United States Solid Waste and
Environmental Protection Emergency Response EPA/530-SW-91-019
Agency (OS-305) January 1991
E PA Test Method 8290
Procedures for the Detection
and Measurement of PCDDs
and PCDFs
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METHOD 8290
POLYCHLQRINATED DIBENZQDIOXINS fPCDDs1 AND POLYCHLQRINATED DIBENZQFURANS fPCDFsl
BY HIGH-RESOLUTION GAS CHRQMATOGRAPHY/HIGH-RESQLUTIQN
MASS SPECTRQMETRY (HRGC/HRMS1
1.0 SCOPE AND APPLICATION
1.1 This method provides procedures for the detection and quantitative
measurement of polychlorinated dibenzo-p-dioxins (tetra- through octachlorinated
homologues; PCDDs), and polychlorinated dibenzofurans (tetra- through
octachlorinated homologues; PCOFs) 1n a variety of environmental matrices and
at part-per-trillion (ppt) to part-per-quadrillion (ppq) concentrations. The
following compounds can be determined by this method:
Compound Name
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDO)
1,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDO)
1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCOO)
1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)
1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDO)
l,2,3,4,6,7,8-Heptachlorodibenzo-p-d1ox1n (HpCDO)
2,3,7,8-Tetrachlorodibenzofuran (TCOF)
1,2,3,7,8-Pentachlorodibenzofuran (PeCOF)
2,3,4,7,8-Pentachlorodibenzofuran (PeCOF)
1,2,3,6,7,8-Hexachlorodibenzofuran (HxCOF)
1,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF)
1,2,3,4,7,8-Hexachlorodibenzofuran (HxCOF)
2,3,4,6,7,8-Hexachlorodibenzofuran (HxCOF)
1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCOF)
1,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCOF)
1.2 The analytical method calls for the use of high-resolution gas
chromatography and high-resolution mass spectrometry (HRGC/HRMS) on purified
sample extracts. Table 1 lists the various sample types covered by this
analytical protocol, the 2,3,7,8-TCDO-based method calibration limits (MCLs),
and other pertinent Information. Samples containing concentrations of specific
congeneric analytes (PCDDs and PCOFs) considered within the scope of this method
that are greater than ten times the upper HCLs must be analyzed by a protocol
designed for such concentration levels, e.g., Method 8280. An optional method
for reporting the analytical results using a 2,3,7,8-TCDD toxlcity equivalency
factor (TEF) Is described.
1.3 The sensitivity of this method 1s dependent upon the level of inter-
ferences within a given matrix. The calibration range of the method for a 1 L
water sample 1s 10 to 2000 ppq for TCDD/TCDF and PeCDO/PeCOF, and 1.0 to 200 ppt
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for a 10 g soil, sediment, fly ash, or tissue sample for the same analytes
(Table 1). Analysis of a one-tenth aliquot of the sample permits measurement
of concentrations up to 10 times the upper MCL. The actual limits of detection
and quantitation will differ from the lower MCL, depending on the complexity of
the matrix.
1.4 This method 1s designed for use by analysts who are experienced with
residue analysis and skilled in HRGC/HRMS.
1.5 Because of the extreme toxlcity of many of these compounds, the
analyst must take the necessary precautions to prevent exposure to materials
known or believed to contain PCODs or PCDFs. It 1s the responsibility of the
laboratory personnel to ensure that safe handling procedures are employed.
Section 11 of this method discusses safety procedures.
2.0 SUMMARY OF METHOD
2.1 This procedure uses matrix specific extraction, analyte specific
cleanup, and HRGC/HRMS analysis techniques.
2.2 If Interferences are encountered, the method provides selected
cleanup procedures to aid the analyst in their elimination. A simplified
analysis flow chart is presented at the end of this method.
2.3 A specified amount (see Table 1) of soil, sediment, fly ash, water,"
sludge (including paper pulp), still bottom, fuel oil, chemical reactor residue,
fish tissue, or human adipose tissue 1s spiked with a solution containing
specified amounts of each of the nine isotopically (13C12) labeled PCOOs/PCDFs
listed in Column 1 of Table 2. The sample 1s then extracted according to a
matrix specific extraction procedure. Aqueous samples that are judged to contain
1 percent or more solids, and solid samples that show an aqueous phase, are
filtered, the solid phase (Including the filter) and the aqueous phase extracted
separately, and the extracts combined before extract cleanup. The extraction
procedures are:
a) Toluene:Soxhlet extraction for soil, sediment, fly ash and paper pulp
samples;
b) Methylene chloride:!iquid-Hquld extraction for water samples;
c) Toluene: Dean-Stark extraction for fuel oil and aqueous sludge samples;
d) Toluene extraction for still bottom samples;
e) Hexane/methylene chloride:Soxhlet extraction or methylene
chloride:Soxhlet extraction for fish tissue samples; and
f) Methylene chloride extraction for human adipose tissue samples.
g) As an option, all solid samples (wet or dry) can be extracted with
toluene using a Soxhlet/Oean Stark extraction system.
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The decision for the selection of an extraction procedure for chemical
reactor residue samples 1s based on the appearance (consistency, viscosity) of
the samples. Generally, they can be handled according to the procedure used
for still bottom (or chemical sludge) samples.
2.4 The extracts are submitted to an acid-base washing treatment and
dried. Following a solvent exchange step, the extracts are cleaned up by column
chromatography on alumina, silica gel, and AX-21 activated carbon on Celite 545*
(or equivalent).
2.4.1 The extracts from adipose tissue samples are treated with
silica gel impregnated with sulfurlc add before chromatography on acidic
silica gel, neutral alumina, and AX-21 on CelUe 545» (or equivalent).
2.4.2 F1sh tissue and paper pulp extracts are subjected to an acid
wash treatment only, prior to chromatography on alumina and
AX-2l/Cel1te 545* (or equivalent).
2.5 The preparation of the final extract for HRGC/HRMS analysis is
accomplished by adding, to the concentrated AX-2l/Cel1te 545* (or equivalent)
column eluate, 10 to 50 ni (depending on the matrix type) of a nonane solution
containing 50 pg/jxL of each of the two recovery standards 13C12-1,2,3,4-TCDD and
13C12-l,2,3,7,8,9-HxCDD (Table 2). The former 1s used to determine the percent
recoveries of tetra- and pentachlorinated PCDO/PCDF congeners, while the latter
is used to determine the percent recoveries of the hexa-, hepta- and
octachlorinated PCOO/PCOF congeners.
2.6 One to two ML of the concentrated extract are Injected into an
HRGC/HRMS system capable of performing selected Ion monitoring at resolving
powers of at least 10,000 (10 percent valley definition).
2.7 The Identification of OCDO and nine of the fifteen 2,3,7,8-
substituted congeners (Table 3), for which a 13C-labeled standard is available
in the sample fortification and recovery standard solutions (Table 2), 1s based
on their elutlon at their exact retention time (within 0.005 retention time units
measured in the routine calibration) and the simultaneous detection of the two
most abundant Ions 1n the molecular Ion region. The remaining six 2,3,7,8-
substltuted congeners (I.e., 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-
HxCOF; 1,2,3,7,8,9-HxCDF; 2,3,4,6.,7,8-HxCOF, and 1,2,3,4,7,8,9-HpCDF), for which
no carbon-labeled Internal standards are available 1n the sample fortification
solution, and all other Identified PCDO/PCDF congeners are Identified by their
relative retention times falling within their respective PCOD/PCDF retention time
windows, as established from the routine calibration data, and the simultaneous
detection of the two most abundant Ions in the molecular ion region. The
Identification of OCDF 1s based on Us retention time relative to 13C12-OCDD and
the simultaneous detection of the two most abundant Ions In the molecular ion
region. Confirmation 1s based on a comparison of the ratios of the Integrated
ion abundance of the molecular ion species to their theoretical abundance ratios.
2.8 Quantltatlon of the Individual congeners, total PCDDs and total
PCDFs 1s achieved 1n conjunction with the establishment of a multipoint (five
points) calibration curve for each horaologue, during which each calibration
solution 1s analyzed once. .
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3.0 INTERFERENCES
3.1 Solvents, reagents, glassware and other sample processing hardware
may yield discrete artifacts or elevated baselines that may cause misinter-
pretation of the chromatographic data (see references 1 and 2.) All of these
materials must be demonstrated to be free from Interferants under the conditions
of analysis by performing laboratory method blanks. Analysts should avoid using
PVC gloves.
3.2 The use of high purity reagents and solvents helps minimize
interference problems. Purification of solvents by distillation 1n all-glass
systems may be necessary.
3.3 Interferants coextracted from the sample will vary considerably from
matrix to matrix. PCDDs and PCDFs are often associated with other interfering
chlorinated substances such as polychlorinated blphenyls (PCBs), polychlorinated
diphenyl ethers (PCDPEs), polychlorinated naphthalenes, and polychlorinated
alkyldibenzofurans that may be found at concentrations several orders of
magnitude higher than the analytes of Interest. Retention times of target
analytes must be verified using reference standards. These values must
correspond to the retention time windows established 1n Section 8.1.1.3. While
certain cleanup techniques are provided as part of this method, unique samples
may require additional cleanup steps to achieve lower detection limits.
3.4 A high-resolution capillary column (60 m DB-5, J&W Scientific, oc
equivalent) 1s used in this method. However, no single column 1s known t»
resolve all Isomers. The 60 m OB-5 GC column 1s capable of 2,3,7,8-TCOD isomer
specificity (Section 8.1.1). In order to determine the concentration of the
2,3,7,8-TCOF (if detected on the DB-5 column), the sample extract must be
reanalyzed on a column capable of 2,3,7,8-TCOF Isomer specificity (e.g., DB-225,
SP-2330, SP-2331, or equivalent). When a column becomes available that resolves
all isomers, then a single analysis on this column can be used instead of
analyses on more than one column.
4.0 APPARATUS AND MATERIALS
4.1 High-Resolution Gas Chromatooraph/Hiah-Resolutlon Mass
Spectrometer/Data System fHRGC/HRMS/DS) - The GC must be equipped for temperature
programming, and all required accessories must be available, such as syringes,
gases, and capillary columns.
4.1.1 GC !n.1ect1on Port - The GC Injection port oust be designed
for capillary columns. The use of splitless Injection techniques is
recommended. On column 1 Ml Injections can be used on the 60 m DB-5
colimn. The use of a moving needle Injection port 1s also acceptable.
When using the Mthod described 1n this protocol, a 2 ML Injection volume
Is used consistently (I.e., the Injection volumes for all extracts, blanks,
calibration solutions and the performance check samples are 2 ML). One ML
Injections are allowed; however, laboratories must remain consistent
throughout the analyses by using the same Injection volume at all times.
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4.1.2 Gas Chromatoaraph/Mass Spectrometer (GC/HS) Interface - The
GC/MS Interface components should withstand 350°C. The Interface must be
designed so that the separation of 2,3,7,8-TCDD from the other TCDO isomers
achieved 1n the gas chromatographlc column 1s not appreciably degraded.
Cold spots or active surfaces (adsorption sites) 1n the GC/MS Interface
can cause peak tailing and peak broadening. It 1s recommended that the
GC column be fitted directly Into the mass spectrometer 1on source without
being exposed to the Ionizing electron beam. Graphite ferrules should be
avoided 1n the Injection port because they may adsorb the PCDOs and PCOFs.
Vespel™, or equivalent, ferrules are recommended.
4.1.3 Mass Spectrometer - The static resolving power of the
instrument must be maintained at a minimum of 10,000 (10 percent valley).
4.1.4 Data System - A dedicated data system 1s employed to control
the rapid multiple-ion monitoring process and to acquire the data.
Quantltation data (peak areas or peak heights) and SIH traces (displays
of intensities of each ion signal being monitored Including the lock-mass
ion as a function of time) must be acquired during the analyses and stored.
Quantitations may be reported based upon computer generated peak areas or
upon measured peak heights (chart recording). The data system must be
capable of acquiring data at a minimum of 10 ions in a single scan. It is
also recommended to have a data system capable of switching to different
sets of ions (descriptors) at specified times during an HRGC/HRMS
acquisition. The data system should be able to provide hard copies of
individual ion chromatograms for selected gas chromatographlc time
Intervals. It should also be able to acquire mass spectral peak profiles
(Section 8.1.2.3) and provide hard copies of peak profiles to demonstrate
the required resolving power. The data system should permit the
measurement of noise on the base line.
NOTE: The detector ADC zero setting must allow peak-to-peak measurement of the
noise on the base line of every monitored channel and allow for good
estimation of the instrument resolving power. In Figure 2, the effect of
different zero settings on the measured resolving power is shown.
4.2 GC Columns
4.2.1 In order to have an isomer specific determination for 2,3,7,8-
TCDO and to allow the detection of OCDO/OCOF within a reasonable time
interval in one HRGC/HRMS analysis, use of the 60 m OB-5 fused silica
capillary column Is recommended. Minimum acceptance criteria must be
demonstrated and documented (Section 8.1.1). At the beginning of each 12
hour period (after mass resolution and GC resolution 1s 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 acceptable results with the recommended column are shown
in Section 7.6.
4.2.2 Isomer specificity for all 2,3,7,8-substltuted PCDDs/PCDFs
cannot be achieved on the 60 m OB-5 GC column alone. In order to determine
the proper concentrations of the Individual 2,3,7,8-substltuted congeners,
the sample extract must be reanalyzed on another GC column that resolves
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the isomers. When such a column becomes available, and the isomer
specificity can be documented, the performing laboratory will be required
to use it.
4.2.3 30 m DB-225 fused silica capillary column, (Jiw Scientific)
or equivalent.
4.3 Miscellaneous Equipment and Materials - The following list of items
does not necessarily constitute an exhaustive compendium of the equipment needed
for this analytical method.
4.3.1 Nitrogen evaporation apparatus with variable flow rate.
4.3.2 Balances capable of accurately weighing to 0.01 g and 0.0001 g.
4.3.3 Centrifuge.
4.3.4 Water bath, equipped with concentric ring covers and capable
of being temperature controlled within + 2°C.
4.3.5 Stainless steel or glass container large enough to hold
contents of one pint sample containers.
4.3.6 Glove box.
4.3.7 Drying oven.
4.3.8 Stainless steel spoons and spatulas.
4.3.9 Laboratory hoods.
4.3.10 Pipets, disposable, Pasteur, 150 mm long x 5 mm ID.
4.3.11 Pipets, disposable, serologlcal, 10 ml, for the preparation
of the carbon columns specified in Section 7.5.3.
4.3.12 Reaction vial, 2 ml, silanized amber glass (Reacti-vial, or
equivalent).
4.3.13 Stainless steel meat grinder with a 3 to 5 mm hole size inner
plate.
4.3.14 Separatory funnels, 125 iL and 2000 ML.
4.3.15 Kuderna-Danlsh concentrator, 500 al, fitted with 10 ml
concentrator tube and three ball Snyder column.
4.3.16 Teflon™ or carborundum (silicon carbide) boiling chips (or
equivalent), washed with hexane before use.
NOTE: Teflon™ boiling chips may float In nethylene chloride, may not work in
the presence of any water phase, and may be penetrated by nonpolar organic
compounds.
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4.3.17 Chromatographic columns, glass, 300 mm x 10.5 mm, fitted with
Teflon™ stopcock.
4.3.18 Adapters for concentrator tubes.
4.3.19 Glass fiber filters.
4.3.20 Dean-Stark trap, 5 or 10 ml, with T-jo1nts, condenser and
125 mi flask.
4.3.21 Continuous liquid-liquid extractor.
4.3.22 All glass Soxhlet apparatus, 500 ml flask.
4.3.23 Soxhlet/Dean Stark extractor (optional), all glass, 500 ml
flask.
4.3.24 Glass funnels, sized to hold 170 ml of liquid.
4.3.25 Desiccator.
4.3.26 Solvent reservoir (125 ml), Kontes; 12.35 cm diameter (special
order Item), compatible with gravity carbon column.
4.3.27 Rotary evaporator with a temperature controlled water bath.
4.3.28 High speed tissue homogenizer, equipped with an EN-8 probe,
or equivalent.
4.3.29 Glass wool, extracted with methylene chloride, dried and
stored in a clean glass jar.
4.3.30 Extraction jars, glass, 250 ml, with teflon lined screw cap.
4.3.31 Volumetric flasks, Class A - 10 mL to 1000 ml.
4.3.32 Glass vials, 1 dram (or metric equivalent).
NOTE: Reuse of glassware should be minimized to avoid the risk of contamination.
All glassware that 1s reused must be scrupulously cleaned as soon as
possible after ust, according to the following procedure: Rinse glassware
with the last solvent used in it. Hash with hot detergent water, then
rinse with copious amounts of tap water and several portions of organic-
free reagent water. Rinse with high purity acetone and hexane and store
it Inverted or capped with solvent rinsed aluminum foil in a clean
environment.
5.0 REAGENTS AND STANDARD SOLUTIONS
5.1 Organic-free reagent water - All references to water in this method
refer to organic-free reagent water, as defined in Chapter One.
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5.2 Column Chromatography Reagents
5.2.1 Alumina, neutral, 80/200 mesh (Super 1, Woelm*, or
equivalent). Store In a sealed container at room temperature, in a
desiccator, over self-Indicating silica gel.
5.2.2 Alumina, acidic AG4, (Bio Rad Laboratories catalog 1132-1240,
or equivalent). Soxhlet extract with methylene chloride for 24 hours 1f
blanks show contamination, and activate by heating in a foil covered glass
container for 24 hours at 190°C. Store 1n a glass bottle sealed with a
Teflon™ lined screw cap.
