and Dibenzof urans
< 8 an enzof ura
and Sediments by Gag Chromatography/Masa SpectromeSJ
Ann L. Alford-Stevens
James w. Eichelberger
Thomas A. Bellar
William X,. Budde
December 15, 1986
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Office of Research and Development
0. S. Environmental Protection Agency
26 West St. Clair Street
Cincinnati/ Ohio 45268
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INDEX
Section
Number Subject
1 Scope and Application
2 Summary of Method
3 Definitions
4 Interferences
5 Safety
6 Apparatus and Equipment
7 Reagents and Consumable Materials
8 Sample Collection/ Preservation and Handling
9 Calibration
10 Quality Control
11 Procedures
12 Calculations
13 Confirmatory Analyses
14 Automated Identification and Measurement
15 Method Performance
16 References
Tables
1 GC Operating Conditions
2 Composition of Concentration Calibration Solutions
3 Composition of Retention Time Calibration Solution
4 Ions for Selected Ion Monitoring Data Acquisition
5 Relative Abundance Criteria for Ions Monitored to Identify and
Measure CDDs and CDPs
6 Known Relative Abundances of Ions in CDD/CDF Molecular Ion Clusters
7 Precision of Triplicate Determinations of CDDs and CDPs in
Composite Ash Extract
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DETE1 iINATION OF CHLORINATED DIBENZO-P—DIOXINS AND DIBENZOFURANS IN
SOILS AND S )IMENTS BY GAS CHROMATOGRAPHY/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1 • 1 This method provides procedures for mass spectroinetric analysis of extracts
of soils and se iments to deterutLne chlorinated dibenzo—p—dioxins (CDDs)
and dibenzofurans (CDFs) containing four to eight chlorine atoms. The
existence of 49 pose ibile CDD method analytes and 87 CDF method analytes
makes impractical the Listing of the Chemical Abstracts Service Registry
Number for each method analyte. Because CDDe and CDFs are identified and
measured as isomer groups, the non—specific CAS 1 for each level of chlori-
nation is used to describe method analytes.
Isomer No. of
Group Isomers Formula CASRN
C1 4 —CDDs 22 C 12 C 1 4 H 4 0 2 4 1903—57—5
C1 5 —cDD S 14 C 12 C 1 5 H 3 0 2 36088—22—9
C 1 6 —CDDs 10 C 12 C 1 6 H 2 0 2 55684—94—1
C 1 7 —cDDs 2 C 12 C 1 7 R0 2 37871—00—4
C1 8 —cDD 5 1 C12C 1 8 O 2 3268—87—9
C 1 4 —CDFs 38 C 12 C 1 4 H 4 O 55722—27—5
C1 5 —CDFs 28 C 12 C 1 5 H 3 0 30402—15—4
C 1 6 —CDFs 16 C 12 Cl B 2 O 34465-46—8
C 1 7 —cDFs 4 C 12 C 1 7 H0 38998—75—3
C 1 8 —CDFs I C 12 C 1 8 0 39001—02—0
1 • 2 Detection Limits vary a ng method analytee and with sample matrix, sample
pr aration procedures, condition of the GC/MS system, and individual
samples. The detector sensitivity decreases with increasing level of -
chlorination, and estimated method detection l(m.its range from 1 ug/kg
for each C1 4 -CDD or C1 4 -CDF to 3 uq/kg for C1 9 -CDD or C1 8 -CDF.
I • 3 Because some De and Fs may be extremely toxic, safety procedures
described in Section 5 should be followed to prevent exposure of laboratory
personnel to materials containing these compounds.
2 • SW MA OF ksi- O
2.1 Four isotapically—labeled surrogate compounds (SCs) are added to each
sample before sample extraction or other preparation procedur!s. These
compounds are 3 C 12 —2,3,7,8 -Cl 4 -CDD, 13 C 12 —2,3,7,8-C1 4 -CDF, 1 C 12 —Cl 9 -CDD,
and 3 C 12 -Cl 8 —CDF. After the SCa are added to a 10—g aliquot of soil, or
s lim nt sample, the wet soil or se tm nt is mixed with 20 g of anhydrous
sodium sulfate and is extracted with a mixture of hexane and methanol
while the sample aliqu.ot and solvent are agitated continually in a glass
jar. Colu chromatographic procedures are used to help eliminate sample
components that may interfere with detection and measurement of CDD5 and
CDFs. The sample extract is concentrated to 50 uL, and an internal standard
(IS) is added before a 2-.uL aliquot is analyzed. In this method, dibromo—
octafluorobiphenyl (DBOFB, CASRN 10386—84—2) is used as the IS. Sample
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—2—
extract components are separated with capillary coluim gas chromatography
(GC) and selected ions are 1 nitored with low resolution, electron ioni-
zation mass spectrometry (MS). An interfaced data system (DS) to control
data acquisition and to store, retrieve, and manipulate mass spectral data
is essential.
2.2 Aiialytee are identified and measured as isomer groups (i.e., by level of
chlorination). Ten selected CDD and CDF congeners, one representing each
level of chlorination, are used as concentration calibration standards. a
concentration is measured for each CDD and CDF isomer group, total COD and
total CDF concentrations in each sample extract are obtained by tamiiii ng
appropriate isomer group concentrations. Method bias for SCs is calculated
to ind.tcate method performance with each sample.
3. DEPINITIONS
3.1 CONCENTRATION CALIBRATION SOLZJrION (CAL) -- A solution of selected CODs
and COPs used to calibrate the ease spectrometer response.
3 • 2 INTE AL STR11DA (IS) -— A pure compound added to a sample extract in known
amounts and used to calibrate concentration measurements of other compounds
that are sample components. The IS must be a compound that is not a
sample component.
3 • 3 LABORATORY DUPi .ICATE — Two aliquots of the same environmental sample
treated identically throughout a laboratory analytical procedure. Each
aliquot is carried through the entire laboratory analytical method. Ana-
lytical results indicate precision associated with analytical procedures
but not with sample collection, preservation or storage procedures.
3 • 4 LABORATORY PER7O W1CE CRECK SOL J ION -- A solution of elected method
“alytes, surrogate compounds, and internal stanitard used to evaluate the
performance of the GC/MS/D6 with respect to criteria defined in a method.
3 • 5 LABORATORY RZA(ZNT BLANK (LRB) -- An aLtquot of reagent blank so lid
material that is treated as a sample in all, aspects in the laboratory,
including addition of all method reagents, internal standards, and surrogate
compounds, and exposure to all glassware, apparatus, and equipment. If a
reagent blank solid material is not available, the entire method is followed,
except that no solid material is added to the extraction jar.
3 • 6 LABOR ORY SURROGATE SPI -- A surrogate compound concentration measured
in a .se 1. extract with the same procedures used to measure sample compo-
nents. The known value is determined from standard gravimetric and/or
volumetric techniques used during sample fortification.
3.8 PERIOPMANCZ EVALUATION (PB) SAMPLE — A sample that contains Iciown concen-
trations of method analytes and has been analyzed by multiple laboratories
to det (ne statistically the accuracy and precision that can be expected
when a method is performed by a competent analyst. Analyte concentrations
are unknown to the analyst.
3.9 QUALITY CONTROL (QC) CHECK SAMPLE -— A sample containing known concentrations
of analytes prepared by a laboratory other than the laboratory performing
the analysis. The analyzing laboratory uses this sample to demonstrate
that it can obtain acceptable identifications and measurements with
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—3—
procedures to be used to analyze environn ental samples containing the
same or similar analytes. Analyte concentrations are known by the analyst.
3.10 RESPONSE FACTOR (RE’) —— MS response to a known amount of a concentration
calibration compound relative to a known amount of an internal standard.
3 • 11 SIGNAL-TO—NOISE RATIO (S/N) -— The ratio of the area of the analyte signal
to the area of the random background signal, it is determined by integra-
ting the signal for a characteristic ion in a region of the selected ion
current profile where only random noise is observed and relating that area
to the area measured for a positive response for the same ion. The same
number of spectra must be integrated for both areas. (The ratio of peak
heights may be used instead of peak areas.)
3 • 12 SURROGATE COMPOUNDS (SCa) — Compounds that are not expected to be found in
the sample, are added to each environmental sample to indicate method per-
formance, and are measured with the same procedures used to measure sample
components. In this method, four isotopically labeled diomins and furans
( 13 C 12 —Cl 4 —!rCDD, CASRN 80494—19—5i 13 C 12 -Cl 4 —TCDF, CASRN unknown;
C1 9 -CDD, CAS 1 80479—48—7; and 3 C 12 -Cl 8 -OCDF, CAS i unknown) are added to
the soil/sediment sample aliquot before any sample extraction or other
preparation procedures • Their concentrations are measured in each sample,
and method bias is calculated for each to indicate acceptable method
performance with each sample.
