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The Truth About Dioxin Analysis: Using High Resolution GC-TOFMS and GCxGC-TOFMS to
Uncover the Pollutants Ignored by Targeted Uncover the Pollutants Ignored by Targeted Analytical Approaches
Peter Gorst-Allman1, Jack Cochran2, Jayne de Vos3 and Eric Reiner4
1LECO Africa (Pty) Ltd., 2Restek Corporation, 3NMISA, and 4Ministry of the Environment, Toronto
Common Abbreviations used in the PresentationCommon Abbreviations used in the Presentation
TCDD Tetrachloro-dibenzo-p-dioxinPCDD Pentachloro-dibenzo-p-dioxinHeCDD Hexachloro-dibenzo-p-dioxinHpCDD Heptachloro-dibenzo-p-dioxinOCDD Octachloro-dibenzo-p-dioxinPCDD Polychloro -dibenzo -p-dioxin
TOFMS Time of Flight Mass Spectrometry
HRT High Resolution Time of Flight Mass Spectrometry
EPA Environmental Protection AgencyPCDD Polychloro -dibenzo -p-dioxin
TCDF Tetrachloro-dibenzofuranPCDF Pentachloro-dibenzofuranHeCDF Hexachloro-dibenzofuranHpCDF Heptachloro-dibenzofuranOCDF Octachloro-dibenzofuranPCDF Polychloro-dibenzofuranGC-HRMS Gas Chromatography – High
Resolution Mass Spectrometry
GCxGC Comprehensive Gas Chromatography
AgencySIM Selected Ion MonitoringPOP Persistent Organic PollutantRRF Relative Response FactorEI Electron IonisationeV Electron VoltageLOD Limit of DetectionMS/MS Tandem Mass Spectrometryppm Parts per millionDa Daltonsm/z Mass to charge ratio
The Current SituationThe Current Situation
Globally, chlorinated dioxins and furans are analysed by expensive gas chromatography –gas chromatography –high resolution mass
spectrometry (GC-HRMS) using EPA
Methods such as 8290A or 1613B, or local
methodology based on these
Why does HRMS have the edge?Why does HRMS have the edge?
Dioxins usually occur at low levels in the presence of much higher matrix interferences
The selectivity and sensitivity of HRMS is needed: selectivity is achieved by working at a resolution of +/ - 10 000, sensitivityresolution of +/ - 10 000, sensitivityby using SIM (selected ion monitoring)
Ion abundance ratios are used to confirm identity (e.g. for TCDD 319.8965/321.8936, theoretically 0.77, allowed 0.65 – 0.89)
Disadvantages of HRMSDisadvantages of HRMSTo achieve the sensitivity required by regulatory methods, the HRMS approach uses selected ion monitoring (SIM)
This is a target molecule technique, and requires a list of the selected analytes before running the analysis to set up the experimental parametersexperimental parameters
Only the targeted compounds are located – all other information about the sample is forfeited
Using high res systems to do SIM is also expensive and requires a high degree of operator sophistication
Can we fashion a different approach?Can we fashion a different approach?