5.2.3 Silica gel, high purity grade, type 60, 70-230 mesh; Soxhlet
extract with methylene chloride for 24 hours 1f blanks show contamination,
and activate by heating in a foil covered glass container for 24 hours at
190°C. Store 1n a glass bottle sealed with a Teflon™ lined screw cap.
5.2.4 Silica gel Impregnated with sodium hydroxide. Add one part
(by weight) of 1 H NaOH solution to two parts (by weight) silica gel
(extracted and activated) in a screw cap bottle and mix with a glass rod
until free of lumps. Store in a glass bottle sealed with a Teflon™ lined
screw cap.
5.2.5 Silica gel impregnated with 40 percent (by weight) sulfuric
add. Add two parts (by weight) concentrated sulfuric add 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. Store in
a glass bottle sealed with a Teflon™ lined screw cap.
5.2.6 Cellte 545* (Supelco), or equivalent.
5.2.7 Active carbon AX-21 (Anderson Development Co., Adrian, MI),
or equivalent, prewashed with methanol and dried in vacuo at 110°C. Store
1n a glass bottle sealed with a Teflon™ lined screw cap.
5.3 Reagents
5.3.1 Sulfuric add, H,S04, concentrated, ACS grade, specific gravity
1.84.
5.3.2 Potassium hydroxide, KOH, ACS grade, 20 percent (w/v) in
organic-free reagent water.
5.3.3 Sodium chloride, NaCI, analytical reagent, 5 percent (w/v) in
organic-free reagent water.
5.3.4 Potassium carbonate, KjC03, anhydrous, analytical reagent.
5.4 Desiccating agent
5.4.1 Sodium sulfate (powder, anhydrous), Na2S04. Purify by heating
at 400°C for 4 hours 1n a shallow tray, or by precleanlng the sodium
sulfate with methyl ene .chloride. If the sodium sulfate 1s precleaned with
*
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methylene chloride, a method blank must be analyzed, demonstrating that
there 1s no Interference from the sodium sulfate.
5.5 Solvents
5.5.1 Methylene chloride, CHjClj. High purity, distilled 1n glass
or highest available purity.
5.5.2 Hexane, C9H14. High purity, distilled 1n glass or highest
available purity.
5.5.3 Methanol, CH3OH. High purity, distilled 1n glass or highest
available purity.
5.5.4 Nonane, CjHj,,. High purity, distilled 1n glass or highest
available purity.
5.5.5 Toluene, C9HsC93. High purity, distilled 1n glass or highest
available purity.
5.5.6 Cyclohexane, C8H12. High purity, distilled 1n glass or highest
available purity.
5.5.7 Acetone, CH3COCH3. High purity, distilled in glass or highest
available purity.
5.6 High-Resolution Concentration Calibration Solutions (Table 5) - Five
nonane solutions containing unlabeled (totaling 17) and carbon-labeled (totaling
11) PCOOs and PCOFs at known concentrations are used to calibrate the Instrument.
The concentration ranges are homologue dependent, with the lowest values for the
tetrachlorlnated dioxin and furan (1.0 pg/ML) and the highest values for the
octachlorinated congeners (1000 pg/jiL).
5.6.1 Depending on the availability of materials, these high-
resolution concentration calibration solutions may be obtained from the
Environmental Monitoring Systems Laboratory, U.S. EPA, Cincinnati, Ohio.
However, additional secondary standards must be obtained from commercial
sources, and solutions must be prepared In the analyst's laboratory.
Traceability of standards must b« verified against EPA-suppl1ed standard
solutions. It 1s the responsibility of the laboratory to ascertain that
the calibration solutions received (or prepared) are indeed at the
appropriate concentrations before they are used to analyze samples.
5.6.2 Store the concentration calibration solutions in 1 ml minivials
at roo* temperature In the dark.
5.7 SC Column Performance Check Solution - This solution contains the
first and last fluting isomers for each homologous series from tetra- through
heptachlorinated congeners. The solution also contains a series of other TCOO
isomers for the purpose of documenting the chromatographlc resolution. The
13C12-2,3,7,8-TCOO 1s also present. The laboratory 1s required to use nonane as
the solvent and adjust the volume so that the final concentration does not exceed
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100 pg/ML per congener. Table 7 summarizes the qualitative composition (minimum
requirement) of this performance evaluation solution.
5.8 Sample Fortification Solution - This nonane solution contains the
nine Internal standards at the nominal concentrations that are listed in Table 2.
The solution contains at least one carbon-labeled standard for each homologous
series, and It 1s used to measure the concentrations of the native substances.
(Note that 13C12-OCDF 1s not present 1n the solution.)
5.9 Recovery Standard Solution - This nonane solution contains two
recovery standards, "C12-1,2,3,4-TCDD and 13C17-l,2,3,7,8,9-HxCDD, at a nominal
concentration of 50 pg/ML per compound. 10 to 50 ML of this solution will be
spiked into each sample extract before the final concentration step and HRGC/HRMS
analysis.
5.10 Matrix Spike Fortification Solution - Solution used to prepare the
MS and HSO samples. It contains all unlabeled analytes listed 1n Table 5 at con-
centrations corresponding to the HRCC 3.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the Introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Sample Collection •
6.2.1 Sample collection personnel should, to the extent possible,
homogenize samples 1n the field before filling the sample containers.
This should minimize or eliminate the necessity for sample homogenization
in the laboratory. The analyst should make a judgment, based on the
appearance of the sample, regarding the necessity for additional mixing.
If the sample 1s clearly not homogeneous, the entire contents should be
transferred to a glass or stainless steel pan for mixing with a stainless
steel spoon or spatula before removal of a sample portion for analysis.
6.2.2 Grab and composite samples must be collected in glass
containers. Conventional sampling practices must be followed. The bottle
must not be prewashed with sample before collection. Sampling equipment
must be free of potential sources of contamination.
6.3 Grinding or Blending of F1sh Samples - If not otherwise specified
by the U.S. EPA, the whole fish (frozen) should be blended or ground to provide
a homogeneous sample. The use of a stainless steel meat grinder with a 3 to 5
mm hole size Inner plate 1s recommended. In some circumstances, analysis of
fillet or specific organs of fish may be requested by the U.S. EPA. If so
requested, the above whole fish requirement 1s superseded.
6.4 Storage and Holding Times - All samples, except fish and adipose
tissue samples, must be stored at 4°C in the dark, extracted within 30 days and
completely analyzed within 45 days of collection. Fish and adipose tissue
samples must be stored at -20°C In the dark, extracted within 30 days and
completely analyzed within 45 days of collection. Whenever samples are analyzed
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after the holding time expiration date, the results should be considered to be
minimum concentrations and must be Identified as such.
Note: The holding times listed In Section 6.4 are recommendations. PCDOs and
PCDFs are very stable 1n a variety of matrices, and holding times under
the conditions listed in Section 6.4 may be as high as a year for certain
matrices. Sample extracts, however, should always be analyzed within 45
days of extraction.
6.5 Phase Separation - This is a guideline for phase separation for very
wet (>25 percent water) soil, sediment and paper pulp samples. Place a 50 g
portion in a suitable centrifuge bottle and centrifuge for 30 minutes at
2,000 rpm. Remove the bottle and mark the interface level on the bottle.
Estimate the relative volume of each phase. With a disposable pipet, transfer
the liquid layer into a clean bottle. Mix the solid with a stainless steel
spatula and remove a portion to be weighed and analyzed (percent dry weight
determination, extraction). Return the remaining solid portion to the original
sample bottle (empty) or to a clean sample bottle that is properly labeled, and
store it as appropriate. Analyze the solid phase by using only the soil,
sediment and paper pulp method. Take note of, and report, the estimated volume
of liquid before disposing of the liquid as a liquid waste.
6.6 Soil. Sediment, or Paper Sludge (Pulp) Percent Drv Weight
Determination - The percent dry weight of soil, sediment or paper pulp samples
showing detectable levels (see note below) of at least one 2,3,7,8-substltuted
PCDD/PCDF congener is determined according to the following procedure. Weigh:
a 10 g portion of the soil or sediment sample (+ 0.5 g) to three significant
figures. Dry it to constant weight at 110°C in an adequately ventilated oven.
Allow the sample to cool in a desiccator. Weigh the dried solid to three
significant figures. Calculate and report the percent dry weight. Do not use
this solid portion of the sample for extraction, but instead dispose of it as
hazardous waste.
NOTE: Until detection limits have been established (Section 1.3), the lower MCLs
(Table 1) may be used to estimate the minimum detectable levels.
X dry weight • Q of drv sample x 100
g of sample
CAUTION: Finely divided soils and sediments contaminated with PCDDs/PCDFs are
hazardous because of the potential for Inhalation or ingestion of
particles containing PCDDs/PCDFs (including 2,3,7,8-TCDO). Such samples
should be handled in a confined environment (I.e., a closed hood or a
glove box).
6.7 Lipid Content Determination
6.7.1 Fish Tissue - To determine the I1p1d content of fish tissue,
concentrate 125 ml of the fish tissue extract (Section 7.2.2), in a tared
200 ml round bottom flask, on a rotary evaporator until a constant weight
(W) 1s achieved.
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DRMT
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100 (W)
Percent Hp1d • -
10
Dispose of the lipid residue as a hazardous waste if the results of the
analysis Indicate the presence of PCDDs or PCDFs.
6.7.2 Ad-|pose Tissue - Details for the determination of the adipose
tissue lipid content are provided in Section 7.3.3.
7.0 PROCEDURE
7.1 Internal standard addition
7.1.1 Use a portion of 1 g to 1000 g (± 5 percent) of the sample to
be analyzed. Typical sample size requirements for different matrices are
given in Section 7.4 and in Table 1. Transfer the sample portion to a
tared flask and determine Us weight.
7.1.2 Except for adipose tissue, add an appropriate quantity of the
sample fortification mixture (Section 5.8) to the sample. All samples
should be spiked with 100 ML of the sample fortification mixture to give
internal standard concentrations as Indicated in Table 1. As an example,
for 13C12-2,3,7,8-TCDD, a 10 g soil sample requires the addition of 1000 pg
of 13C12-2,3,7,8-TCDD to give the required 100 ppt fortification level. The
fish tissue sample (20 g) must be spiked with 200 ML of the Internal
standard solution, because half of the extract will be used to determine
the 11p1d content (Section 6.7.1).
7.1.2.1 For the fortification of soil, sediment, fly ash,
water, fish tissue, paper pulp and wet sludge samples, mix the sample
fortification solution with 1.0 mL acetone,
7.1.2.2 Do not dilute the nonane solution for the other
matrices.
7.1.2.3 The fortification of adipose tissue 1s carried out
at the time of homogenlzatlon (Section 7.3.2.3).
7.2 Extraction and Purification of F1sh and Paper Pulp Samples
7.2.1 Add 60 g anhydrous sodium sulfate to a 20 g portion of a
homogeneous fish sample (Section 6.3) and mix thoroughly with a stainless
steel spatula. After breaking up any lumps, place the fish/sodium sulfate
mixture In the Soxhlet apparatus on top of a glasswool plug. Add 250 mL
methylene chloride or hexane/methylene chloride (1:1) to the Soxhlet
apparatus and reflux for 16 hours. The solvent must cycle completely
through the system five times per hour. Follow the same procedure for the
partially dewatered paper pulp sample (using a 10 g sample, 30 g of
anhydrous sodium sulfate and 200 mL of toluene).
NOTE: As an option, a Soxhlet/Dean Stark extractor system may be used, with
toluene as the solvent: No sodium sulfate 1s added when using this option.
8290 • 12 Revision 0
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7.2.2 Transfer the fish extract from Section 7.2.1 to a 250 mL
volumetric flask and fill to the mark with methylene chloride. Mix well,
then remove 125 ml for the determination of the lipid content (Section
6.7.1). Transfer the remaining 125 ml of the extract, plus two 15 mL
hexane/methylene chloride rinses of the volumetric flask, to a KD apparatus
equipped with a Snyder column. Quantitatively transfer all of the paper
pulp extract to a KD apparatus equipped with a Snyder column.
NOTE: As an option, a rotary evaporator may be used 1n place of the KD apparatus
for the concentration of the extracts.
7.2.3 Add a Teflon™, or equivalent, boiling chip. Concentrate the
extract 1n a water bath to an apparent volume of 10 mL. Remove the
apparatus from the water bath and allow to cool for 5 minutes.
7.2.4 Add 50 mL hexane and a new boiling chip to the KD flask.
Concentrate in a water bath to an apparent volume of 5 mL. Remove the
apparatus from the water bath and allow to cool for 5 minutes.
NOTE: The methylene chloride must have been completely removed before proceeding
with the next step.
7.2.5 Remove and Invert the Snyder column and rinse it into the KD
apparatus with two 1 mL portions of hexane. Decant the contents of the
KD apparatus and concentrator tube Into a 125 mL separatory funnel. Rinse
the KD apparatus with two additional 5 mL portions of hexane and add the-
rinses to the funnel. Proceed with the cleanup according to the
Instructions starting in Section 7.5.1.1, but omit the procedures described
1n Sections 7.5.1.2 and 7.5.1.3.
7.3 Extraction and Purification of Human Adipose Tissue
7.3.1 Human adipose tissue samples must be stored at a temperature
of -208C or lower from the time of collection until the time of analysis.
The use of chlorinated materials during the collection of the samples must
be avoided. Samples are handled with stainless steel forceps, spatulas,
or scissors. All sample bottles (glass) are cleaned as specified in the
note at the end of Section 4.3. Teflon™ lined caps should be used.
NOTE: The specified storage temperature of -20°C 1s the maximum storage
temperature permissible for adipose tissue samples. Lower storage
temperatures are recommended.
7.3.2 Adipose Tissue Extraction
7.3.2.1 Weigh, to the nearest 0.01 g, a 10 g portion of a
frozen adipose tissue sample Into a culture tube (2.2 x 15 cm).
NOTE: The sample size may be smaller, depending on availability. In such a
situation, the analyst 1s required to adjust the volume of the internal
standard solution added to the sample to meet the fortification level
stipulated 1n Table 1.
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7.3.2.2 Allow the adipose tissue specimen to reach room
temperature (up to 2 hours).
7.3.2.3 Add 10 ml methylene chloride and 100 ML of the sample
fortification solution. Homogenize the mixture for approximately
1 minute with a tissue homogenizer.
7.3.2.4 Allow the mixture to separate, then remove the
methylene chloride extract from the residual solid material with a
disposable pipet. Percolate the methylene chloride through a filter
funnel containing a clean glass wool plug and 10 g anhydrous sodium
sulfate. Collect the dried extract 1n a graduated 100 ml volumetric
flask.
7.3.2.5 Add a second 10 ml portion of methylene chloride to
the sample and homogenize for 1 minute. Decant the solvent, dry
it, and transfer 1t to the 100 ml volumetric flask (Section 7.3.2.4).
7.3.2.6 Rinse the culture tube with at least two additional
portions of methylene chloride (10 ml each), and transfer the entire
contents to the filter funnel containing the anhydrous sodium
sulfate. Rinse the filter funnel and the anhydrous sodium sulfate
contents with additional methylene chloride (20 to 40 ml) Into the
100 ml flask. Discard the sodium sulfate.
•
7.3.2.7 Adjust the volume to the 100 ml mark with methylene
chloride.
7.3.3 Adipose Tissue Lipid Content Determination
7.3.3.1 Preweigh a clean 1 dram (or metric equivalent) glass
vial to the nearest 0.0001 g on an analytical balance tared to zero.
7.3.3.2 Accurately transfer 1.0 ml of the final extract
(100 ml) from Section 7.3.2.6 to the vial. Reduce the volume of the
extract on a water bath (50-60°C) by a gentle stream of purified
nitrogen until an oily residue remains. Nitrogen blowdown is
continued until a constant weight Is achieved.
Note: When the sample size of the adipose tissue 1s smaller than 10 g, then the
analyst may use a larger portion (up to 10 percent) of the extract defined
in Section 7.3.2.7 for the 11p1d determination.
7.3.3.3 Accurately weigh the vial with the residue to the
nearest 0.0001 g and calculate the weight of the lipid present in
tht vial based on the difference of the weights.
7.3.3.4 Calculate the percent lipid content of the original
sample to the nearest 0.1 percent as shown below:
U x V
L1p1d content, LC (X) - - - - — x 100
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DRtfT
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where:
«„ - weight of the Hpid residue to the nearest 0.0001 g calculated
from Section 7.3.3.3,
Vrt - total volume (100 ml) of the extract 1n mL from
Section 7.3.2.6,
W« - weight of the original adipose tissue sample to the nearest
0.01 g from Section 7.3.2.1, and
V., - volume of the aliquot of the final extract 1n ml used for the
quantitative measure of the llpld residue (1.0 ml).
7.3.3.5 Record the I1p1d residue measured In Section 7.3.3.3
and the percent Hpid content from Section 7.3.3.4.
7.3.4 Adipose Tissue Extract Concentration
7.3.4.1 Quantitatively transfer the remaining extract
(99.0 ml) to a 500 ml Erlenmeyer flask. Rinse the volumetric flask
with 20 to 30 ml of additional methylene chloride to ensure
quantitative transfer.
7.3.4.2 Concentrate the extract on a rotary evaporator and
a water bath at 40°C until an oily residue remains. .-
7.3.5 Adipose Tissue Extract Cleanup
7.3.5.1 Add 200 ml hexane to the lipld residue in the 500 mL
Erlenmeyer flask and swirl the flask to dissolve the residue.
7.3.5.2 Slowly add, with stirring, 100 g of 40 percent (w/w)
sulfuric acid-impregnated silica gel. Stir with a magnetic stirrer
for two hours at room temperature.
