This presentation describes the operation and application of the Waters APGC (Atmospheric Pressure Gas Chromatography) ion source which provides a highly sensitive GC-MS, MS/MS capability for tandem quadrupole and time of flight MS systems. It is very easy to swap between APGC, Electrospray (for UPLC) and other ion sources without instrument venting in minutes. APGC provides significant performance advantages over traditional GC/MS ionisation methods, giving high sensitivity and less fragmented spectra.

1. ©2015 Waters Corporation 1
Ultra-Trace Level Quantitative Analysis of Food
Contaminants using LC and GC coupled to MS

2. ©2015 Waters Corporation 2
Overview
 Ultra-trace residue analysis in food
– What are the challenges?
 Persistent organic pollutants in foods
 LC & GC source interfaces & options
– Universal source interface
– Introduction to the Xevo-TQ-S
 Principles of APGC
– How does it work?
– Multi-residue pesticide analysis (LC & GC amenable)
– Commission Regulation 589/2014
– APGC for dioxins
 Summary and conclusions

3. ©2015 Waters Corporation 3
Residue & contaminant analysis challenges?
Matrix &
analyte
complexity
Sample type &
extract
preparation
Ease of use &
implementation
Compliance with
regulatory
performance
criteria
Validation &
AQC
requirements

4. ©2015 Waters Corporation 4
Analysis of POPs
Requirement for both LC & GC
 Stockholm convention
– Global agreement to reduce environmental POPs levels
– 12 classes of compounds listed
– Persistent environmental pollutants
 Dioxins: HR-GC-MS (magnetic sector)
 BFRs: PBDEs: HR-GC-MS or GC-MS/MS
 PFCs: LC-MS/MS
 Endocrine disruptors: LC-MS/MS & GC-MS/MS
 Nitrosamines: GC-MS/MS
 Legacy pesticides: LC-MS/MS & GC-MS/MS
O
BrBr
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
O
O
Cl
Cl
F
F
F
F
F
F
F
F
F
S
O
OF F
F
F
F
F
FF OH
Br
Br
Br
Br
Br
Br
Cl
Cl
O
Cl
Cl
Cl Cl

5. ©2015 Waters Corporation 5
LC and GC Source Interfaces and
Options

6. ©2015 Waters Corporation 6
The Universal Source – configurations
and options
LC

7. ©2015 Waters Corporation 7
APGC Overview
 Waters APGC is an optional ion source for Xevo systems that provides
a highly sensitive GC-MS, MS/MS capability
 Very easy to swap between APGC, UPLC, other ion sources without
instrument venting in minutes
 APGC ionisation is soft (cf. APCI) and molecular ions are readily
detected
 Fragmentation can be induced (CID) to provide information for
structural elucidation
 Xevo tandem quad (Xevo TQ-S) is a powerful tool for routine
quantitation coupled to both LC & GC
– Capability of using the RADAR for exploratory experiments

8. ©2015 Waters Corporation 8
APGC coupled to Xevo TQ-S
- enhanced sensitivity tandem quadrupole-
MS

9. ©2015 Waters Corporation 9
Xevo TQ
Xevo TQ-S
StepWave
ScanWave
(Collision cell)
How did we increase sensitivity?
Hexapole
(Transfer Optics)
1st Quadrupole
(Precursor ion selection)
2nd Quadrupole
(Product ion selection)
Larger sampling cone aperture = 200x increase in ion flux

10. ©2015 Waters Corporation 10
ElectricField
Diffuse
Ion
Cloud
Maximising signal
Maximising robustness
Designed to deal with problems
associated with a larger sampling orifice
Compact
ion cloud

11. ©2015 Waters Corporation 11
LC and GC on one instrument
Sensitive and robust
GC
LC

12. ©2015 Waters Corporation 12
LC and GC on one instrument
Is it possible?
ESI

13. ©2015 Waters Corporation 13
LC and GC on one instrument
Is it possible?
APGC

14. ©2015 Waters Corporation 14
Atmospheric Pressure Gas Chromatography
(APGC)
How does it work?

