Method 1668C

Chlorinated Biphenyl Congeners in Water, Soil, Sediment, Biosolids, and
Tissue by HRGC/HRMS

April 2010



U.S. Environmental Protection Agency

Office of Water

Office of Science and Technology

Engineering and Analysis Division (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC  20460

EPA-820-R-10-005

Method 1668C Chlorinated Biphenyl Congeners in Water, Soil, Sediment,
Biosolids, and Tissue by HRGC/HRMS April 2010

The Office of Science and Technology (OST) in EPA’s Office of Water
developed Method 1668C (Method 1668C; the “Method”) for use in Clean
Water Act (CWA) programs.  EPA is publishing this Method for users who
wish to measure PCBs as congeners now, and in 2010, EPA expects to
publish a proposal in the Federal Register for public comment to add
this Method to other CWA Methods published at 40 CFR Part 136.

This Method determines chlorinated biphenyl congeners in environmental
samples by isotope dilution and internal standard high-resolution gas
chromatography/high-resolution mass spectrometry, HRGC/HRMS.  EPA
developed this Method for use in wastewater, surface water, soil,
sediment, biosolids and tissue matrices.  Other applications and
matrices may be possible, which may or may not require modifications of
sample preparation, chromatography, etc.

EPA used the results of an interlaboratory validation study of Method
1668A, a peer review of that study, user suggestions and additional
interlaboratory data to write this version, 1668C, of Method 1668.  
Method 1668C, the validation study report, Method1668A Interlaboratory
Validation Study Report (EPA-821-08-021), and the addendum describing
the revised QC acceptance criteria, Method 1668A Interlab Study Report
Addendum, are available at EPA’s CWA methods website at
www.epa.gov/waterscience/methods.

This “C” version of Method 1668 revises the quality control (QC)
acceptance criteria in EPA Method 1668B to allow the upper recovery
limit for some congeners to be above 100 percent, to revise the
estimated method detection limits (EMDLs) and estimated minimum levels
of quantitation (EMLs) to MDLs and MLs, and to makes other changes
summarized below.  The QC acceptance criteria developed in the
interlaboratory method validation study of 1668A, and published in
version B of the Method, did not allow the upper recovery limit for some
congeners to be above 100 percent.  The criteria have been revised based
on data from the interlaboratory study and data from two laboratories
with extensive experience in use of Method 1668A.  TestAmerica,
Knoxville, Tennessee and AXYS Analytical Services, Ltd., Sidney, British
Columbia, Canada provided this new data.  These two laboratories and
Battelle-Columbus provided MDLs for the congeners and congener groups,
which EPA pooled and used to replace the EMDLs and EMLs in Table 2 of
Method 1668B with the MDLs and MLs in Method 1668C.

The detection limits and quantitation levels in this Method are usually
dependent on the level of interferences and laboratory background levels
rather than instrumental limitations.  The method detection limits
(MDLs) and minimum levels of quantitation (MLs) in Table 2 are
concentrations at which a congener can be measured with no interferences
present.  In water, MDLs range from approximately 7 to 30 parts per
quadrillion (picograms per liter, pg/L).  

Interface, Inc. and CSC prepared this Method under EPA Contract
EP-C-06-085.  AXYS Analytical provided the single-lab data in Method
1668A that was later replaced by multi-lab data from laboratories that
participated in EPA's  inter-laboratory validation of 1668A (six labs
for water and tissue, four for biosolids).  

Summary of changes between EPA Method 1668B (January 2009) and 1668C
(April 2010)

(	Additional information on the concentration of extracts has been
included in Section 4.2.

The following note has been added to Section 10.1, “RTs, RRTs, and RRT
limits may differ slightly from those in Table 2.” This statement has
also been added to the footnotes to Table 2.

The note in Section 10.2.1 has been modified to inform the analyst that
careful selection of the grade and purity of PFK may help minimize
interferences with the dichlorobiphenyl secondary quantitation ion.

The diluted combined 209 congener solution is now used for calibration
verification, in place of the VER-3 solution.  This allows all
verification tests to be performed with a single solution.

(	Section 17.2.1 has been changed to clarify that concentrations of
native compounds other than those in the native toxics/LOC standard, in
the labeled cleanup standard, and in the labeled injection internal
standard (except for labeled CB 178) should be determined using the
response factors from Section 10.5 or Section 15.4.2.3.

Section 17.6.5 has been added to provide information on the use of
optional data qualifier flags for reporting coeluting congeners.

(	Based on data from the interlab validation study and data from two
laboratories, the QC acceptance criteria in Table 6 have been revised to
be consistent among tests for calibration verification (VER), initial
precision and recovery (IPR), on-going precision and recovery (OPR), and
labeled compound recovery from samples.

(	Reference 22 has been added to cite the Addendum to the
interlaboratory validation study report.

Sections 1.3, 4.1, 4.6, 9.1.2.1, 9.5.2, 10.3.3, 17.6.1.4.1, 17.6.1.4.2,
17.6.1.4.3, and Table 2 been revised to change estimated method
detection limits (EMDLs) and estimated minimum levels of quantitation
(EMLs) to MDLs and MLs.

Reference 23  has been added to cite the MDL data from AXYS,
TestAmerica-Knoxville, and Battelle-Columbus, and to explain how these
data were processed to produce the pooled MDLs in Table 2.

A sentence was added to Section 11.4.2.1 to require weighing the sample
bottle after emptying, and to determine the volume using the density of
water.

ML definition revised to cite the ML procedure.

A note was added to Section 10.3.3 to state that MDLs and MLs lower than
those in Table 2 may be established per Section 17.6.1.4.1.

Section 17.6.1.4.1 expanded to state how MDLs and MLs lower than those
in Table 2 may be established.

A footnote was added to Table 2 to cite Reference 23.

Summary of changes between EPA Method 1668A (8-20-03) and 1668B (January
2009) (excluding typographical and grammatical error corrections, and
section insertions or deletions necessitated by the following changes).

(	Based  on the interlaboratory validation study, single-laboratory QC
acceptance criteria are replaced with interlaboratory criteria (Table
6).  A new footnote 1 to Table 6 references the EPA interlaboratory
study report, and the other footnote numbers are incremented.

(	Section 1.5, the performance-based discussion, describes additional
flexibility to modify CWA Methods that is allowed by 40 CFR Part 136.6.

(	Section 2.5.2 now indicates that internal standards are the labeled
congeners spiked into the sample.

(	Section 2.5.3 now indicates that injection internal standards are
labeled compounds spiked into the extract.

(	Section 5.4 is an added section on biohazards.

(	Section 7.8 notes that Method 1668A part numbers are valid for Method
1668B.

(	Section 8.1 allows use of alternate sample collection techniques, if
documented. 

(	Section 8.2 adds that one liter, or a larger or smaller volume of
sample, may be collected.

(	Section 12.3 adds a note to indicate that SDS extraction may cause
loss of some mono- through tri-chloro congeners.

(	Section 12.5.6 states that a macro concentration device is to be used
to concentrate extracts, and deletes the requirement for collection of
the extract in a round-bottom flask because any macro concentration
device may be used.

(	Section 16.2 requires an expert spectrometrist to determine analyte
presence when an interference precludes meeting the signal-to-noise
requirement for dichloro-CB congeners.

(	Section 21 cites the validation studies, and that performance data are
in the interlaboratory validation study report.

(	Reference 1 was updated to the 2006 World Health Organization paper on
toxicity equivalency factors.

(	References 4 and 17 add titles to the papers in these references.

(	Reference 21 cites the Method 1668A Interlaboratory Validation Study
Report.

(	Tables 2 and A-1 revised the elution order for congeners 107-109.

(	Table 4 defines the solutions containing congeners 107, 108, and 109.

(	Table 6 contains revised QC acceptance criteria for performance tests,
and footnote 1 to Table 6 references the Method 1668A Interlaboratory
Validation Study Report.

(	Table 7 adds footnote 2 to require meeting the 10:1 signal-to-noise
specification at the CS-2 calibration level.

Summary of corrections and changes to EPA Method 1668A as of August 20,
2003 (excluding typographical and grammatical error corrections, and
section insertions or deletions necessitated by the following changes).

Throughout:  All references to IUPAC have been deleted. We have been
informed that IUPAC does not assign congener numbers. Therefore, all
references to congeners by number are to “congener number.” The
congener naming system given by Guitart, et al. (Guitart R., Puig P.,
Gomez-Catalan J., Chemosphere 27 1451-1459, 1993) has been used in EPA
Method 1668A since its inception and continues in this version.

Sections 2.1.3, 12.4.2., 12.4.3, 12.4.5, and 12.4.9:  Hexane has be
deleted from the extraction solvent for fish and other tissue to
preclude loss of the more volatile CBs.

Section 7.7:  A note has been added to reference the two known suppliers
of labeled compounds.

Section 7.15:  A statement has been added to include certified reference
materials (CRMs) from the National Resource Council of Canada.

Sections 8.2.3, 8.3.2, and 8.4.2:  The preservation temperature for
shipment of samples has been changed to <6 °C to encompass the 4 ± 2
°C used by some organizations (e.g., USGS).

Section 8.2.3:  The requirement to preserve aqueous samples with
sulfuric acid has been deleted because PCBs are stable in environmental
samples, and the storage temperature for aqueous samples has been
changed to <6 °C.

Section 9.1.2.1:  A statement has been added that a modification may be
used routinely after it has been demonstrated to meet the QC acceptance
criteria of the performance tests, so long as the other requirements in
the Method are met (e.g., labeled compound recovery).

Section 10.1.2.3:  The word “approximately” has been inserted in the
requirement to meet the retention times in Table 2 to reflect that
slight changes in GC columns will produce slightly different retention
times.

Section 10.1.2.4:  A statement has been added to indicate that the
absolute and relative retention times in Table 2 were obtained under the
GC conditions given in Section 10.1.1.

Section 10.2.2:  The text has been changed to clarify that the deviation
between each monitored exact m/z and the theoretical m/z (Table 7) must
be less than 5 ppm.

Section 10.5:  The text has been corrected to state that the diluted
combined 209 congener solution (Section 7.10.2.2 and Table 5) is used
for single-point calibration of the Native Toxics/LOC CBs.

Section 12.4:  A note has been added to allow use of a separate aliquot
for percent lipid determination.

Section 12.4.1:  The minimum time required to dry the sample has been
reduced from 12-24 hours to 30 minutes.

Section 15.6:  A requirement has been added to analyze one or more
aliquots of solvent after the OPR if the CBs would be carried into the
Method blank.

Section 16.4:  RRT QC limits may be based on the limits in Table 2 or
limits developed from calibration data.

Section 17.2.2:  The units have been corrected to ng/mL

Section 17.4: A  multiplier of 1000 has been inserted in the equation to
convert ng in extract to pg in sample.

Section 18.5:  A section has been added to suggest that the carbon
column should be used if interferences preclude identification and/or
quantitation of the Toxics.

Table 2:  The relative retention times have been changed to correct
errors and reference each compound to the correct retention time and
quantitation reference. The RT and RRT windows have been adjusted to
attempt to unambiguously identify each congener in the presence of other
congeners. Footnotes 7 and 8 have been revised to reflect this changes.

Table 3:  Units for the diluted combined 209 congener solution have been
corrected to ng/mL as have the concentrations of the native compounds in
the diluted combined 209 congener solution.

Table 6:  The lower QC acceptance criteria limit for the labeled
monochloro- and dichloro-CBs has been lowered for the IPR, OPR, and
recovery from samples to reflect that these compounds can be lost by
evaporation.

