vvEPA
United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 15027
Las Vegas NV 89114-5027
EPA 600/4-86-004
January 1986
Research and Development
Protocol for the
Analysis of 2,3,7,8-
Tetrachlorodibenzo-p-Dioxin
by High-Resolution Gas
Chromatography/
High-Resolution
Mass Spectrometry
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PROTOCOL FOR THE ANALYSIS OF 2,3,7,8-TETRACHLORODIBENZO-£-DIOXIN BY
HIGH-RESOLUTION GAS CHROMATOGRAPHY/HIGH-RESOLUTION MASS SPECTROMETRY
by
John S. Stanley and Thomas M. Sack
Midwest Research Institute
Kansas City, Missouri 64110
Contract Number SAS 1576X
I
Project Officer
Werner F. Beckert
Quality Assurance Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
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NOTICE
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency under Contract Number
SAS 1576X to the Midwest Research Institute, Kansas City, Missouri. It has
been subject to the Agency's peer and administrative review, and it has been
approved for publication as an Environmental Protection Agency document.
Mention of trade names or commercial products does not constitute endorse-
ment or recommendation for use.
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PREFACE
This report describes the activities completed as part of a single-
laboratory evaluation of a high-resolution gas chromatography/high-
resolution mass spectrometry method for the determination of tetrachloro-
dibenzo-£-dioxins in water, soil, and sediment samples. The work described
in this report was completed at the Midwest Research Institute under con-
tract to Viar and Company (Special Analytical Services SAS 1576X) for the
U.S. Environmental Protection Agency, Environmental Monitoring Systems
Laboratory, Quality Assurance Division, Las Vegas, Nevada. The revision of
the protocol to allow for lower quantitation limits for tetrachlorodibenzo-
£-dioxins was carried out at the Environmental Monitoring Systems Laboratory-
Las Vegas.
This report was prepared with assistance from M. McGrath. The authors
acknowledge the technical project monitor, W. F. Beckert, as well as R. K.
Hitchum and S. Billets of the Environmental Monitoring Systems Laboratory-
Las Vegas and, especially, Y. Tondeur of the Environmental Research Center,
University of Nevada, Las Vegas for guidance provided during this study.
111
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ABSTRACT
This report provides the results of the single-laboratory evaluation of
a high-resolution gas chromatography/high-resolution mass spectrometry
method for the determination of 2,3,7,8-tetrachlorodibenzo-£-dioxin and
total tetrachlorodibenzo-£-dioxins at concentrations ranging from 10 to
200 pg/g (ppt) in soils and 100 to 2,000 pg/L (ppq) in water. The report
summarizes the data for the precision and accuracy of triplicate measure-
ments of five solid and five aqueous samples. The results indicate that the
method is capable of generating accurate and precise data within the concen-
tration limits specified above and within absolute recoveries of 40 to 120
percent with 50 percent precision. An attempt to reach a quantitation limit
for TCDD of 2 ppt (or less) for soil and 20 ppq (or less) for aqueous sam-
ples was not successful. Based on the data generated during this study and
based on discussions at the Environmental Monitoring Systems Laboratory-
Las Vegas, the Environmental Monitoring Systems Laboratory-Las Vegas revised
certain parts of the protocol to lower the quantitation limit for tetra-
chlorodibenzo-£-dioxins to 2 ppt in soil and 20 ppq in water samples.
IV
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CONTENTS
Preface iii
Abstract iv
Figures vi
Tables vii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Experimental Procedures 7
Sample description 7
Sample preparation 7
Reagents 9
HRGC/HRMS instrumentation 9
Mass measurement accuracy 11
Chromatographic resolution 13
Injection technique 13
5. Results and Discussion 14
Approach to cleanup column evaluation 14
Final method evaluation 22
References 42
Appendices 43
A. Validated Analytical Protocol
B. Proposed Analytical Protocol
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FIGURES
Number Page
1 Column cleanup procedures specified in the protocol 15
2 Column cleanup procedures proposed by the EMSL-LV 16
3 Background levels of 1,3,6,8- and 1,3,7,9-TCDD observed over
the single-laboratory evaluation study 41
VI
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TABLES
Table Page
1 Solid Samples Used for HRGC/HRMS Method Evaluation 8
2 Aqueous Samples Used for HRGC/HRMS Method Evaluation 8
3 TCDD Isomers Used for HRGC/HRMS Method Evaluation 10
4 Composition of Concentration Calibration Solutions (pg/pL) . . 10
5 HRGC/HRMS Operating Conditions 12
6 Recovery (%) of Several TCDD Isomers from Cleanup Option A . . 18
7 Recovery (%) of Several TCDD Isomers from Cleanup Option B . . 19
8 Recovery (%) of Several TCDD Isomers from Cleanup Option C . . 20
9 Recovery (%) of Several TCDD Isomers from Cleanup Option D . . 21
10 Initial Calibration Summary 23
11 HRGC and Mass Resolution Check Summary 24
12 TCDD Data Report Form 26
13 Accuracy and Precision of the HRGC/HRMS Analysis for
2,3,7,8-TCDD from Laboratory Aqueous Matrix Spikes 33
14 Precision of the HRGC/HRMS Analysis for 2,3,7,8-TCDD of
Soil and Fly Ash Samples 34
15 Accuracy of the HRGC/HRMS Method for the Determination of
TCDD Isomers Spiked into Aqueous Matrices 35
16 Accuracy of the HRGC/HRMS Method for the Determination of
TCDD Isomers Spiked into Soil Matrices 36
17 Fortified Field Blank Results 37
VI1
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SECTION 1
INTRODUCTION
The U.S. Environmental Protection Agency's (EPA) strategy for dealing
with dioxin requires the development and validation of an analytical method
capable of achieving detection of the tetrachlorodibenzo-£-dioxins (TCDD),
specifically 2,3,7,8-TCDD, at the parts-per-trillion (ppt) level in soil and
sediment and parts-per-quadrillion (ppq) level in water.1 This validated
method will be used by qualified contract laboratories to extend the analyt-
ical capabilities for such analyses to all EPA regional and program offices.
This report deals specifically with the single-laboratory evaluation of
a high-resolution gas chromatography/high-resolution mass spectrometry
(HRGC/HRMS) analysis method for TCDDs in soil, sediment, and water. The
method (Appendix A) is intended to provide quantitative determination of
TCDD at levels of 10 to 200 pg/g (soil and sediment) and 100 to 2,000 pg/L
(water) at a mass resolution of 10,000. This single-laboratory evaluation
has been completed as part of the validation process recommended by EPA.2
The proposed method was prepared after several candidate methods were
reviewed and their best features were selected. After peer review, the pro-
posed method was refined for completeness, technical accuracy, clarity, and
regulatory applicability. The single-laboratory evaluation of the proposed
analytical method has been accomplished through three tasks. The first task
involved preliminary performance testing of the method using TCDD-
contaminated soils and TCDD-spiked aqueous samples. The results of this
study indicated that the proposed method required modification to achieve
the target method detection limits and the accuracy and precision criteria.
The second task focused on ruggedness testing of the chromatographic cleanup
procedures. The results of this study were used to modify the proposed
method. This report is focused on the results of the triplicate analysis
of five solid and five aqueous samples completed under the third task of
the evaluation, using the modified method.
Section 2 of this report summarizes the conclusions based on the
single-laboratory evaluation of this method using TCDD-contaminated soils
and TCDD-spiked aqueous samples. Section 3 presents recommendations that
should be considered for inclusion in the method before proceeding with
collaborative testing. Section 4 presents some specific experimental con-
ditions, and Section 5 summarizes the analytical data for the triplicate
analysis of four soil, one fly ash, and five aqueous samples completed in
the third task of the single-laboratory evaluation. Triplicate analyses of
a 1-pg/pL calibration solution did not give satisfactory results. In order
to achieve a quantitation limit of 2 ppt for soil (using a 10-g sample) and
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20 ppq for water (using a 2.0-L sample), the protocol evaluated in this
study was modified. The rationale for the modifications and the revised
protocol are included as Appendix B.
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SECTION 2
CONCLUSIONS
The single-laboratory evaluation of the analytical method for the
determination of 2,3,7,8-TCDD in soil and aqueous samples demonstrates that
the method as described is capable of achieving the target detection limits
of 10 pg/g (ppt) for soils and 100 pg/L (ppq) for water.
The relative response factors (RRF) determined for native 2,3,7,8-TCDD
versus the internal standard l3C12-2,3,7,8-TCDD, and the RRF of the internal
standard versus the recovery standard 13C12~1|2,3,4-TCDD over the five-point
concentration calibration curve demonstrate that the HRGC/HRMS method main-
tains a linear response for 2,3,7,8-TCDD from 10 to 200 ppt for soils and
100 to 2,000 ppq for water.
The results of the analysis of spiked aqueous samples demonstrate that
internal standard (isotope dilution) quantitation provides an accurate mea-
surement of 2,3,7,8-TCDD. The accuracy of the 2,3,7,8-TCDD measurement for
triplicate analysis of four water samples spiked at various concentrations
was quite good. The accuracy of measurement for 2,3,7,8-TCDD averaged 104
percent for three aqueous matrices prepared as laboratory matrix spikes.
The absolute recovery of the internal standard 13C!2-2,3,7,8-TCDD did not
significantly affect the accuracy of the 2,3,7,8-TCDD determination. The
precision of the analyses for 2,3,7,8-TCDD ranged from 3.6 to 16 percent for
replicate analyses of the five aqueous samples. The precision of the trip-
licate analyses of the soil samples was somewhat higher than determined for
aqueous samples. The precision of triplicate analyses of the four soil
samples ranged from 19 to 50 percent. The difference in precision from that
of the aqueous samples may be attributable to the potential for TCDD adsorp-
tion on the soil samples.
The results from the analyses of soil and aqueous samples spiked with
additional TCDD isomers demonstrate that the internal standard quantitation
gives good estimates of total TCDD values. The accuracy of the analyses of
fortified distilled water and influent and effluent wastewaters averaged
101 ± 14 percent for five TCDD isomers. The accuracy of the measurements of
these isomers for the four fortified soil samples averaged 87 t 24 percent.
The results of the analyses demonstrate that the requirements for abso-
lute recovery of the internal standard (40 to 120 percent) and precision of
replicate analyses (RPD < 50 percent) can be achieved for relatively clean
samples.
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The sample matrix can severely impact the performance of the analytical
method. This is evidenced by the consistent low recovery of the internal
standard from the fifth aqueous sample, an industrial wastewater, and from a
fly ash sample. The low recovery from the industrial wastewater is possibly
due to the effect of coextractants on the elution sequence from alumina.
The low recoveries observed for the fly ash sample, on the other hand, may
be attributed to adsorption by the sample matrix.
One of the most critical variables in the analytical method is the com-
pleteness of removal of the benzene from the extract before proceeding with
the acidic alumina column fractionation. The cleanup column ruggedness
testing experiments demonstrated that the recoveries of 2,3,7,8-TCDD and the
other TCDDs are affected by the presence of benzene in the alumina column
fractionation step.
The analyst must be aware of the potential problem of interferences
arising from background contamination. For example, the 1,3,6,8- and
1,3,7,9-TCDD isomers were present in the fortified field blanks in this
work. From other referenced activities it becomes clear that these isomers
may present problems in other laboratories as well. The fortified field
blanks are important tools in assessing the background contamination prob-
lems over time.
Although the 1.0-pg/pL standard did not yield satisfactory results in
this study, due to unacceptable ion ratios, the response factors are within
the established curve. The data for the triplicate analyses of the 1.0-|Jg/pL
standard demonstrate that the characteristic ions for TCDD were greater than
20:1 for the m/z 322 S/N and approximately 10:1 for m/z 259 S/N. Thus, it
should be possible to extend the detection limit to 1 pg/pL if an allowance
for abundance ratios based on ion statistical errors is incorporated.
Based on the column performance and bleed characteristics, the column
of choice for the analysis for TCDD at ppt (for soils and sediments) and ppq
(for water) levels appears to be the 50-m CP-Sil 88 with a 0.2-pm film
thickness. To preserve the performance characteristics of the HRGC columns,
an injection technique that excludes any air is highly recommended.
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SECTION 3
RECOMMENDATIONS
1. Mass measurement accuracy should properly be determined relative to the
lock mass (if any), rather than m/z 254.9856, because it is that rela-
tionship which will determine how accurately the masses of the TCDD
ions will be measured.
2. It is recommended that the chromatographic resolution check be per*
formed on the summed ion chromatograms of m/z 259 + m/z 320 + m/z 322.
This yields a chromatogram which is less noisy and more representative
of the true column performance.
3. The 5 percent peak width criterion for mass resolution should be the
selected mass/1,000 mmu rather than 31.9 nunu because the protocol
allows peaks other than m/z 319 to be used for resolution measurement
(e.g., 31.7 mmu if m/z 317 is used).
4. It is recommended that the mass measurement accuracy be recorded and
reported along with the resolution check summary table.
5. The addition of the recovery standard 13C!2-1,2,3,4-TCDD should be
achieved by using a spike volume of 25 to 50 pL rather than 5 |JL to
minimize errors resulting from volume measurement.
6. The recommended temperature program settings in the method should be
converted to those presented in the experimental section of this re-
port. These conditions were established for analysis with tridecane as
the solvent.
7. Lower limits of detection can be achieved by allowing the analyst to
concentrate the final extract to as low as 10 pL. It may be necessary
to use the smaller final volume with other HRMS instruments to achieve
the same levels of detection.
8. The method should recommend several techniques to break up emulsions
resulting from extraction of aqueous samples. In this evaluation the
emulsion phase was put through a column packed with glass wool, which
was then rinsed with additional metbylene chloride. Other options
might include stirring or centrifugation of the emulsion phase.
9. The method should specify the procedure to deal with aqueous samples
containing high levels of suspended solids. In this study it was
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necessary to centrifuge the soil extract sample before proceeding with
the extraction.
10. It is highly recommended that the method be modified such that the ben-
zene extract is completely exchanged to hexane prior to cleanup on the
silica column since this is apparently one of the most critical factors
leading to successful sample analysis.
11. It may be worthwhile to evaluate a cleanup procedure in which the char-
coal column precedes the alumina column as a means to improve method
recovery.
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SECTION 4
EXPERIMENTAL PROCEDURES
SAMPLE DESCRIPTION
Five solid samples were provided by the Environmental Monitoring
Systems Laboratory-Las Vegas (EMSL-LV) to the Midwest Research Institute
(MRI) for analysis for 2,3,7,8-TCDD and total TCDD using the analytical
method in Appendix A. A description of the five solid samples and the esti-
mated 2,3,7,8-TCDD concentrations from previous analyses by an independent
laboratory are provided in Table 1. Each sample was analyzed in triplicate
as specified in the protocol. One of the triplicate samples for each soil
sample was spiked with the seven TCDD isomers (1,3,6,8-; 1,3,7,9-; 1,2,3,7-;
1,2,3,8-; 1,2,3,4-; 1,2,7,8-; and 1,2,8,9-TCDD) at approximately 10 times
the estimated level of 2,3,7,8-TCDD specified in Table 1.
Five aqueous samples were generated for the evaluation of the analyt-
ical method at the ppq detection level. Table 2 presents a description of
each water type and lists the fortification levels of 2,3,7,8-TCDD and seven
additional TCDD isomers (1,3,6,8-; 1,3,7,9; 1,2,3,7-; 1,2,3,8-; 1,2,3,4-;
1,2,7,8-; and 1,2,8,9-TCDD) in each sample.
The influent and effluent wastewater samples were collected from a
sewage treatment facility in metropolitan Kansas City, Missouri. The indus-
trial wastewater was obtained from a holding pond within a hazardous waste
area that was known to be highly contaminated with PCBs and possibly other
chlorinated aromatic compounds (chlorobenzenes). This aqueous sample was
very acidic (pH < 1) and was dark in color.
The soil extract was prepared from 30 g of a soil sample, Hyde Park 002
(H2), and 1 gallon of distilled water. The mixture was stirred constantly
(at least 24 hrs) until just prior to subsampling of 1.0-L aliquots.
SAMPLE PREPARATION
All samples listed in Tables 1 and 2 were extracted and analyzed in
triplicate according to the protocol provided in Appendix A. As indicated
in Tables 1 and 2, one aliquot of each sample matrix was fortified with
additional TCDD isomers, which represent the compounds that elute first
(1,3,6,8-TCDD), last (1,2,8,9-TCDD), and within the approximate retention
window of 2,3,7,8-TCDD (1,2,3,7-; 1,2,3,8-; and 1,2,3,4-TCDD) from the HRGC
columns used for sample analysis.
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TABLE 1. SOLID SAMPLES USED FOR HRGC/HRMS METHOD EVALUATION
Approximate
sample
EPA sample no. Matrix size3
B25-Piazza Road (B5) Soil 10 g
Hyde Park 001 (HI) Soil 10 g
B52-Shenandoah (Bl) Soil 1 g
Hyde Park 003 (H3) Soil 1 g
RRAI-5,7,8 (FA) Fly ash 10 g
Estimated
2,3,7,8-TCDD
concentration
(FPt)5
50
70
360
1,700
NRd
Spike level
(ppt) of
TCDD isomers
100
140
720
1,700
e
.Approximate sample size of each replicate sample.
Estimated level of endogenous 2,3,7,8-TCDD reported to MRI by
CW. Beckert in letters dated April 19, 1985 and August 30, 1985.
.Approximate fortification level of each of seven additional TCDD isomers.
^o estimate of 2,3,7,8-TCDD concentration was reported.
Additional TCDD isomers were not spiked into this matrix.
TABLE 2. AQUEOUS SAMPLES USED FOR HRGC/HRMS METHOD EVALUATION
Sample type
Distilled water (DW)
POTW influent (IWW)
POTW effluent (EWW)
Industrial wastewater (IND)
Hyde Park 002; soil extract
Approximate
sample
size
1.0 L
1.0 L
1.0 L
1.0 L
(H2W) 1.0 L
Fortification
level of
2,3,7,8-TCDD
(ppq)
250
500
1,000
500
c
Fortification
level of
TCDD isomers
(ppq)B
500
1,000
2,000
1,000
c
a
.Approximate sample size of each replicate sample.
Approximate fortification level of each of seven additional TCDD isomers
This aqueous sample was not fortified with TCDD isomers.
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All samples were fortified with 500 pg 13C12-2,3,7,8-TCDD in 1.5 ml
acetone. The solid samples were extracted continuously for 24 hr in a
Soxhlet apparatus with benzene and the 1.0-L aqueous samples were batch-
extracted using 2.0-L separately funnels and three 60-mL portions of methy1-
ene chloride. The extractions of the influent wastewater (IWW) and effluent
wastewater (EWW) and the soil extract (H2W) were complicated by the forma-
tion of emulsions. In each case, the emulsion was removed by passing the
methylene chloride and emulsion layer through a column of glass wool pre-
rinsed with methylene chloride. The extract and resulting aqueous layers
were collected in a sample bottle and the glass wool plug was rinsed with an
additional 10 ml methylene chloride. Following the complete extraction of
the aqueous sample, the contents of the bottle were transferred to a clean
250-mL separatory funnel and the methylene chloride was removed from the
aqueous phase that was transferred with the emulsion. All extracts were
concentrated with Kuderna-Danish evaporators and nitrogen evaporation to
approximately 1.0 ml. Each extract was taken through the entire cleanup
procedure including the acidic silica, acidic alumina, and Carbopak C as
specified in the protocol (Appendix A). The HRGC/HRMS analysis of each ex-
tract was completed as specified below.
REAGENTS
All solvents for extraction and cleanup were obtained as "Burdick and
Jackson distilled-in-glass" quality. The tridecane (99 percent purity) was
obtained from Aldrich (TS, 740-1). The chromatographic materials, acidic
alumina (100-200 nesh AG-4, Biorad Laboratories 132-1340), silica (70-230
mesh Kieselgel 60, EM Reagent, American Scientific Products C5475-2), sodium
sulfate, potassium carbonate, Celite 545® (Fisher Scientific Company), and
the silanized glass wool and Carbopak C (80-100 mesh Supelco 1-1025) were
prepared for use as specified in Section 7 of the protocol (Appendix A).
Table 3 provides the sources of standards used to prepare the calibra-
tion solutions, sample fortification solutions, recovery standard spiking
solution, internal standard spiking solutions, field fortification solu-
tions, and TCDD isomer fortification solutions.
Table 4 is a summary of the concentration calibration standards pre-
pared for the HRGC/HRMS method evaluation. These standards were prepared as
specified in the protocol (Appendix A). The standard HRCC6 was included in
the final evaluation of the HRGC/HRMS method as a means to demonstrate the
lower limit of detection under optimum instrumental conditions.
HRGC/HRMS INSTRUMENTATION
Sample extracts and calibration standards were analyzed using a Carlo
Erba Mega Series gas chromatograph (GC) which was coupled to a Kratos MS50
TC double-focusing mass spectrometer (MS). The GC/MS interface was simply 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
processing.
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TABLE 3. TCDD ISOMERS USED FOR HRGC/HRMS METHOD EVALUATION
Isomer
Stock
concentration
Source
Standard code
2,3,7,8-TCDD
13C12-2,3,7,8-TCDD
1,2,3,4-TCDD
13C12-1,2,3,4-TCDD
1,3,6,8-/1,3,7,9-TCDD
1,2,3,7-/1,2,3,8-TCDD
1,2,7,8-TCDD
1,2,8,9-TCDD
Column performance
standard
7.87 ± 0.79 Mg/mL
50 t 5 Mg/mL
2.7 mg/mL
50 ± 5 jJg/mL
0.82 mg/mL
0.5 mg/mL
0.39 mg/mL
1.46 mg/mL
10 |Jg/mL
EPA QA Reference
Materials
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
Cambridge Isotope
Laboratories
20603
R00201 (Lot
AWN-1203-65)
ED-915C (Lot
6578)
R00212 (Lot
AWN-1203-93)
ED-913C (Lot
F2086)
ED-905C (Lot
7371)
ED-915C (Lot
7184)
ED-916C (Lot
MLB-682-26)
ED-908 (Lot
No. R00215)
Mixture of TCDD isomers including 2,3,7,8-; 1,2,3,4-; 1,2,3,7-/1,2,3,8-;
1,2,7,8-; and 1,4,7,8-TCDD.
TABLE 4. COMPOSITION OF CONCENTRATION CALIBRATION SOLUTIONS (pg/pL)
HRCC1
HRCC2
HRCC3
HRCC4
HRCC5
HRCC63
Recovery standard
13C12-1,2,3,4-TCDD
2.5
5.0
10.0
20.0
40.0
1.0
Analyte
2,3,7,8-TCDD
2.5
5.0
10.0
20.0
40.0
1.0
Internal standard
l3C12-2,3,7,8-TCDD
10.0
10.0
10.0
10.0
10.0
10.0
This solution is not specified in the analytical method in Appendix A.
10
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The HRGC/HRMS operating conditions used in the final phase of this work
are summarized in Table 5. The GC operating conditions recommended in the
protocol were not used for these analyses for three reasons. First, the
TCDDs hr.ve rather long retention times, and the solvent (tridecane) boils at
235°C. Thus no benefit could be realized with a low initial temperature.
Second, past experience at MR I has indicated that 200°C is an acceptable
starting temperature for these types of analyses when tridecane is used as
a solvent. Finally, since the CP-Sil 88 and SP-2330 phases are both very
polar and thinly coated, it has been recommended that they not be subjected
to rapid heating or cryogenic cooling to prevent thermal shock to the
column.3
The MS 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 (typical
calibration range was 255 to 605 amu). 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.
MASS MEASUREMENT ACCURACY
For this work, mass measurement accuracy was measured relative to PFK
m/z 254.986, as required by the protocol, by applying the mass correction,
Am, to the entire spectrum, which yields an error of 0 ppm at m/z 254.986.
In this way, it was possible to meet routinely the 5 ppm accuracy criterion
at m/z 330.979. However, if a lock mass other than 254.986 is used, the
mass measurement accuracy should be measured relative to that lock mass,
since it is that peak which is used to maintain magnet alignment and will
ultimately control the mass measurements during the selected ion monitoring
(SIM) experiments.
Mass Resolution
Mass resolution at m/z 316.983 was documented by an output of the Incos
PROF program. However, the computer-generated value for resolution was
found to be significantly higher than the value measured manually. Thus,
the manually determined resolution, which was nearly identical to the value
measured by using the peak matching unit, is reported. Closer inspection of
the PROF source code revealed that resolution is computed via a statistical
method, not as m/Am at 5 percent height. Incos users should therefore be
aware of this discrepancy, because the computer-generated value can be as
much as 20 percent over the proper value.
Following calibration, the SIM experiment descriptor was updated to re-
flect the new calibration. Six masses (see Table 5) were monitored by scan-
ning ^ m/10,000 amu over each mass. The total cycle time was kept to 1 sec.
The m/z 280.983 ion from PFK was used as a lock mass because it is the most
abundant PFK ion within the range of m/z 255 to 334 and therefore permits
the use of low partial pressures of PFK, which minimizes PFK interferences
at the analytical masses.
11
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TABLE 5. HRGC/HRMS OPERATING CONDITIONS
Mass spectrometer
Accelerating voltage:
Trap current:
Electron energy:
Electron multiplier voltage:
Source temperature:
Resolution:
Ions monitored
258.930
319.897
321.894
331.937
333.934
280.9825 (lock mass)
Overall SIM cycle time
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:
= 1 sec
8,000 V
500 MA
70 eV
2,000 V
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 pm
50 m x 0.22 mm ID
* 25 cm/sec
1.75 kg/cm2 (25 psi)
Splitless, 45 sec
30 ml/min
6 ml/min
270°C
240°C
2 HL
200°C
1 min
200°C to 240°C at 4°C/min
12
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CHROMATOGRAPHIC RESOLUTION
. «
Chromatographic resolution values were measured for the SIM plot of m/z
320. However, it may be advantageous to measure chromatographic resolution
from a plot of the sum of m'z 259, 320, and 322. The sum trace has better
signal-to-noise ratio (S/N) and peak definition than the SIM plots, which
permits a more accurate measurement of resolution.
Selection of the HRGC Column
Three different HRGC columns were evaluated in the course of this proj-
ect: SP-2330 (60 m x 0.24 mm); DBS (60 m x 0.22 mm); and CP-Sil 88 (50 m x
0.22 mm). By evaluating the mass spectra of the bleed from each column at
240 to 250°C, it became apparent that the column background may be the lim-
iting factor in achieving the desired detection limit for this method. The
DBS column provided the least amount of background at 250°C, and the SP-2330
had the worst. This coincides with the fact that quantitation at the detec-
tion limit (i.e., 2.5 pg/|JL) with the SP-2330 column was difficult at best.
The CP-Sil 88 column appeared to offer less bleed than the SP-2330 column
and indeed does permit more accurate quantitation due to reduced background
contribution.
The chromatographic performance afforded by these columns is a further
issue, since the column best suited for low detection limits, DB-S, cannot
meet the 25 percent valley chromatographic resolution criteria in all cases.
Both the SP-2330 and CP-Sil 88 columns can easily resolve the 2,3,7,8-TCDD.
However, based on the bleed considerations discussed above, the 50-m CP-Sil
88 column is recommended for the best combination of low bleed and good
isomer separation.
It may also be advisable that other HRGC columns (including SP-2340,
Silar IOC, and SP-2331) that have been used for 2,3,7,8-TCDD analysis at the
1-ppb soil level be evaluated for background contribution and their applica-
tion for HRMS analysis at ppt and ppq concentrations.
INJECTION TECHNIQUE
The HRGC column performance can degrade very quickly if proper injec-
tion techniques are not used. Specifically, the SP-2330 and CP-Sil 88
phases are very sensitive to ©2 and will decompose rapidly at 200°C if any
trace of 02 is present. Therefore, the common practice of using 1 pL of air
to flush the syringe and effect reproducible injections is to be avoided,
since even that small amount of air per injection can cause column perfor-
mance to degrade in less than one week of continued use.
The following injection technique is recommended. First rinse the sy-
ringe copiously with isooctane (or other volatile solvent, such as toluene).
Dry the syringe by drawing air through it. Pull up and expel several vol-
umes of tridecane until all bubbles are gone, and leave 1 pL of tridecane in
the barrel. Finally, pull up 2 pL of the sample solution and inject. This
technique has worked very well and yields injection reproducibility compar-
able to that of the air purge method, without introducing air onto the ana-
lytical GC column.
13
-------
SECTION 5
RESULTS AND DISCUSSION
The primary purpose of any method validation process is to assure that
the method under consideration is adequate to meet testing and monitoring
requirements.1 The single-laboratory evaluation of the analytical protocol
presented in this report has been preceded by several evaluation and im-
provement steps. These have included the preparation of a written protocol,
technical review of the protocol for completeness, technical accuracy, and
clarity; preliminary testing to evaluate performance of the analytical
method; and revision and refinement of the written protocol based on the
results of the preliminary testing.
Prior to the assessment of the refined protocol presented in Appendix A,
the proposed analytical method had been evaluated for performance through
the analysis of several duplicate samples. The results of the preliminary
evaluation indicated that problems existed in the design and approach to the
extract cleanup steps, which greatly affected the method detection limit,
accuracy, and precision.
This section presents a summary of the studies that have led to the
refinement of the analytical protocol as provided in Appendix A and also
summarizes the single-laboratory evaluation of this protocol.
APPROACH TO CLEANUP COLUMN EVALUATION
The initial method evaluation completed under the first task resulted
in very low recoveries of the internal standard, 13Cl2-2,3,7,8-TCDD, and the
accuracy and precision of duplicate sample analyses were poor. After re-
viewing the data, it was apparent that the problems were the result of poor
chromatographic separation in the cleanup columns. The initial protocol in-
volved reducing sample extract volumes to 1.0 mL in benzene, elution through
the acidic silica column with hexane, and collection of the total eluent
which was then added to the acidic alumina column. The alumina column was
further eluted with hexane/20-percent methylene chloride. The eluate was
concentrated and cleaned further using a Carbopak C/Celite column, and the
TCDDs were eluted with 2 mL toluene.
Column cleanup techniques were revised and further evaluated following
the procedures depicted in Figures 1 and 2. The column evaluations were
completed with triplicate measurements at three spike levels (0.10, 1.0, and
10 ng) equivalent to 10, 100, and 1,000 ppt of TCDD in solids with several
TCDD isomers (2,3,7,8-; 1,3,6,8-; 1,3,7,9-; 1,2,3,4-; 1,4,7,8-; 1,2,3,7-;
1,2,3,8-; and 1,2,8,9-TCDD).
14
-------
OPTION A
1 ml Benzene Exfrocf
I
H2SO4 - SJO2
4.0g
Si 02
l.Og
Acidic AI2O3
6.0g
30ml 20% CH2Cl2/H«xon«
Concentrate fo
I
Corfaopok C/C*lit«
6ml Toluene
HRGC/HRMS
OPTION B
1 ml Benzene extract
I
H2SO4 - SIO2
4.09
SiO2
l.Og
Acidic AI2O3
6.0g
Carfaopok C/C«m«
6 mi Toluene
HRGC/HRMS
•xan«
Figure 1. Column cleanup procedures specified in the protocol
15
-------
OPTION C
1 ml Benzene Extract
H2SO4 - SiO2
4.0g
/.Og
4
>
Concentrate fo
0.5mL
I
Acidic AI2C>3
6.0g
30 mC 20% CH2Cl2/H«*ane
Concentrate fo
I
Corbopak C/Cei!te
6mL Toluene
HRGC/HRMS
OPTION 0
I ml Senzene Extract
H2S04 - Si02
4.09
Si02
l.09
Concentrate to 0.5mL
I
Acidic AI2O3
o.Og
30mL 20% CH2Cl2/Hexane
Carbopok C/CeHte
6mi Toluene
HRGC/HRMS
Figure 2. Column cleanup procedures proposed by the EMSL-LV.
16
-------
The TCDD isomers were added to 1-mL portions of benzene and were taken
through the four sample cleanup sequences depicted in Figures 1 and 2. One
of the replicates for each procedure was also spiked with 100 ng of Aroclor
1260.