4, INTERFERENCES
4.1 interferences may be caused by contaminants in solvents, reagents, glass-
ware, and other sample processing equipment. High purity reagents and
solvents must be used and all, equipment must be scrupulously cleaned • All
of these materials must be deisonstrated to be free of interferences under
the analytical conditions by routine analysis of laboratory reagent
blanks.
4 • 2 Most frequently encountered interferences are other sample components that
are extracted along with CDD5 and COPs. Because very low levels of CODs
and COPe must be measured, eliM 4 nation of interference is highly desirable.
Colunm chr togra hic procedures are used to remove some coextracted
sample components; these procedures must be performed carefully to minimize
loss of amalytes during attmepts to enrich their concentrations relative
to other le components. Capillary colunsi CC RTs and the compound-specific
tharacteritios of mass spectra eliminate many interferences • Sample com-
ponents t produce the same quantitat.ton and confirmation ions and have
the same as a method analyte will interfere. These interferences will
not produce false positives, however, unless the abundance of the confir-
mation ions relative to the quantitation ions is very similar to that of a
method analyte.
4 • 3 Chlorinated diphenyl ethers are compounds that may cause false positive
identifications of COPs • Loss of two chlorine atoms from a chlorinated
diphenyl ether produces an ion cluster with ions of the same masses and
relative abundances as a solecular ion cluster produced by a COP containing
two less chlorine atoms than the chlorinated diphenyl ether. Because of
the lack of standards of chlorinated diphenyl ethers, their RTs are unknown
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-4-
with the GC coluimt and analytical conditions used. To prevent false
positives, DF identification criteria include a check to determine the
absence of at least one ion that would indicate the presence of an
interfering chlorinated diphenyl ether.
4.4 Because one of the SCa, 13 C 12 -C1 4 -CDF, produces ions at m/z 320 and 322,
this ccxnpound will interfere -with any coeluting C14—CDD. Such coelution has
not been observed, but analysis of all 22 C1 4 —Ct)Ds along with 13 C-C1 4 -CDF
with the analytical conditions to be used for samples is necessary to
eliminate that possibility.
5 • SAFETY
5 • 1 The to d.city or carcinogenicity of each ch ical used in this method has
not been precisely defined. Some CDDs and CDFs have been identified as
suspected human or mAmMalian carcinogens. Therefore, each should be
treated as a potential health hazard, and exposure should be reduced to
the lowest feasible level • Each laboratory is responsible for maintaining
current awareness of OSHA regulations regarding safe handling of the -
chmnicals used in this method. A reference file of material data handling
sheets should be made available to all, personnel involved in analyses.
Additional information on laboratory safety is available (1—3).
5 • 2 Each laboratory must develop a strict safety program for handling CDDs
and CDFs. The following laboratory practices are recrI!ml nded:
‘5 • 2 • 1 Contamination of the laboratory will be minimized by conducting
all manipulations in a hood.
5 • 2 • 2 The effluents of sample splitters for the GC and roughing pumps
on the GC/MS should pass through either a coluzm of activated
tharcoal or through a trap containing oil or high-boiling alcohols.
5.3 The following precautions for safe Ita ling of CDDe and Fa in the labor-
atory are presented as guidelines only. Precautions for safe handling
and use are necessarily general. in nature because detailed, specific
recr m ndations can be made only for particular exposures or circumstances.
Assistance in evaluating the health hazards of particular laboratory
conditions may be obtained from certain consulting laboratories and from
state departments of health or labor, many of which have an industrial
health service. Techniques used in handling radioactive and infectious
materials re generally applicable to CDDa and Pa.
5 3 1 Protective Equipment: Throw-away plastic gloves, apron or lab
coat, safety glasses and lab hood adequate for radioactive work.
5 • 3 • 2 Training: Workers must be trained to remeve contA nated gloves
and clothing without contacting the exterior surfaces.
5 • 3 • 3 Personal Hygiene: Thorough washing of hands and forearms after
each manipulation and before breaks (coffee, lunch, and shift)
with a mild soap and plenty of scrubbing action.
5 • 3 • 4 Confinenent: Isolated work area, posted with signs, segregated
glassware and tools, plastic—backed absorbent paper on benchtops.
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—5—
5.3.5 Waste: Contaminated waste should be minimized. Plastic bag liners
should be used in waste cans. Janitors should not handle wastes.
5.3.6 Disposa.1 of Wastes: Low—level waste such as absorbent paper and
plastic gloves may be burned in a good incinerator • Water containing
gross quantities (milligrams) of CDDs and CDFS should be packaged
securely and disposed through cIv ercial or governmental channels
that are capable of handling high—level radioactive wastes or
extremely toxic wastes. Liquids should be allowed to evaporate
in a good hood and l it a disposable container; residues may then
be incinerated.
5 • 3 • 7 Glassware, Tools, and Surfaces —— Satisfactory cleaning may be
accosplished by rinsing with solvents (such as acetone, ntethylene
chloride, and trichioroethylene), followed by washing with detergent
and water. Dish water may be disposed to the sewer. (Also see
Sect. 6.5.)
5 • 3 • 8 Laundry: Clothing known to be contaminated should be disposed with
the precautions described under Sect. 5.3.6. Lab coats or other
clothing may be laundered. Clothing should be collected in 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 a cycle before
being used again for other clothing. Disposable garments may be
used to avoid a laundry problem, but they must be properly disposed
or incinerated.
5.3.9 Wipe Tests: A useful method to determine cleanliness of work
surfaces and tools is to wipe the surface with a piece of filter
paper, which is extracted and analyzed by GC or GC/MS. Less than
4 ng/m of a D or F indicates acceptable laboratory cleanliness;
anything higher warrants further cleaning. More than 10 ug on a
1-rn 2 wipe sample indicates an acute hazard that requires prcrn t
cleaning before further use of the equipment or work space and
indicates that unacceptable work practices have been employed in
the past.
5 • 3 • 10 Inhalation: Any procedure that may produce airborne contamination
meat be done with good ventilation. Gross losses to a ventilation
system meat not be allowed. Handling of the dilute solutions nor-
mally used in analytical and animal work presents no inhalation
hazards except in case of an accident. Finely divided soils contain—
mated with Ds and CDF may be hazardous because of the potential
for inhalation. Such samples should be handled in a confined environ-
ment, such as a hood or glove box, or laboratory personnel should
wear masks fitted with a particulate filter and charcoal sorbent.
5.3.11 Accidents: Reii ve contaminated clothing i-mi ediate1y, taking pre-
cautions not to contaminate skin or other articles. Wash exposed
skin vigorously and repeatedly until medical attention is obtained.
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-6-
6. APp A ’tys AND EQUIPMENT
6. 1 COMP 7rERIZED CC/MS SYSTDI
6.1.1 The CC must be capable of temperature programming and be equipped with
all required accessories, such as syringes, gases, and a capillary
column. The GC injection port must be designed for capillary columns.
Manual splitless injections were used to acquire data used as the basis
for quality control requirements. An automatic injector, however, is
desirable, because it should provide more precise RTs and areas. On-
column injection techniques are encouraged, because they minimize high
mass descrisination and analyte degradation problems. If the low
initial column temperature required for on—column injections cannot be
reproduced, RTs will not be reproducible within acceptable limits.
That may cause ion sets to be changed at inappropriate times during
data acquistion. With on—column injection, an uncoated precolumn is
rec i nded. Splitting injections are not appropriate.
6.1.2 Mass spectral data are obtained with electron ionization at a nominal
electron energy of 70 eV. To ensure sufficient precision of nasa
spectral data, the required MS scan rate must allow acquisition of
at least five data points for each monitored ion while a sample
component elutes from the CC.
6.1.3 An interfaced OS is required to acquire, store, reduce, and output
mass spectral data • The DS must be capable of searching a data file
for specific ions and plotting ion abundances versus time or spectrum
number to produce selected ion current profiles (SICPs) and extracted
ion current profiles (EICPe) • Also required is the capability to
obtain chromatographic peak areas between specified times or spectrum
numbers in SICPs or EXCPs • Total data acquisition time per cycle
should be 20.5 a and jl.0 s. The OS must be equipped with software
capable of acquiring data for six aipe of <12 ions, and the OS
must allow autamate4 and rapid changes of the set of ions being
monitored. The fim s spent monitoring ions during sample analyiea
must be the same as the times used when CAL were analyzed.
