good separation efficiency •  Choice of detectors •  Easy coupling with MS for increased

selectivity •  EI/CI spectra for identification of analytes

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Pesticide Analysis: Triple Quad GC-MS

Highly Selective Reaction Monitoring (SRM)   Improved detection limits

  Longer column lifetime   Less frequent inlet maintenance

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Selectivity: Selected Ion Monitoring (SIM) and SRM

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

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Selectivity: SIM and SRM

DDE-p,p’, 0.001 mg/kg in green tea, 1.0 uL splitless injection

9 The world leader in serving science

Proprietary & Confidential

Key Factors in the GC-MS/MS Method

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Hot split/splitless

Programmed Temperature

PTV

Typical GC Injector Choices for pesticides

•  Liquid introduction by syringe

“GC Injection is the Achilles Heel in GC”

Bertsch 1983, Univ. Alabama

•  Most commonly used technique •  Split/Splitless injection (SSL) •  Programmed temperature (PTV)

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GC Inlets •  Splitless

•  maximum sensitivity •  excellent repeatability for low volumes •  simple, probably most wide used

•  Split

•  reproducible •  less discrimination (short residence time) •  Shoot and Dilute

•  Programmed Temperature Vaporising (PTV) injector •  versatile and excellent performance if optimised •  reduced discrimination •  many liner types (baffled, dimpled, packed, etc) •  packed liners (possible discrimination) •  large volume injection (solvent removal/exchange in liner)

•  Cool on-column (not widely used)

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GC Liner Selection for Pesticides

•  In pesticide analyisis QuEChERS extractions are typical and result in extractions in acetonitrile.

•  Many labs use acetonitrile as GC injection solvent •  Requires careful method optimisation

•  Considerations in liner selection for acetonitrile injections are: •  Internal diameter •  Type of injection •  Packing of liner •  Other liner features ie baffles....

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GC liners – Type of Injection

•  Split •  Typically open ended at the bottom •  Enables split flow to pass across the bottom of the liner removing a portion of the

sample, allowing a split injection to be performed

•  Splitless

•  Typically tapered at the bottom with the column inserted into the taper •  Funnels sample onto the column and minimizes sample contact with reactive metal

components

•  PTV •  Generally used with very active compounds such as pesticides •  Good option for acetonitrile injection solvents •  Thermally liable compounds protected

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PTV Injector: Key Points

•  Minimal thermal mass for fast cooling and heating

•  Injection volumes from nano liter up to largevolume

•  Cold injection technique

•  Clean step possibility for keeping the liner inert

•  Multiple injection modes

OVEN column

Liner

Cooling by fan Heater element

Inlet Carrier Septum Purge

Split line

Slide courtesy of Thermo Fisher Scientific

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System Contamination with Heavy Matrices

9.03 min (start of run)

1.0 µg/ml dimethoate in crude extracts of lettuce - 3 µl splitless

9.06 (Injection ~20)

GC Liner

Slide courtesy of Fera, UK

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Backflush Injection

(Thermo GCQ Quantum)

Pre-column (2 m x 0.53 mm i.d., deactivated)

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PTV Backflush of Pear Extracted with AcEt

•  No BKFL

•  BKFL ON 10 min after injection of sample

•  BKFL ON 10 min after injection of standard

 xc21_estratto_plus40ppbmixpestethaccy... 16/04/2009 19.02.57

RT: 5.07 - 24.68

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Extract

BKF at 20 min (No BKF) Vitamine E

Sitosterol (area)

Octadecanoic Acid 14.68

14.1921.1513.39

19.9017.707.6421.47

18.3011.769.738.15 16.52

12.64 22.037.42 16.868.72 22.1715.38 19.455.81 11.23 22.516.74 23.47

Extract

BKF at 10 min

Vitamine E

Sitosterol (area)

Octadecanoic Acid14.20

13.7813.04

7.65 19.08

11.619.739.38 17.00

12.387.42 15.938.72

5.83 6.88 15.2711.15 17.5919.24 19.98 23.0520.84 23.69

Standard Mix

BKF at 10 min

Pesticides14.88

15.9712.95

13.72 16.7812.32

14.5117.07

17.827.29 7.80 10.41 11.539.08 18.4411.29

18.657.18 8.39 21.30 23.6520.799.266.45 21.44

NL: 5.69E9TIC MS xc21_estratto_plus40ppbmixpestethaccyc_120ul_ptvbkf_02mindelay_85cto260ptv_r3