The results of the sample analyses are provided in Tables 6 through 9.
As noted in Tables 6 and 7, recoveries of the TCDD isomers were low and
quite variable for the early eluting isomers 1,3,6,8- and 1,3,7,9-TCDD as
compared to 1,2,8,9-TCDD. Recovery of 1,2,8,9-TCDD was still low and vari-
able (approximately 60 percent recovery with an RSD of * 20 percent). These
results were generated using the procedures specified in the original proto-
col (see Figure 1). The results of the analyses following the cleanup op-
tions A and B demonstrate that accurate quantitation of all TCDD isomers is
not possible using only the 13Cl2-2,3,7,8-TCDD surrogate standard. The low
recoveries measured for options A and B are obviously a result of the pres-
ence of benzene in the eluent from the acid-modified silica column that is
taken directly through the acidic alumina column.
In contrast, options C and D (Tables 8 and 9) demonstrate quantitative
recovery of the TCDD isomers. Some background contamination has been noted
from the acidic alumina for the 1,3,6,8- and 1,3,7,9-TCDD isomers. This
material had previously been prepared by Soxhlet extraction with methylene
chloride and activation at 190°C prior to use. As noted in Tables 8 and 9,
the average recovery of the other spiked TCDD isomers was greater than 84
percent.
When the recoveries of the different isomers and the 13Ci2-2,3,7,8-TCDD
are compared, the average relative percent difference ranges from 1 percent
for 2,3,7,8-TCDD (Table 3) to 24 percent for 1,2,3,4-TCDD (Table 4). These
results demonstrate that either of these cleanup procedures (options C and
D) will provide good recovery and reliable quantitation of 2,3,7,8-TCDD and
very good estimates of the concentrations of the other TCDD isomers present
in the samples. No interferences were observed in the samples spiked with
100 ng Aroclor 1260. The lack of PCB interferences was especially noted in
the extracts of samples spiked at 0.10 ng/TCDD isomer.
In addition to the evaluations of the cleanup procedures presented above,
the acid-modified silica gel/acidic alumina columns and the Carbopak C/Celite
column were evaluated separately. Evaluation of the silica/alumina at the
0.10-ng spike level as shown in Figure 2 resulted in an average recovery of
120 percent for 1,2,3,4-, 1,2,3,7-, 1,2,3,8-, and 1,4,7,8-TCDD; 114 percent
for 2,3,7,8-TCDD; 118 percent for 13Cl2-2,3,7,8-TCDD; and 118 percent for
1,2,8,9-TCDD. The results for the recovery of 1,3,6,8- and 1,3,7,9-TCDD
indicated that some contamination originated from the acidic alumina.
Replicate analyses of the Carbopak C/Celite column at the 0.10-ng spike
level resulted in average recoveries of 97 percent for 1,3,6,8-TCDD; 88 per-
cent for 1,3,7,9-TCDD; 81 percent for 1,2,3,4-, 1,2,3,7-, 1,2,3,8-, and
1,4,7,8-TCDD; 75 percent for 2,3,7,8-TCDD; 96 percent for 13C12-2,3,7,8-TCDD;
and 90 percent for 1,2,8,9-TCDD. Elution of the Carbopak C/Celite column
with additional toluene beyond 6 ml did not improve recoveries even for the
samples spiked at 10 ng/TCDD isomer.
17
-------
TABU 6. KECOVKKV (1) OF SEVERAL TCIM) ISWIKRS H!UM CI.EANIII* 01TION A
00
_..... _ .1 1 —
l.l»*M~.t.l..f
1
HJSO« - siQ}
4-0.
$10}
i
AcMUAIjOj
iJO.t M«
CMMWMM* «• IflO/it
*
U.I IclMK
HRGC/IIRMS
Spike
level 1,3,6,8
1 ng 4.6
1 it a 12
1....* •» •
10 ug 12
10 ng 6.2
""" 10 ng' 6.3
He a u 7.4
IO »L
2,3,7/
2,3,8
14
ll
• 7
31
20
20
23
TCDI) iioaei
2,3,7,8
19
11
94
44
31
29
31
r
l3C12-2.3l7.ft
27
4B
11
38
36
34
36
1,2,8,9
38
64
An
76
61
59
56
Sanple was also spiked with Kill ng »l Arorlor IJ60.
-------
TABI.K 7. RECOVERY (1) OF SKVKRAI. TCDI1 ISOHKRS FROM CLEANUP OPTION B
Recovery (%) of TCOI) I»CNK
1 .1 I>M«M tmUta
J
MjSO< - JIOj
4.0,
IIOj
, 1
A*l*c AljOj
•Of
130.1 »«
Cntijil C/C.III,
Spike
level 1,3,6,8
1 ng 7.6
1 ng 2.4
1 ng* 6.4
10 ng 0.9
10 ng 1.8
CHjCiykum
10 nga II
J».tl.h.M
Mean 5.0
HRGC/HRMS
1 USD 79
1,3.7.9
15
21
25
7.6
II
17
16
40
I.2.3.4/
1,4.7.8
35
31
34
14
36
44
32
31
I.2.3.7/
1.2,3,8
14
17
22
21
25
31
22
28
2,3.7,8
37
34
40
50
47
59
45
21
r
'3Clj-2,3,7,8
38
40
41
44
48
52
44
12
1,2,8,9
48
52
57
70
59
78
61
19
JSan|)le was also spiked witli 100 ng ol Arurlor 1260.
-------
TAHIJi 8. kecOVKKY (1) UK SKVEKAI. TCOO ISONEMS FKOH C.l£MtUP OPTION C
ro
O
, ..._.,_. . ..... , . , ___________ _ . __
Recovery (1) of TCI1I) inomfr
Spike
level 1.3.6.8"
1 «4 •••••<• I rtrari
M,I04 . JIO,
SIOj
1
J
AtMcAJjOj
Itt»t30%
C~«*«.I.MOjU
J
c.wu eye.**
1 4«t I.I-.
HRGC/HRMS
0
0
0
1
1
1
10
citjciykiuw
10
10
. 10 ug
.10 ng
. 10 ugd
.0 ng
.0 ng
.0 ngd
.0 ng
.0 ng
.0 ngj
Mean
m
, .
300
310
340
158
168
171
157
130
210
39
1
1.3.7,9" 1
390
420
440
165
162
183
155
140
130
240
54
.2.3,47
,«./,8
l'"u"
97"
140
98
136
118
130
102
115
14
1.2,3,77
c
c
c
116
96
120
116
130
99
112
12
107
102
88
94
80
96
110
130
100
101
14
'C|2*2 378
89
89
92
95
75
90
88
106
85
90
9.1
1289
81
84
91
89
80
94
107
122
110
95
15
*Tlie 1,3,6,8- and 1,3,7,9-TCOD iuoaers were also noted in reagent blanks fro» the acidic aluaina
coluwi. No «ucIt interferences we-re noted fron tlu> dcidificd Milieu gel or the Carbopak C/Celite
. coluwi.
Resolution of 1,2,3,4-, 1,2.J,7-/1.2, I.8-, and 1,4,7,8-TCIM} was not achieved. This value
represents recovery of the four isoaers.
^Recovery reported with I ,^,3,4-/l,4,7,fl-TCIin.
Sa>ple was also spiked with 100 ng of Aroclor 1260.
-------
TABLE 9. RKCOVtKY (1) OF SEVERAL TCIID ISOHERS FKOM Ol.tAHUI' OITION D
Recovery (D ol
Spike
level 1
1 «l (••»« biracl
I
H,5O< - JIO,
4.0.
JIOj
1.0,
1
I
AcMkAljOj
«.o.
1 M»l 10%
C.WH.C/IC.MI.
1 *ml T«h»
HRGC/HRMS
0
0
0
1
1
10
CHaCI»ftln»m If)
10
M
H
t
.10 ng
.10 ng
. 10 ng*1
.0 ng
• 0 ng
.0 ,,gd
.0 ng
.0 ng
.Ongd
can
RSI)
.3,6,8'
147
290
260
90
180
135
81
114
126
158
46
1 1 1 9s 14
197
200
360
93
185
158
107
72
138
168
51
,3.4/
7 8
in"
•21"
nob
106
95
85
50
105
89
97
21
TCDI) iiioaer
1,2, 3,77
c
c
c
103
121
79
104
108
95
101
14
72
71
84
85
109
47
101
107
91
85
23
C12-2.3.7,8
84
90
97
86
90
60
78
82
92
84
13
1.2.B.9
70
71
e
53
108
63
84
118
112
85
29
The 1,3,6,8- ami 1,3,7,9-T4'l)0 f suiters were also noted in reagent blanks fro* Hie acidic (liraina
coluMi. No such in! i:r liMern es were nutnl 11 un I lie uciililied silica gel or Hie Carliopdli C/Celitc
. colimn.
Resolution ol I.2.J.4-, 1,2,:i,7-/l .2,3,8- , and I ,4, 7,B-TCI)I> was not achieved. This value
represents recovery ol I lie lour isowrH.
^Recovery reported with I,2,3,4-/l,4,7,8-TCW).
Sunple was also spiked with 100 ng of Aroclor 1260.
MHIJC/IIKHS analysis was interrupted pi ior to Hie olutiun of lliis isnner.
-------
Three additional experiments were completed to evaluate the efficiency
of reverse elution of the carbon column. The Carbopak C/Celite was placed
in a 5-mL disposable pipette packed at both ends with glass wool plugs. The
column was eluted in one direction for the hexane, cyclohexane/methylene
chloride, and the methylene chloride/methanol/benzene mixture. The column
was then turned over and eluted with 6 ml toluene. Triplicate analyses at
the 0.10 ng/TCDD isomer spike level demonstrated average recoveries of 98
percent for 1,3,6,8-TCDD; 91 percent for 1,3,7,9-TCDD; 104 percent for
1,2,3,4-, 1,2,3,7-, 1,2,3,8-, and 1,4,7,8-TCDD; 116 percent for 2,3,7,8-
TCDD; 102 percent for 13C12-2,3,7,8-TCDD; and 93 percent for 1,2,8,9-TCDD.
FINAL METHOD EVALUATION
Based on the results of the column evaluation study, the analytical
method was revised to specify the cleanup procedure presented as Option D in
Figure 2. The final protocol, as presented in Appendix A, was then evalu-
ated as described below.
The data presented in Tables 10 through 17 are summaries of the initial
column calibration, HRGC and HRMS resolution checks, and the results of the
sample analysis.
Calibration
Table 10 summarizes the RRF data for the concentration calibration
standards from the initial calibration and the routine monitoring of the RRF
values over the time required to complete the sample analyses. The RRF(I)
as specified in the protocol is a measure of the response of 2,3,7,8-TCDD
versus the internal standard, 13Ci2-2,3,7,8-TCDD. The value for RRF(I) var-
ied ±9.4 percent over the five concentration levels of 2,3,7,8-TCDD ranging
from 2.5 pg/pL to 40 pg/pL. The RRF(II) is used to calculate the absolute
recovery of the internal standard as compared to the recovery standard
13C12-1,2,3,4-TCDD. The average RRF(II) was determined to vary by ± 19.3
percent over the calibration curve. The variability of the RRF(I) and
RRF(II) were determined to be less than ± 10 percent and ± 18 percent,
respectively, over all data points required to complete the sample analysis.
In addition to the analysis of calibration standards specified in the
protocol, solution HRCC6 was analyzed in triplicate to determine the lower
limit of sensitivity (1 pg/pL). Although the calculated RRF(I) and RRF(II)
values and the S/N are within the specified criteria, the ion ratio for the
native compound and recovery standard indicate that these measurements fall
outside the acceptable calibration window.
22
-------
ro
to
TARU 10
Calibration
atandard
imcci
imcci
IIRCCI
mice*
IIRCC2
IIRCC2
IIRCC2
HRCC3
IIRCC3
HRCC3
HRCC4
HRCC4
NRCC4
IIRCC5
HRCC5
IIRCC5
IIRCC6
HRCC6
IIRCC6
HRCCI
IIRCCI
IIRCC2
IIRCC2
IIRCC2
imcc2
HRCC2
IIRCC2
IIRCC2
IIRCC2
IIRCC2
Date
09/12/85
09/12/85
09/12/85
09/12/85
09/12/85
09/12/85
09/12/85
09/13/85
09/13/85
09/13/85
09/13/85
09/13/85
09/13/85
09/13/85
09/13/85
09/13/85
09/16/85
09/16/85
09/16/85
09/16/85
09/16/85
09/20/85
09/23/85
09/23/85
09/24/85
09/25/85
09/26/85
09/27/85
09/30/85
10/03/85
Tlae
09:05
09:44
12:31
13:00
13:27
13:53
15:39
10:31
10:57
11:23
13:02
13:29
13:56
14:22
14:49
15:15
10:43
11:19
13:44
12:42
14:40
10:46
08:47
10:46
10:47
08 : .19
08:56
09:33
09:19
08:53
•/» 320/322 •/*
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
,
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
82
84
84
73
80
92
78
78
78
73
77
73
77
78
75
76
32
18
86
87
81
86
82
88
89
76
77
78
83
68
332/334
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
77
80
73
77
73
66
77
7»
80
83
80
76
78
80
82
78
71
8.1
80
8J
79
73
80
80
69
78
80
84
80
75
. IMITIAL CALIBRATION SUHHART
(IS) aj/z 332/334 (RS) S/H 259 S/N 322 S/N 334(IS) RJ)F(I)
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
80 41:1 > 65:
73 48:1 > 65:
87 24:1 > 65:
83 36:1 > 65:
81 58:1 > 65:
69 78:1 > 65:
73 76:1 > 65:
79 97:1 > 65:
78 111:1 > 65:
78 110:1 > 65:
76 96:1 > 65:
78 > 144:1 > 65:
76 > 144:1 > 65:
78 > 144:1 > 65:
83 > 144:1 > 65:
79 > 144.1 > 65:
84 10: 21:
14 9.6: 25:
01 18: 30:
75 36: 63:
85 48: > 6.1:
80 > 75: > 63:
70 > 75: > 63:
83 42: > 63:
69 26: > 63:
89 58: > 63:
72 73: > 63:
81 49: > 63:
85 7.1: > 63:
81 29: > 63:
1 > 65:1 0.783
1 > 65:1 0.794
1 > 65:1 0.750
Nea> 0.776
t RSD 2.9t
1 > 65:1 0.829
> 65:1 0.853
1 > 65:1 0.799
1 > 65:1 0.861
Mean 0.848
X RSD 2.01
1 > 65:1 0.974
1 > 65:1 0.965
1 > 65:1 0.972
Head 0.970
1 RSD 0.5t
t
1 > 65:1 0.945
1 > 65:1 0.935
1 > 65:1 0.967
Mean 0.949
I RSO 1 . 7X
1 > 65:1 0.964
1 > 65:1 1.01
1 > 65:1 0.989
Mean 0.987
I RSD 2.2X
Overall llran (RRF) 0.906
X RSD 9.4X
> 65: 0.917
> 65: 0.878
> 65: 0.935
> 65: 0.876
> 65: 0.850
> 63: 0.835
> 63: 0.832
> 63: 0.941
> 63: 1.01
> 63: 0.949
> 63: 0.941
> 63: 1.04
> 63: 0.854
> 63: 0.955
RRF(II)
2
2
2
2
2
1
1
2
2
1
U
,
1
I
I
1
1
1
1
1
1
1
I
1
1
3
1
19
1
1
1
1
2
I
2
1
1
1
1
1
1
.18
.27
.28
.24
• 4X
.76
.93.
.03*
.27
.99
• OX
.53
.57
.58
.56
• 9X
.49
.52
.49
.50
• IX
.52
.42
.43
.46
• 9X
.75
• 3X
.44
.09
.58
.86
.11
.98h
.65
.41
.68
.96
.57
.68
.67
.53
.Nnl Inrliiilrd in «ran RRK rnaniitat Ion.
Nnl witliin allowable limit* lor roiil iiir ral dual Ion.
-------
TABLE 11. HRCC AND MASS RESOLUTION CHECK SUMMARY
Date
9/12/85
9/12/85
9/12/85
9/12/85
9/13/85
9/13/85
9/13/85
9/13/85
9/16/85
9/16/85
9/16/85
9/16/85
9/17/85
9/17/85
9/18/85
9/18/85
9/19/85
9/19/85
9/20/85
9/20/85
9/20/85
9/20/85
9/23/85
9/23/85
9/23/85
9/23/85
9/24/85
9/24/85
9/24/85
9/24/85
last.
ID
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
NSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
NSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
MSSO
Sol.
ID
.
PC
PC
-
-
PC
PC
-
-
PC
PC
-
PC
-
-
PC
-
PC
-
PC
PC
-
. •
PC
PC
-
PC
PC
"
Tiae
07:51
08:32
16:05
16:47
08:26
08:45
15:48
16:23
09:25
10:45
15:16
15:57
10:26
10:14
07:58
08:18
11:14
12:56
08:00
08:16
15:44
16:14
07:55
08:15
16:01
16:45
09:51
10:15
16:01
16:33
TCDD isomer
File resolution
name (% valley)
MID254I12X1
83671 12XQ1
83671 12XQ9
HID2S4I12X2
MID2S4I13X1
83671 13XQ1
83671 13XQ12
HID2S4I13X2
MID2S4I16X1
8367I16XQ1
83671 16XQ8
MID254I16X2
8367I17XQ1
MID2S4I17X1
MID254I18X1
8367I18XQ1
MID254I19X1
6367I19XQ1
MID2S4I20X1
8367I20XQ1
8367I20XQ5
MID254I20X3
MID254I23X1
8367I23XQ1
8367I23XQ6
Manual check*
MID2S4I24X1
8367I24XQ1
8367I24XQ3
MID2S4I24X2
-
5.9
2.9
-
-
6.9
11.4
-
-
11.9
23.0
-
13.3
-
-
20
-
3.5
-
6.7
4.1
-
-
8.8
12.5
-
12.2
13.1
Mass
resolution
at 10X valley
10,774
-
-
10,450
10,230
-
-
10,384
10.294
-
-
10,388
-
10,824
11,019
-
11,679
-
12,068
-
-
10,777
10,096
-
12,500
10,374
-
-
10,567
Mass
Measurement
error
5 ppn
-
-
-
0 ppm
-
-
-
4 ppn
-
-
-
-
2 ppm
1 ppm
-
4 ppn
-
3 ppm
-
-
-
1 ppn
-
—
3 ppa
-
-
*A aianual resolution cheek was performed due to data syitesi failure.
(continued)
24
-------
TABLE 11. (continued)
Date
9/25/85
9/25/85
9/25/85
9/25/85
9/26/85
9/26/85
9/26/85
9/26/85
9/27/85
9/27/85
9/27/85
9/27/85
9/30/65
9/30/85
9/30/85
9/30/85
10/3/85
10/3/85
10/3/85
10/3/85
lost.
ID
HS50
HS50
MS50
HS50
MS50
HS50
MS50
MS50
HS50
MS50
HS50
HS50
HS50
MS50
HS50
MS50
asso
HSSO
.1S50
MS50
Sol.
ID
.
PC
PC
-
-
PC
PC
-
-
PC
PC
-
-
PC
PC
-
-
PC
PC
'
Tine
07:50
08:05
16:13
16:45
08:07
08:21
15:49
16:21
08:19
09:02
16:01
16:29
08:15
08:33
15:10
15:41
07:59
08:20
15:56
16:29
File
nine
HID254I25X1
8367I25XQ1
8367I25XQ3
MID254I25X2
MID254I26X1
8367I26XQ1
8367I26XQ3
MID2S4I26X2
MID254I27X1
8367I27XQ1
8367I27XQ3
HID254I27X2
MID254I30X1
8367I30XQ1
8367I30XQ3
HID254I30X2
MID254J03X1
8367J03XQ1
8367J03XQ3
HID254J03X2
TCDD if oner
resolution
(* vallev)
.
11.1
6.5
-
-
8.3
13.2
-
-
11.9
11.8
-
-
< 25
< 25
-
-
17
12
"
Mass
resolution
at 10% valley
11.165
-
-
11,419
10,989
-
-
10,499
11,564
-
-
10,639
11,149
-
-
11,321
10.567
-
-
10,442
Mais
Measurement
error
0 ppm
-
-
-
3 ppm
-
-
-
1 ppm
-
-
-
5 pp«
-
-
•»
0 ppm
-
-
'
A manual resolution check was performed due to data system failure.
25
-------
TABLE 12. TCDD DATA REPORT FORM
Aliquot
Sample Air-dry wt. (g) TCDD
Mo. or Vol. (L) Isomer
8367-83- 1576X-DVD 1.0 L
2,3,7
,8-
1,3,6,8-
8367-82-1576X-DV 1.0 L
8367-89- 1576X-EV.VD 1.0 L
8367-88-1576X-EWW 1.0 L
8367-90-1576X-EWN 1.0 L
8367-85-1576X-IND 1.0 L
8367-92- 1576X-IWWD 1.0 L
-
8367-87-1576X-INDN 1.0 L
8367-84- 1576X-DWH 1.0 L
-
1,3,7
2,3,7
2,3,7
1.3,6
1,3,7
c
2,3,7
c
2,3,7
1,3,6
1,3,7
1,2,3
1,2,3
1,2,3
1,2,7
1,2,8
2.3,7
2,3,7
1,3,6
1,3,7
1,2,7
2,3,7
1,2,7
2,3,7
1,3,6
1,3,7
1,2,3
,9-
,8-
,8-
,8-
,9-
TCDD (ppt
Retention time or ppq)
TCDD
21:35
16:43
17:54
21:35
21:40
16:48
17:58
24:10
,8-
21:29
23:57
,8-
,8-
,9-
,7/
,8-
,4-
,8-
,9-
,8-
,8-
,8-
,9-
,8-
,8-
,8-
,8-
,8-
,9-
,11
21:
59
17:02
18:14
22:
22:
24:
30:
21:
22:
17:
18:
24:
22:
24:
22:
17:
18:
22:
18
31
30
01
55
01
06
16
33
00
31
01
06
18
21
1JC,2-2,3,7,8 Meas. DL
21:34 228
117
86.5 -
21:33 196
21:37 2,277
134
282
137
21:27 1,090
75.9 -
21:56 1,010
502
766
1,860
1,840
3,430
1,330
21:55 1,290
21:59 508
191
208
55.2 -
21:59 1,520
586
22:00 234
512
395
403
HRGC/HRMS Analysis
Instr. Relative loo Abundance Ratios
ID Date Time 320/322 332/334(15)
MS50 09/20/85 11:27 0.
78 0.63
0.69
0.80
MS50 09/20/85 13:02 0.58 0.69
MS50 09/20/85 14:41 0.
1.
0.
0.
MS50 09/20/85 15:11 0.
0.
MS50 09/23/85 11:17 0.
0.
0.
0.
0.
0.
0.
MS50 09/23/85 12:51 0.
MS50 09/23/85 13:29 0.
0.
0.
0.
MS50 09/23/85 14:00 0.
0.
MS50 09/23/85 14:31 0.
0.
0.
0.
79 0.72
02
84
90
77 0.71
89
74 0.72
85
83
81
71
78
80
81 0.90
74 0.80
85
75
89
87 0.78
71
75 0.71
80
74
72
332/334(RS) X Rec. m/r 259
0.68 40 73:
64:
41:
0.72 14 21:
0.71 96 > 146:
10:
28:
6:
0.72 61 146:
7:
0.82 91 27:
22:
30:
54:
45:
78:
22:
0.81 23 37:
0.71 75 49:
25:
27:
8:
0.86 20 49:
21:
0.74 82 23:
73:
53:
42:
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
S/N
m/z 322 m/2 334(IS) Comments
> 63:
50:
30:
62:
> 63:
> 5:
> 11:
63:
> 63:
31:
34:
22:
33:
> 63:
60:
> 63:
18:
63:
63:
29:
29:
16:
63:
31:
.
63:
50:
45:
1 > 49:1 Ratio unacceptable. Rerun.
1
1
1 > 20:1 Ratio unacceptable; low recovery. Rerun.
1 > 63:1 Sample spiked at twice requested level.
1 Rerun.
1
1
1 > 63:1
1
1 > 63:1
1
1
1
1
1
1
1 30:1 Low recovery.
1 > 63:1
1
1
1
1 23:1 Low recovery.
1
> 63:1
1
1
1
1,2,3,8-
1,2,3,4-
1,2,7.8-
1,2,8,9-'
22:
24:
30:
35
32
02
616
840
328
0.
0.
0.
72
80
79
61:
70:
23:
1
1
1
55:
63:
20:
1
1
1
^Aqueous sample data reported as ppq and_soil sample data'presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
Isooer could not be identified.
~ (continued)
26
-------
TABLE 12. (continued)
Aliquot
Sample Air-dry wt. (g) TCDD
No. or Vol. (L) Isomer
8367-9 1-1576X-IWW
8367-86-1576X-INDD
8367-93- 1576X-IWWN
8367-70-1576X-H1N
-
8367-65-1576X-B5 -
8367-66-1576X-B5D
8367-67-1576X-B5N
_
"•
_
~
- • -
1.0 L 2,3,7,8-
1,3.6,8-
1,3,7,9-
1,2.7,8-
1.0 L 2,3,7,8-
1.0 L 2,3,7,8-
1,3.6,8-
1,3,7,9-
1.2.3.7/
1,2.3,8-
1,2,3,4-
1,2,7,8-
1,2,8,9-
.10.01 g~ 2,3,7,8-
1,3,6,8-
1,3,7,9-
1.2.3.7/
1,2,3,8-
1,2,3,4-
1,2,7,8-
1,2,8,9-
10.00 g 2,3,7,8-
1.3,7,9-
9.85 g 2,3,7,8-
1,3,7,9-
10.00 g 2,3,7.8-
1,3,6,8-
— 1,3,7,9-
1.2.3.7/
1.2,3,8-
. — 1,2,3,4-
1,2,7,8-
1,2,8,9-
HRCC/HRMS Analysis
TCDD (ppt
Retention tine or ppq) Instr. Relative Ion Abundance Ratios
TCDD lJC12-2,3,7,8 Meas. DL ID Date Time
22:00
17:03
18:14
24:30
21:50
21:39
16:47
17:57
21:58
21:11
24:08
29:35
21:38
16:45
17:55
21:58
22:10
24:09
29:36
21:47
18:05
21:44
18:03
21:47.
16:55
18:05
22:07
22:19 ~
24:16
29:45
21:57 534 - MS50 09/23/85 15:01
246
222
54.7 -
21:48 1,430 - MS50 09/23/85 15:31
21:38 530 - MS50 09/24/85 11:19
582
690
940
1,180
1,790
691
21:37 " 30.3 - MS50 09/24/85 12:55
29 .0 -
51.1 -
125
. _
118
252 - —
100
21:45 18.2 - MS50 -09/24/85 13:27
8.5 -
21:43 15.1 - MS50 09/24/85 13:58
4.2 -
21:46 12.9 - MS50 09/24/85 14:29
ND 9.2
15.2 -
61.8 -
— 54.1 - " —
147
63.9 -
320/322 332/334(15)
0.87 0.83
0.71
0.78
0.70
0.80 0.76
0.76 0.72
0.82
0.80
0.72
0.79
0.82
0.77
0.85 0.78
0.63
0.87
0.81
0.69
0.81
0.85
0.87 0.78
0.85
0.67 0.80
0.87
0.86 0.73
0.59
0.67
0.77
0.90
0.75
0.83
332/334(RS) \ Rec. m/z 259
0.81 77 72:1
47:1
' 36:1
7:1
0.79 29 49:1
0.73 71 23:1
42:1
43:1
49:1
50:1
72:1
22:1
0.77 56 12:1
15:1
24:1
46:1
38:1
73:1
20:1
0.75 73 12:1
9.4:1
0.72 85 18:1
6.3:1
0.71 48 9:1
11:1
14:1
48:1
35:1
73:1
26:1
S/N
o/z 322 ra/z 334(IS) Comments
> 62:1 > 63:1
48:1
32:1
16:1
63:1 > 33:1 Low recovery.
49:1 > 63:1
40:1
41:1
49:1
64:1
63:1
20:1
15:1 > 63:1
26:1
33:1
63:1
58:1
> 63:1
23:1
42:1 > 63:1
25:1
31:1 > 63:1
12:1
16:1 > 63:1
15:1 Ratio unacceptable.
20:1
63:1
58:1
63:1
22:1
.Aqueous cample data reported as ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
Isomer could not be identified.
_• - - (continued)
-27
-------
TABLE 12. (continued)
Aliquot
Sample Air-dry wt. (g)
No. or Vol. (L)
8367-68- 1576X-H1
8367-69-1576x-HlD
8367-71-1576x-Bl
8367-72-1576x-BlD
8367-73-1576x-BlN
—
8367-74-1576x-H3
8367-77-1576x-FA
TCDD
Isomer
9.67 g 2,3
1
10.00 g 2
1
1
1.02 g 2
1
1
c
1.05 g 2
1
1
c
1.03 g 2
1
1
c
1
1
1
1
1
1.15 g 2
1
1
c
1
c
9.94 g 2
1
1
c
,3
,3
,3
,3
,3
,3
,3
.3
,3
,3
,3
,3
,3
,2
,2
,2
,2
,2
,3
,3
,3
,2
,3
,3
,3
,4
,6
,7
,6
,7
,7
,6
,7
,7
,6
,7
,7
,6
,7
,3
,3
,3
,7
,8
,7
,6
,7
,7
,7
,6
,7
,7-
,8-
.8-
,8-
,9-
,8-
,8-
,9-
,8-
,8-
.9-
,8-
,8-
,9-
,6/
,8-
,4-
,8-
,9-
,8-
,8-
,9-
,8-
,8-
,8-
,9-
TCDD (ppt
Retention time or ppq) Instr
TCDD
21:
42
16:48
21:33
16:
17:
21:
16:
17:
19:
21:
16:
17:
19:
21:
16:
17:
19:
22:
22:
24:
29:
21:
16:
18:
19:
24:
26:
21:
16:
17:
42
52
37
44
54
04
35
42
53
02
41
48
59
09
01
15
12
41
44
49
00
10
58
03
36
42
53
19:02
lJC12-2,3,7,8 Meas. DL ID
21:40 34.3 - MS50
4.5 -
21:31 36.6 - MS50
5.2 -
10.0 -
21:35 937 - MS50
160
312
50.6 -
21:33 785 - MS50
201
308
ND 28.9
21:41 1,280 - MS50
333
635
52
518
695
1,170
463
21:42 2,020 - MS50
164 - -
237
70.6 -
31.7 -
27.3 -
21:34 1,720 - MS50
1,880
1,750
1,250
HRGC/HRMS Analysis
Relative Ion Abundance Ratios
Date Time 320/322 332/334(15) 332/334(RSj 1 Rec.
09/24/85 15:02 0.
0.
09/24/85 15:32 0.
0.
0.
09/25/85 10:01 0.
0.
0.
0.
09/25/85 10:30 0.
0.
0.
0.
09/25/85 11:27 0.
0.
0.
0.
0.
0.
0.
0.
09/25/85 13:00 0.
0.
0.
0.
0.
0.
09/25/85 13:30 0.
0.
0.
0.
82 0.67 0.75 73
67
70 0.74 0-.81 46
69
88
83 0.83 0.83 95
85
84
69
78 0.85 0.80 75
87
82
65
77 0.83 0.81 80
77
85
81
72
78
82
83
81 0.81 0.81 79
86
81
74
92
68
80 0.82 0.82 4
83
81
80
m/z 259
29:1
11:1
39:1
11:1
15:1
97:1
34:1
44:1
7.3:1
73:1
36:1
40:1
6.5:1
97:1
47:1
79:1
6.7:1
57:1
51:1
87:1
23:1
> 145:1
"22:1
24:1
9:1
2.5:1
3:1
67:1
109:1
94:1
60:1
S/N
ra/z 322 m/z 334(IS) Comments
63:1 > 63:1
14:1
31:1 > 54:1
5:1
10:1
63:1 > 63:1
14:1
24:1
4:1
63:1 > 63:1
22:1
28:1
3:1 Ratio unacceptable.