6.2 CC C LU — A 60 a X 0.32 mm ID fused silica capillary column coated with
a 0.25 um or thicker film crosslinked phenyl methyl silicone (such as
Durabond—5 (OS—S) , J and W Scientific, Rancho Cordova, CA) or polydiphenyl.
vinyl &b’ thyl. silo, ne (such as SE—54, Alltech Associates, Deerfield, IL)
is required. Operating conditions known to produce acceptable results with
these colu are shown in Table 1, but conditions must be optimized at
each laboratory with each CC and CC column.
6 • 3 MISC LMIEO SQUIPI NT
6 • 3 • 1 Nitrogen evaporation apparatus with variable flow rate fran approxi-
mately 30 aL/win to 150 aL/mm.
6 • 3 • 2 Mechanical. shaker — A magnetic stirrer or a wrist—action or platform—
type shaker that produces vigorous agitation. Agitation conditions
must be determined and demonstrated.
6 • 3 • 3 Analytical balance capable of accurately weighing 0.01g.
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—7—
6.3.4 Analytical balance capable of accurately weighing 0.0001 g.
6.3.5 centrifuge capable of operating at 2000 rpm.
6.3.6 Water bath -— equipped with concentric ring cover and temperature
controlled within + 2°C. (A sand bath may be used.)
6.3.7 Stainless steel spatulas or spoons.
6.3.8 Stainless steel (or glass) pan large enough to hold contents
of 1—pt sample containers.
6.3.9 Glove box.
6.3.10 Boiling Chips —— appro (m*tely 10/40 mesh. Reat at 400°C for
30 m.tn or extract with methylene chloride in a Soxhlet apparatus.
6.4 GLASSWARE
6.4.1 Volumetric flasks - various standard sizes with ground glass stoppers.
6 • 4,2 Microsyringes — various standard sizes.
6.4.3 Extraction jars —— amber glass with Teflon—lined screw cap; minimum
capacity of approximately 500 m l .; imast be compatible with mechanical
shaker to be used.
6 • 4 • 4 Xud.rna—Danish apparatus —— 50 0—mL evaporating flask, 10-mI. graduated
concentrator tubes with ground—glass stoppers, and 3—ball macro
Snyder columu.
6.4.5 Culture tubes — 8-aL glass.
6.4 • 6 Mini—vials —— 1—aL amber borosilicate glass with conical-shaped
reservoir and screw caps lined with Teflon—faced silicone disks.
6.4.7 Funnels —— glass; appropriate size to acc”°” date filter paper used
to filter jar extract (volume of approximately 170 a ! .).
6.4.8 Chromatography columts -- 1. an ID x 20 an long and 3. an ID X
30 long.
6.5 CAuTION: se of glassware should be minimized to avoid contamination.
All glassware that is reused must be scrupulously cleaned as soon as
possibl. after use. Rinse glassware with the last solvent used in it.
Wash with hot water containing detergent. Rinse with copious amounts of
tap water and several portions of distilled water. Drain dry and heat in
a muffle furnace at 450C for a at least 4 h. Volumetric glassware should
not be heated in a muffle furnace, and some thermally stable materials
(such as PCBs) may not be removed by heating in a muffle furnace • In
these cases, rinsing with high-purity acetone and hexane may be substituted
for muffle furnace heating. After glassware is dry and cool, store inverted
or capped with aluminum foil in a clean environment. Before using, rinse
each piece with an appropriate solvent.
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—8—
7 • REAGENTS AND CONSUMABLE MATERIALS
7 • 1 COLUP I CB MATOGRAPIiY REAGENTS
7.1.1 PJ.umina, acidic —— Extract in a Soxh].at apparatus with iuethylene
chloride for 21. h and activate by heating in a foil covered glass
container for 24 h at 190°C.
7.1.2 Silica gel -— high purity grade, type 60, 70—230 mesh, Soxhiet
extract with inethylene chloride for 21 h and activate by heating in
a foil-covered glass container for 24 h at 130°C.
7.1.3 Silica gel. impregnated with 40% (by weight) sulfuric acid —- Add two
parts (by weight) concentrated sulfuric acid to three parts (by
weight) silica gel (extracted and activated), mix with a glass rod
%mtil free of lumps, and store in a screw—capped glass bottle.
7.1.4 Silica gel impregnated with sodium hydroxide - - Add one part (by
weight) 1 U sodium hydroxide to two parts (by weight) silica gel
(extracted and activated) • Mix with a glass rod until free of
lumps arid store in a screw—capped glass bottle.
7.1.5 Sulfuric acid, concentrated — ACS grade, epecific gravity 1.84.
7.1.6 Sodium hydroxide, I N.
7.1.7 Graphtt.tzad carbon black (Carbopack C or equivalent), surface area
of appr’ 4 ’ °tely 12 m 2 /g, 80/100 u sh. Mix 3.6g of Carbopack C (or
equivalent) with 16.4g of reagent grade Celite 54 S (or equivalent)
in a 40—mL vial and activate by heating in an oven at 130°C for 6h.
Store in a desiccator.
CAIfl’XCNI Check each new batch of mixed Carbopack/CeliteR to ensure
recovery of 50% of each D and CD ! ’ in CAL *3.
7 • 2 FILTER PAPER - — pore sine of < 20 to 2 S us rinse with hexane before use.
7 • 3 GLASS l OL - ailanised; extract with methylene chloride and he ne before
7.4 SODXVM 3LFATE — Gramilar, anhydrouas before use, extract with methylene
chloride and dxy for 4 it in a shallow tray placed in an oven heated to
120°C.
7.5 SOLvarITS .gtt purity, j t.tl1eding1aSs hexana, methanol, methylene
chloride, 2— opsnol, and decane.
7.6 S SOW!ION — Prepare a 2.5 rag/uL solution of 4,4 ’ _dib 1 )octafLUr0btPbeT Y1
(DE ’B} in decane. Add a 50—u i. aliquot to each sample extract just before
concentration of the extract to final volu .
7 • 7 CALa —— Five iscane solutions containing the ten- calibration congeners,
the four SC ., and the IS are needed. Compositions are shown in Table 2.
Sadi of CALa *l—*5 contains a .conatait concentration of the 19 and varying
concentrations of calibration congenere and SCa. In a single solution,
concentrations of calibration congeners and SCa increase with increasing
nur ar of chlorine atoms because the MS detector sans it.tvity decreases with
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—9—
increasing number of chlorine atoms. Concentrations of CALs are equivalent
to concentrations ranging from 1 to 50 ug/kg for C14—analytes and from 3
to 150 uafkg for C1 8 —analytes in 50—uL extracts of I0-q samples. Store
concentration calibration solutions in small amber mini—vials.
cMYrIoN: Each time a Vial containing a small volume of solution is warmed
to room tamperature and opened, a small volume of solvent in the vial
headspace evaporates, significantly affecting concentration • Solutions
should be stored with the smallest possible volume of headapace, and vials
should be opened only when necessary.
7.9 SAMPLE FORTIFICATION SOLEJ ION — Prepare a methanol solution containing the
each of the Cl 4 SCa at a concentration of 0.5 ng/uL, and each of the Cl 8
SCe at 1.5 rzg/uL. A 100-u !. aliquot of this solution is added to each
sample aliquot.
7.10 z . o o y PERFO IANCE ECX SOL YrIOw — For both full-range data acquisition,
and the SIN data acquisition aption, C A l *3 is used as the laboratory perfor-
mance d eck solution (LPC) to verify response factors and to demonstrate
adequate CC resolution and MS performance.
7 • 11 RT CALIBRATION SOWr 1014 - Prepar. a eolution containing the first eluting
co at each level of chlorination from Cl 4 through Cl 8 (Table 3) • This
solution will be used to determin, when sets of monitored ions must be
changed. Component concentrations are not critical but should be at least
as great as those of analogous compounds in CAL t (Table 2).
7 • 12 T ABDryw4paIxuM LFITB REAGENT — Dissolve 3 • 39 g of tetrabutyl-
nium hydrogen sulfate in 100 aL of distilled water. To remove impur-
ities, extract this ealution three 41 with 20—EL portions of hexane.
Discard the he, ne extracts, and add 25 g of sodium sulfite to the water
solution. Store the resulting solution in an amber bottl. with a Teflon—
lined screw cap. The solution can be stored at room temperature for at
least one month.
