advances in the analysis of dioxin and environmental toxicants

Advances in the Analysis of Dioxin and Environmental Toxicants

2 2

Co-Authors

Eric Reiner, Terry Kolic, Karen MacPherson, Sheng Yang, Alina Muscalu, Adrienne Boden,

Karl Jobst, Patrick Crozier, Vin Khurana, Teresa Gobran, Liad Haimovici, , Li Shen

Christy Hartley, Rob Brunato, Vince Taguchi, Jeff Scott, Paul Helm, Tony Chen,

Vadrana Pantelic, Gerry Ladwig

Frank Dorman, Jack Cochran,

Peter Gorst-AllmanJoe Binkley, David E Alonso

Ian Brindle, Chris Marvin

3 3

What is Dioxin?

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated furans (PCDFs) • A group of 210 compounds with chlorines 0 to 8 chlorines

2,3,7,8-tetrachlorodibenzo-p-dioxin 2,3,7,8-tetrachlorodibenzofuran

44

What is Dioxin?

• Most toxic group of chemicals with LD50 as low as 1 ug/kg body weight

• Persistent, bioacummulative and toxic

• Health effects include: immune impairment, reproductive disorders, weight loss, development impairment, cancer

• Toxicity through the AhR binding

LD50: Lethal dose (50% test population)

6 6

NOEL = 3g/kg LD50 = 1ug/kg

TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin NOEL: No-observable effect-level LD50: Lethal dose (50% test population)

Dioxin Toxicity

8

Most toxic are planar with 3Å x 10Å dimensions

Dioxin-Like Toxicity

Toxic Equivalent Quantity (TEQ)

• Results reported in TEQ normalized to 2,3,7,8-TCDD

• TEQ = Σ[PCDDi×TEFi] + Σ[PCDFj×TEFj] + Σ[PCBk×TEFk] + Σ[DLCl×TEFl]

Where: i = 1−7, j = 1−10, k = 1−12, l = 1−?

10

Dioxin/PCB TEFs Eadon Ontario Germany California USEPA Nordic NATO-I WHO WHO WHO 1982 1984 1985 1986 1987 1988 1989 1994 1998 2005

PCDDs 2,3,7,8-TCDD 1 1 1 1 1 1 1 1 1 1,2,3,7,8-PeCDD 1 0.1 0.1 1 0.5 0.5 0.5 1 1 1,2,3,4,7,8-HxCDD 0.03 0.1 0.1 0.03 0.04 0.1 0.1 0.1 0.1 1,2,3,6,7,8-HxCDD 0.03 0.1 0.1 0.03 0.04 0.1 0.1 0.1 0.1 1,2,3,7,8,9-HxCDD 0.03 0.1 0.1 0.03 0.04 0.1 0.1 0.1 0.1 1,2,3,4,6,7,8-HpCDD 0 0.01 0.01 0.03 0.001 0.01 0.01 0.01 0.01

1,2,3,4,6,7,8,9-OCDD 0 0.0001 0.001 0 0 0.001 0.001 0.0001 0.0003

PCDFs 2,3,7,8-TCDF 0.33 0.5 0.1 1 0.1 0.1 0.1 0.1 0.1 1,2,3,7,8-PeCDF 0.33 0.5 0.1 1 0.1 0.01 0.05 0.05 0.03 2,3,4,7,8-PeCDF 0.33 0.5 0.1 1 0.1 0.5 0.5 0.5 0.3 1,2,3,4,7,8-HxCDF 0.01 0.1 0.01 0.03 0.01 0.1 0.1 0.1 0.1 1,2,3,6,7,8-HxCDF 0.01 0.1 0.01 0.03 0.01 0.1 0.1 0.1 0.1 1,2,3,7,8,9-HxCDF 0.01 0.1 0.01 0.03 0.01 0.1 0.1 0.1 0.1 2,3,4,6,7,8-HxCDF 0.01 0.1 0.01 0.03 0.01 0.1 0.1 0.1 0.1 1,2,3,4,6,7,8-HpCDF 0 0.01 0.01 0.03 0.001 0.01 0.01 0.01 0.01 1,2,3,4,7,8,9-HpCDF 0 0.01 0.01 0.03 0.001 0.01 0.01 0.01 0.01 1,2,3,4,6,7,8,9-OCDF 0 0.0001 0 0 0 0.001 0.001 0.0001 0.0003

dl-PCBs PCB-077 (3,3',4,4'-TCB) 0.0005 0.0001 0.0001

PCB-081 (3,4,4',5-TCB) 0 0.0001 0.0003

PCB-105 (2,3,3',4,4'-PeCB) 0.0001 0.0001 0.00003

PCB-114 (2,3,4,4',5-PeCB) 0.0005 0.0005 0.00003

PCB-118 (2,3',4,4',5-PeCB) 0.0001 0.0001 0.00003

PCB-123 (2',3,4,4',5-PeCB) 0.0001 0.0001 0.00003

PCB-126 (3,3',4,4',5-PeCB) 0.1 0.1 0.1

PCB-156 (2,3,3',4,4',5-HxCB) 0.0005 0.0005 0.00003

PCB-157 (2,3,3',4,4',5'-HxCB) 0.0005 0.0005 0.00003

PCB-167 (2,3',4,4',5,5'-HxCB) 0.00001 0.00001 0.00003

PCB-169 (3,3',4,4',5,5'-HxCB) 0.01 0.01 0.03

PCB-189 (2,3,3',4,4',5,5'-HpCB) 0.0001 0.0001 0.00003

11

Agent Orange

3× > miscarriagesin sprayed areas

12

Seveso, Italy – 1976

Chlorophenol plant explosion

•Normal sex ratio 106 males to 100 females• Seveso parents produced only 26 boys to 48 girls

Mocarelli et al, Lancet, 1996

13

Yuschenko Poisoning – 2002

14

Yuschenko Poisoning – 2009

15

16 16

Dioxin Sources

• Open burning/wood burning• Medical and waste incineration • Chemical fires• Auto exhaust• Chemical production• Tanneries• Metal reclamation• Industrial emissions