7.3.5.3 Allow the solid phase to settle, and decant the liquid
through a filter funnel containing 10 g anhydrous sodium sulfate on
a glass wool plug, Into another 500 mL Erlenmeyer flask.
7.3.5.4 Rinse the solid phase with two 50 ml portions of
hexane. Stir each rinse for 15 minutes, decant, and dry as described
under Section 7.3.5.3. Combine the hexane extracts from Section
7.3.5.3 with the rinses.
7.3.5.5 Rinse the sodium sulfate in the filter funnel with
an additional 25 mL hexane and combine this rinse with the hexane
extracts from Section 7.3.5.4.
7.3.5.6 Prepare an acidic silica column as follows: Pack a
2 cm x 10 en chromatographlc column with a glass wool plug, add
approximately 20 mL hexane, add 1 g silica gel and allow to settle,
then add 4 g of 40 percent (w/w) sulfuric add-Impregnated silica
gel and allow to settle. Elute the excess hexane from the column
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until the solvent level reaches the top of the chromatographic
packing. Verify that the column does not have any air bubbles and
channels.
7.3.5.7 Quantitatively transfer the hexane extract from the
Erlenmeyer flask (Sections 7.3.5.3 through 7.3.5.5) to the silica
gel column reservoir. Allow the hexane extract to percolate through
the column and collect the eluate In a 500 ml KO apparatus.
7.3.5.8 Complete the elutlon by percolating 50 ml hexane
through the column Into the KG apparatus. Concentrate the eluate
on a steam bath to approximately 5 ml. Use nitrogen blowdown to
bring the final volume to about 100 nl.
NOTE: If the silica gel Impregnated with 40 percent sulfuric add is highly
discolored throughout the length of the adsorbent bed, the cleaning
procedure must be repeated beginning with Section 7.3.5.1.
7.3.5.9 The extract 1s ready for the column cleanups described
1n Sections 7.5.2 through 7.5.3.6.
7.4 Extraction and Purification of Environmental and Waste Samples
7.4.1 Sludge/Wet Fuel 011
7.4.1.1 Extract aqueous sludge or wet fuel oil samples by;
refluxing a sample (e.g., 2 g) with 50 ml toluene in a 125 mL flask
fitted with a Dean-Stark water separator. Continue refluxing the
sample until all the water 1s removed.
7.4.1.2 Cool the sample, filter the toluene extract through
a glass fiber filter, or equivalent, Into a 100 mL round bottom
flask.
7.4.1.3 Rinse the filter with 10 ml toluene and combine the
extract with the rinse.
7.4.1.4 Concentrate the combined solutions to near dryness
on a rotary evaporator at 50°C. Use of an Inert gas to concentrate
the extract Is also pemltted. Proceed with Section 7.4.4.
NOTE: If the sludge or fuel oil sample dissolves In toluene, treat it according
to tht Instructions 1n Section 7.4.2 below. If the labeled sludge sample
originates fro« pulp (paper mills), treat it according to the Instructions
starting In Section 7.2, but without the addition of sodium sulfate.
7.4.2 Still Bottom/011
7.4.2.1 Extract still bottooi or oil samples by mixing a sample
portion (e.g., 1.0 g) with 10 mi toluene in a small beaker and
filtering the solution through a glass fiber filter (or equivalent)
Into a 50 mi round bottom flask. Rinse the beaker and filter with
10 el toluene.
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7.4.2.2 Concentrate the combined toluene solutions to near
dryness on a rotary evaporator at 50°C. Proceed with Section 7.4.4.
7.4.3 Fly Ash
Note: Because of the tendency of fly ash to "fly", all handling steps should
be performed in a hood in order to minimize contamination.
7.4.3.1 Weigh about 10 g fly ash to two decimal places and
transfer to an extraction jar. Add 100 jiL sample fortification
solution (Section 5.8), diluted to 1 ml with acetone, to the sample.
Add 150 mi. of 1 M HC1 to the fly ash sample. Seal the jar with the
Teflon lined screw cap and shake for 3 hours at room temperature.
7.4.3.2 Rinse a glass fiber filter with toluene, and filter
the sample through the filter paper, placed in a Buchner funnel, into
all flask. Wash the fly ash cake with approximately 500 mi
organic-free reagent water and dry the filter cake overnight at room
temperature 1n a desiccator.
7.4.3.3 Add 10 g anhydrous powdered sodium sulfate, mix
thoroughly, let sit in a closed container for one hour, mix again,
let sit for another hour, and mix again.
7.4.3.4 Place the sample and the filter paper Into an
extraction thimble, and extract in a Soxhlet extraction apparatus
charged with 200 ml toluene for 16 hours using a five cycle/hour
schedule.
NOTE: As an option, a Soxhlet/Dean Stark extractor system may be used, with
toluene as the solvent. No sodium sulfate 1s added when using this option.
7.4.3.5 Cool and filter the toluene extract through a glass
fiber filter Into a 500 ml round bottom flask. Rinse the filter
with 10 ml toluene. Add the rinse to the extract and concentrate
the combined toluene solutions to near dryness on a rotary evaporator
at 50°C. Proceed with Section 7.4.4.
7.4.4 Transfer the concentrate to a '125 ml separatory funnel using
15 ml hexane. Rinse the flask with two 5 ml portions of hexane and add
the rinses to the funnel. Shake the combined solutions 1n the separatory
funnel for two Minutes with 50 «L of 5 percent sodium chloride solution,
discard the aqueous layer, and proceed with Section 7.5.
7.4.5 Aqueous samples
7.4.5.1 Allow the sample to come to ambient temperature, then
•ark the water meniscus on the side of the 1 L sample bottle for
1 later determination of the exact sample volume. Add the required
acetone diluted sample fortification solution (Section 5.8).
7.4.5.2 When the sample 1s judged to contain 1 percent or
more solids, the sample must be filtered through a 0.45 urn glass
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DRAFT
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fiber filter that has been rinsed with toluene. If the suspended
solids content is too great to filter through the 0.45 urn filter,
centrifuge the sample, decant, and then filter the aqueous phase.
7.4.5.3 Combine the solids from the centrifuge bottle(s) with
the particulates on the filter and with the filter itself and proceed
with the Soxhlet extraction as specified in Sections 7.4.6.1 through
7.4.6.4. Remove and Invert the Snyder column and rinse it down into
the KD apparatus with two 1 ml portions of hexane.
7.4.5.4 Pour the aqueous filtrate Into a 2 L separatory
funnel. Add 60 ml methylene chloride to the sample bottle, seal
and shake for 30 seconds to rinse the Inner surface. Transfer the
solvent to the separatory funnel and extract the sample by shaking
the funnel for two minutes with periodic venting.
7.4.5.5 Allow the organic layer to separate from the water
phase for a minimum of 10 minutes. If the emulsion Interface between
layers 1s more than one third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase
separation (e.g., glass stirring rod).
7.4.5.6 Collect the methylene chloride Into a KD apparatus
(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 anhydrous sodium sulfate. •
NOTE: As an option, a rotary evaporator may be used in place of the KD apparatus
for the concentration of the extracts.
7.4.5.7 Repeat the extraction twice with fresh 60 ml portions
of methyl ene chloride. After the third extraction, rinse the sodium
sulfate with an additional 30 ml methyl ene chloride to ensure quanti-
tative transfer. Combine all extracts and the rinse in the KD
apparatus.
NOTE: A continuous liquid-liquid extractor may be used 1n place of a separatory
funnel when experience with a sample from a given source Indicates that
a serious emulsion problem will result or an emulsion 1s encountered when
using a separatory funnel. Add 60 iL methylene chloride to the sample
bottle, seal, and shake for 30 seconds to Hnse the Inner surface.
Transfer the solvtnt to the extractor. Repeat the rinse of the sample
bottle with an additional 50 to 100 ml portion of methylene chloride and
add tht rinse to the extractor. Add 200 to 500 ml methylene chloride to
the distilling flask, add sufficient organic-free reagent water (Section
5.1) to ensure proper operation, and extract for 24 hours. Allow to cool,
then detach the distilling flask. Dry and concentrate the extract as
described in Sections 7.4.5.6 and 7.4.5.8 through 7.4.5.10. Proceed with
Section 7.4.5.11.
7.4.5.8 Attach a Snyder column and concentrate the extract
on a water bath until the apparent volume of the liquid is 5 ml.
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Remove the KO apparatus and allow It to drain and cool for at least
10 minutes.
7.4.5.9 Remove the Snyder column, add 50 ml hexane, add the
concentrate obtained from the Soxhlet extraction of the suspended
sol Ids (Section 7.4.5.3), if applicable, re-attach the Snyder column,
and concentrate to approximately 5 mL. Add a new boiling chip to
the KD apparatus before proceeding with the second concentration
step.
7.4.5.10 Rinse the flask and the lower joint with two 5 mL
portions of hexane and combine the rinses with the extract to give
a final volume of about 15 ml.
7.4.5.11 Determine the original sample volume by filling the
sample bottle to the mark with water and transferring the water to
a 1000 ml graduated cylinder. Record the sample volume to the
nearest 5 ml. Proceed with Section 7.5.
7.4.6 So11/Sediment
7.4.6.1 Add 10 g anhydrous powdered sodium sulfate to the
sample portion (e.g., 10 g) and mix thoroughly with a stainless
steel spatula. After breaking up any lumps, place the soil/sodium
sulfate mixture in the Soxhlet. apparatus on top of a glass wool plug
(the use of an extraction thimble is optional). -
NOTE: As an option, a Soxhlet/Dean Stark extractor system may be used, with
toluene as the solvent. No sodium sulfate Is added when using this option.
7.4.6.2 Add 200 to 250 ml toluene to the Soxhlet apparatus
and reflux for 16 hours. The solvent must cycle completely through
the system five times per hour.
NOTE: If the dried sample Is not of free flowing consistency, more sodium sulfate
must be added.
7.4.6.3 Cool and filter the extract through a glass fiber
filter Into a 500 mL round bottom flask for evaporation of the
toluene. Rinse the filter with 10 ml of toluene, and concentrate
the combined fractions to near dryness on a rotary evaporator at
50°C. Remove the flask from the water bath and allow to cool for
5 minutes.
7.4.6.4 Transfer the residue to a 125 mL separatory funnel,
using 15 ml of hexane. Rinse the flask with two additional portions
of hexane, and add the rinses to the funnel. Proceed with
Section 7.5.
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7.5 Cleanup
7.5.1 Partition
7.5.1.1 Partition the hexane extract against 40 ml of
concentrated sulfuHc add. Shake for two minutes. Remove and
discard the sulfurlc add layer (bottom). Repeat the add washing
until no color 1s visible 1n the add layer (perform a maximum of
four add washings).
7.5.1.2 Omit this step for the fish sample extract. Partition
the extract against 40 ml of 5 percent (w/v) aqueous sodium chloride.
Shake for two minutes. Remove and discard the aqueous layer
(bottom).
7.5.1.3 Omit this step for the fish sample extract. Partition
the extract against 40 ml of 20 percent (w/v) aqueous potassium
hydroxide (KOH). Shake for two minutes. Remove and discard the
aqueous layer (bottom). Repeat the base washing until no color 1s
visible in the bottom layer (perform a maximum of four base
washings). Strong base (KOH) 1s known to degrade certain
PCDDs/PCOFs, so contact time must be minimized.
7.5.1.4 Partition the extract against 40 mL of 5 percent
(w/v) aqueous sodium chloride; Shake for two minutes. Remove and
discard the aqueous layer (bottom). Dry the extract by pouring 1t
through a filter funnel containing anhydrous sodium sulfate on a
glass wool plug, and collect 1t 1n a 50 ml round bottom flask.
Rinse the funnel with the sodium sulfate with two 15 ml portions of
hexane, add the rinses to the 50 ml flask, and concentrate the hexane
solution to near dryness on a rotary evaporator (35°C water bath),
making sure all traces of toluene (when applicable) are removed.
(Use of blowdown with an Inert gas to concentrate the extract 1s also
permitted.)
7.5.2 S111ca/Alum1na Column Cleanup
7.5.2.1 Pack a gravity column (glass, 30 cmx 10.5 mm), fitted
with a Teflon™ stopcock, with silica gel as follows: Insert a glass
wool plug Into the bottom of the column. Place 1 g silica gel 1n
the column and tap the column gently to settle the silica gel. Add
2 g sodium hydroxide-Impregnated silica gel, 4 g sulfurlc acid-
Impregnated silica gel, and 2 g silica gel. Tap the column gently
after each addition. A small positive pressure (5 ps1) of clean
nitrogen may be used If needed. Elute with 10 ml hexane and close
the stopcock just before exposure of the top layer of silica gel to
air. Discard the eluate. Check the column for channeling. If
channeling 1s observed, discard the column. Do not tap the wetted
column.
7.5.2.2 Pack a gravity column (glass, 300 mm x 10.5 mm),
fitted with a Teflon™ stopcock, with alumina as follows: Insert
a glass wool plug Into the bottom of the column. Add a 4 g layer
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DRAFT
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of sodium sulfate. Add a 4 g layer of Woelm* Super 1 neutral
alumina. Tap the top of the column gently. Woelm* Super 1 neutral
alumina need not be activated or cleaned before use, but it should
be stored 1n a sealed desiccator. Add a 4 g layer of anhydrous
sodium sulfate to cover the alumina. Elute with 10 ml hexane and
close the stopcock just before exposure of the sodium sulfate layer
to air. Discard the eluate. Check the column for channeling. If
channeling 1s observed, discard the column. Do not tap a wetted
column.
NOTE: Optionally, acidic alumina (Section 5.2.2) can be used in place of neutral
alumina.
7.5.2.3 Dissolve the residue from Section 7.5.1.4 in 2 mi
hexane and apply the hexane solution to the top of the silica gel
column. Rinse the flask with enough hexane (3-4 ml) to complete
the quantitative transfer of the sample to the surface of the silica
gel.
7.5.2.4 Elute the silica gel column with 90 ml of hexane,
concentrate the eluate on a rotary evaporator (35°C water bath) to
approximately 1 ml, and apply the concentrate to the top of the
alumina column (Section 7.5.2.2). Rinse the rotary evaporator flask
twice with 2 ml of hexane, and add the rinses to the top of the
alumina column.
7.5.2.5 Add 20 ml hexane to the alumina column and elute
until the hexane level is just below the top of the sodium sulfate.
Do not discard the eluted hexane, but collect it in a separate flask
and store it for later use, as 1t may be useful in determining where
the labeled analytes are being lost if recoveries are not
satisfactory.
>
7.5.2.6 Add 15 ml of 60 percent methylene chloride in hexane
(v/v) to the alumina column and collect the eluate in a conical
shaped (15 ml) concentration tube. With a carefully regulated stream
of nitrogen, concentrate the 60 percent methylene chloride/hexane
fraction to about 2 ml.
7.5.3 Carbon Column Cleanup
7.5.3.1 Prepare an AX-2l/Cel1te 545* column as follows:
Thoroughly i1x 5.40 g active carbon AX-21 and 62.0 g Celite 545*
to produce an 8 percent (w/w) mixture. Activate the mixture at
130CC for 6 hours and store 1t 1n a desiccator.
7.5.3.2 Cut off both ends of a 10 ml disposable serological
pi pet to give a 10 cm long column. Fire polish both ends and flare,
if desired. Insert a glass wool plug at one end, then pack the
column with enough Celite 545* to fom a 1 cm plug, add 1 g of the
AX-21/Cellte 545* mixture, top with additional Celite 545» (enough
for a 1 en plug), and cap the packing with another glass wool plug.
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NOTE: Each new batch of AX-21/Celite 545* must be checked as follows: Add 50 ML
of the continuing calibration solution to 950 Ml hexane. Take this
solution through the carbon column cleanup step, concentrate to 50 ML and
analyze. If the recovery of any of the analytes 1s <80 percent, discard
this batch of AX-2l/Cel1te 545*.
7.5.3.3 Rinse the AX-21/Celite 545* column with 5 ml of
toluene, followed by 2 ml of 75:20:5 (v/v) methylene
chlorlde/methanol/toluene, 1 ml of 1:1 (v/v) cyclohexane/methylene
chloride, and 5 mL hexane. The flow rate should be less than
0.5 ml/min. Discard the rinses. While the column 1s still wet with
hexane, add the sample concentrate (Section 7.5.2.6) to the top of
the column. Rinse the concentrator tube (which contained the sample
concentrate) twice with 1 ml hexane, and add the rinses to the top
of the column.
7.5.3.4 Elute the column sequentially with two 2 ml portions
of hexane, 2 ml cyclohexane/methylene chloride (50:50, v/v), and 2 ml
methylene chlorlde/methanol/toluene (75:20:5, v/v). Combine these
eluates; this combined fraction may be used as a check on column
efficiency.
7.5.3.5 Turn the column upside down and elute the PCOO/PCOF
fraction with 20 ml toluene. .Verify that no carbon fines are present
in the eluate. If carbon fines are present 1n the eluate, filter
the eluate through a glass fiber filter (0.45 MID) and rinse the"
filter with 2 mi toluene. Add the rinse to the eluate.
7.5.3.6 Concentrate the toluene fraction to about 1 ml on a
rotary evaporator by using a water bath at 50°C. Carefully transfer
the concentrate Into a 1 ML minlvlal and, again at elevated
temperature (50°C), reduce the volume to about 100 ni using a stream
of nitrogen and a sand bath. Rinse the rotary evaporator flask three
times with 300 ML of a solution of 1 percent toluene in methylene
chloride, and add the rinses to the concentrate. Add 10 ML of the
nonane recovery standard solution for soil, sediment, water, fish,
paper pulp and adipose tissue samples, or 50 ML of the recovery
standard solution for sludge, still bottom and fly ash samples.