B • SAIGLE PRESsavaTION AIID STORAGE
8 • I SAMPLE PRZ waTXON
8.1.1 When received, most samples will be contained in a 1-pt glass jar
•i round. by vermiculite in a sealed metal paint can. Dfltil a
portion is to be remcivad for analysis, store the sealed paint cans
i locked 4 ted-acceas area where *mkjeflt t erature is j —
ta4 4 above freezing bat below 35C. After a portion is roved
for ‘ ‘ ysis, return the v1 ed portion of sample to its original
container and store as stated above • Do not freeze samples; they
may contain sufficient water to break the sample ar if frozen.
8.1.2 To avoid photodscompositjor*, protect samples from light.
9. CALIBRATION
9 • I Initial c libratton is required before any samples are analyzed and is
required intermittently throughout sample analyses as dictated by results
of cont.trin.tng calibration d ecks • No samples are to be analyzed until
acceptable initiaL calibration is demonstrated and documented. After
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—10—
initial calibration is successfully performed, a continuing calibration
check is required at the beginning and end of each 12-h period during
which analyses are performed.
9.2. INITIAL CALIBRATION
9.2.1 Calibrate and tune the MS with 8tandards and procedures prescribed
by the manufacturer with any necessary n dificationg to meet USEPA
requl rements.
9.2.2 Determination of RTs —— Inject a 2—uL aliquot of the R’r calibration
solution (RT CAL, Table 3) and acquire SIM data with the six ion
sets shown in Table 4.
CAUTION: Because CDDs and a)Fs are mass deficient (i.e., accurate
masses are less than un.tt naninaj. masses),
accurate masses (Table 5) may need to be considered when establishing
SIll data acquisition conditions to ensure correct mass assignments.
Begin data
acquisition with ion Bet 1 as soon as feasible after injection. As
soon as the IS has eluted, collect data with ion set 2 until after
the Cl 4 -CDF el ites. Then collect data with ion set 3 until after
the C1 5 —CDF elutes, ion set 4 until, after the C1 6 -CDP eluteg, ion set
5 until after the C1 7 -CDF eluteg, and, ion set 6 until after the
Clg-CDF eluteg. Use the same CC conditions and SIM data
acquisition conditions that will be used to collect data for samples..
Acquire at least five scans for each solution canponent during
elutjon of each GC peak. Total time per scan should be >0.5 s and
.: .1.0 5.
9.2.3 Usa the s (scan numbers) observed in Sect. 9.2.2 to determine
when ions sets should be switched for subsequent analyses.
9.2.3.1 Begin data acquisition with ion set 1 as soon as feasible
after injection.
9.2.3.2 Begin acquisition with ion set 2 soon after elution of
the IS.
9.2.3.3 Stop data acquisition with ion set 2 and begin ion set 3
<15 s before elution of the C1 5 —CDF RT calibration congener.
CAUTION: This time is critical because of the small
difference in p1’s of late eluting C14—analytes and early
eluting C1 5 —analytes.
9.2.3.4 Begin acquisition with each remaining ion set <30 s before
elution of each additional CDF RT calibration congener.
9.2.4 Optimi e thranatographjc conditions to obtain syinmetricai peak
shapes and reproducible peak areas. (A tailing IS peak indicates
inadequate GC conditions and will prevent measurement of reproducible
GC peak areas. Multiple analyses of the W I ’ CAL may be necessary.
9.2.5 Inject a 2—uL aliquot of CAL f 3 (Table 2) and acquire SIN data with
the same conditions used to analyze the RT calibration solution and
ensure that each calibration congener’ is observed.
CAUTION: CC operating conditions must be carefully reproduced for
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—11—
each analysis to provide reproducible RT37 if not, ions wil]. not be
monitored at the appropriate times.
9.2.6 Performance Criteria
9.2.6.1 CC performance -— Baseline separation of C1 8 -CDD and
C1 8 —CDF; symmetrical peak for each with peak width
at half height equivalent to <5 9.
9.2.6.2 MS s sitivity -— S/N ratio of >15 for m/z 468 measured
for 3 C 12 -C1 9 -CDD.
9.2.6.3 MS calibration -— For 3 C 12 -Cl 9 —CDD, abundance of m/z 470
relative to m/z 472 is >77% and <97%; abundance of in/z 474
relative to m/z 472 is >56% and <76%. Abundance of in/z 457
relative to m/z 456 (produced by the IS) is 3—23%.
9.2.7 If all performance criteria are met, analyze one 2—uL aliquot of
each of the other four CALs.
9 • 2 • 8 Response Factor Calculation —— Calculate response factors (RPS)
for each CDD and CD ! concentration calibration congenar, and SC
relative to the IS:
ftp — A Qig/AisQ,c
where A — integrated ion abundance of quantitation
ione (Table 4) for a CDD or CD ! calibration
congener or SC,
Aj 5 — integrated ion abundance of the IS quantitation
ions (Table 4),
Qis — injected quantity of IS,
— injected quantity of CDD or CD! calibration
congener or SC.
RP is a wLttless number, units used to express quantities
must be equivalent.
9 • 2 • 9 Response Factor Reproducibility —— For each concentration cali-
bration congener and SC, calculate five RPs (one for each CAL) and
the mean RF • If the relative standard deviation (RSD) exceeds
20%, analyze additional aliquots of appropriate CALS to obtain
<20% R of RYe over the entire concentration range, or take action
to improve CC/MS performance.
9.2.10 r Reproducibility —— Absolute RTs of each calibration congener
should not vary by re than ± s frc a one analysis to the next
and muat not continue either to increase or decrease
9.2.11 Calculate the mean RF for 2,3,7,8C1 4 CDD, 2,3,7,8C1 4 CDF, C1 9 -CDD,
and Cl 8 -CDF: using the equation in Sect. 9.2.6, where the appropriate
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—12—
13 C 12 —labeled cc npound .s used as the TS. (These RFs will, be used
to calculate concentrations of specific canpounds with isotopic
dilution procedures, Sect. 12.6)
9.2.12 Calculate the mean area measured for C]. 8 -CDD, Clg- .CDF and the IS.
9.3. C NTINUING CALIBRATION CifECK
9.3.1 With the following procedures, verify initial calibration at the
beginning and end of each 12—h period during which analyses are to
be performed.
9.3.2 Calibrate the MS with standards and procedures prescrthed by the
manufacturer.
9.3.3 Inject a 1—u i . or 2—uL aliquot of CAL *3 and collect SIM data with
the same conditions uaed during initial calibration.
9.3.4 Demonstrate acceptable performance for criteria described in Sect.
9.2.6.
9.3.5 Determine that neither the area measured for m/z 456 for 13 C 12 —C1 9 -CDF
nor that for in/a 472 for 13 C 12 -C1 8 -CDD has decreased or increased
by more than 30% from the area measured in the most recent previous
analysts of a CAL or by more than 50% fr the mean area measured
during initial calibration.
Note: Variability in the ratio of these areas is an indication of
chrosiatogtaphy or MS calibration probL ia. When sufficient data
are available, guidelines will be added to this method.
9.3.6 RE’ Reproducibility —— For an acceptable continuing calibration
check, the measured RE’ for each calibration congener/SC must be
within +20% of the mean value calculated (Sect. 9.2.9) during
initial calibration. If not, remedial action must be taken;
recalibration may be necessary.
9 • 3 • 7 RT Reproducibility — Demonstrate and document acceptable repro-
ducibility of absolute RTa of RT calibration congeners (Sect.
9.2.10).
9.3.8 Re&tal. actions must be taken if criteria are not met; possible
rmedies are:
9.3.8.1 Check and adjust GC and/or MS operating conditions.
9.3.8.2 Clean or replace injector liner.
9.3.8.3 Flush column with solvent according to manufacturers
instructions.
9.3.8.4 Break off a short portion (approximately 0.33 in) of the
column, check column performance by analysis of CAL #3,
and ensure that RTs have not changed.
9.3.8.5 Replace GC column; performance of all initial caU.bratien
procedures La then required.
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-13—
9.3.8.6 Adjust MS for greater or lesser resolution.
9.3.8.7 Calibrate MS mass scale.
9.3.8.8 Prepare and analyze new CAts.
10. QUALITY CONTROL -
10.1 LABORATORY REAGENT BLANK (LRB) -— Perform all steps in the analytical
procedure (Section 11) using all reagents, standards, SCa, equipment,
apparatus, glassware, and solvents that would be used for a sample analysis,
but c3nit an aliquot of soil/sediment sample. If available, substitute
EPA-provided reagent blank solid material for an aliquot of soil/sediment
sample.