Pulp and paper plant lagoon 17

Open Barrel Burning

18

Hagarsville Tire Fire – 1990

19

Hagarsville Tire Fire – 1990

20

212121

Plastimet Recycling Plant Fire – 1997

21

22 22

POPs Food Contamination

• BB-153 FeedMaster/FireMaster• Belgian dairy – Dioxin/PCBs1999• Irish Chicken – Dioxin 2006• German chicken – Dioxin 2009

BB = Brominated Biphenyl

23 23

Method Attributes

• Sensitivity • Selectivity

• (Precision and Accuracy)

• Speed • $ (Cost)

Dioxin analysis requires selective/sensitive detection (≤ 100 fg) which requires concentration factors of 105 to 106

24 24

Dioxin Guidelines

• Food: 1–6 pg/g lipid weight (lw) – (EU)• Feed: 0.75–6 ng/kg (EU)• Air: 5 pg/M3 (ON)• Drinking Water 15 pg/L (ON)• Surface Water: 0.015 pg/L (ON)• Effluent: 60 pg/L – 5 pg/g 2,3,7,8-TCDD (ON)• Stack Emission: 80 pg/M3 (CAN)• Soils: 7 pg/g (ON)• Sediments: 0.85 pg/g (CAN)• Fish: 2.3 pg/g (ww) (ON)

ON = Ontario, CAN = Canada, EU = European Union, lw = lipid weight ww = wet weight

25 25

Dioxin Daily Intake Guidelines

• Average Daily intake: 1 to 3 pg/kg/day

Guidelines:U.S. EPA: 0.7 pg/kg/dayEU: 1–4 pg/kg/dayCanada: 10 pg/kg/day

26 26

POPs in Environmental Samples:the Analytical Challenge

• Ensure all labware, reagents and analytical instruments are free of contamination and interferences before beginning analysis

• Detect analytes at sub picogram level – to meet sub picogram detection limits for dioxin, e.g., every piece of labware should be prechecked (<500 fg) or labware segregated for high and low samples

• Determination of a representative sub-sample including gravimetric or volumetric determination for analysis. May require adjusting sample size or replicate analysis

• Addition of all internal and surrogate standards to the sample such that they will behave as the natural analytes in the sample during the analysis

• Quantitative extraction of analytes from matrix

• Extract may contain a gram or more of coextractable organic material including: Dioxins, Furans, PCBs PCNs, PCDEs, polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCs), brominated flame retardants (BFRs), and many other organic compounds including lipids, humic material and sulfur

27 27


* Cleaning of extracts to remove interfering coextractables to the degree where DQOs and QC limits can be met for dioxin analysis

* Not all coextractable compounds need to be removed, but they should not affect the separation or detection systems with respect to the analytes of interest

* Separation of target analytes from non-target or non-toxic isomers, congeners or interfering compounds (GC-HRMS)

* There are many congeners per analyte group* dioxins/furans: 210; PCBs: 209, PCNs 75

* Separate and accurately quantify all toxic congeners* dioxins/furans: 17, PCBs: 12, PCNs: 8–10

* Ensure method and instrument selectivity and sensitivity meet DQOs and QC limits

* Accredited laboratory (to ISO 17025), Quality System, trained/experienced analysts, proper instrument and analytical procedures, validated and documented methods and SOPs, control charting, non conformance and root cause determination

28 28


* Ensure quantitative accuracy* Calibration, blanks, spiked samples, CRMs, ILS, Performance Evaluation, surrogates and

internal standards, standard validation.

* Other Considerations:* Toxicity of compounds can range up to 6 orders of magnitude* T4CDD toxicity can range from: NOEL = 3 g/kg to LD50 = 1 ug/kg – must identify correct isomers

using proper GC columns and conditions.* Range of concentrations – fg/g (10-15 g/g ) to % levels – potential lab contamination and

instrument issues from carryover.* Range of sample types and complexities – biota, air, water, soil, hazardous waste which have

different matrix dependent and method requirement issues. Use method fit for purpose that has been validated for specific matrix being analyzed.

* Are the patterns of congeners representative of samples being analyzed, e.g., does an ash sample have a combustion source pattern or does a biota sample have only the toxic congeners present?

Analytical Approach

• Selection of appropriate method • Fit for Purpose

• Quantitative extraction of analytes from matrix • Cleanup of sample extract to remove

interfering matrix and coextractable compounds

• Chromatographic separation – selective detection

29

Analytical Approach• Gold standard – regulatory compliance

• Isotope dilution with13C labeled internal standards• Capillary gas chromatography separation• High resolution mass spectrometry detection at a resolution >

10,000 (10% valley definition) using selected ion monitoring

• Many more compounds in samples and grouped analyses desirable• Alternate methods like multidimensional chromatography,

tandem mass spectrometry and time-of-flight mass spectrometry can enhance selectivity. These techniques may not have the required sensitivity.

30

Horwitz Uncertainty Curve

from Eppe, Chemosphere 71 379, (2008) 31

32 32 32

Extraction

• Classical Method • Aqueous: filter particles, Soxhlet extract filter paper,

liquid/liquid extract aqueous phase and combine extracts

• Solid – Soxhlet extraction

• Green Methods • Solid phase extraction with particles together • Pressurized liquid extraction – PLE

33

Solid Phase Extraction

34


35

d Phase Extraction


37

Pressurized Liquid Extraction – PLE

38


39

Pressurized Liquid Extraction – PLE 40


41

42 4242

Extract Cleanup and Fractionation

• Multi-analyte groups are extracted together (dioxins/furans, PCNs , PCBs, PBDEs, HFRs) but cannot be separated in a single GC column analysis

• Coeluting / interfering compounds can be separated physically (into fractions) or by using multiple GC column phases (e.g., PCB 77 and 110)