> 63:1
23:1
35:1
4:1
27:1
29:1
63:1
29:1
> 63:1 > 63:1
> 6:1
> 10:1
> 3:1
6:1
6:1
28:1 6:1 Lou recovery.
38:1
34:1
22:1
.Aqueous sample data reported as "ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
Isomer could not be identified.
(continued)
28
-------
TABLE 12. (continued)
Aliquot
Sample ' Air-dry we. (g) TCOD
No. or Vol. (L) Isomer
8367-77- 15 76X-FA c
(concluded) c
c
1,2,3,7/
1,2,3,8-
1,2,3,4-
c
1,2,7,8-
c
c
c
c
c
c
8367-96- 1576X-H2W 1.0 L_ 2,3,7,8-
1,3,6,8-
1,3,7,9-
c
c
c
1,2,7,8-
c
c
c
c
8367-83-1576X-DWD 1.0 L 2,3,7,8-
1,3,6,8-
1,3,7,9-
c
8367-82- 1576X-DW 1.0 L 2,3,7,8-
1,3,6,8-
1,3,7,9-
TCDD (ppt
Reteation tine or ppq) Instr
TCDD
20:00
20.43
21:25
21:54
22:08
22:35
24:05
24:49
25:23
25:55
27:18
28:07
29:41
21:42
16:47
17:58
19:08
20:06
20:49
24:13
24:55
26:01
27:25
28:13
21:11
16:25
17:34
23:39
21:10
16:22
17:32
lJCl2-2,3,7,8 Meas. DL ID
1,220 - MS50
274
109
675
4,640
879
720
3,460
155
3,430
441
2,920
ND 58
21:41 NC - MS50
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
21:10 265 - MS50
ND 167
125
50.7 -
21:09 300 - MS50
140
106
HRGC/HRMS Analysis
Relative Ion Abundance Ratios
Date Time 320/322 332/334(15)
09/25/85 13:30 0.84
0.75
0.77
0.80
0.81
0.88
0.88
0.80
0.75
0.78
0.79
0.68
0.57
09/25/85 15:18 0.86 8.3
0.81
0.84
0.79
0.98
0.68
0.75
0.80
0.72
0.82
0.81
09/26/85 09:56 0.79 0.73
0.91
0.71
0.71
09/26/85 10:26 0.72 0.70
0.81
0.96
332/334(RS) X Rec. m/z 259
44:1
16:1
6:1
32:1
146:1
31:1
23:1
80:1
7:1
86:1
14:1
68:1
7:1
0.82 ND > 154:1
18:1
12:1
20:1
6:1
3:1
3:1
37:1
63:1
9:1
30:1
0.70 42 18:1
18:1
14:1
3.2:1
0.74 16 10:1
6.3:1
3.8:1
S/N
m/z 322 m/z 334(IS) Cooaents
15:1
5:
2.5:
11:
63:
14:
15:
56:1
5:1
63:1
9:1
46:1
4:1 Ratio unacceptable.
> 65:1 4:1 332/334(13) Ratio unacceptable; no amount
computations performed.
55:1
40:1
63:1
20:1 -
11:1
3:1
35:1
63:1
8:1
25:1
31:1 47:1
24:1 - Ratio unacceptable.
21:1
7:1
21:1 22:1 Low recovery. Rerun.
12:1
11:1
^Aqueous sample~data reported as ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
Isomer could not be identified.
(continued)
29
-------
TABLE 12. (continued)
Aliquot
Sample Air-dry wt. (g)
No. or Vol. (L)
8367-88-1576X-EWW 1.0 L
8367-95-1576X-H2WD 1.0 L
8367-96-1576X-H2WN 1.0 L
-
—
-
•
-
TCDD
Isomer
2,3.7,8-
1,3,6,8-
1,3,7,9-
1,2,7,8-
2,3,7,8-
1,3,6,8-
1,3,7,9-
c
c
c
c
1,2,7,8-
c
c
c
c
c
c
c
2,3,7,8-
1,3,6,8-
1,3,7,9-
c
c
c
c
1,2,7,8-
c
c
c
c
c
c
c
Retention time
TCDD '•JC12-2,3,7I8
21:18 21:15
16:28
17:38
23:44
21:13 21:12
16:25
17:35
18:43
19:38
20:20
22:11
23:39
24:23
25:27
26:47
27:37
29:34
30:12
31:07
21:14 21:13
16:26
17:35
18:44
19:41
20:22
21:13
23:40
24:24
25:30
26:51
27:39
29:36
30:14
31:12
TCDD (ppt
or ppq) Instr.
Meas. DL ID
1,030 - MS50
119
221
119
NC - MS50
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC - MS50
NC
NC -
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
HRCC/HRMS Analysis
Relative Ion Abundance Ratios
Date Time 320/322 332/334(15) 332/334(RS)
09/26/85 12:32 0.76 0.79 0.75
0.71
0.68
0.76
09/26/85 13:05 0.93 13.3 0.67
0.77
0.82
0.81
0.87
0.79
0.84
0.70
0.76
0.76
0.79
0.73
0.71
0.81
0.58
09/26/85 14:21 0.87 9.47 0.87
0.79
0.78
0.78
0.89
0.63
0.59
0.79
0.73
0.75
0.76
0.70
0.77
0.74
0.33
% Rec. m/z 259
66 73:1
12:1
17:1
6:1
NC > 146:1
76:1
42:1
46:1
15:1
9:1
5:1
5:1
54:1
79:1
11:1
36:1
7:1
20:1
4:1
NC > 145:1
40:1
22:1
34:1
11:1
7:1
4:1
5:1
58:1
89:1
11:1
39:1
9:1
23:1
3:1
S/N
m/z 322 m/z -334(15) Comments
> 63:1 > 63:1
> 10:1
> 15:1
31:1
> 63:1 32:1 332/334(15) Ratio unacceptable; no amount
computations performed.
> 26:1
> 14:1
> 17:1
> 5:1
> 4:1
> 2:1
3.5:1
39:1
63:1
8:1
25:1
5:1
15:1
2.5:1
> 63:1 3:1 332/334(15) Ratio unacceptable; no amount
computations performed.
> 23:1
> 15:1
> 21:1
> 6:1
> 5:1 - Ratio unacceptable.
> 6:1 - Ratio unacceptable.
> 3:1
> 41:1
> 69:1
> 10:1
> 28:1
> 6:1
> 20:1
> 25:1 - Ratio unacceptable.
^Aqueous-sample data reported as ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
clsooer could not be identified.
(continued)
30
-------
TABLE 12. (continued)
Aliquot
Sample Air-dry wt. (g)
No. or Vol. (L)
8367-75-1576X-H3D 1.16 g
8367-76-1576-X-H3N 1.14 g
8367-78-1576X-FAD 10.04 g
-
8367-99-1576X-FAN 9.93 g
"
2
1
1
c
2
1
1
c
1
1
1
1
1
2
1
1
c
c
c
1
1
1
c
c
c
. c
c
c
c
2
1
1
c
c
c
1
1
1
TCDD
I some r
.3
,3
,3
,3
,3
,3
,2
,2
,2
,2
,2
,3
,3
,3
,2
,2
,2
,3
.3
.3
,2
,2
,2
,7
,6
,7
,7
,6
,7
,3
,3
.3
,7
,8
,7
,6
,7
,3
,3
,3
,7
,6
,7
,3
,3
,3
.8-
.8-
.9-
,8-
,8-
,9-
,77
,8-
,*-
,8-
,9-
,8-
,8-
,9-
,11
,8-
,4-
,8-
.8-
,9-
,7/
,8-
,4-
TCDD (ppt
Retention time or ppq) Instr
TCDD
21:20
16:31
17:41
18:49
21:39
16:45
17:56
19:07
21:58
22:11
24:10
29:36
21:33
16:41
17:51
19:01
19:57
20:41
21:52
22:05
22:33
24:01
24:45
25:51
27:14
28:03
30:01
21:39
16:45
17:56
19:06
20:01
20:47
21:58
22:11
lJCl2-2,3,7,8 Meas. DL ID
21:19 2,260 - MS50
116
163
21:39 1,800 - MS50
383
367
ND 49.5
825
855
2,330
952
21:30 1,020 - HS50
926
747
610
557
146
286
2,260
MD 329
356
3,520
3,680
558
3,120
3,270
21:38 1,160 - MS50
1,390
1,160
881
888
194
423
3,620
HT.CC/HRMS Analysis
Relative Ion Abundance Ratios
Date Time 320/322 332/334(13)
09/27/85 15:31 0
0
0
09/30/85 09:57 0
0
0
1
0
0
0
0
09/30/85 10:29 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
09/30/85 13:00 0
0
0
0
0
0
0
0
.79 0.88
.90
.67
.80 0.79
.80
.81
.00
.81
.83
.85
.77
.73 0.71
.77
.80
.87
.89
.71
.67
.79
.91
.74
.78
.79
.83
.76
.79
.80 0.84
.78
.79
.85
.74
.76
•80
.86
332/334(RS) I Rec. ra/z 259
0.80 99 > 145:1
14:1
20:1
6:1
0.80 86 > 143:1
44:1
41:1
5:1
79:1
60:1
145:1
46:1
0.88 6.7 45:1
55:1
42:1
32:1
23:1
8.7:1
17:1
75:1
16:1
13:1
92:1
97:1
17:1
80:1
, 83:1
0.88 5 56:1
94:1
80:1
60:1
35:1
13:1
29:1
131:1
32:1
S/H
m/z 322 m/z 334(IS) Comments
> 63:
> 5:
> 7:
> 2.5
> 63:
> 15:
> 17:
> 2.5:
> 29:
> 24:
63:
23:
32:
37:
25:
22:
14:
6:
10:
63:
11:
8:
56:
63:
10:
48:
47:
25:
40:
28:
18:
17:
5:
10:
63:
10:
I > 63:1
1
1
:1
1 > 63:1
I
1
1 Ratio unacceptable.
1
\
1
1
1 9:1 Recovery low.
I
1
1
1
1
I
1
1 Ratio unacceptable.
1
1
1
1
1
1
1 6:1 Low recovery.
1
1
1
1
1
1
1
1
.Aqueous sample data reported as ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
IsoBer could not be identified..
(continued)
- 31
-------
TABLE 12. (concluded)
Aliquot
Sample Air-dry wt. (g) TCDD
No. or Vol. (L) Isomer
8367-99-1576X-FAN
(concluded)
8367-100-1576X-DWD 1.0 L
8367-102-1576X-IND 500 mL
8367-101-1576X-EWD 1.0 mL
8367-103-1576X-IND 500 mL
8367-104-1576X-H2W 430 mL
~
8367- 105- 1576X-H2W 430 mL
-
c
1,2,7,8-
c
c
c
c
c
c
2,3,7,8*
1,3,6,8-
1,3,7,9-
2,3,7,8-
2,3,7,8-
1,3,6,8-
1,3,7,9-
2,3,7,8-
1,3,6,8-
1,3,7,9-
2,3,7,8-
1,3,6,8-
1,3,7,9-
c
c
c
c
c
2,3,7,8-
1,3,6,8-
1,3,7,9-
c
c
c
c
c
TCDD (ppt
Retention time or ppq) Instr
TCDD
22:38
24:08
24:52
25:58
27:20
28:10
30:09
30:47
21:04
16:19
17:26
21:02
20:58
16:14
17:23
20:57
16:17
17:23
21:00
16:18
17:27
18:32
24:06
25:09
27:16
29:49
20:56
16:15
17:22
18:30
24:03
25:07
24:17
29:45
1JCl2-2,3,7,8 Meas. DL ID
562 - MS50
516
4,310
4,530
ND 632
3,980
4,080
1,170
21:01 246 - MS50
637
489 -
21:03 604 - MS50
20:55 1,050 - MS50
157
384
20:56 628 - MS50
ND 45
87
20:59 27,100 - MS50
ND 71
164 -
391 -
ND 427
531 -
575 -
313 -
20:56 28,100 - MS50
224 -
302 -
453 -
564 -
730 -
518 -
347 -
HRCC/HRMS Analysis
Relative Ion Abundance Ratios
Date Time 320/322 332/334(13) 332/334(RS)
09/30/85 13:00 0.81
0.82
0.75
0.73
0.95
0.77
0.88
0.71
10/03/85 11:32 0.86 0.82 0.74
0.77
0.80
10/03/85 12:58 0.71 0.87 0.85
10/03/85 13:28 0.69 0.82 0.90
0.81
10/03/85 14:01 0.71 0.75 0.78
0.52
0.69
10/03/85 14:33 0.74 0.84 0.74
1.05
0.68
0.85
0.65
0.70
0.81
0.86
10/03/85 15:17 0.80 0.84 0.71
0.85
0.88
0.73
0.80
0.81
0.80
0.79
% Rec. m/z 259
5 32:1
20:1
127:1
146:1
20:1
24:1
122:1
26:1
68.5 21:1
73:1
51:1
59.5 16:1
80 72:1
17:1
28:1
57 18:1
6:1
8:1
78 > 145:1
9:1
16:1
30:1
19:1
32:1
28:1
18:1
96 > 145:1
16:1
17:1
30:1
20:1
33:1
23:1
13:1
S/N
m/z 322 m/z 334(IS) Comments
10:1
10:1
54:1
63:1
7:1 Ratio unacceptable.
46:1
44:1
14:1
18:1 > 63:1
63:1
47:1
31:1 > 63:1
63:1 > 63:1
13:1
26:1
42:1 > 63:1
13:1
17:1
> 63:1 > 63:1
18:1 Ratio unacceptable.
31:1
55:1
21:1 Ratio unacceptable.
32:1
26:1
27:1
> 63:1 > 63:1
19:1
21:1
34:1
14:1
31:1
14:1
10:1
.Aqueous sample data reported as ppq and soil sample data presented as ppt.
Criteria for positive identification require that the ion ratios fall between 0.67 and 0.90.
Isomer could not be identified.
32
-------
TABU I). ACCUUCT AND HKCtStON OT THE HUGC/Him AMLTSIS TO* 2,3,7,8-TCDO
FROM UBORATOBT AQUEOUS HATRIK SPIKES
U>
CO
2.3,7,8-Troo 2.3,7,8-Tcno
Saaple •*trl« Spike level (ppq) Delected (ppq)
Dlatilled water (DW)
Efflmnt traatewater (CW)
Influeot matevater (IW)
Indnitrlil waatevater (IHD)
Industrial vaatevater (IHD)
Soil extract (H2H)
ISO
250
250
1,000
1,000
1,000
SOO
500
SOO
SOO
SOO
SOO
-
-
.
-
Average cone
RPR
Average cone
RPR
Average cone
RPR
Average cone
RPR
Average cone
RPDD
Average cone.
RPD
2)4
26S
2*6
. 2*8
12.5
1,090, 1,030
1,010
1,050
. 1,050
7.6
53*
SOS
S30
. 524
5.0
1,2*0
1,520
1,430
. 1,410
16
604
628
. 616
3.9
27,100
28,100
27,600
3.6
2,3.7,8-TCOO
Recovery (X)
•3.6
106
103
Average ree. 101
RPR 9.3
109, 10.1
101
IDS
Average rec. 105
RPR 7.6
107
102
106
Average rec. IDS
RPR 4.8
2S8
304
286
Average rec. 283
RPD 16
.
-
.
-
"C.,-2
AbaolMte
Average rec.
RPR
Average rec.
RPR
Average ree.
RPR
Average rec.
RPR
Average rec.
RPD
Average rec.
RPD
,3,7,8-TCOO
recovery (X)
•2
42
69
64
6]
61, 66
91
80
75
40
77
75
71
74
8.1
23
20
29
24
38
60
57
58
5.2
78
96
87
25
Relative percent raage (calculated from the difference of the high and low values divided by the average of all value* and
.•ulllplled by 100 percent).
Relative percent difference.
-------
TABLE 14. PRECISION OF THE IIRGC/IIRNS ANALYSIS FOR 2,3,7,8-TCDD
OF SOIL AND FLY ASH SAMPLES
Saaple •atrin
B25-Plazza Road (BS)
Hyde Park 001 (III)
B52-Shen«ndoah (Bl)
Hyde Park 003 (111)
Fly ash - RRAI
Endogenous
2,3,7,8-TCI)»
level (Pl>l)a
50
Average
RPR°
70
Average
RPR
360
Average
RPR
1,700
Average
RPR
.
Average
RPR
2,3,7,8-TCDD
Detected (ppt)
18.2
15.1
12.9
rone. 15.4
34
34.3
36.6
30.3
cone . 33 . 7
19
937
785
1,280
cone. 1,000
50
2,020
2,260
1,800
cone. 2,0.10
23
1,720
1,020
1,160
eonc. 1,300
54
•
>3C,2-2
Absolute
Average rec.
RPR
Average rec.
RPR
Average rec.
RPR
Average rec.
RPR
Average rec.
RPR
,3,7,8-TCDD
recovery (J.)
73
85
48
69
54
73
46
56
58
47
95
75
80
83
24
79
99
86
88
23
4
7
5
5.3
57
"Estimated level of endogenous 2,3,7,8-TCDD reported to NRI by Dr. W. Beckert in letters dated
bApril 19, 1985, and August .10, 1985.
Relative percent range (calculated frno the difference of the high and low values, divided by
the average ol all values, and 1111111111 led by 100 percent.
-------
TABLE IS. ACOimCT Of THE HRCC/ICWO IgtllOO FOB THE DCTtRHIMTIOH Of TCTO ISCTBO SPIKED HTTP AQUEOUS HATHICES
Efflueat vaatc
fCDD
analyte
1.3.6.8
1,3,7.9
1.2.3.7/1,2,3,8
1,2.3.4
1.2.7,8
1.2,8.9
2.3.7.8
"Clt-2,3. 7,8
Spike
(PI)
1.840
840
1,680
2,440
3,080
1,200
1,000
500
Heaaiired
(PR)
502
766
1,860
1.840
3,430
1.330
1,010
455
water
Recovery
U)
27
91
110
75
III
III
101
91
Dlatllled water
Spike
(PI)
460
210
420
610
770
300
250
500
Heaanred
(PS)
512
395
403
616
840
328
234
410
Recovery
(1)
III
190
96
101
110
110
94
82
Inl
Spite
(PI)
920
420
840
1,220
1.540
600
500
500
TlMnt vaatevater
Heamred
(PI)
582
690
940
1.180
1.790
691
530
355
Recovery
(I)
63
164
112
97
116
IIS
106
71
Indnatrlal vaatevater
Spike
(PI)
920
420
840
1.220
1,540
600
500
500
(featured
(PI)
HD'
HD
HD
RD
586
HD
904"
100
Recovery
(t)
0
0
0
0
38
0
181
20
jHot detected.
Meaaured value corrected for eedogenoaa 2,3,7,8-TCDD content (averaged 616 pi/L).
Oi
-------
ARS36B(V), I
TABU 16. ACCURACY OP THE HHCC/HRM3 METHOD Km THE PETERrllHATIO*! Of TCTO ISOHERS SPIKED IHTO SOU HATH I Ct 3
Hyde Park 001
TCOO
•oalfte
1,3,6,8
I.3.M
1,2,3,7/1,2,3.8
1,2,3.4
1,2,7.8
1,2.8.9
2.3,7.8
l3Clt-2.3.7.8
Spike
(PI)
130
60
120
170
220
84
-
500
Heasored
(PI)
29.0
Sl.l
125
118
252
100
30.3
280
(HI)
Recovery
(M
22
86
106
69
117
119
-
56
B25-Piazza Road
Spike
_j£il
92
42
84
120
ISO
60
-
500
Measured
(PI)
m> (9.2)*
IS. 2
61.8
54.1
147
63.9
12.9
240
JMJ
Recovery
(1)
0
36
74
44
95
107
-
48
B52-Sbenandoah
Spike
(pi)
660
300
600
880
1,110
430
-
500
Measured
(PI)
333
63S
SIB
695
1.170
463
1.280
400
m
Recovery
(t)
SO
210
87
79
106
108
-
80
HT«
iplkV
(Pi)
1.560
710
1.430
2,070
2.620
1.020
-
500
le Park 003
Heaaured
(PI)
383
367
825
855
2,330
9S2
1,800
430
(H3)
Recovery
24
SI
58
41
89
93
-
86
to
*ND - not detected. The val«e U parentheses reflects the cstiuted detection limit.
-------
TABLE 17. FORTIFIED FIELD BLANK RESULTS
Sample
Ho.
8367-62- 1576X-FFV3
8367-64-1576X-FFSB
8367-63- 15 76X-FFSA
8367-61-1576X-FFWA
8367-81-1576X-FFB
8367-80-1576X-FFA
8367-97-1576X-FFA
8367-98- 1576X-FFB
Aliquot
Air-dry wt. (g)
or Vol. (L)
1.0 L
10 g
10 g
1.0 L
10.01 g
10.01 g
1.0 L
1.0 L
Retention tine
Native
23:38
23:38
23:40
23:40
22:17
21:37
21:37
21:43
13C
23:35
23:38
23:38
23:39
22:15
21:35
21:37
21:43
Instr.
ID
MS50
MS50
MS50
MS50
MS50
MS50
MS50
MS50
Relative Ion Abundance Ratios
Date
09/11/85
09/11/85
09/12/85
09/12/85
09/20/85
09/20/85
09/20/85
09/20/85
Time
14:08
14:42
14:47
15:14
13:33
14:09
09:10
09:24
320/322
0.78
0.79
0.77
0.80
0.88
0.86
0.82
0.80
332/334(15)
-
-
0.78
0.76
-
0.73
0.83
-
332/334(RS)
0.71
0.80
-
-
0.74
-
-
0.82
% Rec.
65
68
71
79
29
83
50
48
m/z 259
> 145:1
> 145:1
145:1
145:1
144:1
145:1
23:1
145:1
S/N
ra/z 322
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
m/z 334(IS)
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
> 63:1
Comments
Low recovery
37
-------
HRGC and Mass Resolution
Table 11 presents a summary of all chromatographic and mass resolution
checks completed during the final method evaluation. As per the protocol
requirements the required mass resolution was demonstiated as the first and
last quality control requirements for each day. The column performance
check mixture was also analyzed before the first sample analysis and after
the final sample analysis each day as a QC measure to assure that speci-
ficity for 2,3,7,8-TCDD was maintained. The mass measurement accuracy at
m/z 330.979 is also included in this table, as it was verified on a daily
basis prior to any sample analyses.
Sample Analysis
The results from the analysis of the aqueous and soil samples are pro-
vided in Table 12. The data in Table 12 are presented in the format speci-
fied as Form B-l in the protocol reporting requirement. The data are re-
corded in the chronological order that they were obtained by HRGC/HKMS.
As indicated in Table 12, several samples required reanalysis due to
low recovery of the internal standard, unacceptable ion ratios for 320/322,
or the result of interferences at the internal standard. Two of the dis-
tilled water samples demonstrated responses for the characteristic ions at
m/z 259, 320, and 322 for 2,3,7,8-TCDD. However, the ion ratio for the
native 2,3,7,8-TCDD in one replicate and the ion ratio for the internal
standard in another required that both samples be reanalyzed. Although both
samples met all the qualitative criteria, recoveries were noted to be low
(< 20 percent) for one of the samples and complete reanalysis of the repli-
cate was required.
Significant problems were encountered with the aqueous soil extract,
H2W, and the fly ash sample. The problems with the soil extract resulted
from an interference at m/z 332 that coeluted with the internal standard,
13C12-2,3,7,8-TCDD. No accurate quantitative measurements could be achieved
for TCDD responses observed for this sample. The original sample contained
a large amount of suspended particulate in each of the three replicates.
Problems with the extraction were noted with the first portion of methylene
chloride. A large amount of particulate matter was noted at the interface
of the aqueous and organic phases. Hence, the interference at m/z 332 and
TCDD responses observed in these replicates were probably due to direct ex-
traction of the suspended soil particulate rather than the actual water-
soluble TCDD.
The remaining aqueous sample for H2W was centrifuged for 10 min at ap-
proximately 2,000 rpm, and the aqueous phase was decanted from the settled
particulate. The resulting aqueous sample was divided into duplicate 430-mL
samples and each was analyzed according to the protocol. The HRGC/HRMS
analysis was successful for both replicates with absolute recoveries of
78 percent and 96 percent of the internal standard.
The triplicate analyses of the fly ash sample resulted in absolute
recoveries less than 10 percent for the internal standard in each aliquot
38
-------
analyzed. These low recoveries may be associated with the total fixed car-
bon content of the fly ash material. Previous work in this laboratory with
fly ash from coal-fired power plants has demonstrated low recoveries of ana-
lytes from materials with high carbon content.4
The only other sample for which successful analysis was not achieved as
specified in the protocol on first analysis was the industrial wastewater
(IND). The triplicate analysis of the sample resulted in absolute internal
standard recoveries of 23, 20, and 29 percent. The criteria for successful
analysis for TCDD as discussed in the protocol require an absolute recovery
of 40 to 120 percent. In addition to the observed low recoveries, the level
of 2,3,7,8-TCDD detected in the sample averaged 1,410 ppq as compared to the
500-ppq spike level. Two 500-mL aliquots of the unspiked industrial waste-
water sample were reanalyzed to determine the background level of 2,3,7,8-
TCDD. The results of the duplicate analysis yielded an average 2,3,7,8-TCDD
concentration of approximately 620 ppq and the absolute recoveries were
noted to be 60 percent and 57 percent. The increase in absolute recovery of
the internal standard in the unspiked sample by approximately a factor of
two is possibly due to the preparation of samples one half the size of that
used for the original analysis. This suggests that the sample matrix has a
considerable impact on the effectiveness of the cleanup procedure.
Table 13 provides a summary of the accuracy and precision of the analy-
ses of the five aqueous sample types for 2,3,7,8-TCDD. Only the data points
from Table 12 that demonstrate compliance with all QC criteria (ion ratios,
absolute recovery of the internal standard, etc.) are included in Table 13.
These data demonstrate that the isotope dilution method of quantitation pro-
vides accurate and precise quantitation of 2,3,7,8-TCDD in the aqueous sam-
ples. It should be noted that even when the absolute recovery of the 13Ci2~
2,3,7,8-TCDD internal standard varies by as much as 66 percent (RPR) for the
triplicate distilled water samples, the accuracy of the measurement of the
spiked 2,3,7,8-TCDD averaged 101 percent with less than 10 percent variabil-
ity. Table 13 summarizes data for both the spiked and unspiked aliquots of
industrial wastewater. The high recovery noted for the 2,3,7,8-TCDD value
in the spiked samples is a result of the presence of this compound at ap-
proximately 620 ppq in the original matrix.
Table 14 presents a similar summary for the five solid samples ana-
lyzed. The precision of the measurements is not quite as good as noted for
the aqueous samples and may reflect the difference in adsorption of the
endogenous 2,3,7,8-TCDD and the spiked internal standard to the matrices.
Tables 15 and 16 provide data dealing with the accuracy of the HRGC/
HRMS methods for the determination of total TCDD isomers in aqueous and
solid samples. In general, the data support the use of the internal stan-
dard method of quantitation for all but the earliest eluting isomers,
1,3,6,8- and 1,3,7,9-TCDD. The accuracy for the additional isomers is very
good and more consistent than is observed for the solid samples. This may
be partially due to the differences in adsorption to the soil particles.
39
-------
Fortified Field Blanks
As part of the overall quality assurance/quality control (QA/QC) pro-
gram identified in the HRGC/HRMS protocol, the analyst is required to ana-
lyze fortified field blanks to demonstrate (a) that the extraction and
cleanup procedure will provide recovery of the 2,3,7,8-TCDD within the cri-
teria of greater than 40 percent specified in the protocol and (b) that the
reagents are free from contamination with TCDD isomers.
Table 17 provides the results of the fortified field blanks run before
proceeding with sample analysis and also those of an additional set of
blanks prepared along with the actual samples. The analyses of the forti-
fied field blanks at the outset of the study demonstrated that the recover-
ies of 2,3,7,8-TCDD and 1,2,3,4-TCDD ranged from 65 to 79 percent. No de-
tectable levels of other TCDD isomers were found in this preliminary study.
The field fortification blanks analyzed with the actual samples resulted in
recoveries of 29 percent and 83 percent. More importantly, these analyses
demonstrated some interferences arising from 1,3,6,8- and 1,3,7,9-TCDD.
Previous studies involving evaluation of the cleanup procedure indicated
that these isomers are associated with the acidic alumina cleanup.
Figure 4 is a plot of the ratio of response of 1,3,6,8- and 1,3,7,9-
TCDD and the response of the recovery standard 13Cj2-l,2,3,4-TCDD versus the
time elapsed since the acidic alumina was cleaned and activated at 190°C.
The results of the analyses of the fortified field blanks and the samples
not spiked with the 1,3,6,8- and 1,3,7,9-TCDD isomers are presented in Fig-
ure 4. As noted from this plot, these TCDD isomers were not initially de-
tected in the acidic alumina immediately following cleanup by Soxhlet ex-
traction. The first set of fortified field blanks was taken through the
acidic alumina column 7 days later. Although response was observed at m/z
320 and 322 at the retention time for these isomers, the ion ratios did not
indicate presence of the compounds. Since the detectable levels were well
below 10 pg/g of alumina, the sample analyses were initiated. The data for
the fortified field blanks and samples taken through alumina from 14 to 30
days from activation indicate that the contamination of the 1,3,6,8- and
1,3,7,9-TCDD isomers apparently occurs over time using this particular oven.
The background contamination of 1,3,6,8- and 1,3,7,9-TCDD isomers has also
been recently addressed by the Center for Disease Control.5
Note Added in Proof
A second magnetic sector instrument (built in 1976) from a different
manufacturer was tested and found to be incapable of achieving sufficient
sensitivity at 10,000 resolving power to be used in experiments for this
study.
40
-------
Fortified Field Blanks
[J Aqueous and Environmental Samp I
10 20
Time (DaysJ Elapsed from Cleanup and
Activation of Acidic Alumina "~
Figure 3.
Background levels of 1,3,6,8- and 1,3,7,9-TCDD observed
over the single-laboratory evaluation study.
41
-------
REFERENCES
1. U.S. Environmental Protection Agency, "Dioxin Strategy," prepared by the
Office of Water Regulations and Standards and the Office of Solid Waste
and Emergency Response in conjunction with the Dioxin Strategy Task
Force, Washington, D.C., November 28, 1983.
2. L. R. Williams, Validation of Testing/Measurement Methods.
EPA 600/X-83-060, 1983.
3. GC Bulletin 793C, Supelco Inc., Beliefonte, Pennsylvania, 1983.
4. C. L. Haile, J. S. Stanley, T. Walker, 6. R. Cobb, and B. A. Boomer,
"Comprehensive Assessment of the Specific Compounds Present in Combus-
tion Processes. Volume 3. National Survey of Organic Emissions from
Coal-Fired Utility Boiler Plants," EPA-560/5-83-006, September 1983.
5. J. S. Heller, D. G. Patterson, L. R. Alexander, D. F. Groce, R. P.
O'Connor, and C. R. Lapeza, "Control of Artifacts and Contamination in
the Development of a Dioxin Analytical Program," presented at the 33rd
Annual Conference on Mass Spectrometry and Allied Topics, May 26-31,
1985, San Diego, California.
42
-------
APPENDICES
43
-------
APPENDIX A
VALIDATED ANALYTICAL PROTOCOL
for the Determination of
2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and Total
TCDDs in Soil/Sediment and Water by High-Resolution Gas
Chromatography/High-Resolution Mass Spectrometry
September 10, 1985
This analytical protocol has been written in the format used in the
Superfund program, as "Exhibit D" of a Statement of Work which in turn is part
of an Invitation-for-Bid package under the Superfund Contract Laboratory Program.
The other exhibits of the Statement of Work, although cited in Exhibit D, do
not pertain to this method evaluation study.
