determination of dioxins and pcbs in feed and food using ... · sample extracts and standards...

Determination of Dioxins and PCBs in Feed and

Food using APGC-MS/MS

[email protected]

RIKILT- Institute of Food Safety, Wageningen, The Netherlands

RIKILT

Mission

RIKILT contributes to the safety and health of the food supply

Running projects on Dioxins and PCBs

Monitoring animal feed, primary agricultural products and fish

Monitoring hot spots

Monitoring free range eggs

Carry-over experiments

● Feed animal products

Quality assurance of external Labs (PTs, Audits)

Training

External customers

Database (within EURL/NRL network)

Method development in close cooperation with EURL and NRLs

Initial start of MS-MS project

Jef Focant

Difference 2001-2003

EU project dedicated to alternative techniques for dioxin analysis such as:

• Bio-assay • Elisa • GC-MS

• single quad • Ion Trap

• GC-ECD • GC x GC –ECD

• Overall conclusions in 2003

• CALUX ok • GC-HRMS only tool for analysis

EU-Reference Laboratories (EU-RL)

National Reference Laboratories (NRL)

Routine Field Laboratories (RFL)

EU food safety monitoring system is based on a network of laboratories

EU Reference Laboratories

The overall objective of the EURLs and NRLs is to improve the quality, accuracy and comparability of the results at official control laboratories.

Activities of EURL/NRL network

Activities past and present via core working group e.g:

● Description of CEN standard for the determination of dioxins

and DL-PCBs in feed (EN 162150)

● Criteria for Bio-assays

● Possible use of new MS techniques

● Measurement uncertainty

● Database related to dioxin en PCB patterns

EU regulations on analytical criteria 2001 –mid 2014

Methods of analysis

Screening methods:

Selection of samples exceeding maximum or action levels

cost-effective high sample-throughput, aim to avoid false- compliant results

e.g. GC-MS methods, bio -analytical methods

Confirmation of results in samples with significant level (above ML) with a confirmatory method

Confirmatory methods:

Determination of low background levels

Identification and quantification of PCDD/Fs and DL-PCBs at level of interest

GC/HRMS methods

Application of MS/MS

Several articles (e.g EU project Difference) up till 2011 showed :

● lack of sensitivity of GC-MS/MS in comparison to GC-HRMS (food, feed)

● Possible use of GC-MS/MS as screening method

● Until mid 2014 GC-HRMS still to be used as confirmatory method

Application of MS/MS

Recent developments showed that sensitivity has been significantly improved:

● Comparison of GC-MS/MS and GC-HRMS (food, feed) Peter Furst et al. 2011, Ingelido et al. 2011, Kotz et al. 2011

● Application notes from different vendors

APGC Source

Ionization

http://www.crl-freiburg.eu/dioxin/index.html

Ionization

Benefits of APGC

SOFT IONIZATION SOURCE techniques in GC overcomes extensive fragmentation

Source (APGC) can be coupled to triple quad

Compared to an EI source; M+· and/or MH+ is often the base peak of the spectrum and fragmentation is low

Resulting in high sensitivity comparable to GC-HRMS

First test of the APGC

EURL-RIKILT-WATERS

Sample previous used by the EURL in PTs were again

extracted and purified by EURL

Standards and prepared extracts were analysed during

a two day visit at Waters

GC parameters

Column DB-5MS UI 60 m x 0.25 mm, 0.25 µm (Agilent)

Carrier gas Helium

Injector mode Pulsed Splitless

Injector liner Deactivated single gooseneck splitless liner, 4mm

Column pneumatics Constant flow

Injection volume (µL) 1

Column flow (mL/min) 2.0

Injector temperature (°C) 280

Pulse Time (min) 2

Pulse Pressure (kPa) 450

Purge time (min) 2

Purge flow (mL/min) 80

Rate (°C/min) Temperature (°C) Hold Time (min) Total Time (min)