10.1.1 An LRB must contain the same amounts of IS and SCs that are added
to each sample before extraction.
10 • I • 2 Analyze an LRB before any samples are extracted and analyzed.
10 • I • 3 Before a new batch of solvents or reagents is used for sample
extraction or for colusm dir iatoqraphic procedures, analyze an
LRB • In addition, analyze a laboratory solvent blank (LSB), which
is the earns as an LRB except that no SC or IS is added; this
demonstrates that reagents contain no impurities producing an ion
current above the level of background noise for quantitation ions
for those c npounds.
10.1.4 Analyze an LRB along with each batch of <20 samples.
10.1.5 Acceptance criteria for an LRB have not been specifically defined.
Until criteria are defined, analysts should exercise judgement
and fully document results. Obviously, only ions produced by the
IS and SC5 are desirable in LRB data.
10 • I • 6 Corrective action for imaccaptable LRB — Check solvents, reagents,
apparatus and glassware to locate and elEminate the source of con-
tamination before any samples are extracted and analyzed. Purify
or discard contaminated reagents and solvents. If the LRB that
s extracted along with a batch of samples is contaminated, the
entire batch of samples must be reextracted and reanalyzed.
10 • 2 CALIBRS’PION — Included among initial and continuing calibration procedures
are ‘‘ roue quality control checks to ensure that valid data are acquired
(See Sect. 9). Continuing calibration checks are acc nplished with results
fras analysis of one solution, CAL *3.
10 • 2 • I If same criteria are not met for a continuing calibration check
after a 12—h period during which samples were analyzed, those
samples must be reanalyzed. Those criteria are: GC performance,
calibration, and f sensitivity. (See Sect. 9.2.6.)
10 • 2 • 2 When other criteria in Sect • 9.2 are not met, results for affected
analytes must be labeled to alert the data user of the observed
problem. Included among those criteria are: reproducibility of
RTs and RFs for calibration congeners and SC5.
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—14—
10.3 I 8ORATORY PERFORMANCE CNECI( SOLTYrION -— In this method, CAL *3 also
serves as a laboratory performance check solution.
10.4 LABORATORY SURROGATE SPIKE
10.4.1 Measure the concentration and calculate method bias for each of
the four SCs in every sample and blank.
10.5.2 Acceptance limits have not been established for SC measurements,
report LU results.
10.5 QUALITY CONTROL CRECK SAMPLE —- Not yet available.
10.6 LABORATORY DCJPLXCATE SAMPLE —— Select one sample from each batch of <20
samples of similar type. If sufficient information is available, select
a sample that is expected to produce positive results • After addition of
SCe, extract and analyze (Sect. 11) these two aliquots. Relative difference
(RD) of duplicate results for surrogate compound concentrations should be
<30%. (RD — (C 1 — C 2 / 0.5 (C 1 + C 2 )J 100 ) Insufficient data are currently
available to provide guidance for acceptable EDs of measured concentrations
of CDD and CD? is r groups; report all results.
10.7 p pjiAMcg EVALUATION SAMPLE -— Not yet available, to be analyzed
periodically when available.
11 • PROCEDURES
11.1 SAMPLE HANDLI*
11.1 • 1 CAIJTION: Pinely divided soils contaminated with CDD5 or CDFS are
hazardous because of the potential, for inhalation or ingestion of
particles. Such samples should be handled in a confined environment,
(e.g., a closed hood or a glove box) whenever possible; otherwise,
laboratory personnel should wear masks fitted with a particulate
filter and chgrcoaj. sorbent.
I I • 2 PRE-Ex’rRAcT ION SAMPLE TREA’fl4ENT
11.2.1 Ucmogen.tzation -- Although sampling personnel will. attempt to collect
ha geneoue samples, examine each sample and udge if it needs
fi ther mixing. Stirring is rec nded when possible.
11 • 2 • 2 Camtrifugation —— If a sample contains an obvious aqueous liquid
phee., centrifuge it to separate liquid and solid phases. Place the
entire sample in a suitable centrifuge bottle and centrifuge for 30
mm at 2000 rpm. Re ve bottle fros centrifuge. With a disposable
pipet, remove liquid phase and discard.
CAUTION: This liquid may contain Os and CDFs and should be disposed
as a potentially hazardous waste.
Mix solid layer with stainless steel spatula and r ve a portion t0
be weighed and analyzed. Return the remaining solid portion to -
original sample bottle and store.
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-15—
11.3 SAMPLE EXTR CTION
11.3.1 CAUTION: See Section 5 for safety gu.tdeLtneg and recommendations.
11.3.2 Note: Extremely wet samples may require centrifuging to remove
water before addition of sodium sulfate; see Section 11.2.2.
11.3.3 Accurately weigh to three significant figures a l0—g (4 0.5 g) portion
of the wet soil or sediment sample, and transfer it to the extraction
jar.
11.3.4 Add 100 uL of the solution containing SCs to the soil or sediment in
the extraction jar. Add small portions of the solutions at several
sites on the surface of the soil or sediment.
11.3.5 Add 20 g of purified anhydrous sodium sulfate and mix thoroughly
using a stainless steel spoon or spatula.
11 • 3 • 6 Allow the mixture of soil and sodium sulfate to set for two hours at
ambient tmaperature; mix again, break all, visible lumps, and allow
to set for at least four more hours.
11.3.7 Mix again and add 20 mL of methanol: mix again and add 150 mL of
hexane.
11 • 3 • S Place the extraction jar containing the soil, sodium sulfate and
solvents in the shaker and shake for at least 3 h.
11 • 3 • 9 Remove the jar from the shaker and allow soU.ds to settle • Decant
the solvent through a glass f nmel containing hexane -rinsed filter
paper • Rinse the jar, solid sample residue, and filter the residue
with four 5—mL portions of hexane.
11.3. 10 For sediment samples, r val of elemental, sulfur is recrr mended.
11 • 3 • 10 • 1 Concentrate the extract volume to approximately 10 mL with
a Xuderna-Dan.tah apparatus, and transfer the extract to a
50—mL clear glass bottle or vial with a Teflon—lined screw
cap. Rinse the extract container with 1 • 0 mL of hexane,
adding the rinse to the 50-aL bottle.
11.3. 10.2 Add 1 mL of tetrabuty1 1m r n.tuin —sulfite reagent (Sect. 7.12)
and 1 aL 2-propanol, cap the bottle, and shake for at least
I sin. If the sample is colorless or if the initial color
is uncthanged, and if clear crystals (precipitated sodium
sulfite) are observed, sufficient sodium sulfite is present.
If the precipitated sodium sulfite disappears, add more
crystalline sodium sulfite in approximately 100—mg portions
imtil a solid residue r na4ns after repeated shaking.
11.3.10.3 Add 5 sL of distilled water and shake for at least 1 sin.
Allow the sample to stand for 5—10 sin and remove the
hexane layer (top) for analysis. Dry the extract by passing
it through a 1 0—om column containing hexane-’washed sodium
sulfate. Rinse the sodium sulfate with about 30 mL of
hexane and add this hexane to the extract.
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—16—
11.3.11 Concentrate the extract volume to approximately 2—3 mL with a
Xuderna—Danish apparatus. Note: Glassware used for more than
one sample must be careful.y cleaned betwCén samples to prevent
cross contamination.
11.3.12 Transfer the concentrated extract to an 8—mL glass culture tube.
Rinse the evaporator flask with three 5—mL portions of hexane, and
transfer each rinse to the culture tube. Between additions of
hexane rinse, reduce the extract volume in the culture tube enough
to allow addition of another 5—mL volume of rinse. To reduce the
volume, place the culture tube in a water or sand bath adjusted to
operate at 50°C and position the tube so that the surfaces of the
extract and the water are at about the same level. Evaporate the
solvent with a stream of nitrogen with the tip of the nitrogen
delivery tube 2 cm above the solution.
11.3.13 After the final rinse has been added, reduce the extract volume to
approximately 1 ui !.
11,4 LUMN CR R ATOGRAPHY
11.4.1 Column Preparation
11.4.1.1 Column 1: Place 1.0 g of silica gel into a 1 cm x 20 cm
column and tap the column gently to settle the silica
gel. Md 2 g sodium hydroxide—impregnated silica gel, I
g silica gel 4.0 g of suif uric acid—impregnated silica
gel, and 2 g silica gel. Tap column gently after each
addition.
11.4.1.2 Column 2: Place 6.0 g of alumina into a 1 cm x 30 cm
column and tap the column gently to settle the alumina.
