GC-MS/MS determination of PCDD/Fs and PCBs ... - Thermo Fisher

State Institute for Chemical and

Veterinary Analysis of Food

CVUA Freiburg1 / GC-MS/MS

GC-MS/MS determination

of PCDD/Fs and PCBs in Food and Feed

—

Comparison to GC-HRMS

European Union Reference Laboratory for Dioxins and PCBs in Feed and Food

State Institute for Chemical and Veterinary Analysis of Food (CVUA)

Freiburg, Germany

Alexander Kotz, Helmut Winterhalter and Rainer Malisch

6th Thermo Scientific Symposium on Recent Advances in POPs Analysis

Niagara-on-the-Lake, Canada, April 28-29, 2011




Content

European Union Reference Laboratory (EU-RL)

Criteria for screening and confirmatory methods

GC-MS/MS method for determination of PCDD/Fs

Comparison with GC-HRMS

Modified clean-up




EU-RL for Dioxins and PCBsin Feed and Food

Tasks of EU-RL:

Technical and scientific support to the EU Commission, NRLs

Development and validation of analytical methodology

Co-operation with NRLs

Organisation of workshops

Analyses of official samples, ...

Important contribution of EU-RL, NRLs to EU policy:

Ensure harmonized application of EU legislation for extraction/analysis

Provide independent advice on screening and confirmatory methods

...

State Institute for Chemical and Veterinary Analysis of Food (CVUA) Freiburg designated as Community Reference Laboratory (CRL) for Dioxins and PCBs in Feed and Food(Commission Regulation (EC) No. 776/2002 on May 23, 2006)

After entry into force of Treaty of Lisbon in 2009 change of name to European Union Reference Laboratory (EU-RL)(Commission Regulation (EU) No 208/2011)




Confirmatory and Screening MethodsEU Regulations

Application of methods for analysisCommission Regulation (EC) No 152/2009 (Feed), No 1883/2006 (Food)

Screening methods:

Detect the presence of PCDD/Fs and DL-PCBs at the level of interest, capacity for high

sample throughput, aim to avoid false-compliant results

e.g. GC-MS, GC-MS/MS methods, bioanalytical methods

Confirmation of results in samples with significant level with a confirmatory method

Confirmatory methods:

Methods providing full or complementary information, unequivocally identification and

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

GC/HRMS methods

Screening methods Confirmatory methods

False negative rate < 1 %

Trueness – 20 % to + 20 %

Precision (RSDR) < 30 % < 15 %




Criteria for methodsGC/MS as confirmatory/screening method

Definition of ...

Detection method

Criteria for identification

Working range / limit of quantification




Detection methods

Identification requirements for different types of mass spectrometers

System of identification points (Commission Decision 2002/657/EC)

Available methods (SANCO/10684/2009, pesticide residue analysis):

MS mode Single MS (standard mass resolution) Single MS (high

resolution/high mass

accuracy)

MS/MS

Typical systems quadrupole, ion trap, TOF TOF, Orbitrap, FTMS,

magnetic sector

Triple quadrupole iontrap,

hybride MS (e.g. Q-TOF, Q-

trap)

Acquisition Full scan, limited m/z range, SIM Full scan, limited m/z range,

SIM

SRM, MRM, full-scan

product-ion spectra

Requirements for

identification

≥ 3 diagnostic ions (preferably

including quasi molecular ion)

≥ 2 diagnostic ions (preferably

including quasi molecular

ion). Mass accuracy < 5 ppm.

At least one fragment ion.

≥ 2 product ions

Ion ratios identical with 2002/657/EC (maximum permitted tolerances)




Suggested criteria for identification

Requirements for identification

HRMS: ≥ 2 diagnostic ions (preferably molecular ion)

MSn: ≥ 2 precursor ions, each with 1 product ion

Criteria for maximum permitted tolerances for relative

ion intensities (referring to 2002/657/EC, EPA 1613 B)

Relative intensity of

qualifier ion(s)

compared to target ion

GC-EI-MS GC-CI-MS, GC-MSn

(relative deviation)

> 50 % ± 10 % ± 20 %

> 20 % to 50 % ± 15 % ± 25 %

> 10 % to 20 % ± 20 % ± 30 %

≤ 10 % ± 50 %*) ± 50 %

QC limits around the

theoretical ion abundance

rations: ± 15 %




Working range / LOQCommission Regulation (EC) No. 1883/2006

Performance of a method in the range of the level of interest, (e.g. 0.5x, 1x and 2x) with an acceptable CV

Limit of quantification for a confirmatory method:in the range of about 1/5 of the level of interest,

Difference between upper bound and lower bound ≤ 20 % for foodstuffs with ca. 1 pg WHO-TEQ/g fat.[LOQ for GC/MS screening methods not defined]

Specific limit of quantification of an individual congener:concentration of an analyte in the extract of a sample which produces an instrumental response at two different ions to be monitored with an S/N (signal/noise) ratio of 3:1 for the less sensitive signal

TEQ

Congener




Signal-to-Noise ratio

GC-MS/MS:

Chemical / electronical noise?