15. ©2015 Waters Corporation 15
Universal Source
ESI and APGC modifications fitted
Sampling
cone
Electrospray Configuration
Vial containing
modifier
Ion chamber &
corona pin
APGC Configuration
APGC source door
Entrance for transfer line

16. ©2015 Waters Corporation 16
Source and Ion Chamber

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Remove the source housing
Plinth mounting – helps
ensure correct alignment of
the transfer line to the source
GC 7890
Exchange between LC and GC
No venting required

18. ©2015 Waters Corporation 18
Slide the GC

19. ©2015 Waters Corporation 19
Ready for GC/MS

20. ©2015 Waters Corporation 20
Mass Spec
Corona Pin
Atmospheric
Region
Heated Transfer Line
Capillary
GC Column
Ionization
Chamber
APGC Source Schematic

21. ©2015 Waters Corporation 21
Corona Pin
Creates
corona
discharge at
the needle
Atmospheric Pressure Source
Capillary
GC Column
Ionization Chamber
Ionisation occurs either by charge transfer
or proton transfer depending on the
conditions in the source
Heated Transfer Line
Transfer Line GC
Make up (sheath) gas
N2 250 ml/min
together with cone gas
supplies nitrogen for
the plasma
Mass
Spectrometer
APGC – How it works

22. ©2015 Waters Corporation 22
Mass Analyser GC Oven
Corona discharge
at needle creates plasma
N2 make-up
(250ml/min) gas
delivered
through transfer line
interior
N2 meets GC eluent flow
at transfer line tip
Analyte Molecules are ionised after
GC elution and directed to the mass analyser
APGC – How it works

23. ©2015 Waters Corporation 23
APGC Ionisation Modes & Advantages

24. ©2015 Waters Corporation 24
Mechanism of Ionisation (I)
N2
+●
N2e-
2e-
2N2
N4
+● M●+
M
Corona Pin
M●+
M
Charge Transfer
 “Dry” source conditions
 Favored by relatively non-polar compounds

25. ©2015 Waters Corporation 25
Mechanism of Ionisation (II)
N2
+●
N4
+●
H2O
H2O+●
H2O
H3O+●
+OH●
[M+H]+
M
Protonation
 Modified source conditions eg. with water or methanol present
 Favored by relatively polar compounds
Corona Pin

26. ©2015 Waters Corporation 26
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00
%
0
100
ANAPGC240409TEST009 TOF MS AP+
278.025 0.02Da
671
8.75
0.01µg/ml BF Std
Time
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
%
0
100
CSL_200306_204 TOF MS EI+
277.018 0.02Da
298
15.98
EI GC
5uL Injection
APGC
1uL Injection
298cps
EI+
671cps
AP+
Faster run possible due
to atm pressure source
and higher res MS
15.98
8.75
~ 10X higher
response for APGC
Chromatographic Performance
Flow rate

27. ©2015 Waters Corporation 27
EI GC/MS compared to APGC-MS
Parameter GC/MS APGC
Linearity ✔ ✔
Sensitivity ✔ ✔
NIST Library Spectra ✔ ✘
Ease of Use ✘ ✔
Matrix Tolerance ✘ ✔
Low fragmentation spectra ✘ ✔
Access to latest MS innovations ✘ ✔

28. ©2015 Waters Corporation 28
APGC-TQ-S
Complete solution for targeted multi-class
pesticide residue analysis
Circa 60% of pesticides tested in EU require LC-MS &
40% require GC-MS

29. ©2015 Waters Corporation 29
NIST EI
Spectrum
APGC
Spectrum
Endosulphan
M+.
The challenge...

30. ©2015 Waters Corporation 30
Strategy for analysis of LC & GC
amenable pesticides

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SPE for GC-MS Tea Analysis

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“Difficult” pesticide comparison
EI-GC & APGC
tR Compounds Molecular formula M EI APGC APGC+H2O
M•+ M•+ MH+ MH+
4.70 Dichlorvos C4H7Cl2O4P 220 +++ +++
5.97 Mevinphos C7H13O6P 224 ++ + ++
6.96 Molinate C9H17NOS 187 + +++ +++
8.00 Dicrotophos C8H16NO5P 237 + ++ ++ +++
8.24 Monocrotophos C7H14NO5P 223 + ++ ++ +++
8.95 Terbufos C9H21O2PS3 288 +
9.80 Phosphamidon C10H19ClNO5P 299 + ++ +++
9.76 Endosulfanether C9H6Cl6O 340 + + ++ ++
9.94 Chlorpyriphosmethyl C7H7Cl3NO3PS 321 ++ +++
10.77 Chlorpyriphos C9H11Cl3NO3PS 349 + +++ +++
10.85 Aldrin C12H8Cl6 362 + + ++
11.39 Isodrin C12H8Cl6 362 ++ ++ ++
11.56 Chlorfenvinphos C12H14Cl3O4P 358 ++ +++
11.56 Oxychlordane C10H4Cl8O 420 + + ++
11.56 HeptachlorepoxideB C10H5Cl7O 386 + + ++
12.23 EndosulfanI C9H6Cl6O3S 404 ++ ++
12.72 Buprofezin C16H23N3OS 305 + ++ +++ +++
12.73 Dieldrin C12H8Cl6O 378 + ++ ++ ++
13.10 Endrin C12H8Cl6O 378 ++ ++ ++
13.36 Ethion C9H22O4P2S4 384 + +++ +++
14.01 Endosulfansulfate C9H6Cl6O4S 420 ++ ++
15.63 Azinphosmethyl C10H12N3O3PS2 317 +
15.66 Pyriproxyfen C20H19NO3 321 ++ +++
16.04 Fenarimol C17H12Cl2N2O 330 + ++ +++ +++
16.17 Azinphosethyl C12H16N3O3PS2 345 +
+, very small peak; ++, clearly identifiable peak (>20%); +++, base peak (or >80%)

