Dioxin/Furan Screening Method
John D. Ragsdale, GC/MS Product Specialist
GC/MS/MS Dioxin Screening
In The Beginning… Current Methodology The Plan Sample Preparation The GC/MS/MS Configuration The data
In The Beginning…
Interest in the analysis of dioxins and furans have increased due to the contamination of feed for livestock and poultry in Europe. The local sales force has responded by requesting application notes and other literature for this analysis on PolarisQ.
Similar requests have come from our Pan-Pacific and Asian sales force. This group also includes a current Finnigan customer directly involved in the analysis of dioxin and furan contaminated samples.
Dioxin/Furan Analysis EPA Methods
Low Resolution MS– 8270 ( full scan )– 8280 ( SIM )– Part per million and
part per billion detection limits
High Resolution MS– 8290 ( SIM )– 1613 ( SIM )– Part per trillion
detection limits
The Plan
Sample Preparation– Minimal cleanup required
Chromatographic Resolution– BPX-5; 25M; 0.15mm ID
column
Robust Chromatography– Split injections (2 ul injection)
Specificity– MS/MS
Deliverables– Write SOPs
Sample Preparation
25 grams of material mixed with 50 grams of sodium sulfate.
Soxhlet extraction for 24 hours with 50:50 hexane : methylene chloride.
Partition against 4 X 40ml concentrated sulfuric acid.
Concentrate to 100 ul of 5X dilution of CS3 (2pg/ul of tetra-isomers)
Additional Sample Preparation
Gel Permeation Chromatography– Removes high molecular weight interferences
Acid, neutral, and basic silica gel and carbon– Removes nonpolar and polar interferences
Alumina, Florosil– Removes chlorodiphenyl ethers
Any additional cleanup will lower ultimate detection limits but is not required for this MS/MS screening technique
Dioxin Screening GC/MS/MS System
Trace GC with split/splitless inlet PPKD DPFC 700kPa with Manual Split Flow 250 Liter Turbo pump PolarisQ EI only / Vacuum Interlock AS2000 Liquid Autosampler SGE BPX-5, 25 meter, 0.15 mm ID, 0.25
micron film column Restek Silco-Steel split liners w/glass wool
plug
Configuration Rationale
Split Injections allow for “dirtier” sample introduction.
Extracts can be screened without the time and cost of extensive cleanup.
MS/MS affords the selectivity to remove matrix interference from “dirty” extracts.
Chromatographic Conditions
Injector: – 250C
Column Flow: – 0.9 ml/min
Split flow: – 10 ml/min
Ramp:
– 150C (1min) to 200C @ 20C/min (1.5min)– to 300C @ 3C/min (1.7min) : 40min total
Chromatographic Resolution