-------
EXHIBIT D
Analytical Methods
2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and Total
TCDDs in Soil/Sediment and Water by High-Resolution Gas
Chromatography/High-Resolution Mass Spectrometry
-------
EXHIBIT D
Section Subject
1 Scope and Application D-l
2 Summary of Method D-l
3 Definitions D-2
A Interferences D-3
5 Safety D-3
6 Apparatus and Equipment D-3
7 Reagents and Standard Solutions D-6
8 System Performance Criteria D-8
9 Quality Control Procedures D-l3
10 Sample Preservation and Handling ...... D-l3
11 Sample Extraction D-14
12 Analytical Procedures D-l7
13 Calculations D-18
-------
1. SCOPE AND APPLICATION
1.1 This method provides procedures for the detection and quantitative
measurement of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD; CAS
Registry Number 1746-01-6; Storet number 3475) at concentrations of
10 pg/g (10 parts per trillion) to 200 pg/g (200 parts per trillion)
in 10-g portions of soil and sediment and at 100 pg/L (100 parts per
quadrillion) to 2000 pg/L (2 parts per trillion) in 1-L samples of
water. The use of 1-g or 100-mL portions permits measurements of
concentrations up to 2,000 pg/g (2 parts per billion) or 20 ng/L,
respectively. This method also allows the estimation of quantities
of total TCDD present in the sample. Samples containing concentrations
of 2,3,7,8-TCDD greater than 2 ppb or 20 ng/L must be analyzed by a
protocol designed for such concentration levels, with an appropriate
instrument calibration range.
1.2 The minimum measurable concentration is estimated to be 10 pg/g (10
parts per trillion) for soil and sediment samples and 100 pg/L for
water samples, but this depends on kinds and concentrations of
interfering compounds in the sample matrix.
1.3 This method is designed for use by analysts who are experienced in
the use of high-resolution gas chromatography/high-resolution mass
spectroraetry.
CAUTION: TCDDs are extremely hazardous. It is the laboratory's responsi-
bility to ensure that safe handling procedures are employed.
2. SUMMARY OF METHOD
Five hundred pg of 13C,2~2,3,7,8-TCDD (internal standard) are added to a
10-g portion of a soil/sediment sample (weighed to 3 significant figures)
or a 1-L aqueous sample and the sample is extracted with 200 to 250 mL
benzene using a Soxhlet apparatus with a minimum of 3 cycles per hour or a
continuous liquid-liquid extractor for 24 hours. A separatory funnel and
3 x 60 mL methylene chloride may also be used for aqueous samples. After
appropriate concentration and cleanup, 50 uL of tridecane are added to the
extract. Before HRGC-HRMS analysis, 500 pg of a recovery standard ( Cj2~
1,2,3,4-TCDD) are added to the extract which is then concentrated to a
final volume of 50 uL. A 2-uL aliquot of the concentrated extract is
injected into a gas chromatograph with a capillary column interfaced to a
high-resolution mass spectrometer capable of rapid multiple ion monitoring
at resolutions of at least 10,000 (10 percent valley).
Identification of 2,3,7,8-TCDD is based on the detection of the ions m/z
319.897 and 321.894 at the same GC retention time and within -1 to +3
seconds GC retention time of the internal standard masses of m/z 331.937
and 333.934. Confirmation of 2,3,7,8-TCDD (and of other TCDD isomers) is
based on the ion m/z 258.930 which results from loss of COCL by the parent
ion.
D-l
-------
3. DEFINITIONS
3.1 Concentration calibration solutions — solutions containing known
amounts of the analyte (unlabeled 2,3,7,8-TCDD), the internal standard
13C12-2,3,7,8-TCDD and the recovery standard 13C,2-l,2,3,4-TCDD;
they are used to determine instrument response of the analyte
relative to the internal standard and of the internal standard
relative to the recovery standard.
3.2 Field blank — a portion of soil/sediment or water uncontaminated with
2,3,7,8-TCDD and/or other TCDDs.
3.3 Rinsate — a portion of solvent used to rinse sampling equipment; the
rinsate is analyzed to demonstrate that samples have not been contami-
nated during sampling.
3.4 Internal standard — ^Cj2~2,3,7,8-TCDD, which is added to every
sample (except the blanks described in Sections 4.2.1 and 4.2.3 of
Exhibit E) and is present at the same concentration in every labora-
tory method blank, quality control sample, and concentration calibra-
tion solution. It is added to the soil/sediment or aqueous sample
before extraction and is used to measure the concentration of each
analyte. Its concentration is measured in every sample, and percent
recovery is determined using an internal standard method.
3.5 Recovery standard — ^12-l»2,3,4-TCDD which is added to every sample
(except for the blanks discussed in Sections 4.2.1.A.2 and 4.2.3.6,
Exhibit E) extract just before HRGC-HRMS analysis.
3.6 Laboratory method blank — this blank is prepared in the laboratory
through performing all analytical procedures except addition of a
sample aliquot to the extraction vessel.
3.7 GC column performance check mixture — a mixture containing known
amounts of selected standards; it is used to demonstrate continued
acceptable performance of the capillary column, i.e., separation
(£ 25% valley) of 2,3,7,8-TCDD isomer from all other 21 TCDD isomers
and to define the retention time window.
3.8 Performance evaluation sample — a soil, sediment or aqueous sample
containing a known amount of unlabeled 2,3,7,8-TCDD and/or other
TCDDs. It is distributed by EPA to potential contractor laboratories
who must analyze it and obtain acceptable results before being awarded
a contract for sample analyses (see IFB Pre-Award Bid Confirmations).
It may also be included as an unspecified ("blind") QC sample in any
sample batch submitted to a laboratory for analysis.
3.9 Relative response factor — response of the mass spectrometer to a
known amount of an analyte relative to a known amount of an internal
standard.
3.10 Mass resolution check — standard method used to demonstrate static
resolution of 10,000 minimum (10Z valley definition).
D-2
-------
4. INTERFERENCES
Chemicals which elute from tbe GC column within ^10 scans of the internal
and/or recovery standard (m/z 331.937 and 333.934) and which produce ions
at any of the masses used to detect or quantify TCDD are potential inter-
ferences. Most frequently encountered potential interferences are other
sample components that are extracted along with TCDD, e.g. PCBs, methoxy-
biphenyls, chlorinated hydroxydiphenylethers, benzylphenylethers, chlori-
nated naphthalenes, DDE, DDT, etc. The actual incidence of interference
by these chemicals depends also upon relative concentrations, mass spectro-
metric resolution, and chromatographic conditions. Because very low
levels of TCDD must be measured, the elimination of interferences is
essential. High-purity reagents and solvents must be used and all equip-
ment must be scrupulously cleaned. Laboratory reagent blanks (Exhibit E,
Quality Control, Section 4) must be analyzed to demonstrate absence of
contamination that would interfere with TCDD measurement. Column chromato-
graphic procedures are used to remove some coextracted sample components;
these procedures must be performed carefully to minimize loss of TCDD
during attempts to increase its concentration relative to other sample
components.
5. SAFETY
•
The toxicity or carcinogenicity of each reagent used in this method has
not been precisely defined; however, each chemical compound should be
treated as a potential health hazard. From this viewpoint, exposure to
these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a file of
current OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material data handling
sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are identi-
fied d~3) (page D-38). 2,3,7,8-TCDD has been identified as a suspected
human or mammalian carcinogen. The laboratory is responsible for ensuring
that safe handling procedures are followed.
6. APPARATUS AND EQUIPMENT
6.1 High-Resolution Gas Chromatograph/High-Resolution Mass
Spectrometer/Data System (HRGC/HRMS/DS)
6.1.1 The GC must be equipped for temperature programming, and all
required accessories must be available, such as syringes, gases,
and a capillary column. The GC injection port must be designed
for capillary columns. The use of splitless injection tech-
niques is recommended. On-column injection techiques can be
used but this may severely reduce column lifetime for non-
chemical ly bonded columns. When using the method in this
protocol, a 2-uL injection volume is used consistently. With
some GC injection ports, however, 1-uL injections may produce
improved precision and chromatographic separation. A 1-uL
D-3
-------
injection volume may be used if adequate sensitivity and
precision can be achieved.
NOTE: If 1 uL is used at all as injection volume, the injection
volumes for all extracts, blanks, calibration solutions and
the performance check sample must be 1 uL.
6.1.2 Gas Chromatograph-Mass Spectrometer Interface
The GC-MS interface may include enrichment devices, such as
a glass jet separator or a silicone membrane separator, or
the gas chromatograph can be directly coupled to the mass
spectrometer source. The interface may include a diverter
valve for shunting the column effluent and isolating the mass
spectrometer source. All components of the interface should
be glass or glass-lined stainless steel. The interface com-
ponents should be compatible with 300°C temperatures. The
GC/MS interface must be appropriately designed so that the
separation of 2,3,7,8-TCDD from the other TCDD isomers which
is achieved in the gas chromatographic column is not appreci-
ably degraded. Cold spots and/or active surfaces (adsorption
sites) in the GC/MS interface can cause peak tailing and peak
broadening. It is recommended that the GC column be fitted
directly into the MS source. Graphite ferrules should be
avoided in the GC injection area since they may adsorb TCDD.
Vespel* or equivalent ferrules are recommended.
6.1.3 Mass Spectrometer
The static resolution of the instrument must be maintained at
a minimum 10,000 (10 percent valley). The mass spectrometer
must be operated in a selected ion monitoring (SIM) mode with
total cycle time (including voltage reset time) of one second
or less (Section 8.3.4.1). At a minimum, the following ions
which occur at these masses must be monitored: m/z 258.930,
319.897, 321.894, 331.937 and 333.934.
6.1.4 Data System
A dedicated hardware or data system is employed to control the
rapid multiple ion monitoring process and to acquire the data.
Quantification data (peak areas or peak heights) and SIM traces
(displays of intensities of each m/z being monitored as a
function of time) must be acquired during the analyses.
Quantifications may be reported based upon computer-generated
peak areas or upon measured peak heights (chart recording).
NOTE: Detector zero setting must allow peak-to-peak measurement of the noise
on the base line.
6.2 GC Columns
D-4
-------
For isomer-specific determinations of 2,3,7,8-TCDD, the following two
fused silica capillary columns are recommended: a 60-m SP-2330 column
and a 50-tn CP-Sil 88 column. However, any capillary column which
separates 2,3,7,8-TCDD from all other TCDDs may be used for such
analyses, but this separation must be demonstrated and documented.
Minimum acceptance criteria must be determined per Section 8.1. At
the beginning of each 12-hour period (after mass resolution has been
demonstrated) during which sample extracts or concentration calibra-
tion solutions will be analyzed, column operating conditions must be
attained for the required separation on the column to be used for
samples. Operating conditions known to produce acceptable results
with the recommended columns are shown in Table 2 at the end of this
Exhibit.
6.3 Miscellaneous Equipment
6.3.1 Nitrogen evaporation apparatus with variable flow rate.
6.3.2 Balance capable of accurately weighing to 0.01 g.
6.3.3 Centrifuge capable of operating at 2,000 rpm.
6.3.4 Water bath — equipped with concentric ring cover and capable
of being temperature-controlled within *2°C.
6.3.5 Stainless steel spatulas or spoons.
6.3.6 Stainless steel (or glass) pan large enough to hold contents
of 1-pint sample containers.
6.3.7 Glove box.
6.3.8 Drying oven.
6.4 Glassware
6.4.1 Soxhlet apparatus — all-glass, Kontes 6730-02 or equivalent;
90 mm x 35 mm glass thimble; 500-mL flask; condenser of appro-
priate size.
6.4.2 Kuderna-Danish apparatus — 500-mL evaporating flask, 10-mL
graduated concentrator tubes with ground-glass stoppers, and
3-ball macro Snyder column (Kontes K-570001-0500, K-503000-
0121 and K-569001-0219 or equivalent).
6.4.3 Mini-vials — 1-mL borosilicate glass with conical-shaped
reservoir and screw caps lined with Teflon-faced silicone disks.
6.4.4 Funnels — glass; appropriate size to accommodate filter
paper used to filter jar extract (volume of approximately 170 mL)
6.4.5 Separatory funnel — 2000 mL with Teflon stopcock.
D-5
-------
6.A.6 Continuous liquid-liquid extractors equipped with Teflon or
glass connecting joints and stopcocks requiring no lubrication
(Hershberg-Wolf Extractor - Ace Glass Company Vineland, NJ,
P/N 6841-10 or equivalent).
6.4.7 Chromatographic columns for the silica and alumina chroma-
tography — 1 cm ID x 10 cm long and 1 cm ID x 30 cm long.
6.4.8 Chromatography column for the Carbopak cleanup — disposable
5-mL graduated glass pipets, 7 mm ID.
6.4.9 Desiccator.
6.4.10 Glass rods.
NOTE: Reuse of glassware should be minimized to avoid the risk of
cross contamination. All glassware that is reused must be
scrupulously cleaned as soon as possible after use, applying
the following procedure.
Rinse glassware with the last solvent used in it then with
high-purity acetone and hexane. Wash with hot water containing
detergent. Rinse with copious amounts of tap water and several
portions of distilled water. Drain dry and heat in a muffle
furnace at 400°C for 15 to 30 minutes. Volumetric glassware
should not be heated in a muffle furnace, and some thermally
stable materials (such as PCBs) may not be removed by heating
in a muffle furnace. In these cases, rinsing with high-purity
acetone and hexane may be substituted for muffle furnace
heating. After the glassware is dry and cool, rinse with hexane,
and store inverted or capped with solvent-rinsed aluminum foil
in a clean environment.
7. REAGENTS AND STANDARD SOLUTIONS
7.1 Column Chromatography Reagents
7.1.1 Alumina, acidic — Extract the alumina in a Soxhlet with
methylene chloride for 6 hours (minimum of 3 cycles per hour)
and activate it by heating in a foil-covered glass container
for 24 hours at 190°C.
7.1.2 Silica gel — high-purity grade, type 60, 70-230 mesh; extract
the silica gel in a Soxhlet with methylene chloride for 6 hours
(minimum of 3 cycles per hour) and activate it by heating in a
foil-covered glass container for 24 hours at 130°C.
7.1.3 Silica gel impregnated with 40 percent (by weight) g-ulfuric
acid — add two parts (by weight) concentrated sulfuric acid
to three parts (by weight) silica gel (extracted and activated),
mix with a glass rod until free of lumps, and store in a
screw-capped glass bottle.
D-6
-------
7.1.A Sulfuric acid, concentrated — ACS grade, specific gravity 1.84.
7.1.5 Graphitized carbon black (Carbopack C or equivalent), surface
of approximately 12 m^/g, 80/100 mesh — mix thoroughly 3.6
grams Carbopak C and 16.4 grams Celite 545® in a 40-mL vial.
Activate at 130° C for six hours. Store in a desiccator.
7.1.6 Celite 545®, reagent grade, or equivalent.
7.2 Membrane filters or filter paper with pore size of <25 urn; rinse with
hexane before use.
7.3 Glass wool, silanized — extract with methylene chloride and hexane
and air-dry before use.
7.4 Desiccating Agents
7.4.1 Sodium sulfate — granular, anhydrous; before use, extract it
with methylene chloride for 6 hours (minimum of 3 cycles per
hour) and dry it for >4 hours in a shallow tray placed in an
oven operated at 120°C. Let it cool in a desiccator.
7.4.2 Potassium carbonate—anhydrous, granular; use as such.
7.5 Solvents -- high purity, distilled in glass: methylene chloride,
toluene, benzene, cyclohexane, methanol, acetone, hexane; reagent
grade: tridecane.
7.6 Concentration calibration solutions (Table 1) — five tridecane
solutions containing unlabeled 2,3,7,8-TCDD and 13C,a~l»2,3,4-TCDD
(recovery standard) at varying concentrations, and C,9-2,3,7,8-TCDD
(internal standard, CASRN 80494-19-5) at a constant concentration
must be used to calibrate the instrument. These concentration calibra-
tion solutions must be obtained from the Quality Assurance Division,
US EPA Environmental Monitoring Systems Laboratory (EMSL-LV), Las Vegas,
Nevada. However, additional secondary standards may be obtained from
commercial sources, and solutions may be prepared in the contractor
laboratory. Traceability of standards must be verified against EPA-
supplied standard solutions. Such procedures will be documented by
laboratory SOPs as required in IFB Pre-award Bid Confirmations, part
2.f.(4). It is the responsibility of the laboratory to ascertain that
the calibration solutions received are indeed at the appropriate
concentrations before they are injected into the instrument. Serious
overloading of the instrument may occur if the concentration calibra-
tion solutions intended for a low-resolution MS are injected into the
high-resolution MS.
7.6.1 The five concentration calibration solutions contain unlabeled
2,3,7,8-TCDD and labeled 13C,2~1,2,3,4-TCDD at nominal concen-
trations of 2.5, 5.0, 10.0, 20.0 and 40.0 pg/uL, respectively,
and labeled 1^C12~2»3,7,8-TCDD at a constant nominal concen-
tration of 10.0 pg/uL.
D-7
-------
7.6.2 Store the concentration calibration solutions in 1-mL mini-
vials at 4°C.
7.7 Column performance check mixture — this solventless mixture must be
obtained fvora the Quality Assurance Division, Environmental Monitoring
Systems Laboratory, Las Vegas, Nevada, and dissolved by the Contractor
in 1 mL tridecane. This solution will then contain the following
components (including TCDDs (A) eluting closely to 2,3,7,8-TCDD, and
the first- (F) and last-eluting (L) TCDDs when using the columns
recommended in Section 6.2) at a concentration of 10 pg/uL of each of
these isomers:
Analyte Approximate Amount Per Ampule
Unlabeled 2,3,7,8-TCDD 10 ng
13C12-2,3,7,8-TCDD 10 ng
1,2,3,4-TCDD (A) 10 ng
1,4,7,8-TCDD (A) 10 ng
1,2,3,7-TCDD (A) 10 ng
1,2,3,8-TCDD (A) 10 ng
1,2,7,8-TCDD 10 ng
1,3,6,8-TCDD (F) 10 ng
1,2,8,9-TCDD (L) 10 ng
7.8 Sample fortification solution — an isooctane solution containing
the internal standard at a nominal concentration of 5 pg/uL.
7.9 Recovery standard spiking solution — an isooctane solution contain-
ing the recovery standard at a nominal concentration of 100 pg/uL.
Five uL of this solution will be spiked into the extract before
HRGC/HRMS analysis.
7.10 Internal standard spiking solution — an isooctane solution containing
the internal standard at a nominal concentration of 100 pg/uL. Five
uL of this solution will be added to a fortified field blank extract
(Section 4.2.1.A.2, Exhibit E).
8. SYSTEM PERFORMANCE CRITERIA
System performance criteria are presented in two sections. One section
deals with GC column performance criteria while the other section consists
of initial calibration criteria. The laboratory may use either of the
recommended columns described in Section 6.2. It must be documented that
D-8
-------
all applicable system performance criteria specified in Sections 8.1, 8.2
and 8.3 have been met before analysis of any sample is performed. Table 2
provides recommended conditions that can be used to satisfy the required
criteria. Table 3 provides a typical 12-hour analysis sequence.
8.1 GC Column Performance
8.1.1 Inject 2 uL (Section 6.1.1) of the column performance check
solution (Section 7.7) and acquire selected ion monitoring
(SIM) data for m/z 258.930, 319.897, 321.894, 331.937 and
333.934 within a total cycle time of
-------
NOTE: Excessive PFK may cause background noise problems and contami-
nation of the source resulting in an increase in "downtime"
for source cleaning.
Using a PFK molecular leak, tune the instrument to meet the
minimum required mass resolution of 10,000 (10Z valley) at
m/z 254.986 (or any other mass reasonably close to ra/z 259).
Calibrate the voltage sweep at least across the mass range m/z
259 to m/z 334 and verify that m/z 330.979 from PFK (or any
other mass close to m/z 334) is measured within _+5 ppm (i.e.,
1.7 mmu) using m/z 254.986 as a reference. Documentation of the
mass resolution must then be accomplished by recording the
peak profile of the PFK reference peak m/z 318.979 (or any
other reference peak at a mass close to m/z 320/322). The
format of the peak profile representation must allow manual
determination of the resolution, i.e., the horizontal axis
must be a calibrated mass scale (amu or ppm per division).
The result of the peak width measurement (performed at 5
percent of the maximum) must appear on the hard copy and
cannot exceed 31.9 mmu or 100 ppm.
8.3 Initial Calibration
Initial calibration is required before any samples are analyzed for
2,3,7,8-TCDD. Initial calibration is also required if any routine
calibration does not meet the required criteria listed in Section 8.6.
8.3.1 All concentration calibration solutions listed in Table 1 must
be utilized for the initial calibration.
8.3.2 Tune the instrument with PFK as described in Section 8.2.2.
8.3.3 Inject 2 uL of the column performance check solution (Section
7.7) and acquire SIM mass spectral data for m/z 258.930,
319.897, 321.894, 331.937 and 333.934 using a total cycle time
of <_ 1 second (Section 8.3.4.1). The laboratory must not
perform any further analysis until it has been demonstrated
and documented that the criterion listed in Section 8.1.2 has
been met.
8.3*4 Using the same GC (Section 8.1) and MS (Section 8.2) conditions
that produced acceptable results with the column performance
check solution, analyze a 2-uL aliquot of each of the 5 concen-
tration calibration solutions in triplicate with the following
MS operating parameters.
8.3.4.1 Total cycle time for data acquisition must be <_ 1
second. Total cycle time includes the sum of all the
dwell times and voltage reset times.
8.3.4.2 Acquire SIM data for the following selected
characteristic ions:
D-10
-------
m/z Compound
258.930 TCDD - COC1
319.897 Unlabeled TCDD
321.894 Unlabeled TCDD
331.937 13C12-2,3,7,8-TCDD, 13C12-1,2,3,4-TCDD
333.934 13C12-2,3,7,8-TCDD, 13C12-1,2,3,4-TCDD
8.3.4.3 The ratio of integrated ion current for m/z 319.897 to
m/z 321.894 for 2,3,7,8-TCDD must be between 0.67 and
0.90.
8.3.4.4 The ratio of integrated ion current for m/z 331.937 to
m/z 333.934 for 13C1?-2,3,7,8-TCDD and 13C12-1,2,3,4-
TCDD must be between 0.67 and 0.90.
8.3.4.5 Calculate the relative response factors for unlabeled
2,3,7,8-TCDD [RRFCI)] relative to 13C12-2,3,7,8-TCDD
and for labeled 13C12-2,3,7,8-TCDD [RRF(II)] relative
to 13C2-1,2,3,4-TCDD as follows:
RRF(I) - —
AIS
RRF(II)
AIS
where
* sum of the integrated ion abundances of m/z 319.897 and m/z 321.894
for unlabeled 2,3,7,8-TCDD.
• sum of the integrated ion abundances of m/z 331.937 and m/z 333.934
sum o te ntegrate
for 13C12-2,3,7,8-TCDD.
* sum of the integrated ion abundances for m/z 331.937 and m/z
333.934 for 13C12-1,2,3,4-TCDD.
QIS - Quantity of 13C12~2,3, 7,8-TCDD injected (pg).
QRg • quantity of 13C12~1 ,2,3,4-TCDD injected (pg).
Qx » quantity of unlabeled 2,3,7,8-TCDD injected (pg).
D-ll
-------
RRF is a dimensionless quantity; the units used to express QJS» QRS an<* Qx
must be the same*
8.4 Criteria for Acceptable Calibration
The criteria listed below for acceptable calibration must be met
before analysis of any sample is performed.
8.4.1 The percent relative standard deviation (RSD) for the response
factors from each of the triplicate analyses for both unlabeled
and C12-2,3,7,8-TCDD must be less than ^20 percent.
8.4.2 The variation of the 5 mean RRFs for unlabeled 2,3,7,8-TCDD
obtained from the triplicate analyses must be less than ^20
percent RSD.
8.4.3 SIM traces for 2,3,7,8-TCDD must present a signal-to-noise
ratio of >2.5 for m/z 258.930 and MO for m/z 321.894.
8.4.4 SIM traces for 13Cj2-2,3,7,8-TCDD must present a signal-to-
noise ratio MO f°* 333.934.
8.4.5 Isotopic ratios (Sections 8.3.4.3 and 8.3.4.4) must be within
the allowed range.
NOTE: If the criteria for acceptable calibration listed in Sections
8.4.1 and 8.4.2 have been met, the RRF can be considered inde-
pendent of the analyte quantity for the calibration concentra-
tion range. The mean RRF from 5 triplicate determinations for
unlabeled 2,3,7,8-TCDD and for 13C12-2,3,7,8-TCDD will be used
for all calculations until routine calibration criteria (Section
8.6) are no longer met. At such time, new mean RRFs will be
calculated from a new set of five triplicate determinations.
8.5 Routine Calibrations
Routine calibrations must be performed at the beginning of a 12-hour
period after successful mass resolution and GC column performance
check runs.
8.5.1 Inject 2 uL of the concentration calibration solution which
contains 5.0 pg/uL of unlabeled 2,3,7.8-TCDD, 10.0 pg/uL
of 13C12-2,3,7,8-TCDD and 5.0 pg/uL 13C12-1,2,3,4-TCDD.
Using the same GC/MS/DS conditions as used in Sections 8.1,
8.2 and 8.3, determine and document acceptable calibration as
provided in Section 8.6.
8.6 Criteria for Acceptable Routine Calibration
The following criteria must be met before further analysis is per-
formed. If these criteria are not met, corrective action must be
taken and the instrument must be recalibrated.
D-12
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8.6.1 The measured RRF for unlabeled 2,3,7,8-TCDD must be within ^20
percent of the mean values established (Section 8.3.4.6) by
triplicate analyses of concentration calibration solutions.
8.6.2 The measured RRF for C,2~2,3,7,8-TCDD must be within +20 per-
cent of the mean value established by triplicate analysis
of the concentration calibration solutions (Section 8.3.A.6).
8.6.3 Isotopic ratios (Sections 8.3.4.3 and 8.3.4.4) must be within
the allowed range.
8.6.4 If one of the above criteria is not satisfied, a second attempt
can be made before repeating the entire initialization process
(Section 8.3).
NOTE: An initial calibration must be carried out whenever any HRCC
solution is replaced.
9. QUALITY CONTROL PROCEDURES
See Exhibit E for QA/QC requirements.
10. SAMPLE PRESERVATION AND HANDLING
10.1 Chain-of-custody procedures — see Exhibit G.
10.2 Sample Preservation
10.2.1 When received, each soil or sediment sample will be contained
in a 1-pint glass jar surrounded by vermiculite in a sealed
metal paint can. Until a portion is to be removed for analysis,
store the sealed paint cans in a locked limited-access area
where the temperature is maintained between 25° and 35°C.
After a portion of a sample has been removed for analysis,
return the remainder of the sample to its original container
and store as stated above.
10.2.2 Each aqueous sample will be contained in a 1-liter glass
bottle. The bottles with the samples are stored at 4°C in a
refrigerator located in a locked limited-access area.
10.2.3 To avoid photodecomposition, protect samples from light.
10.3 Sample Handling
CAUTION: Finely divided soils contaminated with 2,3,7,8-TCDD are hazardous
because of the potential for inhalation or ingestion of particles
containing 2,3,7,8-TCDD. Such samples should be handled in a
confined environment (i.e., a closed hood or a glove box).
10.3.1 Pre-extraction sample treatment
D-13
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10.3.1.1 Homogenization — Although sampling personnel will
attempt to collect homogeneous samples, the contrac-
tor shall examine each sample and judge if it needs
further mixing.
NOTE: Contractor personnel have the responsibility to take a
representative sample portion; this responsibility
entails efforts to make the sample as homogeneous as
possible. Stirring is recommended when possible.
10.3.1.2 Centrifugation — When a soil or sediment sample
contains an obvious liquid phase, it must be
centrifuged to separate the liquid from the solid
phase. Place the entire sample in a suitable centri-
fuge bottle and centrifuge for 10 minutes at 2000 rptn.
Remove the bottle from the centrifuge. With a dis-
posable pipet, remove the liquid phase and discard
it. Mix the solid phase with a stainless steel
spatula and remove a portion to be air-dried, weighed
and analyzed. Return the remaining solid portion to
the original sample bottle and store it as described
in 10.2.1.
CAUTION: The removed liquid may contain TCDD and should be
disposed as a liquid waste.
10.3.1.3 Weigh between 9.5 and 10.5 g of the air-dried soil
sample (+0.5 g) to 3 significant figures. Dry it to
constant weight at 100°C. Allow the sample to cool
in a desiccator. Weigh the dried soil to 3 signifi-
cant figures. Calculate and report percent moisture
on Form B-l.
11. SAMPLE EXTRACTION
11.1 Soil Extraction
11.1.1 Immediately before use, the Soxhlet apparatus is charged
with 200 to 250 mL benzene which is then refluxed for 2 hours.
The apparatus is allowed to cool, disassembled and the benzene
removed and retained as a blank for later analysis if required.
11.1.2 Accurately weigh to 3 significant figures a 10-g (9.50 g to
10.50 g) portion of the wet soil or sediment sample. Mix 100
uL of the sample fortification solution (Section 7.8) with
1.5 mL of acetone (500 pg of C,2~2,3,7,8-TCDD) and deposit
the entire mixture in small portions on several sites on the
surface of the soil or sediment.
11.1.3 Add 10 g anhydrous sodium sulfate and mix thoroughly using a
stainless steel spoon spatula.
D-14
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11.1.4 After breaking up any lumps, place the soil-sodium sulfate
mixture in the Soxhlet apparatus using a glass wool plug (the
use of an extraction thimble is optional). Add 200 to 250 tnL
benzene to the Soxhlet apparatus and reflux for 24 hours. The
solvent must cycle completely through the system at least 3
times per hour.
11.1.5 Transfer the extract to a Kuderna-Danish apparatus and
concentrate to 2 to 3 uL. Rinse the column and flask with 5 mL
benzene and collect the rinsate in the concentrator tube.
Reduce the volume in the concentrator tube to 2 to 3 uL.
Repeat this rinsing and concentrating operation twice more.
Remove the concentrator tube from the K-D apparatus and care-
fully reduce the extract volume to approximately 1 mL with a
stream of nitrogen using a flow rate and distance such that
gentle solution surface rippling is observed.
NOTE: Glassware used for more than one sample must be carefully
cleaned between uses to prevent cross-contamination (Note on
page D-10).
11.2 Extraction of Aqueous Samples
11.2.1 Mark the water meniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume. Pour
the entire sample (approximately 1 L) into a 2-L separatory
funnel.
11.2.2 Mix 100 uL of the sample fortification solution with 1.5 mL
of acetone (500 pg of C\2~2,3,7,8-TCDD) and add the mixture
to the sample in the separatory funnel.
NOTE: A continuous liquid-liquid extractor may be used in place of
a separatory funnel.
11.2.3 Add 60 mL mcthylene chloride to the sample bottle, seal and
shake 30 seconds to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 minutes with periodic venting.
Allow the organic layer to separate from the water phase for
a minimum of 10 minutes. If the emulsion interface between
layers is more than one-third the volume of the solvent
layer, the analyst must employ mechanical techniques to
complete the phase separation. Collect the methylene
chloride (3 x 60 mL) directly into a 500 mL Kuderna-Danish
concentrator (mounted with a 10 mL concentrator tube) by
passing the sample extracts through a filter funnel packed
with a glass wool plug and 5 g of anhydrous sodium sulfate.
After the third extraction, rinse the sodium sulfate with an
additional 30 mL of methylene chloride to ensure quantitiative
transfer.
D-15
-------
11.2.4 Attach a Snyder column and concentrate the extract until
the apparent volume of the liquid reaches 1 mL. Remove the
K-D apparatus and allow it to drain and cool for at least
10 minutes. Remove the Snyder column, add 50 mL benzene,
reattach the Snyder column and concentrate to approximately
1 mL. Rinse the flask and the lower joint with 1 to 2 mL
benzene. Concentrate the extract to 1.0 mL under a gentle
stream of nitrogen.
11.2.5 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 mL.
11.3 Cleanup Procedures - Column Cleanup
11.3.1 Prepare an acidic silica column as follows: Pack a 1 cm x 10
cm chromatographic column with a glass wool plug, a layer (1
cm) of ^804/1^03(1:1), 1.0 g silica gel (Section 7.1.2} and
4.0 g of 40-percent w/w sulfuric acid-impregnated silica gel
(Section 7.1.3). Pack a second chromatographic column (1 cm x
30 cm) with a glass wool plug, 6.0 g acidic alumina (Section
7.1.1) and top with a 1-cm layer of sodium sulfate (Section
7.4). Add hexane to the columns until they are free of
channels and air bubbles.