Table 7:  Cl-3 scan descriptors have been added to Function 2 and the
m/z types for the 13C12 Cl-4 PCBs have been corrected in Function 4.

Table 8:  The m/z’s forming the ratio, the ratio, and the QC limits
have been corrected for decachlorobiphenyl.

Table A1:  The header has been corrected to delete reference to EMDLs
and EMLs.

Disclaimer

Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

Contact

Please address questions, comments, or suggestions to:

Richard Reding or Brian Englert

c/o The OST CWA Methods Team 

Engineering and Analytical Support Branch 

Engineering and Analysis Division (4303T)

Office of Science and Technology

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, DC  20460

E-mail: OSTCWAMethods@epa.gov

Method 1668C

Chlorinated Biphenyl Congeners in Water, Soil, Sediment, Biosolids,

and Tissue by HRGC/HRMS

April 2010

1.0	Scope and Application

1.1	Method 1668C (the Method) is for determination of chlorinated
biphenyl congeners (CBs) in wastewater and other matrices by
high-resolution gas chromatography/high resolution mass spectrometry
(HRGC/HRMS).

1.1.1	The CBs that can be determined by this Method are the 12
polychlorinated biphenyls (PCBs) designated as toxic by the World Health
Organization (WHO):  congeners 77, 81, 105, 114, 118, 123, 126, 156,
157, 167, 169, and 189.  The Method also determines the remaining 197
CBs, approximately 125 of which are resolved adequately on an SPB-octyl
gas chromatographic column to be determined as individual congeners. 
The remaining approximately 70 congeners are determined as mixtures of
isomers (co-elutions).

1.1.2	The 12 PCBs designated as toxic by WHO (the “Toxics”; also
known as dioxin-like PCBs; DLPCBs), and the earliest and latest eluted
congener at each level of chlorination are determined by the isotope
dilution quantitation technique; the remaining congeners are determined
by the internal standard quantitation technique.

1.1.3	This Method allows determination of the PCB toxicity equivalent
(TEQPCB) for the Toxics in a sample using toxicity equivalency factors
(TEFs; Reference 1) and allows unique determination of 19 of 21 CBs of
interest to the National Oceanic and Atmospheric Administration (NOAA;
Reference 2).  A second-column option is provided for resolution of the
two toxic PCB congeners (congener 156 and 157) that are not resolved on
the SPB-octyl column and for resolution of other CB congeners.

1.1.4	This Method also allows estimation of homolog totals by level of
chlorination (LOC) and estimation of total CBs in a sample by summation
of the concentrations of the CB congeners and congener groups.

1.1.5	The list of 209 CBs (Table 1) identifies the Toxics, the CBs of
interest to NOAA, and the LOC CBs.

1.2	EPA developed this Method for use in Clean Water Act (CWA) programs
and for wastewater, surface water, soil, sediment, biosolids and tissue
matrices.  Other applications and matrices may be possible, which may or
may not require modifications of sample preparation, chromatographic
conditions, etc.  Method 1668C is a revision of previous versions of
Method 1668 all of which  are based on a compilation of methods from the
technical literature (References 3 and 4), and EPA’s dioxins and
furans Method, Method 1613.

1.3	The detection limits and quantitation levels in this Method are
usually dependent on the level of interferences and laboratory
background levels rather than instrumental limitations.  The method
detection limits (MDLs; 40 CFR 136, appendix B) and minimum levels of
quantitation (MLs; 68 FR 11790) in Table 2 are the levels at which the
CBs can be determined with no interferences present.  The MDL for CB 126
in water is 16 pg/L (picograms-per-liter; parts-per-quadrillion).

1.4	The GC/MS portions of this Method are for use only by analysts
experienced with HRGC/HRMS or under the close supervision of such
qualified persons.  Each laboratory that uses this Method must
demonstrate the ability to generate acceptable results using the
procedure in Section 9.2.

1.5	This Method is “performance-based,” which means that you may
make modifications without additional EPA review to improve performance
(e.g., overcome interferences, or improve the sensitivity, accuracy or
precision of the results) provided that you meet all performance
criteria in this Method.  Requirements for establishing equivalency are
in Section 9.1.2, and include 9.1.2.2.3 – explaining the reason for
your modifications.  For CWA uses, additional flexibility is described
at 40 CFR 136.6.  You must document changes in performance, sensitivity,
selectivity, precision, recovery, etc., that result from modifications
within the scope of Part 136.6, and Section 9 of this Method, and how
these modifications compare to the specifications in this Method. 
Changes outside the scope of Part 136.6 and Section 9 of this Method may
require prior review or approval.

2.0	Summary of Method

Flow charts summarize procedures for sample preparation, extraction, and
analysis for aqueous and solid samples, multi-phase samples, and tissue
samples (Figures 1, 2 and 3, respectively.)

2.1	Extraction

2.1.1	Aqueous samples (samples containing less than one percent solids)
– Stable isotopically labeled analogs of the Toxics and labeled LOC
CBs are spiked into a 1-L sample.  The sample is extracted using
solid-phase extraction (SPE), separatory funnel extraction (SFE), or
continuous liquid/liquid extraction (CLLE).

2.1.2	Solid, semi-solid, and multi-phase samples (excluding tissue) –
The labeled compounds are spiked into a sample containing 10 g (dry
weight) of solids.  Samples containing multiple phases are pressure
filtered and any aqueous liquid is discarded.  Coarse solids are ground
or homogenized.  Any non-aqueous liquid from multi-phase samples is
combined with the solids and extracted in a Soxhlet/Dean-Stark (SDS)
extractor.  The extract is concentrated for cleanup.

2.1.3	Fish and other tissue – A 20-g aliquot of sample is homogenized,
and a 10-g aliquot is spiked with the labeled compounds.  The sample is
mixed with anhydrous sodium sulfate, allowed to dry for 12 - 24 hours,
and extracted for 18-24 hours using methylene chloride in a Soxhlet
extractor.  The extract is evaporated to dryness, and the lipid content
is determined.

2.2	After extraction, a labeled cleanup standard is spiked into the
extract which is then cleaned up using back-extraction with sulfuric
acid and/or base, and gel permeation, silica gel, or Florisil
chromatography.  Activated carbon and high-performance liquid
chromatography (HPLC) can be used for further isolation of specific
congener groups.  Prior to the cleanup procedures cited above, tissue
extracts are cleaned up using an anthropogenic isolation column.

2.3	After cleanup, the extract is concentrated to 20 µL.  Immediately
prior to injection, labeled injection internal standards are added to
each extract and an aliquot of the extract is injected into the gas
chromatograph (GC).  The analytes are separated by the GC and detected
by a high-resolution ((10,000) mass spectrometer.  Two exact m/z’s are
monitored at each level of chlorination (LOC) throughout a
pre-determined retention time window.

2.4	An individual CB congener is identified by comparing the GC
retention time and ion-abundance ratio of two exact m/z’s with the
corresponding retention time of an authentic standard and the
theoretical or acquired ion-abundance ratio of the two exact m/z’s. 
Isomer specificity for certain of the CB congeners is achieved using GC
columns that resolve these congeners.

2.5	Quantitative analysis is performed in one of two ways using selected
ion current profile (SICP) areas:

2.5.1	For the Toxics and the LOC CBs, the GC/MS is multi-point
calibrated and the concentration is determined using the isotope
dilution technique.

2.5.2	For all congeners other than the Toxics and LOC CBs, the GC/MS is
calibrated at a single concentration and the concentrations are
determined using the internal standard technique.  The internal
standards are the labeled congeners spiked into the sample, thus
affording recovery correction for all congeners.

2.5.3	For the labeled Toxics, labeled LOC CBs, and the cleanup
standards, the GC/MS is calibrated using replicates at a single
concentration and the concentrations of these labeled compounds are
determined using the internal standard technique.  The labeled injection
internal standards are determined using the internal standard technique.


2.6	The quality of the analysis is assured through reproducible
calibration and testing of the extraction, cleanup, and HRGC/HRMS
systems.

3.0	Definitions

Definitions are in the glossary at the end of this Method.

4.0	Contamination and interferences

4.1	Solvents, reagents, glassware, and other sample processing hardware
may yield artifacts, elevated baselines, and/or lock-mass suppression
causing misinterpretation of chromatograms.  Specific selection of
reagents and purification of solvents by distillation in all-glass
systems may be required.  Where possible, reagents are cleaned by
extraction or solvent rinse.  Environmentally abundant CBs have been
shown to be very difficult to completely eliminate from the laboratory
at levels lower than the MDLs in this Method (Table 2), and baking of
glassware in a kiln or furnace at 450 - 500 ºC may be necessary to
remove these and other contaminants.

4.2	Proper cleaning of glassware is extremely important, because
glassware may not only contaminate the samples but may also remove the
analytes of interest by adsorption on the glass surface.

4.2.1	Glassware should be rinsed with solvent and washed with a
detergent solution as soon after use as is practical.  Sonication of
glassware containing a detergent solution for approximately 30 seconds
may aid in cleaning.  Glassware with removable parts, particularly
separatory funnels with fluoropolymer stopcocks, must be disassembled
prior to detergent washing.

4.2.2	After detergent washing, glassware should be rinsed immediately,
first with methanol, then with hot tap water.  The tap water rinse is
followed by another methanol rinse, then acetone, and then methylene
chloride.

4.2.3	Baking of glassware in a kiln or other high temperature furnace
(300 - 500 ºC) may be warranted after particularly dirty samples are
encountered.  The kiln or furnace should be vented to prevent laboratory
contamination by CB vapors.  Baking should be minimized, as repeated
baking of glassware may cause active sites on the glass surface that may
irreversibly adsorb CBs.

4.2.4	Immediately prior to use, the Soxhlet apparatus should be
pre-extracted with toluene for approximately 3 hours (see Sections
12.3.1-12.3.3).  The extraction apparatus (Section 6.4) should be rinsed
with methylene chloride/toluene (80/20 mixture).

4.2.5	A separate set of glassware may to necessary to effectively
preclude contamination when low-level samples are analyzed.

4.2.6	Concentration of extracts by Kuderna-Danish (K-D) concentrator
and/or final concentration using nitrogen evaporation may help reduce
levels of background PCBs in samples. 

4.3	All materials used in the analysis must be demonstrated to be free
from interferences by running reference matrix Method blanks (Section
9.5) initially and with each sample batch (samples started through the
extraction process on a given 12-hour shift, to a maximum of 20
samples).

4.3.1	The reference matrix must simulate, as closely as possible, the
sample matrix under test.  Ideally, the reference matrix should not
contain the CBs in detectable amounts, but should contain potential
interferents in the concentrations expected to be found in the samples
to be analyzed.

4.3.2	When a reference matrix that simulates the sample matrix under
test is not available, reagent water (Section 7.6.1) can be used to
simulate water samples; playground sand (Section 7.6.2) or white quartz
sand (Section 7.3.2) can be used to simulate soils; filter paper
(Section 7.6.3) can be used to simulate papers and similar materials;
and corn oil (Section 7.6.4) can be used to simulate tissues.