Time of Flight (TOF) mass spectrometers do not use SIM. All analyses provide full range mass spectra for all the analytes in the sample
This is an important advantage, and provided the This is an important advantage, and provided the mandated sensitivities can be obtained using TOF, we should then have a method which can locate and identify all components of the sample, while also being able to quantify targeted PCDDs and PCDFs, all in one run
This applies both to the Pegasus 4D (GCxGC-TOFMS) and to the Pegasus HRT (High Resolution TOFMS)
The Pillars of the The Pillars of the TOFMS TOFMS approachapproach
For the GCxGC-TOFMS, Selectivity would be Selectivity would be
provided by the increased peak capacity of GCxGC, and Sensitivity by the enhanced
S/N due to the focussing (improved peak sharpness) provided by the modulator
For the HRT, Selectivity would be provided by the accurate mass
capability (< 1 ppm), and Sensitivity by the removal of
chemical noise due to the high resolution capability
The GoalThe Goal
Determine if GCXGC-TOFMS and GC-HRT can provide the selectivity necessary
Determine if the techniques can identify other POP components present in real world samples, which are not located by the target approach
Determine if GCXGC-TOFMS and GC-HRT has the sensitivity to calibrate 2378-TCDD and 2378-TCDF down to 0.5 pg/µl as required by EPA Method 1613, and to analyse samples at this level
selectivity necessary to measure 17 dioxins and furans (from Method 1613, assigned due to their toxicity) in a variety of sample matrices
by the target approach
The ApproachThe Approach
Use these Spike and run Data mine the Run the relevant 13C
labelled standard set
for 1613B
Use these results to
obtain response
factors for the PCDD/Fs
Spike and run the sample set and calculate quantitative
values for the PCDD/Fs
Data mine the samples for other POPs
which may be present in the
samples
Sample SetSample SetSix soil and sediment samples (provided by Prof Eric Reiner from the Ministry of the Environment, Toronto)
These samples had been extracted and worked up according to the procedures outlined in:
Sample Preparation for POPs Analysis Sample Preparation for POPs Analysis
• Concentrated to 20 µL • GC-HRMS
– EI at lower eV (35eV)
• Air-dry samples • Addition of labelled
standards – EI at lower eV (35eV)– 10,000 Resolution – SIM
• LECO Pegasus GC-HRT • LECO Pegasus 4D
GCxGC-TOFMS
standards• Soxhlet extraction • Column clean-up
– Acid-base-AgNO 3 Si – Alumina – Carbon
Analysis Conditions GCAnalysis Conditions GC--HRMSHRMS
Inlet 280°CInlet 280°C
Constant Flow He at 0.8 mL/minConstant Flow He at 0.8 mL/min
40m x 0.18mm x 0.18 µm DB-540m x 0.18mm x 0.18 µm DB-540m x 0.18mm x 0.18 µm DB-540m x 0.18mm x 0.18 µm DB-5
140°C (1min), 52°C/min to 200°C, 2.9°C/min to 235°C (3 min), 3°C/min to 267°C (3 min), 7°C/min to 310°C (Hold until OCDD el utes) 140°C (1min), 52°C/min to 200°C, 2.9°C/min to 235°C (3 min), 3°C/min to 267°C (3 min), 7°C/min to 310°C (Hold until OCDD el utes)
Transfer Line 280°CTransfer Line 280°C
HRMS, EI+, SIM, 35 eVHRMS, EI+, SIM, 35 eV
Analysis Conditions GCAnalysis Conditions GC--HRTHRT
Inlet 280°CInlet 280°C
Corrected Constant Flow He at 1 mL/minCorrected Constant Flow He at 1 mL/min