Add a 1—cm layer of purified sodium sulfate to the top of
the alumina.
11.4.1.3 Add hexane to columns 1 and 2 until the packing is free
of d annels and air bubbles. A small positive pressure
(5 psi) of clean nitrogen can be used if needed.
11.4.1.4 Column 3 (carbon) —— Insert a small plug of glass wool
into a silanized glass column approximately 7 cm long by
10 ma O.D. Apply suction with a vacuum aspirator, and
add the Carbcpack/CeliteR mixture until a 2 cm column
is obtained.
11.4.2 Quantitatively transfer the hexane sample extract from the culture
tube to the top of the silica gel in Column 1 • If extract tends to
adhere to culture tube walls, the tube can be placed in a sonic bath
to suspend undissolved material. Rinse the culture tube with two
0.5—mL portions of hexane and transfer rinses to Column 1.
11.4.3 With 90 mL of hei ne, elute the extract from Column 1 directly into
Column 2 containing alumina and sodium sulfate.
11.4.4 Add 20 mL of hexane to Column 2 and elute until the hexane level is
just below the top of the sodium sulfate7 discard the eluted hexane.
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—17—
11.4.5 Add 20 mL of 20% methylene chloride/80% hexane (volume/volume) to
Column 2 and collect the eluate.
11.4.6 With a gentle stream of filtered dry nitrogen, reduce the volume of
the eluate to about 1 to 2 iL.
11.4.7 Preelute column 3 (the Carbopack/Celite column) with:
11.4.7.1 2 mL of toluene,
11.4.7.2 1 m l. of a mixture of 75% (by volume) niethy].ene chloride,
20% methanol, and 5% benzene,
11.4.7.3 1 aX. of 50% (by volume) cyclohexane and 50% inethylene
chloride, and
11.4.7.4 2 inL of hexane.
11.4.8 While the column is still wet with hexane, add the sample extract.
Elute the column with the following sequence of solvents and discard
eluates.
11.4.8.1 2 ml. of hexane,
11.4.8.2 1 aL of 50% (by volume) cycichexane and 50% methylene
chloride, and
11.4.8.3 1 mL of 75% (by volume) methylene chloride, 20% methanol
and 5% benzene,
11.4.9 Invert the column and elute (backflush) with 5 mL of toluene.
Collect the entire eluate.
11 • 4.10 Reduce the eluate volume to approximately 1—2 mL. Transfer aliquots
to a 1—mi. amber minivial with conical reservoir. Concentrate and
add additional aliquots with further concentration until entire
eluate is transferred. Rinse eluate container with two 0.5—mL
portions of hexane and transfer rinses to the minivial with further
concentration as necessary.
CAUTION: not evaporate eluate (sample extract) to dryness.
11.4.11 With the final sample extract volume at approximately 1 mL, store the
sctract in a dark place at 4C until just before CC/MS analysis.
11 • 5 CC/MS ANALYSIS
11 • 5.1 Renove the sample extract or blank frcxi storage and allow it to warm
to * 4ent laboratory temperature. Add a SO—uL aliquot of the 2 • 5
ng/uL solution of the IS. Immediately before CC/MS analysis,
reduce the extract or blank volume to 50 uL with a stream of dry,
filtered nitrogen. To prevent extract cooling, the extract
container can be placed in a water bath at itth4 temperature.
11.5.2 Inject a 2—ui. aliquot of the extract into the CC operated under
conditions previously used (Sect. 9) to produce acceptable results
with the performance check solution.
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—18—
11.5.3 Acquire mass spectral data using the same data acquisition time and
operating conditions previously used to determine response factors.
11.5.4 Demonstrate acceptable GC/MS system performance.
11.5.4.1 For the IS, determine that the area measured in the sample
ctract has- riot increased or decreased by >30% from the area
measured during the most recent previous analysis of a CAL or
by >50% from the mean area measured during initial calibration.
If either criterion is riot met, remedial action must be taken
to improve reproducibility and the sample extract must be
reanalyzed.
11.5.4.2 Document that GC/MS system performance criteria in Sect.
9.2.6 are met.
11.6 I NTIFICATION PROCEDURES
11.6.1 Using the ions shown in Table 4, obtain appropriate selected ion
current profiles (SICP5) for IS quantitation and confirmation ions
and for each ion monitored to detect CDDs/CDFs, the SCs, and the
IS. The integrated ion current measured for each ion monitored to
detect an analyte or SC ton must be at least 3 times background noise
and must not have saturated the detector.
11.6.2 Internal Standard
11.6.2.1 The abundance of m/z 454 relative to ni/z 456 must be >40%
and <60%, and these ions must maximize within 1 s.
11.6.2.2 RT must be within +10 a of that observed during the most
recent acceptable calibration.
11.6.3 Surro te compounds
11.6.3.1 Absolute 2s of SCa must be within +5 s of that measured
during the last previous continuing calibration check.
11.6.3.2 All ions monitored for each compound (Table 4) must be
weaent, mavlnitinize within 1 a, and meet relative abundance
criteria in Table 5.
11.6.4 Ds and CDFs
11.6.4.1 For each candidate for a particular CDD or CDF isomer
group, the integrated peak areas measured for quantitation
and confirmation ions must maximize within 1 a.
11 • 6.4.2 Use SICP data to calculate the relative abundance of measured
quantitation and confirmation ions, and compare to criteria
in Table 5. If criteria are not met, a coeluting or partially
coeluting compound may be interfering. Examination of data
from several scans may provide information that will allow
application of additional background corrections to improve
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—19-.
the ion ratio. known relative abundances of analyte ions
are shown in Table 6.
11.6.4.3 Ions monitored to demonstrate the absence of a potentially
interfering chlorinated diphenyl ether must be <5% abundance
relative to appropriate CDF ions; all other ions for a
CDD or CDF candidate must be present, maximize within 1 s,
and meet relative abundance criteria in Table 5. For
C1 8 —CDD and C1 8 -CDF, in addition to above criteria, the
candidate spectrum must elute within I s of the relevant
isotopicalJ .y labeled Sc.
11.6.4.4 Determine if 2,3,7,8-C1 4 —CDD and 2,3,7,8-C1 4 -CDF can be
present an ng sample components. In addition to all
other identification criteria, a candidate spectrum
must maximize within 1 s of its isotopically—labeled
hou log.
11.6.4.5 Determine if any c1 4 —analytee could have eluted after
initiation of data acquistion with ion set 3. If C1 4 -CDD
Lone (320 and 322) or C1 4 -CDF ions (304 and 306) maximize
within 1 s of each other at the apex of a peak and meet
the relative ab mdance criteria in Table 5, estimate
possible concentration with the equation in Sect • 12 • 3,
and contact data user or use judgement as to whether the
sample ctract ehould be reanalyzed or data (including
estimated concentration of possible late eluting Cl 4
analytes) should be reported without reanalysis.
12. CALCULATIONS
12.1 Fran appropriate SICPs of quantitation ions, obtain and record the spectrum
number of the chromatographic peak apex and the area of the entire
chranatographic peak.
12.2 For CDDs and C)Fs, sum the areas for all isomers identified at each level
of chlorination (e • g., sum all quantitation ion areas for C1 4 —CDDs).
1 2.,3 Calculate the concentration of each SC and CDD/CDF isomer group using the
formula:
c, — (A, 1 • Qj 5 )/(Aj 5 • RF • 11)
where C concentration (nanograins per gram or micrograms per
kilogram) of SC or CDD/CDF isomer group,
Ax — the sum of the areas of quantitation ions for each SC or
the sum of areas of quantitation ions for all CDD/CDF
isomers at a particular level of chlorination,
Aj 3 — the sum of the area of the quantitation ions for the
appropriate IS,
Qj 5 — quantity (nanograms) of IS added to the sample extract
before GC/MS analysis,
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—20—
RF = mean response factor calculated during initial cali-
bration for a SC or a calibration c npound for a CDD/CDF
isomer group, and
W weight (grams) of sample extracted.
12.3.1 CAUTION: For these analyses with automated data interpretation
a linear fit algorithm will produce erroneous concentration
data.
12.3.2 If the calculated concentration of any analyte exceeds the concen-
tration of CAL #5, the linear range may have been exceeded, and a
smaller aliquot of that sample must be analyzed. Accurately weigh
to two significant figures a 1—g aliquot of the wet soil/sediment.
Add 100 iiL of the solution containing SCs before sample extraction
and other preparation procedures. Add 50 uL of the 2.5 ng/uL IS
solution to the extract and analyze.
