Chromatography: Pesticide Residue Analysis Webinar Series: Part 3 of 4: Maximizing Analysis Efficiency through GC-MS Approaches

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

2
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

3
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

4
•  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
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
6.32
6.59
4.63
7.00 11.13
10.12
11.99
25.20
14.969.50 23.13 29.61
17.38
8.89
12.207.31
5.77 19.1315.57 15.84
8.20
26.4712.87 17.95
13.77 23.58
28.7125.3620.625.09
21.70
23.8319.25
27.77
24.12
35.78
34.8134.3932.92
30.11 31.00
NL:
3.94E8
TIC MS
2july2104_0
11

5
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

6
Pesticide Analysis: Triple Quad GC-MS
Highly Selective Reaction Monitoring (SRM)
 Improved detection limits
 Longer column lifetime
 Less frequent inlet maintenance

7
Selectivity: Selected Ion Monitoring (SIM) and SRM
DDE-p,p’, 0.05 mg/kg in green tea, 1.0 uL splitless injection

8
Selectivity: SIM and SRM
DDE-p,p’, 0.001 mg/kg in green tea, 1.0 uL splitless injection

9
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Key Factors in the GC-MS/MS Method

10
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)

11
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)

12
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....

13
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

14
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

15
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

16
Backflush Injection
(Thermo GCQ Quantum)
Pre-column (2 m x 0.53 mm i.d., deactivated)

17
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
6 8 10 12 14 16 18 20 22 24
Time (min)
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
Extract
BKF at 20 min (No BKF) Vitamine E
Sitosterol (area)
Octadecanoic Acid 14.68
14.19
21.1513.39
19.9017.707.64
21.47
18.30
11.76
9.738.15 16.52
12.64 22.037.42 16.868.72 22.1715.38
19.45
5.81 11.23 22.516.74 23.47
Extract
BKF at 10 min
Vitamine E
Sitosterol (area)
Octadecanoic Acid
14.20
13.78
13.04
7.65 19.08
11.61
9.739.38 17.00
12.38
7.42 15.93
8.72
5.83 6.88 15.2711.15 17.59
19.24 19.98 23.0520.84 23.69
Standard Mix
BKF at 10 min
Pesticides
14.88
15.9712.95
13.72
16.78
12.32
14.51
17.07
17.827.29 7.80 10.41 11.539.08 18.44
11.29
18.65
7.18 8.39 21.30 23.6520.799.266.45 21.44
NL: 5.69E9
TIC MS
xc21_estratto_p
pbmixpestethac
20ul_ptvbkf_02m
y_85cto260ptv_r
NL: 5.12E9
TIC MS
XC21_EstrattoP
mandi_120ulEtA
_PTVBKF_Clean
n
NL: 2.65E9
TIC MS
xc21_400ppbmix
rmandi_120uleta
_ptvbkf_clean10
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

18
Effect of Back-Flush on Carryover
RT: 0.00 - 35.10 SM: 7G
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
29.45 30.21
33.99
32.4129.14
28.70
28.45
27.99
27.25
26.98
26.76
26.26
26.04
25.30
24.84
24.49
14.2510.44 23.7122.359.10
8.80 10.79 21.62
7.90 19.7112.64 18.9217.60
12.17
20.71
12.64
28.23
29.22 33.7932.99
27.0310.17
13.3810.07 11.52
14.219.32
9.05
8.64
7.90 14.92 24.96
16.62 17.46 24.34
18.11 21.46
NL:
2.50E5
TIC MS
51845023
NL:
2.50E5
TIC MS
51845038
Fresh Podded Peas
RT: 0.00 - 35.10 SM: 7G
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
28.88
29.59
28.66
27.58
30.12
27.34
30.98
32.1027.02
33.34
26.49
34.95
26.25
25.73
24.98
24.8014.25
24.53
24.09
9.02 23.839.35
7.39 23.1710.32
11.84 20.5619.40
12.068.64 19.01
17.23
16.11
12.62 23.11
22.92
30.79
31.70
14.06
32.34
27.50 32.79
27.18
21.99
12.08
32.97
9.63
9.34
8.75 11.86 25.75
20.30 25.45
14.59 20.187.86
15.05 16.89 18.60
NL:
2.00E5
TIC MS
51865013
NL:
2.00E5
TIC 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