40m x 0.18mm x 0.18 µm Rtx -Dioxin2 ( Arylene backbone -modified 40m x 0.18mm x 0.18 µm Rtx -Dioxin2 ( Arylene backbone -modified 40m x 0.18mm x 0.18 µm Rtx -Dioxin2 ( Arylene backbone -modified siloxane column selective for coplanars) 40m x 0.18mm x 0.18 µm Rtx -Dioxin2 ( Arylene backbone -modified siloxane column selective for coplanars)
140°C (1min), 50°C/min to 200°C, 3°C/min to 260°C, 1°C/min to 280°C, 6°C/min to 310°C (5 min) 140°C (1min), 50°C/min to 200°C, 3°C/min to 260°C, 1°C/min to 280°C, 6°C/min to 310°C (5 min)
Transfer Line 300°CTransfer Line 300°C
HRT, EI+, 140 to 520 Da, 3 spectra/sec, 50 eVHRT, EI+, 140 to 520 Da, 3 spectra/sec, 50 eV
40m x 0.18mm x 0.18 µm Rtx-Dioxin2 (Arylene backbone-modified siloxane column selective for coplanars) 40m x 0.18mm x 0.18 µm Rtx-Dioxin2 (Arylene backbone-modified siloxane column selective for coplanars)
120°C (2min), 20°C/min to 200°C, 5°C/min to 320°C ( 3 min) 120°C (2min), 20°C/min to 200°C, 5°C/min to 320°C ( 3 min)
1.0m x 0.15mm x 0.15 µm Rxi-17Sil MS (50% phenyl (silarylene) / 50% methyl type siloxane ) 1.0m x 0.15mm x 0.15 µm Rxi-17Sil MS (50% phenyl (silarylene) / 50% methyl type siloxane )
Analysis Conditions GCxGCAnalysis Conditions GCxGC--TOFMSTOFMS
methyl type siloxane ) methyl type siloxane )
+ 5°C offset from primary column + 5°C offset from primary column
Corrected Constant Flow He at 1.4 mL/min (Speed optimized flow)Corrected Constant Flow He at 1.4 mL/min (Speed optimized flow)
Thermal modulation, 2.0 sec (Hot time 0.70 sec, Cool time 0.30 sec) Thermal modulation, 2.0 sec (Hot time 0.70 sec, Cool time 0.30 sec)
TOFMS, EI+, 45 to 750 Da, 100 spectra/sec TOFMS, EI+, 45 to 750 Da, 100 spectra/sec
Sample 1Sample 1INSTRUMENT
2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 3.5 3.0 2.7 4.5 1.46 2.2 1.7 ND ND
GC-HRMS 3.7 2.6 1.7 5.6 1.1 5.4 1.7 1.5 0.81
GCxGC-TOFMS
ND ND ND ND ND 11 ND ND ND
GCxGC-3.5 5.6 6.6 12 ND 9.8 5.7 6.5 ND
GCxGC-TOFMS (x5)
3.5 5.6 6.6 12 ND 9.8 5.7 6.5 ND
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT 3.1 ND ND 12 33 ND 19 150
GC-HRMS 1.7 1.6 0.60 15 24 1.5 24 170
GCxGC-TOFMS
ND ND ND ND 31 16 ND 160
GCxGC-TOFMS (x5)
6.3 ND ND ND 31 16 31 170
pg/g
Sample 2Sample 2
INSTRUMENT2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 20 28 21 21 2.8 76 32 23 10
GC-HRMS 26 32 18 19 2.8 85 35 17 3.7
GCxGC-TOFMS 22 48 18 20 ND 90 40 32 NDTOFMS
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT 13 14 7.9 280 170 55 2400 1200
GC-HRMS 9.3 6.0 2.3 300 160 47 2600 1900
GCxGC-TOFMS ND ND ND 410 230 120 2400 1500
pg/g
Sample 3Sample 3
INSTRUMENT 2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 48 55 22 140 10 220 37 42 17
GC-HRMS 46 69 14 180 3.1 240 21 14 5.2
GCxGC-TOFMS 40 73 32 150 21 210 46 41 18TOFMS 40 73 32 150 21 210 46 41 18
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT 18 28 13 620 240 51 980 790
GC-HRMS 14 8.6 1.5 990 230 17 1100 1300
GCxGC-TOFMS 41 32 36 950 210 58 980 990
pg/g
Sample 4Sample 4
INSTRUMENT2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 52 3.2 120 39 5.1 280 190 8.9 5.1
GC-HRMS 59 3.9 140 55 4.7 330 210 28 4.1
GCxGC-TOFMS 48 5.8 130 36 14 210 210 75 23TOFMS
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT 13 20 86 710 65 380 6500 170
GC-HRMS 9.9 8.1 50 1000 70 470 5200 220
GCxGC-TOFMS 14 18 100 1000 66 460 5100 190
pg/g
Sample 5Sample 5