Store the sample at room temperature 1n the dark.
7.6 Chroraatographic/Mass Spectrometrlc Conditions and Data Acquisition
Parameters
7.6.1 Gas Chromatograph
Column coating: DB-5
Fill thickness: 0.25 M«
Column dimension: 60 a x 0.32 mm
Injector temperature: 270°C
Splltless valve time: 45 s
Interface temperature: Function of the final temperature
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Temperature program:
Stage
1
2
3
In1t.
Temp.
200
Init.
Hold Time
(min)
2
Temp.
Ramp
5
5
5'
Final
Temp.
(8C)
220
235
330
Final
Hold
Time (min)
16
7
5
Total time: 60 min
7.6.2 Mass Spectrometer
7.6.2.1 The mass spectrometer must be operated in a selected
ion monitoring (SIM) mode with a total cycle time (Including the
voltage reset time) of one second or less (Section 7.6.3.1). At a
minimum, the Ions listed in Table 6 for each of the five SIM
descriptors must be monitored. Note that with the exception of the
last descriptor (OCDO/OCOF), all descriptors contain 10 ions. The
selection (Table 6) of the molecular Ions M and M+2 for 13C-HxCDF and
13C-HpCOF rather than M+2 and M+4 (for consistency) was made to
eliminate, even under high-resolution mass spectrometric conditions,
Interferences occurring 1n these two ion channels for samples
containing high levels of native HxCDOs and HpCOOs. It is important
to maintain the same set of Ions for both calibration and sample
extract analyses. The selection of the lock-mass ion 1s left to the
performing laboratory.
Note: At the option of the analyst, the tetra- and pentachlorinated dioxins and
furans can be combined Into a single descriptor.
7.6.2.2 The recommended mass spectrometer tuning conditions
are based on the groups of monitored Ions shown in Table 6. By using
a PFK molecular leak, tune the Instrument to meet the minimum
required resolving power of 10,000 (10 percent valley) at m/z
304.9824 (PFK) or any other reference signal close to m/z 303.9016
(from TCOF). By using peak matching conditions and the
aforementioned PFK reference peak, verify that the exact mass of m/z
380.9760 (PFK) Is within 5 ppm of the required value. Note that the
selection of the low- and high-mass Ions must be such that they
provide tht largest voltage jump performed in any of the five mass
descriptors (Table 6).
7.6.3 Data Acquisition
7.6.3.1 The total cycle time for data acquisition must be <
1 second. The total cycle time Includes the sum of all the dwell
times and voltage reset times.
7.6.3.2 Acquire SIN data for all the Ions listed 1n the five
descriptors of Table 6.
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7.7 Calibration
7.7.1 Initial Calibration - Initial calibration Is required before
any samples are analyzed for PCDDs and PCDFs. Initial calibration Is also
required 1f any routine calibration (Section 7.7.3) does not meet the
required criteria listed in Section 9.4.
7.7.1.1 All five high-resolution concentration calibration
solutions listed in Table 5 must be used for the Initial calibration.
7.7.1.2 Tune the Instrument with PFK as described in
Section 7.6.2.2.
7.7.1.3 Inject 2 ML of the GC column performance check
solution (Section 5.7) and acquire SIM mass spectral data as
described earlier in Section 8.1. The total cycle time must be < 1
second. The laboratory must not perfom any further analysis until
it 1s demonstrated and documented that the criterion listed in
Section 8.1.2 was met.
7.7.1.4 By using the same GC (Section 7.6.1) and MS
(Section 7.6.2) conditions that produced acceptable results with
the column performance check solution, analyze a 2 ML portion of
each of the five concentration calibration solutions once with the
following mass spectrometer operating parameters.
7.7.1.4.1 The ratio of Integrated 1on current for the
Ions appearing 1n Table 8 (homologous series quant nation Ions)
must be within the Indicated control limits (set for each
homologous series).
7.7.1.4.2 The ratio of Integrated 1on current for the
Ions belonging to the carbon-labeled Internal and recovery
standards must be within the control limits stipulated in
Table 8. ., . .
NOTE: Sections 7.7.1.4.1 and 7.7.1.4.2 require that 17 Ion ratios from Section
7.7.1.4.1 and 11 1on ratios from Section 7.7.1.4.2 be within the specified
control Units simultaneously In one run. It 1s the laboratory's
responsibility to take corrective action If the 1on abundance ratios are
outside the limits.
7.7.1.4.3 For each SICP and for each GC signal
corresponding to the elutlon of a target analyte and of Us
labeled standards, the signal-to-no1se ratio (S/N) must be
better than or equal to 2.5. Measurement of S/N 1s required
for any GC peak that has an apparent S/N of less than 5:1.
The result of the calculation must appear on the SICP above
the GC peak 1n question.
7.7.1.4.4 Referring to Table 9, calculate the 17
relative response factors (RRF) for unlabeled target analytes
[RRF(n); n - 1 to 17] relative to their appropriate Internal
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17
standards (Table 5) and the nine RRFs for the labeled nC
Internal standards [RRF(ra); m - 18 to 26)] relative to the two
recovery standards according to the following formulae:
RRF(n) -
A,x Q,
Q, x A,
RRF(m)
where:
()„
(}„
sum of the Integrated ion abundances of the
quantitatlon Ions (Tables 6 and 9) for unlabeled
PCDDs/PCDFs,
sum of the Integrated 1on abundances of the
quantitatlon Ions (Tables 6 and 9) for the labeled
Internal standards,
sum of the Integrated ion abundances of the
quantitatlon Ions (Tables 6 and 9) for the labeled
recovery standards, •
quantity of the Internal standard Injected (pg),
quantity of the recovery standard Injected (pg),
and
Q, - quantity of the unlabeled PCDD/PCDF analyte
Injected (pg).
The RRF(n) and RRF(m) are dimensionless quantities; the units
used to express Q», Q,, and Q, must be the same.
7.7.1.4.5 Calculate the ERT and their respective
percent relative standard deviations (XRSO) for the five
calibration solutions:
RRF(n) - 1/5 I RRFj(n)
J-i
where n represents a particular PCDO/PCDF (2,3,7,8-substltuted)
congener (n • 1 to 17; Table 9), and j 1s the Injection number
(or calibration solution number; j « 1 to 5).
7.7.1.4.6 The relative response factors to be used for
the determination of the concentration of total Isomers in a
homologous series (Table 9) are calculated as follows:
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7.7.1.4.6.1 For congeners that belong to a
homologous series containing only one Isomer (e.g.,
OCOD and OCDF) or only one 2,3,7,8-substltuted isomer
(Table 4; TCDD, PeCOD, HpCDD, and TCDFJ, the mean RRF
used will be the same as the mean RRF determined 1n
Section 7.7.1.4.5.
NOTE: The calibration solutions do not contain 13C,,-OCDF as an Internal standard.
This 1s because a minimum resolving power or 12,000 1s required to resolve
the [M+6]* 1on of %2-OCDF from the [M+2]* ion of OCDO (and [M+4]* from
13C12-OCDF with [M]* of OCDD). Therefore, the RRF for OCDF 1s calculated
relative to 13C12-OCOO.
7.7.I.4.6.2 For congeners that belong to a
homologous series containing more than one
2,3,7,8-substltuted Isomer (Table 4), the mean RRF used
for those homologous series will be the mean of the RRFs
calculated for all Individual 2,3,7,8-substituted
congeners using the equation below:
1 t
RRF(k) - - I RRFn
t n-l
where:
•7
k - 27 to 30 (Table 9), with 27 - PeCDF; 28 -
HxCDF; 29 • HxCDD; and 30 • HpCOF,
t • total number of 2,3,7,8-substltuted isomers
present 1n the calibration solutions (Table
5) for each homologous series (e.g., two for
PeCOF, four for HxCOF, three for HxCDD, two
for HpCDF).
NOTE: Presumably, the HRGC/HRMS response factors of different Isomers within
a homologous series are different. However, this analytical protocol
will make the assumption that the HRGC/HRMS responses of all Isomers in
a homologous series that do not have the 2,3,7,8-substitution pattern
are the same as the responses of one or more of the 2,3,7,8-substltuted
isomer(s) in that homologous series.
7.7.1.4.7 Relative response factors [RRF(m)] to be
used for the determination of the percent recoveries for the
nine Internal standards are calculated as follows:
V x 0.
RRF(i) -
0." x A,,
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1 5
RRF(m) - - z RRFj(m),
5 J-l
where:
• • 18 to 26 (congener type) and j - 1 to 5
(Injection number),
V - sum of the Integrated ion abundances of the
quantltatlon Ions (Tables 6 and 9) for a given
Internal standard (m • 18 to 26),
A,, • sum of the Integrated ion abundances of the
quantltatlon Ions (Tables 6 and 9) for the
appropriate recovery standard (see Table 5,
footnotes),
Qr,. Q»m -quantities of, respectively, the recovery
standard (rs) and a particular internal
standard (1s • m) Injected (pg),
RRF(m) • relative response factor of a particular
Internal standard (m) relative to an
appropriate recovery standard, as determined
from one Injection, and
R~R7(m) • calculated mean relative response factor of
a particular Internal standard (m) relative
to an appropriate recovery standard, as
determined from the five Initial calibration
Injections (j).
7.7.2 Criteria for Acceptable Calibration - The criteria listed
below for acceptable calibration must b« met before the analysis is
performed.
7.7.2.1 The percent relative standard deviations for the mean
response factors [RRF(n) and RRF(m)] from the 17 unlabeled standards
must not exceed ± 20 percent, and these for the nine labeled
reference compounds must not exceed ± 30 percent.
7.7.2.2 The S/N for the GC signals present in every SICP
(Including the ones for the labeled standards) must be > 10.
7.7.2.3 The 1sotop1c ratios (Table 8) must be within the
specified control limits.
NOTE- If the criterion for acceptable calibration listed In Section 7.7.2.1 is
met, the analyte specific RRF can then be considered Independent of the
analyte quantity for the calibration concentration range. The mean RRFs
will be used for all calculations until the routine calibration criteria
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(Section 7.7.4) are no longer met. At such time, new mean RRFs will be
calculated from a new set of Injections of the calibration solutions.
7.7.3 Routine Calibration (Continuing Calibration Check) - Routine
calibrations must be performed at the beginning of a 12 hour period after
successful mass resolution and GC resolution performance checks. A routine
calibration 1s also required at the end of a 12 hour shift.
7.7.3.1 Inject 2 nl of the concentration calibration solution
HRCC-3 standard (Table 5). By using the same HRGC/HRMS conditions
as used in Sections 7.6.1 and 7.6.2, determine and document an
acceptable calibration as provided In Section 7.7.4.
7.7.4 Criteria for Acceptable Routine Calibration - The following
criteria must be met before further analysis 1s performed.
7.7.4.1 The measured RRFs [RRF(n) for the unlabeled standards]
obtained during the routine calibration runs must be within ± 20
percent of the mean values established during the Initial calibration
(Section 7.7.1.4.5).
7.7.4.2 The measured RRFs [RRF(m) for the labeled standards]
obtained during the routine calibration runs must be within
+ 30 percent of the mean values established during the Initial
calibration (Section 7.7.1.4.7).
C
7.7.4.3 The Ion-abundance ratios (Table 8) must be within the
allowed control limits.
7.7.4.4 If either one of the criteria 1n Sections 7.7.4.1
and 7.7.4.2 1s not satisfied, repeat one more time. If these
criteria are still not satisfied, the entire routine calibration
process (Section 7.7.1) must be reviewed. It 1s realized that it
may not always be possible to achieve all RRF criteria. For example,
1t has occurred that the RRF criteria for 13C12-HpCDO and 13C1Z-OCDO
were not met, however, the RRF values for the corresponding unlabeled
compounds were routinely within the criteria established in the
method. In these cases, 24 of the 26 RRF parameters have met the
QC criteria, and the data quality for the unlabeled HpCOO and OCOO
values were not compromised as a result of the calibration event.
In these situations, the analyst must assess the effect on overall
data quality as required for the data quality objectives and decide
on appropriate action. Corrective action would be in order, for
example, 1f the compounds for which the RRF criteria were not met
Included both the unlabeled and the corresponding Internal standard
coapounds. If the Ion-abundance ratio criterion (Section 7.7.4.3)
1s not satisfied, refer to the note in Section 7.7.1.4.2 for
resolution.
NOTE: An Initial calibration must be carried out whenever the HRCC-3, the sample
fortification or the recovery standard solution 1s replaced by a new
solution from a different lot.
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7.8 Analysis
7.8.1 Remove the sample extract (from Section 7.5.3.6) or blank from
storage. With a stream of dry, purified nitrogen, reduce the extract
volume to 10 /*(. to 50 /il.
Note: A final volume of 20 ^L or more should be used whenever possible. A 10 ML
final volume Is difficult to handle, and Injection of 2 ML out of 10 ML
leaves little sample for confirmations and repeat Injections, and for
archiving.
7.8.2 Inject a 2 ML aliquot of the extract into the GC, operated
under the conditions that have been established to produce acceptable
results with the performance check solution (Sections 7.6.1 and 7.6.2).
7.8.3 Acquire SIM data according to Sections 7.6.2 and 7.6.3. Use
the same acquisition and mass spectrometer operating conditions previously
used to determine the relative response factors (Sections 7.7.1.4.4 through
7.7.1.4.7). Ions characteristic for polychlorlnated diphenyl ethers are
included in the descriptors listed in Table 6.
NOTE: The acquisition period must at least encompass the PCOO/PCDF overall
retention time window previously determined (Section 8.1). Selected ion
current profiles (SICP) for the lock-mass Ions (one per mass descriptor)
must also be recorded and included in the data package. These SICPs must.
be true representations of the evolution of the lock-mass ions amplitudes
during the HRGC/HRMS run (see Section 8.2.2 for the proper level of
reference compound to be metered into the ion chamber.) The analyst may
be required to monitor a PFK ion, not as a lock mass, but as a regular ion,
in order to meet this requirement. It 1s recommended to examine the lock-
mass ion SICP for obvious basic sensitivity and stability changes of the
instrument during the 6C/HS run that could affect the measurements [Tondeur
et al., 1984, 1987]. Report any discrepancies in the case narrative.
7.8.4 Identification Criteria - For a gas chromatographic peak to
be identified as a PCOD or PCOF, 1t must meet all of the following
criteria:
7.8.4.1 Retention Times
7.8.4.1.1 For 2,3,7,8-substituted congeners, which
have an Isotopically labeled Internal or recovery standard
present In the sample extract (this represents a total of 10
congeners Including OCDO; Tables 2 and 3), the retention time
(RRT; at maximum peak height) of the sample components (I.e.,
the two Ions used for quantUatlon purposes listed in Table
6) must be within -1 to +3 seconds of the Isotopically labelled
standard.
7.8.4.1.2 For 2,3,7,8-substituted compounds that do
not have an Isotopically labeled Internal standard present in
the sample extract (this represents a total of six congeners;
Table 3), 'the retention time must fall within 0.005 retention
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time units of the relative retention times measured in the
routine calibration. Identification of OCDF is based on its
retention time relative to 13C12-OCDD as determined from the
dally routine calibration results.
7.8.4.1.3 For non-2,3,7,8-substituted compounds (tetra
through octa; totaling 119 congeners), the retention time must
be within the corresponding homologous retention time windows
established by analyzing the column performance check solution
(Section 8.1.3).
7.8.4.1.4 The ion current responses for both ions used
for quantitative purposes (e.g., for TCOOs: m/z 319.8965 and
321.8936) must reach maximum simultaneously (± 2 seconds).
7.8.4.1.5 The 1on current responses for both Ions used
for the labeled standards (e.g., for %,-TCDD: m/z 331.9368
and m/z 333.9339) must reach maximum simultaneously (± 2
seconds).
NOTE: The analyst 1s required to verify the presence of 1,2,8,9-TCOO and
1,3,4,6,8-PeCDF (Section 8.1.3) 1n the SICPs of the dally performance
checks. Should either one compound be missing, the analyst 1s required
to take corrective action as 1t may Indicate a potential problem with the
ability to detect all the PCDDs/PCDFs.
7.8.4.2 Ion'Abundance Ratios
7.8.4.2.1 The Integrated ion current for the two Ions
used for quantitatlon purposes must have a ratio between the
lower and upper limits established for the homologous series
to which the peak 1s assigned. See Sections 7.7.1.4.1 and
7.7.1.4.2 and Table 8 for details.
7.8.4.3 Signal-to-No1se Ratio
7.8.4.3.1 All 1on current Intensities must be > 2.5
times noise level for positive Identification of a PCOO/PCOF
compound or a group of coelutlng Isomers. Figure 6 describes
the procedure to bt followed for the determination of the S/N.
7.8.4.4 Polychlorinated Dlphenyl Ether Interferences
7.8.4.4.1 In addition to the above criteria, the
Identification of a GC peak as a PCDF can only be made if no
signal having a S/N > 2.5 1s detected, at the same retention
time (± 2 seconds), In the corresponding polychlorinated
diphenyl ether (PCDPE, Table 6) channel.
7.9 Calculations
7.9.1 For gas chromatographlc peaks that have met the criteria
outlined 1n Sections 7.8.4.1.1 through 7.8.4.3.1, calculate the concen-
tration of the PCOO or PCDF compounds using the formula:
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A. x Q*
Cx - —
A,, x W x RRF(n)
where:
C, - concentration of unlabeled PCDD/PCDF congeners (or group of
coeluting Isomers within an homologous series) 1n pg/g,
A, - sum of the Integrated 1on abundances of the quantltation Ions
(Table 6) for unlabeled PCDOs/PCOFs,
A,, • sum of the Integrated Ion abundances of the quantitatlon ions
(Table 6) for the labeled internal standards,
()„ - quantity, in pg, of the internal standard added to the sample
before extraction,
U • weight, in g, of the sample (solid or liquid), and
. RRF- calculated mean relative response factor for the analyte
[RRF(n) with n - 1 to 17; Section 7.7.1.4.5].