Tetra-Dioxins Tetra-FuransRT: 16.93 - 20.35 SM: 3G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Relati
ve Ab
unda
nce
NL:2.25E5
m/z= 321.50-322.50 MS pcb1248-20psi-05
NL:4.70E4
m/z= 333.50-334.50 MS pcb1248-20psi-05
RT: 16.93 - 20.35 SM: 5G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Relati
ve Ab
unda
nce
NL:2.66E5
m/z= 305.50-306.50 MS pcb1248-20psi-05
NL:5.20E4
m/z= 317.50-318.50 MS pcb1248-20psi-05
2,3,7,8TCDD-IS
2,3,7,8TCDF-IS
2,3,7,8TCDD 2,3,7,8
TCDF
Full Scan, SIM, or MS/MS
Full Scan – 10pg/ul in 25g Cow’s milkRT: 0.00 - 40.04
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
7.927.75
7.26
8.26
27.28
6.99
26.788.42
27.4625.34
29.00
25.0829.31
6.46 24.8437.3621.60 29.72
23.3829.96
8.73 20.8420.09 30.4019.85
35.1318.1331.288.99 17.49 34.5112.23 12.78 38.0036.126.08 9.76
5.69
NL:2.93E7
TIC MS CS3-MILK-PCDD-PCDF-FS-402
Dioxins: EPA Method 1613
Linearity to 500 fg/uL to 200 pg/uL for 2,3,7,8-TCDD
Dioxins: Typical 500 fg/ul injection
RT: 17.09 - 20.19 SM: 7G
17.5 18.0 18.5 19.0 19.5 20.0
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
500fg 2,3,7,8-TCDD
100pg C13-2,3,7,8-TCDD
NL: 3.60E1
m/z= 256.5-257.5+258.5-259.5 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS ICIS CS1-MSMS-012001-102
NL: 7.11E3
m/z= 267.5-268.5+269.5-270.5 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS ICIS CS1-MSMS-012001-102
Dioxins:Typical Precision for 1613
COMPOUND % RSD
2,3,7,8-TCDD 10.8
1,2,3,7,8-PeCDD 6.4
1,2,3,4,7,8-HxCDD 6.9
1,2,3,6,7,8-HxCDD 9.1
1,2,3,7,8,9-HxCDD 8.7
1,2,3,4,6,7,8-HpCDD 4.9
OCDD 5.2
Dioxins:Typical Precision for 1613 ISTD
COMPOUND % RSD
C13-2,3,7,8-TCDD 4.2
C13-1,2,3,7,8-PeCDD 5.6
C13-1,2,3,4,7,8-HxCDD 8.6
C13-1,2,3,6,7,8-HxCDD 6.9
C131,2,3,7,8,9-HxCDD 7.0
C13-1,2,3,4,6,7,8-HpCDD
8.9
C13-OCDD 9.2
Dioxins: Isotope Ratio StabilityCOMPOUND
IONS RATIO % RSD
2,3,7,8-TCDD
259/257 0.97 22.7
1,2,3,7,8-PeCDD
275/277 0.83 10.0
1,2,3,4,7,8-HxCDD
325/327 0.69 20.1
1,2,3,6,7,8-HxCDD
325/327 0.66 13.15
1,2,3,7,8,9-HxCDD
325/327 0.69 14.4
1,2,3,4,6,7,8-HpCDD
361/363 1.35 9.5
OCDD 395/397 1.14 5.7
Dioxins: Full Scan – 10pg/ul std vs. cow’s milk
RT: 16.93 - 20.35 SM: 7G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Relati
ve Abu
ndance
NL:2.82E3
m/z= 321.5-322.5 MS CS3--PCDD-PCDF-FS-404
NL:2.07E4
m/z= 333.5-334.5 MS CS3--PCDD-PCDF-FS-404
RT: 16.93 - 20.35 SM: 7G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Relativ
e Abun
dance
NL:1.91E4
m/z= 321.5-322.5 MS CS3-MILK-PCDD-PCDF-FS-402
NL:3.41E4
m/z= 333.5-334.5 MS CS3-MILK-PCDD-PCDF-FS-402
Nonane Cow’s milk
2,3,7,8TCDD
2,3,7,8TCDD-IS
2,3,7,8-TCDD ??