INSTRUMENT2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 40 ND 35 24 ND 97 35 23 ND
GC-HRMS 50 0.65 40 31 1.9 120 48 20 2.9
GCxGC-TOFMS 43 ND 43 30 ND 120 47 29 NDTOFMS
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT ND 14 17 290 71 72 1100 370
GC-HRMS 5.8 3.9 4.7 420 75 79 1200 550
GCxGC-TOFMS 10 15 38 400 78 90 1200 480
pg/g
Sample 6Sample 6
INSTRUMENT2378-TCDF
2378-TCDD
12378-PCDF
23478-PCDF
12378-PCDD
123678-HxCDF
123478-HxCDF
123789-HxCDF
123478-HxCDD
GC-HRT 12 15 16 17 5.2 71 30 7.2 5.8
GC-HRMS 18 20 19 17 2.5 73 28 14 2.9
GCxGC-TOFMS 15 21 21 18 ND 59 28 17 7.8TOFMS
INSTRUMENT123678-HxCDD
123789-HxCDD
234678-HxCDF
1234678-HpCDF
1234678-HpCDD
1234789-HpCDF
OCDF OCDD
GC-HRT 10 12 6.7 180 120 30 1800 1100
GC-HRMS 8.3 5.2 2.7 250 130 38 1800 1500
GCxGC-TOFMS 10 ND 15 220 110 31 1900 1400
pg/g
Comparison of HRT and HRMS DataComparison of HRT and HRMS DataRegression ComparisonRegression Comparison
HRT (pg/g)
HR
MS
(pg
/g)
Regression Comparison Regression Comparison –– Reduced Reduced Concentration RangeConcentration Range
Removed points above 400 for weighting
HR
MS
(pg
/g)
HRT (pg/g)
Comparative Results for TCDFComparative Results for TCDF
y = 1.058x + 2.409R² = 0.963
50.00
60.00
70.00
HR
MS
(pg
/g)
2378-TCDF
0.00
10.00
20.00
30.00
40.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00
HR
MS
(pg
/g)
HRT (pg/g)
Overview of Quantitative ResultsOverview of Quantitative Results
• Values from the three instruments correlate well, considering the low levels analysed
• At the lowest level the Pegasus 4D (GCxGC-TOFMS) system is not capable of detecting analytes
• This can be partially offset using concurrent solvent re-• This can be partially offset using concurrent solvent re-condensation
• The ease of use and low cost of the Pegasus 4D suggest a role as a screening tool, where access to high resolution systems is limited
• The results from the two high resolution systems are in excellent agreement showing that the HRT can achieve the requirements of EPA 1613B
Limits of Detection: GCLimits of Detection: GC--HRTHRT
The results for the standard set can be used as an indication of the LOD possible, bearing in mind that, in dioxin work, the achievable LOD depends critically on matrix interference
The low level standard which contains TCDD and TCDF at 0.5 pg/µl was used to confirm that the GC-HRT could achieve the levels required by 1613B
Standard Results for CS1Standard Results for CS1HRT HRT Extracted Extracted Ion Chromatograms at 500 fg/µlIon Chromatograms at 500 fg/µl
TCDF TCDD
Limits of Detection: GCxGCLimits of Detection: GCxGC--TOFMSTOFMS
Two sterilized soil samples were spiked with native dioxins at different concentration levels
After adding labeled material, extraction by accelerated solvent extraction, clean -up with Gel Permeation solvent extraction, clean -up with Gel Permeation Chromatography and concentration, we could calculate LODs for PCDD/Fs in the soil samples
Using a S/N ratio of 3:1, LODs for 2378-TCDD were:Sample 1: 353 fg and Sample 2: 322 fg
This is consistent and indicates that the method can attain the low levels for 2378-TCDD required by EPA 1613 (500 fg)
Comprehensive Sample EvaluationComprehensive Sample Evaluation
As mentioned previously,
with SIM (and also MS/MS)
As mentioned previously,