If the analyte is identified as one of the 2,3,7,8-substltuted PCDDs or
PCOFs, RRF(n) is the value calculated using the equation in
Section 7.7.1.4.5. However, if 1t is a non-2,3,7,8-subst1tuted congener,:
the RRT(k) value is the one calculated using the equation in
Section 7.7.1.4.6.2. [RRF(k) with k - 27 to 30].
7.9.2 Calculate the percent recovery of the nine internal standards
measured in the sample extract, using the formula:'
Internal standard percent recovery - - — — x 100
(}„ x A,, x RRT(m)
where:
A* - sum of the integrated Ion abundances of the quantitatlon
Ions (Table 6) for the labeled internal standard,
A_ • sun of the integrated ion abundances of the quantitatlon
Ions (Table 6) for the labeled recovery standard; the
selection of the recovery standard depends on the type of
congeners (see Table 5, footnotes),
Q^ « quantity, In pg, of the Internal standard added to the
sample before extraction,
Q • quantity, 1n pg, of the recovery standard added to the
" cleaned-up sample residue before HRGC/HRMS analysis, and
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RRF(m) - calculated mean relative response factor for the labeled
Internal standard relative to the appropriate (see Table
5, footnotes) recovery standard ._rh 1s represents the mean
obtained in Section 7.7.1.4.7 [RRF(m) with m - 18 to 26].
NOTE: For human adipose tissue, adjust the percent recoveries by adding
1 percent to the calculated value to compensate for the 1 percent of the
extract diverted for the lipld determination.
7.9.3 If the concentration 1n the final extract of any of the fifteen
2,3,7,8-substituted PCOO/PCDF compounds (Table 3) exceeds the upper method
calibration limits (MCL) listed 1n Table 1 (e.g., 200 pg//iL for TCOO in
soil), the linear range of response versus concentration may have been
exceeded, and a second analysis of the sample (using a one tenth aliquot)
should be undertaken. The volumes of the Internal and recovery standard
solutions should remain the same as described for the sample preparation
(Sections 11.1 to 11.9.3). For the other congeners (including OCOO),
however, report the measured concentration and Indicate that the value
exceeds the MCL.
7.9.4 The total concentration for each homologous series of PCDO
and PCOF is calculated by summing up the concentrations of all positively
identified isomers of each homologous series. Therefore, the total should
also include the 2,3,7,8-substituted congeners. The total number of GQ
signals Included in the homologous total concentration value must be
specified in the report.
7.9.5 Sample Specific Estimated Detection Limit - The sample specific
estimated detection limit (EDL) 1s the concentration of a given analyte
required to produce a signal with a peak height of at least 2.5 times the
background signal level. An EOL 1s calculated for each
2,3,7,8-substituted congener that 1s not Identified, regardless of whether
or not other non-2,3,7,8-subst1tuted Isomers are present. Two methods of
calculation can be used, as follows, depending on the type of response
produced during the analysis of a particular sample.
7.9.5.1 Samples giving a response for both quantltation Ions
(Tables 6 and 9) that 1s less than 2.5 times the background level.
7.9.5.1.1 Use the expression for EDL (specific
2,3,7,8-substituted PCDO/PCDF) below to calculate an EDL for
each absent 2,3,7,8-substituted PCDO/PCDF (I.e., S/N < 2.5).
The background level is determined by measuring the range of
the noise (peak to peak) for the two quantltation Ions (Table
6) of a particular 2,3,7,8-substituted Isomer within an
homologous series, In the region of the SICP trace
corresponding to the elutlon of the Internal standard (if the
congener possesses an Internal standard) or in the region of
the SICP where the congener 1s expected to elute by comparison
with the routine calibration data (for those congeners that
do not have a 13C-labeled standard), multiplying that noise
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height by 2.5, and relating the product to an estimated
concentration that would produce that peak height.
Use the formula:
2.5 x A. x Q,,
EDL (specific 2,3,7,8-subst. PCDD/PCOF)
where:
x U x R"RF(n)
EDL • estimated detection limit for homologous
2,3,7,8-substltuted PCOOs/PCDFs.
A,, A^ W, P.RF(n), and Q,. retain the same meanings as
defined in Section 7.9.1.
7.9.5.2 Samples characterized by a response above the
background level with a S/N of at least 2.5 for both quantitation
Ions (Tables 6 and 9).
7.9.5.2.1 When the response of a signal having the
same retention time as a 2,3,7,8-substltuted congener has a
S/N 1n excess of 2.5 and does not meet any of the other
qualitative Identification criteria listed 1n Section 7.8.4,
calculate the "Estimated Maximum Possible Concentration" (EMPC)
according to the expression shown 1n Section 7.9.1, except that
A" in Section 7.9.1 should represent the sum of the area under
the smaller peak and of the other peak area calculated using
the theoretical chlorine Isotope ratio.
7.9.6 The relative percent difference (RPO) 1s calculated as follows:
I S, - S, |
RPD • x 100
( S, + S, ) / 2
S1 and S, represent sample and duplicate sample results.
7.9.7 The 2,3,7,8-TCDD toxlclty equivalents (TE) of PCDDs and PCDFs
present 1n the sample are calculated, 1f requested by the data user,
according to the method recommended by the Chlorinated Dioxins Workgroup
(CDWG) of the EPA and the Center for Disease Control (CDC). This method
assigns a 2,3,7,8-TCDO toxlclty equivalency factor (TEF) to each of the
fifteen 2,3,7,8-substltuted PCDOs and PCDFs (Table 3) and to OCDD and
OCDF, as shown 1n Table 10. The 2,3,7,8-TCDD equivalent of the PCDDs and
PCDFs present 1n the sample 1s calculated by sunning the TEF times their
concentration for each of the compounds or groups of compounds listed in
Table 10. The exclusion of other homologous series such as mono-, dl-,
and tr1- chlorinated d1benzod1ox1ns and dlbenzofurans does not mean that
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they are non-toxic. However, their toxldty, as known at this time, is
much lower than the toxicity of the compounds listed in Table 10. The
above procedure for calculating the 2,3,7,8-TCOO toxicity equivalents is
not claimed by the COWG to be based on a thoroughly established scientific
foundation. The procedure, rather, represents a "consensus recommendation
on science policy". Since the procedure may be changed in the future,
reporting requirements for PCDD and PCOF data would still include the
reporting of the analyte concentrations of the PCDD/PCDF congener as
calculated 1n Sections 7.9.1 and 7.9.4.
7.9.7.1 Two GC Column TEF Determination
7.9.7.1.1 The concentration of 2,3,7,8-TCDO (see note
below), 1s calculated from the analysis of the sample extract
on the 60 m DB-5 fused silica capillary column. The
experimental conditions remain the same as the conditions
described previously In Section 7.8, and the calculations are
performed as outlined In Section 7.9. The chromatographic
separation between the 2,3,7,8-TCOO and Us close eluters
(1,2,3,7/1,2,3,8-TCOO and 1,2,3,9-TCDD) must be equal or less
than 25 percent valley.
7.9.7.1.2 The concentration of the 2,3,7,8-TCDF is
obtained from the analysis of the sample extract on the 30 m
06-225 fused silica capillary column. However, the GC/MS
conditions must be altered so that: (1) only the first three.
descriptors (I.e., tetra-, penta-, and hexachlorinated
congeners) of Table 6 are used; and (2) the switching time
between descriptor 2 (pentachlorinated congeners) and
descriptor 3 (hexachlorinated congeners) takes place following
the elution of 13C12-l,2,3,7,8-PeCDD. The concentration
calculations are performed as outlined in Section 7.9. The
chromatographic separation between the 2,3,7,8-TCOF and its
close eluters (2,3,4,7-TCOF and 1,2,3,9-TCOF) must be equal
or less than 25 percent valley.
NOTE: The confirmation and quantltatlon of 2,3,7,8-TCOO (Section 7.9.7.1.1) may
be accomplished on the SP-2330 GC column Instead of the DB-5 column,
provided the criteria listed in Section 8.1.2 are met and the requirements
described in Section 17.2.2 are followed.
7.9.7.1.3 For a gas chromatographic peak to be
Identified as a 2,3,7,8-substituted PCDO/PCOF congener, it
must neet the Ion abundance and signal-to-no1se ratio criteria
listed In Sections 7.8.4.2 and 7.8.4.3, respectively. In
addition, the retention time Identification criterion described
In Section 7.8.4.1.1 applies here for congeners for which a
carbon-labeled analogue is available in the sample extract.
However, the relative retention time (RRT) of the
2,3,7,8-substituted congeners for which no carbon-labeled
analogues are available nust fall within 0.006 units of the
carbon-labeled standard RRT. Experimentally, this 1s
accomplished by using the attributions described in Table 11
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height by 2.5, and relating the product to an estimated
concentration that would produce that peak height.
Use the formula:
2.5 x A, x Q,.
EDL (specific 2,3,7,8-subst. PCOO/PCDF)
where:
x U x SRF(n)
EDL « estimated detection limit for homologous
2,3,7,8-substltuted PCDDs/PCOFs.
A,, A,,, W, RRF(n), and (}„ retain the same meanings as
defined in Section 7.9.1.
7.9.5.2 Samples characterized by a response above the
background level with a S/N of at least 2.5 for both quantltation
Ions (Tables 6 and 9).
7.9.5.2.1 When the response of a signal having the
same retention time as a 2,3,7,8-substltuted congener has a
S/N In excess of 2.5 and does not meet any of the other
qualitative Identification criteria listed 1n Section 7.8.4,
calculate the "Estimated Maximum Possible Concentration" (EMPC)
according to the expression shown in Section 7.9.1, except that
A" 1n Section 7.9.1 should represent the sum of the area under
the smaller peak and of the other peak area calculated using
the theoretical chlorine Isotope ratio.
7.9.6 The relative percent difference (RPD) 1s calculated as follows:
I S, - S, |
( S, + S, ) / 2
x 100
S, and S, represent sample and duplicate sample results.
7.9.7 The 2,3,7,8-TCDD toxldty equivalents (TE) of PCDDs and PCDFs
present in the sample are calculated, if requested by the data user,
according to the method recommended by the Chlorinated Dioxins Workgroup
(CDWS) of the EPA and the Center for Disease Control (CDC). This method
assigns a 2,3,7,8-TCDO toxldty equivalency factor (TEF) to each of the
fifteen 2,3,7,8-substltuted PCDOs and PCDFs (Table 3) and to OCDD and
OCDF, as shown In Table 10. The 2,3,7,8-TCDO equivalent of the PCDDs and
PCOFs present In the sample 1s calculated by summing the TEF times their
concentration for each of the compounds or groups of compounds listed in
Table 10. The exclusion of other homologous series such as mono-, di-,
and tri- chlorinated d1benzod1ox1ns and dlbenzofurans does not mean that
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they are non-toxic. However, their toxlcity, as known at this time, is
much lower than the toxidty of the compounds listed in Table 10. The
above procedure for calculating the 2,3,7,8-TCDD toxicity equivalents is
not claimed by the COWG to be based on a thoroughly established scientific
foundation. The procedure, rather, represents a "consensus recommendation
on science policy". Since the procedure may be changed in the future,
reporting requirements for PCOO and PCOF data would still Include the
reporting of the analyte concentrations of the PCDD/PCDF congener as
calculated 1n Sections 7.9.1 and 7.9.4.
7.9.7.1 Two GC Column TEF Determination
7.9.7.1.1 The concentration of 2,3,7,8-TCOO (see note
below), 1s calculated from the analysis of the sample extract
on the 60 m DB-5 fused silica capillary column. The
experimental conditions remain the same as the conditions
described previously in Section 7.8, and the calculations are
performed as outlined in Section 7.9. The chromatographic
separation between the 2,3,7,8-TCOO and Us close eluters
(1,2,3,7/1,2,3,8-TCDD and 1,2,3,9-TCDO) must be equal or less
than 25 percent valley.
7.9.7.1.2 The concentration of the 2,3,7,8-TCDF is
obtained from the analysis of the sample extract on the 30 m
OB-225 fused silica capillary column. However, the GC/HS
conditions must be altered so that: (1) only the first thre»
descriptors (I.e., tetra-, penta-, and hexachlorinated
congeners) of Table 6 are used; and (2) the switching time
between descriptor 2 (pentachlorlnated congeners) and
descriptor 3 (hexachlorinated congeners) takes place following
the elutlon of 13C12-l,2,3,7,8-PeCDD. The concentration
calculations are performed as outlined 1n Section 7.9. The
chromatographic separation between the 2,3,7,8-TCDF and its
close eluters (2,3,4,7-TCDF and 1,2,3,9-TCDF) must be equal
or less than 25 percent valley.
NOTE: The confirmation and quantltatlon of 2,3,7,8-TCDD (Section 7.9.7.1.1) may
be accomplished on the SP-2330 GC column Instead of the 06-5 column,
provided the criteria listed in Section 8.1.2 are met and the requirements
described 1n Section 17.2.2 are followed.
7.9.7.1.3 For a gas chromatographic peak to be
Identified as a 2,3,7,8-substUuted PCOD/PCDF congener, it
must Met the ion abundance and signal-to-noise ratio criteria
listed In Sections 7.8.4.2 and 7.8.4.3, respectively. In
addition, the retention time Identification criterion described
1n Section 7.8.4.1.1 applies here for congeners for which a
carbon-labeled analogue 1s available in the sample extract.
However, the relative retention time (RRT) of the
2,3,7,8-substUuted congeners for which no carbon-labeled
analogues are available must fall within 0.006 units of the
carbon-labeled standard RRT. Experimentally, this 1s
accomplished by using the attributions described in Table 11
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and the results from the routine calibration run on the SP-2330
column.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control (QC) procedures.
Quality control to validate sample extraction 1s covered 1n Method 3500. If
extract cleanup was performed, follow the QC 1n Method 3600 and 1n the specific
cleanup method.
8.2 System Performance Criteria - System performance criteria are
presented below. The laboratory may use the recommended GC column described in
Section 4.2. It must be documented that all applicable system performance
criteria (specified 1n Sections 8.2.1 and 8.2.2) were met before analysis of any
sample is performed. Section 7.6 provides recommended GC conditions that can
be used to satisfy the required criteria. Figure 3 provides a typical 12 hour
analysis sequence, whereby the response factors and mass spectrometer resolving
power checks must be performed at the beginning and the end of each 12 hour
period of operation. A GC column performance check 1s only required at the
beginning of each 12 hour period during which samples are analyzed. An HRGC/HRMS
method blank run 1s required between a calibration run and the first sample run.
The same method blank extract may thus be analyzed more than once if the number
of samples within a batch requires more than 12 hours of analyses.
8.2.1 GC Column Performance ~.
8.2.1.1 Inject 2 ML (Section 4.1:1} of the column performance
check solution (Section 5.7) and acquire selected ion monitoring
(SIM) data as described in Section 7.6.2 within a total cycle time
of < 1 second (Section 7.6.3.1).
8.2.1.2 The chromatographic separation between 2,3,7,8-TCDD
and the peaks representing any other unlabeled TCOO isomers must be
resolved with a valley of < 25 percent (Figure 4), where:
Valley percent • (x/y) (100)
x • measured as in Figure 4 from the 2,3,7,8-closest TCOO
elutlng isomer, and
y - the peak height of 2,3,7,8-TCDO.
It 1s the responsibility of the laboratory to verify the conditions
suitable for the appropriate resolution of 2,3,7,8-TCDD from all
other TCOO Isomers. The GC column performance check solution also
contains the known first and last PCDO/PCOF eluters under the
conditions specified 1n this protocol. Their retention times are
used to determine the eight homologue retention time windows that
are used for qualitative (Section 7.8.4.1) and quantitative purposes.
All peaks (that Includes 13C12-2,3,7,8-TCDD) should be labeled and
Identified on the chromatograms. Furthermore, all first eluters of
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a homologous series should be labeled with the letter F, and all last
eluters of a homologous series should be labeled with the letter L
(Figure 4 shows an example of peak labeling for TCOO isomers). Any
individual selected ion current profile (SICP) (for the tetras, this
would be the SICP for m/z 322 and m/z 304) or the reconstructed
homologue ion current (for the tetras, this would correspond to m/z
320 + m/z 322 + m/z 304 + m/z 306) constitutes an acceptable form
of data presentation. An SICP for the labeled compounds (e.g., m/z
334 for labeled TCDO) is also required.
8.2.1.3 The retention times for the switching of SIM ions
characteristic of one homologous series to the next higher homologous
series must be indicated in the SICP. Accurate switching at the
appropriate times is absolutely necessary for accurate monitoring
of these compounds. Allowable tolerance on the daily verification
with the GC performance check solution should be better than 10
seconds for the absolute retention times of all the components of
the mixture. Particular caution should be exercised for the
switching time between the last tetrachlorinated congener (i.e.,
1,2,8,9-TCOO) and the first pentachlorinated congener (I.e.,
1,3,4,6,8-PeCOF), as these two compounds elute within 15 seconds of
each other on the 60 m OB-5 column. A laboratory with a GC/HS system
that is not capable of detecting both congeners (1,2,8,9-TCOO and
1,3,4,6,8-PeCOF) within one analysis must take corrective action.
If the recommended column 1s not used, then the first and last
eluting isomer of each homologue must be determined experimentally
on the column which is used, and the appropriate isomers must then-'
be used for window definition and switching times.