NL: 5.12E9TIC MS XC21_EstrattoPeraArmandi_120ulEtAcCycl_PTVBKF_Clean10min

NL: 2.65E9TIC MS xc21_400ppbmixpestarmandi_120uletaccycl_ptvbkf_clean10min

Full scan data acquisition – Trace GC w PTV-BKF – 30 m TR-Pesticides, 5 m pre-column 0.53 mm ID

Area of high boiling matrix

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Effect of Back-Flush on Carryover RT: 0.00 - 35.10 SM: 7G

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29.45 30.2133.99

32.4129.14

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27.2526.98

26.76

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24.8424.49

14.2510.44 23.7122.359.108.80 10.79 21.62

7.90 19.7112.64 18.9217.60

12.17

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28.23

29.22 33.7932.99

27.0310.1713.3810.07 11.52

14.219.32

9.058.64

7.90 14.92 24.96

16.62 17.46 24.3418.11 21.46

NL:2.50E5TIC MS 51845023NL:2.50E5TIC MS 51845038

Fresh Podded Peas

RT: 0.00 - 35.10 SM: 7G

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28.8829.5928.66

27.5830.12

27.3430.98

32.1027.0233.34

26.4934.95

26.25

25.73

24.9824.8014.2524.53

24.099.02 23.839.35

7.39 23.1710.3211.84 20.5619.40

12.068.64 19.0117.23

16.11

12.62 23.1122.92

30.79

31.7014.06

32.3427.50 32.79

27.1821.99

12.0832.97

9.63

9.348.75 11.86 25.75

20.30 25.4514.59 20.187.86

15.05 16.89 18.60

NL:2.00E5TIC MS 51865013NL:2.00E5TIC MS 51865028

Molasses

Chromatograms for EtAc solvent after injection (n=10) of QuEChERS extracts with back-flush and without back-flush

Slide courtesy of Fera, UK

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Guard columns

•  Analytical columns with a length of 5-10 m of deactivated fused silica. They can be purchased already integrated or joined by a union.

•  Provides the benefit of protecting the analytical column from

contamination of non-volatile residues. Very important when working with dirty sample extracts eg. QuEChERS.

•  Can also act as a retention gap to improve analyte focussing. •  Maintain retention time of analytes and SRM segments in the method. •  Can be a source of leaks if using a connection. •  Added maintenance

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:

Thermo Scientific™ TSQ ™ 8000 Evo GC-MS/MS Thermo Scientific™ TRACE™ 1310 GC Thermo Scientific™ 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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Increasing Laboratory Productivity

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

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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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Decrease the Analysis Time RT: 4.04 - 10.89

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5Time (min)

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7.687.65 8.45

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8.788.23

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8.677.40

6.03 7.467.036.63 7.25

7.026.619.306.826.39 7.20

9.026.17 8.02 10.349.216.35 8.036.57

5.884.65 9.609.944.89 9.44

5.424.72 9.61

9.985.245.21 10.5710.04

4.09 9.73 10.175.85 10.675.504.20 5.684.27 5.11

NL:1.39E9TIC MS 2july2104_048

Full Scan 144 pesticides in baby food @ 0.2 mg/kg TG-5 SILMS, 20m x 0.18 mm x 0.18 µm GC run time: <11 min

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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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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.

32

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

15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6Time (min)

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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

35

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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Thermo Scientific TSQ 8000 EVO Pesticide Analyzer

A complete pesticide method implementation, management and maintenance solution to drive unstoppable result productivity

TSQ 8000 EVO PA designed to create powerful pesticide methods that are:

1.  Self-customized 2.  Auto-optimized

Pesticide Analyzer

37

Powering the TSQ 8000 Pesticide Analyzer

•  Preconfigured performance leading TSQ 8000 EVO GC-MS/MS system featuring the award winning TRACE1310 GC

•  Pre-loaded acquisition methods •  Thermo Scientific TraceGOLD GC Column

and consumable technology •  Tracefinder 3.2 EFS Data Processing

software •  600+ Pesticide compound database (CDB)

with 1500 + SRM transitions •  AutoSRM & timed SRM (t-SRM) •  Pesticide Analyzer installation guide

GC High Resolution Mass Spectrometry for Pesticide Analysis

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Q Exactive GC for Pesticide Analysis

•  Launched at ASMS June 2015. •  Screen (qualitative and quantitative) samples for pesticides within a single

analysis, fast and at a competitive cost.