with SIM (and also MS/MS)
TOF mass spectrometers
do not use
TOF mass spectrometers
do not use
We should then be able to locate and
identify all components
We should then be able to locate and
identify all components
Lets look at the samples above and
see if
Lets look at the samples above and
see if MS/MS) analyses only the targeted
compounds are located – all other
information about the sample is forfeited
MS/MS) analyses only the targeted
compounds are located – all other
information about the sample is forfeited
do not use SIM. All
analyses provide full range mass
spectra for all the analytes in
the sample
do not use SIM. All
analyses provide full range mass
spectra for all the analytes in
the sample
components of the
sample, while also being
able to quantify targeted
PCDDs and PCDFs, all in
one run
components of the
sample, while also being
able to quantify targeted
PCDDs and PCDFs, all in
one run
see if additional
POPs can be located, which have not been seen in
the GC-HRMS
method
see if additional
POPs can be located, which have not been seen in
the GC-HRMS
method
GCxGCGCxGC--TOFMS Chromatogram (Sample 2)TOFMS Chromatogram (Sample 2)
Extracted ion plot showing m/z 360
Identification of m/z 360 PeakIdentification of m/z 360 Peak
GCGC--HRT Results for the Same PeakHRT Results for the Same Peak
Measured Mass = 359.89648 Da
Calculated Mass = 359.89668 Da
(for C16H8Br2)
Mass Accuracy = -0.55 ppm
GCGC--HRT Chromatogram (Sample 6)HRT Chromatogram (Sample 6)
Extracted Ion plot showing m/z 485.7111
Identification of m/z 485.7111 PeakIdentification of m/z 485.7111 Peak(significant ions)(significant ions)
Measured Mass = 481.71496 Da
Calculated Mass = 481.71521 Da
(for C12H6Br4O)
Mass Accuracy = -0.52 ppmMass Accuracy = -0.52 ppm
GCGC--HRT Chromatogram (Sample 2)HRT Chromatogram (Sample 2)
Extracted Ion plot showing m/z 291.9188
Identification of m/z 289.9218 PeakIdentification of m/z 289.9218 Peak(significant ions)(significant ions)
Measured Mass = 289.92181 Da
Calculated Mass = 289.92163 Da
(for C12H6Cl4)
Mass Accuracy = -0.63 ppm
Some Other Components in the SamplesSome Other Components in the SamplesSample Compound Similarity Mass Accuracy (ppm)
1 2,4,4’,6- Tetrachlorobiphenyl 516 2.20
1 7H-Benz[de]anthracen-7-one 816 -0.66
2 Perylene 832 -0.71
2 1,6,8-Trichloropyrene 702 0.29
2 2,5,4’-Trichloroterphenyl 608 -1.74
2 Benz[a]anthracene 942 -0.22
2 9,10-Dichloroanthracene 958 -0.11
3 3,3’,4,4’-Tetrachlorobiphenyl 854 -0.15
4 Benzo[e]pyrene 824 0.23
5 Coronene 860 -0.16
5 2,2’,4,4’-Tetrachlorobiphenyl 750 1.57
5 2,3,3’,4,4’-Pentachlorobiphenyl 769 0.51
6 2,3’,4,4’,6-Pentachlorobiphenyl 664 0.84
6 2,3,3’,4,4’,5-Hexachlorobiphenyl 853 1.82
6 Benzyl butyl phthalate 927 -0.41
6 Tetrabromodiphenylether 653 0.52
Some Other Components in Sample 1Some Other Components in Sample 1
2,4,4’,6- TetrachlorobiphenylMass Accuracy = 2.20 ppm
7H-Benz[de]anthracen-7-oneMass Accuracy = -0.66 ppm
Perylene
2,5,4’’-TrichloroterphenylMass Accuracy = -1.74 ppm
Some Other Components in Sample 2Some Other Components in Sample 2
PeryleneMass Accuracy = -0.71 ppm 1,6,8-Trichloropyrene
Mass Accuracy = 0.29 ppm
Benz[a]anthraceneMass Accuracy = -0.22 ppm 9,10-Dichloroanthracene
Mass Accuracy = -0.11 ppm
Some Other Components in Sample 3 & 4Some Other Components in Sample 3 & 4