8.2.2 Mass Spectrometer Performance
8.2.2.1 The mass spectrometer must be operated in the electron
ionization mode. A static resolving power of at least 10,000 (10
percent valley definition) must be demonstrated at appropriate masses
before any analysis 1s performed (Section 7.8). Static resolving
power checks must be performed at the beginning and at the end of
each 12 hour period of operation. However, it Is recommended that
a check of the static resolution be made and documented before and
after each analysis. Corrective action must be Implemented whenever
the resolving power does not meet the requirement.
8.2.2.2 Chromatography time for PCDOs and PCDFs exceeds the
long term mass stability of the mass spectrometer. Because the
Instrument Is operated In the high-resolution mode, mass drifts of
a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on
Instrument performance. Therefore, a mass drift correction is
mandatory. To that effect, 1t 1s recommended to select a lock-mass
1on from the reference compound (PFK Is recommended) used for tuning
the mass spectrometer. The selection of the lock-mass ion is
dependent on the masses of the Ions Monitored within each descriptor.
Table 6 offers some suggestions for the lock-Mass Ions. However,
an acceptable lock-lass Ion at any MSS between the lightest and
heaviest Ion in each descriptor can be used to monitor and correct
mass drifts. The level of the reference compound (PFK) metered into
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the 1on chamber during HRGC/HRMS analyses should be adjusted so that
the amplitude of the most intense selected lock-mass ion signal
(regardless of the descriptor number) does not exceed 10 percent of
the full scale deflection for a given set of detector parameters.
Under those conditions, sensitivity changes that might occur during
the analysis can be more effectively monitored.
NOTE: Excessive PFK (or any other reference substance) may cause noise problems
and contamination of the ion source resulting in an Increase in downtime
for source cleaning.
8.2.2.3 Documentation of the Instrument resolving power must
then be accomplished by recording the peak profile of the high-mass
reference signal (m/z 380.9760) obtained during the above peak
matching experiment by using the low-mass PFK ion at m/z 304.9824
as a reference. The minimum resolving power of 10,000 must be
demonstrated on the high-mass 1on while 1t 1s transmitted at a lower
accelerating voltage than the low-mass reference ion, which is
transmitted at full sensitivity. The format of the peak profile
representation (Figure 5) 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, which corresponds to the 10
percent valley definition) must appear on the hard copy and cannot
exceed 100 ppm at m/z 380.9760 (or 0.038 amu at that particular
mass).
8.3 Quality Control Samples
8.3.1 Performance Evaluation Samples - Included among the samples
in all batches may be samples (blind or double blind) containing known
amounts of unlabeled 2,3,7,8-substltuted PCDOs/PCOFs or other PCDD/PCDF
congeners.
8.3.2 Performance Check Solutions
1 8.3.2.1 At the beginning of each 12 hour period during which
samples are to be analyzed, an aliquot of the 1) GC column
performance check solution and 2) high-resolution concentration
calibration solution No. 3 (HRCC-3; see Table 5) shall be analyzed
to demonstrate adequate GC resolution and sensitivity, response
factor reproduclblHty, and mass range calibration, and to establish
the PCOD/PCDF retention time windows. A mass resolution check shall
also be performed to demonstrate adequate mass resolution using an
appropriate reference compound (PFK 1s recommended). If the required
criteria are not met, remedial action must be taken before
any samples are analyzed.
8.3.2.2 To validate positive sample data, the routine or
continuing calibration (HRCC-3; Table 5) and the mass resolution
check must be performed also at the end of each 12 hour period during
which samples are analyzed. Furthermore, an HRGC/HRMS method blank
run must be recorded following a calibration run and the first sample
run.
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8.3.2.2.1 If the laboratory operates only during one
period (shift) each day of 12 hours or less, the GC performance
check solution must be analyzed only once (at the beginning
of the period) to validate the data acquired during the period.
However, the mass resolution and continuing calibration checks
must be performed at the beginning as well as at the end of
the period.
8.3.2.2.2 If the laboratory operates during consecutive
12 hour periods (shifts), analysis of the GC performance check
solution must be performed at the beginning of each 12 hour
period. The mass resolution and continuing calibration checks
from the previous period can be used for the beginning of the
next period.
8.3.2.3 Results of at least one analysis of the GC column
performance check solution and of two mass resolution and continuing
calibration checks must be reported with the sample data collected
during a 12 hour period.
8.3.2.4 Deviations from criteria specified for the GC
performance check or for the mass resolution check invalidate all
positive sample data collected between analyses of the performance
check solution, and the extracts from those positive samples shall
be reanalyzed.
If the routine calibration run falls at the beginning of a 12 hour
shift, the Instructions In Section 7.7.4.4 must be followed. If
the continuing calibration check performed at the end of a 12 hour
period falls by no more than 25 percent RPD for the 17 unlabelled
compounds and 35 percent RPD for the 9 labelled reference compounds,
use the mean RRFs from the two dally routine calibration runs to
compute the analyte concentrations, Instead of the RRFs obtained from
the Initial calibration. A new Initial calibration (new RRFs) is
required Immediately (within two hours) following the analysis of
the samples, whenever the RPO fron the end-of-shift routine
calibration exceeds 25 percent or 35 percent, respectively. Failure
to perform a new Initial calibration Immediately following the
analysis of the samples will automatically require reanalysls of all
positive sample extracts analyzed before the failed end-of-shift
continuing calibration check.
8.3.3 The GC column performance check mixture, high-resolution
concentration calibration solutions, and the sample fortification solutions
may be obtained from the EMSL-CIN. However, If not available from the
EMSL-CIM, standards can be obtained from other sources, and solutions can
be prepared 1n the laboratory. Concentrations of all solutions containing
2,3,7,8-substUuted PCDOs/PCDFs, which are not obtained from the EMSL-CIN,
must be verified by comparison with the EPA standard solutions that are
available from the EMSL-CIN.
8.3.4 Field Blanks • Each batch of samples usually contains a field
blank sample of uncontaminated soil, sediment or water that 1s to be
fortified before analysis according to Section 8.3.4.1. In addition to
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this field blank, a batch of samples may include a rinsate, which is a
portion of the solvent (usually trichloroethylene) that was used to rinse
sampling equipment. The rinsate is analyzed to assure that the samples
were not contaminated by the sampling equipment.
8.3.4.1 Fortified Field Blank
8.3.4.1.1 Heigh a 10 g portion or use 1 L (for aqueous
samples) of the specified field blank sample and add 100 ML
of the solution containing the nine internal standards
(Table 2) diluted with 1.0 mL acetone (Section 7.1).
8.3.4.1.2 Extract by using the procedures beginning
1n Sections 7.4.5 or 7.4.6, as applicable, add 10 ML of the
recovery standard solution (Section 7.5.3.6) and analyze a
2 ML aliquot of the concentrated extract.
8.3.4.1.3 Calculate the concentration (Section 7.9.1)
of 2,3,7,8-substituted PCOOs/PCOFs and the percent recovery
of the Internal standards (Section 7.9.2).
8.3.4.1.4 Extract and analyze a ne'w simulated fortified
field blank whenever new lots of solvents or reagents are used
for sample extraction or for column chromatographlc procedures.
8.3.4.2 Rinsate Sample '"
8.3.4.2.1 The rinsate sample must be fortified like
a regular sample.
8.3.4.2.2 Take a 100 ml (+ 0.5 mL) portion of the
sampling equipment rinse solvent (rinsate sample), filter, if
necessary, and add 100 ML of the solution containing the nine
Internal standards (Table 2).
8.3.4.2.3 Using a KO apparatus, concentrate to
approximately 5 mL.
NOTE: As an option, a rotary evaporator may be used in place of the KO apparatus
for the concentration of the rinsate.
8.3.4.2.4 Transfer the 5 mL concentrate from the KO
concentrator tube 1n 1 mL portions to a 1 mL minivial, reducing
the volume 1n the minivial as necessary with a gentle stream
of dry nitrogen.
8.3.4.2.5 Rinse the KO concentrator tube with two
0.5 «L portions of hexane and transfer the rinses to the 1 mL
•inivial. Blow down with dry nitrogen as necessary.
8.3.4.2.6 Just before analysis, add 10 ML recovery
standard solution (Table 2) and reduce the volume to Us final
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volume, as necessary (Section 7.8.1). No column chromatography
1s required.
8.3.4.2.7 Analyze an aliquot following the same
procedures used to analyze samples.
8.3.4.2.8 Report percent recovery of the Internal
standard and the presence of any PCDO/PCDF compounds In ng/l
of rlnsate solvent.
8.3.5 Duplicate Analyses
8.3.5.1 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, or an appropriate amount of the
type of matrix under consideration.
8.3.5.1.1 The results of the laboratory duplicates
(percent recovery and concentrations of 2,3,7,8-substHuted
PCDO/PCDF compounds) should agree within 25 percent relative
difference (difference expressed as percentage of the mean).
Report all results.
8.3.5.1.2 Recommended actions to help locate problems:
•
8.3.5.1.2.1 Verify satisfactory Instrument
performance (Sections 8.2 and 8.3).
8.3.5.1.2.2 If possible, verify that no error was
made while weighing the sample portions.
8.3.5.1.2.3 Review the analytical procedures with
the performing laboratory personnel.
8.3.6 Matrix Spike and Matrix Spike Duplicate
8.3.6.1 Locate the sample for the MS and MSD analyses (the
sample may be labeled "double volume").
8.3.6.2 Add an appropriate volume of the matrix spike
fortification solution (Section 5.10) and of the sample fortification
solution (Section 5.8), adjusting the fortification level as
specified 1n Table 1 under IS Spiking Levels.
8.3.6.3 Analyze the MS ind MSD samples as described 1n
Section 7.
8.3.6.4 The results obtained from the MS and MSD samples
(concentrations of 2,3,7,8-substltuted PCDDs/PCDFs) should agree
within 20 percent relative difference.
8.4 Percent Recovery of the Internal Standards - For each sample, method
blank and rlnsate, calculate the percent recovery (Section 7.9.2). The percent
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recovery should be between 40 percent and 135 percent for all 2,3,7,8-substituted
Internal standards.
NOTE: A low or high percent recovery for a blank does not require discarding
the analytical data but 1t may indicate a potential problem with future
analytical data.
8.5 Identification Criteria
8.5.1 If either one of the Identification criteria appearing in
Sections 7.8.4.1.1 through 7.8.4.1.4 is not met for an homologous series,
it is reported that the sample does not contain unlabeled
2,3,7,8-substituted PCDD/PCOF isomers for that homologous series at the
calculated detection limit (Section 7.9.5)
8.5.2 If the first initial Identification criteria (Sections
7.8.4.1.1 through 7.8.4.1.4) are met, but the criteria appearing in
Sections 7.8.4.1.5 and 7.8.4.2.1 are not net, that sample 1s presumed to
contain Interfering contaminants. This Must be noted on the analytical
report form, and the sample should be rerun or the extract reanalyzed.
8.6 Unused portions of samples and sample extracts must be preserved
for six months after sample receipt to allow further analyses.
8.7 Reuse of glassware is to be minimized to avoid the risk of
contamination. •
9.0 METHOD PERFORMANCE
9.1 Data are currently not available.
10.0 REFERENCES
1. "Control of Interferences in the Analysis of Human Adipose Tissue for
2,3,7,8-Tetrachlorod1benzo-p-d1ox1n". D. 6. Patterson, J.S. Holler, D.F.
Grote, L.R. Alexander, C.R. Lapeza, R.C. O'Connor and J.A. Uddle. Environ.
Toxlcol. Chen. 5, 355-360 (1986).
2. "Method 8290: Analytical Procedures and Quality Assurance for Multimedia
Analysis of Polychlor1 ruled D1benzo-p-D1ox1ns and Dlbenzofurans by High-
Resolution Gas Chro«atography/H1gh-Resolut1on Mass Spectrometry". Y.
Tondeur and W.F. Beckert. U.S. Environmental Protection Agency,
Environmental Monitoring Systems Laboratory, Las Vegas, NV.
3 "Cardnogtns - 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,
August 1977.
4 "OSHA Safety and Health Standards, General Industry", (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (revised January
1976).
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5. "Safety 1n Academic Chemistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety (3rd Edition, 1979.)
6. "Hybrid HRGC/MS/MS Method for the Characterization of Tetrachlorinated
Dibenzo-p-d1oxins 1n Environmental Samples." Y. Tondeur, W.J. Niederhut,
S.R. Mlssler, and J.E. Campana, Mass Spectrom. 14, 449-456 (1987).
11.0 SAFETY
11.1 The following safety practices are excerpts from EPA Method 613,
Section 4 (July 1982 version) and amended for use in conjunction with this
method. The 2,3,7,8-TCDD Isomer has been found to be acnegenic, carcinogenic,
and teratogenic in laboratory animal studies. Other PCOOs and PCOFs containing
chlorine atoms 1n positions 2,3,7,8 are known to have tox1c1t1es comparable to
that of 2,3,7,8-TCDD. The analyst should note that finely divided dry soils
contaminated with PCDDs and PCDFs are particularly hazardous because of the
potential for Inhalation and ingestion. It 1s recommended that such samples be
processed in a confined environment, such as a hood or a glove box. Laboratory
personnel handling these types of samples should wear masks fitted with charcoal
filters to prevent Inhalation of dust.
11.2 The toxidty or carcinogenicity of each reagent used in this method
is not precisely defined; however, each chemical compound should be treated as
a potential health hazard. From this viewpoint, exposure to these chemicals must
be kept to a minimum. The laboratory 1s responsible for maintaining a current
awareness file of OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material safety data sheets should
be made available to all personnel involved in the chemical analysis of samples
suspected to contain PCODs and/or PCOFs. Additional references to laboratory
safety are given in references 3, 4 and 5.
11.3 Each laboratory must develop a strict safety program for the handling
of PCODs and PCOFs. The laboratory practices listed below are recommended.
11.3.1 Contamination of the laboratory will be minimized by
conducting most of the manipulations In a hood.
11.3.2 The effluents of sample splitters for the gas
chromatograph and roughing pumps on the HRGC/HRMS system should pass
through either a column of activated charcoal or be bubbled through a trap
containing oil or high boiling alcohols.
11.3.3 Liquid waste should be dissolved 1n methanol or ethanol
and Irradiated with ultraviolet light at a wavelength less than 290 nm for
several days (use F 40 BL lamps, or equivalent). Using this analytical
method, analyze the Irradiated liquid wastes and dispose of the solutions
when 2,3,7,8-TCDO and -TCOF congeners can no longer be detected.
11.4 The following precautions were Issued by Dow Chemical U.S.A. (revised
11/78) for safe handling of 2,3,7,8-TCDO In the laboratory and amended for use
in conjunction with this method.
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11.4.1 The following statements on safe hand! ing are as complete
as possible on the basis of available toxicological information. The
precautions for safe handling and use are necessarily general in nature
since detailed, specific recommendations can be made only for the
particular exposure and circumstances of each individual use. Assistance
in evaluating the health hazards of particular plant conditions may be
obtained from certain consulting laboratories and from State Departments
of Health or of Labor, many of which have an industrial health service.
The 2,3,7,8-TCDD Isomer is extremely toxic to certain kinds of laboratory
animals. However, 1t has been handled for years without injury in
analytical and biological laboratories. Many techniques used in handling
radioactive and Infectious materials are applicable to 2,3,7,8-TCDD.
11.4.1.1 Protective Equipment: Throw away plastic gloves,
apron or lab coat, safety glasses and laboratory hood adequate for
radioactive work. However, PVC gloves should not be used.
11.4.1.2 Training: Workers must be trained in the proper
method of removing contaminated gloves and clothing without
contacting the exterior surfaces.
11.4.1.3 Personal Hygiene: Thorough washing of hands and
forearms after each manipulation and before breaks (coffee, lunch,
and shift).
11.4.1.4 Confinement: Isolated work area, posted with signs,,
segregated glassware and tools, plastic backed absorbent paper on
benchtops.
11.4.1.5 Waste: Good technique Includes minimizing
contaminated waste. Plastic bag liners should be used in waste cans.
11.4.1.6 Disposal of Hazardous Wastes: Refer to the November
7, 1986 Issue of the Federal Register on Land Ban Rulings for details
concerning the handling of dioxin containing wastes.
11.4.1.7 Decontamination: Personnel - apply a mild soap with
plenty of scrubbing action. Glassware, tools and surfaces -
Chlorothene NU Solvent (Trademark of the Dow Chemical Company) is
the least toxic solvent shown to be effective. Satisfactory cleaning
may be accomplished by rinsing with Chlorothene, then washing with
a detergent and water. Dish water may be disposed to the sewer after
percolation through a charcoal b«d filter. It 1s prudent to minimize
solvent wastes because they require special disposal through
commercial services that are expensive.
11.4.1.8 Laundry: Clothing known to be contaminated should
be disposed with the precautions described under "Disposal of
Hazardous Hastes". Laboratory coats or other clothing worn in
2,3,7,8-TCDO work area may be laundered. Clothing should be
collected 1n plastic bags. Persons who convey the bags and launder
the clothing should be advised of the hazard and trained in proper
handling. The clothing may be put Into a washer without contact if
the launderer knows the problem. The washer should be run through
one full cycle before being used again for other clothing.
8290 - 43 Revision 0
November 1990
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11.4.1.9 Wipe Tests: A useful method for determining
cleanliness of work surfaces and tools Is to wipe the surface with
a piece of filter paper, extract the filter paper and analyze the
extract.
NOTE: A procedure for the collection, handling, analysis, and reporting
requirements of wipe tests performed within the laboratory 1s described
in Attachment A. The results and decision making processes are based on
the presence of 2,3,7,8-substituted PCDOs/PCDFs.
11.4.1.10 Inhalation: Any procedure that may generate
airborne contamination must be carried out with good ventilation.
