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Determination of Dioxins and PCBs in Feed and
Food using APGC-MS/MS
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
APGC Source
APGC
Ionization
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
E1L E1L E2L E2L E3L E3L E4L E4L E5L E5L
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
E1L E1L E2L E2L E3L E3L E4L E4L E5L E5L
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
F1:MRM of 8 channels,AP+
332 > 268
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
2.052e+00619.1087639.6
22113.5
min
%
0
100
F1:MRM of 8 channels,AP+
322 > 259
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
1.061e+004
471.0
1.2 +11.7%
107.6
18.83 19.79
min
%
0
100
F1:MRM of 8 channels,AP+
320 > 257
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
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
F3:MRM of 8 channels,AP+
388 > 324
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
2.780e+00624.5124.42 59488.2
1.5 -0.2%
5483.225.69
min
%
0
100
F3:MRM of 8 channels,AP+
386 > 322
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
4.179e+00624.5124.4292021.4
9897.425.69
min
%
0
100
F3:MRM of 8 channels,AP+
376 > 313
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
1.375e+00424.43 24.51388.3
1.4 -12.7%
131.9
25.70
min
%
0
100
F3:MRM of 8 channels,AP+
374 > 311
14MAR12_008 Smooth(Mn,1x1)
12.5 fg/µL TCDD 1/5 E1L
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
F1:MRM of 8 channels,AP+
334>270
14MAR12_010 Smooth(Mn,1x1)
pork sausage PT09
8.358e+005
36773.5
1.0 -2.5%
8542.019.11
min
%
0
100
F1:MRM of 8 channels,AP+
332 > 268
14MAR12_010 Smooth(Mn,1x1)
pork sausage PT09
8.567e+00537289.4
9334.319.11
min
%
0
100
F1:MRM of 8 channels,AP+
322 > 259
14MAR12_010 Smooth(Mn,1x1)
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
F1:MRM of 8 channels,AP+
320 > 257
14MAR12_010 Smooth(Mn,1x1)
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?