United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas NV 89114
Research and Development
EPA/600/S4-86/004 Apr. 1986
c/EPA Project Summary
Protocol for the Analysis of
2,3,7,8-Tetrachlorodibenzo-p-
Dioxin by High-Resolution
Gas Chromatography/High-
Resolution Mass Spectrometry
W.F. Beckert
An analytical protocol for the deter-
mination of 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) and total TCDDs in
soil, sediment, and aqueous samples
using high-resolution gas chroma-
tography/high-resolution mass spec-
trometry (HRGC/HRMS) was devel-
oped using the best features of several
candidate methods and input from
experts in the field. After extensive peer
review, the protocol was tested on
TCDD-contaminated soil and TCDD-
spiked aqueous samples. The results of
these tests led to ruggedness testing
and refinements of the chromatographic
cleanup procedures and corresponding
changes in the protocol. A final, single-
laboratory evaluation of the refined
protocol consisting of triplicate anal-
yses of five solid and five aqueous
samples showed that the method is
useful for the determination of 2,3,7,8-
TCDD and total TCDDs at concentra-
tions ranging from 10 to 200 pg/g (ppt)
in soils and 100 to 2,000 pg/L(ppq) in
aqueous samples. Absolute recoveries
ranged from 40 to 120 percent with
better than 50 percent precision. An
attempt to reach a quantitation limit for
2.3,7,8-TCDD of 5 ppt (or less) for solid
samples and 50 ppq (or less) for
aqueous samples was not successful.
Based on the data generated during this
study and on the evaluation of several
options, sections of the protocol were
modified at the EPA's Environmental
Monitoring Systems Laboratory, Las
Vegas, Nevada, to lower the quantita-
tion limit for TCDD to 2 ppt in soil/
sediment samples and to 20 ppq in
aqueous samples.
This Project Summary was devel-
oped by EPA's Environmental Monitor-
ing Systems Laboratory, Las Vegas,
NV, to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering in-
formation at back).
Introduction
Methods for the determination of low
levels of 2,3,7,8-tetrachlorodibenzo-p-
dioxin (2,3,7,8-TCDD) and total TCDDs in
soil/sediment samples and aqueous
samples by HRGC/HRMS are required to
support the National Dioxin Program. A
project was therefore initiated with the
objective to provide an analytical proto-
col, based on the best features of several
candidate methods, for the determina-
tion of 2,3,7,8-TCDD and total TCDDs in
soils and sediments from 10 ppt to 200
ppt and in water from 100 ppq to 2 ppt.
This protocol, after incorporation of tech-
nical comments from EPA and other
experts, was to be written in the format
used in the Invitation for Bid for the
Superfund Contract Laboratory Program
(CLP) and then was to be tested and im-
proved in a single laboratory. Based on
the results of the evaluation study, the
protocol was to be modified to allow (1)
detection and quantitation of 2,3,7,8-
TCDD concentrations of 2 pg/g (2 ppt) to
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1.2 ng/g (1.2 ppb) in soil/sediment
samples and 20 pg/L (20 ppq) to 12 ng/L
(12 ppt) in aqueous samples, and (2)
estimation of quantities of total TCDDs
present in the samples.
Procedure
Protocol Development
A protocol for HRGC/HRMS deter-
mination of 2,3,7,8-TCDD and total
TCDDs in soil/sediment samples and
aqueous samples was written based on
methods published by EPA (including
Method 613 and the Region 7 low-
resolution protocol) and in the open
literature, and on input from experts in
the field. The resulting protocol, which
was written in the format used for the
Superfund CLP and which includes the
stringent quality assurance/quality con-
trol features required by this program,
was extensively reviewed and refined
before being tested in the laboratory. The
protocol, as written, is designed to allow
quantitation of 2,3,7,8-TCDD at 10 ppt for
soil/sediment samples and 100 ppq for
aqueous samples.
