Use of APGC coupled to Tandem Quadrupole Mass Spectrometry for the analysis of Pesticides and Dioxins - Waters Corporation Food Safety

©2015 Waters Corporation 1

Ultra-Trace Level Quantitative Analysis of Food

Contaminants using LC and GC coupled to MS


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


Residue & contaminant analysis challenges?

Matrix & analyte

complexity

Sample type & extract

preparation

Ease of use & implementation

Compliance with regulatory

performance criteria

Validation & AQC

requirements


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

BrBr

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

FFOH

Br

Br

Br

Br

Br

Br

Cl

Cl

OCl

Cl

Cl Cl

http://10.2.240.144/cgi-bin/ImageFolio31/imageFolio.cgi?direct=MS_Technologies/AutoSpec_Premier/AutoSpec_Premier_PHOTOS&img=16


LC and GC Source Interfaces and

Options


The Universal Source – configurations

and options

LC


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


APGC coupled to Xevo TQ-S

- enhanced sensitivity tandem quadrupole-

MS


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


Ele

ctr

ic F

ield

Diffuse Ion

Cloud

Maximising signal

Maximising robustness

Designed to deal with problems associated with a larger sampling orifice

Compact ion cloud


LC and GC on one instrument Sensitive and robust

GC

LC


LC and GC on one instrument Is it possible?

ESI


LC and GC on one instrument Is it possible?

APGC


Atmospheric Pressure Gas Chromatography

(APGC)

How does it work?


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


Source and Ion Chamber


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


Slide the GC


Ready for GC/MS


Mass Spec

Corona Pin

Atmospheric Region

Heated Transfer Line

Capillary GC Column

Ionization Chamber

APGC Source Schematic


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


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


APGC Ionisation Modes & Advantages


Mechanism of Ionisation (I)

N2+●

N2 e-

2e-

2N2

N4+● M●+

M Corona Pin

M●+

M

Charge Transfer

“Dry” source conditions

Favored by relatively non-polar compounds


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

©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

6718.75

0.01µg/ml BF Std

Time8.00 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


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 ✘ ✔


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


NIST EI Spectrum

APGC Spectrum

Endosulphan

M+.

The challenge...


Strategy for analysis of LC & GC amenable pesticides


SPE for GC-MS Tea Analysis


“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%)


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


% Recovery

%

Freq

uen

cy

% % %

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


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


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


APGC linearity Azinphos-methyl in strawberry extract, triplicate injection internally standardised

R2 = 0.9975


APGC – Repeatability in matrix Matrix matched standards at 1 μg/kg n=10


Dioxins & furans

EU legislation revision for screening &

confirmation June 2014


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


Analytical criteria for use of MS/MS for determination of dioxin-like PCBs in feed & food

NEW EU LEGISLATION 02 June 2014


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


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

13C-2378-TCDD 5

Sensitivity - TCDD mix


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


1613 CSL 10-fold dilution 0.01 ng/mL

S/N ≈ 20

Precision - TCDD mix


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

Re

sp

on

se

-0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0


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

TCDF

1237

8 PCDF

2347

8-PC

DF

1234

78 H

xCDF

1236

78 H

xCDF

2346

78 H

xCDF

1237

89 H

xCDF

1234

678 HpC

DF

1234

789 HpC

DF

OCDF

TCDD

PCDD

1234

78 H

xCDD

1236

78 H

xCDD

1237

89 H

xCDD

1234

678 HpC

DD

OCDD

APGC-MS/MS Consensus PT

Fish meal

0.00

0.50

1.00

1.50

2.00

2.50

TCDF

1237

8 PCDF

2347

8-PC

DF

1234

78 H

xCDF

1236

78 H

xCDF

2346

78 H

xCDF

1237

89 H

xCDF

1234

678 HpC

DF

1234

789 HpC

DF

OCDF

TCDD

PCDD

1234

78 H

xCDD

1236

78 H

xCDD

1237

89 H

xCDD

1234

678 HpC

DD

OCDD

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

TCDF

1237

8 PCDF

2347

8-PC

DF

1234

78 H

xCDF

1236

78 H

xCDF

2346

78 H

xCDF

1237

89 H

xCDF

1234

678 HpC

DF

1234

789 HpC

DF

OCDF

TCDD

PCDD

1234

78 H

xCDD

1236

78 H

xCDD

1237

89 H

xCDD

1234

678 HpC

DD

OCDD

APGC-MS/MS Consensus PT

EU NRL comparison between Consensus PT data and APGC


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%


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!


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

Science

Use of APGC coupled to Tandem Quadrupole Mass Spectrometry for the analysis of Pesticides and Dioxins - Waters Corporation Food Safety