Possibility of calculation of signal-to-noise ratio?

Differences between different GC-MS/MS systems

Very low noise level or „flat“ baseline

GC/HRMS systems

„Background noise“ in extracts calculation of signal-to-noise ratio possible

With increasing electronical „optimization“ further reduction of noise

Calculated signal-to-noise ratios very highand LOQs partly unrealisticly low

Comparability of different GC/HRMS systems?




Legal limits for PCDD/Fsin Food and Feed

Maximum levels(Commission Regulation (EC) No. 1881/2006, Commission Directive 2006/13/EC)

Action levels(Commission Recommendation 2006/88/EC, Commission Directive 2006/13/EC)

Maximum levels WHO-PCDD/F-TEQ

Action levels WHO-PCDD/F-TEQ

Food(Meat, liver, eggs, milk

products, fats and oils, fish)

0.75 – 6 pg/g fat

4 pg/g fresh weight (fish)

0.5 – 4 pg/g fat

3 pg/g fresh weight (fish)

Feed(Compound feed,

premixtures, additives ...)

0.75 – 6 ng/kg product(12 % moisture content)

0.5 – 5 ng/kg product(12 % moisture content)




Extraction and Clean-up

Indicator PCBs

n-Hexane fraction

Mono-ortho-PCBs

n-Hexane/toluene fraction

Non-ortho-PCBs

Toluene fraction

Carbon column for PCB

n-Heptane fraction

PCDD/Fs

Multi-layer silica column

Toluene fraction

Carbon column for PCDD/F

Toluene fraction

Florisil column


Gel permation chromatography

Determination of lipid content

Extraction

Sample pre-treatment

Food/Feed sample

GC-HRMS

GC-MS/MS




GC-MS/MS systemMS/MS settings

TSQ Quantum XLS Ultra

Triple-Quadrupole GC-MS/MS

Optimization of Q2 gas pressure,

collision energy for PCDD/F measurement

Source temperature 250 °C

Electron energy 40 eV

Emission current 50 µA Q2 Gas pressure (Argon) 1.5 mTorr

Collision Energy 22 V

Q1 peak width 0.7 amu

Q3 peak width 0.7 amu

Precursor ion M+

Product ion M-COCl+

2 precusor ions, each with 1 product ion




GC-MS/MSGC settings

Indentical PTV and GC settings as for GC-HRMS method

Adjustment of final oven temperature necessary (HRMS: 340 °C)

PTV injection

Injected volume 5 µl (toluene)

Injection speed 5µl/s

Liner Open silcosteel liner

Injection temperature 100 °C

Transfer temperature 340 °C

GC oven method

GC column DB-5MS (60m, 0.25 µm, 0.25 mm)

Initial temperature 120 °C

Rate 1 17°C/min to 250 °C

Rate 2 2.5°C/min to 285 °C

Final temperature 285 °C for 13 min




Content



GC-MS/MS determination of PCDD/Fs


Modified clean-up




PCDD/F analysis using GC-MS/MS

Calibrations (standard solution)

Ion abundance ratios

Relative response factors

Calculation of LOQ

Spiked samples

Comparison with spiked concentrations

Comparison with GC/HRMS results




GC-MS/MS ion abundance ratioCalculation of theoretical value

Depending on ...