• Sample extracts are separated using silica, carbon and alumina

• Dioxins/furans, PCNs and non-ortho PCBs in reverse carbon fraction

• Ortho substituted PCBs, PBDEs and other HFRs in forward carbon fraction

• Combining these analyses can save significant time and costs

Classical Open Column Sample Preparation

43

Classical Open Column Sample Preparation

44

FMS PowerPrep

45 FMS = fluid management systems

45

FMS PowerPrep

46

FMS – WMF-01 – 10 replicates

0

20

40

60

80

100

120

140

13C-23

78-T

CDF

13C-12

378-P

eCDF

13C-23

478-P

eCDF

13C-12

3478

-HxC

DF

13C-12

3678

-HxC

DF

13C-23

4678

-HxC

DF

13C-12

3789

-HxC

DF

13C-12

3467

8-HpC

DF

13C-12

3478

9-HpC

DF

13C-23

78-T

CDD

13C-12

378-P

eCDD

13C-12

3478

-HxC

DD

13C-12

3678

-HxC

DD

13C-12

3467

8-HpC

DD

13C-O

CDD

0

20

40

60

80

100

120

140

13C-23

78-T

CDF

13C-12

378-P

eCDF

13C-23

478-P

eCDF

13C-12

3478

-HxC

DF

13C-12

3678

-HxC

DF

13C-23

4678

-HxC

DF

13C-12

3789

-HxC

DF

13C-12

3467

8-HpC

DF

13C-12

3478

9-HpC

DF

13C-23

78-T

CDD

13C-12

378-P

eCDD

13C-12

3478

-HxC

DD

13C-12

3678

-HxC

DD

13C-12

3467

8-HpC

DD

13C-O

CDD0

20

40

60

80

100

120

140

13C-23

'4'5-te

tra pc

b (70

)

13C-34

4'5-te

tra PCB (8

1)

13C-33

'44'te

tra PCB (7

7)

13C-2'

344'5

-penta

PCB (123

)

13C-23

'44'5-

penta

PCB (118

)

13C-23

44'5-

penta

PCB (1

14)

13C-23

3'44'-

penta

PCB (105

)

13C-33

'44'5-

penta

PCB (126

)

13C-23

'44'55

'-hex

a PCB (1

67)

13C-23

3'44'5

hexa

PCB (1

56)

13C-23

3'44'5

'-hex

a PCB (1

57)

13C-33

'44'55

'-hex

a PCB (1

69)

13C-23

3'44'5

5'-he

pta P

CB (189

)

0

20

40

60

80

100

120

140

13C-23

'4'5-te

tra pc

b (70

)

13C-34

4'5-te

tra PCB (8

1)

13C-33

'44'te

tra PCB (7

7)

13C-2'

344'5

-penta

PCB (123

)

13C-23

'44'5-

penta

PCB (118

)

13C-23

44'5-

penta

PCB (1

14)

13C-23

3'44'-

penta

PCB (105

)

13C-33

'44'5-

penta

PCB (126

)

13C-23

'44'55

'-hex

a PCB (1

67)

13C-23

3'44'5

hexa

PCB (1

56)

13C-23

3'44'5

'-hex

a PCB (1

57)

13C-33

'44'55

'-hex

a PCB (1

69)

13C-23

3'44'5

5'-he

pta P

CB (189

)

020406080

100120140

13C-24

4'-tri

BDE 28

13C-22

'44'-te

tra B

DE 47

13C-22

'44'5-

penta

BDE 99

13C-22

'44'56

'-hex

a BDE 15

4

13C-22

'44'55

'-hex

a BDE 15

3

13C-22

'344'5

'6-he

pta B

DE 183

13C-de

ca BDE 20

9 0

20406080

100120140

13C-24

4'-tri

BDE 28

13C-22

'44'-te

tra B

DE 47

13C-22

'44'5-

penta

BDE 99

13C-22

'44'56

'-hex

a BDE 15

4

13C-22

'44'55

'-hex

a BDE 15

3

13C-22

'344'5

'6-he

pta B

DE 183

13C-de

ca BDE 20

9 0

20

40

60

80

100

120

140

13C-1,

3,5,7C

N-PCN(42

)

13C-1,

2,3,4C

N-PCN(27

)

13C-1,

2,3,5,

7CN-P

CN(52)

13C-1,

2,3,4,

5,7CN-P

CN(64)

13C-oc

taCN-P

CN(75)

0

20

40

60

80

100

120

140

13C-1,

3,5,7C

N-PCN(42

)

13C-1,

2,3,4C

N-PCN(27

)

13C-1,

2,3,5,

7CN-P

CN(52)

13C-1,

2,3,4,

5,7CN-P

CN(64)

13C-oc

taCN-P

CN(75)

PCDD/F DLPCB

BDEs PCNs

47 CRM = certified reference material 47

RM-EDF 2525 RM-EDF 2526 Compound Acceptable range Result Acceptable range Result pg/g pg/g pg/g pg/g PCDD/F 2378-TCDF 24.3 ± 4.74 26.7 18.7 ± 5.58 22.8 12378-PeCDF 4.58 ± 1.42 5.00 39 ± 7.36 31.0 23478-PeCDF 14.5 ± 4.04 13.4 37.8 ± 10.2 32.0 123478-HxCDF 5.95 ± 1.52 5.56 83.8 ± 23.0 64.9 123678-HxCDF 1.73 ± 0.54 3.94 62.8 ± 19.6 47.7 234678-HxCDF 1.04 ± 0.30 0.91 58.6 ± 14.2 44.2 123789-HxCDF 0.10 ± 0.20 0.17 57.3 ± 10.9 42.4 1234678-HpCDF 0.59 ± 0.44 0.60 81.6 ± 13.7 59.5 1234789-HpCDF 0.16 ± 0.32 0.24 76.7 ± 26.6 55.8 OCDF 0.38 ± 0.50 0.46 185 ± 57.4 134 2378-TCDD 17 ± 3.9 16.7 19.7 ± 4.18 18.8 12378-PeCDD 3.71 ± 0.90 4.47 39.9 ± 10.6 32.5 123478-HxCDD 0.33 ± 0.18 0.41 54.9 ± 7.8 40.1 123678-HxCDD 2.03 ± 0.60 1.96 51.1 ± 19.3 39.6 123789-HxCDD 0.30 ± 0.14 0.34 52.9 ± 18.1 42.1 1234678-HpCDD 0.48 ± 0.36 0.56 70.7 ± 23.2 57.5 OCDD 1.71 ± 1.38 1.86 181 ± 53.4 150

