1. Dioxin analysis in water by Isotope Dilution using Triple Quadrupole GC/MS
Presentation Title
11/28/2018
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Regulatory statement (if applicable))
Diana Wong, Ph.D.
GC/MS Scientist
National Environmental Monitoring Conference
New Orleans, Louisiana
August 6, 2018

2. Overview Method Introduction and Project Outline
Presentation Title
11/28/2018
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Current EPA Method 1613 Revision B
Requires GC/HRMS to achieve a high resolution of ≥10,000
Performance based method
Investigate GC/MS/MS for dioxin analysis per Method 1613B
Replace HRMS with MS/MS to simplify analysis and cost of operation/maintenance
Goal
Isomer specificity
Calibration Linearity
Calibration Verification (VER)
Relative Retention Time
Initial Precision and Accuracy (IPR)
Instrument Detection Limit (IDL)
Analysis of spiked real water sample
Equivalency Requirements

3. Nomenclature and Isomers
Presentation Title
11/28/2018
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Dioxins
Polychlorinated dibenzodioxin (CDDs)
Furans
Polychlorinated dibenzofurans (CDFs)
Cl Atoms
CDD Isomers
CDF Isomers 1 2 4 10 16 3 14 28 22 38 5 6 7 8
Total 75
135 cv Cl Cl cv Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl
2,3,7,8 – TCDD
(2,3,7,8-Tetrachlorinated dibenzodioxin)
2,3,4,7,8 – PCDF
(2,3,4,7,8-Pentachlorinated dibenzofurans)
17 toxic isomer
75 total CDD isomers
135 total CDF isomers

4. Toxic Equivalent Factors (TEF)
Presentation Title
11/28/2018
17 Toxic CDDs/CDFs Investigated
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Toxic CDDs
I-TEF
WHO2005-TEF
2,3,7,8-TCDD 1
1,2,3,7,8-PCDD
0.5
1,2,3,4,7,8-HxCDD
0.1
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
0.01
1,2,3,4,6,7,8,9-OCDD
0.001
0.0003
Toxic CDFs
I-TEF
WHO2005-TEF
2,3,7,8-TCDF
0.1
1,2,3,7,8-PCDF
0.05
0.03
2,3,4,7,8-PCDF
0.5
0.3
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
0.01
1,2,3,4,7,8,9-HpCDF
1,2,3,4,6,7,8,9-OCDF
0.001
0.0003
TEQ = 𝒊=𝟏 𝟏𝟕 [𝑪𝑫𝑫/𝑪𝑫𝑭 ] 𝒊 ( 𝒏𝒈 𝑳 )× 𝑻𝑬𝑭 𝒊 ( 𝑻𝑬𝑸 𝒏𝒈 𝑳 )

5. EPA Method 1613B Sample preparation
Presentation Title
11/28/2018
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13C Internal Standard
Clean-up
standard
Injection
standard
Filtration
(~1 L water)
Soxlet >16 h
(filter paper)
Liquid/Liquid Extraction
(filtrate)
Sample Clean-up
(silica, alumina, florisil, or carbon column)
GC/HRMS
vs GC/MS/MS
EPA 1613B is a performance based method

6. GC/MS/MS System Parameters
11/28/2018
2mm Dimpled, splitless, UI
Inlet liner
MMI Inlet  MSD
Constant Flow
Flow 1.1 mL/min
GC Parameters
100 °C (2 min)
30 °C/min to 220 °C (16 min)
2 °C/min to 240 °C (5 min)
5 °C/min to 270 °C (4 min)
15 °C/min to 330 °C (6 min)
Oven program
Automatic
Liquid Sampler
Pulsed Splitless
1 µL injection
62 °C (0.31 min)  ramp to 330 °C at 600 °C/min
Multimode Inlet (MMI)
7890 GC
7010 EI MS/MS
DB-5ms UI
60m x 0.25mm ID
0.25 µm film
Helium Carrier Gas
HES Source
Source: 300 °C
Quads: 150 °C
Transfer Line: 350 °C
He Quench Gas:
4 mL/min
N2 Collision Gas:
1.5 mL/min
Oven