33. ©2015 Waters Corporation 33
142 multi-class method for pesticides in
fruits & vegetables
 QuEChERS including a 10-fold dilution of the final
acetonitrile extract  direct injection splitless
 [M+H]+ precursor ions for most (90%) compounds
 3 SRM transitions /compound
Validation
 Spiked samples 0.01 and 0.1 mg/kg
 Quantification  matrix-matched calibration
 Ion ratio tolerances achieved
Application Of Gas Chromatography-(Triple Quadrupole) Mass Spectrometry With Atmospheric Pressure
Chemical Ionization For The Determination Of Multiclass Pesticides In Fruits And Vegetables, Laura Cherta et
al (2013) Journal of Chromatography A (1292) 132-141

34. ©2015 Waters Corporation 34
% Recovery
%
Frequency
% % %
0
20
40
60
80
100
<70 70-110 110-120 >120
-10
10
30
50
70
90
110
130
<70 70-110 110-120 >120
0.01 mg/kg
0.1 mg/kg
% Recovery
(n=6)
1 pg injected
10 pg injected
Performance Data
Validation 142 pesticides in three different matrices

35. ©2015 Waters Corporation 35
Authors’ Conclusions…
 APCI allows a “universal” soft-ionization for GC-amenable
compounds, which provides more selectivity and sensitivity
due to the selection of the molecular ion/protonated molecule
as precursor ion.
 GC-APCI-(QqQ) MS/MS presents good analytical
characteristics regarding linearity, precision and limits of
detection for determination of target residues in food and
environmental samples.
 The novel APCI source reveals itself as a good
choice to consider in GC-MS/MS for pesticide
residue analysis, as well as a suitable
alternative to HRMS in the dioxins field

36. ©2015 Waters Corporation 36
Waters pesticide GC method
Summary of conditions & performance
Compound
MRM
Quantification
Retention time
(min)
Limit of
detection
(ng/mL)
Correlation
Coefficient (R2)
Aldrin 363>159 13.4 0.5 0.992
Azinphos-Ethyl 289>261 14.2 0.05 0.993
Azinphos-Methyl 261>125 20.0 0.5 0.990
Buprofezin 306>106 15.9 0.05 0.991
Chlorfenvinphos 359>170 14.3 0.05 0.994
Chlorpyriphos 350>198 13.2 0.1 0.995
Chlorpyriphos-Methyl 322>125 12.1 0.05 0.991
Dichlorvos 221>145 6.3 0.01 0.995
Dicrotophos 238>112 9.6 0.05 0.990
Dieldrin 379>325 16.0 0.1 0.995
Endosulfan I 405>323 15.3 0.1 0.992
Endosulfan-Ether 341>205 18.7 0.01 0.995
Endosulfan-Sulphate 323>217 17.7 0.05 0.992
Endrin 379>243 16.5 0.05 0.997
Ethion 385>143 16.8 0.05 0.996
Fenarimol 331>139 20.7 0.1 0.997
Heptachlor Epox B 387>217 17.7 0.1 0.992
Mevinphos 225>127 7.5 0.05 0.995
Phenthoate 321>135 14.4 0.05 0.993
Phosphamidon 300>127 12.0 0.1 0.993
Wet source conditions used [APGC +H2O], internal standard used
Application note reference: 720004776en

37. ©2015 Waters Corporation 37
APGC linearity
Azinphos-methyl in strawberry extract,
triplicate injection internally standardised
R2 = 0.9975