4.4	Interferences co-extracted from samples will vary considerably from
source to source, depending on the diversity of the site being sampled. 
Interfering compounds may be present at concentrations several orders of
magnitude higher than the CBs.  The most frequently encountered
interferences are chlorinated dioxins and dibenzofurans, methoxy
biphenyls, hydroxydiphenyl ethers, benzylphenyl ethers, brominated
diphenyl ethers, polynuclear aromatics, polychlorinated naphthalenes,
and pesticides.  Because very low levels of CBs are measured by this
Method, elimination of interferences is essential.  The cleanup steps
given in Section 13 can be used to reduce or eliminate these
interferences and thereby permit reliable determination of the CBs at
the levels shown in Table 2.

4.5	Each piece of reusable glassware should be numbered to associate
that glassware with the processing of a particular sample.  This will
assist the laboratory in tracking possible sources of contamination for
individual samples, identifying glassware associated with highly
contaminated samples that may require extra cleaning, and determining
when glassware should be discarded.

4.6	Contamination of calibration solutions – The MDLs and MLs in Table
2 are the levels that can be achieved in the absence of laboratory
backgrounds.  Many of the MLs are greater than the equivalent
concentrations of the calibration solutions.  To prevent contamination,
calibration solutions must be prepared in an area free from CB
contamination using glassware free from contamination.  If these
requirements cannot be met or are difficult to meet in the laboratory,
the laboratory should prepare the calibration solutions in a
contamination-free facility or have a vendor prepare the calibration
standards and guarantee freedom from contamination.

4.7	Cleanup of tissue – The natural lipid content of tissue can
interfere in the analysis of tissue samples for the CBs.  The lipid
contents of different species and portions of tissue can vary widely. 
Lipids are soluble to varying degrees in various organic solvents and
may be present in sufficient quantity to overwhelm the column
chromatographic cleanup procedures used for cleanup of sample extracts. 
Lipids must be removed by the anthropogenic isolation column procedure
in Section 13.6, followed by the gel permeation chromatography procedure
in Section 13.2.  Florisil (Section 13.7) is recommended as an
additional cleanup step.

4.8	If the laboratory air is a potential source of CB contamination,
samples, reagents, glassware, and other materials should be dried in a
glove box or other area free from contamination.

5.0	Safety

5.1	The toxicity or carcinogenicity of each chemical used in this Method
has not been precisely determined; however, each compound should be
treated as a potential health hazard.  Exposure to these compounds
should be reduced to the lowest possible level.

5.1.1	PCBs have been tentatively classified as known or suspected human
or mammalian carcinogens.  On the basis of the available toxicological
and physical properties of the CBs, pure standards should be handled
only by highly trained personnel thoroughly familiar with handling and
cautionary procedures and the associated risks.

5.1.2	It is recommended that the laboratory purchase dilute standard
solutions of the analytes in this Method.  However, if primary solutions
are prepared, they must be prepared in a hood, and a NIOSH/MESA approved
toxic gas respirator must be worn when high concentrations are handled.

5.2	The laboratory is 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 (MSDSs) should also be made available to all personnel involved
in these analyses.  It is also suggested that the laboratory perform
personal hygiene monitoring of each analyst who uses this Method and
that the results of this monitoring be made available to the analyst. 
Additional information on laboratory safety can be found in References
5-8.  The references and bibliography at the end of Reference 7 are
particularly comprehensive in dealing with the general subject of
laboratory safety.

5.3	The pure CBs and samples suspected to contain these compounds are
handled using essentially the same techniques employed in handling
radioactive or infectious materials.  Well-ventilated, controlled access
laboratories are required.  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.  Each laboratory must
develop a strict safety program for handling these compounds.  The
practices in Reference 9 for handling chlorinated dibenzo-p-dioxins and
dibenzofurans (CDDs/CDFs) are also recommended for handling the CBs.

5.3.1	Facility – When finely divided samples (dusts, soils, dry
chemicals) are handled, all operations (including removal of samples
from sample containers, weighing, transferring, and mixing) should be
performed in a glove box demonstrated to be leak tight or in a fume hood
demonstrated to have adequate air flow.  Gross losses to the laboratory
ventilation system must not be allowed.  Handling of the dilute
solutions normally used in analytical and animal work presents no
inhalation hazards except in the case of an accident.

5.3.2	Protective equipment – Disposable plastic gloves, apron or lab
coat, safety glasses or mask, and a glove box or fume hood adequate for
radioactive work should be used.  During analytical operations that may
give rise to aerosols or dusts, personnel should wear respirators
equipped with activated carbon filters.  Eye protection (preferably full
face shields) must be worn while working with exposed samples or pure
analytical standards.  Latex gloves are commonly used to reduce exposure
of the hands.  When handling samples suspected or known to contain high
concentrations of the CBs, an additional set of gloves can also be worn
beneath the latex gloves.

5.3.3	Training – Workers must be trained in the proper method of
removing contaminated gloves and clothing without contacting the
exterior surfaces.

5.3.4	Personal hygiene – Hands and forearms should be washed
thoroughly after each manipulation and before breaks (coffee, lunch, and
shift).

5.3.5	Confinement – Isolated work areas posted with signs, segregated
glassware and tools, and plastic absorbent paper on bench tops will aid
in confining contamination.

5.3.6	Effluent vapors – The effluent of the sample splitter from the
gas chromatograph (GC) and from roughing pumps on the mass spectrometer
(MS) should pass through either a column of activated charcoal or be
bubbled through a trap containing oil or high-boiling alcohols to
condense CB vapors.

5.3.7	Waste Handling – Good technique includes minimizing contaminated
waste.  Plastic bag liners should be used in waste cans.  Janitors and
other personnel should be trained in the safe handling of waste.

5.3.8	Decontamination

5.3.8.1	Decontamination of personnel – Use any mild soap with plenty
of scrubbing action.

5.3.8.2	Glassware, tools, and surfaces – Chlorothene NU Solvent is a
less toxic solvent that should be effective in removing CBs. 
Satisfactory cleaning may be accomplished by rinsing with Chlorothene,
then washing with any detergent and water.  If glassware is first rinsed
with solvent, the wash water may be disposed of in the sewer.  Given the
cost of disposal, it is prudent to minimize solvent wastes.

5.3.9	Laundry – Clothing known to be contaminated should be collected
in plastic bags.  Persons that 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
of the potential problem.  The washer should be run through a cycle
before being used again for other clothing.

5.3.10	Wipe tests – A useful method of determining cleanliness of work
surfaces and tools is to perform a wipe test of the surface suspected of
being contaminated.

5.3.10.1	Using a piece of filter paper moistened with Chlorothene or
other solvent, wipe an area approximately 10 x 10 cm.

5.3.10.2	Extract and analyze the wipe by GC with an electron capture
detector (ECD) or by this Method.

5.3.10.3	Using the area wiped (e.g., 10 x 10 cm = 0.01 m2), calculate
the concentration in µg/m2.  A concentration less than 1 µg/m2
indicates acceptable cleanliness; anything higher warrants further
cleaning.  More than 100 µg/m2 constitutes an acute hazard and requires
prompt cleaning before further use of the equipment or work space, and
indicates that unacceptable work practices have been employed.

5.4	Biosolids samples may contain high concentrations of biohazards, and
must be handled with gloves and opened in a hood or biological safety
cabinet to prevent exposure.  Laboratory staff should know and observe
the safety procedures required in a microbiology laboratory that handles
pathogenic organisms when handling biosolids samples.

6.0	Apparatus and materials

Note:	 Brand names, suppliers, and part numbers are for illustration
purposes only and no endorsement is implied.  Equivalent performance may
be achieved using apparatus and materials other than those specified
here.  Meeting the performance requirements of this Method is the
responsibility of the laboratory. ADVANCE \d7 

6.1	Sampling equipment for discrete or composite sampling

6.1.1	Sample bottles and caps

6.1.1.1	Liquid samples (waters, sludges and similar materials containing
5 percent solids or less) – Sample bottle, amber glass, 1.1-L minimum,
with screw cap.

6.1.1.2	Solid samples (soils, sediments, sludges, paper pulps, filter
cake, compost, and similar materials that contain more than 5 percent
solids) – Sample bottle, wide mouth, amber glass, 500-mL minimum.

6.1.1.3	If amber bottles are not available, samples must be protected
from light.

6.1.1.4	Bottle caps – Threaded to fit sample bottles.  Caps must be
lined with fluoropolymer.

6.1.1.5	Cleaning

6.1.1.5.1	Bottles are detergent water washed, then solvent rinsed before
use.

6.1.1.5.2	Liners are detergent water washed and rinsed with reagent
water (Section 7.6.1).

6.1.2	Compositing equipment – Automatic or manual compositing system
incorporating glass containers cleaned per bottle cleaning procedure
above.  Only glass or fluoropolymer tubing must be used.  If the sampler
uses a peristaltic pump, a minimum length of compressible silicone
rubber tubing may be used in the pump only.  Before use, the tubing must
be thoroughly rinsed with methanol, followed by repeated rinsing with
reagent water to minimize sample contamination.  An integrating flow
meter is used to collect proportional composite samples.

6.2	Equipment for glassware cleaning

Note:	 If blanks from bottles or other glassware or with fewer cleaning
steps than required above show no detectable CB contamination,
unnecessary cleaning steps and equipment may be eliminated. ADVANCE \d7 

6.2.1	Laboratory sink with overhead fume hood

6.2.2	Kiln – Capable of reaching 450 ºC within 2 hours and
maintaining 450 - 500 ºC within ±10 ºC, with temperature controller
and safety switch (Cress Manufacturing Co., Santa Fe Springs, CA, B31H,
X31TS, or equivalent).  See the precautions in Section 4.2.3.

6.3	Equipment for sample preparation

6.3.1	Laboratory fume hood of sufficient size to contain the sample
preparation equipment listed below.

6.3.2	Glove box (optional)

6.3.3	Tissue homogenizer – VirTis Model 45 Macro homogenizer (American
Scientific Products H-3515, or equivalent) with stainless steel
Macro-shaft and Turbo-shear blade.

6.3.4	Meat grinder – Hobart, or equivalent, with 3- to 5-mm holes in
inner plate.

6.3.5	Equipment for determining percent moisture

6.3.5.1	Oven – Capable of maintaining a temperature of 110 ±5 ºC

6.3.5.2	Desiccator

6.3.6	Balances

6.3.6.1	Analytical – Capable of weighing 0.1 mg

6.3.6.2	Top loading – Capable of weighing 10 mg

6.4	Extraction apparatus

6.4.1	Water samples

6.4.1.1	pH meter, with combination glass electrode

6.4.1.2	pH paper, wide range (Hydrion Papers, or equivalent)

6.4.1.3	Graduated cylinder, 1-L capacity

6.4.1.4	Liquid/liquid extraction – Separatory funnels, 250-, 500-, and
2000-mL, with fluoropolymer stopcocks

6.4.1.5	Solid-phase extraction

6.4.1.5.1	1-L filtration apparatus, including glass funnel, frit
support, clamp, adapter, stopper, filtration flask, and vacuum tubing
(Figure 4).  For wastewater samples, the apparatus should accept 90 or
144 mm disks.  For drinking water or other samples containing low
solids, smaller disks may be used.

6.4.1.5.2	Vacuum source – Capable of maintaining 25 in. Hg, equipped
with shutoff valve and vacuum gauge

6.4.1.5.3	Glass-fiber filter – Whatman GMF 150 (or equivalent), 1
micron pore size, to fit filtration apparatus in Section 6.4.1.5.1

6.4.1.5.4	Solid-phase extraction disk containing octadecyl (C18) bonded
silica uniformly enmeshed in an inert matrix – Fisher Scientific
14-378F (or equivalent), to fit filtration apparatus in Section
6.4.1.5.1

6.4.1.6	Continuous liquid/liquid extraction (CLLE) – Fluoropolymer or
glass connecting joints and stopcocks without lubrication, 1.5-2 L
capacity (Hershberg-Wolf Extractor, Cal-Glass, Costa Mesa, California,
1000 mL or 2000 mL, or equivalent).