Ion abundance ratios of selected precursor ion

Probability of loss of CO35Cl and CO37Cl for formation of

each product ion

12C 13C 12C 13C

1234-TCDD 1.04 2378-TCDF 1.03 1.04

2378-TCDD 1.03 1.04 12378-PeCDF 0.48 0.48

12378-PeCDD 0.48 0.48 23478-PeCDF 0.48 0.48

123478-HxCDD 0.62 0.63 123478-HxCDF 0.63 0.63

123678-HxCDD 0.62 0.63 123678-HxCDF 0.63 0.63

123789-HxCDD 0.62 0.63 234678-HxCDF 0.63 0.63

1234678-HpCDD 0.80 0.79 123789-HxCDF 0.63 0.63

OCDD 1.04 1.04 1234678-HpCDF 0.80 0.79

1234789-HpCDF 0.80 0.79

OCDF 1.04 1.04




Ion abundance ratioComparison with calibration

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

2378

-TCDD

1237

8-PeC

DD

1234

78-H

xCDD

1236

78-H

xCDD

1237

89-H

xCDD

1234

678-

HpC

DD

OCDD

2378

-TCDF

1237

8-PeC

DF

2347

8-PeC

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

Ion

rati

o

Calculated Calibration

QC limits around the

theoretical ion abundance

rations: ± 15 %




CalibrationPCDD/F

Conc. in pg/µl Cal 1 Cal 2 Cal 3 Cal 4 Cal 5

2,3,7,8-TCDD 0.0125 0.025 0.05 0.2 0.5

1,2,3,7,8-PeCDD 0.025 0.05 0.1 0.4 1

1,2,3,4,7,8-HxCDD 0.025 0.05 0.1 0.4 1

1,2,3,6,7,8-HxCDD 0.0625 0.125 0.25 1 2.5

1,2,3,7,8,9-HxCDD 0.025 0.05 0.1 0.4 1

1,2,3,4,6,7,8-HpCDD 0.125 0.25 0.5 2 5

OCDD 0.25 0.5 1 4 10

2,3,7,8-TCDF 0.0125 0.025 0.05 0.2 0.5

1,2,3,7,8-PeCDF 0.0125 0.025 0.05 0.2 0.5

2,3,4,7,8-PeCDF 0.0625 0.125 0.25 1 2.5

1,2,3,4,7,8-HxCDF 0.025 0.05 0.1 0.4 1

1,2,3,6,7,8-HxCDF 0.025 0.05 0.1 0.4 1

1,2,3,7,8,9-HxCDF 0.0125 0.025 0.05 0.2 0.5

2,3,4,6,7,8-HxCDF 0.0125 0.025 0.05 0.2 0.5

1,2,3,4,6,7,8-HpCDF 0.025 0.05 0.1 0.4 1

1,2,3,4,7,8,9-HpCDF 0.0125 0.025 0.05 0.2 0.5

OCDF 0.05 0.1 0.2 0.8 2

Calibration standards of CVUA/EU-RL used for GC-HRMS




CalibrationCalculation of TEQ

Calculation of TEQ of calibration solutions (on fat basis)

Analysis of 3 g of fat

20 µl final volume (Injection of 5 µl)

Additional dilutions for calulation of LOQ (GC-MS/MS)

Calibration WHO-PCDD/F-TEQ [pg/g fat]

Cal 1 1:5 0.12

Cal 1 1:2 0.3

Cal 1 0.6

Cal 2 1.2

Cal 3 2.4

Cal 4 9.7

Cal 5 24




Calculation of LOQ from Calibration (1)

Criteria:

LOQ as lowest calibration point with acceptable ion

abundance ratio and deviation of relative response factor

Ion abundance ratio: QC limits around the theoretical ion

abundance ratios: ± 15 %

Relative response factors: CV ≤ 20 % for whole calibration

(individual calibration points ≤ 40 % deviation of mean value)

Calculation of LOQ from signal-to-noise-ratio

due to very low noise level not possible !




Calculation of LOQ from Calibration (2)Data from 10 GC-MS/MS sequences

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

2378

-TCDD

1237

8-PeC

DD

1234

78-H

xCDD

1236

78-H

xCDD

1237

89-H

xCDD

1234

678-

HpC

DD

OCDD

2378

-TCDF

1237

8-PeC

DF

2347

8-PeC

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

fg a

bso

lute

(o

n c

olu

mn

)

Range of LOQ Conc of Cal 1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

WHO-PCDD/F-TEQ pg/g

fat (1 g fat)

WHO-PCDD/F-TEQ pg/g

fat (3 g fat)