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
1,2,3,4TCDD-RS
Dioxins: MS/MS – 10pg in nonaneCS3--PCDD-PCDF-MSMS-403 01/17/2001 06:14:33 PM1613 CS3 MSMS :: 120 :: WFON
RT: 16.93 - 20.35 SM: 7G
17.0 17.5 18.0 18.5 19.0 19.5 20.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bun
danc
e
18.97
18.44
19.24 19.4617.46 18.75 19.83 20.0717.9517.2518.95
18.45
19.44 19.7617.80 20.2317.9417.5517.06
NL: 1.00E3m/z= 256.50-257.50+258.50-259.50 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS CS3--P CDD-P CDF-MSMS-403
NL: 9.38E3m/z= 267.50-268.50+269.50-270.50 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS CS3--P CDD-P CDF-MSMS-403
CS3--P CDD-P CDF-MSMS-403 #966-1043 RT: 18.83-19.12 AV: 16 NL: 1.53E2F: + c SRM ms2 [email protected] [ 253.00-263.00]
253 254 255 256 257 258 259 260 261 262 263m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bun
danc
e
257
259
261
263258253
260
256254 262255
264261
2,3,7,8TCDD
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
Dioxins: MS/MS – 10pg/ul in cow’s milkCS3-MILK-PCDD-PCDF-MSMS-403 01/17/2001 06:58:07 PM
1613 CS3 MILK MSMS :: 120 :: WFON
RT: 16.93 - 20.35 SM: 7G
17.0 17.5 18.0 18.5 19.0 19.5 20.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
nda
nce
19.01
18.4720.0019.5617.63 17.7417.25 18.16 18.64
19.00
18.49
19.33 19.56 20.0217.87 18.3017.14 17.53
NL: 1.32E3m/z= 256.50-257.50+258.50-259.50 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS CS3-MILK-P CDD-P CDF-MSMS-403
NL: 1.36E4m/z= 267.50-268.50+269.50-270.50 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS CS3-MILK-P CDD-P CDF-MSMS-403
CS3-MILK-P CDD-P CDF-MSMS-403 #1263-1342 RT: 18.93-19.15 AV: 16 NL: 2.53E2F: + c SRM ms2 [email protected] [ 253.00-263.00]
253 254 255 256 257 258 259 260 261 262 263m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bun
danc
e
257
259
263
253
261
258
260 262
254255
256264
2,3,7,8TCDD
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
Furans: EPA Method 1613
Linearity from 500 fg/uL to 200 pg/uL for 2,3,7,8-TCDF
Furans: Typical 500 fg/ul InjectionRT: 17.09 - 20.19 SM: 7G
17.5 18.0 18.5 19.0 19.5 20.0
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
500fg 2,3,7,8-TCDF
100pg C13-2,3,7,8-TCDF
NL: 7.27E1
m/z= 240.5-241.5+242.5-243.5 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS ICIS CS1-MSMS-012001-102
NL: 8.90E3
m/z= 251.5-252.5+253.5-254.5 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS ICIS CS1-MSMS-012001-102
Furans:Typical Precision for 1613
COMPOUND %RSD
2,3,7,8-TCDF 17.41,2,3,7,8-PeCDF 5.52,3,4,7,8-PeCDF 6.21,2,3,4,7,8-HxCDF 3.51,2,3,6,7,8-HxCDF 4.02,3,4,6,7,8-HxCDF 5.61,2,3,7,8,9-HxCDF 6.41,2,3,4,6,7,8-HpCDF 4.51,2,3,4,7,8,9-HpCDF 5.8OCDF 5.4
Furans:Typical Precision for 1613 ISTD
COMPOUND %RSD
C13-2,3,7,8-TCDF 4.0C13-1,2,3,7,8-PeCDF 4.5C13-2,3,4,7,8-PeCDF 4.7C13-1,2,3,4,7,8-HxCDF 7.2C13-1,2,3,6,7,8-HxCDF 5.5C13-2,3,4,6,7,8-HxCDF 6.2C13-1,2,3,7,8,9-HxCDF 7.7C13-1,2,3,4,6,7,8-HpCDF 8.1C13-1,2,3,4,7,8,9-HpCDF 9.4C13-OCDD 9.2
Furans: Isotope Ratio StabilityCOMPOUND IONS RATIO % RSD2,3,7,8-TCDF 243/241 0.81 22.71,2,3,7,8-PeCDF 275/277 0.81 7.12,3,4,7,8-PeCDF 275/277 0.82 7.01,2,3,4,7,8-HxCDF
309/311 0.67 10.4
1,2,3,4,7,8-HxCDF
309/311 0.67 8.1
1,2,3,6,7,8-HxCDF
309/311 0.65 5.5
2,3,4,6,7,8-HxCDF
309/311 0.65 7.9
1,2,3,4,6,7,8-HpCDF
345/347 1.26 7.3
1,2,3,4,7,8,9-HpCDF
345/347 1.29 7.0
OCDF 379/381 1.14 5.6
Furans: Full Scan – 10pg/ul std vs. cow’s milk
RT: 16.93 - 20.35 SM: 7G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100Re
lative
Abunda
nceNL:2.96E3
m/z= 305.5-306.5 MS CS3--PCDD-PCDF-FS-404
NL:2.53E4
m/z= 317.5-318.5 MS CS3--PCDD-PCDF-FS-404
RT: 16.93 - 20.35 SM: 7G
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Relati
ve Ab
undanc
e
NL:1.36E4
m/z= 305.5-306.5 MS CS3-MILK-PCDD-PCDF-FS-402
NL:4.10E4
m/z= 317.5-318.5 MS CS3-MILK-PCDD-PCDF-FS-402
Nonane Cow’s Milk
2,3,7,8TCDF ???