- 140 2 -

50 200 - -

4 260 - -

6.5 300 10 34.25

GC parameter

GC oven program

MS parameters

Ionisation API+

Source Dry N2

Source temperature

(°C) 150

Corona (µA) 2.0

Sampling cone (V) 30

Cycle time ca. 0.5 s

Dwell time 0.058 s (function TCDD/F -

HpCDD/F)

0.079 s (function OCDD/F)

Compound

Precursor

Ion

Product

Ion

Collision Energy

(eV)

Precursor

Ion

Product

Ion

Collision Energy

(eV)

TCDF 304 241 40 306 243 40

TCDF C13 316 252 40 318 254 40

TCDD 320 257 30 322 259 30

TCDD C13 332 268 30 334 270 30

PCDF 338 275 40 340 277 40

PCDF C13 350 286 40 352 288 40

PCDD 354 291 30 356 293 30

PCDD C13 366 302 30 368 304 30

HxCDF 374 311 40 376 313 40

HxCDF C13 386 322 40 388 324 40

HxCDD 390 327 30 392 329 30

HxCDD C13 402 338 30 404 340 30

HpCDF 408 345 40 410 347 40

HpCDF C13 420 356 40 422 358 40

HpCDD 424 361 30 426 363 30

HpCDD C13 436 372 30 438 374 30

OCDF 442 379 40 444 381 40

OCDD C13 470 406 30 472 408 30

OCDD 458 395 30 460 397 30

MS parameters Multiple Reaction Monitoring (MRM) Conditions


Sample extracts and standards

Calibration standard 2,3,7,8-TCDD concentration

[pg/µl]

E1L 1:5 12.5

E1L 1:2 31.25

E1L 62.5

E2L 125

E3L 250

E4L 1000

E5L 2500

Sample extracts

Grass meal

Fish meal

Fish (Salmon filet)

Fuller Earth

Pork sausage

Fish oil A

Normal clean-up (GPC; silica, florisil, carbon column)

Fish oil B

Reduced clean-up (without carbon column)

Fish oil C

Reduced clean-up (without carbon and florisil column)

Injection of 1 µl splitless (equivalent to 5 µl PTV)

Calibration Relative response factors

Relative response factor E1L 1:5 - E5L

0

0.2

0.4

0.6

0.8

1

1.2

1.4

E1L

1:5

E1L

1:5

E1L

1:2

E1L

1:2

E1L E1L E2L E2L E3L E3L E4L E4L E5L E5L

RR

F

Realtive response factor E1L - E5L

0

0.2

0.4

0.6

0.8

1

1.2


RR

F

Relative response factors for all 17 congeners E1L 1:5 - E5L

CV: 6 – 16 % E1L - E5L

CV: 5 – 13 %

Calibration Ion abundance ratio

Ion abundance ratio E1L 1:5 - E5L

CV: 1.5 – 8 %

Ion Ratio (Quan Ion / Target Ion)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

E1L

1:5

E1L

1:5

E1L

1:2

E1L

1:2


min18.20 18.30 18.40 18.50 18.60 18.70 18.80 18.90 19.00 19.10 19.20 19.30 19.40 19.50 19.60 19.70 19.80 19.90

%

0

100

F1:MRM of 8 channels,AP+

334>270

14MAR12_008 Smooth(Mn,1x1)

12.5 fg/µL TCDD 1/5 E1L

1.940e+00619.10

84867.3

1.0 -0.7%

30269.9

min

%

0

100


332 > 268



2.052e+00619.1087639.6

22113.5

min

%

0

100


322 > 259



1.061e+004

471.0

1.2 +11.7%

107.6

18.83 19.79

min

%

0

100


320 > 257



1.055e+004542.1

90.5

18.9018.32 18.8418.49

Calibration Chromatograms

TCDD 12.5 fg on column

min24.30 24.40 24.50 24.60 24.70 24.80 24.90 25.00 25.10 25.20 25.30 25.40 25.50 25.60 25.70 25.80