12.3.3 Report calculated concentrations to two significant figures if
<10 ng/gi report to three significant figures if >10 ng/g.-
12.4 Calculate the maximum possible concentration of 2,3,7,8—C1 4 —CDD and
2,3,7,8-C1 4 —CDF that could be present. Use the equation in Sect. 12.3,
but terms are defined as:
C, — maximum possible concentration (nanograms per gram) of
2,3,7,8—C1 4 —CDD or 2,3,7,8—C1 4 -CDF,
the sum of the quantitation ions for 2,3,7,8-C1 4 -CDD or
2,3,7,8-C1 4 -CDF fran the candidate spectrum,
— the sum of the quantitation ions for the appropriate
13 C 12 —labeled analogue (SC),
the quantity (nanograms) of appropriate SC added to the
sample before extraction,
RF = calculated mean response factor (Sect. 9.2.9) for
2,3,7,8-C1. 4 —CDD relative to 13 C 12 —2,3,7,8-C1 4 -CDD or
2,3,7,8—C1 4 —CDF relative to 3 C 12 —2,3,7,8—Cl 4 —CDF, and
W weight (grams) of sample extracted.
12.5 For eadi SC, calculate the percent method bias using the equation:
B 100 (C 5 — Ct)! C.
where C 5 — measured concentration (in micrograms per kilogram
or micrograms per liter),
Ct — theoretical concentration (i.e., the quantity added to
the sample aliquot/weight or volu of sample aliquot).
Note: The bias value retains a positive or negative sign.
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—21—
12.6 When C1 9 —CDD or C1 8 —CDF is identified as a sample component, calcu .ate
its concentration using the mean RF relative to its 13 C—labeled analog
(determined during initial calibration, Sect. 9.2. 12). Because this
concentration is calculated with a ‘ 3 C—labeled analogue, the calculated
value is corrected for losses during sample preparation and analysis.
For each compound, this concentration should be essentially the same as
that calculated relative to the IS and corrected with the appropriate
bias value. If not, the cause of the discrepancy should be sought, a
likely cause is deterioration of chromatographic performance.
13 • CONFIBMATORY ANALYSES
13.1 For each sample extract in which 2,3,7,8-C1 4 -CDD may be present, analyze
a second 2—uL aliquot with a 60—rn X 0.32 me SP-2330 or 50 m X 0.32 mm
CP—SIL 88 column coated with a film of appoximately 0.25 urn with helium
flow at 28—29 om/sec at approximately 250C. A solution containing
1,4,7,8—, 2,3,7,8—, and 1,2,3,7/1,2,3,8-C1 4 —CDDs should be analyzed.
With both columns, 2,3,7,8—isomer elutes between the 1,4,7,8—peak and
the combined 1,2,3,7/1,2,3,8—peak. GC conditions should be optimized
to separate 2,3,7,8-C1 4 —CDD from 1,4,7,8— and and 1,2,3,7/1,2,3,8 with
a valley <25%. (For this confirmatory analysis, detection and measure-
ment of the IS, DB ’B, is not necessary.)
13.1 • I Confirmation requi s elution of the candidate sample component
within 1 s of the C 1 2 -Cl 4 - D SC and measured ion abundances
meeting all criteria in Table 5.
13. 1 • 2 If identification is confirmed, calculate and report concentratior
as specified in Sect. 12.4.
Note: Because this concentration is obtained with an isotope
dilution procedure, the value obtained accounts for 2,3,7, 8—C1 4 —CDD
losses during sample preparation and analysis. Labeled and
unlabeled analogues are presumed to behave in the same way.
13.2 For each sample extract in which 2,3,7,8-Cl 4 —CDF may be present, analyze
a 2—uL aliquot with a 60—rn X 0.32 me SP-2330 column.
13.2.1 the candidate sample component elutes within 1 s of the
C 12 —2,3,7,8—Cl 4 —CDF and all criteria in Table 5 are met,
the candidate may be 2,3,7,8-Clg-CDF or 1,2,6,9—Cl 4 -CDP,
which are not resolved with the chromatographic condititions.
13.2.2 If criteria in Sect. 13.2.1 are met, calculate and report the
mexizsun poe sib is concentration of 2,3,7,8 -C l 4 -CDF as specified
in Sect. 12.4.
14. AUTOMATED IDENTIFICATION AND MEASUREMENT
Special software can be used for automated identification and measurement of
CDDs and CDFs • Unprocessed GC/MS data are handled without human interaction
with the software operating on the dedicated computer. A concentration for each
CDD/CDF isomer group is calculated automatically. Contact EMSL-Cincinnati for
further information.
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—22—
15 • METHOD PERFORMANCE
Because no soil or sediment samples containing known concentrations of a
variety of CDDs and CDFs are available to assess method performance, accuracy
data are not available. To assess precision of concentration measurements,
triplicate 2—ut aliquots of a composite extract of an ash from a municipal
incinerater was analyzed with identification and measurement procedures
described in this method. RSDa of measured concentrations rari ed from 2—14%,
and SC recoveries ranged from 29—74%. This low recovery for L C_C1 9 CDD
indicates that better column chromatography procedures are needed to achieve
adequate sensitivity for the higher chlorinated congeners. Because
CDF was not available when this sample extract was prepared, no data for this
SC are available. Because triplicate a.liquots of a composite extract were
analyzed, data reported here reflect precision of measured concentrations but
do not reflect variability associated with sample preparation.
16 • REFERENCES
1. “Carcinogens — - Working with Carcinogens”, Department of Health Service,
Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77—206, August 1977.
2. “OSHA Safety and Health Standards, General Industry”, 29 CFR 1910,
Occupational Safety and Health Adm.tnistratiori, OSHA 2206, Revised
January 1976.
3. “Safety in Academic Chenistry Laboratories”, American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
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—23—
Table 1 • CC Operating Cond.ttionsa
Column type: - DB—5 or SE—54
Film thickness: 0.25 urn
Column 1 4 mønsio na: 60 m X 0 • 32 mm
Helium Linear velocity: 28—29 c u/sec
at 250°c
Injector temperature 290°C
Transfer Line temperature 290°C
Temperature program (spl.ttless injection): Inject at 80°C and hold 1 miii;
increase as rapidly as possible
to 160°C and hold for 4 sin;
increase frau 160°C to 300°C at
3°C/sin; hold until after elution
of C1 8 —CDD. (Begin data acquisition
about 10 sin after injection.)
a These operating conditions produced acceptable results in one laboratory
but different conditions may be required in other laboratories and may vary
with instrunt and CC column.
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—24—
Table 2. Ccxnposition of Concentration Calibration Solutions
Concentration (na/uL
Cônu øund
CAL 1 CAL 2 CAL 3 CAL 4 CAL 5
2 4 10
3 6 15
4 8 20
5 10 25
6 12 30
2 4 10
3 6 15
4 8 20
5- 10 25
6 12 30
1
3
1
3
CDI) and CDF Cal. Congeners
C1 4 —CDD ( 2378 )a
0.2
1
C1 5 —CDD (1,2,3,7,8)
0.3
1.5
C1 6 —CDD (1,2,3,4,7,8)
0.4
2
C1 7 —CDD (1,2,3,4,6,7,8)
0.5
2.5
C 1 8 —CDD
0.6
3
C1 4 —CDF (2,3,7,8)
0.2
1
C1 5 —CDP (1,2,3,7,8)
0.3
1.5
C1 6 —CDF (2,3,4,6,7,8)
0.4
2
C1 7 —CDF (1,2,3,4,6,7,8)
0.5
2.5
C 1 8 —CDF
0.6
3
Surrogate Compounds
13 C 12 —C1 4 —CDD (2,3,7,8)
0.2
0.4
3 C 12 -Cl 8 —CDD
0.6
1.2
3 C 12 —Cl 4 —CDP (2,3,7,8)
0.2
0.4
13 C 12 —C1 8 —CT)F
0.6
1.2
Internal Standard
4,4’—dibromooctafluoro—
biphenyl
2.5
2.5
1.8
2.4
0.6
0.8
1.8
2.4
2.5
2.5 2.5
athiorine aubetitution positions.
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—25—
Table 3. Composition of Retention Time (RT) Calibration So]. ition
RRTa of
Compound (Chlorine Sub.) RT Congener
C1 4 —CDF (1,3,6,8) 1.974
C1 5 —CDF (1,3,4,6,8) 2.302
C1 6 —CDP (1,2,3,4,6,8) 2.665
Cl —CDF (1,2,3,4,6,7,8) 3.022
C1 8 —CDF (1,2,3,4,6,7,8,9) 3.431
aRetent.ton ‘ 4’ relative to the IS, dibranooctafluorobiphanyl, which elutes
abcat 12 mm after injection with GC conditions described in Table 1. These are
provided for information only; RRTs will vary with particular GC conditions
used to analyze the solution.