2,3,7,8TCDF-IS
2,3,7,8TCDF
2,3,7,8TCDF-IS
Furans: MS/MS – 10pg/ul in nonaneCS3--PCDD-PCDF-MSMS-403 01/17/2001 06:14:33 PM
1613 CS3 MSMS :: 120 :: WFON
RT: 16.93 - 20.35 SM: 7G
17.0 17.5 18.0 18.5 19.0 19.5 20.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bun
danc
e
18.44
17.82 18.7517.5017.31 19.05 19.2217.98 19.81 20.0318.43
18.9017.7917.47 19.2118.22 19.55 19.75 20.0417.32
NL: 1.25E3m/z= 240.50-241.50+242.50-243.50 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS CS3--P CDD-P CDF-MSMS-403
NL: 1.22E4m/z= 251.50-252.50+253.50-254.50 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS CS3--P CDD-P CDF-MSMS-403
CS3--P CDD-P CDF-MSMS-403 #838-895 RT: 18.35-18.54 AV: 11 NL: 3.14E2F: + c SRM ms2 [email protected] [ 237.00-247.00]
237 238 239 240 241 242 243 244 245 246 247m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bun
danc
e
241
243
245
242
244240
247237 246238 239
2,3,7,8TCDF
2,3,7,8TCDF-IS
Furans: MS/MS – 10pg/ul in cow’s milk
CS3-MILK-PCDD-PCDF-MSMS-403 01/17/2001 06:58:07 PM1613 CS3 MILK MSMS :: 120 :: WFON
RT: 16.93 - 20.35 SM: 7G
17.0 17.5 18.0 18.5 19.0 19.5 20.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
18.49
17.65
17.9218.6317.24 18.10 19.60 20.0219.5019.08
18.48
18.74 19.0317.84 19.12 19.4817.57 18.30 19.84 20.1717.3117.19
NL: 1.64E3m/z= 240.50-241.50+242.50-243.50 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS CS3-MILK-P CDD-P CDF-MSMS-403
NL: 1.65E4m/z= 251.50-252.50+253.50-254.50 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS CS3-MILK-P CDD-P CDF-MSMS-403
CS3-MILK-P CDD-P CDF-MSMS-403 #1086-1159 RT: 18.41-18.61 AV: 15 NL: 3.85E2F: + c SRM ms2 [email protected] [ 237.00-247.00]
237 238 239 240 241 242 243 244 245 246 247m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
241
243
247
245237
246
244
242238
239240
248
2,3,7,8TCDF
2,3,7,8TCDF-IS
Spiked Matrix @ 2pg/ul or 4 pg/g (ppt)
Cow’s milk Soil Coal fly ash
Dioxin: 2 pg/ul in 25g Cow’s Milk RT: 15.23 - 20.92 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100Re
lative
Abu
ndan
ceNL: 1.95E2
m/z= 256.5-257.5+258.5-259.5 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS CS3-5X-MILK-012101-212
NL: 1.89E3
m/z= 267.5-268.5+269.5-270.5+271.5-272.5 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS CS3-5X-MILK-012101-212
RT: 15.09 - 20.74 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
NL: 2.08E2
m/z= 240.5-241.5+242.5-243.5 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS CS3-5X-MILK-012101-212
NL: 2.25E3