19
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

21
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

22
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

23
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

24
Increasing Laboratory Productivity
•  Decrease the analysis time by shortening the GC run times.
•  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

25
Decrease the Analysis Time
RT: 4.57 - 37.12
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
6.32
6.59
4.63
7.00 11.13
10.12
11.99
25.20
14.969.50 23.13 29.61
17.38
8.89
12.207.31
5.77 19.1315.57 15.84
8.20
26.4712.87 17.95
13.77 23.58
28.7125.3620.625.09
21.70
23.8319.25
27.77
24.12
35.78
34.8134.3932.92
30.11 31.00
NL:
3.94E8
TIC MS
2july2104_0
11
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

26
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 10.5
Time (min)
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
8.73
8.26
8.95
7.68
7.65 8.45
8.00
8.788.23
8.50
7.80
8.67
7.40
6.03 7.46
7.036.63
7.25
7.026.61
9.306.826.39
7.20
9.026.17 8.02
10.349.21
6.35 8.036.57
5.884.65 9.60
9.944.89
9.44
5.42
4.72 9.61
9.985.24
5.21 10.5710.04
4.09 9.73 10.175.85 10.675.504.20 5.684.27 5.11
NL:
1.39E9
TIC MS
2july2104_0
48
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

27
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.

28
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

29
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

30
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

31
Increase Laboratory Productivity
•  Decrease the analysis time by shortening the GC run times.
•  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

32
More SRM Transitions/Compound for More
Confidence
Tecnazene in baby food at 0.01 mg/kg level

33
RT: 14.95 - 16.71
15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
RT: 15.79
AA: 4093580
SN: INF
RT: 15.79
AA: 1901556
SN: INF
NL: 2.60E6
TIC F: + c EI SRM
ms2
246.000@cid28.00
[176.095-176.105]
MS Genesis
19May2014_03
NL: 1.21E6
TIC 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 16.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
RT: 15.79
AA: 3837740
RT: 15.79
AA: 1690756
NL: 2.35E6
TIC F: + c EI SRM
ms2
246.000@cid28.00
[176.095-176.105]
MS Genesis
19May2014_05
NL: 1.03E6
TIC 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 ms27 ms

34
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

36
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

39
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.

41
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).

42
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.

44
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%

45
TraceFinder Screening Browser Positively Identified
Pesticides

48
D:Dom Data...21jan_038 01/22/15 03:37:38
21jan_026 #3957 RT: 5.78 AV: 1 NL: 6.28E6
T: FTMS + p EI Full ms [50.00-500.00]
127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034
m/z
0
20
40
60
80
100
RelativeAbundance
127.02067
21jan_030 #2240 RT: 5.78 AV: 1 NL: 2.39E6
T: FTMS + p EI Full ms [50.00-500.00]
127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034
m/z
0
20
40
60
80
100
RelativeAbundance
127.02164
127.01821
21jan_034 #1192 RT: 5.79 AV: 1 NL: 3.61E6
T: FTMS + p EI Full lock ms [50.00-500.00]
127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034
m/z
0
20
40
60
80
100
RelativeAbundance
127.02117127.01826
21jan_038 #623 RT: 5.79 AV: 1 NL: 5.49E6
T: FTMS + p EI Full lock ms [50.00-500.00]
127.008 127.010 127.012 127.014 127.016 127.018 127.020 127.022 127.024 127.026 127.028 127.030 127.032 127.034
m/z
0
20
40
60
80
100
RelativeAbundance
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

50
Maintaining Sensitivity with Resolution

51
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
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Chromatography: Pesticide Residue Analysis Webinar Series: Part 3 of 4: Maximizing Analysis Efficiency through GC-MS Approaches

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Chromatography: Pesticide Residue Analysis Webinar Series: Part 3 of 4: Maximizing Analysis Efficiency through GC-MS Approaches