pg

/g W

HO

-PC

DD

/F-T

EQ

Range of TEQ TEQ of Cal 1




Calibration GC-MS/MSCal 1

C:\Xcalibur\...\cdxq_040411-13 4/4/2011 21:33:06 Eich_1L

RT: 7.97 - 18.43

8 9 10 11 12 13 14 15 16 17 18

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

RT: 15.68

AA: 2780

RT: 15.94

AA: 14.12

RT: 12.39

AA: 10.72

RT: 8.79

AA: 10.70

RT: 13.79

AA: 14.42 RT: 14.43

AA: 6.33

RT: 9.81

AA: 4.44

RT: 15.47

AA: 2.43

RT: 15.69

AA: 3181

RT: 16.02

AA: 9.71

RT: 14.51

AA: 11.89

RT: 11.57

AA: 12.60

RT: 16.90

AA: 9.35

RT: 15.04

AA: 13.15RT: 14.36

AA: 4.44

RT: 9.49

AA: 3.18

RT: 15.68

AA: 138860

RT: 15.82

AA: 3348RT: 16.74

AA: 518

RT: 15.00

AA: 445

RT: 15.68

AA: 145483

RT: 15.81

AA: 4296

RT: 15.02

AA: 748

RT: 16.88

AA: 96.12

NL: 1.40E3

TIC F: + c EI SRM

ms2 319.900

[256.895-

256.905] MS

ICIS

cdxq_040411-13

NL: 1.32E3

TIC F: + c EI SRM

ms2 321.890

[258.885-

258.895] MS

ICIS

cdxq_040411-13

NL: 4.98E4

TIC F: + c EI SRM

ms2 331.940

[267.965-

267.975] MS

ICIS

cdxq_040411-13

NL: 5.21E4

TIC F: + c EI SRM

ms2 333.930

[269.965-

269.975] MS

ICIS

cdxq_040411-13

x100Magnification of noise range

62.5 fg 2,3,7,8-TCDD

Range of LOQ:

Acceptable ion ratio

(± 15 %)

Acceptable relative

response factor

(CV ≤ 20 %)

319.9 > 256.9

321.9 > 258.9

S/N 1500:1(noise range 12 – 15.5 min)





Spiked fat sample GC-MS/MSComparison with calibration

C:\Xcalibur\...\cdxq_040411-50 4/5/2011 22:02:09 Schweineschmalz E1L A

RT: 7.97 - 18.43

8 9 10 11 12 13 14 15 16 17 18

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

RT: 15.69

AA: 2467

RT: 15.78

AA: 56.79

RT: 14.57

AA: 10.15

RT: 15.57

AA: 1.84

RT: 15.69

AA: 2441

RT: 10.85

AA: 19.03RT: 15.94

AA: 11.41

RT: 15.69

AA: 147862

RT: 15.82

AA: 3787

RT: 15.02

AA: 172RT: 16.63

AA: 66.30

RT: 15.69

AA: 141785

RT: 15.82

AA: 4737

RT: 15.01

AA: 400

RT: 16.83

AA: 81.01

NL: 8.92E2

TIC F: + c EI SRM

ms2 319.900

[256.895-

256.905] MS

ICIS

cdxq_040411-50

NL: 9.69E2

TIC F: + c EI SRM

ms2 321.890

[258.885-

258.895] MS

ICIS

cdxq_040411-50

NL: 5.10E4

TIC F: + c EI SRM

ms2 331.940

[267.965-

267.975] MS

ICIS

cdxq_040411-50

NL: 5.16E4

TIC F: + c EI SRM

ms2 333.930

[269.965-

269.975] MS

ICIS

cdxq_040411-50

x100

62.5 fg 2,3,7,8-TCDD

Magnification of noise range







chromatogramC:\Xcalibur\...\cdx_060411\cdx_060411-35 4/8/2011 9:23:10 AM Eich_1L

RT: 13.97 - 18.47 SM: 3G

14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

RT: 17.41

AA: 1014

RT: 17.45

AA: 105RT: 15.73

AA: 79.19RT: 16.89

AA: 65.63

RT: 17.12

AA: 69.74RT: 14.16

AA: 60.60RT: 18.39

AA: 50.12

RT: 17.40

AA: 1392

RT: 16.02

AA: 127RT: 18.35

AA: 114RT: 14.17

AA: 172

RT: 17.34

AA: 83.14RT: 18.12

AA: 68.67

RT: 15.30

AA: 130

RT: 17.64

AA: 55.08RT: 16.60

AA: 86.05

RT: 17.14

AA: 38485

RT: 17.39

AA: 36686

RT: 17.48

AA: 1013RT: 15.43

AA: 713

RT: 16.95

AA: 635

RT: 17.93

AA: 970RT: 18.39

AA: 855RT: 16.78

AA: 747

RT: 16.27

AA: 521RT: 14.34

AA: 450

RT: 14.51

AA: 489

RT: 14.99

AA: 527

RT: 15.97

AA: 664

RT: 17.14

AA: 50498 RT: 17.40

AA: 48085

RT: 17.53

AA: 624RT: 15.55

AA: 417RT: 16.63

AA: 265

RT: 17.76

AA: 224

RT: 14.23

AA: 197

RT: 18.35

AA: 153

RT: 15.12

AA: 185

RT: 16.21

AA: 136RT: 14.80

AA: 112

NL:

4.56E2

m/z=

319.8962-

319.8968

MS ICIS

cdx_06041

1-35

NL:

6.02E2

m/z=

321.8934-

321.8940

MS ICIS

cdx_06041

1-35

NL:

1.60E4

m/z=

331.9365-

331.9371

MS ICIS

cdx_06041

1-35

NL:

2.12E4

m/z=

333.9335-

333.9341

MS ICIS

cdx_06041

1-35

62.5 fg 2,3,7,8-TCDD

Chromatogram from

routine analysis:

Acceptable relative

response factors as

low as the range of

1:5 dilution of Cal 1

319.896

321.893






Content





Modified clean-up




Comparison of results (1)GC-HRMS – GC-MS/MS

-100

-80

-60

-40

-20

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10

WHO-PCDD/F-TEQ [pg/g fat]

Devia

tio

n o

f M

S/M

S r

esu

lts o

f H

RM

S

Butter Eggs Fat Meat Human milk

Measurement of same

extracts using GC-HRMS

and GC-MS/MS

Food samples

Maximum levels for butter, eggs, meat and fat

Deviation of MS/MS

from HRMS

[%]




Comparison of chromatogramsHRMS – MS/MSc:\Xcalibur\...\cdx_260111\cdx_260111-11 1/26/2011 7:28:03 PM 109.1 (Gefäß-Nr./ Untersuchungs.-Nr. )_T

8

RT: 14.52 - 15.84 SM: 3G

14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

RT: 15.32

AA: 3826RT: 14.88

AA: 3329RT: 14.59

AA: 2404RT: 14.95

AA: 2536 RT: 15.00

AA: 1970RT: 15.43

AA: 1985RT: 15.16

AA: 1504

RT: 15.65

AA: 434RT: 15.69

AA: 510

RT: 15.08

AA: 446RT: 15.51

AA: 349RT: 14.77

AA: 199

RT: 14.62

AA: 164

RT: 15.32

AA: 4443RT: 14.94

AA: 4411RT: 14.59

AA: 3481RT: 14.89

AA: 4060

RT: 15.00

AA: 2003RT: 15.43

AA: 2433RT: 15.17

AA: 1782RT: 15.65

AA: 1050

RT: 15.08

AA: 743RT: 15.53

AA: 696RT: 14.83

AA: 534RT: 15.73

AA: 445

RT: 15.37

AA: 156

RT: 15.31

AA: 61877 RT: 15.52

AA: 54607

RT: 15.35

AA: 1340RT: 14.74

AA: 1803

RT: 14.99

AA: 1695

RT: 15.12

AA: 1234

RT: 15.67

AA: 1234

RT: 14.91

AA: 1078

RT: 15.73

AA: 1140RT: 15.25

AA: 906

RT: 14.54

AA: 1216

RT: 15.46

AA: 752RT: 15.59

AA: 528

RT: 15.31

AA: 78906

RT: 15.52

AA: 58838

RT: 15.57

AA: 655

RT: 15.65

AA: 990

RT: 15.36

AA: 456RT: 14.93

AA: 545

RT: 15.44

AA: 357

RT: 14.73

AA: 316

RT: 14.68

AA: 503

RT: 15.19

AA: 333

RT: 14.99

AA: 271RT: 14.83

AA: 309

NL:

1.53E3

m/z=

319.8962-

319.8968

MS ICIS

cdx_26011

1-11

NL:

1.87E3

m/z=

321.8934-

321.8940

MS ICIS

cdx_26011

1-11

NL:

2.84E4

m/z=

331.9365-

331.9371

MS ICIS

cdx_26011

1-11

NL:

3.78E4

m/z=

333.9335-

333.9341

MS ICIS

cdx_26011

1-11

c:\Xcalibur\...\cdxq_240311-26 3/25/2011 07:14:21 Gruenmehl

RT: 14.59 - 15.89

14.6 14.7 14.8 14.9 15.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

RT: 15.02

AA: 1975RT: 14.94

AA: 1742RT: 15.10

AA: 1629RT: 15.44

AA: 1137RT: 15.28

AA: 733 RT: 15.56

AA: 663RT: 14.61

AA: 668RT: 15.39

AA: 358RT: 15.16

AA: 469 RT: 15.68

AA: 222 RT: 15.82

AA: 87.42

RT: 15.57

AA: 1780RT: 15.02

AA: 2080 RT: 15.45

AA: 1625RT: 14.95

AA: 1499RT: 15.11

AA: 1444 RT: 15.29

AA: 1019

RT: 14.62

AA: 782

RT: 15.19

AA: 169RT: 15.85

AA: 174RT: 15.68

AA: 68.86RT: 14.85

AA: 23.80

RT: 15.69

AA: 71910

RT: 15.45

AA: 53510

RT: 15.82

AA: 1051

RT: 15.59

AA: 690RT: 15.02

AA: 301

RT: 15.09

AA: 150

RT: 15.69

AA: 62340RT: 15.44

AA: 47770

RT: 15.57

AA: 627RT: 15.83

AA: 1429

RT: 15.03

AA: 342RT: 15.23

AA: 28.15

NL: 7.68E2

TIC F: + c EI SRM

ms2 319.900

[256.895-

256.905] MS

ICIS

cdxq_240311-26

NL: 7.97E2

TIC F: + c EI SRM

ms2 321.890

[258.885-

258.895] MS

ICIS

cdxq_240311-26

NL: 2.75E4

TIC F: + c EI SRM

ms2 331.940

[267.965-

267.975] MS

ICIS

cdxq_240311-26

NL: 2.34E4

TIC F: + c EI SRM

ms2 333.930

[269.965-

269.975] MS

ICIS

cdxq_240311-26

HRMS

MS/MS

Mass chromatogramm of

TCDD of grass meal sample





0

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HRMS MS/MS

Difference between upper and

lower bound TEQ calculation:

Criteria:

Acceptable range ≤ 20 % for foodstuffs

with ca. 1 pg WHO-TEQ/g fat

Food samplesMaximum levels for butter, eggs, meat and fat




Comparison of results (3)Spiked level – GC-MS/MS

-40

-20

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120

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Devia

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co

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on

[%

]

Butter fat Pig fat QC sample 2 QC sample 6

Spiked sample:

Spiking level Cal1 – Cal 3

Spiked fat samples

Maximum levels for fat from different animals

Deviation of MS/MS

from spiked concentration




Comparison of results (4)Spiked level – GC-MS/MS

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Butter fat Pig fat QC sample 2 QC sample 6

Spiked fat samples

Maximum levels for fat from different animals

Difference between upper and

lower bound TEQ calculation:

Criteria:

Acceptable range ≤ 20 % for

foodstuffs with ca. 1 pg WHO-

TEQ/g fat





-40

-30

-20

-10

0

10

20

30

40

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

WHO-PCDD/F-TEQ [pg/g fresh weight]

Devia

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f M

S/M

S r

esu

lts o

f H

RM

S

Fish

-40

-30

-20

-10

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30

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0 0.1 0.2 0.3 0.4 0.5

WHO-PCDD/F-TEQ [ng/kg product 12% moisture content]

Devia

tio

n o

f sp

iked

co

ncen

trati

on

[%

]

Mineral clay

Fish

Mineral feed additiv (spiked)

Maximum level

Maximum level 1.0 ng/kg

Deviation of MS/MS

from HRMS, spiked

concentration

[%]




Content





Reduced clean-up




Modified clean-up

Reduction of number of clean-up

steps

Skip of carbon column for

cleaning of PCDD/F fraction

Comparison between normal and

reduced clean-up

Check of possibility of further

reduction of clean-up steps for fast

screening method

PCDD/Fs

Toluene fraction

Florisil column


Gel permation chromatography

Determination of lipid content

Extraction

Sample pre-treatment

Food/Feed sample

Carbon

column




Modified Clean-upComparison of QC fat sample

No significant differences between WHO-PCDD/F-TEQ with/ without reduced clean-up (and HRMS results)

Occurence of additional peaks, no increasing of noise observed

C:\Xcalibur\...\cdxq_110411-14 4/11/2011 20:54:37 QK2 II Verk

RT: 11.10 - 16.97

11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5

Time (min)

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100

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Ab

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0

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40

60

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RT: 16.33

AA: 17072

RT: 16.84

AA: 12479

RT: 15.66

AA: 5309

RT: 14.33

AA: 1881 RT: 14.77

AA: 720RT: 15.29

AA: 232RT: 12.79

AA: 16.96

RT: 13.04

AA: 16.45

RT: 15.75

AA: 15.36

RT: 16.34

AA: 17526

RT: 16.84

AA: 12616

RT: 15.67

AA: 4489RT: 14.75

AA: 2653RT: 14.34

AA: 1748RT: 11.78

AA: 36.43

RT: 15.99

AA: 31.36

RT: 15.65

AA: 86454

RT: 15.41

AA: 59988

RT: 15.75

AA: 1334RT: 14.76

AA: 173

RT: 16.57

AA: 68.98

RT: 11.30

AA: 37.49

RT: 15.66

AA: 79440

RT: 15.42

AA: 60308

RT: 15.79

AA: 1681RT: 15.01

AA: 177

NL: 6.90E3

TIC F: + c EI SRM

ms2 319.900

[256.895-

256.905] MS

ICIS

cdxq_110411-14

NL: 7.19E3

TIC F: + c EI SRM

ms2 321.890

[258.885-

258.895] MS

ICIS

cdxq_110411-14

NL: 3.19E4

TIC F: + c EI SRM

ms2 331.940

[267.965-

267.975] MS

ICIS

cdxq_110411-14

NL: 3.04E4

TIC F: + c EI SRM

ms2 333.930

[269.965-

269.975] MS

ICIS

cdxq_110411-14

C:\Xcalibur\...\cdxq_110411-05 4/11/2011 14:58:53 QK2 C

RT: 11.10 - 16.97

11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5

Time (min)