m/z= 251.5-252.5+253.5-254.5+255.5-256.5 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS CS3-5X-MILK-012101-212
TCDD TCDF
2,3,7,8TCDD
2,3,7,8TCDF
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
2,3,7,8TCDF-IS
Dioxin: 2 pg/ul in 25g Soil RT: 15.23 - 20.92 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
NL: 3.81E2
m/z= 256.5-257.5+258.5-259.5 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS CS3-5X-SOIL-012101-212
NL: 1.99E3
m/z= 267.5-268.5+269.5-270.5+271.5-272.5 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS CS3-5X-SOIL-012101-212
RT: 15.09 - 20.74 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
NL: 2.39E2
m/z= 240.5-241.5+242.5-243.5 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS CS3-5X-SOIL-012101-212
NL: 2.17E3
m/z= 251.5-252.5+253.5-254.5+255.5-256.5 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS CS3-5X-SOIL-012101-212
2,3,7,8TCDD
2,3,7,8TCDF
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
2,3,7,8TCDF-IS
TCDD TCDF
2 pg/ul in 25g Coal Fly Ash RT: 15.23 - 20.92 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
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50
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70
80
90
1000
10
20
30
40
50
60
70
80
90
100Re
lativ
e Ab
unda
nce
NL: 1.87E2
m/z= 256.5-257.5+258.5-259.5 F: + c SRM ms2 [email protected] [ 253.00-263.00] MS CS3-5X-FLYASH-012101-212
NL: 1.83E3
m/z= 267.5-268.5+269.5-270.5+271.5-272.5 F: + c SRM ms2 [email protected] [ 264.00-274.00] MS CS3-5X-FLYASH-012101-212
RT: 15.09 - 20.74 SM: 7G
16 17 18 19 20
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
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80
90
100
Rela
tive
Abun
danc
e
NL: 2.29E2
m/z= 240.5-241.5+242.5-243.5 F: + c SRM ms2 [email protected] [ 237.00-247.00] MS CS3-5X-FLYASH-012101-212
NL: 2.54E3
m/z= 251.5-252.5+253.5-254.5+255.5-256.5 F: + c SRM ms2 [email protected] [ 248.00-258.00] MS CS3-5X-FLYASH-012101-212
TCDD TCDF
2,3,7,8TCDD
2,3,7,8TCDF
2,3,7,8TCDD-IS
1,2,3,4TCDD-RS
2,3,7,8TCDF-IS
Summary of analyte recovery
Matrix Average (pg/ul)
% RSD
(n = 12)
Nonane 2.18 10.2Soil 1.91 9.7Milk 2.28 7.6Coal Fly Ash 2.05 10.2
Matrix Average (pg/ul)
% RSD
(n = 12)
Nonane 1.84 7.8Soil 1.90 11.5Milk 1.92 11.5Coal Fly Ash 1.95 5.6
2,3,7,8-TCDD(2 pg/ul spike)
2,3,7,8-TCDF(2 pg/ul spike)
Summary of isotope ratios
Matrix Average (cali = 0.97)
% RSD
(n = 12)
Nonane 0.93 11.6Soil 0.83 17.1Milk 1.06 20.4Coal Fly Ash 0.99 24.3
Matrix Average (cali = 0.81)
% RSD
(n = 12)
Nonane 0.80 20.8Soil 0.82 20.6Milk 0.87 17.6Coal Fly Ash 0.89 19.7
2,3,7,8-TCDD259/257
2,3,7,8-TCDF243/241