11.3.2 Quantitatively transfer the benzene extract (1 mL) from the
concentrator tube to the top of the silica gel column. Rinse
the concentrator tube with two 0.5-mL portions of hexane.
Transfer the rinses to the top of the silica gel column.
11.3.3 Elute the extract from the silica gel column with 90 mL hexane
directly into a Kuderna-Danish concentrator. Concentrate the
eluate to 0.5 mL, using nitrogen blow-down as necessary.
11.3.4 Transfer the concentrate (0.5 mL) to the top of the alumina
column. Rinse the K-D assembly with two 0.5-mL portions of
hexane and transfer the rinses to the top of the alumina
columns. Elute the alumina column with 18 mL hexane until the
hexane level is just below the top of the sodium sulfate.
Discard the eluate. Columns must not be allowed to reach
dryness (i.e., a solvent "head" must be maintained.)
11.3.5 Place 30 mL of 20-percent (v/v) methylene chloride in hexane
on top of the alumina and elute the TCDDs from the column.
Collect this fraction in a 50-mL Erlenmeyer flask.
11.3.6 Certain extracts, even after cleanup by column chromatography,
contain interferences which preclude determination of TCDD
at low parts-per-trillion levels. Therefore, a cleanup step
is included using activated carbon which selectively retains
planar molecules such as TCDD. The TCDDs are then removed
D-16
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from the carbon by elution with toluene. Proceed as follows:
Prepare a 18-percent Carbopak C/Celite 545® mixture by
thoroughly mixing 3.6 grams Carbopak C (80/100 mesh) and 16.4
grams Celite 545® in a 40-mL vial. Activate at 130°C for 6
hours. Store in a desiccator. Cut off a clean 5-mL disposable
glass pipet at the 4-mL mark. Insert a plug of glass wool
(Section 7.3) and push to the 2-mL mark. Add 340 mg of the
activated Carbopak/Celite mixture followed by another glass
wool plug. Using two glass rods, push both glass wool plugs
simultaneously towards the Carbopak/Celite mixture and gently
compress the Carbopak/Celite plug to a length of 2 to 2.5 cm.
Preelute the column with 2 mL toluene followed by 1 mL of
75:20:5 methylene chloride/methanol/benzene, 1 mL of 1:1
cyclohexane in methylene chloride, and 2 mL hexane. The flow
rate should be less than 0.5 mL min.~^. While the column is
still wet with hexane, add the entire eluate (30 mL) from the
alumina column (Section 11.3.5) to the top of the column.
Rinse the Erlenmeyer flask which contained the extract twice
with 1 mL hexane and add the rinsates to the top of the column.
Elute the column sequentially with two 1-mL aliquots hexane, 1
mL of 1:1 cyclohexane in methylene chloride, and 1 mL of
75:20:5 methylene chloride/ methanol/benzene. Turn the column
upside down and elute the TCDD fraction with 6 mL toluene into
a concentrator tube. Warm the tube to approximately 60°C and
reduce the toluene volume to approximately 1 mL using a stream
of nitrogen. Carefully transfer the residue into a 1-mL
mini-vial and again at elevated temperature, reduce the volume
to about 100 uL using a stream of nitrogen. Rinse the concen-
trator tube with 3 washings using 200 uL of 1% toluene in
CH2C12* Add 50 uL tridecane and store the sample in a refrig-
erator until GC/MS analysis is performed.
12. ANALYTICAL PROCEDURES
12.1 Remove the sample extract or blank from storage, allow it to warm to
ambient laboratory temperature and add 5 uL recovery standard solution.
With a stream of dry, purified nitrogen, reduce the extract/blank
volume to 50 uL.
12.2 Inject a 2-uL aliquot of the extract into the GC, operated under the
conditions previously used (Section 8.1) to produce acceptable results
with the performance check solution.
12.3 Acquire SIM data according to 12.3.1. Use the same acquisition time
and MS operating conditions previously used (Section 8.3.4) to deter-
mine the relative response factors.
12.3.1 Acquire SIM data for the following selected characteristic ions:
D-17
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m/z Compound
258.930 TCDD - COC1
319.897 Unlabeled TCDD
321.894 Unlabeled TCDD
331.937 13C12-2,3,7,8-TCDD, 13C12~1 ,2,3,4-TCDD
333.934 13C12-2,3,7,8-TCDD, 13C12-1 ,2,3 ,4-TCDD
12.4 Identification Criteria
12.4.1 The retention time (RT) (at maximum peak height) of the sample
component m/z 319.897 must be within -1 to +3 seconds of the
retention time of the peak for the isotopically labeled internal
standard at m/z 331.937 to attain a positive identification of
2,3,7,8-TCDD. Retention times of other tentatively identified
TCDDs must fall within the RT window established by analyzing
the column performance check solution (Section 8.1). Retention
times are required for all chroma tograms.
12.4.2 The ion current responses for m/z 258.930, 319.897 and 321.894
must reach maximum simultaneously (+_ 1 scan), and all ion
current intensities must be ^ 2.5 times noise level for
positive identification of a TCDD.
12.4.3 The integrated ion current at m/z 319.897 must be between 67
and 90 percent of the ion current response at m/z 321.894.
12.4.4 The integrated ion current at m/z 331.937 must be between 67
and 90 percent of the ion current response at m/z 333.934.
12.4.5 The integrated ion currents for m/z 331.937 and 333.934 must
reach their maxima within +_ 1 scan.
12.4.6 The recovery of the internal standard 13C12-2,3,7,8-TCDD must
be between 40 and 120 percent.
13. CALCULATIONS
13.1 Calculate the concentration of 2,3,7,8-TCDD (or any other TCDD isomer)
using the formula:
cx>
AX
AIg * W • RRF(I)
D-18
-------
where:
Cx • unlabeled 2,3,7,8-TCDD (or any other unlabeled TCDD isomer) concen-
tration in pg/g for soil/sediment and pg/L for aqueous samples.
AX •= sum of the integrated ion abundances determined for m/z 319.897
and 321.894.
AJS «= sura of the integrated ion abundances determined for m/z 331.937
and 333.934 of *3C12-2,3,7 ,8-TCDD (IS - internal standard).
QIS » quantity (in picograms) of 13Cj2-2,3,7 ,8-TCDD added to the
sample before extraction (Qxs * 500 pg) .
W * weight (in grams) of dry soil or sediment sample or volume of
aqueous sample (in liters).
RRF(I)
calculated mean relative response factor for unlabeled 2,3,7,8TCDD
relative to C, 2-2,3,7,8-TCDD. This represents the grand
mean of the RRF(l)'s obtained in Section 8. 3. A. 5.
13.2 Calculate the recovery of the internal standard 13C12-2, 3,7,8-TCDD,
measured in the sample extract, using the formula:
Internal standard A™ * Qr,g
percent recovery « • x 100
ARS ' RRF(II) ' QIS
where Ajg and Qjg have the same definitions as above (Section 13.1)
» sum of the integrated ion abundances determined for m/z 331.937
and 333.934 of r3C12-l,2,3,4-TCDD (RS - recovery standard).
QRg " quantity (in picograms) of Cj2~l ,2,3,4-TCDD added to the sample
residue before HRGOHRMS analysis.
(QRS - 500 pg).
RRF(II) • calculated mean relative response factor for labeled 3Cj2~2,3, 7,8-
TCDD relative to C12-l ,2,3,4-TCDD. This represents the grand
mean of the RRF(II)'s calculated in Section 8.3.4.5.
13.3 If the calculated concentration of unlabeled 2,3,7,8-TCDD exceeds
200 pg/g for soils or sediments, or 2000 pg/L for aqueous samples,
the linear range of response vs. concentration may have been exceeded
and a smaller portion of that sample must be analyzed. Accurately
weigh to three significant figures a 1-g portion of the wet soil/
sediment. Add the sample fortification solution (Section 11.1.2),
extract and analyze as discussed for the 10-g sample. Similarly,
add the sample fortification solution (Section 11.2.2) to 100 mL of
the aqueous sample, extract and analyze.
D-19
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13.4 Total TCDD concentration — all positively identified isoraers of TCDD
must be within the RT window and meet all identification criteria
listed in Sections 12.4.2, 12.4.3 and 12.4.4. Use the expression
in Section 13.1 to calculate the concentrations of the other TCDD
isomers, with Cx becoming the concentration of any unlabeled TCDD
isomer.
Total TCDD - Sum of the concentrations of the individual TCDDs.
13.5 Estimated Detection Limit — For samples in which no unlabeled
2,3,7,8-TCDD was detected, calculate the estimated minimum detectable
concentration. The background area is determined by integrating the
ion abundances for m/z 319.897 and 321.894 in the appropriate region
of the selected ion monitoring trace, multiplying that area by 2.5,
and relating the product area to an estimated concentration that
would produce that product area.
Use the formula:
(2.5) • (Ax) ' (QIS)
(AIS) ' (RRF(I)) ' (W)
where
CE • estimated concentration of unlabeled 2,3,7,8-TCDD required to
produce Ax.
Ax » sum of integrated ion abundance for m/z 319.897 and 321.894 in the
same group of >5 scans used to measure AI§.
AIS • sum of integrated ion abundance for the appropriate ion character-
istic of the internal standard, m/z 331.937 and m/z 333.934.
QlS, RRF(I), and W retain the definitions previously stated in Section 13.1.
Alternatively, if peak height measurements are used for quantification, measure
the estimated detection limit by the peak height of the noise in the TCDD RT
window.
13.6 The relative percent difference (RPD) is calculated as follows:
| 8j - 82 | | Si - 82 |
RPD - —_——^———— = x 100
Mean Concentration (Sj + 82)/2
S} and $2 represent sample and duplicate sample results.
References
1. "Carcinogens - Working with Carcinogens", Department of Health, Education
and Welfare, Public Health Service, Center for Disease Control, National
Institute for Occupational Safety and Health, Publication No. 77-206, Aug.
1977.
' D-20
-------
2. "OSHA Safety and Health Standards, General Industry" (29 CFR1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised January
1976).
3. "Safety in Academic Che'nistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition 1979*
D-21
-------
TABLE 1. COMPOSITION OF CONCENTRATION CALIBRATION SOLUTIONS
HRCC1
HRCC2
HRCC3
HRCC4
HRCC5
Recovery Standard
13C12-1,2,3,4-TCDD
2.5 pg/uL
5.0 pg/uL
10.0 pg/uL
20.0 pg/uL
40. 0 pg/uL
Analyte
2,3,7,8-TCDD
2.5 pg/uL
5.0 pg/uL
10.0 pg/uL
20.0 pg/uL
40.0 pg/uL
Internal Standard
13C12-2,3,7,8-TCDD
10.0 pg/uL
10.0 pg/uL
10.0 pg/uL
10.0 pg/uL
10.0 pg/uL
Sample Fortification Solution
5.0 pg/uL of 13C12-2,3,7,8-TCDD
Recovery Standard Spiking Solution
'12'
100 pg/uL 13C,2-1,2,3,4-TCDD
Field Blank Fortification Solutions
A) 5.0 pg/uL of unlabeled 2,3,7,8-TCDD
B) 5.0 pg/uL of unlabeled 1,2,3,4-TCDD
Internal Standard Spiking Solution
100 pg/uL of 13C1?-2,3,7,8-TCDD
(Used only in Section 4.2.1.A.2, Exhibit E)
D-22
-------
TABLE 2. RECOMMENDED GC OPERATING CONDITIONS
Column coating
Film thickness
Column dimensions
Helium linear velocity
Initial temperature
Initial time
Temperature program
2,3,7,8-TCDD retention
time
SP-2330
0.2 urn
60 m x 0.24 mm
28-29 cm/sec
at 240°C
70"C
4 min
Rapid increase to 200°C
200°C to 250°C
at 4°C/min
24 min
CP-SIL 88
0.22 urn
50 m x 0.22 mm
28-29 cm/sec
at 240°C
45°C
3 min
Rapid increase to 190°C
190°C to 240°C
at 5°C/min
26 min
D-23
-------
TABLE 3. TYPICAL 12-HOUR SEQUENCE FOR 2,3,7,8-TCDD ANALYSIS
1. Static mass resolution check
2. Column performance check
3. HRCC2
4. Sample 1 through Sample "N"
5. Column performance check
6. Static mass resolution check
10/20/84
10/20/84
10/20/84
10/20/84
10/20/84
10/20/84
0700 hrs.
0730 hrs.
0800 hrs.
0830 hrs.
1800 hrs.
1830 hrs.
D-24
-------
K>
l/i
IM
M
2378
1294
«M
I '
•M
CT-SIt 88
(SO.)
1278
12(7
•M
Figure 1. Selected ion current profile for n/z 320 and 322 produced by MS analysis for
performance check solution using a 50-in CP Si 1-88 fused silica capillary
column and conditions listed in Table 2.
-------
IM
7
K>
10
2371
— 1234
U7i
CT-2330
(60 •)
1271
311 IM
44*
4M
Figure 2. Selected ion current profile for ra/z 320 and 322 produced by MS analysts of performance
check solution using a 60-m SP-2330 fused silica capillary column and conditions
listed in Table 2.
-------
APPENDIX B
PROPOSED ANALYTICAL PROTOCOL
for the Determination of
2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and Total
TCDDs in Soil/Sediment and Water by High-Resolution
Gas Chromatography/High-Resolution Mass Spectrometry
December 1, 1985
This analytical protocol has been written in the format used in the
Superfund program, as "Exhibit D" of a Statement of Work which in turn is part
of an Invitation-for-Bid package under the Superfund Contract Laboratory Program.
Also included are other exhibits listed below for the Statement of Work which
have been tailored to meet the specific requirements of this protocol:
EXHIBIT B: Reporting Requirements and Deliverables
EXHIBIT C: Sample Rerun Requirements
EXHIBIT D: Analytical Method
EXHIBIT E: Quality Assurance/Quality Control Requirements
-------
This protocol (Protocol B) is a modification of the protocol presented as
Appendix A (Protocol A). Examination of the results from the single-laboratory
evaluation of Protocol A had shown that the minimum amount of 2,3,7,8-TCDD that
could be quantified under the conditions specified in Protocol A was 5 pg.
However, a requirement existed to lower the quantitation limits to 2 ppt for
soil and sediment samples and to 20 ppq for aqueous samples. The sample size
should stay at 10 g for soil and sediments and at 1 L for aqueous samples,
since the effect of larger sample sizes on the extract cleanup efficiencies is
not known. Also, the range of the method should overlap with the 1-ppb lower
limit of the low-resolution analytical method for TCDD used in the Superfund
Contract Laboratory Program without necessitating second extractions for samples
containing higher levels of TCDDs.
After careful evaluation by EMSL-LV of the requirements and the options,
the following protocol changes were made:
o In Protocol B, the following calibration solutions will be used:
HRCC1: 2 pg/uL 2,3,7,8-TCDD and 13C12-1,2,3,4-TCDD
10 pg/yL 13C12-2,3,7,8-TCDD
HRCC2: 10 pg/yL 2,3,7,8-TCDD and 13C12-1,2,3,4-TCDD
10 pg/yL 13C12-2,3,7,8-TCDD
HRCC3: 50 pg/yL 2,3,7,8-TCDD and 13C12-1,2,3,4-TCDD
10 pg/yL 13C12-2,3,7,8-TCDD
-------
HRCC4: 100 pg/yL 2,3,7,8-TCDD and 13C12-1,2,3,4-TCDD
10 pg/yL 13C12-2,3,7,8-TCDD
o In Protocol B, the final extract volume will be 10 yL. The decision
to select a final volume of 10 yL was necessary in order to comply
with the above requirements. It is realized that such a small volume
may pose technical difficulties for the analyst.
0 In Protocol B, the fortification level of the internal standard
13C12-2,3,7,8-TCDD was raised from 500 pg/sample to 1,000 pg/sample.
This allows analysis of soil and sediment samples containing between
100 ppt and 1.2 ppb of any TCDD isomer and of water samples containing
between 1 ppt and 12 ppt of any TCDD isomer by diluting a 2-yL aliquot
of the remaining extract concentrate by a factor of 12 with a solution
of the recovery standard (100 pg/yL of 13C12-1,2,3,4-TCDD in tridecane),
Recoveries will be reported using the data generated from the first
injection. Thus, the decision to dilute an aliquot of the 10-yL final
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 in the 10-yL final extract volume. This will eliminate un-
necessary dilutions of the sample extract and analyses for samples
containing between 100 ppt and 250 ppt for soil and sediment and 1 ppt
and 2.5 ppt for water samples of a TCDD isomer, but for which the
recoveries were low.
-------
EXHIBIT B
Reporting Requirements and Deliverables
-------
1. SCOPE AND APPLICATION
The Contractor shall provide reports and other deliverables as specified
in the Contract Reporting Schedule. These reports are described below.
All reports shall be submitted in legible form or resubmission shall be
required. All reports and documentation required, including selected ion
current profiles (also called selected ion monitoring traces), shall be
clearly labeled with the Sample Management Office Case number and associated
Sample/Traffic Report number(s). If documentation is submitted without
the required identification, as specified above, resubmission shall be
required.
The Contract Reporting Schedule (Section 2) specifies the numbers of
copies required, the delivery schedule and the distribution of all required
deliverables.
1.1 Sample data package — Hard copy analytical data and documentation
are required as described below.
NOTE: This analytical protocol is designed for the receipt and analysis
of samples by batches. Therefore, it is desired that sample data
from samples in the same batch be reported together, i.e., on the
same reporting form. However, contract accounting and billing are
based on the sample unit.
1.1.1 Case narrative: Contains the Case number, Dioxin Shipment
Record numbers, Contract number and detailed documentation of
any quality control, sample, shipment and/or analytical pro-
blems encountered in a specific Case. Also included should be
documentation of any internal decision tree process used along
with a summary of corrective actions taken. The Case narrative
must be signed in original signature by the Laboratory Manager
or his designate.
1.1.2 Results of initial triplicate analyses of four (4) concentration
calibration solutions (Form H-2), routine calibration solutions,
(Form H-3), including all selected ion current profiles or
selected ion monitoring (SIM) traces, calculated relative
response factors (RRF), and computer-generated quantification
reports (or manual calculations).
1.1.3 Completed data reporting sheets (Forms H-l, H-4, and H-5, H-8
and H-9) with appropriate SIM traces (including the lock mass
SIM traces). Data results for levels less than 10 ppt but
above the quantitation limit (Section 1.1, Exhibit D) attained
for that sample shall be reported to two (2) significant
figures; results greater than 10 ppt shall be reported to three
(3) significant figures. Apply the rounding rules found in
Section 7.2.2, "Handbook for Analytical Quality Control in Water
and Wastewater Laboratories," EPA-600/4-79-019. Each SIM trace
shall include computer-generated header information indicating
instrumental (GC and MS) operating parameters during data
B-l
-------
acquisition. When samples are analyzed more than once, all
sample data shall be reported. Rejected sample runs must be
separated and attached to the back of the data package and
marked on the SIM trace as "Rejected," with an explanation of
the reasons for the rejection.
1.1.4 SIM traces generated during each GC column performance check
analysis; peak profile outputs of the reference signal used
to document the nass resolution.
1.1.5 Documentation of acceptable MS calibration (Section 8, Exhibit
D, and Exhibit E) for each confirmatory analysis. As
applicable, submit peak matching box settings and calculations
for accurate mass assignments and any other related printouts.
State, in ppm, the level of mass accuracy achieved (Section
8.2.2, Exhibit D).
1.1.6 A chronological list of all analyses performed (Form H-6). If
more than one GC/MS system is used, a chronological list is
required for each system. The list must provide the Data
System File name, the EPA sample number, and (if appropriate)
the contractor laboratory sample number for each sample,
blank, concentration calibration solution, performance check
solution, or other pertinent analytical data. This list shall
specify date and time of beginning of analysis. All sample/
blank analyses performed during a 12-hour period must be
accompanied by two GC column performance check solution ana-
lyses, one preceding and one following the sample/blank ana-
lyses. If multiple shifts are used, the ending GC column
performance check sample analysis from one 12-hour period
shall serve as the beginning analysis for the next 12-hour
period; see Exhibit D, Section 8, for system performance
criteria. The same schedule applies to the mass resolution
check analysis. See Section 8.2.2, Exhibit D.
1.1.7 Verification of recovery of TCDDs from cleanup columns
(Section 11.3, Exhibit D, and Section 4.2.1.2.2, Exhibit E).
1.2 Sample extracts and unused sample portions — Unused portions of
samples and sample extracts shall be retained by the Contractor for
a period of six months after receipt. When directed in writing by
the Project Officer (PO) or Sample Management Office (SMO), the
Contractor shall ship (not at Contractor's expense but in accordance
with Department of Transportation Regulations) specific samples
and/or extracts to specified locations and persons. After six months,
upon obtaining PO or SMO clearance, remaining samples and extracts
shall be disposed of by the Contractor at Contractor's expense, in
accordance with applicable regulations concerning the disposal of
such materials.
1.3 Document Control and Chain-of-Custody Package — The Document Control
and Chain-of-Custody Package includes all laboratory records received
B-2
-------
or generated for a specific case, that have not been previously
submitted to EPA as a deliverable. These items include but are not
limited to: sample tags, custody records, sample tracking records,
analysts logbook pages, bench sheets, chromatographic charts, computer
printouts, raw data summaries, instrument logbook pages, corre-
spondence, and the document inventory (Exhibit G).
NOTE: Pages from logbooks or bench sheets kept exclusively in a high-
hazard area (containment facility) need not be copied.
1.4 Monthly Sample Status Report — The Monthly Sample Status Report
shall provide the status of all samples the Contractor has received
or has had in-house during the calendar month. Required status
information includes: samples received, samples extracted, samples
analyzed, samples rerun, and samples which required special cleanup.
All samples shall be identified by the appropriate EPA sample, case
and batch/shipment numbers.
1.5 Daily Sample Status Report — In response to a verbal request from the
Sample Management Office or the Project Officer, the Contractor must
verbally provide sample status information on a same-day basis.
Should written confirmation be requested, the Contractor must send the
daily sample status information in a written form that same day using
first-class mail service. The required Daily Sample Status informa-
tion shall include the items noted for the Monthly Sample Status
Report and, in addition, shall require information on sample analysis
reports in progress and analysis reports submitted/mailed.
2. In accordance with applicable delivery requirements, the Contractor shall
deliver specified items per the following Contract Reporting Schedule
(Section 2.1). Recipients include the CLP Sample Management Office, the
EMSL/LV QA Division, the appropriate Regional Technical Officer and NEIC.
2.1 Contract Reporting Schedule
CONTRACT REPORTING SCHEDULE
Item Delivery Report Distribution
No. Report No. Copies Schedule SMO EMSL/LV Region NEIC
1 Sample Data 3 30 days after validated XXX
Package sample receipt date
-OR-
10 days after initial XXX
data due date
2 Sample Extracts Within 180 days after As directed
analysis, 7 days after
request by Project Officer
or SMO
(Continued)
B-3
-------
CONTRACT REPORTING SCHEDULE (Continued)
Item
No.
Report
No.
Copies
Delivery
Schedule
Report Distribution
SMO
EMSL/LV
Region
NEIC
Document 1
Control & Pkg
Chain-of-
Custody Package
Monthly 2
Sample Status
Report
7 days after request by
Project Officer
or SMO
5 days following end of
each calendar month
Daily Sample
Status Report
Verbal and/or written
upon request by SMO or PO;
maximum frequency is daily.
As directed
NOTE: All results shall be reported total and complete.
2.2 Addresses for distribution
SMO
EMSL-LV
NEIC
CLP Sample Management Office
P. 0. Box 818
Alexandria, VA 22313
For overnight deliveries, use
street address:
300 N. Lee St., Suite 200
Alexandria, VA 22314
US EPA EMSL-LV QA Division
Box 15027
Las Vegas, NV 89114
Attn: Data Audit Staff
US EPA NEIC
Bldg. 53
Box 25227
Denver Fed. Center
Denver, CO 80225
For overnight deliveries, use
street address:
944 E. Harmon Ave.
Executive Center
Las Vegas, NV 89109
Regional Technical Officer — Following contract award and prior to
Contractor's receipt of the first batch of samples, the Sample Manage-
ment Office will provide the Contractor with the list of Technical
Officers for the ten EPA Regions. SMO will provide the Contractor
with updated Regional address/name lists as necessary throughout the
period of the contract.
3. FORM INSTRUCTION GUIDE
This section includes specific instructions for the completion of all
required forms. These include instructions on header information as
well as specific details to the bodies of individual forms. Instructions
are arranged in the following order:
B-4
-------
Data Summary (Form H-l)
Initial Calibration Summary (Form H-2; 2 pages)
Routine Calibration Summary (Form H-3)
GC and Mass Resolution Check Summary (Form H-A)
Quality Control Summary (Form H-5)
Chronological List of All Analyses Performed (Form H-6)
GC Operating Conditions (Form H-7)
HRMS TCDD Calibration Report Form (Form H-8)
High-Resolution MS TCDD Data Report Form (Form H-9)
3.1 Data Summary (Form H-l)— This form is used for summarizing the
results from all samples in the batch. The detailed results are
available on Form H-8 for each sample.
Complete the header information at the top of the page, including
laboratory name, case number and batch/shipment number (from the
dioxln shipment record), and matrix (soil, sediment, water).
Complete the form using one horizontal row for each sample.
The SMO sample number should be suffixed with the appropriate letter
code as needed.
The TCDD retention time should be reported in minutes and seconds.
TCDD levels are reported as parts per trillion (ppt) regardless of
the matrix. Total TCDD concentration (in ppt) is the sum of the
concentrations of all TCDDs reported on Form H-9.
The S/N criteria apply to m/z 259, 320, 322 (for unlabeled TCDD)
and m/z 322 and 334 (internal and recovery standards). The symbols
used are: (+) all S/N ratios are 2.5 or greater including all TCDDs
present, (-) S/N ratio for native 2,3,7,8-TCDD, the internal or the
recovery standard are less than 2.5, (0) other suspected TCDDs are
present but did not meet the S/N criteria.
The file name is the HRGC/HRMS file name and is used for tracking
results and raw data.
The comments column should be used for any remarks specific to a
particular sample.
3.2 Initial Calibration Summary (Form H-2): Page 1 — The header infor-
mation should be filled in. The column headings are similar to those
on Form H-l.
Ax ' QlS
RRF(I) =
QX ' AIS
B-5
-------
Aj s * QRg
RRF(II) = (Section 8.3.4.5, Exhibit D)
QlS * ARS
Page 2 — The header information should be filled in. For each RRF,
the mean, percent relative standard deviation (%RSD) and number of
runs (N) are reported; N must be at least three (3) for each HRCC
solution. The grand means (RRFs) are the mean of the individual
means and are reported with their %RSD and N. The routine calibra-
tion relative response factor permissible ranges are also reported
(Section 8.3.4.8, Exhibit D).
3.3 Routine Calibration Summary (Form H-3) — The header information
Includes case and batch numbers in addition to the laboratory and
instrument identification.
The columns are the same as on Page 1 of Form H-2. The results
reported are for the routine calibration runs rather than the initial
calibration. The calculated RRF(I) and RRF(II) must be within the
routine calibration relative response factor permissible ranges
(Section 8.3.4.8, Exhibit D) and other criteria listed in Section
8.6, Exhibit D must be met before further analysis is performed.
3.4 GC and Mass Resolution Check Summary (Form H-4) — The header informa-
tion should be filled in. The TCDD isomer resolution (% valley) is
measured from the column performance check solution (Section 8.1.2,
Exhibit D). The resolving power and mass measurement error are measured
using PFK (or equivalent) (Section 8.2, Exhibit D).
3.5 Quality Control Summary (Form H-5) — The items should be completed
as indicated. The "other interferences" should be included even if
they only occur at one mass.
Form H-5 in conjunction with Form H-9 is used to report results
relative to the fortified field blank pair and rinsate analyses.
The total TCDD retention time window is a window that includes all of
the TCDD isomers and is based on the first and last eluting isomers
in the GC column performance check solution using the conditions sum-
marized in Form H-7. All materials used should be recorded in the
standard/reagent QC table. Standards provided by EPA should be
listed, however, the QC columns may be left blank as these are refer-
ence materials.
3.6 Chronological List of All Analyses Performed (Form H-6) — The
header information should be filled in. If more than one instrument
is used, use one form per instrument.
The "Analysis Identification" column should contain enough information
for the data user to clearly identify the analysis, I.e., HRCC 2
Routine Calibration, Fortified Field Blank A, Fortified Field Blank B,
B-6
-------
Reanalysis of Sanple //I, 2, 3, A, etc. The "SMO //" column should be
used only for samples etc. which have an assigned SMO sample number.
3.7 GC Operating Conditions (Form H-7) — This form must be filled out to
describe the GC operating conditions used to analyze a batch of
samples and to analyze the GC performance evaluation check solution.
3.8 HRMS TCDD Calibration Report (Form H-8) — This form is to be filled
in for each initial and routine calibration analysis made. It will be
the first page of the chromatograms and calculations for that analysis.
It is suggested that this form be used as a worksheet for completing
Forms H-2 and H-3. S/N ratios greater than five (5) may be reported
with a (+); S/N ratios of five or less must have a numerical value
reported with accompanying chromatograms scaled so that the measure-
ments may be checked by the data user.
3.9 High-Resolution MS TCDD Data Report (Form H-9) — This form contains
the details of the data reported in sunmary on Form H-l. It will be
the first page of the chromatograms and calculations for each sample
including the fortified field blank pair samples. All data presented
(retention times, areas, and S/N ratios) must also be available on
the accompanying chromatograms. The chromatograms must be scaled
so that the data user may check any S/N ratios that are near or below
five (5). It is suggested that this form be used as a worksheet for
completing Form H-l.
A. REPORTING REQUIREMENTS SUMMARY:
Items that must be included with the data package:
4.1 Complete identification of the samples analyzed (sample numbers and
type).
A.2 The dates and times at which all analyses were accomplished. This
information should also appear on each selected ion current profile
Included with the report.
A.3 Raw mass chromatographic data which consist of the absolute peak
heights or peak areas of the signals observed for the ion masses
monitored.
A.A The calculated ratios of the intensities of the M+0 to (M+2)+0
molecular ions for all TCDD isomers detected.
A.5 The calculated concentrations of native 2,3,7,8-TCDD and other TCDD
isomers for each sample analyzed, expressed in picograms TCDD per gram
of sample (that is, parts per trillion), as determined from the raw
data. If no TCDDs are detected, the notation "Not Detected" or
"N.D." is used, and the minimum detectable concentrations (or detection
limits) are reported.
B-7
-------
HIGH RESOLUTION
FORM H-l DATA SUMMARY
HRGC/HRMS DIOXIN ANALYSIS
Lab:
Cawff
B0tch/Shtpni8ftt w.
Matrix:
SMO
Number
TCOO
2.3.7.8 (IS)
PPt
2.3.7.B-TCDD
Meaa. DL
Relative km
pot Abundance Ratio*
Total 320 332 332 %Rec. S/N m*t. Anaryei* Re
TCDO 322 334(18) 334(RS| (IS) Criteria ID Date Time Name Comment*
B>
I
00
RB- Reagent Blank
N- UnUbetod TCDO Spfta
D - Duplicata
FB- Raid Blank
8R • Sampto Rerun
ER -
NO- NotDatscted
DL- DatactionLimh
MB - Mattwd Blank
Rac- Recovery
Matrix: 8- Soil
W- Water
O- Other
S/N Criteria: report (») rl all S/N > 2.S
report (-) if 2.3.7.8 TCDD.