0

20

40

60

80

100

0

20

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60

80

100

0

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60

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Re

lative

Ab

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0

20

40

60

80

100

RT: 15.67

AA: 3102

RT: 14.34

AA: 954

RT: 14.88

AA: 231 RT: 15.51

AA: 27.86RT: 13.89

AA: 12.73

RT: 12.44

AA: 10.74RT: 15.99

AA: 8.95

RT: 15.67

AA: 3857

RT: 14.35

AA: 1283

RT: 14.88

AA: 34.16

RT: 16.32

AA: 32.97RT: 15.26

AA: 17.22

RT: 14.47

AA: 25.61

RT: 11.79

AA: 14.08

RT: 12.36

AA: 13.37

RT: 15.66

AA: 66644

RT: 15.41

AA: 53627

RT: 15.79

AA: 2353RT: 16.85

AA: 64.54

RT: 14.99

AA: 58.94

RT: 15.66

AA: 66564RT: 15.41

AA: 54291

RT: 15.78

AA: 962RT: 15.00

AA: 133

RT: 16.72

AA: 42.89

NL: 1.30E3

TIC F: + c EI SRM

ms2 319.900

[256.895-

256.905] MS

ICIS

cdxq_110411-05

NL: 1.62E3

TIC F: + c EI SRM

ms2 321.890

[258.885-

258.895] MS

ICIS

cdxq_110411-05

NL: 2.57E4

TIC F: + c EI SRM

ms2 331.940

[267.965-

267.975] MS

ICIS

cdxq_110411-05

NL: 2.45E4

TIC F: + c EI SRM

ms2 333.930

[269.965-

269.975] MS

ICIS

cdxq_110411-05

125 fg 2,3,7,8-TCDD

Normal clean-up,

GC-MS/MS Modified clean-up,

GC-MS/MS




Adjustment of

Q1 peak width


RT: 19.96 - 23.47

20.0 20.5 21.0 21.5 22.0 22.5 23.0

Time (min)

0

20

40

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100

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Re

lative

Ab

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0

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RT: 21.54

AA: 659RT: 20.95

AA: 1010

RT: 20.85

AA: 755 RT: 20.99

AA: 316

RT: 20.83

AA: 2846

RT: 20.96

AA: 1405RT: 21.52

AA: 1722

RT: 20.83

AA: 60632

RT: 21.50

AA: 29550 RT: 22.47

AA: 23629

RT: 23.33

AA: 394RT: 22.59

AA: 307

RT: 20.83

AA: 96204

RT: 21.50

AA: 46182 RT: 22.47

AA: 38067

RT: 22.57

AA: 403

RT: 21.63

AA: 331

NL: 4.41E2

TIC F: + c EI SRM

ms2 371.820

[308.855-

308.865] MS

ICIS

cdxq_120411-28

NL: 1.30E3

TIC F: + c EI SRM

ms2 373.820

[310.855-

310.865] MS

ICIS

cdxq_120411-28

NL: 1.89E4

TIC F: + c EI SRM

ms2 383.860

[319.895-

319.905] MS

ICIS

cdxq_120411-28

NL: 2.88E4

TIC F: + c EI SRM

ms2 385.860

[321.885-

321.895] MS

ICIS

cdxq_120411-28


RT: 19.96 - 23.47

20.0 20.5 21.0 21.5 22.0 22.5 23.0

Time (min)

0

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100

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RT: 20.82

AA: 4302

RT: 21.52

AA: 2588RT: 20.96

AA: 2291

RT: 20.33

AA: 235RT: 21.60

AA: 152

RT: 21.52

AA: 6569RT: 20.85

AA: 5689

RT: 20.14

AA: 367

RT: 20.82

AA: 132980

RT: 21.50

AA: 71042 RT: 22.47

AA: 55522

RT: 20.94

AA: 220502

RT: 21.50

AA: 110112 RT: 22.47

AA: 94254

RT: 21.10

AA: 1767

RT: 22.64

AA: 1181RT: 21.63

AA: 188

NL: 1.60E3

TIC F: + c EI SRM

ms2 371.820

[308.855-

308.865] MS

ICIS

cdxq_120411-26

NL: 2.01E3

TIC F: + c EI SRM

ms2 373.820

[310.855-

310.865] MS

ICIS

cdxq_120411-26

NL: 4.03E4

TIC F: + c EI SRM

ms2 383.860

[319.895-

319.905] MS

ICIS

cdxq_120411-26

NL: 6.58E4

TIC F: + c EI SRM

ms2 385.860

[321.885-

321.895] MS

ICIS

cdxq_120411-26


RT: 19.96 - 23.47

20.0 20.5 21.0 21.5 22.0 22.5 23.0

Time (min)