6.4.2	Soxhlet/Dean-Stark (SDS) extractor (Figure 5 and Reference 10) for
filters and solid/sludge samples

6.4.2.1	Soxhlet – 50-mm ID, 200-mL capacity with 500-mL flask
(Cal-Glass LG-6900, or equivalent, except substitute 500-mL round-bottom
flask for 300-mL flat-bottom flask)

6.4.2.2	Thimble – 43 ( 123 to fit Soxhlet (Cal-Glass LG-6901-122, or
equivalent)

6.4.2.3	Moisture trap – Dean Stark or Barret with fluoropolymer
stopcock, to fit Soxhlet

6.4.2.4	Heating mantle – Hemispherical, to fit 500-mL round-bottom
flask (Cal-Glass LG-8801-112, or equivalent)

6.4.2.5	Variable transformer – Powerstat (or equivalent), 110-volt,
10-amp

6.4.3	Beakers – 400- to 500-mL

6.4.4	Spatulas – Stainless steel

6.5	Filtration apparatus

6.5.1	Pyrex glass wool – Solvent-extracted using a Soxhlet or SDS
extractor for 3 hours minimum

6.5.2	Glass funnel – 125- to 250-mL

6.5.3	Glass-fiber filter paper – Whatman GF/D (or equivalent), to fit
glass funnel in Section 6.5.2.

6.5.4	Drying column – 15- to 20-mm ID Pyrex chromatographic column
equipped with coarse-glass frit or glass-wool plug

6.5.5	Buchner funnel – 15-cm

6.5.6	Glass-fiber filter paper for Buchner funnel above

6.5.7	Filtration flasks – 1.5- to 2.0-L, with side arm

6.5.8	Pressure filtration apparatus – Millipore YT30 142 HW, or
equivalent

6.6	Centrifuge apparatus

6.6.1	Centrifuge – Capable of rotating 500-mL centrifuge bottles or
15-mL centrifuge tubes at 5,000 rpm minimum

6.6.2	Centrifuge bottles – 500-mL, with screw-caps, to fit centrifuge

6.6.3	Centrifuge tubes – 12- to 15-mL, with screw-caps, to fit
centrifuge

6.7	Cleanup apparatus

6.7.1	Automated gel permeation chromatograph (Analytical Biochemical
Labs, Inc, Columbia, MO, Model GPC Autoprep 1002, or equivalent)

6.7.1.1	Column – 600-700 mm long ( 25 mm ID glass, packed with 70 g of
200-400 mesh SX-3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or
equivalent)

6.7.1.2	Syringe – 10-mL, with Luer fitting

6.7.1.3	Syringe filter holder – stainless steel, and glass-fiber or
fluoropolymer filters (Gelman 4310, or equivalent)

6.7.1.4	UV detectors – 254-nm, preparative or semi-preparative flow
cell (Isco, Inc., Type 6; Schmadzu, 5-mm path length; Beckman-Altex
152W, 8-µL micro-prep flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow
cell; LDC Milton-Roy UV-3, monitor #1203; or equivalent).

6.7.2	Reverse-phase high-performance liquid chromatograph (Reference 4)

6.7.2.1	Pump – Perkin-Elmer Series 410, or equivalent

6.7.2.2	Injector – Perkin-Elmer ISS-100 Autosampler, or equivalent

6.7.2.3	6-Port switching valve – Valco N60, or equivalent

6.7.2.4	Column – Hypercarb, 100 x 4.6 mm, 5 µm particle size,
Keystone Scientific, or equivalent

6.7.2.5	Detector – Altex 110A (or equivalent) operated at 0.02 AUFS at
235 nm

6.7.2.6	Fraction collector – Isco Foxy II, or equivalent

6.7.3	Pipets

6.7.3.1	Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific
13-678-6A, or equivalent)

6.7.3.2	Disposable, serological, 50-mL (8- to 10- mm ID)

6.7.4	Glass chromatographic columns

6.7.4.1	150-mm long x 8-mm ID, (Kontes K-420155, or equivalent) with
coarse-glass frit or glass-wool plug and 250-mL reservoir

6.7.4.2	200-mm long x 15-mm ID, with coarse-glass frit or glass-wool
plug and 250-mL reservoir

6.7.4.3	300-mm long x 22-mm ID, with coarse-glass frit, 300-mL
reservoir, and glass or fluoropolymer stopcock

6.7.5	Oven – For baking and storage of adsorbents, capable of
maintaining a constant temperature ( ± 5 ºC) in the range of 105-250
ºC

6.8	Concentration apparatus

6.8.1	Rotary evaporator – Buchi/Brinkman-American Scientific No.
E5045-10 or equivalent, equipped with a variable temperature water bath

6.8.1.1	Vacuum source for rotary evaporator equipped with shutoff valve
at the evaporator and vacuum gauge

6.8.1.2	A recirculating water pump and chiller are recommended, as use
of tap water for cooling the evaporator wastes large volumes of water
and can lead to inconsistent performance as water temperatures and
pressures vary.

6.8.1.3	Round-bottom flask – 100-mL and 500-mL or larger, with
ground-glass fitting compatible with the rotary evaporator

6.8.2	Kuderna-Danish (K-D) concentrator

6.8.2.1	Concentrator tube – 10-mL, graduated (Kontes K-570050-1025, or
equivalent) with calibration verified.  Ground-glass stopper (size 19/22
joint) is used to prevent evaporation of extracts.

6.8.2.2	Evaporation flask – 500-mL (Kontes K-570001-0500, or
equivalent), attached to concentrator tube with springs (Kontes
K-662750-0012 or equivalent)

6.8.2.3	Snyder column – Three-ball macro (Kontes K-503000-0232, or
equivalent)

6.8.2.4	Boiling chips

6.8.2.4.1	Glass or silicon carbide – Approximately 10/40 mesh,
extracted with methylene chloride and baked at 450 ºC for one hour
minimum

6.8.2.4.2	Fluoropolymer (optional) – Extracted with methylene chloride

6.8.2.5	Water bath – Heated, with concentric ring cover, capable of
maintaining a temperature within ± 2 ºC, installed in a fume hood

6.8.3	Nitrogen evaporation apparatus – Equipped with water bath
controlled in the range of 30 - 60 ºC (N-Evap, Organomation Associates,
Inc., South Berlin, MA, or equivalent), installed in a fume hood

6.8.4	Sample vials

6.8.4.1	Amber glass, 2- to 5-mL with fluoropolymer-lined screw-cap

6.8.4.2	Glass, 0.3-mL, conical, with fluoropolymer-lined screw or crimp
cap

6.9	Gas chromatograph – Must have splitless or on-column injection
port for capillary column, temperature program with isothermal hold, and
must meet all of the performance specifications in Section 10.

6.9.1	GC column – Any GC column or column system (2 or more columns)
that provides unique resolution and identification of the Toxics for
determination of a TEQPCB using TEFs (Reference 1).  Isomers may be
unresolved so long as they have the same TEF and response factor and so
long as these unresolved isomers are uniquely resolved from all other
congeners.  For example, the SPB-octyl column (Section 6.9.1.3) achieves
unique GC resolution of all Toxics except congeners with congener
numbers 156 and 157.  This isomeric pair is uniquely resolved from all
other congeners and these congeners have the same TEF and response
factor.

6.9.1.1	If an SPB-octyl column is used, it must meet the specification
in Section 6.9.1 and the following additional specifications:

6.9.1.1.1	The retention time for decachlorobiphenyl (DeCB; PCB 209) must
be greater than 55 minutes.

6.9.1.1.2	The column must uniquely resolve congeners 34 from 23 and 187
from 182, and congeners 156 and 157 must co-elute within 2 seconds at
the peak maximum.  Unique resolution means a valley height less than 40
percent of the shorter of the two peaks that result when the Diluted
combined 209 congener solution (Section 7.10.2.2) is analyzed (see
Figures 6 and 7).

6.9.1.1.3	The column must be replaced when any of the criteria in
Sections 6.9.1 - 6.9.1.1.2 are not met.

6.9.1.2	If a column or column system alternate to the SPB-octyl column
is used, specifications similar to those for the SPB-octyl column
(Sections 6.9.1 - 6.9.1.1.2) must be developed and be functionally
equivalent to those specifications.

Suggested column – 30 ± 5-m long x 0.25 ± 0.02-mm ID; 0.25-µm film
SPB-octyl (Supelco 2-4218, or equivalent).  This column is capable of
meeting the requirements in Sections 6.9.1 - 6.9.1.1.2.

Note:	The SPB-octyl column is subject to rapid degradation when exposed
to oxygen.  The analyst should exclude oxygen from the carrier gas,
should eliminate air leaks, and should cool the injector, column, and
transfer line before opening the column to the atmosphere.  For further
information on precluding oxidation, contact the column manufacturer.
ADVANCE \d7 

6.9.1.4	Column for resolution of additional congeners – See Appendix A
for details on the DB-1 column.  The DB-1 column is optional and is
capable of uniquely resolving the congener pair with congener numbers
156 and 157.  When used in combination with the SPB-octyl column
(Section 6.9.1.3), the two-column system is capable of resolving a total
of approximately 180 CB congeners.

6.10	Mass spectrometer – 28- to 40-eV electron impact ionization, must
be capable of selectively monitoring a minimum of 22 exact m/z’s
minimum at high resolution ((10,000) during a period less than 1.5
seconds, and must meet all of the performance specifications in Section
10.

6.11	GC/MS interface – The mass spectrometer (MS) must be interfaced
to the GC such that the end of the capillary column terminates within 1
cm of the ion source but does not intercept the electron or ion beams.

6.12	Data system – Capable of collecting, recording, storing, and
processing MS data

6.12.1	Data acquisition – The signal at each exact m/z must be
collected repetitively throughout the monitoring period and stored on a
mass storage device.

6.12.2	Response factors and multipoint calibrations – The data system
must record and maintain lists of response factors (response ratios for
isotope dilution) and multipoint calibrations.  Computations of relative
standard deviation (RSD) are be used to test calibration linearity. 
Statistics on initial (Section 9.4) and ongoing (Section 15.5.4)
performance should be computed and maintained, either on the instrument
data system, or on a separate computer system.

7.0	Reagents and standards

7.1	pH adjustment and back-extraction

7.1.1	Potassium hydroxide – Dissolve 20 g reagent grade KOH in 100 mL
reagent water.

7.1.2	Sulfuric acid – Reagent grade (specific gravity 1.84)

7.1.3	Hydrochloric acid – Reagent grade, 6N

7.1.4	Sodium chloride – Reagent grade, prepare at 5% (w/v) solution in
reagent water

7.2	Solution drying and evaporation

7.2.1	Solution drying – Sodium sulfate, reagent grade, granular,
anhydrous (Baker 3375, or equivalent), rinsed with methylene chloride
(20 mL/g), baked at 400 ºC for 1 hour minimum, cooled in a desiccator,
and stored in a pre-cleaned glass bottle with screw-cap that prevents
moisture from entering.  If, after heating, the sodium sulfate develops
a noticeable grayish cast (due to the presence of carbon  in the crystal
matrix), that batch of reagent is not suitable for use and should be
discarded.  Extraction with methylene chloride (as opposed to simple
rinsing) and baking at a lower temperature may produce sodium sulfate
that is suitable for use.