PLE – PowerPrep – CRMs

48

RM-EDF 2525 RM-EDF 2526 Compound Acceptable range Result Acceptable range Result pg/g pg/g pg/g pg/g DLPCBs 344'5-tetra PCB (81) 161 ± 74.0 199 3.0 ± 5.60 2.5 33'44'-tetra PCB (77) 1850 ± 834 2040 451 ± 179 546 2'344'5-penta PCB (123) 3280 ± 2020 6670 7.38 ± 9.58 25.6 23'44'5-penta PCB (118) 122000 ± 38000 122000 348 ± 392 468 2344'5-penta PCB (114) 3410 ± 1550 3890 7.73 ± 4.36 13.7 233'44'-penta PCB (105) 50100 ± 15700 5600 108 ± 73 178 33'44'5-penta PCB (126) 628 ± 242 654 431 ± 17.9 522 23'44'55'-hexa PCB (167) 7060 ± 3020 8070 12 ± 9.54 21.4 233'44'5-hexa PCB (156) 13100 ± 2620 14000 23.3 ± 23.8 34.2 233'44'5'-hexa PCB (157) 3380 ± 1010 3670 9.3 ± 9.16 12.1 33'44'55'-hexa PCB (169) 52.1 ± 14.0 53.8 512 ± 160 592 233'44'55'-hepta PCB (189) 1440 ± 498 1586 3.51 ± 2.76 5.1


49

RM-EDF 2525

Compound Acceptable range Result pg/g pg/g

BDEs 244'-tri BDE 28 312 ± 202 330 22'45'-tetra BDE 49 524 ± 274 550 22'44'-tetra BDE 47 9080 ± 2620 9610 23'44'-tetra BDE 66 262 ± 81.0 336 22'44'6-penta BDE 100 1720 ± 566 1540 22'44'5-penta BDE 99 2280 ± 472 2490 22'44'56'-hexa BDE 154 2550 ± 1000 2930 22'44'55'-hexa BDE 153 2030 ± 506 2040 22'344'5'6-hepta BDE 183 137± 47.8 133 Deca BDE 209 545 ± 1999 2440


50

51

CRM WMF-01 Wellington Labs – Fish

Quasimeme Biota Round 50

Target MOE Target MOE

BDE 28 3.1 ± 0.29 2.9 ± 0.3 0.014 0.019

BDE 47 123 ± 25 119 ± 35 0.35 0.36

BDE 100 35.9 ± 14.5 31.0 ± 5.3 0.11 0.11

BDE 99 37.5 ± 4.2 34.8 ± 3.8 0.54 0.51

BDE 154 19.8 ± 2.9 20.3 ± 2.3 0.088 0.083

BDE 153 17 ± 3 14.8 ± 0.5 0.10 0.074

BDE 183 0.093 0.089

BDE 209 5.2 4.3

N=4


Method

Solvent Usage (mL)

Time (days)*

Wages

Cost ($CDN)

Solvent

Column Packing

Total

Extraction Acid digestion 3620 2 40.00 18.40 10.77 29.17

ASE 960 1 20.00 4.04 3.83 7.87

Sample Cleanup Column Chromatography 7788 3 60 48.53 7.61 56.14

FMS 8176 2 40 38.30 68.85 107.15

Combined Extraction and Cleanup Acid digestion + Column Chromatography

11408

5

100

66.93

18.38

185.31

ASE + FMS 9136 3 60 42.34 72.68 175.02

Cost Comparison

52

Cape Sample Prep System

53 53

Cape Technologies – Spiked Surrogates n=10

54

WMF-01-A

WMF-01-B Certified Range EDF

2525 - A EDF

2525 - B Certified Range

Dioxins 2378-TCDF 11 11 13.1 ± 4.9 25 25 24.3 ± 4.74 12378-PeCDF 1.2 <1.0 1.53 ± 1.4 4.3 4.2 4.58 ± 1.42 23478-PeCDF 4.8 5.3 7.15 ± 2.2 14 13 14.5 ± 4.04 123478-HxCDF 1.8 2.1 0.86 ± 1.0 6.0 6.7 5.95 ± 1.52 123678-HxCDF 1.4 <0.34 0.51 ± 0.7 1.2 2.0 1.73 ± 0.54 234678-HxCDF <0.75 0.67 0.68 ± 1.2 0.79 <0.69 1.04 ± 0.30 123789-HxCDF <0.59 <0.55 0.25 ± 0.4 <0.41 <0.33 0.10 ± 0.20 1234678-HpCDF <0.50 <0.30 1.01 ± 1.9 <0.37 <0.28 0.59 ± 0.44 1234789-HpCDF <0.54 <0.61 0.30 ± 0.5 <0.34 <0.29 0.16 ± 0.32 OCDF <0.43 <0.56 1.38 ± 2.1 <0.39 <0.27 0.38 ± 0.50 2378-TCDD 11 11 13.1 ± 4.4 18 18 17.0 ± 3.90 12378-PeCDD 2.0 2.2 2.72 ± 1.3 3.9 3.4 3.71 ± 0.90 123478-HxCDD <0.35 <0.40 0.22 ± 0.3 <0.35 <0.28 0.33 ± 0.18 123678-HxCDD <0.59 0.6 0.88 ± 0.4 1.8 1.6 2.03 ± 0.60 123789-HxCDD <0.46 <0.55 0.27 ± 0.4 <0.48 <0.38 0.30 ± 0.14 1234678-HpCDD <0.51 <0.73 0.59 ± 0.7 <0.37 <0.39 0.48 ± 0.36 OCDD <0.81 <0.59 3.91 ± 6.2 <1.4 <1.1 1.71 ± 1.38

