Verbesserte GC/MS/MS Analyse von Dioxinen und ... - Agilent · Verbesserte GC/MS/MS Analyse von...

Food and Environmental

Solutions Seminars 2011

Moritz Hebestreit, PhD

Applications Chemist

Agilent Technologies

Europe, Middle East, Africa and India

Verbesserte GC/MS/MS Analyse von Dioxinen und PAHs

mit dem Agilent 7000B Triple-Quadrupol System



Sensitive Determination of Dioxins and PCBs in Food and Feed

The Chemical and Veterinary Analytical Institute

Münsterland-Emscher-Lippe, CVUA-MEL

Münster,Germany

Prof. Dr. Peter Fürst

in collaboration with

Agilent Technologies

Chris Sandy

EMEA Food Team GC-MS Applications Chemist



Objectives of the analytical approach

• To assess the performance of GC-MS/MS in EI mode for the analysis of Dioxins and PCBs in food and feed

• To check whether (l) GC-MS/MS can be used as a sensitive screening method and (ll) eventually as a reliable and cost-effective alternative for GC-HRMS for confirmatory purposes in official food and feed control

• To investigate whether GC-MS/MS is capable to determine dl-PCBs and especially ndl-PCBs at the level of interest dicussed in the framework of setting harmonized MRL-values for PCBs



Introduction / Background

2 3

4

56

2'3'

4'

5' 6' Cl xCl y

PCDDs PCDFs

PCBs



Polychlorinated dibenzo-p-dioxins (PCDD)

and Polychlorinated dibenzofurans (PCDF)

PCDD PCDF

• ‘Dioxins’ = PCDD and PCDF

• 210 different congeners with 1 - 8 chlorine atoms

• No use nor technical and industrial production

• Formation as unwanted by-products during thermal and industrial processes

• Increase of environmental levels in parallel to the beginning of industrial chlorine chemistry

# Isomers # Isomers

# Cl Atoms

PCDD PCDF

1 2 4

2 10 16

3 14 28

4 22 38

5 14 28

6 10 16

7 2 4

8 1 1

Total 75 135



Polychlorinated Biphenyls (PCBs) # Cl atoms Congeners

1 3

2 12

3 24

4 42

5 46

6 42

7 24

8 12

9 3

10 1

Total 209

Industrial production since 1929 (> 1.5 M tons)

Properties:

- lipophilic

- low flamability

- chemical and physical stability

- limited biological degradation

2 3

4

56

2'3'

4'

5' 6' Cl xCl y

Brand names: Clophen, Arochlor, Kanechlor, Phenochlor, Fenchlor,

Pyralen



Dioxin-like (dl-) PCBs

According to their free

rotability around the

axis, non-ortho and

mono-ortho substituted

PCBs may adopt a similar

spatial structure to 2,3,7,8-

TCDD and therefore

exhibit comparable toxic

effects as dioxins

ortho-position



Important PCB congeners (Nomenclature according to IUPAC)

• Dioxin-like PCBs

(Food and Feed: Regulated EU wide)

Non-ortho PCBs PCB 77, 81, 126, 169

Mono-ortho PCBs PCB 105, 114, 118, 123, 156, 157, 167, 189

• Non dioxin-like (Indicator) PCBs

(Food and Feed: EU Regulation in preparation, Sum 6 ndl-PCBs) PCB 28, 52, 101, 138, 153, 180

Concentration of dl-PCBs (esp. of non-ortho PCBs) in food

generally 1-2 orders of magnitude lower than indicator-PCBs



7890-7000B GC-MS/MS Configuration for Dioxins

with Pre-column Backflush

7000B

TQ

7890A

MS-Tee G3186B

PCM 2 µL cold pulsed splitless injection

N2 MMI

2.0 m x 0.25 mm ID

Uncoated deactivated

Fused silica

60m x 0.25 mm ID x 0.25 µm

DB5-MSUI (122-5662UI)

