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

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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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Penalty for Private Use $300

EPA/600/S4-86/004

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