Dioxin Data Comparison

55

WMF-01-A

WMF-01-B Certified Range EDF

2525 - A EDF

2525 - B Certified Range

dlPCBs

344'5-tetra PCB (81) 122 127 201 ± 58 142 131 161 ± 74.0

33'44'-tetra PCB (77) 1716 1723 2233 ± 720 1678 1888 1850 ±834

2'344'5-penta PCB (123) 3258 3459 4233 ± 2620 4193 4086 3280 ± 2020

23'44'5-penta PCB (118) 107266 107901 130100 ± 32500 103411 91634 122000 ± 38000

2344'5-penta PCB (114) 3027 3049 3523 ± 1670 3698 3517 3410 ± 1550

233'44'-penta PCB (105) 47817 46705 49050 ± 14200 56116 54329 50100 ± 15700

33'44'5-penta PCB (126) 615 618 739 ± 260 638 608 628 ± 242

23'44'55'-hexa PCB (167) 9244 9027 9750 ± 3090 7773 7479 7060 ± 3020

233'44'5-hexa PCB (156) 13906 13504 14890 ± 5020 13727 13083 13100 ± 2620

233'44'5'-hexa PCB (157) 3405 3329 3488 ± 870 3688 3534 3380 ± 1010

33'44'55'-hexa PCB (169) 68 65 76 ± 30 52 50 52.1 ± 14.0

233'44'55'-hepta PCB (189) 1932 1911 2016 ± 611 1505 1483 1440 ± 498

dlPCB Data Comparison

56

The automated extraction/cleanup method: • Is simple, non-labour intensive, time- and cost-saving • Decreases the possibility of human health hazards from solvent

exposure as well as solvent usage • Minimizes background contamination • Combines four analyte groups (PCDDs/PCDFs, DLPCBs, PCNs

and BDEs) in a single extraction using only 2 or 3 columns for clean-up and fractionation

• Reduces the time of sample preparation from 15 to 3 working days and increases productivity

• Is robust, repeatable, accurate, has lower method detection limits and greater analytical precision than the separate classical methods

Automated Sample Prep Summary

57

58

Analyte Potential Coeluting Compounds on 5% phenyl

2,3,7,8-T4CDF 1,2,4,9- T4CDF, 1,2,7,9- T4CDF, 2,3,4,6- T4CDF, 2,3,4,7- T4CDF, 2,3,4,8- T4CDF 1,2,3,7,8-P5CDF 1,2,3,4,8- P5CDF 2,3,4,7,8- P5CDF 1,2,4,8,9- P5CDF, 1,2,3,6,9- P5CDF, 1,2,6,7,9- P5CDF 1,2,3,4,7,8-H6CDF 1,2,3,4,6,7- H6CDF 2,3,7,8-T4CDD 1,2,3,7- T4CDD*, 1,2,3,8- T4CDD*, 1,2,3,9- T4CDD* (*nearest neighbors) 1,2,3,7,8- P5CDD PCB-169 (fragment ion) 1,2,3,7,8,9- H6CDD 1,2,3,4,6,7- H6CDD PCB-77 PCB-110 PCB-81 PCB-87 PCB-123 PCB-149 PCB-126 PCB-178, PCB-129 PCB-156 PCB-171 PCB-157 PCB-201

Gas Chromatographic Separation

Gold standard for dioxins: • 60 m, 0.25 mm id, 0.25 um ft, 5% phenyl-methyl column for

analysis • 30 m, 0.25 mm id, 0.25 um ft, 50% cyanopropyl,

50% phenylmethyl polysiloxane column for confirmation of coeluting congeners

GC Column comparison

DB-5, Rtx-5MS HP5-MS, Equity-5

CP-Sil 8 CB/MS

DB-5MS ZB-5MS

Rtx- Dioxin2

ZB5UMS DB-XLB DB-225 SP-2331

PCDDs

2,3,7,8-TCDD + + + – + + + + + + + – + – + – 1,2,3,7,8-PeCDD + + + – + – – – – – – – – – – – 1,2,3,4,7,8-HxCDD + + + + + + + + + + + + + + + + 1,2,3,6,7,8-HxCDD + + + + + + + + + + + + + + + + 1,2,3,7,8,9-HxCDD – – + – + – + + + + + + + + + + 1,2,3,4,6,7,8-HpCDD + + + + + + + + + + + + + + + + 1,2,3,4,6,7,8,9-OCDD + + + + + + + + + + + + + + + + PCDFs 2,3,7,8-TCDF – – + – + – ++ ++ + + + + + – 1,2,3,7,8-PeCDF + + + + + + ++ ++ + + – – – – 2,3,4,7,8-PeCDF – – – – – – – – – – – – + + + + 1,2,3,4,7,8-HxCDF – – + + + + + + + + + + + + – – 1,2,3,6,7,8-HxCDF + + + + + + + + + + + + – – + + 1,2,3,7,8,9-HxCDF + + – – – – – – – – + + ++ + + 2,3,4,6,7,8-HxCDF + – – – – – – – – – + – – – + + 1,2,3,4,6,7,8-HpCDF + + + + + + + + + + + + ++ + + 1,2,3,4,7,8,9-HpCDF + + + + + + + + + + + + ++ + + 1,2,3,4,6,7,8,9-OCDF + + + + + + + + + + + + ++ + +

++ = Baseline separation, +- = partial cocelution, - - = complete coelution 59

from Tondeur, Biomed. Environ. Mass Spectrom. 14 449 (1987)