0

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100

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Re

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RT: 20.84

AA: 6035 RT: 21.53

AA: 4079

RT: 21.50

AA: 3307RT: 20.96

AA: 4307

RT: 22.80

AA: 1121RT: 22.45

AA: 692RT: 20.39

AA: 724RT: 23.35

AA: 518RT: 23.03

AA: 489RT: 21.77

AA: 717RT: 21.20

AA: 499

RT: 21.51

AA: 11236RT: 20.83

AA: 10126

RT: 20.35

AA: 1246

RT: 23.17

AA: 1096RT: 22.85

AA: 829

RT: 22.43

AA: 932

RT: 20.75

AA: 687 RT: 21.23

AA: 502

RT: 20.15

AA: 459

RT: 20.95

AA: 209340

RT: 21.50

AA: 92318 RT: 22.47

AA: 78921

RT: 22.58

AA: 1832

RT: 20.94

AA: 317187

RT: 21.50

AA: 157916 RT: 22.47

AA: 140428

RT: 21.09

AA: 2948RT: 22.75

AA: 698

RT: 21.77

AA: 467

NL: 2.10E3

TIC F: + c EI SRM

ms2 371.820

[308.855-

308.865] MS

ICIS

cdxq_120411-27

NL: 4.11E3

TIC F: + c EI SRM

ms2 373.820

[310.855-

310.865] MS

ICIS

cdxq_120411-27

NL: 6.17E4

TIC F: + c EI SRM

ms2 383.860

[319.895-

319.905] MS

ICIS

cdxq_120411-27

NL: 9.42E4

TIC F: + c EI SRM

ms2 385.860

[321.885-

321.895] MS

ICIS

cdxq_120411-27

Q1: 0.4 amu

Q1: 1.0 amu

Q1: 2.0 amu

Increasing peak intensity and noise with

increasing Q1 peak width

Effects especially visible for Dibenzofurans




Adjustment of Q3 peak widthC:\Xcalibur\...\cdxq_120411-17 4/13/2011 03:52:35 Eich_4L

RT: 20.00 - 23.47

20.5 21.0 21.5 22.0 22.5 23.0

Time (min)

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

Re

lative

Ab

un

da

nce

20

40

60

80

100

RT: 21.76

AA: 224203RT: 20.83

AA: 193757

RT: 21.51

AA: 94870RT: 22.07

AA: 90131 RT: 22.47

AA: 75769

RT: 21.76

AA: 378994

RT: 22.06

AA: 140534RT: 21.65

AA: 132561RT: 20.95

AA: 60686 RT: 21.51

AA: 27304RT: 22.47

AA: 24508RT: 22.92

AA: 1745

RT: 21.74

AA: 507418

RT: 21.64

AA: 229050

RT: 22.05

AA: 132934

RT: 22.25

AA: 1643

RT: 22.65

AA: 917

RT: 21.74

AA: 801366

RT: 21.64

AA: 347409

RT: 22.05

AA: 206762

RT: 22.37

AA: 1517RT: 20.96

AA: 1417

RT: 22.81

AA: 1052

NL: 7.07E4

TIC F: + c EI SRM

ms2 387.820

[324.815-

324.825] MS

ICIS

cdxq_120411-17

NL: 1.11E5

TIC F: + c EI SRM

ms2 389.820

[326.815-

326.825] MS

ICIS

cdxq_120411-17

NL: 1.44E5

TIC F: + c EI SRM

ms2 399.860

[335.855-

335.865] MS

ICIS

cdxq_120411-17

NL: 2.25E5

TIC F: + c EI SRM

ms2 401.860

[337.855-

337.865] MS

ICIS

cdxq_120411-17

Q3: 2.0 amu

387.8 > 324.8

389.8 > 326.8

HxCDD

HxCDF

13C-labeled Dibenzofurans visible

in Dibenzo-p-dioxins mass traces

with Q3 at 2.0 amu




Summary

GC-MS/MS systems in principle applicable for PCDD/F

analysis in food and feed samples

Higher deviations of HRMS results in the range

below 1pg WHO-PCDD/F-TEQ/g fat depending on

limited amount of fat for clean-up

Calculation of LOQ from calibration

not possible from signal-to-noise-ratio (as for HRMS)




Thank you very much for

your attention !

Documents

GC-MS/MS determination of PCDD/Fs and PCBs ... - Thermo Fisher