38. ©2015 Waters Corporation 38
APGC – Repeatability in matrix
Matrix matched standards at 1 μg/kg n=10

39. ©2015 Waters Corporation 39
Dioxins & furans
EU legislation revision for screening &
confirmation June 2014

40. ©2015 Waters Corporation 40
 Dioxins & dioxin-like compounds (DLC)
are by-products of various industrial
processes, and are commonly regarded
as highly toxic compounds that
are environmental and persistent
pollutants (POPs)
 Analysis must comply with legislative
requirements. EPA1613 in USA and (EC)
No 152/2009 & 589/2014 in Europe
 ‘Gold Standard’ is magnetic sector MS
(AutoSpec Premier)
 Regulation 589/2014 permits the use of
GC-MS/MS for both screening &
confirmatory purposes
Commission Regulation 589/2014
Screening & confirmatory methods

41. ©2015 Waters Corporation 41
Analytical criteria for use of MS/MS for
determination of dioxin-like PCBs in feed & food
NEW EU LEGISLATION 02 June 2014

42. ©2015 Waters Corporation 42
APGC application for dioxins & furans
 Charge transfer [M+·] precursor ions for all compounds
 2 SRM transitions / compound + labelled standards
Validation
 Analysis of certified reference material
 Quantification using commercial standard solutions

43. ©2015 Waters Corporation 43
1368-TCDD
min. 2 fg
on column
1379-TCDD
1378-TCDD
1478-TCDD
1234-TCDD
2378-TCDD
TF-TCDD-MXB;
Components and Concentrations
(fg/µl, ± 5% in nonane)
Component Conc (fg/µl)
1368-TCDD 2
1379-TCDD 5
1378-TCDD 10
1478-TCDD 25
1234-TCDD 50
2378-TCDD 100
13
C-2378-TCDD 5
Sensitivity - TCDD mix

44. ©2015 Waters Corporation 44
1368-TCDD
min. 2 fg
on column
1379-TCDD
1379-TCDD
1368-TCDD
1378-TCDD
1378-TCDD
1478-TCDD
1478-TCDD
1234-TCDD
1234-TCDD
2378-TCDD
2378-TCDD
GC-HRMS
GC-MS/MS (QqQ)
Sensitivity – comparison to GC-HRMS

45. ©2015 Waters Corporation 45
1613 CSL 10-fold dilution 0.01 ng/mL
S/N ≈ 20
Precision - TCDD mix

46. ©2015 Waters Corporation 46
Quantitative performance
Linearity over range 10 fg > 40 pg on-column
Compound %RSD Calibration
RRF
Coeff. Of
Determination
TCDD 3.3 >0.999
TCDF 7.9 >0.999
PCDD 2.8 >0.999
1,2,3,7,8-PCDF 2.8 >0.999
2,3,4,7,8-PCDF 2.1 >0.999
1,2,3,4,7,8-HxCDD 4 >0.999
1,2,3,6,7,8-HxCDD 4.1 >0.999
1,2,3,7,8,9-HxCDD 3.5 >0.999
1,2,3,4,7,8-HxCDF 6.3 >0.999
1,2,3,6,7,8-HxCDF 5 0.999
2,3,4,6,7,8-HxCDF 3 >0.999
1,2,3,7,8,9-HxCDF 8 0.998
1,2,3,4,6,7,8-HpCDD 5.8 >0.999
1,2,3,4,6,7,8-HpCDF 4 >0.999
1,2,3,4,7,8,9-HpCDF 3.3 >0.999
OCDD 1.8 >0.999
OCDF 8.3 0.998
Compound name: TCDD
Correlation coefficient: r = 0.999887, r^2 = 0.999774
Calibration curve: 1.02895 * x + -0.00220016
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
pg/µL
-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
Response
-0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0