Potential interferences in TCDD analysis

60

Compound Ion Resolution Interferent Needed

Nonachlorobiphenyl 321.8491 7,236 No Pentachlorobiphenylene 321.8677 12,432 Yes Heptachlorobiphenyl 321.8678 12,480 Yes Hydroxytetrachlorodibenzofuran 321.8936 Infinity Yes – removed in cleanup DDE 321.9292 9,044 No Polychloroxanthenes 321.9114 18,089 Yes Tetrachloromethoxybiphenyl 321.9299 8,870 No

61

Potentially interfering compounds to consider

62

Column Performance

win/res mix-f dlp-cs4 back

15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50Time0

100

%

jan10_02std3 2: Voltage SIR 25 Channels EI+ 321.8936

1.89e7

1289-tcdd

\1237/1238-tcdd

2378-tcdd

1234-tcdd

5% phenyl-methyl column

63

TCDF Ion Channel for Flyash Extract c101863-0004

21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50Time0

100

%

0

100

%

may12_res15 2: Voltage SIR 20 Channels EI+ 303.9016

5.90e523.60

22.52

21.67

20.8821.08

21.53

21.76

21.9022.20

22.03

22.40

23.3323.14

22.69 22.84

23.01

23.67

23.8724.57

may12_res15 2: Voltage SIR 20 Channels EI+ 317.9389

9.97e623.49

13C12-2,3,7,8-tcdf

Flyash Extract

64 64

Comparison of GC Columns Column Length (m) 20 60 40

Inner diameter (mm) 0.1 0.25 0.18

Film thickness (µm) 0.1 0.25 0.18

Theoretical plates/m 8,600 3,300 5,300

Total plates (for column) 172,000 198,000 212,000

Calculated no. plates (T4CDD) 176,000 230,000 285,000

Relative Efficiency 0.93 1 1.03

Relative Anal. Time 0.33 1 0.55

Need 175,000 plates to separate critical pair – 1,2,3,7/1,2,3,8 and 2,3,7,8 TCDD

65

Changes in parameters • shorter & narrower columns • thinner stationary phase films • faster oven temperature programming rates • higher pressures, faster carrier gas flow rates

Faster analyses ⇒ increased sample throughput Narrower peak widths place new demands on detection systems

Fast GC – Method Attributes

66

What is Fast GC ?

GC ramp rates >50ºC/min. GC column diameters <0.18 mm Stationary phase <0.18 µm GC column head pressures >60 psi Phase ratio: β = r / df r = column diameter df = film thickness

67

Handbook of GC/MS – H.J. Huebschmann

Comparison of Different Column Dimensions

68

25% Valley

25% Valley

60m – 0.25μm, 0.25mm 5% phenyl

20m – 0.1 μm, 0.1mm 5% phenyl

RTA = 38%

RT = 23.4

RT = 9.00

Fast GC Chromatogram

How many data points are necessary to correctly characterize a chromatographic peak?

69

Seven to ten points per

peak is optimum

70

71

Reduction in Analysis Time Using Microbore Columns

60 m (.25/.25)

40 m (.18/.18)

30 m (.25/.25)

20 m (.10/.10)

10 m (.10/.10)

Dioxins [50] 28 (44%) 14 (72%)

PCB Congeners [90] 18 (80%)

PAH [40] 22 (55%) 14 (65%)

OC Pesticides [55] 12* (78%)

72 72

Parallel Columns

• Most compound classes cannot be uniquely separated on a single chromatographic phase – e.g., PCBs, dioxins, PAHs

• Extracts must be separated or analyzed on 2 phases• Parallel columns can be used to analyze multiple

fractions simultaneously• e.g., dioxins/furans/coplanar PCBs and ortho PCBs

• Extracts can also be analyzed separately• Analysis and confirmation in the same run • Both columns must be temperature compatible

73 73

Parallel Column Advantages

Mono-ortho PCBs elute from 20m column well before PCDD/Fs elute from 40 m column

DPEs, and interfering PCBs in mono-ortho DLPCB sample fraction

Avoid potential interferences: 1) Furan formation in ion source 2) Reduces coelution of higher chlorinated PCBs with

coplanars (i.e., PCB 110 with PCB 77)

74 74

GC Configuration

HP6890 + model GC • 2 injection ports and 2 autosamplers

2 GC Columns • Dioxin/furan & coplanar PCBs injected on

40 m Rtx5, 0.18 mm x 0.20 µm

• Mono-ortho PCBs – 20 m Rtx5, 0.1 mm x 0.1 µm

75 75

The Gas Chromatograph

76 76 76 76

77 77

123 \

118/ 114

/

105/

167156 \

157/

81 \

77/

126 169

2378-TCDD

12378-PCDD 123478-HxCDD \

123678-HxCDD /123789-HxCDD

/ 123478-HpCDD/

OCDD

Fraction 1: 20 mMono Ortho PCBs

Fraction 2: 40 mCoplanar PCBs

Fraction 2: 40 mTetra – Octa PCDDs

189/

78 78

7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00Time0

100

%

0

100

%

0

100

%

7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00Time0

100

%

P5CBs on 20 m column (PCB105/114/118/123)

H6CBs on 20 m column (PCB156/157/167)

H7CBs on 20 m column (PCB189)

Tetra to octa dioxins on a 40 m column

PCDFs and coplanar PCBs (77/81/126/169) not shown

T4CDDs P5CDDs

H6CDDs

H7CDDs OCDD

Dioxins and WHO PCBs in NIST 1944

79

Dioxin/Furan Results Comparison

NIST 1944

MARINE SED.

Single

(pg/g)

Dual

(pg/g)

Ref. Value

(pg/g) 2378-TCDD 136 128 133 ± 9

12378-PCDD 19 22 19 ± 2

123478-HxCDD 29 27 26 ± 3

23478-PCDF 48 45 45 ± 4

OCDF 1300 1200 1100 ± 100

79

80

DLPCB Results Comparison

NIST 1944

MARINE SED.