%

0

100


388 > 324



2.780e+00624.5124.42 59488.2

1.5 -0.2%

5483.225.69

min

%

0

100


386 > 322



4.179e+00624.5124.4292021.4

9897.425.69

min

%

0

100


376 > 313



1.375e+00424.43 24.51388.3

1.4 -12.7%

131.9

25.70

min

%

0

100


374 > 311



2.173e+00424.5124.43525.5

131.6

25.70

Calibration Chromatograms

2,3,4,6,7,8-HxCDF 12.5 fg on column

Sample extracts Chromatogram

min16.20 16.40 16.60 16.80 17.00 17.20 17.40 17.60 17.80 18.00 18.20 18.40 18.60 18.80 19.00 19.20 19.40 19.60 19.80

%

0

100


334>270


pork sausage PT09

8.358e+005

36773.5

1.0 -2.5%

8542.019.11

min

%

0

100


332 > 268


pork sausage PT09

8.567e+00537289.4

9334.319.11

min

%

0

100


322 > 259


pork sausage PT09

1.430e+004

645.8

1.2 +13.6%

93.7

17.67

17.4117.83 19.2818.5318.4818.2318.02 18.58 18.92 19.65 19.81

min

%

0

100


320 > 257


pork sausage PT09

1.598e+004755.5

121.5

17.6616.5516.18 17.43

16.8817.83

18.4918.2218.08 18.68 19.2619.0518.91 19.67

TCDD 36 fg on column

Calibration

Stability in sequence

Comparison of 13

C-peak area in calibration

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

2 3 4 5 6 8 9 25 26 27 28 29 30 31

Position in sequence

Are

a Q

uan

Io

n

Stability of response of the system: Comparison of absolute peak areas of the

13C-labeled standards in all calibration standards in a complete sequence of samples

Range of CV was between 8 and 20 %, without the calibration point on position 29 (Cal3)


Sample extracts Comparsion of WHO-PCDD/F-TEQ

Comparison of WHO-PCDD/F-TEQ

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Pork

sausage

Fullers

Earth

Fish Fish meal Grass

meal

Fish oil A Fish oil B Fish oil C

pg

/g f

at/

fresh

weig

ht,

ng

/kg

88%

dry

weig

ht

APGC-MS/MS Consensus

Sample type Deviation of

APGC-MS/MS

from consensus

Grass meal 4.3 %

Fish meal - 3.3 %

Fish (Salmon filet) 17.9 %

Pork sausage 2.4 %

Fish oil A 11.5 %

Fish oil B 10.8 %

Fish oil C 136 %


EU legislation from mid 2014

Monitoring of at least 2 specific precursor ions, each with one specific corresponding transition product ion for all labelled and unlabelled analytes (comparable to GC-HRMS)

Maximum permitted tolerance of relative ion intensities of ± 15% for selected transition product ions in comparison to calculated or measured values (average from calibration standards)

Resolution for each quadrupole equal to or better than unit mass resolution (unit mass resolution)

Further criteria as described, for example, in standard EN 16215 and/or in EPA methods 1613 and 1668 as revised, except the obligation to use GC-HRMS have to be followed

Pesticides

APGC full scan 100 pg standard on column

Conclusions of Tania Portolés Nicolau

GC-APCI- (QqQ)MSMS presents good analytical characteristics regarding linearity,

precision and limits of detection for determination of target residues in food,

environment and biofluids

APGC installed at RIKILT on 25-08-2014

First results (Friday 29-08)

20 fg on column

Acknowledgment

Colleagues at both RIKILT and EURL for preparation of samples and support

Rainer Malisch, Alexander Kotz and Helmut Winterthaler (EURL, Freiburg, Germany) as well as colleagues in the WG

Bert van Bavel and co-workers (University of Orebro, Sweden)

Jody Dunstan (Waters, Manchester, UK)

Wim Broer and Jeroen Markesteijn(Nofalab, Schiedam, The Netherlands)

Thank you for

your attention

Questions?

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