the truth about dioxin analysis: using high resolution gc ... · tcdf tetrachloro-dibenzofuran pcdf...

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

[email protected]

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


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


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


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


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


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)



(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)



(for C12H6Cl4)


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


PeryleneMass Accuracy = -0.71 ppm 1,6,8-Trichloropyrene

Mass Accuracy = 0.29 ppm

Benz[a]anthraceneMass Accuracy = -0.22 ppm 9,10-Dichloroanthracene


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


CoroneneMass Accuracy = -0.16 ppm

2,2’,4,4’-TetrachlorobiphenylMass Accuracy = 1.57 ppm

2,3,3’,4,4’-PentachlorobiphenylMass Accuracy = 0.51 ppm


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

the truth about dioxin analysis: using high resolution gc ... · tcdf tetrachloro-dibenzofuran pcdf...

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