Single

(pg/g)

Dual

(pg/g)

Ref. Value

(pg/g)

PCB 105 24,000 26,000 24,500 ± 1100

PCB 118 54,500 62,600 58,000 ± 4300

PCB 156 5900 6150 6520 ± 660

PCB 126 270 262 N/A

PCB 169 16 14 N/A

81 81

Two-Dimensional GC (GC×GC)

• Produces higher peak capacity (more chromatographic peaks per space). Increases peak capacity to 50 × 20 = 1000 compounds

• Eliminates the need for second column confirmation. Can do multiple analyte groups in same run and may eliminate need for extract fractionation

• Fast analysis – requires fast detector – e.g., time-of-flight mass spectrometer (TOFMS), ECD

• Provides structured chromatograms for excellent selectivity

• Provides much more information

• Results in increased sensitivity

82 82

Injector

1D 2DModulator

TOFMS

1tR

PM

Y

X

2tRY2tRX

X+Y

InjectorInjector

1D 2DModulator

TOFMSTOFMS

1tR

PM

Y

X

2tRY2tRX

X+Y

PM = modulation time D = dimension

GC×GC Schematic Diagram

Retention Time (tR)

Modulator

82

83 83

1tR

2tR

Signal

YX

XY1tRX

1tRY

2tRY2tRX

1tR

2tR

Signal

YX

XY1tRX

1tRY

2tRY2tRX

Second Dimension Modulation

Second dimension retention time

Inside GC Oven

84

2nd Oven

Modulator 85

86 86

Peak Modulation

15440.74

15440.72

15460.70

• Original peak is “chopped” into 3 peaks

• Sensitivity enhancement occurs through focusing

• Second dimension peaks are only 400 ms wide

• Need detector capable of defining peaks– Hundreds of spectra/sec

ms = millisecond

87 87

Comprehensive GC×GC

Form of 2DGC: Orthogonal Column Setup

1st Dimension (Column) Standard (10–60 m, 0.25 mm, 0.25 um) Non-polar (DB-1, Rtx-5)

2nd Dimension (Column)

Very short (1–2 m) Narrow bore, thin film (0.10–0.18 mm, 0.10–0.18 um) Polar or shape selective (DB-1701, Rtx-PCB)

88 88

PCBs Challenging Separations DB5-10M/0.18/0.18_DB17-2M/0.1/0.1

89 89

PCB Standard by GC×GC-ECD Orthogonal Elution

206*209

194205*

208*199*

189

169

157156170

126

183*

128167

177171

202

191*

201

180

187178*

158138

149

188*

123

153168*

8177

114

118

105

95

155*

10199

119

87 110*

151*49

104*

44

3774 70

18*

15

54*

33

28

22

52

819

PCB STD (BP-MS)

*to be confirmed by GCxGC/TOF-MS

Deca-

Nona-Octa-

Hepta-

Hexa-

Penta-

Tetra-

Tri-

Di-

90 90

Dioxin-Like PCBs vs. Other PCBs

81

m/z 292 326 360 394

Rtx-1 >

Rtx

-PC

B >

123118

114

189169

157156

167

126

10577

91

Sediments by GC×GC-ECD

676

1

2176 1676 1176

2

3

0

2nd D

imen

sion

(s)

1st Dimension (s)

Unknown compounds

PCBs/OCs

CBz PCNs, PCDEs

Dioxins/Furans

PCA bands

GCxGC-TOFMS of sediment sample C for chlorinated dioxins and furans

40m x 0.18mm x 0.18µm Rtx-Dioxin2

1m x

0.1

5mm

x 0

.15µ

m R

xi-1

7Sil

MS

GCxGC-TOFMS of sediment sample C for chlorinated dioxins and furans


1m x

0.1

5mm

x 0

.15µ

m R

xi-1

7Sil

MS

m/z ions plotted: 306 322 340 356 374 390 408 424 444 460

Dioxin/furan elution area…

GCxGC-TOFMS of sediment sample C for chlorinated dioxins and furans, zoom

1m x

0.1

5mm

x 0

.15µ

m R

xi-1

7Sil

MS


2378 TCDD

Only m/z 322 is plotted. Note isobaric interferences eluting below 2378 TCDD that would interfere in 1D GC.

GCxGC-TOFMS of sediment sample B for chlorinated dioxins and furans, zoom

1m x

0.1

5mm

x 0

.15µ

m R

xi-1

7Sil

MS


While looking for Cl6 PCBs (m/z 360), we see some interesting later-eluting peaks… Cl6

PCBs

GCxGC-TOFMS of sediment sample B for chlorinated dioxins and furans, zoom

1m x

0.1

5mm

x 0

.15µ

m R

xi-1

7Sil

MS


Dibromopyrenes!

97 97

Background

Muir & Howard, ES&T, 2006, 40, 7157-7166US TSCA – United States Toxic Substances Control Act

98

Stockholm Agreement Compounds

Original 12 Added 2009 Under Discussion aldrin, chlordane, dieldrin, DDT, endrin, heptachlor, hexachlorobenzene (HCB), mirex, toxaphene, polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF)

chlordecone, α-hexachlorocyclohexane, β-hexachlorocyclohexane, hexabromobiphenyl, tetra- to hepta-bromodiphenylether, lindane, (δ-hexachlorocyclohexane), pentachlorobenzene, perfluoroctanesulfonic acid (PFOS), its salts and perfluoroctanesulfonyl fluoride

short chain chlorinated paraffins (SCCPs), endosulfan, hexabromocyclododecane (HBCD)

99

Sediment CRM – Lake Ontario

Analysis Time: 7.3 minutes

CRM = certified reference material

100 100

Sediment CRM – Target Compounds

2 1. Phenanthrene 2. Anthracene 3. Fluoranthene 4. Pyrene 5. Benzofluoranthenes 6. Benzo[e]pyrene 7. Benzo[a]pyrene 8. Perylene