•  To increase the scope of the analysis, by using high resolution full scan mass spectrometry.

•  Untargeted analysis where a generic full scan acquisition is run, followed by targeted data processing of a list of compounds.

•  Retrospective data analysis is possible to identify new compounds that were not screened for at the time of acquisition.

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The Power of Accurate Mass

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An Example Study

•  To evaluate the performance of Thermo Scientific Q Exactive GC hybrid quadrupole-Orbitrap mass spectrometer for the reliable screening of GC amenable pesticides.

•  To screen for a wide range of pesticides in different sample matrices with the highest level of confidence possible.

•  To determine if a pesticide is present in a sample above the MRL which is typically 10 ng/g (ppb).

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Experimental •  Sample introduction was performed using a Thermo Scientific™ TriPlus™ RSH

autosampler, and chromatographic separation was obtained with a Thermo Scientific™ TRACE™ 1310 GC. Thermo Scientific™ TraceGOLD TG-5SilMS 15 m x 0.25 mm I.D. x 0.25 µm film capillary column.

•  Q Exactive GC hybrid quadrupole-orbitrap mass spectrometer was used. The system was

operated in EI using full scan and 15k, 30k, 60K and 120k resolution (FWHM, m/z 200). Data was acquired with a minimum of 10 points/peak.

•  Data was acquired and processed using the TraceFinder version 3.3 software.

43

RESULTS

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Screening Criteria Used for Positive Identification

Iden%fica%on  Point   Tolerance   Primary  ID   Confirmatory  ID  

Reten%on  %me   20  seconds  

Accurate  Mass   2  ppm  

Fragment  ions   2  ppm  

Isotopic  pa8ern   >70%  

NIST  Library  match   >600  

Ion  ra%o   30%  

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TraceFinder Screening Browser Positively Identified Pesticides

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Sensitivity: Wheat

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Sensitivity: Horse Feed

48

D:\Dom Data\...\21jan_038 01/22/15 03:37:38

21jan_026 #3957 RT: 5.78 AV: 1 NL: 6.28E6T: FTMS + p EI Full ms [50.00-500.00]

127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034m/z

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127.02067

21jan_030 #2240 RT: 5.78 AV: 1 NL: 2.39E6T: FTMS + p EI Full ms [50.00-500.00]

127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034m/z

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127.02164127.01821

21jan_034 #1192 RT: 5.79 AV: 1 NL: 3.61E6T: FTMS + p EI Full lock ms [50.00-500.00]

127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034m/z

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127.02117127.01826

21jan_038 #623 RT: 5.79 AV: 1 NL: 5.49E6T: FTMS + p EI Full lock ms [50.00-500.00]

127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034m/z

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127.01833

127.02118

127.02261

30K  

60K  

120K   Chlorpropham   Matrix  

Mass  difference  =  18.4  ppm  

Mass  difference  =  0.9  ppm  

Mass  difference  =  0.5  ppm  

15K  

Mass  difference  =  0  ppm  

Effect of Resolving Power on Mass Accuracy Chlorpropham in Leek (10 ng/g)

49

Scan Speed and Accurate Mass Across Peaks 60K

•  XIC  of  diazinon(m/z  179.11789  ±5  ppm  mass  window)  in  wheat  at  10  ng/g  showing  ~11  scans/peak  (peak  width  1.8  sec).

Average = 0.33 ppm RMS

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Maintaining Sensitivity with Resolution

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Linearity

•  XIC (quan and confirm ions) and calibration curve for Fenpropimorph in leek.

•  Triplicate injections of the calibration series was performed with good linearity across (0.5 – 50 ng/g).

•  No internal standard correction.

R2 = 0.9999

52

Conclusions

•  Careful method optimisation focussing on the injection parameters.

•  Routine pesticides analysis with the EVO offers sensitivity, high analysis speed and easy database management at low cost

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

•  Q Exactive GC system improves efficiency by increasing the scope of the analysis:

•  Full scan non-targeted acquisition. •  Provides the required sensitivity and selectivity in complex matrices for routine

pesticide screening and quantification. •  Enables the detection and identification of unknown compounds.

Efficient and robust pesticide analysis can be achieved by:

53

Thermo Scientific Food and Environmental Communities: Resources •  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

54

Thank You for Listening

Questions?

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