"C,J-2.3.7.8-TCOO or
"C,,-1.2.3.4-TCODS/N < 2.S
report (0) if other TCDO* arc •uapectad
not to meet criteria
-------
HIGH RESOLUTION
FORM H-2 INITIAL CALIBRATION SUMMARY
Iof2
Lab:
Contract ff:
Instrument ID:
w
VO
Catibratfon Rte m/s m/t m/t S/N
Standard Name Date Tlnw 320/322* 332/334(18)* 332/334(RS»* Criteria RRF|l)b RRF(II)C Comments
* km ratio* mint be hi the rang* of 0.67 to 0.90
b 2.3.7.8-TCOD ww»u« '»C1Z-2.3.7.B TCDD
c "C12-2.3.7.8 TCDD vvrau* "C12-1.2.3.4 TCDD
S/N Criteria: report f+) if greater than 2.6
report HH torn than 2.5
-------
HIGH RESOLUTION
FORM H-2 INITIAL CALIBRATION SUMMARY
page2of2
Lab:
Date of Intitial Calibration:
Contract #:
Instrument #:
RRF (I) Mean
%RSD
RRF (II) Mean
%RSO
HRCC1
HRCC2
HRCC3
HRCC4
w
I
RRF (I) Grand Mean:
%RSD:
N:
RRF (II) Grand Mean:
*RSD:
N:
Rotitrn* CcNbratkm P»rm)Mib>» Rwtge:
RRF (I) = 2.3.7.8-TCDD w«
"C,,-2.3.7.8-TCDD
Routine Calibration PonrriMible Range:
RRF (II) = «»C -2.3.7.8-TCDD w»
-------
HIGH RESOLUTION
FORM H-3 ROUTINE CALIBRATION SUMMARY
Lab
w
i
Calibration Ffla m/z m/i m/i S/N
Standard Nama Oat* Tfcna 320/322* 332/334(15)* 332/334(RS|* Criteria RRF(l)b RRFfllF Comnwnta
• l«« r_Mna m.n* ha In tha >mnn_ «« A AT tn H On •! /M OriMri. nnnrt III H 111.. 1.1 than ? K
• 2.3.7.8-TCOO VWMM "€,,-2.3.7.8-1000
e "Ct2-2.3.7.8-TCDO vw*tn "C12-1.2.3.4-TCDD
report (•) tf ton thfln 2.5
-------
HIGH RESOLUTION
FORM H-4 GC AND MASS RESOLUTION CHECK SUMMARY
Lab
TCDD Isomer Revolving 'Man
Intt. Sol. File Revolution Power Measurement
Oat* ID IO Time Name (% Valley) at 10% Valley Error (PPM)
Mttf Utttf *?» "«•»» iH^murmmM* MMT ealeuliiian
B-12
-------
HIGH RESOLUTION
FORM H-5 QUALITY CONTROL SUMMARY
Lab:
Case*
Batch #
Number of samples in batch: _
Mean S of recovery for the I.S.:
* of data points:
Fortified field blank A. K recovery ("C,2-2.3.7.8-TCDD):
Contamination by 1,2.3.4-TCDD
'»C12-1.2.3.4-TCDD
SMO Sample ft:
Yes
Estimated
Concentration (ppt)
Other interferences
Retention times:
Estimated concentrations (ppt):
Fortified field blank B, % recovery ("C.,-1.2.3.4-TCDD|: _
. SMO Sample ft:
Contamination by 2.3.7.8 TCDD
"C12-2.3,7.8-TCDD
[_J []]
Estimated
Concentration (ppt)
Other interferences
Retention times:
Estimated concentration* (ppt):
Rinsate, S recovery:
SMO sample ft:
Contamination by 2.3.7.8-TCDD
Other TCDD
Concentre lion
(pg/mL)
Duplicate analysis, SMO sample ft:
"C12-2,3,7.8-TCDD Mean Recovery.
Percent Relative difference "C12-2.3.7.8-TCDD (Recovery)
Percent relative difference iaC12-2.3.7.8-TCDD (Concentration)
Percent relative difference Total TCDD (Concentration)
Method blank file name
HRMS Lab.
Standard /Reagent Number or Origin Date of OC File Results of
Type Mfg. ft OC Name QC
Continue as needed
B-13
-------
HIGH RESOLUTION
FORM H-6 CHRONOLOGICAL LIST OF ALL ANALYSES PERFORMED
Lab:
Instrument ID:
Case*
Batch #
File
Name
Analysis
Identification
SMO
Number
Date
Time
B-14
-------
HIGH RESOLUTION
FORM H-7 GC OPERATING CONDITIONS
Lab: Instrument ID:
GC Column:
Film Thickness:
Column Dimensions.
Initial Column Temperature:
Temperature Program:
Injector Temperature:
Interface Temperature:
Injection Mode:
Injection Volume:
Splittess Valve Closed Time:
Septum Purge Flow:
Injector Sweep Flow:
Carrier Gas Flow Rate (ml/min or cm/sec):
B-15
-------
HIGH RESOLUTION
FORM H-8 HRMS TCDD CALIBRATION REPORT FORM
Lab:
Case*:
Batch/Shipment #:
Instrument ID:
Calibration:
Initial
Routine
2.3.7.8-TCDD
m/z 258.930
319.897
321.894
»C12-2.3.7.8-TCDD
m/i331.937
333.934
«C12-1.2.3.4-TCDD
m/z 331.937
m/z 333.934
Calibration Solution:
GC Column: —
Date of Initial Calibration: .
Analysis Date: Time:
File Name
Retention
Time
Area
Ratios
320
322
334
332
334
(•) H S/N i* graatar than 8. amar (+); H la** than S. »nt»r tha tnaaaurvd ratio
B-16
-------
HIGH RESOLUTION
FORM H-9 HIGH RESOLUTION MS TCDD DATA REPORT FORM
Lab:
Ca*e#:
Batch/Shipmentft:
I ID:
SMO Sample ft:
Matrix:
Soil
Aliquot
Circle
One
g L
Percent
Time:
(xtrmrtinn D»Wi
MMtured ppt 2.3.7.8-TCDD:
E«tim»ted Toul TCDO (ppt):
2.3.7.8-TCDD
m/i 258.930
319.897
321.894
Detection Limit 2.3.7.8-TCDD:
Report Date:
Retention
Time
Area
Ratios
S/N'
'«C12-2.3.7.B-TCDD
m/i331.937
333.934
320.
322
332
334
'»C,j-1.2.3.4-TCDD
m/t 331 .937
m/i 333.934
Percent Recovery "C12-2.3.7.8-TCDD:
Other TCDDs
Retention 320 S/N* 269 S/N* 320 S/N* 322 Concentration
Time 322 (ppt)
*H S/N is greater than S. enter |»); H lew than S enter the measured ratio
B-17
-------
4.6 The same raw and calculated data which are provided for the actual
samples will also be reported for the duplicate analyses, the method
blank analyses, the fortified field blank pair and rinsate analyses,
and any other QA or performance sample analyzed in conjunction with
the actual sample set(s).
4.7 The recoveries of the internal standard ( C12~2»3»7»8~TCDD) in percent.
4.8 The calibration data, including relative response factors calculated from
the calibration procedure described in Section 8.3, Exhibit D. Data
showing that these factors have been verified at least once during each
12-hour period of operation must be included (Section 8.5, Exhibit D).
Exact mass measurement error. Include peak matching box settings
and calculations as appropriate.
4.9 The calculated dry weight of the original soil or sediment sample portion
based on the dry weight determination of another sample portion of approxi-
mately equal wet weight. The exact volumes of the water and rinsate
samples analyzed.
4.10 Documentation of the source of all TCDD standards used and available
specifications on purity.
4.11 In addition, each report of analyses will include the following selcted
ion current profiles: 1) those obtained from all samples analyzed, 2)
those from each GC column performance check, and 3) those from the
calibration solutions. The peak profile from each mass resolution
check must also be part of the data package.
4.12 Identify which HRGC/HRMS system was used for the analyses (manufacturer
and laboratory identification number of system - 01, 02, 03, etc.).
4.13 GC operating conditions such as type of GC column, film thickness, column
dimensions, initial column temperature, temperature program, injector
temperature, interface temperature, injection mode and volume, valve time
(valve flush), septum purge flow, flow rate, and total injector flow
should be provided (Form H-7).
B-18
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EXHIBIT C
Sample Rerun Requirements
-------
1. SCOPE AND APPLICATION
The Contractor shall be required to reextract and/or perform additional
cleanup and reanalyze certain samples or batches of samples in a variety
of situations that may occur in the process of contract performance.
(For purposes of this contract, the term "sample rerun" shall indicate
sample extraction of a fresh 10-g soil or sediment portion or 1-L aqueous
sample, followed by cleanup and analysis, and the term "extract reanalysis"
shall indicate analysis of another aliquot of the final extract.
In situations where the sample rerun is required due to matrix effects,
interferences or other problems encountered because of very complex samples,
the Government will pay the Contractor for the sample reruns. Such sample
reruns shall be billable and accountable under the specified contract
allotment of automatic reruns.
In situations where the sample rerun or the extract reanalysis Is required
due to Contractor materials, equipment or instrumentation problems, or
lack of contractor's adherence to specified contract procedures, the
sample rerun or extract reanalysis shall not be billable under the terms
of the contract.
Contractor's failure to perform any of the sample reruns or extract re-
analyses specified herein, either billable or non-billable, shall be
construed as Contractor nonperf ormance and may result in termination of
the contract for default by the Contractor.
2. Required Sample Reruns and Extract Reanalyses
2.1 Automatic sample reruns and extract reanalyses that may be billable
as such under the contract.
2.1.1 If the percent recovery for the internal standard C12~2, 3,7,8-
TCDD was outside of the acceptance limits of >bQ percent and
£120 percent, the Contractor shall reextract ITnd reanalyze the
sample. If the percent recovery for the sample rerun is still
outisde the acceptance limits, then both analyses can be billed
if the recoveries from both analyses are either <40% or >120%.
If, however, the percent recovery for the sample rerun is
within the acceptance limits, or if it is still outside the
acceptance limits but the percent recoveries from the original
analysis and the sample rerun are not both either <40% or
>120%, then the sample rerun may not be billed.
2.1.2 If the internal standard was not found upon monitoring m/z
331.937 and 333.934, the Contractor shall reextract and
reanalyze the sample. If the internal standard is not
found in the sample rerun, the sample rerun is billable. If
the internal standard is found in the sample rerun, then the
sample rerun is not billable.
C-l
-------
2.1.3 If either one of the isotope abundance ratios for m/z 319.897/
321.894 or for 331.937/333.934 is less than 0.67 or greater
than 0.90 and all other criteria contained in Section 12.4 of
Exhibit D are met, then the extract shall be reanalyzed. If
both ion abundance ratios now meet the criterion, these values
shall be reported as the isotope abundance ratios, and the
Contractor shall not bill the Government for the extract
reanalysis. If the ratio in question is still outside the
criterion, the Contractor shall rerun the sample (Section 7.2,
Exhibit E). If either one of the ratios determined from the
sample rerun is still outside the acceptance limits, then
both runs and the extract reanalysis can be billed if the
corresponding isotope abundance ratios from both runs are
either <0.67 or >0.90. If, however, both isotope abundance
ratios from the sample rerun meet the criteria, or if both
corresponding isotope abundance ratios from the original run
and the sample rerun are not both either <0.67 or >0.90,
then the extract reanalysis and the sample rerun may not be
billed.
2.1.4 If the recoveries of 2,3,7,8-TCDD (Section 4.2.1.1.3.1,
Exhibit E) and/or 1,2,3,4-TCDD (Section 4.2.1.2, Exhibit E)
in the fortified field blank pair are <40% or >120%, the
Contractor shall reextract and reanalyze a second portion of
the field blank sample (Section 4.2, Exhibit E). If the
percent recoveries for the sample rerun are still outside the
acceptance limits, then both analyses can be billed as long
as the recoveries from both analyses are either <40% or >120%.
If, however, the percent recoveries for the sample rerun
are within the acceptance limits, or if they are still outside
the acceptance Units but the percent recoveries from the
original run and the sample rerun are not both either <40%
or >120%, then the sample rerun may not be billed.
NOTE: Fortified field blanks as described in Sections
4.2.1.1.4 and 4.2.1.2.2, Exhibit E, can never be billed.
2.2 Automatic sample extract dilution and HRGC/HRMS analysis, billable as
such under the Contract.
If any individual or group of coeluting TCDD isomer concentrations in
the 10-uL final extract exceeds 100 pg/uL, the analyst will perform a
dilution as specified in Section 13.3, Exhibit D, and reanalyze the
diluted portion using HRGC/HRMS.
2.3 Sample reruns and/or extract reanalyses to be performed at Contractor's
expense (i.e., not billable under the terns of the contract).
2.3.1 If the method blank contains any signal in the TCDD retention
time window at or above the method quantitation limit (2 ppt
C-2
-------
for soil and sediment and 20 ppq for aqueous samples), the
Contractor shall rerun all positive samples in the batch of
samples (Section 4.1.2, Exhibit E).
2.3.2 If the system performance using the GC column performance
check (PC) solution does not meet specified criteria, the
Contractor shall take corrective action, demonstrate acceptable
GC column performance, and reanalyze the extracts from all
positive samples run during the time period between the last
acceptable PC run and the unacceptable PC run (Section 2.4,
Exhibit E).
2.3.3 If a false positive is reported for an uncontaminated soil
(blind QC) sample, upon notification by the Sample Management
Office the Contractor shall reextract and reanalyze all samples
reported as positive in the associated batch of samples
(Section 8.1.1, Exhibit E).
2.3.A If the analysis results for a performance evaluation blind QC
sample fall outside of EPA-established acceptance windows, upon
notification of the Sample Management Office the Contractor
shall reextract and reanalyze the entire associated batch
of samples (Section 8.4.1, Exhibit E).
2.3.5 If the isotope abundance ratio for m/z 319.897/321.894 or for
331.937/333.934 is less than 0.67 or greater than 0.90, and
all other criteria contained in Section 12.4 of Exhibit D are
met, then the extract shall be reanalyzed. If the ion abundance
ratio in question now meets the criterion, this value shall be
reported as the isotope abundance ratio, and the Contractor
shall not bill the Government for the extract reanalysis.
2.3.6 If the system performance mass resolution check does not meet
the specified criterion, the Contractor shall take corrective
action, demonstrate acceptable mass resolution and reanalyze
the extract from all positive samples analyzed during the time
period between the last acceptable pass resolution check and
the unacceptable mass resolution check (Section 2.4,
Exhibit E).
C-3
-------
EXHIBIT D
Analytical Method
2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and Total
TCDDs in Soil/Sediment and Water by High-Resolution Gas
Chromatography/High-Resolution Mass Spectrometry
-------
EXHIBIT D
Section Subject Page
1 Scope and Application D-l
2 Summary of Method D-l
*
3 Definitions D-2
A Interferences. .......... D-3
5 Safety D-3
6 Apparatus and Equipment. D-4
7 Reagents and Standard Solutions D-6
8 System Performance Criteria D-9
9 Quality Control Procedures D-14
10 Sample Preservation and Handling D-14
11 Sample Extraction D-15
12 Analytical Procedures D-18
13 Calculations D-19
-------
1. SCOPE AND APPLICATION
1.1 This method provides procedures for the detection and quantitative
measurement of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD; CAS
Registry Number 1746-01-6; Storet number 3475) at concentrations of
2 pg/g (2 parts per trillion) to 100 pg/g (100 parts per trillion)
in 10-g portions of soil and sediment and at 20 pg/L (20 parts per
quadrillion) to 1000 pg/L (1 part per trillion) in 1-L samples of
water. Dilution of an aliquot of the final extract permits measure-
ment of concentrations up to 1.2 ng/g (1.2 parts per billion) or 12
ng/L (12 parts per trillion), respectively. This method also allows
the estimation of quantities of total TCDD present in the sample.
Samples containing concentrations of any individual TCDD isomer or
group of coeluting TCDD isomers greater than 1.2 ng/g or 12 ng/L must
be analyzed by a protocol designed for such concentration levels,
with an appropriate instrument calibration range.
1.2 The minimum measurable concentration is estimated to be 2 pg/g (2
parts per trillion) for soil and sediment samples and 20 pg/L (20
parts per quadrillion) for water samples, but this depends on kinds
and concentrations of interfering compounds in the sample matrix.
1.3 This method is designed for use by analysts who are experienced in
the use of high-resolution gas chromatography/high-resolution mass
spectrometry.
CAUTION: TCDDs are assumed to be extremely hazardous. It is the labora-
tory's responsibility to ensure that safe handling procedures are
employed.
2. SUMMARY OF METHOD
One thousand pg of C,2~2,3,7,8-TCDD (internal standard) are added to a
10-g portion of a soil/sediment sample (weighed to 3 significant figures)
or a 1-L aqueous sample, and the sample is extracted with 200 to 250 mL
benzene using a Soxhlet apparatus for soils and sediments with a minimum
of 3 cycles per hour, or with methylene chloride using a continuous liquid-
liquid extractor for aqueous samples for 24 hours. A separatory funnel
and 3 x 60 mL methylene chloride may also be used for aqueous samples.
After appropriate cleanup, 10 uL of a tridecane solution of the recovery
standard ( C12~l>2,3,4-TCDD) are added to the extract which is then
concentrated to a final volume of 10 uL. One to three uL of the concen-
trated extract is injected into a gas chromatograph with a capillary
column interfaced to a high-resolution mass spectrometer capable of rapid
multiple ion monitoring at resolutions of at least 10,000 (10 percent
valley).
Identification of 2,3,7,8-TCDD is based on the detection of the ions n/z
319.897 and 321.894 at the same GC retention time and within -1 to +3
seconds GC retention time of the internal standard masses of m/z 331.937
and 333.934. Confirmation of 2,3,7,8-TCDD (and of other TCDD isomers) is
D-l
-------
based on the ion m/z 258.930 which results from loss of COCL by the parent
molecular ion.
3. DEFINITIONS
3.1 Concentration calibration solutions — solutions containing known
(unlabeled 2,3,7,8-TCDD), the internal standard
the recovery standard C,2-l,2,3,4-TCDD;
they are used to determine instrument response of the analyte
relative to the internal standard and of the internal standard
relative to the recovery standard.
amounts of the analyte
13C12-2,3,7,8-TCDD and *v~ - — '-----•-—' 1J'
3.2 Field blank — a portion of soil/sediment or water uncontaminated with
2,3,7,8-TCDD and/or other TCDDs.
3.3 Rinsate — a portion of solvent used to rinse sampling equipment; the
rinsate is analyzed to demonstrate that samples have not been contami-
nated during sampling.
11
3.4 Internal standard — C12-2,3,7,8-TCDD, which is added to every
sample (except the blank described in Sections 4.2.1 of Exhibit E)
and is present at the same concentration in every method blank and
quality control sample. It is added to the soil/sediment or aqueous
sample before extraction and is used to measure the concentration of
each analyte. Its concentration is measured in every sample, and
percent recovery is determined using an internal standard method.
3.5 Recovery standard — C12~l,2,3,4-TCDD which is added to every sample
extract (except for the blank discussed in Sections 4.2.1, Exhibit E)
just before the final concentration step and HRGC-HRMS analysis.
3.6 Laboratory method blank — this blank is prepared in the laboratory
through performing all analytical procedures except addition of a
sample aliquot to the extraction vessel.
3.7 GC column performance check mixture — a mixture containing known
amounts of selected standards; it is used to demonstrate continued
acceptable performance of the capillary column, i.e., separation
(£ 25% valley) of 2,3,7,8-TCDD isomer from all other 21 TCDD isomers,
and to define the TCDD retention time window.
3.8 Performance evaluation sample — a soil, sediment or aqueous sample
containing a known amount of unlabeled 2,3,7,8-TCDD and/or other
TCDDs. It is distributed by the EMSL-LV to potential contractor lab-
oratories who must analyze it and obtain acceptable results before
being awarded a contract for sample analyses (see IFB Pre-Award Bid
Confirmations). It may also be included as an unspecified ("blind")
QC sample in any sample batch submitted to a laboratory for analysis.
3.9 Relative response factor — response of the mass spectrometer to a
known amount of an analyte relative to a known amount of an internal
standard.
D-2
-------
3.10 Mass resolution check — standard method used to demonstrate static
resolution of 10,000 minimum (10% valley definition).
3.11 Positive response for a blank — defined as a signal in the TCDD
retention time window, at any of the masses monitored, which is
equivalent to or above the method quantitation limit (2 ppt for soil
and sediment, and 20 ppq for aqueous samples).
3.12 Sample rerun — extraction of another 10-g soil or sediment sample
portion or 1-L aqueous sample, followed by extract cleanup and
extract analysis.
3.13 Extract reanalysis — analysis of another aliquot of th final extract.
4. INTERFERENCES
Chemicals which elute from the GC column within ^10 scans of the internal
and/or recovery standard (m/z 331.937 and 333.934) and which produce within
the TCDD retention time window ions at any of the masses used to detect or
quantify TCDD are potential interferences. Most frequently encountered
potential interferences are other sample components that are extracted
along with TCDD, e.g. PCBs, chlorinated methoxybiphenyls, chlorinated
hydroxydiphenylethers, chlorinated benzylphenylethers, chlorinated naphtha-
lenes, DDE, DDT, etc. The actual incidence of interference by these
chemicals depends also upon relative concentrations, mass spectrometric
resolution, and chromatographic conditions. Because very low levels of
TCDDs must be measured, the elimination of interferences is essential.
High-purity reagents and solvents must be used and all equipment must be
scrupulously cleaned. Blanks (Exhibit E, Quality Control, Section 4) must
be analyzed to demonstrate absence of contamination that would interfere
with TCDD measurement. Column chromatographic procedures are used to
remove some coextracted sample components; these procedures must be
performed carefully to minimize loss of TCDDs during attempts to increase
their concentration relative to other sample components.
5. SAFETY
The toxicity or carcinogen!city of each reagent used in this method has
not been precisely defined; however, each chemical compound should be
treated as a potential health hazard. From this viewpoint, exposure to
these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a file of
current OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material data handling
sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are identi-
fied O~3) (page D-21). 2,3,7,8-TCDD has been identified as a suspected
hunan or mammalian carcinogen. The laboratory is responsible for ensuring
that safe handling procedures are followed.
D-3
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6. APPARATUS AND EQUIPMENT
6.1 High-Resolution Gas Chromatograph/High-Resolution Mass
Spectrometer/Data System (HRGC/HRMS/DS)
6.1.1 The GC must be equipped for temperature programming, and all
required accessories must be available, such as syringes, gases,
and a capillary column. The GC injection port must be designed
for capillary columns. The use of splitless injection tech-
niques is recommended. On-column injection techiques can be
used but this may severely reduce column lifetime for
noncheraically bonded columns. When using the method in this
protocol, a 2-uL injection volume is used consistently. With
some GC injection ports, however, 1-uL injections may produce
improved precision and chromatographic separation. A 1- to 3-uL
injection volume may be used if adequate sensitivity and
precision can be achieved.
NOTE: If 1 uL or 3 uL is used at all as injection volume, the injec-
tion volumes for all extracts, blanks, calibration solutions
and the performance check sample must be 1 uL or 3 uL.
6.1.2 Gas Chromatograph-Mass Spectrometer Interface
The GC-MS interface may include enrichment devices, such as a
glass jet separator or a silicone membrane separator, or the
gas chromatograph can be directly coupled to the mass spectrome-
ter ion source. The interface may include a diverter valve
for shunting the column effluent and isolating the mass spec-
trometer ion source. All components of the interface should
be glass or glass-lined stainless steel. The interface com-
ponents should be compatible with 300°C temperatures. The
GC/MS interface must be appropriately designed so that the
separation of 2,3,7,8-TCDD from the other TCDD isoraers which
is achieved in the gas chromatographic column is not appreci-
ably degraded. Cold spots and/or active surfaces (adsorption
sites) in the GC/MS interface can cause peak tailing and peak
broadening. It is recommended that the GC column be fitted
directly into the MS ion source. Graphite ferrules should be
avoided in the GC injection port since they may adsorb TCDD.
Vespel" or equivalent ferrules are recommended.
6.1.3 Mass Spectrometer
The static resolution of the instrument must be maintained at
a minimum 10,000 (10 percent valley). The mass spectrometer
must be operated in a selected ion monitoring (SIM) mode with
total cycle time (including voltage reset time) of one second
or less (Section 8.3.4.1). At a minimum, the following ions
which occur at these masses must be monitored: m/z 258.930,
319.897, 321.894, 331.937 and 333.934.
D-4
-------
6.1.4 Data System
A dedicated hardware or data system is employed to control the
rapid multiple ion monitoring process and to acquire the data.
Quantification data (peak areas or peak heights) and SIM traces
(displays of intensities of each m/z being monitored as a
function of time) must be acquired during the analyses.
Quantifications may be reported based upon computer-generated
peak areas or upon measured peak heights (chart recording).
NOTE: Detector zero setting must allow peak-to-peak measurement of the noise
on the base line.
6.2 GC Columns
For isomer-specific determinations of 2,3,7,8-TCDD, the following
fused silica capillary columns are recommended: a 60-m SP-2330 (SP-
2331) column and a 50-m CP-Sil 88 column. However, any capillary
column which separates 2,3,7,8-TCDD from all other TCDDs may be used
for such analyses, but this separation must be demonstrated and
documented. Minimum acceptance criteria must be determined per
Section 8.1. At the beginning of each 12-hour period (after mass
resolution has been demonstrated) during which sample extracts or
concentration calibration solutions will be analyzed, column operating
conditions must be attained for the required separation on the column
to be used for samples. Operating conditions known to produce accept-
able results with the recommended columns are shown in Table 2 at the
end of this Exhibit.
6.3 Miscellaneous Equipment
6.3.1 Nitrogen evaporation apparatus with variable flow rate.
6.3.2 Balance capable of accurately weighing to +0.01 g.
6.3.3 Centrifuge capable of operating at 2,000 rpm.
6.3.4 Water bath — equipped with concentric ring cover and capable
of being temperature-controlled within +2°C.
6.3.5 Stainless steel spatulas or spoons.
6.3.6 Stainless steel (or glass) pan large enough to hold contents
of 1-pint sample containers.
6.3.7 Glove box.
6.3.8 Drying oven.
6.4 Glassware
6.4.1 Soxhlet apparatus — all-glass, Kontes 6730-02 or equivalent;
D-5
-------
90 mm x 35 mm glass thimble; 500-mL flask; condenser of appro-
priate size.
6.4.2 Kuderna-Danish apparatus — 500-mL evaporating flask, 10-mL
graduated concentrator tubes with ground-glass stoppers, and
3-ball macro Snyder column (Kontes K-570001-0500, K-503000-
0121 and K-569001-0219 or equivalent).
6.4.3 Mini-vials — 1-mL borosilicate glass with conical-shaped
reservoir and screw caps lined with Teflon-faced silicone disks.
6.4.4 Funnels — glass; appropriate size to accommodate filter
paper used to filter jar extract (volume of approximately 170 mL)
6.4.5 Separatory funnel — 2000 mL with Teflon stopcock.
6.4.6 Continuous liquid-liquid extractors equipped with Teflon or
glass connecting joints and stopcocks requiring no lubrication
(Hershberg-Wolf Extractor - Ace Glass Company, Vineland, NJ;
P/N 6841-10 or equivalent).
6.4.7 Chromatographic columns for the silica and alumina chroma-
tography — 1 cm ID x 10 cm long and 1 cm ID x 30 cm long.
6.4.8 Chromatographic column for the Carbopak cleanup — disposable
5-mL graduated glass pipets, 6 to 7 mm ID.
6.4.9 Desiccator.
6.4.10 Glass rods.
NOTE: Reuse of glassware should be minimized to avoid the risk of
cross contamination. All glassware that is reused must be
scrupulously cleaned as soon as possible after use, applying
the following procedure.
Rinse glassware with the last solvent used in it then with
high-purity acetone and hexane. Wash with hot water containing
detergent. Rinse with copious amounts of tap water and several
portions of distilled water. Drain, dry and heat in a muffle
furnace at 400°C for 15 to 30 minutes. Volumetric glassware
must not be heated in a muffle furnace, and some thermally
stable materials (such as PCBs) may not be removed by heating
in a muffle furnace. In these two cases, rinsing with high-
purity acetone and hexane may be substituted for muffle-furnace
heating. After the glassware is dry and cool, rinse with hexane,
and store inverted or capped with solvent-rinsed aluminum foil
in a clean environment.
7. REAGENTS AND STANDARD SOLUTIONS
7.1 Column Chromatography Reagents
D-6
-------
7.1.1 Alumina, acidic — extract the alumina in a Soxhlet with
methylene chloride for 6 hours (minimum of 3 cycles per hour)
and activate it by heating in a foil-covered glass container
for 24 hours at 190°C.
7.1.2 Silica gel — high-purity grade, type 60, 70-230 mesh; extract
the silica gel in a Soxhlet with methylene chloride for 6 hours
(minimum of 3 cycles per hour) and activate it by heating in a
foil-covered glass container for 24 hours at 130°C.
7.1.3 Silica gel impregnated with 40 percent (by weight) sulfuric
acid — add two parts (by weight) concentrated sulfuric acid
to three parts (by weight) silica gel (extracted and activated),
mix with a glass rod until free of lumps, and store in a
screw-capped glass bottle.
7.1.4 Sulfuric acid, concentrated —ACS grade, specific gravity 1.84.
7.1.5 Graphitized carbon black (Carbopack C or equivalent), surface
of approximately 12 m^/g, 80/100 mesh — mix thoroughly 3.6
grams Carbopak C and 16.4 grams Celite 545® in a 40-mL vial.
Activate at 130°C for six hours. Store in a desiccator.
7.1.6 Celite 545®, reagent grade, or equivalent.
7.2 Membrane filters or filter paper with pore size of <25 urn; rinse with
hexane before use.
7.3 Glass wool, silanized — extract with methylene chloride and hexane
and air-dry before use.
7.4 Desiccating Agents
7.4.1 Sodium sulfate — granular, anhydrous; before use, extract it
with methylene chloride for 6 hours (minimum of 3 cycles per
hour) and dry it for >4 hours in a shallow tray placed in an
oven at 120°C. Let it cool in a desiccator.
7.4.2 Potassium carbonate—anhydrous, granular; use as such.
7.5 Solvents — high purity, distilled in glass: methylene chloride,
toluene, benzene, cyclohexane, methanol, acetone, hexane; reagent
grade: tridecane.
7.6 Concentration calibration solutions (Table 1) — four tridecane
solutions containing C^"! f2,3,4-TCDD (recovery standard) and
unlabeled 2,3,7,8-TCDD at varying concentrations, and ^c _2,3,7,8-
TCDD (internal standard, CAS RN 80494-19-5) at a constant concentration
must be used to calibrate the instrument. These concentration calibra-
tion solutions must be obtained from the Quality Assurance Division,
US EPA, Environmental Monitoring Systems Laboratory (EMSL-LV), Las
Vegas, Nevada. However, additional secondary standards may be obtained
D-7
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from commercial sources, and solutions may be prepared in the con-
tractor laboratory. Traceability of standards must be verified
against EPAsupplied standard solutions. Such procedures will be
documented by laboratory SOPs as required in IFB Pre-award Bid Con-
firmations, part 2.f.(4). It is the responsibility of the laboratory
to ascertain that the calibration solutions received are indeed at the
appropriate concentrations before they are injected into the instrument!
NOTE: Serious overloading of the instrument may occur if the concentration
calibration solutions intended for a low-resolution MS are injected
into the high-resolution MS.
7.6.1 The four concentration calibration solutions contain unlabeled
2,3,7,8-TCDD and labeled 13C12~1,2,3,4-TCDD at nominal concen-
trations of 2.0, 10.0, 50.0, and 100 pg/uL, respectively, and
labeled C^2~2,3,7,8-TCDD at a constant nominal concentration
7.7
7.6.2
of 10.0 pg/uL.
Store the concentration calibration solutions in 1-raL mini-
vials at 4°C.