0.9 mL/min

Constant flow

1.0 mL/min

Constant flow

Inlet temp program : 100 (0.05) – 600 °C/min – 300 °C

Oven temp program : 130 (2) – 10 – 220 (16) – 5 – 235 (7) – 5 – 350 (0) °C



GC-MS/MS MRM Settings

PCDD / PCDF Segment # MRM Segment Start Time (min)

Tetra 1 25.0

Penta 2 40.0

Hexa 3 46.0

Hepta 4 51.0

Octa 5 55.0

MRM Segment 1

Compound Name ISTD? Precursor ion Product ion Dwell (ms) CE (V)

13C-2378TCDF X 317.9 253.9 50 33

13C-2378TCDF X 315.9 251.9 50 33

2378-TCDF 305.9 242.9 50 33

2378-TCDF 303.9 240.9 50 33

13C-TCDDs X 333.9 269.9 50 24

13C-TCDDs X 331.9 267.9 50 24

2378-TCDD 321.9 258.9 50 24

2378-TCDD 319.9 256.9 50 24

Source temp. : 280 °C

Electron Energy : -70 eV

Tune : Autotune

MS1 / MS2 Resolution : 1.2u / 1.2u



Full GC-MS/MS MRM Settings TS Segment Start Time (min.) Peak Number Analyte RT (min.) Quant Pre-cursor Product CE (V) Qual Pre-cursor Product CE (V)