7.2.2	Tissue drying – Sodium sulfate, reagent grade, powdered, treated
and stored as in Section 7.2.1

7.2.3	Prepurified nitrogen

7.3	Extraction

7.3.1	Solvents – Acetone, toluene, cyclohexane, hexane, methanol,
methylene chloride, isooctane, and nonane; distilled in glass, pesticide
quality, lot-certified to be free of interferences

Note:	 Some solvents;  e.g., isooctane and nonane, may need to be
re-distilled to eliminate CB backgrounds. ADVANCE \d7 

7.3.2	White quartz sand, 60/70 mesh – For Soxhlet/Dean-Stark
extraction (Aldrich Chemical, Cat. No. 27-437-9, or equivalent).  Bake
at 450 ºC for 4 hour minimum.

7.4	GPC calibration solution – Prepare a solution containing 2.5 mg/mL
corn oil, 0.05  mg/mL bis(2-ethylhexyl) phthalate (BEHP), 0.01 mg/mL
methoxychlor, 0.002 mg/mL perylene, and 0.008 mg/mL sulfur, or at
concentrations appropriate to the response of the detector.

7.5	Adsorbents for sample cleanup

7.5.1	Silica gel

7.5.1.1	Activated silica gel – 100-200 mesh, Supelco 1-3651 (or
equivalent), 100-200 mesh, rinsed with methylene chloride, baked at 180
ºC for a minimum of 1 hour, cooled in a desiccator, and stored in a
precleaned glass bottle with screw-cap that prevents moisture from
entering.

7.5.1.2	Acid silica gel (30% w/w) – Thoroughly mix 44 g of
concentrated sulfuric acid with 100 g of activated silica gel in a clean
container.  Break up aggregates with a stirring rod until a uniform
mixture is obtained.  Store in a screw-capped bottle with
fluoropolymer-lined cap.

7.5.1.3	Basic silica gel – Thoroughly mix 30 g of 1N sodium hydroxide
with 100 g of activated silica gel in a clean container.  Break up 
aggregates with a stirring rod until a uniform mixture is obtained. 
Store in a screw-capped bottle with fluoropolymer-lined cap.

7.5.1.4	Potassium silicate

7.5.1.4.1	Dissolve 56 g of high purity potassium hydroxide (Aldrich, or
equivalent) in 300 mL of methanol in a 750- to 1000-mL flat-bottom
flask.

7.5.1.4.2	Add 100 g of activated silica gel (Section 7.5.1.1) and a
stirring bar, and stir on an explosion-proof hot plate at 60-70 ºC for
1-2 hours.

7.5.1.4.3	Decant the liquid and rinse the potassium silicate twice with
100-mL portions of methanol, followed by a single rinse with 100 mL of
methylene chloride.

7.5.1.4.4	Spread the potassium silicate on solvent-rinsed aluminum foil
and dry for 2-4 hours in a hood.  Observe the precaution in Section 4.8.

7.5.1.4.5	Activate overnight at 200-250 ºC prior to use.

7.5.2	Carbon

7.5.2.1	Carbopak C – (Supelco 1-0258, or equivalent)

7.5.2.2	Celite 545 – (Supelco 2-0199, or equivalent)

7.5.2.3	Thoroughly mix 18.0 g Carbopak C and 18.0 g Celite 545 to
produce a 50% w/w mixture.  Activate the mixture at 130 ºC for a
minimum of 6 hours.  Store in a desiccator.

Note:	The carbon column has been included in this Method to allow
separation of co-planar congeners 77, 126, and 169 from other congeners
and interferences, should such separation be desired. ADVANCE \d7 

7.5.3	Anthropogenic isolation column – Pack the column in Section
6.7.4.3 from bottom to top with the following:

7.5.3.1	2 g silica gel (Section 7.5.1.1)

7.5.3.2	2 g potassium silicate (Section 7.5.1.4)

7.5.3.3	2 g granular anhydrous sodium sulfate (Section 7.2.1)

7.5.3.4	10 g acid silica gel (Section 7.5.1.2)

7.5.3.5	2 g granular anhydrous sodium sulfate

7.5.4	Florisil column

7.5.4.1	Florisil – PR grade, 60-100 mesh (U.S. Silica Corp, Berkeley
Springs, WV, or equivalent).  Alternatively, prepacked Florisil columns
may be used.  Use the following procedure for Florisil activation and
column packing.

7.5.4.1.1	Fill a clean 1- to 2-L bottle ½ to 2/3 full with Florisil and
place in an oven at 130-150 ºC for a minimum of three days to activate
the Florisil.

7.5.4.1.2	Immediately prior to use, dry pack a 300-mm x 22-mm ID glass
column (Section 6.7.4.3) bottom to top with  0.5-1.0 cm of warm to hot
anhydrous sodium sulfate (Section 7.2.1), 10-10.5 cm of warm to hot
activated Florisil (Section 7.5.4.1.1), and 1-2 cm of warm to hot
anhydrous sodium sulfate.  Allow the column to cool and wet immediately
with 100 mL of n-hexane to prevent water from entering.

7.5.4.2	Using the procedure in Section 13.7.3, establish the elution
pattern for each carton of Florisil or each lot of Florisil columns
received.

7.6	Reference matrices – Matrices in which the CBs and interfering
compounds are not detected by this Method

7.6.1	Reagent water – Bottled water purchased locally, or prepared by
passage through activated carbon

7.6.2	High-solids reference matrix – Playground sand or similar
material.  Prepared by extraction with methylene chloride and/or baking
at 450 ºC for a minimum of 4 hours.

7.6.3	Paper reference matrix – Glass-fiber filter, Gelman type A, or
equivalent.  Cut paper to simulate the surface area of the paper sample
being tested.

7.6.4	Tissue reference matrix – Corn or other vegetable oil.

7.6.5	Other matrices – This Method may be verified on any reference
matrix by performing the tests in Section 9.2.  Ideally, the matrix
should be free of the CBs, but in no case must the background level of
the CBs in the reference matrix exceed the minimum levels in Table 2. 
If low background levels of the CBs are present in the reference matrix,
the spike level of the analytes used in Section 9.2 should be increased
to provide a spike-to-background ratio of approximately 5 (Reference
11).

7.7	Standard solutions – Prepare from materials of known purity and
composition or purchase as solutions or mixtures with certification to
their purity, concentration, and authenticity.  If the chemical purity
is 98 % or greater, the weight may be used without correction to
calculate the concentration of the standard.  Observe the safety
precautions in Section 5 and the recommendation in Section 5.1.2.

Note:	Native PCB standards are available from several suppliers. 
13C12-labeled congeners are available from Cambridge Isotope
Laboratories and Wellington Laboratories, and may be available from
other suppliers.  Listing of these suppliers does not constitute a
recommendation or endorsement for use.  Part numbers are for reference
only. ADVANCE \d7 

7.7.1	For preparation of stock solutions from neat materials, dissolve
an appropriate amount of assayed reference material in solvent.  For
example, weigh 10 to 20 mg  of PCB 126 to three significant figures in a
10-mL ground-glass-stoppered volumetric flask and fill to the mark with
nonane.  After the compound is completely dissolved, transfer the
solution to a clean 15-mL vial with fluoropolymer-lined cap.

7.7.2	When not being used, store standard solutions in the dark at room
temperature in screw-capped vials with fluoropolymer-lined caps.  Place
a mark on the vial at the level of the solution so that solvent loss by
evaporation can be detected.  Replace the solution if solvent loss has
occurred.

7.8	Native (unlabeled) stock solutions

Note:	 Some of the part numbers for solutions described below contain
the identifier “1668A.”  These part numbers remain valid for Method
1668C. ADVANCE \d7 

7.8.1	Native Toxics/LOC stock solution – Prepare to contain the native
Toxics and LOC CBs at the concentrations shown in Table 3, or purchase
Accu-Standard M1668A-C-NT-LOC-WD-GCPC, or equivalent.  If additional CBs
are to be determined by isotope dilution (e.g., 170 and 180), include
the additional native compounds in this stock solution.

7.8.2	Native 209 CB congener stock solutions – Solutions containing CB
congeners to calibrate the SPB-octyl column.

Note:	 If a column other than the SPB-octyl column is used, solutions
that will allow separation of all 209 congeners on that column must be
prepared. ADVANCE \d7 

7.8.2.1	Native congener mix stock solutions for separation of individual
congeners on the SPB-octyl column – Prepare the five solutions with
the congeners listed in Table 4 at the concentrations shown in Table 3
or purchase Accu-Standard M-1668A-1, M-1668A-2, M-1668A-3, M-1668-4, and
M-1668-5, or equivalent.

7.8.2.2	Combined 209 congener stock solution – Combine equal volumes
of the standards in Section 7.8.2.1 to form a stock solution containing 
all CB congeners.  This solution will be at 1/5 the concentration of 
the 5 individual solutions.

7.8.3	Stock solutions should be checked for signs of degradation prior
to preparation of calibration or performance test standards.  Reference
standards that can be used to determine the accuracy of standard
solutions are available from several vendors.

7.9	Labeled compound stock solutions (Table 3)

7.9.1	Labeled Toxics/LOC/window-defining stock solution – Prepare in
isooctane or nonane at the concentrations in Table 3 or purchase
Cambridge Isotope Laboratories (CIL) EC-4977, or equivalent.  If
additional CBs are to be determined by isotope dilution (e.g., 170 and
180), include the additional labeled compounds in this stock solution.

7.9.2	Labeled cleanup standard stock solution – Prepare labeled CBs
28, 111, and 178 in iso-octane or nonane at the concentration shown in
Table 3 or purchase CIL EC-4978, or equivalent.

7.9.3	Labeled injection internal standard stock solution – Prepare
labeled CBs 9, 52, 101, 138, and 194 in nonane or isooctane at the
concentrations shown in Table 3, or purchase CIL EC-4979, or equivalent.

7.10	Calibration standards

7.10.1	Calibration standards – Combine and dilute the solutions in
Sections 7.8.1 and 7.9 to produce the calibration solutions in Table 5
or purchase CIL EC-4976, or equivalent, for the CS-1 to CS-5 set of
calibration solutions.  If a 6-point calibration is used, prepare the
CS-0.2 solution or purchase CIL EC-4976-0.2, or equivalent.  These
solutions permit the relative response (labeled to native) and response
factor to be measured as a function of concentration.  The CS-3 standard
(CIL EC-4976-3, or equivalent) is used for calibration verification
(VER).

7.10.2	Solutions of congener mixes

7.10.2.1	Diluted individual solutions

7.10.2.1.1	The 5 individual solutions, when analyzed individually, allow
resolution of all 209 congeners on the SPB-octyl column, and are used
for establishing retention time and other data for each congener.  The
elution order of the congeners present in each of the 5 solutions
(Section 7.8.2.1) is given in Table 4.

7.10.2.1.2	Individually combine an aliquot of each individual mix stock
solution (Section 7.8.2.1) with an aliquot of the Labeled
Toxics/LOC/window-defining stock solution (Section 7.9.1), the Labeled
cleanup standard stock solution (Section 7.9.2), and the Labeled
injection internal standard stock solution (7.9.3) to produce
concentrations of 100 ng/mL for the labeled compounds and 25, 50, and 75
ng/mL for the MoCB-TrCB, TeCB-HpCB, and OcCB-DeCB congeners,
respectively, as shown in Table 3. 