Preliminary Protocol Evaluation
During a preliminary protocol evalua-
tion, low recoveries of the internal
standard 13Ci2-2,3,7,8-TCDD were ob-
tained, and the accuracy and precision of
duplicate sample analyses were poor.
The data seemed to indicate that the
problems resulted from poor chroma-
tographic separation in the extract
cleanup columns. As part of the method,
the sample extract volume was reduced
to 1.0 mL (benzene), the concentrate was
eluted through an acidic silica column
with hexane, and the total collected
eluate was added to an acidic alumina
column. The alumina column was then
eluted with hexane/20-percent methy-
lene chloride, the eluate was concen-
trated and cleaned further using a
Carbopak C/Celite column, and the
TCDDs were eluted from the Carbopak
C/Celite column with 2 mL toluene.
A systematic evaluation of the in-
dividual cleanup steps specified in the
original protocol led to the following
conclusions:
• The eluate from the acidic silica
column must be concentrated (to
0.5 mL) before transfer to the
alumina column.
• The hexane/methylene chloride
eluate from the acidic alumina
column need not be concentrated
prior to application to the Carbopak
C/Celite column.
• The optimum toluene volume for
the elution of the TCDDs from the
Carbopak C/Celite column is ap-
proximately 6 mL.
• Reverse elution of the Carbopak
C/Celite column with toluene
further improves the recovery.
Appropriate changes were incorpo-
rated in the modified protocol.
Evaluation of the improved column
cleanup procedures using 1 -mL portions
of benzene spiked with eight TCDD
isomers gave overall recoveries of 84
percent or better.
Description Of The Method
(Figure 1)
Sample Extraction
Soil/Sediment Samples
A 10-g soil or sediment sample is
spiked with 500 pg 13Ci2-2,3,7,8-TCDD
(internal) standard), is mixed with an-
hydrous sodium sulfate, and is extracted
with benzene in a Soxhlet apparatus for
24 hours. The extract is then concen-
trated to 1 mL.
Soil/Sediment
10g
Water
1 L
Liquid-Liquid
Extraction or
Separatory Funnel
Methylene
Chloride
Methlyene Chloride
Extract
Exchange Solvent,
Concentrate
Reverse Elution
with Toluene
90 mL Hexane
1) 20 mL Hexane
2) 30 mL 20% CH2 CI2/Hexane
Figure 1. Extraction, cleanup, and analysis steps in the refined protocol.
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Aqueous Samples
A 1-L aqueous sample is spiked with
500 pg 13Ci2-2,3,7,8-TCDD (internal)
standard) and is extracted with methy-
lene chloride either in a continuous
liquid-liquid extractor for 24 hours or in a
separatory funnel. The extract is dried,
the solvent is exchanged for benzene,
and the benzene extract is concentrated
to 1 mL.
Extract Cleanup
The concentrated benzene extract is
passed through a column with acid-
impregnated (H2S04) silica gel using
hexane as eluant, the eluate is concen-
trated to 0.5 mL and is transferred to a
column with acidic alumina which is then
eluted with 20 percent methylene
chloride in hexane. The eluate is added to
the top of a column containing a mixture
of activated Carbopak C and Celite 545®
and is eluted sequentially with hexane,
cyclohexane/methylene chloride, and
methylene chloride/methanol/benzene.
The column is then inverted, the TCDD
fraction is eluted with 6 mL toluene, the
toluene volume is reduced, and 50 uL
tridecane is added.
Extract Analysis
The sample extracts and calibration
standards were analyzed with a Carlo
Erba Mega Series GC that was coupled to
a Kratos MS 50TCdouble-focusing mass
spectrometer. The GC/MS interface was
a direct connection of the GC column to
the ion source via a heated interface
oven. A Finnigan 2300 Incos data system
was used for data acquisition and pro-
cessing. The HRGC/HRMS operating
conditions are summarized in Table 1.