7. GC Oven Temperature Program Optimization
Presentation Title
11/28/2018
Slow Oven Program for Isomeric Separation
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Optimal Oven Temperature Program
>25% valley
First eluted
1368-TCDD
<25% valley
Rate
Oven Temperature
Hold time
(Initial)
100 °C
2 min
30 °C/min
220 °C
2 °C/min
250 °C
10 min
260 °C
5 °C/min
280 °C
15 °C/min
330 °C
5 min
Rate
Oven Temperature
Hold time
(Initial)
100 °C
2 min
30 °C/min
220 °C
16 min
2 °C/min
240 °C
5 min
5 °C/min
270 °C
4 min
15 °C/min
330 °C
6 min

8. Order of Isomer Specificity
Presentation Title
11/28/2018
Percent Valley must be less than 25% between toxic 2378-TCDD and the closest eluted isomers
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DB-5 Column
EPA Method 1613B
DB-5ms UI Column
Current Study
1,2,3,9-TCDD
2,3,7,8-TCDD
1,2,3,7/1,2,3,8-TCDD
<25% Valley
<25% Valley
(EPA Method 1613B, 1994)
(Fishman et al., 2004 and 2011; Wilken et al., 2008)

9. MRM Transition and Collision Energy Optimization
Presentation Title
11/28/2018
RRT, Ion Ratios, and S/N meet Method 1613B Criteria
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Calibration Level 3
Method 1613B Criteria
13C-2,3,7,8-TCDD
331.9267.9 CE 26
333.9269.9 CE 26
RT: min
2,3,7,8-TCDD
319.9256.9 CE 26
321.9258.9 CE 26
RT: min
m/z
Two transitions
RRT = 1.001
(Criteria )
Method 1613B Limits
Relative Retention Time (RRT)
QC limits of ±15%
Ion Abundance Ratio
2378-TCDD
Ion Ratio = 104.8
Criteria ( )
13C-2378-TCDD
Ion Ratio = 106.1
Criteria ( )
Sample >2.5
Calibration >10
Signal to Noise (S/N)
s/n 543
s/n 2569

10. Retention Time Window Defining Solution
Presentation Title
11/28/2018
Method 1613B requires all isomers to fall within first and last eluters
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1,2,3,7,8,9 -
EPA Method 1613B (1994): Window Defining Solution defines the beginning (first eluted) and ending (last eluted) retention times for dioxin and furan isomers to demonstrate isomer specificity. Standards must contain compounds listed in this order

11. Tetrachlorinated dibenzodioxins (TCDD)
Presentation Title
11/28/2018
Peaks correlate and are well separate on GC/MS/MS and GC/HRMS
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GC/MS/MS
Abundance
321.9258.9
Toxic
2378-TCDD
First eluted
1368-TCDD
1239-TCDD
Last eluted
1289-TCDD
𝑥+𝑦=4
22 isomers
1 toxic
<25% valley
GC/HRMS
Abundance
First eluted
1368-TCDD
1239-TCDD
Toxic
2378-TCDD
Last eluted
1289-TCDD
<25% valley

12. Tetrachlorinated dibenzofurans (TCDFs)
Presentation Title
11/28/2018
Peaks correlate and are well separate on GC/MS/MS and GC/HRMS
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305.9242.9
GC/MS/MS
Abundance
First eluted
1368-TCDF
Toxic
2378-TCDF
Last eluted
1289-TCDF
𝑥+𝑦=4
38 isomers
1 toxic
Toxic
2378-TCDF
GC/HRMS
Abundance
First eluted
1368-TCDF
Last eluted
1289-TCDF

13. Hexachlorinated dibenzofurans (HxCDFs)
Presentation Title
11/28/2018
Peaks correlate and are well separated on GC/MS/MS and GC/HRMS
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Last eluted HxCDF
Toxic HxCDF
Split peaks between toxic and non-toxic HxCDF
GC/MS/MS
Abundance
373.8310.9
First eluted
HxCDF
Toxic HxCDF
Toxic HxCDF
Toxic HxCDF
Toxic HxCDF
GC/HRMS
Abundance
Toxic HxCDF
Last eluted
HxCDF
𝑥+𝑦=6
16 isomers
4 toxic
First eluted
HxCDF
Toxic HxCDF
Toxic HxCDF

14. Hexachlorinated dibenzodioxins (HxCDDs)
Presentation Title
11/28/2018
Peaks correlate and are well separate on GC/MS/MS and GC/HRMS
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GC/MS/MS
Abundance
391.8328.8
First eluted
HxCDD
Toxic HxCDD
Last eluted and toxic
HxCDD
Toxic HxCDD
𝑥+𝑦=6
10 isomers
3 toxic
Toxic HxCDD
GC/HRMS
Abundance
First eluted
HxCDD
Last eluted and toxic
HxCDD
Toxic HxCDD