7.10.2.2	Diluted combined 209 congener solution

7.10.2.2.1	This solution combines the 5 individual mixes with the
labeled compounds to allow single-point calibration of the congeners not
included in the multi-point calibration, and establishes an average
response factor for the co-eluting isomeric congeners.

7.10.2.2.2	Combine an aliquot of the combined 209 congener solution
(Section 7.8.2.2) with an aliquot of the Labeled
Toxics/LOC/window-defining stock solution (Section 7.9.1), the Labeled
cleanup standard stock solution (Section 7.9.2), and the Labeled
injection internal standard stock solution (7.9.3) to produce the same
concentrations as in the diluted individual mix solutions (Section
7.10.2.1.2 and Table 3).

7.11	Native Toxics/LOC standard spiking solution – Used for
determining initial precision and recovery (IPR; Section 9.2) and
ongoing precision and recovery (OPR; Section 15.5).  Dilute the Native
Toxics/LOC stock solution (Section 7.8.1) with acetone to produce a
concentration of the Toxics at 1 ng/mL, as shown in Table 3.  When 1 mL
of this solution spiked into the IPR (Section 9.2.1) or OPR (Section
15.5) and concentrated  to a final volume of 20 µL, the concentration
in the final volume will be 50 ng/mL (50 pg/µL).  Prepare only the
amount necessary for each reference matrix with each sample batch.

7.12	Labeled Toxics/LOC/window-defining standard spiking solution –
This solution is spiked into each sample (Section 9.3) and into the IPR
(Section 9.2.1), OPR (Section 15.5), and blank (Section 9.5) to measure
recovery.  Dilute the Labeled Toxics/LOC/window-defining stock solution
(Section 7.9.1) with acetone to produce a concentration of the labeled
compounds at 2 ng/mL, as shown in Table 3.  When 1 mL of this solution
is spiked into an IPR, OPR, blank, or sample and concentrated to a final
extract volume  of 20 µL, the concentration in the final extract volume
will be 100 ng/mL (100 pg/µL).  Prepare only the amount necessary for
each reference matrix with each sample batch.

7.13	Labeled cleanup standard spiking solution – This solution is
spiked into each extract prior to cleanup to measure the efficiency of
the cleanup process.  Dilute the Labeled cleanup standard stock solution
(Section 7.9.2) in methylene chloride to produce a concentration of the
cleanup standards at 2 ng/mL, as shown in Table 3.  When 1 mL of this
solution is spiked into a sample extract and concentrated to a final
volume of 20 µL, the concentration in the final volume will be 100
ng/mL (100 pg/µL).

7.14	Labeled injection internal standard spiking solution – This
solution is added to each concentrated extract prior to injection into
the HRGC/HRMS.  Dilute the Labeled injection internal standard stock
solution (Section 7.9.3) in nonane to produce a concentration of the
injection internal standards at 1000 ng/mL, as shown in Table 3.  When 2
µL of this solution is spiked into a 20 µL extract, the concentration
of each injection internal standard will be nominally 100 ng/mL (100
pg/µL).

Note:	  The addition of 2 µL of the Labeled injection internal standard
spiking solution to a 20-µL final extract has the effect of diluting
the concentration of the components in the extract by 10%.  Provided all
calibration solutions and all extracts undergo this dilution as a result
of adding the Labeled injection internal standard spiking solution, the
effect of the 10% solution is compensated, and correction for this
dilution should not be made. ADVANCE \d7 

7.15	QC Check Sample – A QC Check Sample should be obtained from a
source independent of the calibration standards.  Ideally, this check
sample would be a certified Standard Reference Material (SRM) containing
the CBs in known concentrations in a sample matrix similar to the matrix
under test.  The National Institute of Standards and Technology (NIST)
in Gaithersburg, Maryland has SRMs, and the Institute for National
Measurement Standards of the National Research Council of Canada in
Ottawa has certified reference materials (CRMs) for CBs in various
matrices.

7.16	Stability of solutions – Standard solutions used for quantitative
purposes (Sections 7.9 through 7.14) should be assayed periodically
(e.g., every 6 months) against SRMs from NIST (if available), or
certified reference materials from a source that will attest to the
authenticity and concentration, to assure that the composition and
concentrations have not changed.

8.0	Sample collection, preservation, storage, and holding times

8.1	Collect samples in amber glass containers following conventional
sampling practices (Reference 12).  Other sample collection techniques,
or sample volumes may be used, if documented.

8.2	Aqueous samples

8.2.1	Samples that flow freely are collected as grab samples or in
refrigerated bottles using automatic sampling equipment.  Collect one
liter (or a larger or smaller volume) of sample sufficient to meet
project needs.

8.2.2	If residual chlorine is present, add 80 mg sodium thiosulfate per
liter of water.  EPA Methods 330.4 and 330.5 may be used to measure
residual chlorine (Reference 13).

8.2.3	Maintain aqueous samples in the dark at less than 6 ºC from the
time of collection until receipt at the laboratory.  If the sample will
be frozen, allow room for expansion.  Store in the dark at less than 6
ºC.

8.3	Solid, mixed-phase, semi-solid, and oily samples, excluding tissue.

8.3.1	Collect samples as grab samples using wide-mouth jars.

8.3.2	Maintain solid, semi-solid, oily, and mixed-phase samples in the
dark at less than 6 ºC from the time of collection until receipt at the
laboratory.  Store solid, semi-solid, oily, and mixed-phase samples in
the dark at less than -10 ºC.

8.4	Fish and other tissue samples

8.4.1	Fish may be cleaned, filleted, or processed in other ways in the
field, such that the laboratory may expect to receive whole fish, fish
fillets, or other tissues for analysis.

8.4.2	Collect fish, wrap in aluminum foil, and maintain at less than 6
ºC from the time of collection until receipt at the laboratory, to a
maximum time of 24 hours.  If a longer transport time is necessary,
freeze the sample.  Ideally, fish should be frozen upon collection and
shipped to the laboratory on dry ice.

8.4.3	Freeze tissue samples upon receipt at the laboratory and maintain
them in the dark at less than -10 ºC until prepared.  Maintain unused
sample in the dark at less than -10 ºC.

8.5	Holding times

8.5.1	There are no demonstrated maximum holding times associated with
the CBs in aqueous, solid, semi-solid, tissue, or other sample matrices.
 If stored in the dark at less than 6 ºC, aqueous samples may be stored
for up to one year.  Similarly, if stored in the dark at less than -10
ºC, solid, semi-solid, multi-phase, and tissue samples may be stored
for up to one year.

8.5.2	Store sample extracts in the dark at less than -10 ºC until
analyzed.  If stored in the dark at less than -10 ºC, sample extracts
may be stored for one year.

9.0	Quality assurance/quality control

9.1	Each laboratory that uses this Method is required to operate a
formal quality assurance program (Reference 14).  The minimum
requirements of this program consist of an initial demonstration of
laboratory capability, analysis of samples spiked with labeled compounds
to evaluate and document data quality, and analysis of standards and
blanks as tests of continued performance.  Laboratory performance is
compared to established performance criteria to determine if the results
of analyses meet the performance characteristics of the Method.

If the Method is to be applied to sample matrix other than water (e.g.,
soils, filter cake, compost, tissue) the most appropriate alternate
reference matrix (Sections 7.6.2 - 7.6.5  and 7.15) is substituted for
the reagent water matrix (Section 7.6.1) in all performance tests.

9.1.1	The laboratory must make an initial demonstration of the ability
to generate acceptable precision and recovery with this Method.  This
demonstration is given in Section 9.2.

9.1.2	In recognition of advances that are occurring in analytical
technology, and to overcome matrix interferences, the laboratory is
permitted certain options to improve separations or lower the costs of
measurements.  These options include alternate extraction,
concentration, and cleanup procedures, and changes in sample volumes,
columns and detectors.  Alternate determinative techniques, such as
substitution of spectroscopic or immunoassay techniques for HRGC/HRMS
technology, and changes that degrade Method performance, are not allowed
without prior review and approval.  If an analytical technique other
than the techniques specified in this Method is used, that technique
must have a specificity equal to or greater than the specificity of the
techniques in this Method for the analytes of interest. (Note: For
additional flexibility to make modifications without prior EPA review
see 40 CFR Part 136.6.)

9.1.2.1	Each time a modification is made to this Method, the laboratory
is required to repeat the procedure in Section 9.2.  If MDLs would be
affected by the change, the laboratory is required to demonstrate that
the MDLs (40 CFR Part 136, Appendix B) are lower than one-third the
regulatory compliance level or lower than five times the MDLs in this
Method, whichever are greater.  If calibration will be affected by the
change, the instrument must be recalibrated per Section 10.  Once the
modification is demonstrated to produce results equivalent or superior
to results produced by this Method as written, that modification may be
used routinely thereafter, so long as the other requirements in this
Method are met (e.g., labeled compound recovery).

9.1.2.2	The laboratory is required to maintain records of modifications
made to this Method.  These records include the following, at a minimum:

9.1.2.2.1	The names, titles, addresses, and telephone numbers of the
analyst(s) that performed the analyses and modification, and of the
quality control officer that witnessed and will verify the analyses and
modifications.

9.1.2.2.2	A listing of pollutant(s) measured, by name and CAS Registry
number.

9.1.2.2.3	A narrative stating reason(s) for the modifications (see
Section 1.5).

9.1.2.2.4	Results from all quality control (QC) tests comparing the
modified method to this Method, including:

a)	Calibration (Section 10).

b)	Calibration verification (Section 15.3).

c)	Initial precision and recovery (Section 9.2).

d)	Labeled compound recovery (Section 9.3).

e)	Analysis of blanks (Section 9.5).

f)	Accuracy assessment (Section 9.4).

9.1.2.2.5	Data that will allow an independent reviewer to validate each
determination by tracing the instrument output (peak height, area, or
other signal) to the final result.  These data are to include:

a)	Sample numbers and other identifiers.

b)	Extraction dates.

c)	Analysis dates and times.

d)	Analysis sequence/run chronology.

e)	Sample weight or volume (Section 11).

f)	Extract volume prior to each cleanup step (Section 13).

g)	Extract volume after each cleanup step (Section 13).

h)	Final extract volume prior to injection (Section 14).

i)	Injection volume (Section 14.3).

j)	Dilution data, differentiating between dilution of a sample or
extract (Section 17.5).

k)	Instrument and operating conditions.

l)	Column (dimensions, liquid phase, solid support, film thickness,
etc).

m)	Operating conditions (temperatures, temperature program, flow rates).

n)	Detector (type, operating conditions, etc).

o)	Chromatograms, printer tapes, and other recordings of raw data.

p)	Quantitation reports, data system outputs, and other data to link the
raw data to the results reported.

9.1.2.3	Alternate HRGC columns and column systems – See Sections
6.9.1.  If a column or column system alternate to those specified in
this Method is used, that column or column system must meet the
requirements in Section 6.9.1 - 6.9.1.1.3.

9.1.3	Analyses of Method blanks are required to demonstrate freedom from
contamination (Section 4.3).  The procedures and criteria for analysis
of a Method blank are described in Sections 9.5 and 15.6.

9.1.4	The laboratory must spike all samples with labeled compounds to
monitor Method performance.  This test is described in Section 9.3. 
When results of these spikes indicate atypical Method performance for
samples, the samples are diluted to bring Method performance within
acceptable limits.  Procedures for dilution are given in Section 17.5.