47. ©2015 Waters Corporation 47
APGC-TQ-S comparison with
HR-GC/MS
Fish
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
TCD
F
12378
PCDF
23478-PC
DF
123478
H
xCD
F
123678
H
xCD
F
234678
H
xCD
F
123789
H
xCD
F
1234678
H
pCD
F
1234789
H
pCD
F
O
C
DF
TCD
D
PCDD
123478
H
xCD
D
123678
H
xCD
D
123789
H
xCD
D
1234678
H
pCD
D
O
C
DD
APGC-MS/MS Consensus PT
Fish meal
0.00
0.50
1.00
1.50
2.00
2.50
TCD
F
12378
PCDF
23478-PC
DF
123478
H
xCD
F
123678
H
xCD
F
234678
H
xCD
F
123789
H
xCD
F
1234678
H
pCD
F
1234789
H
pCD
F
O
C
DF
TCD
D
PCDD
123478
H
xCD
D
123678
H
xCD
D
123789
H
xCD
D
1234678
H
pCD
D
O
C
DD
APGC-MS/MS Consensus QC material
Pork sausage
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
TCD
F
12378
PCDF
23478-PC
DF
123478
H
xCD
F
123678
H
xCD
F
234678
H
xCD
F
123789
H
xCD
F
1234678
H
pCD
F
1234789
H
pCD
F
O
C
DF
TCD
D
PCDD
123478
H
xCD
D
123678
H
xCD
D
123789
H
xCD
D
1234678
H
pCD
D
O
C
DD
APGC-MS/MS Consensus PT
EU NRL comparison between
Consensus PT data and APGC

48. ©2015 Waters Corporation 48
Name Std. Conc RT Area ng/mL 1º Ratio (Pred) 1º Ratio (Actual)
1º Ratio
Flag
Standard 0.05 ng/mL 0.05 6.28 124.2 0.05 1.442 1.403 -2.7
Standard 0.1 ng/mL 0.1 6.27 194.2 0.07 1.442 1.4 -2.9
Standard 0.5 ng/mL 0.5 6.27 1183.4 0.44 1.442 1.442 *
Standard 1 ng/mL 1 6.27 2448.1 0.92 1.442 1.561 8.3
Standard 5 ng/mL 5 6.27 12255 4.59 1.442 1.474 2.2
Standard 10 ng/mL 10 6.27 23042.4 8.63 1.442 1.526 5.8
Standard 20 ng/mL 20 6.27 57394.2 21.50 1.442 1.539 6.7
Standard 50 ng/mL 50 6.27 134669.3 50.46 1.442 1.438 -0.3
QC 1 ng/mL 1 6.26 3058.5 1.14 1.442 1.542 6.9
QC 1 ng/mL 1 6.27 2863.5 1.07 1.442 1.493 3.5
QC 1 ng/mL 1 6.27 2737.6 1.02 1.442 1.47 1.9
QC 1 ng/mL 1 6.27 2808.3 1.05 1.442 1.465 1.6
QC 1 ng/mL 1 6.27 2904.4 1.09 1.442 1.422 -1.4
QC 1 ng/mL 1 6.27 525.5 0.20 1.442 1.499 4.0
QC 1 ng/mL 1 6.27 1945.1 0.73 1.442 1.428 -1.0
QC 1 ng/mL 1 6.26 2038 0.76 1.442 1.371 -4.9
QC 1 ng/mL 1 6.27 2207.5 0.83 1.442 1.404 -2.6
QC 1 ng/mL 1 6.27 1247.4 0.47 1.442 1.384 -4.0
APGC-TQ-S ion ratio stability
* - 0.5 ng/µL standard was determined to be definitive ion ratio
Deviation < 8.5%

49. ©2015 Waters Corporation 49
Summary and Conclusions
 For many food safety and environmental applications (e.g.
POPs, Pesticides) both LC & GC techniques are required
– APGC very easy source exchange
– Robust and easy to use
– FID like chromatography performance (gas flow)
 Commission Regulation 589/2014 permits the use of GC-MS/MS
as an acceptable approach for screening & confirmatory
methods of analysis for dioxins (June 2014)
 APGC is highly selective for many GC methods
– Often use “precursor->product” transitions (MRM) from EI-GC
 Sensitivity of Xevo TQ-S in GC or LC is uncompromised
– Very sensitive in both modes!

50. ©2015 Waters Corporation 50
Acknowledgments and Collaborators
APGC-Xevo-TQ-S
 Rainer Malisch, Alexander Kotz, Helmut Winterhalter, EU-RL for
Dioxins in Food and Feed, Germany
 Prof. Bert van Bavel, Ingrid Ericson, MTM Research Centre,
Örebro University, Sweden
 Juan Vi Sancho, Tania Portolés, Félix Hernández, Universitat
Jaume I, Castellón, Spain
 Wim Broer, Nofalab, The Netherlands
 Richard Fussell, Martin Rose, FERA, UK
 Brock G. Chittim, Wellington Labs, Canada
 Petr Kukučka, Recetox, Czech Republic