Column performance check mixture — this solventless mixture must be
obtained from the Quality Assurance Division, Environmental Monitoring
Systems Laboratory, Las Vegas, Nevada, and dissolved by the Contractor
in 1 mL tridecane. This solution will then contain the following
components [including TCDDs (A) eluting closely to 2,3,7,8-TCDD, and
the first- (F) and last-eluting (L) TCDDs when using the columns
recommended in Section 6.2] at a concentration of 10 pg/uL of each of
these isomers:
Analyte
Unlabeled 2,3,7,8-TCDD
13C12-2,3,7,8-TCDD
1,2,3,4-TCDD (A)
1,4,7,8-TCDD (A)
1,2,3,7-TCDD (A)
1,2,3,8-TCDD (A)
1,3,6,8-TCDD (F)
1,2,8,9-TCDD (L)
Approximate Amount Per Ampule
10'ng
10 ng
10 ng
10 ng
10 ng
10 ng
10 ng
10 ng
7.8 Sample fortification solution — an isooctane solution containing
the internal standard at a nominal concentration of 10 pg/uL.
D-8
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7.9 Recovery standard spiking solution — a tridecane solution contain-
ing the recovery standard at a nominal concentration of 10 pg/uL.
Ten uL of this solution will be spiked into each sample extract
(except for the fortified field blank A) before the final concentration
step and HRGC/HRMS analysis. It is also used for the dilution of the
extracts from samples with high TCDD levels (Section 13.3, Exhibit D).
7.10 Internal standard spiking solution — a tridecane solution containing
the internal standard ( C12~2,3,7,8-TCDD) at a nominal concentration of
10 pg/uL. Ten uL of this solution will be added to a fortified field
blank extract (Section 4.2.1.1, Exhibit E). This is the only case
where C,22,3,7,8-TCDD is used for recovery purposes.
7.11 Field blank fortification solutions — isooctane solutions containing
the following TCDD isomers:
Solution A: 10.0 pg/uL of unlabeled 2,3,7,8-TCDD
Solution B: 10.0 pg/uL of unlabeled 1,2,3,4-TCDD.
8. SYSTEM PERFORMANCE CRITERIA
System performance criteria are presented below. The laboratory may use
any of the recommended columns described in Section 6.2. It must be
documented that all applicable system performance criteria specified in
Sections 8.1, 8.2, 8.3 and 8.5 have been met before analysis of any sample
is performed. Table 2 provides recommended conditions that can be used to
satisfy the required criteria. Table 3 provides a typical 12-hour analysis
sequence. The GC column performance and mass resolution checks must be
performed at the beginning and end of each 12-hour period of operation.
8.1 GC Column Performance
8.1.1 Inject 2 uL (Section 6.1.1) of the column performance check
solution (Section 7.7) and acquire selected ion monitoring
(SIM) data for m/z 258.930, 319.897, 321.894, 331.937 and
333.934 within a total cycle time of <1 second (Section
8.3.4.1).
8.1.2 The chromatographic peak separation between 2,3,7,8-TCDD and
the peaks representing any other TCDD isomers must be resolved
with a valley of <25 percent, where
Valley Percent = (x/y)(100)
x = measured as in Figure 1
y = the peak height of 2,3,7,8-TCDD.
It is the responsibility of the laboratory to verify the con-
ditions suitable for the appropriate resolution of 2,3,7,8-TCDD
D-9
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from all other TCDD isomers. The column performance check
solution also contains the TCDD isomers eluting first and last
under the analytical conditions specified in this protocol
thus defining the retention time window for total TCDD determi-
nation. The peaks representing 2,3,7,8-TCDD and the first and
the last eluting TCDD isomer must be labeled and identified as
such on the chromatograms (F and L, resp.). Any individual
selected ion current profile or the reconstructed total ion
current (m/z 259 + m/z 320 + m/z 322) constitutes an acceptable
form of data presentation.
8.2 Mass Spectrometer Performance
8.2.1 The mass spectrometer must be operated in the electron (impact)
ionization mode. Static resolving power of at least 10,000
(10 percent valley) must be demonstrated before any analysis
of a set of samples is performed (Section 8.2.2). Static
resolution checks must be performed at the beginning and at
the end of each 12-hour period of operation. However, it is
recommended that a visual check (i.e., not documented) of the
static resolution be made using the peak matching unit before
and after each analysis.
8.2.2 Chromatography time for TCDD may exceed the long-term mass
stability of the mass spectrometer and thus mass drift correc-
tion is mandatory. A reference compound [high-boiling
perfluorokerosene (PFK) is recommended] is introduced into the
mass spectrometer. An acceptable lock mass ion at any mass
between m/z 250 and m/z 334 (m/z 318.979 from PFK is recommended)
must be used to monitor and correct mass drifts.
NOTE: Excessive PFK may cause background noise problems and contami-
nation of the source resulting in an increase in "downtime"
for source cleaning.
Using a PFK molecular leak, tune the instrument to meet the
minimum required resolving power of 10,000 (10% valley) at
m/z 254.986 (or any other mass reasonably close to m/z 259).
Calibrate the voltage sweep at least across the mass range o/z
259 to m/z 334 and verify that m/z 330.979 from PFK (or any
other mass close to m/z 334) is measured within +5 ppn (i.e.,
1.7 mmu, if m/z 331 is chosen) using m/z 254.986 as a reference.
Documentation of the mass resolution must then be accomplished
by recording the peak profile of the PFK reference peak m/z
318.979 (or any other reference peak at a mass close to m/z
320/322). The format of the peak profile representation must
allow manual determination of the resolution, i.e., the hori-
zontal axis must be a calibrated mass scale (amu or ppn per
division). The result of the peak width measurement (performed
at 5 percent of the maximum which corresponds to the 10%
valley definition) must appear on the hard copy and cannot
exceed 100 ppn (or 31.9 nmu if m/z 319 is the chosen reference
ion).
D-10
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8.3 Initial Calibration
Initial calibration is required before any samples are analyzed for
2,3,7,8-TCDD. Initial calibration is also required if any routine
calibration does not meet the required criteria listed in Section 8.6.
8.3.1 All concentration calibration solutions listed in Table 1 must
be utilized for the initial calibration.
8.3.2 Tune the instrument with PFK as described in Section 8.2.2.
8.3.3 Inject 2 uL of the column performance check solution (Section
7.7) and acquire SIM mass spectral data for m/z 258.930,
319.897, 321.894, 331.937 and 333.934 using a total cycle time
of ^ 1 second (Section 8.3.4.1). The laboratory must not
perform any further analysis until it has been demonstrated
and documented that the criterion listed in Section 8.1.2 has
been met.
8.3.4 Using the same GC (Section 8.1) and MS (Section 8.2) conditions
that produced acceptable results with the column performance
check solution, analyze a 2-uL aliquot of each of the 4 concen-
tration calibration solutions in triplicate with the following
MS operating parameters.
8.3.4.1 Total cycle time for data acquisition must be ^ 1
second. Total cycle time includes the sum of all the
dwell times and voltage reset times.
8.3.4.2 Acquire SIM data for the following selected
characteristic ions:
m/z Compound
258.930 TCDD - COC1
319.897 Unlabeled TCDD
321.894 Unlabeled TCDD
331.937 13C12-2,3,7,8-TCDD, 13C12-1,2,3,4-TCDD
333.934 13C12-2,3,7,8-TCDD, 13C12~1,2,3,4-TCDD
8.3.4.3 The ratio of integrated ion current for m/z 319.897 to
m/z 321.894 for 2,3,7,8-TCDD must be between 0.67 and
0.90.
1.3.4.4 The ratio of integrated ion current for m/z 331.937 to
m/z 333.934 for 13C12-2,3,7,8-TCDD and 13C12~1,2,3,4-
8,
TCDD must be between 0.67 and 0.90.
D-ll
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8.3.4.5 Calculate the relative response factors for unlabeled
2,3,7,8-TCDD [RRF(D] relative to * C,2-2,3,7,8-TCDD
and for labeled 13C12-2,3,7,8-TCDD [RRF(II)] relative
to C12-1,2,3,4-TCDD as follows:
'12
A.
RRF(I) = —
RRF(II) =
QX ' AIS
Ais * QRS
QIS * ARS
where
Ax = sun of the Integrated ion abundances of m/z 319.897 and m/z 321.894
for unlabeled 2,3,7,8-TCDD.
AJS = sum of the integrated ion abundances of m/z 331.937 and m/z 333.934
for 13C12-2,3,7,8-TCDD.
A^g = sum of the integrated ion abundances for m/z 331.937 and n/z
333.934 for 13C12-1,2,3,4-TCDD.
QIS = quantity of 13C12~2,3,7,8-TCDD injected (pg).
QRS = quantity of C}2~1,2,3,4-TCDD injected (pg).
Qx = quantity of unlabeled 2,3,7,8-TCDD injected (pg).
RRF is a dimensionless quantity; the units used to express QJS» QRS an(* QX
must be the same.
8.3.4.6 Calculate the four means (RRFs) and their respective
relative standard deviations (%RSD) for the response
factors from each of the triplicate analyses for both
unlabeled and 13C12-2,3,7,8-TCDD (Form H-2).
8.3.4.7 Calculate the grand means RRF(I) and RRF(II) and their
respective relative standard deviations (%RSD) using
the four mean RRFs (Section 8.3.4.6) (Form H-2).
8.3.4.8 Calculate the routine calibration permissible range
for RRF(I) and RRF(II) using a ^20% window from the
grand means RRF(I) and RRF(II) (Section 8.3.4.7)
(Form H-2).
8.4 Criteria for Acceptable Calibration
The criteria listed below for acceptable calibration must be met
before analysis of any sample is performed.
• D-12
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8.A.I The percent relative standard deviation (RSD) for the response
factors from each of the triplicate analyses for both unlabeled
and Cj2~2,3,7,8-TCDD must be less than 20 percent.
1 «1
8.4.2 The variation of the 4 mean RRFs for unlabeled and C^-
2,3,7,8-TCDD obtained from the triplicate analyses must be
less than 20 percent RSD.
8.4.3 SIM traces for 2,3,7,8-TCDD must present a signal-to-noise
ratio of ^2.5 for m/z 258.930, m/z 319.897 and, m/z 321.894.
8.4.4 SIM traces for Cj2~2»3,7,8-TCDD must present a signal-to-
noise ratio >2.5 for m/z 331.937 and m/z 333.934.
8.4.5 Isotopic ratios (Sections 8.3.4.3 and 8.3.4.4) must be within
the allowed range.
NOTE: If the criteria for acceptable calibration listed in Sections
8.4.1 and 8.4.2 have been met, the RRF can be considered inde-
pendent of the analyte quantity for the calibration concentra-
tion range. The mean RRF from 4 triplicate determinations for
unlabeled 2,3,7,8-TCDD and for 13C12-2,3,7,8-TCDD will be used
for all calculations until routine calibration criteria (Section
8.6) are no longer met. At such time, new mean RRFs will be
calculated from a new set of four triplicate determinations.
8.5 Routine Calibrations
Routine calibrations must be performed at the beginning of a 12-hour
period after successful mass resolution and GC column performance
check runs.
8.5.1 Inject 2 uL of the concentration calibration solution which
contains 10 pg/uL of unlabeled 2,3,7.8-TCDD, 10.0 pg/uL
of 13C12-2,3,7,8-TCDD and 10 pg/uL C12~l,2,3,4-TCDD.
Using the same GC/MS/DS conditions as used in Sections 8.1,
8.2 and 8.3, determine and document acceptable calibration as
provided in Section 8.6.
8.6 Criteria for Acceptable Routine Calibration
The following criteria must be met before further analysis is per-
formed. If these criteria are not met, corrective action must be
taken and the instrument must be recalibrated.
8.6.1 The measured RRF for unlabeled 2,3,7,8-TCDD must be within 20
percent of the mean values established (Section 8.3.4.8) by
triplicate analyses of concentration calibration solutions.
8.6.2 The measured RRF for 13C12-2,3,7,8-TCDD must be within 20 per-
cent of the mean value established by triplicate analysis
of the concentration calibration solutions (Section 8.3.4.8).
D-13
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8.6.3 Isotopic ratios (Sections 8.3.4.3 and 8.3.4.4) must be within
the allowed range.
8.6.4 If one of the above criteria is not satisfied, a second attempt
can be made before repeating the entire initialization process
(Section 8.3).
NOTE: An initial calibration must be carried out whenever the HRCC 2
solution is replaced by a new one from a different lot.
9. QUALITY CONTROL PROCEDURES
See Exhibit E for QA/QC requirements.
10. SAMPLE PRESERVATION AND HANDLING
10.1 Chain-of-custody procedures — see Exhibit G.
10.2 Sample Preservation
10.2.1 When received, each soil or sediment sample will be contained
in a 1-pint glass jar surrounded by vermiculite in a sealed
metal paint can. Until a portion is to be removed for analysis,
store the sealed paint cans in a locked limited-access area
where the temperature is maintained between 25° and 35°C.
After a portion of a sample has been removed for analysis,
return the remainder of the sample to its original container
and store as stated above.
10.2.2 Each aqueous sample will be contained in a 1-liter glass
bottle. The bottles with the samples are stored at 4°C in a
refrigerator located in a locked limited-access area.
10.2.3 To avoid photodecomposition, protect samples from light.
10.3 Sample Handling
CAUTION: Finely divided soils and sediments contaminated with 2,3,7,8-TCDD
are hazardous because of the potential for inhalation or ingestion
of particles containing 2,3,7,8-TCDD. Such samples should be
handled in a confined environment (i.e., a closed hood or a
glove box).
10.3.1 Pre-extraction sample treatment
10.3.1.1 Horaogenization — Although sampling personnel will
attempt to collect homogeneous samples, the contrac-
tor shall examine each sample and judge if it needs
further mixing.
NOTE: Contractor personnel have the responsibility to take a
representative sample portion; this responsibility
D-14
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entails efforts to make the sample as homogeneous as
possible. Stirring is recommended when possible.
10.3.1.2 Centrifugation — When a soil or sedimerit sample
contains an obvious liquid phase, it must be
centrifuged to separate the liquid from the solid
phase. Place the entire sample in a suitable centri-
fuge bottle and centrifuge for 10 minutes at 2000 rpn.
Remove the bottle from the centrifuge. With a dis-
posable pipet, remove the liquid phase and discard
it. Mix the solid phase with a stainless steel
spatula and remove a portion to be weighed and analyzed.
Return the remaining solid portion to the original
sample bottle (which must be empty) or to a clean,
empty sample bottle which is properly labeled, and
store it as described in 10.2.1.
CAUTION: The removed liquid may contain TCDD and should be
disposed as a liquid waste.
10.3.1.3 Weigh between 9.5 and 10.5 g of the soil or sediment
sample ^+0.5 g) to 3 significant figures. Dry it to
constant weight at 100eC. Allow the sample to cool
in a desiccator. Weigh the dried soil to 3 signifi-
cant figures. Calculate and report percent moisture
on Form H-9.
11. SAMPLE EXTRACTION
11.1 Soil/Sediment Extraction
11.1.1 Immediately before use, the Soxhlet apparatus is charged
with 200 to 250 mL benzene which is then refluxed for 2 hours.
The apparatus is allowed to cool, disassembled and the benzene
removed and retained as a blank for later analysis if required.
11.1.2 Accurately weigh to 3 significant figures a 10-g (9.50 g to
10.50 g) portion of the wet soil or sediment sample. Mix 100
uL of the sample fortification solution (Section 7.8) with
1.5 mL acetone (1000 pg of Cj2~2>3,7,8-TCDD) and deposit the
entire mixture in small portions on several sites on the
surface of the soil or sediment.
11.1.3 Add 10 g anhydrous sodium sulfate and mix thoroughly using a
stainless steel spoon spatula.
11.1.4 After breaking up any lumps, place the soil-sodium sulfate
mixture in the Soxhlet apparatus using a glass wool plug (the
use of an extraction thimble is optional). Add 200 to 250 mL
benzene to the Soxhlet apparatus and reflux for 24 hours. The
solvent must cycle completely through the system at least 3
times per hour.
D-15
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11.1.5 Transfer the extract to a Kuderna-Danish apparatus and
concentrate to 2 to 3 mL. Rinse the column and flask with 5 mL
benzene and collect the rinsate in the concentrator tube.
Reduce the volume in the concentrator tube to 2 to 3 mL.
Repeat this rinsing and concentrating operation twice more.
Remove the concentrator tube from the K-D apparatus and care-
fully reduce the extract volume to approximately 1 mL with a
stream of nitrogen using a flow rate and distance such that
gentle solution surface rippling is observed.
NOTE: Glassware used for more than one sample must be carefully
cleaned between uses to prevent cross-contamination (Note on
page D-6).
11.2 Extraction of Aqueous Samples
11.2.1 Mark the water meniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume. Pour
the entire sample (approximately 1 L) into a 2-L separatory
funnel.
11.2.2 Mix 100 uL of the sample fortification solution with 1.5 mL
acetone (1000 pg of ^€^"2,3,7,8-TCDD) and add the mixture
to the sample in the separatory funnel.
NOTE: A continuous liquid-liquid extractor may be used in place of
a separatory funnel.
11.2.3 Add 60 raL methylene chloride to the sample bottle, seal and
shake 30 seconds to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 minutes with periodic venting.
Allow the organic layer to separate from the water phase for
a minimum of 10 minutes. If an emulsion interface between
layers exists, the analyst must employ mechanical techniques
(to be described in the final report) to complete the phase
separation. Collect the methylene chloride (3 x 60 mL)
directly into a 500-mL Kuderna-Danish concentrator (mounted
with a 10-mL concentrator tube) by passing the sample extracts
through a filter funnel packed with a glass wool plug and 5
g of anhydrous sodium sulfate. After the third extraction,
rinse the sodium sulfate with an additional 30 mL of methylene
chloride to ensure quantitative transfer.
11.2.4 Attach a Snyder column and concentrate the extract until
the apparent volume of the liquid reaches 1 mL. Remove the
K-D apparatus and allow it to drain and cool for at least
10 minutes. Remove the Snyder column, add 50 mL benzene,
reattach the Snyder column and concentrate to approximately
1 mL. Rinse the flask and the lower joint with 1 to 2 mL
benzene. Concentrate the extract to 1.0 mL under a gentle
stream of nitrogen.
D-16
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11.2.5 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 raL.
11.3 Cleanup Procedures
11.3.1 Prepare an acidic silica column as follows: Pack a 1 cm x 10
cm chromatographic column with a glass wool plug, a layer (1
cm) of Na2S04/K2C(>}(l :1) , 1.0 g silica gel (Section 7.1.2) and
4.0 g of 40-percent w/w sulfuric acid-impregnated silica gel
(Section 7.1.3). Pack a second chromatographic column (1 cm x
30 cm) with a glass wool plug, 6.0 g acidic alumina (Section
7.1.1) and top with a 1-cm layer of sodium sulfate (Section
7.4.1). Add hexane to the columns until they are free of
channels and air bubbles.
11.3.2 Quantitatively transfer the benzene extract (1 tnL) from the
concentrator tube to the top of the silica gel column. Rinse
the concentrator tube with two 0.5-mL portions of hexane.
Transfer the rinses to the top of the silica gel column.
11.3.3 Elute the extract from the silica gel column with 90 mL hexane
directly into a Kuderna-Danish concentrator. Concentrate the
eluate to 0.5 mL, using nitrogen blow-down as necessary.
11.3.4 Transfer the concentrate (0.5 mL) to the top of the alumina
column. Rinse the K-D assembly with two 0.5-mL portions of
hexane and transfer the rinses to the top of the alumina
column. Elute the alumina column with 18 mL hexane until the
hexane level is just below the top of the sodium sulfate.
Discard the eluate. Columns must not be allowed to reach
dryness (i.e., a solvent "head" must be maintained.)
11.3.5 Place 30 mL of 20-percent (v/v) methylene chloride in hexane
on top of the alumina and elute the TCDDs from the column.
Collect this fraction in a 50-mL Erlenmeyer flask.
11.3.6 Prepare an 18-percent Carbopak C/Celite 545® mixture by thoroughly
mixing 3.6 grams Carbopak C (80/100 mesh) and 16.4 grams Celite
545® in a 40-mL vial. Activate at 130°C for 6 hours. Store
in a desiccator. Cut off a clean 5-mL disposable glass pipet
(6 to 7mm ID) at the 4-mL mark. Insert a plug of glass wool
(Section 7.3) and push to the 2-mL mark. Add 340 to 600 mg of
the activated Carbopak/Celite mixture (see NOTE) followed by
another glass wool plug. Using two glass rods, push both
glass wool plugs simultaneously towards the Carbopak/Celite
mixture and gently compress the Carbopak/Celite plug to a
length of 2 to 2.5 cm. Preelute the column with 2 mL toluene
followed by 1 mL of 75:20:5 methylene chloride/methanol/benzene,
1 mL of 1:1 cyclohexane in methylene chloride, and 2 mL hexane.
The flow rate should be less than 0.5 mL/min. While the
D-17
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column is still wet with hexane, add the entire eluate (30 mL)
from the alumina column (Section 11.3.5) to the top of the
column. Rinse the Erlenmeyer flask which contained the extract
twice with 1 mL hexane and add the rinsates to the top of the
column. Elute the column sequentially with two 1-mL aliquots
hexane, 1 mL of 1:1 cyclohexane in methylene chloride, and 1
mL of 75:20:5 methylene chloride/ methanol/benzene. Turn the
column upside down and elute the TCDD fraction with 6 mL tolu-
ene into a concentrator tube. Warm the tube to approximately
60°C and reduce the toluene volume to approximately 1 mL using
a stream of nitrogen. Carefully transfer the concentrate into
a 1-mL mini-vial and, again at elevated temperature, reduce the
volume to about 100 uL using a stream of nitrogen. Rinse the
concentrator tube with 3 washings using 200 uL of 1% toluene
in CH2C12. Add 10 uL of the tridecane solution containing the
recovery standard and store the sample in a refrigerator until
HRGC/HRMS analysis is performed.
NOTE: The amount of activate Carbopak/Celite mixture required
to form a 2-to 2.5-cm plug in the column depends on the
density of the Celite being used.
12. ANALYTICAL PROCEDURES
12.1 Remove the sample extract or blank from storage and allow it to warm
to ambient laboratory temperature. With a stream of dry, purified
nitrogen, reduce the extract/blank volume to 10 uL.
12.2 Inject a 2-uL aliquot of the extract into the GC, operated under the
conditions previously used (Section 8.1) to produce acceptable results
with the performance check solution.
12,3 Acquire SIM data according to 12.3.1. Use the same acquisition and
MS operating conditions previously used (Section 8.3.4) to determine
the relative response factors.
12.3.1 Acquire SIM data for the following selected characteristic ions:
m/z Compound
258.930 TCDD - COC1
319.897 Unlabeled TCDD
*
321.894 Unlabeled TCDD
331.937 13C12-2,3,7,8-TCDD, 13C,2-1,2,3,4-
TCDD
333.934 13C12-2,3,7,8-TCDD, 13C12-1,2,3,4-
TCDD
D-18
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NOTE: The acquisition period must at least encompass the TCDD reten-
tion time window previously determined (Section 8.1.2, Exhibit
D).
12.4 Identification Criteria
12. A.I The retention time (RT) (at maximum peak height) of the sample
component m/z 319.897 must be within -1 to +3 seconds of the
retention time of the peak for the isotopically labeled internal
standard at m/z 331.937 to attain a positive identification of
2,3,7,8-TCDD. Retention times of other tentatively identified
TCDDs must fall within the RT window established by analyzing
the column performance check solution (Section 8.1). Retention
times are required for all chroma tograms .
12.4.2 The ion current responses for m/z 258.930, 319.897 and 321.894
must reach maximum simultaneously (_+ 1 sec), and all ion
current intensities must.be > 2.5 times noise level for
positive identification of a TCDD or group of coeluting TCDD
isomers.
12.4.3 The integrated ion current at m/z 319.897 must be between 67
and 90 percent of the ion current response at m/z 321.894.
12.4.4 The integrated ion current at m/z 331.937 must be between 67
and 90 percent of the ion current response at m/z 333.934.
12.4.5 The integrated ion currents for m/z 331.937 and 333.934 must
reach their maxima within +_ 1 sec.
12.4.6 The recovery of the internal standard *^Cj2~2»3, 7,8-TCDD must
be between 40 and 120 percent.
13. CALCULATIONS
13.1 Calculate the concentration of 2,3,7,8-TCDD (or any other TCDD isomer
or group of coeluting TCDD isomers) using the formula:
cx
AX
AIS * W * RRF(I)
where:
Cx = unlabeled 2,3,7,8-TCDD (or any other unlabeled TCDD isomer or group of
coeluting TCDD isomers) concentration in pg/g.
Ax = sum of the integrated ion abundances determined for m/z 319.897
and 321.894.
= sum of the integrated ion abundances determined for m/z 331.937
and 333.934 of r3C12-2,3, 7,8-TCDD (IS = internal standard).
D-19
-------
QIS = quantity (in picograms) of C12~2,3,7,8-TCDD added to the
sample before extraction (Qjg = 1000 pg).
W = weight (in grams) of dry soil or sediment sample or volume of
aqueous sample converted to grams.
RRF(I) = calculated m<
ean relative response factor for unlabeled 2,3,7,8-TCDD
C12-2,3,7,8-TCDD. This represents the grand mean of
the RRF(I)'s obtained in Section 8.3.4.5.
relative to 12
13.2 Calculate the recovery of the internal standard C12~2,3,7,8-TCDD
measured in the sample extract, using the formula:
Internal standard Ajg
percent recovery = Y • 100
ARg * RRF(II)
Where:
sun of the integrated ion abundances determined for m/z 331.937
and 333.934 of 13C12-2,3,7,8-TCDD (IS = internal standard).
ARS " sum °f c^e integrated ion abundances determined for m/z 331.937
and 333.934 of C12-l ,2,3,4-TCDD (RS = recovery standard).
Y = 0.1 for the "10-yL extract" injection (to be reported on Forms H-l,
H-5 and H-9).
and y = 1.2 for the "24-yL extract" injection (Section 13.3) (to be reported
on Form H-9 used for reporting the diluted extract analysis).
— — 1 3
RRF(II) = calculated mean relative response factor for labeled C|2-2, 3,7,8-
TCDD relative to C12-l ,2 ,3,4-TCDD. This represents the grand
mean of the RRF(II)'s calculated in Section 8.3.4.5.
13.3 If the concentration of the most abundant TCDD isomer (or group of
coeluting TCDD isomers) exceeds 100 pg/uL in the 10 uL final extract,
the linear range of response vs. concentration may have been exceeded,
and a diluted aliquot of the original sample extract must be analyzed.
Accurately dilute 2 uL of the remaining original extract with 22 uL
of the tridecane solution containing 10 pg/uL of the recovery standard
(Section 7.9, Exhibit D).
13.4 Total TCDD concentration — all positively identified isomers of TCDD
must be within the RT window and meet all identification criteria
listed in Sections 12.4.2 and 12.4.3. Use the expression in Section
13.1 to calculate the concentrations of the other TCDD isomers, with
Cx becoming the concentration of any unlabeled TCDD isomer or group
of coeluting TCDD isomers.
C Total TCDD = Sum of the concentrations of the individual TCDDs including
2,3,7,8-TCDD.
D-20
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13.5 Estimated Detection Limit — For samples in which no unlabeled
2,3,7,8-TCDD was detected, calculate the estimated minimum detectable
concentration. The background area is determined by integrating the
ion abundances for n/z 319.897 and 321.894 in the appropriate region
of the selected ion current profiles, multiplying that area by 2.5,
and relating the product area to an estimated concentration that
would produce that product area.
Use the formula:
(2.5) ' (Ax) ' (QIS)
(AIS) • (RRF(I)) • (W)
where
CE = estimated concentration of unlabeled 2,3,7,8-TCDD required to
produce Ax.
Ax = sum of integrated ion abundances for m/z 319.897 and 321.894 in the
same group of >5 scans used to measure
sum °^ integrated ion abundances for the appropriate ion character-
istic of the internal standard, m/z 331.937 and m/z 333.934.
, RRF(I), and W retain the definitions previously stated in Section 13.1.
Alternatively, if peak height measurements are used for quantification, measure
the estimated detection limit by the peak height of the noise in the 2,3,7,8-
TCDD RT window.
13.6 The relative percent difference (RPD) is calculated as follows:
I Si - S2 | I Si - S2 |
RPD - «= x 100
Mean Concentration (S^ + S2>/2
Sj and 82 represent sample and duplicate sample results.
References
1. "Carcinogens - Working with Carcinogens", Department of Health, Education
and Welfare, Public Health Service, Center for Disease Control, National
Institute for Occupational Safety and Health, Publication No. 77-206, Aug.
1977.
2. "OSHA Safety and Health Standards, General Industry" (29 CFR1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised January
1976).
3. "Safety in Academic Chemistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition 1979.
D-21
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TABLE 1. COMPOSITION OF CONCENTRATION CALIBRATION SOLUTIONS
HRCC1
HRCC2
HRCC3
HRCC4
Recovery Standard
13C12-1,2,3,4-TCDD
2.0 pg/uL
10.0 pg/uL
50.0 pg/uL
100.0 pg/uL
Analyte
2,3,7,8-TCDD
2.0 pg/uL
10.0 pg/uL
50.0 pg/uL
100.0 pg/uL
Internal Standard
13C12-2,3,7,8-TCDD
10.0 pg/uL
10.0 pg/uL
10.0 pg/uL
10.0 pg/uL
Sample Fortification Solution
10.0 pg/uL of 13C12-2,3,7,8-TCDD
Recovery Standard Spiking Solution
10.0 pg/uL 13C12-1,2,3,4-TCDD
Field Blank Fortification Solutions
A) 10.0 pg/uL of unlabeled 2,3,7,8-TCDD
B) 10.0 pg/uL of unlabeled 1,2,3,4-TCDD
Internal Standard Spiking Solution
10 pg/uL of 13C12-2,3,7,8-TCDD
(Used only in Section 4.2.1.1, Exhibit E)
D-22
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TABLE 2. RECOMMENDED GC OPERATING CONDITIONS
Column coating
Film thickness
Column dimensions
Helium linear velocity
Initial temperature
Initial time
Temperature program
Approximate 2,3,7,8-TCDD
retention time
SP-2330 (SP-2331)
0.2 urn
60 m x 0.24 mm
28-29 cm/sec
at 240°C
150°C
4 min
Rapid increase to 200°C
(15°C/min)
200°C to 250°C
at 4°C/min
27 min
CP-SIL 88
0.22 urn
50 m x 0.22 mm
28-29 cm/sec
at 240°C
200°C
1 min
Program from 200°C
to 240°C
at 4°C/min
22 min
TABLE 3. TYPICAL 12-HOUR SEQUENCE FOR 2,3,7,8-TCDD ANALYSIS
1. Static mass resolution check and mass
measurement error determination
2. Column performance check
3 . HRCC2
4. Sample 1 through Sample "N"
5. Column performance check
6. Static mass resolution check
10/20/84
10/20/84
10/20/84
10/20/84
10/20/84
10/20/84
0700h
0730h
0800h
0830h
1800h
1830h
D-23
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100-1
80-
CO
g 60H
o
d>
OC
20-
00
(O
CO
(F)
(U
24:00
26:00
28:00
30:00
32:00
Time
Figure 1.
Selected ion current profile for m/z 322 produced by MS analysis of performance check
solution using a 60-m SP-2331 fused silica capillary column and conditions listed in
Table 2.
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EXHIBIT E
QA/QC Requirements
-------
SUMMARY OF QC ANALYSES
o Initial and periodic calibration and instrument performance checks.
o Field blank analyses (Section A.I); a minimum of one fortified field blank
pair shall be analyzed with each sample batch; an additional fortified field
blank pair must be analyzed when a new lot of absorbent and/or solvent is used.
o Analysis of a batch of samples with accompanying QC analyses:
Sample Batch —<2b samples, including field blank and rinsate sample(s).
Additional QC analyses per batch:
Fortified field blanks 2
Method blank (1*)
Duplicate sample 1
TOTAL 3(A)
* A method blank is required whenever a fortified field blank shows a
positive response as defined in Section 3.11, Exhibit D.
o "Blind" OC samples may be submitted to the contractor as ordinary soil,
sediment or water samples included among the batch of samples. Blind samples
include:
Uncontaminated soil, sediment and water,
Split samples,
Unidentified duplicates, and
Performance evaluation samples.