1 25

1 13C-2378-TCDF 35.43 315.9 251.9 33 317.9 253.9 33

2 2378-TCDF 35.47 303.9 240.9 33 305.9 242.9 33

3 13C-1234-TCDD 35.77 331.9 267.9 24 333.9 269.9 24

4 13C-2378-TCDD 36.79 331.9 267.9 24 333.9 269.9 24

5 2378-TCDD 36.80 319.9 256.9 24 321.9 258.9 24

2 40

6 13C-12378-PeCDF 42.55 351.9 287.9 35 349.9 285.9 35

7 12378-PeCDF 42.56 339.9 276.9 35 337.9 274.9 35

8 13C-23478-PeCDF 44.00 351.9 287.9 35 349.9 285.9 35

9 23478-PeCDF 44.02 339.9 276.9 35 337.9 274.9 35

10 13C-12378-PeCDD 44.45 365.9 301.9 25 367.9 303.9 25

11 12378-PeCDD 44.48 355.9 292.9 25 353.9 290.9 25

3 46

12 13C-123478-HxCDF 48.04 385.8 321.9 35 387.8 323.9 35

13 123478-HxCDF 48.06 373.8 310.9 35 375.8 312.9 35

14 13C-123678-HxCDF 48.21 385.8 321.9 35 387.8 323.9 35

15 123678-HxCDF 48.22 373.8 310.9 35 375.8 312.9 35

16 13C-234678HxCDF 48.96 385.8 321.9 35 387.8 323.9 35

17 234678-HxCDF 48.97 373.8 310.9 35 375.8 312.9 35

18 13C-123478-HxCDD 49.17 403.8 339.8 25 401.8 337.9 25

19 123478-HxCDD 49.19 389.8 326.9 25 391.8 328.8 25

20 13C-123678-HxCDD 49.30 403.8 339.8 25 401.8 337.9 25

21 123678-HxCDD 49.32 389.8 326.9 25 391.8 328.8 25

22 13C-123789HxCDD 49.63 403.8 339.8 25 401.8 337.9 25

23 123789-HxCDD 49.65 389.8 326.9 25 391.8 328.8 25

24 13C-123789-HxCDF 50.04 385.8 321.9 35 387.8 323.9 35

25 123789-HxCDF 50.06 373.8 310.9 35 375.8 312.9 35

4 51

26 13C-1234678-HpCDF 51.84 419.8 355.8 36 421.8 357.8 36

27 1234678-HpCDF 51.86 409.8 346.8 36 407.8 344.8 36

28 13C-1234678-HpCDD 53.11 437.8 373.8 25 435.8 371.8 25

29 1234678-HpCDD 53.13 423.8 360.8 25 425.8 362.8 25

30 13C-1234789-HpCDF 53.69 419.8 355.8 36 421.8 357.8 36

31 1234789-HpCDF 53.70 407.8 344.8 36 409.8 346.8 36

5 55

32 13C-OCDD 56.23 469.7 405.8 26 471.7 407.8 26

33 OCDD 56.24 457.7 394.8 26 459.7 396.8 26

34 13C-OCDF 56.41 453.7 389.8 35 455.7 391.8 35

35 OCDF 56.42 441.7 378.8 35 443.7 380.8 35



Native PCDD and PCDF Isomers

Tetra Penta Hexa Hepta Octa

Run time 60 minutes



Chromatographic Separation of HxCDFs and HxCDDs

123478-HxCDF

123678-HxCDF

123789-HxCDF

234678-HxCDF

123478-HxCDD

123678-HxCDD

123789-HxCDD



Cal Curve : 12378-PeCDD 100fg – 10pg Injected

Source : CVUA-MEL

0.05 pg/uL

5.0 pg/uL



7 point ISTD Calibration Curve, Linear Fit

100 fg – 10 pg Injected

Source : CVUA-MEL

Analyte R2

2378-TCDD 0.99934

12378-PeCDD 0.99976

123478-HxCDD 0.99994

123678-HxCDD 0.99905

123789-HxCDD 0.99977

1234678-HpCDD 0.99945

OCDD * 0.99780

Analyte R2

2378-TCDF 0.99984

12378-PeCDF 0.99909

23478-PeCDF 0.99995

123478-HxCDF 0.99971

123678-HxCDF 0.99983

234678-HxCDF 0.99953

123789-HxCDF 0.99972

1234678-HpCDF 0.99971

1234789-HpCDF 0.99991

OCDF 0.99907

* 500fg – 50pg injected



10 Replicate injections of 100fg 2378-TCDD

Peak Area %RSD = 8.3

Noise region



Inj # 2378-TCDF 2378-TCDD 12378-PCDF 23478-PCDF 12378-PCDD

Acq. Date-Time Area* Area* Area* Area* Area*

1 09/04/2010 13:39 1039 668 791 898 480

2 09/04/2010 14:43 1189 702 891 877 565

3 09/04/2010 15:47 1104 728 882 879 606

4 09/04/2010 16:51 1035 756 789 852 563

5 09/04/2010 17:55 993 704 806 948 587

6 09/04/2010 18:58 1071 661 834 897 623

7 09/04/2010 20:02 1013 654 799 1012 568

8 09/04/2010 21:06 975 635 864 953 610

9 09/04/2010 22:10 1085 618 792 835 565

10 09/04/2010 23:13 1076 709 829 873 531

11 10/04/2010 00:17 942 799 745 1003 554

12 10/04/2010 01:21 957 679 853 834 540

13 10/04/2010 02:24 1050 584 877 914 579

14 10/04/2010 03:28 935 606 802 923 666

15 10/04/2010 04:32 1051 661 855 891 642

Mean 1034 678 827 906 579

Stdevn 68 57 42 54 46

%RSD 6.60 8.48 5.11 5.98 8.02

* Raw (uncorrected) peak area

Peak Area* Reproducibility (n=15) Spiked Meat Extract

(100 fg/uL Natives / 1 pg/uL 13C-ISTDs)

2uL Injection : Natives 200 fg injected , 13C-ISTDs 2pg Injected



* Response ratio = (Peak area Quant Transition / Peak area ISTD)