9.1.5	The laboratory must, on an ongoing basis, demonstrate through
calibration verification and the analysis of the ongoing precision and
recovery standard (OPR) and blanks that the analytical system is in
control.  These procedures are given in Sections 15.1 through 15.6.

9.1.6	The laboratory should maintain records to define the quality of
data generated.  Development of accuracy statements is described in
Section 9.4.

9.2	Initial precision and recovery (IPR) – To establish the ability to
generate acceptable precision and recovery, the laboratory must perform
the following operations.

9.2.1	For low solids (aqueous) samples, extract, concentrate, and
analyze four 1-L aliquots of reagent water spiked with 1 mL each of the
Native Toxics/LOC spiking solution (Section 7.11), the Labeled
Toxics/LOC/window-defining standard spiking solution (Section 7.12), and
the Labeled cleanup standard  spiking solution (Section 7.13), according
to the procedures in Sections 11 through 18.  For an alternative sample
matrix, four aliquots of the alternative reference matrix (Section 7.6)
are used.  All sample processing steps that are to  be used for
processing samples, including preparation (Section 11), extraction
(Section 12), and cleanup (Section 13), must be included in this test.

9.2.2	Using results of the set of four analyses, compute the average
percent recovery (X) of the extracts and the relative standard deviation
(RSD) of the concentration for each compound, by isotope dilution for
CBs with a labeled analog, and by internal standard for CBs without a
labeled analog and for the labeled  compounds.

9.2.3	For each CB and labeled compound, compare RSD and X with the
corresponding limits for initial precision and recovery in Table 6.  If
RSD and X for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. 
If, however, any individual RSD exceeds the precision limit or any
individual X falls outside the range for recovery, system performance is
unacceptable for that compound.  Correct the problem and repeat the test
(Section 9.2).

9.3	To assess Method performance on the sample matrix, the laboratory
must spike all samples with the Labeled Toxics/LOC/window-defining
standard spiking solution (Section 7.12) and all sample extracts with
the Labeled cleanup standard spiking solution (Section 7.13).

9.3.1	Analyze each sample according to the procedures in Sections 11
through 18.

9.3.2	Compute the percent recovery of the labeled
Toxics/LOC/window-defining congeners and the labeled cleanup congeners
using the internal standard method (Section 17.2).

9.3.3	The recovery of each labeled compound must be within the limits in
Table 6.  If the recovery of any compound falls outside of these limits,
Method performance  is unacceptable for that compound in that sample. 
Additional cleanup procedures must then be employed to attempt to bring
the recovery within the normal range.  If the recovery cannot be brought
within the normal range after all cleanup procedures have been employed,
water samples are diluted and smaller amounts of soils, sludges,
sediments, and other matrices are analyzed per Section 18.

9.4	It is suggested, but not required, that recovery of labeled
compounds from samples be assessed and records maintained.

9.4.1	After the analysis of 30 samples of a given matrix type (water,
soil, sludge, pulp, etc.) for which the labeled compounds pass the tests
in Section 9.3, compute the average percent recovery (R) and the
standard deviation of the percent recovery (SR) for the labeled
compounds only.  Express the assessment as a percent recovery interval
from R - 2SR to R + 2SR for each matrix.  For example, if R = 90% and SR
= 10% for five analyses of pulp, the recovery interval is expressed as
70 to 110%.

9.4.2	Update the accuracy assessment for each labeled compound in each
matrix on a regular basis (e.g., after each five to ten new
measurements).

9.5	Method blanks – A reference matrix Method blank is analyzed with
each sample batch (Section 4.3) to demonstrate freedom from
contamination.  The matrix for the Method blank must be similar to the
sample matrix for the batch, e.g., a 1-L reagent water blank (Section
7.6.1), high-solids reference matrix blank (Section 7.6.2), paper matrix
blank (Section 7.6.3); tissue blank (Section 7.6.4), or alternative
reference matrix blank (Section 7.6.5).

9.5.1	Spike 1.0 mL each of the Labeled Toxics/LOC/window-defining
standard spiking solution (Section 7.12), and the Labeled cleanup
standard spiking solution (Section 7.13) into the Method blank,
according to the procedures in Sections 11 through 18.  Prepare,
extract, clean up, and concentrate the Method blank.  Analyze the blank
immediately after analysis of the OPR (Section 15.5) to demonstrate
freedom from contamination.

9.5.2	If any CB (Table 1) is found in the blank at greater than two
times the minimum level (Table 2) or one-third the regulatory compliance
limit, whichever is greater; or if any potentially interfering compound
is found in the blank at the minimum level for each CB given in Table 2
(assuming a response factor of 1 relative to the quantitation reference
in Table 2 at that level of chlorination for a potentially interfering
compound; i.e., a compound not listed in this Method), analysis of
samples must be halted until the sample batch is re-extracted and the
extracts re-analyzed, and the blank associated with the sample batch
shows no evidence of contamination at these levels.  All samples must be
associated with an uncontaminated Method blank before the results for
those samples may be reported or used for permitting or regulatory
compliance purposes.

9.6	QC Check Sample – Analyze the QC Check Sample (Section 7.15)
periodically to assure the accuracy of calibration standards and the
overall reliability of the analytical process.  It is suggested that the
QC Check Sample be analyzed at least quarterly.

9.7	The specifications contained in this Method can be met if the
apparatus used is calibrated properly and then maintained in a
calibrated state.  The standards used for calibration (Section 10),
calibration verification (Section 15.3), and for initial (Section 9.2)
and ongoing (Section 15.5) precision and recovery should be identical,
so that the most precise results will be obtained.  A GC/MS instrument
will provide the most reproducible results if dedicated to the settings
and conditions required for determination of CBs by this Method.

9.8	Depending on specific program requirements, field replicates may be
collected to determine the precision of the sampling technique, and
spiked samples may be required to determine the accuracy of the analysis
when the internal standard method is used.

10.0	Calibration

Establish the operating conditions necessary to meet the retention times
(RTs) and relative retention times (RRTs) for the CBs in Table 2.

Note:	RTs, RRTs, and RRT limits may differ slightly from those in Table
2.

10.1.1	Suggested GC operating conditions:

Injector temperature:	270 ºC

Interface temperature:	290 ºC

Initial temperature:	75 ºC

Initial time:	2 minutes

Temperature program:	75-150 ºC at 15 ºC/minute

	150-290 ºC at 2.5 ºC/minute

Final time:	1 minute



Note:	All portions of the column that connect the GC to the ion source
should remain at or above the interface temperature specified above
during analysis to preclude condensation of less volatile compounds.

The GC conditions may be optimized for compound separation and
sensitivity.  Once optimized, the same GC conditions must be used for
the analysis of all standards, blanks, IPR and OPR standards, and
samples.

10.1.2	Retention time calibration for the CB congeners

10.1.2.1	Separately inject each of the diluted individual congener
solutions (Section 7.10.2.1.2).  Establish the beginning and ending
retention times for the scan descriptors in Table 7.  Scan descriptors
other than those listed in Table 7 may be used provided the MLs in Table
2 are met.  Store the retention time (RT) and relative retention time
(RRT) for each congener in the data system.

10.1.2.2	The absolute retention time of CB 209 must exceed 55 minutes on
 the SPB-octyl column; otherwise, the GC temperature program must be
adjusted and this test repeated until the minimum retention time
criterion is met.  If a GC column or column system alternate to the
SPB-octyl column is used, a similar minimum retention time specification
must be established for the alternate column or column systems so that
interferences that may be encountered in environmental samples will be
resolved from the analytes of interest.  This specification is deemed to
be met if the retention time of CB 209 is greater than 55 minutes on
such alternate column.

10.1.2.3	Inject the Diluted combined 209 congener solution (Section
7.10.2.2).  Adjust the chromatographic conditions and scan descriptors
until the RT and RRT for all congeners are approximately within the
windows in Table 2 and the column performance specifications in Sections
6.9.1 - 6.9.1.2 are met.  If an alternate column is used, adjust the
conditions for that column.  If column performance is unacceptable,
optimize the analysis conditions or replace the column and repeat the
performance tests.  Confirm that the scan descriptor changes at times
when CBs do not elute.

10.1.2.4	After the column performance tests are passed (Section 10.1.2.2
- 10.1.2.3), calculate and store the RT and RRT for the resolved
congeners and the RT and RRT for the isomeric congeners that co-elute. 
The windows in Table 2 were developed based on the GC conditions given
in Section 10.1.1.

10.2	Mass spectrometer (MS) resolution

10.2.1	Using perfluorokerosene (PFK) (or other reference substance) and
a molecular leak, tune the instrument to meet the minimum required
resolving power of 10,000 (10% valley) at m/z 330.9792 or any other
significant PFK fragment in the range of 300 to 350.  For each
descriptor (Table 7), monitor and record the resolution and exact
m/z’s of three to five reference peaks covering the mass range of the
descriptor.  The level of PFK (or other reference substance) metered
into the HRMS during analyses should be adjusted so that the amplitude
of the most intense selected lock-mass m/z signal (regardless of the
descriptor number) does not exceed 10% 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:	Different lots and types of PFK can contain varying levels of
contamination, and excessive PFK (or other reference substance) may
cause noise problems and contamination of the ion source necessitating
increased frequency of source cleaning.  A minor PFK mass (223.9872) is
known to interfere with dichlorobiphenyl secondary quantitation ion
(M+2).  Careful selection of the grade and purity of PFK and
minimization of the amount of PFK bled into the HRMS has been shown to
correct this problem.  

10.2.2	The analysis time for CBs may exceed 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,
mass-drift correction is mandatory and a lock-mass m/z from PFK or other
reference substance is used for drift correction.  The lock-mass m/z is
dependent on the exact m/z’s monitored within each descriptor, as
shown in Table 7.  The deviation between each monitored exact m/z and
the theoretical m/z (Table 7) must be less than 5 ppm.

10.2.3	Obtain a selected ion current profile (SICP) at the two exact
m/z’s specified in Table 7 and at (10,000 resolving power at each LOC
for the native congeners  and congener groups and for the labeled
congeners.  Because of the extensive mass range covered in each
function, it may not be possible to maintain 10,000 resolution
throughout the mass range during the function.  Therefore, resolution
must be (8,000 throughout the mass range and must be (10,000 in the
center of the mass range for each function.

10.2.4	If the HRMS has the capability to monitor resolution during the
analysis, it is acceptable to terminate the analysis when the resolution
falls below the minimum (Section 10.2.3) to save re-analysis time.

10.3	Ion abundance ratios, minimum levels, and signal-to-noise ratios. 
Choose an injection volume of either 1 or 2 µL, consistent with the
capability of the HRGC/HRMS instrument.  Inject a 1 or 2 µL aliquot of
the CS-1 calibration solution (Table 5) using the GC conditions in
Section 10.1.1.

10.3.1	Measure the SICP areas for each congener or congener group, and
compute the ion abundance ratios at the exact m/z’s specified in Table
7.  Compare the computed ratio to the theoretical ratio given in Table
8.

10.3.1.1	The exact m/z’s to be monitored in each descriptor are shown
in Table 7.  Each group or descriptor must be monitored in succession as
a function of GC retention time to ensure that the CBs of interest are
detected.  Additional m/z’s may be monitored in each descriptor, and
the m/z’s may be divided among more than the descriptors listed in
Table 7, provided that the laboratory is able to monitor the m/z’s of
all CBs that may elute from the GC in a given LOC window.  The
laboratory must also monitor exact m/z’s for congeners at higher
levels of chlorination to determine if fragments will compromise
measurement of congeners at lower levels of chlorination.