The mass spectrometer was tuned
daily to yield a resolution of at least
10,000 (10 percent valley) and optimal
response at m/z 254.986. This step was
followed by calibration of an accelerating
voltage scan beginning at m/z 254.
Other voltage scans from the same data
file were then used to establish and
document both the resolution at m/z
316.983 and the mass measurement
accuracy at m/z 330.979.
Just before analysis, a recovery
standard spiking solution was added to
the extract (5 uL of a 100-pg///L solution
of 13Ci2-1,2,3,4-TCDD in isooctane).
Final Protocol Evaluation
The refined protocol was evaluated on
five solid samples and five aqueous
samples.
Solid Samples
Four soil samples and one fly ash
sample were analyzed. The four soil
samples were known to contain en-
dogenous 2,3,7,8-TCDD. Each sample
was fortified with 500 pg of 13Ci2-2,3,7,8-
TCDD in 1.5 mL acetone and was anal-
yzed in triplicate as specified in the proto-
col. One of each triplicate was spiked
with other TCDD isomers at varying con-
centrations before analysis.
Aqueous Samples
Distilled water, influent and effluent
wastewater from a sewage treatment
facility, industrial wastewater (pH <1),
and an aqueous extract from a highly
contaminated soil sample were evalu-
ated. The industrial wastewater and the
soil extract were known to contain
endogenous 2,3,7,8-TCDD. Each sample
was fortified with 500 pg of 13C12-2,3,7,8-
TCDD in 1.5 mL acetone and was anal-
yzed in triplicate as specified in the proto-
col. Except for the soi I extract, one of each
triplicate was spiked with 2,3,7,8-TCDD
Table 1. HRGC/HRMS Operating Conditions
and other TCDD isomers at varying con-
centrations before analysis.
Results And Discussion
The results of the evaluation are
summarized in Tables 2 and 3 for 2,3,7,8-
TCDD. Results from the analysis of
standards demonstrate that the method
is capable of achieving quantitation limits
of 12.5 pg/g (ppt) for soil/sediment
samples and 125 pg/L (ppq) for aqueous
samples.
The relative response factors (RRF)
determined for native 2,3,7,8-TCDD
versus the internal standard 13Ciz-
2,3,7,8-TCDD, and the RRF of the internal
standard versus the recovery standard
13Cir1,2,3,4-TCDD over the five-point
concentration calibration curve demon-
strate that the HRGC/HRMS method
maintains a linear response for 2,3,7,8-
TCDD from 12.5 to 200 ppt for soil/
sediment samples and 125 to 2,000 ppq
for aqueous samples.
The results of the analyses of spiked
aqueous samples demonstrate that in-
Mass Spectrometer
Accelerating voltage
Trap current
Electron energy
Electron multiplier voltage
Source temperature
Resolving power
Ions monitored
258.930
319.897
321.894
331.937
333 934
280.9825 (lock mass)
Overall SIM cycle time = 1 sec
Gas Chromatograph
Column coating
Film thickness
Column dimensions
Helium linear velocity
Helium head pressure
Injection type
Split flow
Purge flow
Injector temperature
Interface temperature
Injection size
Initial temperature
Initial time
Temperature program
8,000V
500 uA
70 eV
2,000V
280°C
10,000 (10% valley definition)
Nominal dwell times (sec)
0.15
0.15
0.15
0.15
0.15
0.10
CP-Sil 88
0.2 um
50 m x 0.22 mm ID
~ 25 cm/sec
1 .75 kg/cm2 (25 psi)
Split less, 45 sec
30 mL/min
6 mL/min
270°C
240°C
200°C
1 min
20O°C to 240° at 4°C/min
-------
ternal standard (isotope dilution) quanti-
tation provides an accurate measure-
ment of 2,3,7,8-TCDD. The accuracy of
the 2,3,7,8-TCDD measurement for
triplicate analysis of four out of five
aqueous samples spiked at various con-
centrations was quite good.