1 3 5

4 6

7 8

101 101

Time-of-Flight Mass Spectrometry

PCB-87 PCB-81

p,p’-DDE

PCB-77 PCB-110

PCB-82 PCB-151

Endrin Dieldrin

102

PAHs, PCBs & Organochlorine Pesticides by GC-TOF

g-Chlordane

Pyrene

PCB-101

PCB-99

Endosulphan-1

a-Chlordane

PCB-119

103

GC- HR TOF of Dioxins

800 1000 1200 1400 1600 1800 2000 2200 24000.0e0

1.0e5

2.0e5

3.0e5

4.0e5

Time (s)XIC(305.898±0.005) XIC(321.893±0.005) XIC(339.859±0.005) XIC(355.854±0.005)XIC(373.820±0.005) XIC(389.815±0.005) XIC(407.781±0.005) XIC(423.776±0.005)XIC(423.776±0.005) XIC(443.739±0.005) XIC(459.734±0.005)

1150 1200 1250 1300

CS3WT

RFWHM = 25,000

103

104

GC- HR TOF MS Dioxin Sensitivity

1200 1220 1240 1260 1280 1300 1320 1340 1360 13800.0e0

5.0e2

1.0e3

1.5e3

2.0e3

2.5e3

3.0e3

3.5e3

Time (s)XIC(305.898±0.005) CSL PEIS 70eV XIC(321.893±0.005) CSL PEIS 70eV XIC(305.898±0.005) CS-0.5 PEIS 70eVXIC(321.893±0.005) CS-0.5 PEIS 70eV XIC(305.898±0.005) CS-1 PEIS 70eV XIC(321.893±0.005) CS-1 PEIS 70eV

TCDF TCDF

CS1 (500 fg/µL; 2 µL inj.)

CS0.5 (250 fg/µL; 2 µL inj.)

CSL (100 fg/µL; 2 µL inj.)

TCDD

104

105

HR TOF MS Full Scan Data

OCDF

OCDD

105

106

GC- HRTOF MS – Additional Compounds

700 800 900 1000 1100 1200 1300 14000.0e0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

Time (s)

Name Similarity Formula Mass Accuracy (ppm)1-Chloropyrene 913 C16H9Cl -0.21

4-Phenyldibenzofuran 879 C18H12O 0.35Benzo[2,1-b:3,4-b']bisbenzofuran 891 C18H10O2 0.22

Pyreno[4,5-b]furan (CAS) 918 C18H10O 0.211,3-Dichlorobenzo[c]phenanthrene 821 C18H10Cl2 0.13

106

107

12 14 16 18 200.0e0

3.0e5

6.0e5

9.0e5

1.2e6

1.5e6

1.8e6

2.1e6

2.4e6

2.7e6

Time (minutes)

Name Formula RDBE Mass Accuracy (ppm)Benzo[ghi]fluoranthene C18H10 14 -1.14Triphenylene C18H12 13 -0.89Benz[a]anthracene, 7-methyl- C19H14 13 -1.21Benzo[k]fluoranthene C20H12 15 -0.42

GC- HR TOF MS – Additional Compounds

107

Plastimet Fire – 1997

108

109

110,000 FWHM

2048K data points

29,000 FWHM

512K data points

7,000 FWHM

128K data points

1,500 FWHM

32K data points

Fourier Transform Mass Spectrometry

Ultrahigh resolution (The above mass range is 0.3 Da)

ROMS (Regular old mass spectrometer)

R=100,000

109

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

150 200 250 300 350 400 450 500 550

Ken

dric

k M

ass

Def

ect

Nominal Kendrick Mass

Kendrick plot - vegetation exposed to fallout from the 1997 Plastimet fire

unassignedCHCHClCHClOCHO

Kendrick Plot

Kendrick mass = mass x (35 / 34.9689)

114

118

Summary of Method Enhancements

• The analysis of dioxin and related compounds requires the most sensitive and selective instrumentation

• Most POPs sample preparation procedures can be automated or semi-automated and many can be combined to save money and time

• Fast GC, Parallel GC and GC×GC can significantly reduce sample analysis times and costs while increasing analytical capacity.

• Kendrick Plots can be used to identify unknown compounds.

• GC×GC can increase selectivity and sensitivity and can also be used for analytical triage. Extract fractionation may not be required.

• These enhancements can save time, costs and reduce the use of solvents and reagents.

4th Annual Multidimensional Chromatography Workshop

Toronto, Canada – January 8 & 9, 2013

Confirmed Speakers:

Daniela Cavgnino – Dani Jack Cochran – Restek John Dimandja – Collage Frank Dorman – Penn State Taduesz Gorecki – Waterloo Teruyo Ieda – Gerstel Philip Marriott – Monash University Brian McCarry – McMaster Jef Focant – U Liege John Seeley – Oakland University Heather Bean – Vermont Catherine Rawlinson – Murdoch U No Registration Fee

Contact Eric Reiner at : [email protected]

119

Thank You ! [email protected]

References:

E.J. Reiner, R.E. Clement, A.B. Okey, C.H. Marvin, “Advances in the Analysis of Polychlorinated Dibenzo-p-dioxins, Dibenzofurans and Dioxin-like Polychlorinated Biphenyls in Environmental Samples” Analytical and Bioanalytical Chemistry, 386, 791 (2006).

E.J. Reiner, A.R. Boden, T. Chen, K.A. MacPherson, A.M. Muscalu, Advances in the analysis of persistent halogenated organic compounds, LC GC Europe, February, 23(2), 60 (2010) E.J. Reiner, “Analysis of Dioxin and Related Compounds” Mass Spectrometry Reviews, 29(4), 526 (2010).

V.Y Taguchi, RJ Nieckarz, RE Clement. S Krolik, R Williams, Dioxin Analysis by Gas Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (GC-FTICRMS), J Am Soc Mass Spectrom 21, 1918 (2010)

120

advances in the analysis of dioxin and environmental toxicants

Documents