Inj # 2378-TCDF 2378-TCDD 12378-PCDF 23478-PCDF 12378-PCDD

Acq. Date-Time Ratio Ratio Ratio Ratio Ratio

1 09/04/2010 13:39 0.0898 0.0965 0.0896 0.1007 0.0896

2 09/04/2010 14:43 0.1035 0.0968 0.0989 0.1000 0.1050

3 09/04/2010 15:47 0.0889 0.1003 0.0981 0.0941 0.1062

4 09/04/2010 16:51 0.0854 0.1060 0.0920 0.0939 0.1071

5 09/04/2010 17:55 0.0848 0.0970 0.0929 0.1015 0.1061

6 09/04/2010 18:58 0.0897 0.0929 0.0989 0.0980 0.1092

7 09/04/2010 20:02 0.0875 0.0917 0.0908 0.1125 0.1023

8 09/04/2010 21:06 0.0832 0.0931 0.0975 0.1102 0.1119

9 09/04/2010 22:10 0.0961 0.0898 0.0928 0.0943 0.1064

10 09/04/2010 23:13 0.0966 0.1012 0.0954 0.1008 0.0989

11 10/04/2010 00:17 0.0834 0.1150 0.0845 0.1149 0.1038

12 10/04/2010 01:21 0.0862 0.0981 0.0988 0.0980 0.1002

13 10/04/2010 02:24 0.0943 0.0839 0.0997 0.1052 0.1097

14 10/04/2010 03:28 0.0827 0.0870 0.0940 0.1035 0.1312

15 10/04/2010 04:32 0.0919 0.0957 0.1013 0.1041 0.1200

Mean 0.0896 0.0963 0.0949 0.1021 0.1071

Stdevn 0.0060 0.0076 0.0046 0.0065 0.0094

%RSD 6.65 7.91 4.87 6.33 8.79

Response Ratio* Reproducibility (n=15) Spiked Meat Extract

(100 fg/uL Natives / 1 pg/uL 13C-ISTDs)

2uL Injection : Natives 200 fg injected , 13C-ISTDs 2 pg Injected



Agilent 7000 GC-MS/MS

Performance of the 7000 GC-MS/MS System for the

Determination of Dioxins in Food and Feed

Comparative results vs GC-HRMS

Waters GC-HRMS



PCDFs in Feed / Eggs (Ukraine incident, 2010)

2378-TCDF in feed*

12378-PeCDF in feed*

2378-TCDF in eggs

23478-PeCDF in eggs

*20g feed sample

1.5 ng/Kg

0.37 ng/Kg

16 pg/g Fat

8.4 pg/g Fat



GC-HRMS vs. GC-MS/MS Results (upperbound TEQ)

PCDD/F-TEQ pg/g

LOD animal feed: GC-HRMS: 0.01 - 0.06 pg/g; GC-MS/MS: 0.02 - 0.08 pg/g



PCB 28

PCB 52

PCB 101

PCB 153

PCB 138

PCB 180

Each congener ~ 2mg/kg fat. Total = 12 mg/kg fat



GC-HRMS vs. GC-MS/MS Results

(Sum of 6 indicator ndl-PCBs)

(n = 67)



Dioxins and PCBs by GC-MS/MS Summary - 1

The Agilent 7000 Tandem quadrupole GC/MS system

provides :

• Chromatographic results that meet legislated screening requirements for “EU methods”

• Good linearity and response reproducibility for Dioxins as well as dl- and ndl-PCBs in food and feed over the range of interest

• Detection down to low pg WHO-TEQ/g



TEQ values of GC-HRMS and GC-MS/MS are

comparable and generally in a range of +/- 20%

At lower TEQ values (upperbound concentrations)

the difference between HRMS and MS/MS is due

to the 2-3 times lower LOD of the HRMS system

ndl-PCB analysis < 10 ng/g (sum of 6 individual PCBs)

is achievable

Dioxins and PCBs by GC-MS/MS Summary - 2



just a brief note

PAH analysis by GC-MS/MS



EU + EPA PAH‘s, 500 ng/mL, EIC Scan, 15m Select PAH column



EU + EPA PAH‘s, 10 ng/mL Extracted MRM, 15m Select PAH column



EU + EPA PAH‘s, 10 ng/mL, Extracted MRM, 15m Select PAH column



EU + EPA PAH‘s, 10 ng/mL Extracted MRM, 15m Select PAH column



EU + EPA PAH‘s, 10 ng/mL, Extracted MRM, 15m Select PAH column



EU + EPA PAH‘s, 10 and 1 ng/mL, MRM, 15m Select PAH column



Thank you for your attention!

Questions???

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Verbesserte GC/MS/MS Analyse von Dioxinen und ... - Agilent · Verbesserte GC/MS/MS Analyse von...

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