QUALITY CONTROL
1. Performance Evaluation Samples — Included among the samples in all batches
will be samples containing known amounts of unlabeled 2,3,7,8-TCDD and/or
other TCDDs that may or may not be marked as other-than-ordinary samples.
2. Performance Check Solutions
2.1 At the beginning of each 12-hour period during which samples are to
be analyzed, an aliquot each of the 1) GC column performance check
solution and 2) high-resolution concentration calibration solution
E-l
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No. 2 (HRCC2) shall be analyzed to demonstrate adequate GC resolution
and sensitivity, response factor reproducibility, and mass range
calibration. A mass resolution check shall also be performed to
demonstrate adequate mass resolution using an appropriate
reference compound (PFK is recommended).
These procedures are described in Section 8 of Exhibit D. If the
required criteria are not met, remedial action oust be taken before
any samples are analyzed.
2.2 To validate positive sample data, the GC column performance check
and the mass resolution check must be performed also at the end of
each 12-hour period during which samples are analyzed.
2.2.1 If the contractor laboratory operates only during one period
(shift) each day of 12 hours or less, the GC performance check
solution must be analyzed twice (at the beginning and end of
the period) to validate data acquired during the interim
period. This applies also to the mass resolution check.
2.2.2 If the contractor laboratory operates during consecutive
12-hour periods (shifts), analysis of the GC performance check
solution at the beginning of each 12-hour period and at the
end of the final 12-hour period is sufficient. This applies
also to the mass resolution check.
2.3 Results of at least two analyses of the GC column performance check
solution and the mass resolution check must be reported with the
sample data collected during a 12-hour period.
2.4 Deviations from criteria specified for the GC performance check or
for the mass resolution check (Section 8, Exhibit D) invalidate all
positive sample data collected between analyses of the performance
check solution, and the extract from those positive samples shall be
reanalyzed Exhibit C).
The GC column performance check mixture, concentration calibration solu-
tions, and the sample fortification solutions are to be obtained from the
EMSL-LV. However, if not available from the EMSL-LV, standards can be
obtained from other sources, and solutions can be prepared in the contractor
laboratory. Concentrations of all solutions containing unlabeled 2,3,7,8-
TCDD which are not obtained from the EMSL-LV must be verified by comparison
with the unlabeled 2,3,7,8-TCDD standard solution (concentration of 7.87
ug/raL) that is available from the EMSL-LV. When a lower-concentration
standard solution becomes available from the EMSL-LV, it will be substituted
for the 7.87 ug/mL standard.
E-2
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4. Blanks
4.1 A method blank Is required whenever a positive response (Section 3.11,
Exhibit D) is obtained for a fortified field blank. To that effect,
perform all steps detailed in the analytical procedure (Section 11,
Exhibit D) using all reagents, standards, equipment, apparatus,
glassware, and solvents that would be used for a sample analysis, but
omit addition of the soil, sediment or aqueous sample portion.
13
4.1.1 The method blank must contain the same amount of C]2~2,3,7,8-
TCDD that is added to samples before extraction.
4.1.2 An acceptable method blank exhibits no positive response (Section
3.11, Exhibit D) for any of the characteristic ions monitored.
If the method blank which was extracted along with a batch of
samples is contaminated, all positive samples must be rerun
(Exhibit C).
4.1.2.1 If the above criterion is not met, check solvents,
reagents, fortification solutions, apparatus, and
glassware to locate and eliminate the source of
contamination before any samples are extracted and
analyzed.
4.1.2.2 If new batches of reagents or solvents contain
interfering contaminants, purify or discard them.
4.2 Field blanks — Each batch of samples contains a field blank sample
of uncontaminated soil/sediment or water that is to be fortified
before analysis according to Section 4.2.1, Exhibit E. In addition
to this field blank, a batch of samples may include a rinsate, that
is a portion of solvent (usually trichloroethylene) that was used to
rinse sampling equipment. The rinsate is analyzed to assure that the
samples have not been contaminated by the sampling equipment.
4.2.1 Fortified field blank pair
4.2.1.1 Fortified field blank A: 2,3,7,8-TCDD
4.2.1.1.1 Weigh a 10-g portion or use 1 liter (for aqueous
samples) of the specified field blank sample and
add 100 uL of the solution containing 10.0 pg/uL of
2,3,7,8-TCDD (Table 1, Exhibit D) diluted in 1.5 mL
of acetone (Section 11.1.2, Exhibit D).
4.2.1.1.2 Extract using the procedures beginning in Sections
11.1 or 11.2 of Exhibit D, as applicable, add 10 uL
of the Internal standard solution (Section 7.10,
Exhibit D) and analyze a 2-uL aliquot of the con-
centrated extract.
E-3
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NOTE: This is the only case where the recovery standard is
used for other than recovery purposes.
4.2.1.1.3 Calculate the concentration (Section 13.1, Exhibit
D) of 2,3,7,8-TCDD and the percent recovery of
unlabeled 2,3,7,8-TCDD. If the percent recovery at
the measured concentration of 2,3,7,8-TCDD is <40
percent or >120 percent, report the results and
repeat the fortified field blank extraction and
analysis with a second aliquot of the specified
field blank sample (Exhibit C).
4.2.1.1.4 Extract and analyze a new fortified simulated field
blank whenever new lots of solvents or reagents are
used for sample extraction or for column chromato-
graphic procedures. When a fortified simulated
field blank produces a positive response (Section
3.11, Exhibit D) for any m/z being monitored at the
retention time of 1,2,3,4-TCDD, a method blank
(Section 4.1, Exhibit E) is required.
NOTE: For this purpose only, the Contractor will simulate
field blanks by using clean sand or distilled water.
4.2.1.2 Fortified field blank B: 1,2,3,4-TCDD
4.2.1.2.1 Repeat steps 4.2.1.1.1 to 4.2.1.1.3 using unlabeled
1,2,3,4TCDD (instead of 2,3,7.8-TCDD) and 13C
12-1,2,3,4-TCDD (instead of 13C12-2,3,7,8-TCDD) as
recovery standard.
4.2.1.2.2 Extract and analyze a new fortified simulated field
blank whenever new lots of solvents or reagents are
used for sample extraction or for column chromato-
graphic procedures. When a fortified simulated
field blank produces a positive response (Section
3.11, Exhibit D) for any m/z being monitored at the
retention time of 2,3,7,8-TCDD, a method blank
(Section 4.1, Exhibit E) is required.
4.2.2 Rinsate sample
4.2.2.1 The rinsate sample must be fortified as a regular
sample.
4.2.2.2 Take a 100-mL aliquot of sampling equipment rinse
solvent (rinsate sample), filter, if necessary, and
Id 100 uL of the solution containing 10.0 pg/uL of
>C12-2,3,7,8-TCDD (Table 1, Exhibit D).
E-4
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4.2.2.3 Using a Kuderna-Danish apparatus, concentrate to
approximately 5 mL.
A.2.2.4 Transfer the 5-mL concentrate in 1-mL portions to a 1-
mL mini-vial, reducing the volume as necessary with a
gentle stream of dry nitrogen; see Exhibit D,
Section 11.1.5 for volume reduction procedures.
4.2.2.5 Rinse the container with two 0.5-mL portions of hexane
and transfer the rinses to the 1-mL mini-vial.
4.2.2.6 Just before analysis, add 10 uL tridecane recovery
standard spiking solution (Table 1, Exhibit D), and
reduce the volume to a final volume of 10 uL (no
column chromatography is required).
4.2.2.7 Analyze an aliquot following the same procedures used
to analyze samples (Section 12, Exhibit D).
4.2.2.8 Report percent recovery of the internal standard and
the level of contamination by any TCDD isomer (or
group of coeluting TCDD Isomers) on Form H-5 in pg/mL
of rinsate solvent.
5. Duplicate Analyses
5.1 Laboratory duplicates — in each batch of samples, locate the sample
specified for duplicate analysis and analyze a second 10-g soil or
sediment sample portion or 1-L water sample.
5.1.1 The results of laboratory duplicates (percent recovery and
concentrations of 2,3,7,8-TCDD and total TCDD) must agree
within 50 percent relative difference (difference expressed as
percentage of the mean). If the relative difference is >50
percent, the Contractor shall immediately contact the Sample
Management Office for resolution of the problem. Report all
results.
5.1.2 Recommended actions to help locate problems:
5.1.2.1 Verify satisfactory instrument performance
(Section 8, Exhibit D).
5.1.2.2 If possible, verify that no error was made while
weighing sample portions.
5.1.2.3 Review the analytical procedures with the performing
laboratory personnel.
6. Percent Recovery of the Internal Standard 13C,2~2,3,7,8-TCDD — For each
sample, method blank and rinsate, calculate the
13.2, Exhibit D) of the measured concentration of
E-5
percent recovery (Section
lf Cl2-2,3,7,8-TCDD. If
-------
the percent recovery is <40 percent or >120 percent for a sample, analyze
a second portion of that sample and report both results (Exhibit C).
NOTE: A low or high percent recovery for a blank does not require discarding
analytical data but it may indicate a potential problem with future
analytical data.
7. Identification Criteria
7.1 If either of the two identification criteria (Sections 12.4.1 and
12.4.2, Exhibit D) is not met, it is reported that the sample does
not contain unlabeled 2,3,7,8-TCDD at the calculated detection limit
(Section 13.5, Exhibit D).
7.2 If the first two initial identification criteria are met, but the
third, fourth, fifth or sixth criterion (Sections 12.4.3 through
12.4.6, Exhibit D) is not met, that sample is presumed to contain
interfering contaminants. This must be noted on the analytical
report form and the sample must be rerun or the extract reanalyzed.
Detailed sample rerun and extract reanalysis requirements are
presented in Exhibit C.
8. Blind QC Samples — Included among soil, sediment and aqueous samples may
be QC samples that are not specified as such to the performing laboratory.
Types that may be included are:
8.1 Uncontaminated soil, sediment or water.
8.1.1 If a false positive is reported for such a sample,
the Contractor shall be required to rerun the entire
associated batch of samples (Section 2.3.3, Exhibit C).
8.2 Split samples — composited sample portions sent to more than
one laboratory.
8.3 Unlabeled field duplicates — two portions of a composited
sample.
8.4 Performance evaluation sample — soil/sediment or water sample
containing a known amount of unlabeled 2,3,7,8-TCDD and/or
other TCDDs.
8.4.1 If the performance evaluation sample result falls
outside the acceptance windows established by EPA, the
Contractor shall be required to rerun the entire associ-
ated batch of samples (Exhibit C).
NOTE: EPA acceptance windows are based on previously generated
data.
9. Records - At each contractor laboratory, records must be maintained on
E-6
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site for six months after contract completion to document the quality of
all data generated during the contract performance. Before any records are
disposed, written concurrence from the Contracting Officer must be obtained.
10. Unused portions of samples and sample extracts must be preserved for
six months after sample receipt; appropriate samples may be selected
by EPA personnel for further analyses.
11. Reuse of glassware is to be minimized to avoid the risk of contamination.
LABORATORY EVALUATION PROCEDURES
1. On a quarterly basis, the EPA Project Officer and/or designated
representatives may conduct an evaluation of the laboratory to ascertain
that the laboratory is meeting contract requirements. This section outlines
the procedures which may be used by the Project Officer or his authorized
representative in order to conduct a successful evaluation of laboratories
conducting dioxin analyses according to this protocol. The evaluation
process consists of the following steps: 1) analysis of a performance
evaluation (PE) sample, and 2) on-site evaluation of the laboratory to
verify continuity of personnel, instrumentation, and quality assurance/
quality control functions. The following is a description of these
two steps.
2. Performance Evaluation Sample Analysis
2.1 The PE sample set will be sent to a participating laboratory to
verify the laboratory's continuing ability to produce acceptable
analytical results. The PE sample will be representative of the
types of samples that will be subject to analysis under this contract.
2.2 When the PE sample results are received, they are scored using the
PE Sample Score Sheet shown in Figure 1. If a false positive
(e.g., a PE sample not containing 2,3,7,8-TCDD and/or other TCDDs
but reported by the laboratory to contain it and/or them) is reported,
the laboratory has failed the PE analysis requirement. The Project
Officer will notify the laboratory immediately if such an event
occurs.
2.3 As a general rule, a laboratory should achieve 75 percent or more of
the total possible points for all three categories, and 75 percent or
more of the maximum possible points in each category to be considered
acceptable for this program. However, the Government reserves the
right to accept scores of less than 75 percent.
2.4 If unanticipated difficulties with the PE samples are encountered,
the total points may be adjusted by the Government evaluator in an
impartial and equitable manner for all participating laboratories.
E-7
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Number of Maximum Possible Recommended Passing
PE Samples Score Score (75%)
1 290 218
2 475 356
3 660 495
4 845 634
5 1030 773
On-Site Laboratory Evaluation
3.1 An on-site laboratory evaluation is performed to verify that (1) the
laboratory is maintaining the necessary minimum level in instrumen-
tation and levels of experience in personnel committed to the con-
tract and (2) that the necessary quality control/quality assurance
activities are being carried out. It also serves as a mechanism for
discussing laboratory weaknesses identified through routine data
audits, PE sample analyses results, and prior on-site evaluation.
Photographs may be taken during the on-site laboratory evaluation
tour.
3.2 The sequence of events for the on-site evaluations is shown in
Figure 2. The Site Evaluation Sheet (SES) (Figure 3) is used to
document the results of the evaluation.
E-8
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Laboratory
PERFORMANCE EVALUATION SAMPLE SCORE SHEET
Date
False Positive
False Positive - If a laboratory reports a false
positive on any PE sample, the laboratory may be
disqualified, i.e., rendered ineligible for
contract award based on the failure to pass the
PE sample analysis requirement.
2,3,7,8-TCDD
Other TCDD(s)
( ) Yes ( ) No
( ) Yes ( ) No
Possible
Score
II. Calibration Data
1. Method Blank:
a. Results properly recorded on Forms H-l, H-5 and
H-9.
b. No native TCDD isomers at /or above method
quantitative limit.
c. Results documented by selected ion
monitoring (SIM) traces for m/z being
monitored to detect TCDDs.
d. Percent recovery of 13C,2-2,3,7 ,8-TCDD
and 120%.
5
5
2. Initial Concentration Calibration:
a. Results properly recorded on Forms H-2 5
and H-8.
b. The percent relative standard deviation
(RSD) for the response factors for each
of the triplicate analyses for both unlabeled
and C12-2,3,7,8-TCDD less than 20%. 5
c. The variation of the 4 mean RRFs for both
unlabeled and labeled 2,3,7,8-TCDD obtained
from the triplicate analyses less than 20% RSD. 5
d. For unlabeled 2,3,7,8-TCDD the abundance ratio
must be >Q.67 and £0.90 for m/z 319.897 to
321.894. 5
Figure 1. Performance evaluation sample score sheet,
E-9
Score
Achieved
-------
Possible Score
Score Achieved
e. The abundance ratios must be XK67 and £0.90
for 331.937 to 333.934 for 13C12~2,3,7,¥-TCDD
and 13C12-1,2,3,4-TCDD. 5
f. Results must be documented with appropriate
SIM traces, labeled with the corresponding EPA
sample numbers, and calculations. 5
Performance Checks:
a. GC resolution and MS resolution checks performed
at the beginning and end of each 12-hour period. 5
b. Results of performance checks properly recorded
on Form H-4. 5
c. MS Resolution: PFK (or alternate) tune shows
appropriate mass resolution (Section 8.2,
Exhibit D) with mass assignment accuracy
within +5 ppm. 5
d. GC Resolution: chromatograms meet the criteria
specified in Section 8.1, Exhibit D. 5
Routine Calibration:
a. Performed each 12 hours, after MS and GC
resolution checks, using HRCC2. 5
b. Results of routine calibrations properly
reported on Forms H-3 and H-8. 5
c. For unlabeled 2,3,7,8-TCDD: abundance
ratio must be X).67 and <0.90 for m/z
319.897 to 321.894. 5
d. Abundance ratio correct for isotopically ,
labeled standards (e.g., 331.937/333.934
must be X5.67 and <0.90 for 13C12-2,3,7,8-TCDD
and 13C12-1,2,3,4-TCDD). 5
e. Response factors [RRF(I) and RRF(II)] are
within ^20% of the mean of the respective
initial calibration response factors. 5
f. Signal-to-Noise (S/N) Ratio: SIM traces
for 2,3,7,8-TCDD demonstrate S/N of >2.5. 5
g. Results documented with appropriate SIM
traces and calculations. 5
Subtotal II 105
Figure 1. (Continued).
E-10
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Possible Score
Score Achieved
III. Performance Evaluation (PE) Sample Data
(Scores to be determined for each sample
in the PE set)
1. Forms H-l and H-9 properly filled out for sample. 5
2. Measured concentration of unlabeled
2,3,7,8-TCDD within acceptance window
established by EPA. 40
3. Estimated concentration of total TCDDs
within acceptance window established by
EPA. 20
4. Identification Criteria for 2,3,7,8-TCDD:
a. Retention time (RT) (at maximum peak
height) of the sample component m/z
319.897 is within -1 to +3 seconds
of the m/z 331.937 13C122,3,7,8-TCDD
internal standard peak. 10
b. The ion current responses for m/z
258.930, 319.897 and 321.894 must reach
a maximum simultaneously (+1 second)
and must be >2.5 times noise level. 10
c. The m/z 319.897/321.894 ratio is X>.67
and _<0.90. 10
d. The m/z 331.937/333.934 ratio is X>.67
and _<0.90. 5
e. The S/N ratio for m/z 331.937 and
333.934 is >2.5. 5
5. Identification Criteria for other TCDDs:
a. Retention time must fall into window
established by GC performance check. 5
b. The ion current responses for m/z
258.930, 319.897, and 321.894 reach
a maximum simultaneously (j^l second)
and are 2^«5 times noise level. 10
c. The m/z 319.897/321.894 ratio is
20.67 and ^0.90. 5
Figure 1. (Continued).
E-ll
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Possible Score
Score Achieved
6. Concentrations of unlabeled TCDDs
are calculated according to D-13.1. 10
7. Duplicate analysis values agree within
±50%. 10
8. Estimated detection limits calculated
according to D-13.5. 10
9. Percent recovery of 13C,2~2,3,7,8-TCDD
>40 and O20%. 10
10. Results documented with appropriate
SIM traces and calculations. 20
Subtotal III 185
Total 290
Figure 1. (Continued).
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EVENT SEQUENCE FOR ON-SITE LABORATORY EVALUATION
I. Meeting with Laboratory Manager and Project Manager
Introduction; discuss purpose of visit; discuss problems with data
submitted by the laboratory.
II. Verification of Personnel
Review qualification of contractor personnel in place and committed to
project (Section I, SES).
III. Verification of Instrumentation
Review equipment in place and committeed to project (Section II, SES).
The Contractor must demonstrate adequate equipment redundancy, as defined
in SES, Section II.D., to ensure his capability to perform the required
analyses in the required time.
IV. Quality Control Procedures
Walk through the laboratory to review:
1. Sample receiving and logging procedures,
2. Sample and extract storage area,
3. Procedures to prevent sample contamination,
4. Security procedures for laboratory and samples,
5. Safety procedures,
6. Conforraance to written SOPs,
7. Instrument records and logbooks,
8. Sample and data control systems,
9. Procedures for handling and disposing of hazardous materials,
10. Glassware cleaning procedures,
11. Status of equipment and its availability,
12. Technical and managerial review of laboratory operations and
data package preparations,
13. Procedures for data handling, analysis, reporting and case
file preparation, and
14. Chain-of-custody procedures.
V. Review of Standard Operating Procedures (SOPs)
Review SOPs with the Project Manager to assure that the laboratory under-
stands the dimensions and requirements of the program.
VI. Identification of Needed Corrective Actions
Discuss with the Project Manager the actions needed to correct weaknesses
identified during the site inspection, PE sample analysis or production of
Figure 2. Event Sequence for On-Site Laboratory Evaluation.
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reports (hard copies and, if appropriate, manual calculations) and documen-
tation. Determine how and when corrective actions will be documented,
how and when improvements will be demonstrated, and identify the contractor
employee responsible for corrective actions.
VII. Previously Identified Problems
Check the most recent SES to verify that all previously identified
problems have been corrected.
VIII. Identification of New Problems
a. Discuss any weaknesses identified in the performance evaluation
sample analyses and reports.
b. Discuss any weaknessess identified in this site inspection.
Figure 2. (Continued)
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SITE EVALUATION SHEET
Laboratory: Date:
Location:
EVALUATORS
Name Organization
1.
2.
3.
4.
5.
6.
7.
I. Laboratory Personnel Committed to Project:
A. Project Manager (responsible for overall technical effort)
Name:
Title:
B. GC/MS Operator:
Experience:*
(one year minimum)
C. GC/MS Data Interpreter:
Experience:*
(two year minimum)
D. Person responsible for sample exraction, column chromatography
and extract concentration:
Experience:*
(one year minumum)
E. Person(s) responsible for calculations and report preparation:
Hardcopy Reports:
F. Person responsible for handling, storage and (if appropriate)
preparation of solutions of standard compounds:
*Experience is deemed to mean "more than 50 percent of the person's productive
work time."
Figure 3. Site Evaluation Sheet.
E-15
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G. Person responsible for standards preparation/storage:
H. Person responsible for record keeping:
I. Quality Assurance Officer:
J. Personnel checklist
( ) Yes ( ) No
1. Do personnel assigned to this project have
the appropriate level and type of experience
to successfully accomplish the objectives of
this program?
2. Is the organization adequately staffed to ( ) Yes ( ) No
meet project requirements in a tmely
manner?
3. Does the Laboratory Quality Assurance officer ( ) Yes ( ) No
report to senior management levels?
4. Was the Quality Assurance officer available ( ) Yes ( ) No
during the evaluation?
II. Laboratory Equipment
A. Gas chromatograph(s)*
Manufacturer and Model:
Installation Date:
Type of Capillary Column Injection System:
Capillary Column to be used (length, ID, coating, etc.):
Necessary Ancillary Equipment (gases, syringes, etc.):
B. High Resolution Mass Spectrometer( s)*
Static Resolution Capability (10,000 min.):
Peak matching system:
Manufacturer and Model:
Installation Date:
Pertinent Modifications:
Peak Matching System/Accuracy (Mfg. spec.):
C. Data System(s)*
Manufacturer and Model:
* If more than one GC/MS/DC, indicate system 1,2,3, etc., by numbering
components with 1,2,3, etc.
Figure 3. (Continued).
E-16
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Installation Date:
Software Version Identifier:
Appropriate selected ion monitoring software/hardware ( ) Yes ( ) No
Capability to produce hard copies of computer-
generated information ( ) Yes ( ) No
D. Evidence that at least one GC/MS/DS system can be reasonably
expected to be operating acceptably at any given time:
( ) More than one adequate GC/MS/DS system is available in-house,
(i.e.,meeting requirements specified in SOW Section 6.1,
Exhibit D).
( ) Appropriate in-house replacement parts and trained service
personnel are available.
( ) A service contract is in place with guaranteed response time
(specify type of contract and limitations).
( ) Voltage control devices are used on major instruments; isolated
circuits are used.
( ) Other (specify)
III. Facilities Checklist
A. Does the laboratory appear to have adequate ( ) Yes ( ) No
workspace (120 sq. feet, 6 linear feet of
unencumbered bench space per analyst)?
B. Does the laboratory have a source of distilled/ ( ) Yes ( ) No
demineralized water?
C. Is the analytical balance located away from ( ) Yes ( ) No
draft and areas subject to rapid temperature
changes or vibration?
D. Has the balance been calibrated within one year ( ) Yes ( ) No
by a certified technician?
E. Is the balance routinely checked with class S ( ) Yes ( ) No
weights before each use and the results recorded
in a logbook?
F. Is the laboratory maintained in a clean and ( ) Yes ( ) No
organized manner?
Figure 3. (Continued).
E-17
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G. Is the facility designed for hazardous organic ( ) Yes ( ) No
chemical analysis?
1. Is ventilation provided in the sample ( ) Yes ( ) No
preparation areas?
2. Are vented hoods available and adequately ( ) Yes ( ) No
vented in the sample preparation areas?
3. Are the hoods equipped with charcoal ( ) Yes ( ) No
and HEPA filters?
4. Are instruments, including GC/MS pumps, ( ) Yes ( ) No
vented into hoods or control devices such
as charcoal traps?
H. Are adequate secured facilities provided for ( ) Yes ( ) No
storage of samples, extracts, and calibration
standards, including cold storage?
I. Are the temperatures of the cold storage units ( ) Yes ( ) No
recorded daily in logbooks?
J. Are chemical waste disposal policies/procedures ( ) Yes ( ) No
in place?
K. Is the laboratory secure? ( ) Yes ( ) No
IV. Analysis Control Checklist
A. Do the project personnel have SOPs for the required ( ) Yes ( ) No
activities?
B. Is a logbook maintained for each instrument and ( ) Yes ( ) No
is information such as calibration data and
instrument maintenance continually recorded?
C. Do the analysts record bench data in a neat ( ) Yes ( ) No
and accurate manner?
D. Standards
1. Are fresh analytical standards prepared ( ) Yes ( ) No
at a frequency consistent with good QC?
2. Are reference materials properly labeled with ( ) Yes ( ) No
concentrations, date of preparation, and the
identity of the person preparing the sample?
3. Is a standards preparation and tracking ( ) Yes ( ) No
logbook maintained?
Figure 3. (Continued).
E-18
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4. Are working standards traceable to EPA ( ) Yes ( ) No
standards or validated against EPA
standards?
V. Documentation/Tracking Checklist
A. Is a sample custodian designated? If yes, ( ) Yes ( ) No
name of sample custodian.
Name:
B. Are the sample custodian's procedures and ( ) Yes ( ) No
responsibilities documented? If yes, where
are these documented?
Are the chain-of-custody procedures documented? ( ) Yes ( ) No
C. Are written Standard Operating Procedures (SOPs) ( ) Yes ( ) No
developed for receipt of samples? If yes, where
are the SOPs documented (laboratory manual,
written instructions, etc.)?
D. Are quality assurance procedures documented ( ) Yes ( ) No
and available to the analysts? If yes, where
are these documented?
E. Are written Standard Operating Procedures (SOPs) ( ) Yes ( ) No
developed for compiling and maintaining sample
document files? If yes, where are the SOPs
documented (laboratory manual, written
instructions, etc.)?
F. Are the magnetic tapes stored in a secure area? ( ) Yes ( ) No
G. Are samples that require preservation stored ( ) Yes ( ) No
in such a way as to maintain their integrity?
If yes, how are the samples stored?
Documentation/Notebooks Checklist
A. Is a permanently bound notebook with preprinted, ( ) Yes ( ) No
consecutively numbered pages being used?
B. Is the type of work clearly displayed on the ( ) Yes ( ) No
notebook?
C. Is the notebook maintained in a legible manner? ( ) Yes ( ) No
D. Are entries noting anomalies routinely recorded? ( ) Yes ( ) No
Figure 3. (Continued).
E-19
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E. Has the analyst avoided obliterating entries or the ( ) Yes ( ) No
use of a pencil?
F. Are inserts (i.e. chromatograms, computer print- ( ) Yes ( ) No
outs, etc.) permanently affixed to the notebook
and signed across insert edge and page?
G. Has the supervisor of the individual maintaining the ( ) Yes ( ) No
notebook personally examined and reviewed the notebook
periodically, and signed his/her name therein, together
with the date and appropriate comments as to whether or
not the notebook is being maintained in an appropriate
manner?
H. Where applicable, is the notebook holder ( ) Yes ( ) No
referencing reports or memoranda pertinent to
the contents of an entry?
VI. Quality Control Manual Checklist
Does the laboratory maintain a Quality Assurance/ ( ) Yes ( ) No
Quality Control (QA/QC) Manual?
Does the manual address the important elements ( ) Yes ( ) No
of a QA/QC program, including the following:
A. Personnel ( ) Yes ( ) No
B. Facilities and equipment ( ) Yes ( ) No
C. Operation of instruments ( ) Yes ( ) No
D. Documentation of Procedures ( ) Yes ( ) No
E. Procurement and inventory practices ( ) Yes ( ) No
F. Preventive maintenance ( ) Yes ( ) No
G. Reliability of data ( ) Yes ( ) No
H. Data validation ( ) Yes ( ) No
I. Feedback and corrective action ( ) Yes ( ) No
J. Instrument calibration ( ) Yes ( ) No
K. Recordkeeping ( ) Yes ( ) No
L. Internal audits ( ) Yes ( ) No
Figure 3. (Continued).
E-20
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Are QA/QC responsibilities and reporting relationships ( ) Yes ( ) No
clearing defined?
Have standard curves been adequately documented? ( ) Yes ( ) No
Are laboratory standards traceable? ( ) Yes ( ) No
Are quality control charts maintained for each ( ) Yes ( ) No
routine analysis?
Do QC records show corrective action when ( ) Yes ( ) No
analytical results fail to meet QC criteria?
Do supervisory personnel review the data and QC results? ( ) Yes ( ) No
VII. Data Handling Checklist
Are data calculations checked by a second person? ( ) Yes ( ) No
Are data calculations documented? ( ) Yes ( ) No
Do records indicate corrective action that has ( ) Yes ( ) No
been taken on projected data?
Are limits of detection determined and reported ( ) Yes ( ) No
properly?
Are all data and records retained for the ( ) Yes ( ) No
required amount of time?
Are quality control data (e.g., standard curve ( ) Yes ( ) No
duplicates) accessible for all analytical
results?
VIII. Summary
Do responses to the evaluation indicate that ( ) Yes ( ) No
project and supervisory personnel are aware
of QA/QC and its application to the project?
Do project and supervisory personnel place ( ) Yes ( ) No
positive emphasis on QA/QC?
Have responses with respect to QA/QC aspects of ( ) Yes ( ) No
the project been open and direct?
Has a cooperative attitude been displayed by all ( ) Yes ( ) No
project and supervisory personnel?
Figure 3. (Continued).
E-21
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Does the organization place the proper emphasis ( ) Yes ( ) No
on quality assurance?
Have any QA/QC deficiencies been discussed before ( ) Yes ( ) No
leaving?
Is the overall quality assurance adequate to ( ) Yes ( ) No
accomplish the objectives of the project?
Have corrective actions recommended during ( ) Yes ( ) No
previous evaluations been implemented?
Are any corrective actions required? If so, ( ) Yes ( ) No
list the necessary actions below.
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TECHNICAL REPORT DATA
(fleae read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE 6. REPORT DATE
PROTOCOL FOR THE ANALYSIS OF 2,3,7,8-TETRACHLORODIBENZOt
p-DIOXIN BY HIGH-RESOLUTION GAS CHROMATOGRAPHY/HIGH-
RESOLUTION MASS SPECTROMETRY
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. S. Stanley and T.
B. PERFORMING ORGANIZATION REPORT NO.
M. Sack
>. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
Contract Number SAS 1576X
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring Systems Laboratory - LV, NV
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89114
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
Project Officer - Werner F. Beckert, Environmental Monitoring Systems Laboratory
Las Vegas, NV 89114
16. ABSTRACT
An analytical protocol for the determination of 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD) and total TCDDs in soil, sediment and aqueous samples using high-
resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) was
developed using the best features of several candidate methods and input from experts
in the field. Preliminary tests led to refinements of the chromatographic cleanup
procedures and corresponding changes in the protocol. A final single-laboratory
evaluation of the refined protocol, consisting of triplicate analyses 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 concentrations from 10 to 200 pg/g (ppt) in soils and
100 to 2,000 pg/L (ppq) in aqueous samples. Based on the data generated and on the
evaluation of several options, parts of the protocol were modified at the EMSL-LV to
lower the quantitation limit for TCDD to 2 ppt in soil/sediments and to 20 ppq in
aqueous samples.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFlERS/OPEN ENDED TERMS C. COSATI FieW/GlOUp
16. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (This page I
UNCLASSIFIED
22. PRICE
EPA Pw« 2220.1 («•». 4.77) PNKVIOUI EDITION if OMOLCTC
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