The results of the analyses of samples
spiked with additional TCDD isomers
demonstrate that the internal standard
quantitation gives good estimates of total
TCDD values.
The overall results of the analyses
demonstrate that the requirements for
absolute recovery of the internal stand-
ard (40 to 120 percent) and the precision
of replicate analyses (RPD < 50 percent)
could be achieved for most of the samples
tested.
Significant problems were encount-
ered with the fly ash, the soil extract and
the industrial wastewater samples.
The triplicate analyses of the fly ash
sample resulted in absolute recoveries of
less than 10 percent for the internal
standard in each aliquot analyzed. This
low recovery may be associated with the
total fixed carbon content of the fly ash
material. Previous work with fly ash from
coal-fired power plants has demon-
strated low recoveries of analytes from
materials with high carbon content.
The soil extract contained a large
amount of suspended particulate in each
of the three replicates, and the inter-
ference and TCDD responses observed in
these replicates were probably due to
direct extraction of the suspended soil
particulate material. Centrifugation prior
to analysis resulted in absolute recov-
eries of 78 and 96 percent of the internal
standard. The triplicate analysis of the
industrial wastewater sample resulted in
absolute internal standard recoveries of
23, 20, and 29 percent. Additional ex-
periments indicate that this sample
matrix (which may have contained mis-
cellaneous industrial solvents) had a
considerable impact on the extraction
efficiency and on the effectiveness of the
cleanup procedure. The influence of the
sample pH (<1 for the industrial waste-
water sample) on extraction efficiency is
not known.
Analysis of 2 //L of the 1.0 pg/fiL
standard did not yield satisfactory re-
sults.
Modification Of The Protocol
Examination of the results from the
single-laboratory protocol evaluation
study showed that the minimum amount
of 2,3,7,8-TCDD which could be quanti-
fied under the conditions described
Tab/0 2. Precision of the HRGC/HRMS Analysis for 2.3,7,8-TCDO of Soil and Fly Ash Samples
Sample
Matrix
Soil (85)
Soil (HI)
Soil (B1)
Soil(H3>
Fly Ash
Estimated
Endogenous
2.3.7,8-TCDD
level (pptr
50
Avg. cone.
RPD"
70
Avg. cone.
RPD
360
Avg. cone
RPD
1 ,700
Avg cone.
RPD
Avg. cone.
RPD
2.3,7.8-TCDD
Detected
(ppt)
18.2
15.1
12.9
15.4
34
34.3
36.6
30.3
33.7
19
937
785
1.280
1,000
50
2,020
2,260
1 ,800
2,030
23
1,720
1,020
1,160
1 ,300
54
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
13C -2 ,3 .7 ,8-TCDD
Absolute
Recovery /%)
73
85
48
69
54
73
46
56
53
47
95
75
80
83
24
79
99
86
88
23
4
7
5
5.3
57
"Relative percent difference.
above was 5 pg. To adapt the protocol to
quantitation limits of 2 ppt for soil/
sediment samples and 20 ppq for
aqueous samples (without increasing the
sample sizes, and while still overlapping
the ppb low-resolution method without
necessitating a second extraction for
samples containing higher levels of
TCDD) the protocol was modified as
follows:
• The following calibration solutions
will be used:
HRCC1: 2 pg///L 2,3,7,8-TCDD
and13Ci2-1,2,3,
4-TCDD
10pg//uL 13Ci2-2,3,7,8-
TCDD
HRCC2: 10pg/A«L 2,3,7,8-TCDD
and13C,2-1,2,3,
4-TCDD
10pg/yuL 13Ci2-2,3,7,8-
TCDD
HRCC3: 50 pg/pL 2,3,7,8-TCDD
and13C12-1,2,3,
4-TCDD
10pg//uL 13Ci2-2,3,7,8-
TCDD
HRCC4:100 pg//uL 2,3,7,8-TCDD
and13C12-1,2,3,
4-TCDD
10pg/A(L13Ci2-2,3,7,8-
TCDD
The final extract volume will be
10 fjL. The decision to select such a
small final volume was necessary
to comply with the above require-
ments. It is realized that handling
such small volumes requires spe-
cial technical skills of the operator.