Gross losses to a ventilation system must not be allowed. Handling
of the dilute solutions normally used 1n analytical and animal work
presents no significant Inhalation hazards except in case of an
accident.
11.4.1.11 Accidents: Remove contaminated clothing
Immediately, taking precautions not to contaminate skin or other
articles. Wash exposed skin vigorously and repeatedly until medical
attention is obtained.
8290 - 44 Revision 0
November 1990
DRAFT
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Attachment A
PROCEDURES FOR THE COLLECTION, HANDLING, ANALYSIS, AND
REPORTING OF WIPE TESTS PERFORMED WITHIN THE LABORATORY
This procedure 1s designed for the periodic evaluation of potential contamination
by 2,3,7,8-substituted PCDO/PCDF congeners of the working areas inside the
laboratory.
A.I Perform the wipe tests on surface areas of two inches by one foot
with glass fiber paper saturated with distilled 1n glass acetone using a pair
of clean stainless steel forceps. Use one wiper for each of the designated
areas. Combine the wipers to one composite sample 1n an extraction jar containing
200 mL distilled in glass acetone. Place an equal number of unused wipers in
200 mL acetone and use this as a control. Add 100 pi of the sample
fortification solution to each jar containing used or unused wipers (Section
5.8).
A.2.1 Close the jar containing the wipers and the acetone and extract
for 20 minutes using a wrist action shaker. Transfer the extract into a
KD apparatus fitted with a concentration tube and a three ball Snyder
column. Add two Teflon™ or Carborundum™ boiling chips and concentrate
the extract to an apparent volume of 1.0 mL on a steam bath. Rinse the
Snyder column and the KD assembly with two 1 mL portions of hexane into
the concentrator tube, and concentrate Us contents to near dryness with
a gentle stream of nitrogen. Add 1.0 mL hexane to the concentrator tube"
and swirl the solvent on the walls.
A.2.2 Prepare a neutral alumina column as described in Section
7.5.2.2 and follow the steps outlined 1n Sections 7.5.2.3 through 7.5.2.5.
A.2.3 Add 10 ML of the recovery standard solution as described in
Section 7.5.3.6.
A.3 Concentrate the contents of the vial to a final volume of 10 ni
(either in a m1n1v1al or 1n a capillary tube). Inject 2 ni of each extract
(wipe and control) onto a capillary column and analyze for 2,3,7,8-substituted
PCDDs/PCDFs as specified 1n the analytical method 1n Section 7.8. Perform
calculations according to Section 7.9.
A.4 Report the presence of 2,3,7,8-substituted PCDDs and PCDFs as a
quantity (pg or ng) per wipe test experiment (WTE). Under the conditions out-
lined 1n this analytical protocol, a lower Halt of calibration of 10 pg/WTE is
expected for 2,3,7,8-TCDO. A positive response for the blank (control) is
defined as a signal 1n the TCDD retention time window at any of the masses
monitored which 1s equivalent to or above 3 W of 2,3,7,8-TCDO ptr WTE. For
other congeners, use the multiplication factors listed In Table 1, footnote (a)
(e q , for OCDO, the lower MCL 1s 10 x 5 • 50 pg/WTE and the positive response
for the blank would be 3 x 5 - 15 pg). Also, report the recoveries of the
Internal standards during the simplified cleanup procedure.
8290 - 45 Revision 0
November 1990
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A.5 At a minimum, wipe tests should be performed when there is evidence
of contamination 1n the method blanks.
A.6 An upper limit of 25 pg per TCOO isoroer and per wipe test experiment
1s allowed (use multlplication factors listed 1n footnote (a) from Table 1 for
other congeners). This value corresponds to 21 times the lower calibration limit
of the analytical method. Steps to correct the contamination must be taken
whenever these levels are exceeded. To that effect, first vacuum the working
places (hoods, benches, sink) using a vacuum cleaner equipped with a high
efficiency participate absorbent (HEPA) filter and then wash with a detergent.
A new set of wipes should be analyzed before anyone 1s allowed to work in the
dioxin area of the laboratory after corrective action has been taken.
8290 - 46 Revision 0
November 1990
DRAFT
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Figure 1.
3
Dibenzodioxin
8
6 « 4
Oibenzofuran
Central structures of d1btnzo-p-d1ox1n ind dltenzofuran.
8290 - 47
Rtv1s1on 0
Novtobtr 1990
-------
Figure 2
M/AM
5.600
B
5,600
8,550
400 ppm
Peak profile displays demonstrating tht tfftct of tht detector zero on the
measured resolving power. In this example, the true resolving power 1s 5,600.
A) The zero was set too high; no effect Is observed upon the Masurtnwnt
of the resolving power.
B) The zero was adjusted properly.
C) The zero was set too low; this results 1n overestimating the actual
rtsolving power because the peak-to-peak noise cannot be aeasurtd
accurately.
8290 - 48
Revision 0
Novenber 1990
DRAFT
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Figure 3.
Analytical Procedure
8:00 AM
Mass Resolution
Mass Accuracy
Thaw Sample Extract
I
Concentrate to 10 gL
1
9:00 AM
Initial or
Routine
Calibration
GC Column
Performance
11:00 AM
Samples
Method
Blank
8:00 PM
Mas*
Resolution
Routine
Calibration
Typical 12 hour analysis sequence of events.
8290 - 49
Revision 0
November 1990
DRAFT
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••ft'
e
n
§
'«
N
Stltcttd 1on currtnt proflit for «/z 322 (TCOOs) productd by MS analysis of the
GC ptrfornanct chtck solution on t 60 • 08-5 fustd silica capillary column under
the conditions listed In Section 7.6.
8290 - SO
Revision 0
Hoveofccr 1990
DRAFT
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figure 5.
Ref. mass 304 9824
Span. 200 ppm
Systim file namt
Data file name
Resolution
Group number
lonization modt
Switching
flef. masses
Peak top
YVES150
A 852567
10000
1
El*
VOLTAGE
304 9824
380.9260
M/AM—10.500
Channel B 380.9260 Lock mass
Span 200 ppm
Ptak prof11ts representing two PFK reference Ions it m/z 305 and 381. The
resolution of tht h1gh-Mss signal is 95 ppaj at 5 percent of the peak height;
this corresponds to a resolving power M/ H of 10,500 (10 percent valley
definition).
8290 • 51
w»n
Revision 0
November 1990
-------
Figure 6.
E,
100^
90-
80-
70-
60-
50-
40-
30-
20-
10-
•c >N
20:00
22:00
24:00
2t:00
21:00
30:00
Manual dtttralnation of S/N.
The ptak height (S) Is Masurtd between the Man noise (lines C and 0). These
mean signal values are obtained by tracing the line between the baseline average
noise extremes, El and E2, and between the apex average noise extremes, E3 and
E4, at the apex of the signal.
NOTE: It 1s Imperative that the Instrument Interface amplifier electronic zero
offset be set high enough so that negative going baseline noise 1s
recorded.
8290 - 52
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November 1990
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Table 1.
Types of Matrices, Sample Sizes and 2,3,7,8-TCDD-Based
Method Calibration Limits (Parts per Trillion)
Lower MCL("
Upper MCL(t)
Weight (g)
IS Spiking
Levels (ppt)
Final Extr.
Vol. (ML)W
Water
0.
2
1000
1
10-50
Soil
Sediment
Paper Pulp"
01 1.0
200
10
100
10-50
Fly
Ash
1.0
200
10
100
50
Fish
Tissue
1.0
200
20
100
10-50
Human
Adipose
e Tissue
1.0
200
10
100
10-50
Sludges,
Fuel Oil
5.0
1000
2
500
50
Still-
Bottom
10
2000
1
1000
50
(a) For other congeners multiply the values by 1 for TCDF/PeCDO/PeCDF, by 2.5
for HxCDD/HxCDF/HpCOD/HpCDF, and by 5 for OCDO/OCDF.
(b) Sample dewatered according to Section 6.5.
(c) One half of the extract from the 20 g sample is used for determination of
lipid content (Section 7.2.2).
(d) See Section 7.8.1, Note.
NOTE: Chemical reactor residues are treated as still bottoms if their
appearances so suggest.
8290 - 53
DRAFT
Revision 0
Novenfcer 1990
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Table 2.
Composition of the Sample Fortification
and Recovery Standard Solutions*
Analyte
Sample Fortification
Solution
Concentration
(pg/ML; Solvent:
Nonane)
Recovery Standard
Solution
Concentration
(pg/^L; Solvent:
Nonane)
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
X-1.2.3.4-TCDD
13C12-l,2,3,7,8-PeCDD
13C12-1.2,3,7,8-PeCDF
13C12-l,2,3,6,7,8-HxCDD
13C12-l,2,3,4,7,8-HxCDF
13C12-l,2,3,7,8,9-HxCDD
13C12-l.2,3,4,6,7,8-HpCDD
13C12-l.2,3,4,6,7,8-HpCDF
13C12-OCOO
10
10
10
10
25
25
25
25
50
50
50
(a) These solutions should be made freshly every day because of the possibility
of adsorptlve losses to glassware. If these solutions are to be kept for more
than one day, then the sample fortification solution concentrations should be
increased ten fold, and the recovery standard solution concentrations should be
doubled. Corresponding adjustments of the spiking volumes must then be made.
8290 - 54
DRAFT
Revision 0
November 1990
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Table 3.
The Fifteen 2,3,7,8-SubstUuted PCDD and PCDF Congeners
PCDO PCOF
2,3,7,8-TCDD(*) 2,3,7,8-TCDF(*)
l,2,3,7,8-PeCDD(*) l,2,3,7,8-PeCDF(*)
l,2,3,6,7,8-HxCDD(*) 2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDF
l,2,3,7,8,9-HxCDD(+) 1,2,3,7,8,9-HxCDF
l,2,3,4,6,7,8-HpCDD(*) l,2,3,4,7,8-HxCDF(*)
2,3,4,6,7,8-HxCDF
l,2,3,4,6,7,8-HpCDF(*)
1,2,3,4,7,8,9-HpCDF
(*) The 13C-labeled analogue 1s used as an Internal standard.
(+) The 13C-labeled analogue 1s used as a recovery standard.
8290 • 55 Revision 0
November 1990
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Table 4.
Isomers of Chlorinated Dioxins and Furans as a
Function of the Number of Chlorine Atoms
Number of
Chlorine
Atoms
1
2
3
4
5
6
7
8
Total
Number of
01ox1n
Isomers
2
10
14
22
14
10
2
1
75
Number of
2,3,7,8
Isomers
—
...
1
1
3
1
1
7
Number of
Furan
Isomers
4
16
28
38
28
16
4
1
135
Number of
2,3,7,8
Isomers
—
—
—
1
2
4
2
1
10
8290 - 56
Revision 0
November 1990
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Table 5.
High-Resolution Concentration Calibration Solutions
Compound
HRCC
Concentration foQ/ul. in Nonanel
1
Unlabeled Analytes
2,3,7,8-TCOO
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCOF
1,2,3,4,7,8-HxCOD
1,2,3,6,7,8-HxCOO
1,2,3,7,8,9-HxCDD
1,2,3,4,7,8-HxCOF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDD
OCOF
Internal Standards
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-l,2,3,7,8-PeCDO
13C12-l,2,3,7,8-PeCDF
13C,2-l,2,3,6,7,8-HxCDO
13C12-l,2,3,4,7,8-HxCDF
13C12-l,2,3,4,6,7,8-HpCOO
%2-l,2,3,4,6,7,8-HpCOF
"C^-OCDD
Recovery Standards
%,-1,2,3,4-TCOff*
13c,|-if 2,3,7,8,9-Hxcoo"
200
200
500
500
500
500
500
' 500
500
500
500
500
500
500
500
1,000
1,000
50
50
50
50
125
125
125
125
250
50
125
50
50
125
125
125
125
125
125
125
125
125
125
125
125
125
250
250
50
50
50
50
125
125
125
125
250
50
125
10
10
25
25
25
25
25
25
25
25
25
25
25
25
25
50
50
50
50
50
50
125
125
125
125
250
50
125
2.5
2.5
25
25
25
25
6.25
6.25
6.25
25
25
25
6.25
25
25
12.5
12.5
50
50
50
50
125
125
125
125
250
50
125
1
1
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5
5
50
50
50
50
125
125
125
125
250
50
125
w Used for recovery determinations of TCDO, TCDF, PeCOO and PeCOF internal
standards.
w Used for recovery determinations of HxCDO, HxCDF, HpCDO, HpCDF and OCDO
Internal standards.
8290 - 57
•an
Revision 0
November 1990
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Table 6.
Ions Monitored for HRGC/HRMS Analysis of PCDDs/PCDFs
Descriptor Accurate'" Ion
Mass 10
Elemental
Composition
Analyte
303.9016
305.8987
315.9419
317.9389
319.8965
321.8936
331.9368
333.9338
375.8364
[354.9792]
339.8597
341.8567
351.9000
353.8970
355.8546
357.8516
367.8949
369.8919
409.7974
[354.9792]
373.8208
375.8178
383.8639
385.8610
389.8156
391.8127
401.8559
403.8529
445.7555
[430.9728]
407.7818
409.7788
417.8250
419.8220
423.7767
425.7737
435.8169
437.8140
479.7165
[430.9728]
M
M+2
M
M+2
M
M+2
M
M+2
M+2
LOCK
M+2
M+4
M+2
M+4
M+2
M+4
M+2
M+4
M+2
LOCK
M+2
M+4
M
M+2
M+2
M+2
M+4
LOCK
M+2
M
M+2
M+2
M+2
M+4
c12H4McV7cio
"cA^ftiq,
C12H»C14 CIO
LOCK
TCDF
TCDF
TCDF (S)
TCDF (S)
TCDD
TCDD
TCDD (S)
TCDO (S)
HxCDPE
PFK
(S)
PeCDF
PeCDF
PeCDF (S)
PeCDF
PeCDD
PeCDO
PeCDO (S)
PeCDD (S)
HpCOPE
PFK
HxCDF
HxCDF
HxCDF (S)
HxCDF (S)
HxCDD
HxCDD
HxCDO (S)
HxCDD (S)
OCDPE
PFK
HpCDF
HpCDF
HpCDF (S)
HpCDF
HpCDO
HpCDO
HpCDO (S)
HpCDO (S)
NCOPE
PFK
8290 - 58
DRAFT
Revision 0
November 1990
-------
Descriptor
5
Accurate*'
Mass
441.7428
443.7399
457.7377
459.7348
469.7780
471.7750
513.6775
[442.9278]
Table 6.
Continued
Ion Elemental
ID Composition
M+2 C^CV'CIO
M+4 C-^CV'CljO
M+2 C^CV'CIO,
M+4 C12UC1637C1,02
M+2 13C1j39Cl737C10,
M+4 "C^CV'CljOj
M+4 C12*CVC120
LOCK C10F17
Analyte
OCDF
OCDF
OCDD
OCDO
OCDD (S)
OCDO (S)
DCDPE
PFK
(1) The following nuclldic masses were used:
H - 1.007825 0 - 15.994915
C -12.000000 "Cl - 34.968853
13 C -13.003355 37C1 • 36.965903
F -18.9984
S - internal/recovery standard
8290 - 59 Revision 0
November 1990
DRAFT
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Table 7.
PCDD and PCDF Congeners Present in the GC Performance
Evaluation Solution and Used for Defining the
Homologous GC Retention Time Windows on a
60 m DB-5 Column
No. of
Chlorine
Atoms
. 4W>
5
6
7
8
PCDD Positional
First
Eluter
1,3,6,8
1,2,4,6,8/
1,2,4,7,9
1,2,4,6,7,9/
1,2,4,6,8,9
1,2,3,4,6,7,9
Isomer
Last
Eluter
1,2,8,9
1,2,3,8,9
1,2,3,4,6,7
1,2,3,4,6,7,8
1,2,3,4,6,7,8,9
PCDF Positional
First
Eluter
1,3,6,8
1,3,4,6,8
1,2,3,4,6,8
1,2,3,4,6,7,8
][somer
Last
Eluter
1,2,8,9
1,2,3,8,9
1,2,3,4,8,9
1,2,3,4,7,8,9
1,2,3,4,6,7,8,9
(1) In addition to these two TCDD isomers, the 1,2,3,4-, 1,2,3,7-, 1,2,3,8-, 2,3,7,8-,
13C12-2,3,7,8-, and 1,2,3,9-TCOD Isomers must also be present as a check of column
resolution.
8290 - 60
DRAFT
Revision 0
November 1990
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Table 8.
Theoretical Ion Abundance Ratios and Their Control Limits
for PCDOs and PCDFs
Number of
Chlorine
Atoms
4
5
6
6<«
7 Used only for 13C-HxCDF (IS).
(B) Used only for 13C-HpCDF (IS).
8290 - 61
Revision 0
November 1990
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Table 9.
Relative Response Factor [RRF (number)] Attributions
Number Specific Congener Name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2,3,7
2,3,7
1,2,3
1,2,3
2,3,4
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
2,3,4
1,2,3
1,2,3
1,2,3
OCDD
OCDF
,8-TCOO (and total TCODs)
,8-TCDF (and total TCDFs)
,7,8-PeCDO (and total PeCOOs)
,7,8-PeCOF
,7,8-PeCDF
,4,7,8-HxCDO
,6,7,8-HxCOO
,7,8,9-HxCDO
,4,7,8-HxCOF
,6,7,8-HxCOF
,7,8,9-HxCDF
,6,7,8-HxCDF
,4,6,7,8-HpCDD (and total HpCODs)
,4,6,7,8-HpCDF
,4,7,8,9-HpCDF
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
%,-!