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—26—
Table 4. Ion Sets for Selected Ion Monitoring Data ccpaisition
Set Ion Compound Monitored/Purpo8e
Set Ion Compound Monitored/Purpose
SUMMARY
No. of
Set Zone
1
2
3
4
5
6
6
12
12
8
9
12
Compounds
Monitored
1
257
Set ion range lower limit
4
327
C1. 6 —CDD con.firmation ion
454
IS quantitation ion
372
C1 6 —CDF confirmation ion
456
IS quantitation ion
374
C1 6 —CDF qu ant1tation ion
457
MS resolution check
376
Cl 6 —CDF qu ,antitation ion
458
IS confirmation ion
388
C1 6 —CDD confirmation’ ion
2
514
257
Set ion range upper 1 i& t
C1 4 —CDD confirmation ion
390
392
446
Cl 6 —cDD quantitation ion
C1 6 —CDD quantitation ion
C1 6 —CDF confirmation ion
259
C1 4 —CDD confirmation ion
304
C1 4 —CDF quantitation ion
5
361
C1 7 —CDD confirmation ion
306
C1 4 -CDP quantitation ion
406
C1 7 -CDF confirmation ion
3
308
316
318
320
322
332
334
376
293
C1 4 -CDF confirmation ion
13 C 4 DF quantitat.ton
‘ 3 C-C1 4 -CDF quantitation
94 -CDD quant. ion and
1 C-C3. 4 -CDF confirm. ion
C1 4 -CDD quantitation ion
13 C-C1 4 -CDD cpiantitation
13 C l D quantitation
C1 4 -CDF confirmation ion
C1 5 —CDD confirmation ion
ion
ion
ion
ion
6
408
410
412
422
424
426
480
440
442
444
C1 7 -CDF quantitation ion
C1 7 -CDF quantitation ion
C1 7 -CDF confirmation ion
C1 7 —CDD confirmation ion
C1 7 —CDD quantitation ion
C1 7 —CDD quantitation ion
C1 7 —CDF confirmation ion
C1 9 -CDF confirmation ion
C1 8 -CDF quantitation ion
C1 8 -CDF quantitation ion
304
306
320
322
338
340
342
C1 4 —CDF check ion
C1 4 -CDF check ion
C1 4 -CDD check ion
C1 4 -CDD check ion
C1 5 -CDF quantitation ion
C1 5 -CDF quantitation ion
C1 5 -CDF confirmation ion
446
456
458
460
C1 8 —CDF confirmation ion
3 C—C1 9 -CDF quant. ion
a d C1 8 -CDD confirm. ion
1 C—C1 9 -CDF quant. ion
and C1 9 -CDD quant. ion
3 C-Cl 8 -CDF confirm. ion
and Clg-CDD quant. ion
354
356
358
410
C1 5 -CDD confirmation ion
C1 5 -CDD quantttatiort ion
C1 5 -CDD quantitation ion
C1 5 -CDF confirmation ion
462
470
472
474
514
Th9_CDD quantitat.iori ion
C-C1 9 -CDD confirm. ion
1 C-Cl 8 -CDD quant. ion
C-C1 9 -CDD quant. ion
C1 8 -CDF confirm. ion
IS
C1 4 -analyte s
C1 5 -analyte 9 a
C1 6 —analyte s
Cl 7 —analyte a
C1 8 -analyte a
aSet 3 includes four ions to check that C1 4 —analytes did not elute after data
acquisition with ion set 3 was initiated.
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—27—
Table 5. Relative Abundance Criter .a for Ions Monitored to Identify and
Measure CDDs and Fs -
Ana. lyte
Isomer Group
Acceptance
Criteria
Nominal Exact
m/z
C 1 4 —CDF
304
306
303.9016
305.8987
>66% and <86%
100%
308
376
307.8957
375.8364
>39% and <59%
Absent or <5%
C1 5 —CDF
338
340
342
410
337.8626
339.8597
341.8567
409.7974
>51% and <71%
100%
>55% and <75%
Absent or (5%
C1 6 —CDF
372
374
376
446
371.8236
373.8207
375.8178
445.7554
>41% and <61%
100%
>72% and <92%
Absent or <5%
C1 7 —CDF
.
406
408
410
412
480
405.7847
407.7817
409.7788
411.7758
479.7164
>34% and <54%
100%
>87% and <100%
>42% and <62%
Absent or <5%
C1 8 C0F
440
442
444
446
514
439.7457
441.7428
443.7398
445.7369
513.6774
>24% and <44%
>78% and <98%
100%
>55% and <75%
Absent or (5%
13 C, 2 —C1 4 —CDV
316
318
320
315.9418
317.9389
319.9360
67% and j87%
100%
>39% and <59%
13 C 12 .c1 8 ..cDr
456
458
460
455.7800
457.7771
459.7741
-
100%
>55% and <75%
>16% and <36%
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—28—
Table 5 (cont.). Relative Abundance Criteria for Ions Monitored to
Identify and Measure CODs and CDFs
Analyte
Isomer Group
m/z
Accurate
m/z
Acceptance
Criteria
C 1 4 —cDD
320
319.8965
>67% and <87%
322
321.8936
100%
257
256.9327
>10% and <40%
259
258.9298
>10% and <40%
C1 5 —cDD
354
356
358
293
353.8575
355.8546
357.8516
292.8908
>52% and <72%
100%
and
>10% and <40%
C1 6 —cDD
388
390
392
387.8186
389.8156
391.8127
>41% and <61%
100%
>71% and <91%
.
327
326.8518
Present and <40%
C1 7 —cDD
422
424
426
361
421.7796
423.7766
425.7737
360.8128
>34% and <54%
100%
>87% and <100%
Present and <40%
C1e—c D
456
458
460
462
455.7406
457.7377
459.7347
461.7318
>24% and <44%
>78% and <98%
100%
<55% and >75%
13 C 12 —C1 4 -cDD
332
334
331.9367
333.9338
>66% and <86%
100%
470
472
474
469.7779
471.7749
473.7720
77% and <97%
100%
>55% and <75%
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-29—
Table 6. nown Relative Abundances of Ions in CT)D/CDF riolecular Ion Clusters
mhz nt,z
Isomer Relative Isomer Relative
Group CDDs CDFs Abundance Group CDDs CDFs Abundance
Cl 4 320 304 77 Cl 7 422 406 44
321 305 10 423 407 6
322 306 100 424 408 100
323 307 13 425 409 13
324 308 49 426 410 97
325 309 7 427 411 13
326 310 10 428 412 52
327 311 1 429 413 7
328 312 <1 430 414 17
431 415 2
432 416 3
Cl 5 354 338 62 433 417 <1
355 339 8 434 418 <1
356 340 100
357 341 13 Cl 8 456 440 34
358 342 65 457 441 5
359 343 9 458 442 88
360 344 21 459 443 12
361 345 3 460 444 100
362 346 3 461 445 13
363 347 <1 462 446 65
364 348 <1 463 447 9
464 448 26
Cl 6 388 372 51 465 449 4
389 373 7 466 450 7
390 374 100 467 451 1
391 375 13 468 452 1
392 376 81 469 453 <1
393 377 11 470 454 (1
394 378 35
395 379 5
396 380 9
397 381 1
398 382 1
399 383 <1
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—30—
Table 7. Precision of Triplicate Determinations of CDOs and CDFs in
Composite Ash Extract
Conc. No. of
Analyte ng/g (RSD) Isomers
CDDs
C1 4 —CDDs 37.8 (14) 7
C1 5 —CDDg 101 (4.1) 5
C 1 6 —CDDe 84.5 (4.3) 3
C 1 7 —CDDs 65.9 (2.7) 2
Clg—CDD 59.5 (5.0) 1
Total C )De 349 18
CDFs
C1 4 —CDFs 194 (2.1) 7
C 1 5 —CDFs 108 (2.8) 3
C 1 6 —CDFs 24.3 (1.6) 2
C 1 7 —CDFs 2.93 a 1
C 1 8 -CDF
Total CDFs 329 13
Surro te Canpounda
13 C-C 1 4 -CDD 27.8 ( 24 )b
‘ 3 C-C1 8 —CDD 14.9 ( 1 • 6 )b
13 C 1
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