15. Establishing Method Equivalency
Presentation Title
11/28/2018
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Calibration linearity
Calibration verification
Relative retention time
Initial precision and recovery
Ongoing precision and recovery
Labeled compound recovery
Method detection limit demonstration (Instrument detection limit)
Performed
Analysis of a performance testing (PT) on real samples
Ongoing

16. Calibration (CAL), Linear Range, and Relative Retention Time
Presentation Title
11/28/2018
Met Method 1613B criteria
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Level
Cal (ppt) L1
200 L2
500 L3
1000 L4
4000 L5
10000 L6
50000 L7
250000 L8 L9
Analyte RF
(Avg)
(CS1)
RF Difference
(CS1-Avg)
RF RSD
S/N
RRT
1613B
RRT Criteria
Pass/Fail
2378-TCDD
1.123
1.004
-11% 6 25
1.002
Pass
2378-TCDF
0.97
0.943
-3%
2.9 50
1.001
12378-PCDD
0.985
0.994 1%
3.5 42
12378-PCDF
0.991
1.025 3%
2.8 54
23478-PCDF
1.007
0.997
-1%
2.1 63
1.000
HxCDD
0.999
4.2 21
HxCDF
0.924
0.921 0%
4.4 33
HxCDD
0.929
0.917
3.6
HxCDF
0.908
0.877
4.5 43
HxCDD
1.027
5.3
HxCDF
0.912
0.902
5.2 38
HxCDF
0.983 2%
4.1 48
HpCDD
1.008
1.033 4 83
HpCDF 92
HpCDF
0.948 5% 90
OCDD
1.056
1.040
2.4
150
OCDF
0.913
0.940
148
Note:
RF = 𝑨 𝟐,𝟑,𝟕,𝟖−𝐓𝐂𝐃𝐃,𝑺𝒕𝒅 𝑨 𝟏𝟑𝐂,𝑺𝒕𝒅 × 𝑴 𝟏𝟑𝐂,𝑺𝒕𝒅 (𝒏𝒈) 𝑴 𝟐,𝟑,𝟕,𝟖−𝐓𝐂𝐃𝐃,𝑺𝒕𝒅 (𝒏𝒈)
CS1 Range
RF difference -11% to 5%
RF RSD: 2.1 to 6
S/N: 21 to 15
EPA 1613B
Cal Range
1613B Criteria: RF Difference < 15%; Avg RSD < 10%; S/N (CS1) > 10;
Relative Retention Time (RRT) must meet RRT criteria

17. Verification Standard Recoveries
Presentation Title
11/28/2018
Calibration is verified and passed Method1613B criteria
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18. Initial Precision and Accuracy (IPR)
Presentation Title
11/28/2018
All native and labeled recoveries are within Method 1613B Criteria
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19. Spiked Water Sample (GC/HRMS vs GC/MS/MS)
Presentation Title
11/28/2018
Similar concentration detected in water sample on both systems!
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H7CDD

20. Low working range and sensitivity
Presentation Title
11/28/2018
50 femtogram of 2378-TCDD can be detected by GC/MS/MS
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Primary Native 2378-TCDD S/N = 5.8
Secondary Native 2378-TCDD S/N = 4.6
Method 1613B Criteria
> 2.5 signal to noise ratio
The relative ion intensities is within 15% difference to the calibration average

21. Isomer specificity and Separation Passed Method1613B Performance
Summary
Presentation Title
11/28/2018
GC/MS/MS meets Method 1613B Criteria in water sample
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HRMS to MS/MS
Good correlation
Detected similar concentration in spiked water samples
Isomer specificity and Separation
< 25% valley between toxic 2378-TCDD and closest isomer
Isomer Specificity observed
Analytes elute within the defined retention time window
Passed Method1613B Performance
RF RSD CC
S/N
RRT
VER
IPR

22. Acknowledgements
Project Collaborators Hui Lin, Ph.D. Jessica Betz Jim Dunn Greg Martin David Somerville, Ph.D. Brian Pike Chuck Sueper Agustin Pierri
Agilent Technologies Craig Marvin Dale Walker Tarun Anumol, Ph.D. Jeff Nutter Matt Leyden Tom Barrett
7890 Gas Chromatograph
7010 Triple Quadrupole
Mass Spectrometer

23. Thank you NEMC! Diana Wong, Ph.D. GC/MS Scientist
Agilent Booth #16 and #17