13C -1
**12 *
13C -1
^12 I
13C -1
,^12 •*
13C -1
.^12 J
13C -1
,1 12
,2,3,7,8-PeCDO
,2,3,7,8-PeCDF
,2,3,6,7,8-HxCOO
,2,3,4,7,8-HxCDF
,2,3,4,6,7,8-HpCDO
,2,3,4,6,7,8-HpCDF
13C12-OCOD
Total
Total
Total
Total
PeCOFs
HxCOFs
HxCOOs
HpCOFs
8290 • 62 Revision 0
November 1990
DRAFT
-------
Table 10.
2,3,7,8-TCDD Toxldty Equivalency Factors (TEFs) for the
Polychlorlnated D1benzod1ox1ns and Dlbenzofurans
Number
Compound(s)
TEF
1
2
3
4
5
6
7
2,3,7,8-TCOD
1,2,3,7,8-PeCOD
1,2,3,6,7,8-HxCDO
1,2,3,7,8,9-HxCDD
1,2,3,4,7,8-HxCDO
1,2,3,4,6,7,8-HpCOD
1,2,3,4,6,7,8,9-OCDO
1.00
0.50
0.10
0.10
0.10
0.01
0.001
8
9
10
11
12
13
14
15
16
17
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCOF
1,2,3,4,7,8-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
1,2,3,4,6,7,8,9-OCDF
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.001
8290 - 63
DWtft
Revision 0
November 1990
-------
Table 11.
Analyte Relative Retention Time Reference Attributions
Analyte Analyte RRT Reference'"
1,2,3,4,7,8-HxCDD 13C12-l,2,3,6,7,8-HxCDO
1,2,3,6,7,8-HxCDF 13C12-l.2,3,4,7,8-HxCDF
1,2,3,7,8,9-HxCDF 13C12-l,2,3,4,7,8-HxCOF
2,3,4,6,7,8-HxCOF 13C12-l,2,3,4,7,8-HxCDF
The retention time of 2,3,4,7,8-PeCDF on the DB-5 column 1s measured relative
to 13C12-l,2,3,7,8-PeCOF and the retention time of 1,2,3,4,7,8,9-HpCDF relative
to 13C12-l,2,3,4,6,7,8-HpCDF.
8290 - 64 Revision 0
November 1990
DRAFT
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Million 8290
POI YCIUORINAHD DIIHN/(H)IOXINS (PCDOs) AND POLYCMLORINATEO OIBENZOFURANS (PCDFs)
BY HIGH RlSOlUlION GAS CHROMATOGRAPHY/HIGH RESOLUTION MASS SPEC1ROMEIRY (HRGC/HRHS)
CSTARp
1
7.1 INTERNAL STANDARD AOOI1ION
1
i
7.1.1 Sompto slieof 1 lo 1000
groms. see section 7.4 i Table 1.
Determine wl. on tared llash
7.1.2 Spite samples w/100 ul
forlllkotlon mixture yielding
internal standard cones, of
fable I. except lor adipose tissue
7.1.M for sol. sediment. Ity
ash. water, and llsh Hssue, ml*
1 ml acetone with 100 ul
7.1.2.2 Do not dUulo lor olher
sample malrkes
i
| 7.2 SAMPLE EXTRACTION AND PURIFICATION|
±
±
17.2 Fish and Pop* Pulp| |7 3 Humon Adipose TiuuTj I 7.4 Cnvironmentol ond Wqstel
7.2.1 Mb SO or sodium
tulloU ond 20 gr sample;
ploco mil In Soxhlot; odd
200 mh I: 1 hexone/MeCI;
rellui 12 hours
l
1
7.3.1 Store samples al or
below -20 C. cart taken in
handling
7.2.2 Transfer eilrocl lo a
KO apparatus with a Snyder
column
I
7.2.3 Add lotion boWng
chip; concentrate lo 107
mis In wotor bath; cool lor
5 minutes
7.3.2 Extraction
.1 Wwgn oul sample
.2 Lei stand lo room T
.3 Add MeCI. lortllkaHon
soln.. homogoniie
.4 Separate MeCI layer.
IHter. dry. transfer lo
ml. Hash
.5 Redo step 3. odd to
vol. Itosi
.6 Rinse vomple train.
odd lo vol. flask
.7 Adjust lo mark «/
UeCI
7.2.4 Add new chip. 50 mis
hoxone lo tloslc; concenlroli
lo S mis: cool lor 5 mins.;
assure MeCI oul before noil
slop
7 2.5 Rinse apparatus with
hexane; Ironsler contents
to a separalory tunnel; do
cleanup procedure
ZJ.1
Conlenl
freweigh 1 dram
oss vial
.2 Transfer and reduce 1
ml. eilrocl lo vial II
weight constant
.3 Calculate woighl dried
•itrocl
.4 Calculate X HpkJ content
from eon.
.5 Record lipid extract wl.
and X lipid content
L
7.3.4 Extract Concentration
TTironsler and nnso vol.
flask contents of 7.3.2.7
lo round bottom
.2 Concentrate on rolovap
al 40 C
7.3.5 Extract Cleonui
tP-
Ron
.1 Dissolve 5ecHon 4 eilrocl
wHh hexane
.2 Add acid bnpregnaled
sitteo. stir lor 2 hours
.3 Decant and dry llauld
wHh sodium sulfole
.4 Rinse silica 2x w/hexone
dry w/sodktm sulfale.
combine rinses w/slep 3
.5 Rinse sodium sulfofe,
combine rinse w/slep 4
.6 Prepor* ocidk sika
column
.7 Pass hexane extract
through column. coNecl
eluole in 500 ml. KO
assembly
.8 Rinse column w/hexane.
combine eluole w/slep 7
concentrate total ehwU
lo 100 ul
Note: If column discolored.
repeal cleanup (7.3.5.1)
.9 Extract ready for column
cleanup
8290 65
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November 1990
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METHOD 8290
continued
|7.4 Iiwteonmontol ond Wosto Samples!
7.4.1 SludioAM fuel M
.1 filrod
using DOM-Stork »otor
•ton liber IMor
.5 Mnso IHtor •/toluene.
combine •/eilrecl
.4 Concentrate to near drynoss
treat m bi SecMo* 7.4 2;
mito Irom pub), treat at
Section 7.2
L
7.4.1 SHU JoHom/M
.1 direct sample ./toluene
liter through flait Ifcer
IHUr Into round bottom
.2 Concentrate MI retovap
•IMC
7.4.4 transfer concentrate to sop
' ' using heione; rinse
_ lor. odd to tunnel;
5X MaCI tohv. shake
2
7.4.S
.1 Ut
ttond I* f*»m I:
.2 fltor
N
.1
CMttrtfuw) llnl
7.4 «. I;
«Md II
MCUT
c«nc*nkai« Ml ••!•» koMi
M 5 mt» Ml; rMMM KD
•ttmnMr. •«•• to drata *
.4 Irani* M)>M»UI pKoM to *•?
HUM lanifto biHtoi
A IroMlcr to IUMIM:
wid Hlracl ••**
.5 Ul pKm«t ttporato. UM
MtchMikrt mMm II n««d»d
.1 PM* ItoCI taMf HH«ugli drying
•9Mil. ctwjcl In KO a*SMnbl|r
•/concwihatof tub*
.7 R*p*ol ttop 4-6 2i. ri«M
drftn^ •^Mtl. cembtn* all
In KO
.J iMn*** CMumn; odd n«ion«,
nlracHon cMX»4r«t« •! soMi.
A n«« biBlna cMf ; attach column.
lrato to
S nib
.10 RbiM Itosli ond oMMnMy to llnol
vokim* 15 mb
. 1 1 Ottormin* original fompU volum*
by Ironflorrlno mcnltcut volume to
giodualod cywidor
7.4.) fly Ash
.1 Weigh sample; odd
lortiilcoNon sem. In acetone.
IM HCh shake In eitractton
Klor 1 hours
or mli In •uchneV tunnel:
rinse IWer cahe •/•ater; dry
IWer cahe ol room I
.J Add sodium sutfale lo coke.
mli ond let stand lor I hr..
mli again and let stand
.4 Place sample In eilrocllon
Nilmbto; eitrocl bi Soihlol
for 1C hours •/toluene
.5 Cool ond IHer eitrocl; rinse
containers A combine; retotap
to near drynett at 50 C
j
7.4 t Soil
.1 Add sodium suHoto. mli; fconslor mliluro lo
SoihM oisombly olop gtoss wool plug
.2 Add tohMM. rollui lor 24 hours
Add moro sodhim suiloto II sompto doos not
lloo IfoWy
.J Ironslor oilrocl lo round bottom
.4 Concenlroto to 10 mis on rolovop. oNow lo
cool
5 Ironilor concentrolo and hoiono rlnsos lo KO
oitwnbly: conconlraU lo 10 mis. alow lo
cool
.6 Dins* Snydor column inlo KO; frontier KO
ft conconlrolor lubo liquktt lo top lunnol;
lint* KO ottcmbly o/h*ion« A odd lo funnel
T
8290 - 66
Revision 0
November 1990
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ML 11 KM) 8290
continued
7.5.1 •orMion
.1 PorMM eihocl
•
MM Vtflk Ml
f 4MM 4v
Pii s. rw
•rirad •/Nad Mb.; ttwta.
.2 oun rot risM SAMPii s.
•/Na
.1 0*1 rot]UN SAuHii
d •/KOM Mb*.; sJM
*d »*M toftc rap*
N n* Mbr *M*kod bi
Dry eikoci •/nJhim SuMoto
• • mna* keHani ftotk* rim
"ka***lMMM* seln. 'in
TT reek a w
in •/ heiane.
.2 P*ck • gr*«N»
"
f«r
•/sHcaaol:
to top el bod;
•/olumlne;
to top *f bad.
.3 MtMlM rnUu* •( S^HM 7.5.1.4
bi !MI«I«; Iromltr M!H. to to* »l
.4 Clul* sMco column w/b«OM
«r«cNr onl* otumlM cckmrn
.5 Add h*ian« U otumln* c*tumn;
•kit* to lop •( Mdkmi «uMo»» bi
celtcl OM MM clutod IMIOM
J Add ItoCI/liMon* tain, to alumina
column: caltocl dual* In conccnkator
tub*
75.3 Corbon Column
.f Triiif. A)l"lll
•par* AX-2i/C«lil* 545 column;
•cKvato mbluf* al I JO C lor 6
Hour*; ttor* In dMllcator
.2 Peck • 10 ml strategical plH
•/•r*par*d AX-21/CcW* S4S ml.
Nato: Each kokh ol AX-2I/C«M« J45
musl b« cn«ck*d lor X recovery
el eno%t«*.
J Cencenketo WeCI/ne«on« IrocHon
el SecHen 7.5 2i to 2 mis
•/nHrofl*n; rinse celumn
•/several solns.; add sample
cencenkato and rinses to top
ef column
.4 I Me celumn sequ»nHo<|r
«/: cyctoheione/UeCI; MeCI/
melhanol/loluene: combine eluatos
.5 Turn celumn upside down, ekito
fCOO/rCOf IracHon •/toluene:
IHtor II carbon lines present
.( Cencenkato toluene fraction on
rotovap; lurlker cencenkoto to
100 ul In mhMal using nHroaen
al M C; rinse Hash J. »/IX
toluene In MeCI; add Mdecane
recover* std; store room lamp.
8290 67
Revision 0
November 1990
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HEIHOp 8290
continued
7.1 Cbromaloorophk, Moil Spoctromotrlc. and
Data Acaultillon faram*loft
.
Setecl correct dimiiiiliiii and *»romelirt
•I coluflM. ond tot-MB cl>r»iinl»gfOftilc
.1 Ooorot* matt tMdri
of live SM descriptors
.2 Tuna matt spectrometer
ol SMdotcrMon
hMlotM
Ion*
boiod on lont
TftlfH-
.1 loM cycl* Hm* ol< or = I ,
.1 Aco^lr* SM dot* lor lont ol 5
descriptor
••quired before any sample analysis.
ona H routine coBbraMon dees net
PIM| criteria
.IMS ceibfoHon feln*. mud be
mod for InWol coloration
.2 Tun* most spectrometer •/PfK as
described hi Section 773
.1 Injecl 2 id ol CC column performance
check t*tn. ond acquire SIM data;
assure Section 1.1.2 criterion or* mot
.4 Anotyie *ach of S coRbroHan standards
using MM 10111* MitdWoni. wlh the
Moving MS operating parameters:
.1 Roth *l mtogrotod ton cuff ml lor
tool* • IOM •Nhln control tmMs
.2 Rolle ol Integrated Ion curronl lor
carbon lot oh* Internal ond recovery
standards •UMn control IntH*
Note: CoMrol NmlH muil b* oclwwod In
on* run for •• l*m.
.3 SJowol I* Mb* (S/N) roll* lor oach
lofftot OMlyl* ond h*oM tld. ««lo£l*d
IMI cvrront orolloi (SICP) ond
CC ilonoh > 25
7.7.1 4
.4 Cokul* rolaH** rnponi* laclori (MRf )
lor unlobilid ond bboM larg*l onolyl**
rololb* lo inlornol tldi (lobte 5)
.5 Cakulol* ovoraQ* ond rolalhi* slondord
dMiolion lor In* & cottUoHon tokilioni
.1 MKf s lor concentration determination ol
total ttomor* In o homologous loriM
or* colculolod at:
.1 Congonort In o bomotogoui «orl*t •/on* .
bomor. moon Mf utodli MHM 01 Section
77145
Hole: CoWwation solnt. do not contain
lobotod OCOf ; merelore. »«f OCDT
retoliv* lo labeled OCOO
.2 Cakulotlon lor mean HHf lor congonort
bi o hom*l*goui seriot •/more man one
Hole:
homort M bomologoui serlet */o
oNorUd
2.1.7.1 tubtlHolion oallorn
tamo retoome loclor at amor 2.3.
7.1 rtomort In teriet
.7 Calculation ol MTt used lo determine
X rocoverie* ol nine Inlornol tlondardt
onolysis
.1 Ik* X BSD lor unlg*»l*d sldt. must
b* •HMn «/- 20X; for labiliJ.
«/- 30X
.2 S/N roNo for CC sbinoh > or = 2 5
.3 loM* • bolopk rolbs •MMn HmMi
When criteria lor acceptable coNbrallon
ore met. moan Mf s used lor cofcutoNont
unW rouNno caobroNon criteria ore not
met
Performed at 12 hour periods oiler
succMslul resolution chochs
.1 Inject 2 ul coNbralion sofai. HIICC-1:
us* some MCC/HRMS conditions ol
Sections 7.C.I ond 7.o 2; document
on occeplable coNbraHon
774
.1 1 Uoosurod
Mrs must bo
Note
*/- 20X ol initial calibration value*
.2 Measured labeled Mfs must be «/bi
*/- 30X ol Initial coWxolion values
.3 loot* 8 Ion abundance ratios must b*
w/ln limits
.4 Review routine coftbrollon process II
criteria of slops I and 2 or* not
satisfied
An initial calibration must be done
•hen ne« HRCC-3. sample forlillcolion.
or recovery tld. lobi Irom another lot
i> used
8290 68
Revision 0
November 1990
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7.1 I Reduce ••keel M Wand volume
to 10 or SO ul
1717 tnlocl 2 ul etlauel el the sample!
InUmeCC |
1
1713 Acquire SM data accerdmg to I
Sections 7 > 7 end 7.8.3 |
Acquisition ported mud ot
leesl encompass PCOQ/PCOf
evero*
L
cc
r.iu,i«
.1 Rotative Retention lime*
.1 2.J. 7.8 tub: Sample components
relative retention HIM (Ml) w/ln
-I lo 1 seconds ol retention
Km* of labeled Internal or
recovery sld.
.2 2.1.7.8 tub: Sample Mil
Hm« wlndevt If •/•
Inlwnal tkl.
.3 M«I 2.J.7.8 tub: McUnHon
Hm« •/In Homologous
.4 ten awrwil r*tponM* l*r
^uanlMollon mutl reach moilmum
•/In 2 tecentft
.5 Wn currwil r**^on*M lor toboled
•Mr mutl r««ch meibnum •/!«
2
Hole: VoriTy pf«»«nc« ol ».7.1.1-ICDP and
i.).4.c.8-r«cor i« sect
.2 Ion Abundance Holiot
.1 (lotto ol Integrated Ion current lor
(we lont uted lor quantification
•/In Nmlh ol homologous terlei
.3 Slanal-lo-Nolte RaMo
.1 All Ion current inlentJUet ) = 2.5
.4 PotycMorlnaled Mphenyl tmer
Interference!
.1 Corresponding CCOff channel clear
of signal > = S/N 2.5 ol tame
•••lelnllnn Umel
f vtfjnnvn ntri2 5« nohe lor
.1 Colculale ttNmated Uoibnum PessiMo
Concentration- (CMfCJ •hen signal >
2.5i nobe and retention time me tame
[ 7 ».o »etoTive pofcent dillerence (HfD) lormulo |
Reonobie sample eilracl on (0 meter
SF-2130 column
.1 ConcenkoHont of specified congeners
calculated from analysis done on M-S
column
.2 ConcenlraMent of tpecilled congener t
cakutaUd from analysis done on
SP-2110 column ./dlllerenl CC/US
condllioni
Cr>lirmollon ami quantification of 2.1.7.8-
ICUO done on either column as long at
Section 8.1.2 criteria met
.1 CC pooh must meet criteria ol Sections
78 47 78.4.1 and/or 78.4.1.1 Mlt
ol 2.1.7.8-tub congenert •/no corbon-
labvled analogues referred lo w/ln 0 00»
SHI unMi of carbon -labeled tld
(STOP)
8290 - 69
Revision 0
November 1990
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