The fortification level of the internal
standard 13Ci2-2,3,7,8-TCDD was
raised from 500 pg/sample to
1,000 pg/sample. By diluting a 2-
fjL aliquot of the remaining con-
centrate by a factor of 12 with a
solution of the recovery standard
(10 pg///L of 13Ci2-1,2,3,4-TCDD in
tridecane), this allows analysis of
(1) soil/sediment samples contain-
ing between 100 ppt and 1.2 ppb of
any TCDD isomer and (2) aqueous
samples containing between 1 ppt
and 12 ppt of any TCDD isomer.
Recoveries will be reported using
the data generated from the first
injection. Thus, the decision to
dilute an aliquot of the 10-juL final
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Table 3. Accuracy and Precision of the HRGC/HRMS Analysis for 2,3,7,8-TCDD From Laboratory Aqueous Matrix Spikes
Sample
Matrix
Distilled
water
Effluent
waste -
water
Influent
waste -
water
Industrial
waste -
water
2.3,7,8-TCDD
Spike
level
(PPQ)
250
250
250
1.000
1 ,000
1.000
500
500
500
500
500
500
Avg. cone.
RPD*
Avg. cone.
RPD
Avg. cone.
RPD
Avg. cone.
RPD
2.3.7.8-TCDD
Detected
(PPO.)
234
265
246
248
12.5
1,090, 1,030
1.010
1,050
1,050
7.6
534
508
530
524
5.0
1.290
1.520
1,430
1 ,410
16
2.3,7,8-TCDD
Recovery
<%i
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
93.6
106
103
101
9.3
109,103
101
105
105
7.6
107
102
106
105
4.8
258
304
286
283
16
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
Avg. rec.
RPD
13r
C-I2-
2.3,7,8-TCDD
Absolute
Recovery
(%)
82
42
69
64
63
61,66
91
80
75
40
77
75
71
74
8.1
23
20
29
24
38
Industrial
waste -
water
Avg. cone.
RPD
604
628
616
3.9
Avg. rec.
RPD
60
57
58
5.2
Soil extract
Avg. cone.
RPD
27,100
28,100
27,600
3.6
Avg. rec.
RPD
78
96
87
25
*Relative percent difference.
extract will not be based on the
concentration of 2,3,7,8-TCDD or
total TCDD in the sample but on the
concentration of the most abundant
TCDD isomer or group of coeluting
TCDD isomers in the 10-AtL final
extract volume. This will eliminate
unnecessary dilutions of the sam-
ple extract, and will eliminate
analyses for soil/sediment sam-
ples containing between 100 ppt
and 250 ppt and for aqueous
samples containing between 1 ppt
and 2.5 ppt of a TCDD isomer or
group of coeluting TCDD isomers
but for which the recoveries were
low.
The original and the refined protocol are
included in the full report.
The EPA author W. F. Beckon is with the Environmental Monitoring Systems
Laboratory, Las Vegas, NV 89114.
The complete report, entitled "Protocol for the Analysis of 2,3,7,8-
Tetrachlorodibenzo-p-Dioxin by High-Resolution Gas Chromatography/'High-
Resolution Mass Spectrometry." (Order No. PB 86-161 361 /AS; Cost: $16.95,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
For information contact the EPA author at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
P.O. Box 15027
Las Vegas, NV 89114
U. S. GOVERNMENT PRINTING OFFICE: 1986/646-116/20794
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Environmental Protection
Agency
Center for Environmental Research
Information
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Official Business
Penalty for Private Use $300
EPA/600/S4-86/004
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