Sample 33,3’,4,4’-TetrachlorobiphenylMass Accuracy = -0.15 ppm
Sample 4Benzo[e]pyrene
Mass Accuracy = 0.23 ppm
Some Other Components in Sample 5Some Other Components in Sample 5
CoroneneMass Accuracy = -0.16 ppm
2,2’,4,4’-TetrachlorobiphenylMass Accuracy = 1.57 ppm
2,3,3’,4,4’-PentachlorobiphenylMass Accuracy = 0.51 ppm
Some Other Components in Sample 6Some Other Components in Sample 6
2,3’,4,4’,6-PentachlorobiphenylMass Accuracy = 0.84 ppm
2,3,3’,4,4’,5-HexachlorobiphenylMass Accuracy = 1.82 ppm
Benzyl butyl phthalateMass Accuracy = -0.41 ppm
2,2’,4,4’-TetrabromodiphenyletherMass Accuracy = 0.62 ppm
Relative Isotope AbundanceRelative Isotope Abundance
For compounds with pronounced For compounds with pronounced For compounds with pronounced molecular ion clusters (arising from the presence of Cl or Br in the molecule) measurement of
relative isotopic abundance is an important confirmation of
molecular formula
For compounds with pronounced molecular ion clusters (arising from the presence of Cl or Br in the molecule) measurement of
relative isotopic abundance is an important confirmation of
molecular formula
In general differences of up to 30% are considered acceptable when working with POPs at low
levels
In general differences of up to 30% are considered acceptable when working with POPs at low
levels
Relative Isotope AbundanceRelative Isotope AbundanceMEASURED CALCULATED
ION (Da) HEIGHTRELATIVE
ABUNDANCERELATIVE
ABUNDANCEDIFFERENCE
(%)481.71 19 21.3 17.3 23.1
483.71 67 75.3 67.8 11.1
485.71 89 100.0 100 -
487.71 58 65.2 65.8 -0.9
489.71 14.5 16.3 16.5 1.2
Relative Isotope Abundance
MEASURED CALCULATED
ION (Da) HEIGHTRELATIVE
ABUNDANCE
RELATIVE
ABUNDANCE
DIFFERENCE
(%)
289.92 7903 78.9 78.1 1.0
291.92 10014 100 100 -
293.92 4809 48.0 48.0 0.0
ConclusionsConclusions11
• Not able to locate other POPs in the samples
GC-HRMS or GC-MS/MS analysis of
PCDD/Fs requires a targeted approach
GC-HRMS or GC-MS/MS analysis of
PCDD/Fs requires a targeted approach
• Quantifying at levels mandated for analysis by regulatory authorities
• Locating and identifying other priority pollutants present in the sample in the same analytical run
TOF technology, using either GCxGC or
High Resolution, provides
an alternative approach capable of:
TOF technology, using either GCxGC or
High Resolution, provides
an alternative approach capable of:
ConclusionsConclusions22
• Permits good separation of PCDD/Fsfrom matrix interference (and other POPs)
GCxGC-TOFMS system, (make the correct choice of
column combination)
GCxGC-TOFMS system, (make the correct choice of
column combination)
• HRT results are usually < 1ppm. That allows confident formula assignment
Mass accuracy for PCDD/Fs and other POPs present in the samples is excellent
Mass accuracy for PCDD/Fs and other POPs present in the samples is excellent
• The results obtained with the HRT are in good agreement with the theoretical values
Isotope ratio measurement for the molecular ion cluster
is an additional confirmation of
formula assignment
Isotope ratio measurement for the molecular ion cluster
is an additional confirmation of
formula assignment
AcknowledgementsAcknowledgements
Jack Cochran, Restek,
USA
Eric Reiner, Ministry of the Environment, T
oronto
Jayne de Vos,
NMISA