10.3.1.2	The mass spectrometer must be operated in a mass-drift
correction mode, using PFK (or other reference substance) to provide
lock  m/z’s.  The lock mass for each group of m/z’s is shown in
Table 7.  Each lock mass must be monitored and must not vary by more
than ± 20% throughout its respective retention time window.  Variations
of lock mass by more than 20% indicate the presence of co-eluting
interferences that raise the source pressure and may significantly
reduce the sensitivity of the mass spectrometer.  Re-injection of
another aliquot of the sample extract may not resolve the problem and
additional cleanup of the extract may be required to remove the
interference.  A lock mass interference or suppression in a retention
time region in which CBs and labeled compounds do not elute may be
ignored.

10.3.2	All CBs and labeled compounds in the CS-1 standard must be within
the QC limits in Table 8 for their respective ion abundance ratios;
otherwise, the mass spectrometer must be adjusted and this test repeated
until the m/z ratios fall  within the limits specified.  If the
adjustment alters the resolution of the mass spectrometer, resolution
must be verified (Section 10.2.3) prior to repeat of the test.

10.3.3	Verify that the HRGC/HRMS instrument achieves a minimum level
(ML) for each congener no greater than 2 times the MLs in Table 2.  The
peaks representing the CBs and labeled compounds in the CS-1 calibration
standard must have signal-to-noise ratios (S/N) ( 10; otherwise, the
mass spectrometer must be adjusted and this test repeated until the
minimum levels in Table 2 are met. ADVANCE \d7 

Note:	 The MDLs and MLs in Table 2 are based on the levels of
contamination normally found in laboratories. Lower levels may be
readily achievable if segregation and extensive cleaning of glassware
are employed.  If lower levels are achievable, these lower levels must
be established as described in Section 17.6.1.4.1. ADVANCE \d7 

10.4	Calibration by isotope dilution – Isotope dilution is used for
calibration of the Toxics/LOC CBs.  The reference compound for each
native compound its labeled analog, as listed in Table 2.  A 5- or
6-point calibration encompassing the concentration range is prepared for
each native congener.

10.4.1	For the Toxics/LOC CBs determined by isotope dilution, the
relative response (RR) (labeled to native) vs. concentration in the
calibration solutions (Table 5) is computed over the calibration range
according to the procedures described below.  Five calibration points
are employed for less-sensitive HRMS instruments (e.g., VG 70); five or
six points may be employed for more-sensitive instruments (e.g.,
Micromass Autospec Ultima).

10.4.2	The response of each Toxics/LOC CB relative to its labeled analog
is determined using the area responses of both the primary and secondary
exact m/z’s specified in Table 7, for each calibration standard, as
follows:

 

	where:

	A1n and A2n	=	The measured areas at the primary and secondary m/z’s
for the PCB

			A1l and A2l	=	The measured areas at the primary and secondary m/z’s
for the labeled compound

				Cl	=	The concentration of the labeled compound in the calibration
standard (Table 4)

				Cn	=	The concentration of the native compound in the calibration
standard  (Table 4)

10.4.3	To calibrate the analytical system by isotope dilution, inject
calibration standards CS-1 through CS-5 (Section 7.10 and Table 5) for a
less sensitive instrument or CS-0.2 through CS-5 for a more sensitive
instrument.  Use a volume identical to the volume chosen in Section
10.3, the procedure in Section 14, and the  conditions in Section
10.1.1.  Compute and store the relative response (RR) for each Native
Toxics/LOC CB at each concentration.  Compute the average (mean) RR and
the RSD of the 5 (or 6) RRs.

10.4.4	Linearity – If the RR for any Native Toxics/LOC CB is constant
(less than 20% RSD), the average RR may be used for that congener;
otherwise, the complete calibration curve for that congener must be used
over the calibration range.

10.5	Calibration by internal standard – Internal standard calibration
is applied to determination of the native CBs for which a labeled
compound is not available, determination of the Labeled
Toxics/LOC/window-defining congeners and Labeled cleanup congeners for
performance tests and intra-laboratory statistics (Sections 9.4 and
15.5.4), and determination of the Labeled injection internal standards
except for CB 178.  The reference compound for each compound is listed
in Table 2.  For the native congeners (other than the Native Toxics/LOC
CBs), calibration is performed at a single point using the Diluted
combined 209 congener solution (Section 7.10.2.2 and Table 5).  For the
labeled compounds, calibration is performed using data from the 5 (or 6)
points in the calibration for the Native Toxics/LOC CBs (Section 10.4).

10.5.1	Response factors – Internal standard calibration requires the
determination of response factors (RF) defined by the following
equation:

 

	where:

			A1s and A2s	=	The measured areas at the primary and secondary m/z’s
for the PCB

			A1is and A2is	=	The measured areas at the primary and secondary
m/z’s for the internal standard

				Cis	=	The concentration of the internal standard (Table 5)

				Cs	=	The concentration of the compound in the calibration standard
(Table 5)

10.5.2	To single-concentration calibrate the analytical system for
native CBs other than the Native Toxics/LOC CBs by internal standard,
inject the Diluted combined 209 congener solution (Section 7.10.2.2 and
Table 3).  Use a volume identical to the volume chosen in Section 10.3,
the procedure in Section 14, and the  conditions in Section 10.1.1.

10.5.3	Compute and store the response factor (RF) for all native CBs
except the Native Toxics/LOC CBs.  Use the average (mean) response of
the labeled compounds at each level of chlorination (LOC) as the
quantitation reference, to a maximum of 5 labeled congeners, as shown in
Table 2.  For the combinations of isomeric congeners that co-elute,
compute a combined RF for the co-eluted group.  For example, for
congener 122, the areas at the two exact m/z’s for 104L, 105L, 114L,
118L, and 123L are summed and the total area is divided  by 5 (because
there are 5 congeners in the quantitation reference).

Note:	  All labeled congeners at each LOC are used as reference to
reduce the effect of an interference if a single congener is used as
reference.  Other quantitation references and procedures may be used
provided that the results produced are as accurate as results produced
by the quantitation references and procedures described in this Section.
ADVANCE \d7 

10.5.4	Compute and store the response factor (RF) for the labeled
compounds, except CB 138.  For the Labeled Toxics/LOC/window-defining
compounds and the Labeled cleanup standards, use the nearest eluted
Labeled injection internal standard as the quantitation reference, as
given in Table 2.  The Labeled injection internal standards are
referenced to CB 138, as shown in Table 2.

11.0	Sample preparation

11.1	Sample preparation involves modifying the physical form of the
sample so that the CBs can be extracted efficiently.  In general, the
samples must be in a liquid form or in the  form of finely divided
solids in order for efficient extraction to take place.  Table 9 lists
the phases and suggested quantities for extraction of various sample
matrices.

For samples known or expected to contain high levels of the CBs, the
smallest sample size representative of the entire sample should be used
(see Section 18).  For all samples, the blank and IPR/OPR aliquots must
be processed through the same steps as the sample to check for
contamination and losses in the preparation processes.

11.1.1	For samples that contain particles, percent solids and particle
size are determined using the procedures in Sections 11.2 and 11.3,
respectively.

11.1.2	Aqueous samples – Because CBs may be bound to suspended
particles, the preparation of aqueous samples is dependent on the solids
content of the sample.

11.1.2.1	Aqueous samples containing one percent solids or less are
prepared per Section 11.4 and extracted directly using one of the
extraction techniques in Section 12.2.

11.1.2.2	For aqueous samples containing greater than one percent solids,
a sample aliquot sufficient to provide 10 g of dry solids is used, as
described in Section 11.5.

11.1.3	Solid samples are prepared using the procedure described in
Section 11.5 followed by extraction using the SDS procedure in Section
12.3.

11.1.4	Multi-phase samples – The phase(s) containing the CBs is
separated from the  non-CB phase using pressure filtration and
centrifugation, as described in Section 11.6.  The CBs will be in the
organic phase in a multi-phase sample in which an organic phase exists.

11.1.5	Procedures for grinding, homogenization, and blending of various
sample phases are given in Section 11.7.

11.1.6	Tissue samples – Preparation procedures for fish and other
tissues are given in Section 11.8.

11.2	Determination of percent suspended solids

Note:	This aliquot is used for determining solids content of the sample,
not for determination of CBs.

11.2.1	 Aqueous liquids and multi-phase samples consisting of mainly an
aqueous phase

11.2.1.1	Desiccate and weigh a GF/D filter (Section 6.5.3) to three
significant figures.

11.2.1.2	Filter 10.0 ± 0.02 mL of well-mixed sample through the filter.

11.2.1.3	Dry the filter a minimum of 12 hours at 110 ±5 ºC and cool in
a desiccator.

11.2.1.4	Calculate percent solids as follows:

11.2.2	Non-aqueous liquids, solids, semi-solid samples, and multi-phase
samples in which the main phase is not aqueous; but not tissues

11.2.2.1	Weigh 5 to 10 g of sample to three significant figures in a
tared beaker.

11.2.2.2	Dry a minimum of 12 hours at 110 ± 5 ºC, and cool in a
desiccator.

11.2.2.3	Calculate percent solids as follows:

11.3	Estimation of particle size

11.3.1	Spread the dried sample from Section 11.2.2.2 on a piece of
filter paper or aluminum foil in a fume hood or glove box.

11.3.2	Estimate the size of the particles in the sample.  If the size of
the largest particles is greater than 1 mm, the particle size must be
reduced to 1 mm or less prior to extraction using the procedures in
Section 11.7.

11.4	Preparation of aqueous samples containing one percent suspended
solids or less

11.4.1	Aqueous samples containing one percent suspended solids or less
are prepared using the procedure below and extracted using the one of
the extraction techniques in Section 12.2.

11.4.2	Preparation of sample and QC aliquots

11.4.2.1	Mark the original level of the sample on the sample bottle for
reference.  Weigh the sample plus bottle to ± 1 g.  After extraction
(Section 12.2), re-weigh the sample bottle and convert the weight to
volume assuming a density of 1.00 g/mL.

11.4.2.2	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into the sample bottle.  Cap the bottle
and mix the sample by careful shaking.  Allow the sample to equilibrate
for 1 to 2 hours, with occasional shaking.

11.4.2.3	For each sample or sample batch (to a maximum of 20 samples) to
be extracted during the same 12-hour shift, place two 1.0-L aliquots of
reagent water in clean sample bottles or flasks.

11.4.2.4	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into both reagent water aliquots.  One
of these aliquots will serve as the Method blank.

11.4.2.5	Spike 1.0 mL of the Native Toxics/LOC standard spiking solution
(Section 7.11) into the remaining reagent water aliquot.  This aliquot
will serve as the OPR (Section 15.5).

11.4.2.6	For extraction using SPE, add 5 mL of methanol to the sample
and QC aliquots.  Cap and shake the sample and QC aliquots to mix
thoroughly, and proceed to Section 12.2 for extraction.

11.5	Preparation of samples containing greater than one percent solids

11.5.1	Weigh a well-mixed aliquot of each sample (of the same matrix
type) sufficient to provide 10 g of dry solids (based on the solids
determination in Section 11.2) into a clean beaker or glass jar.

11.5.2	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into the sample.

11.5.3	For each sample or sample batch (to a maximum of 20 samples) to
be extracted during the same 12 hour shift, weigh two 10-g aliquots of
the appropriate reference matrix (Section 7.6) into clean beakers or
glass jars.

11.5.4	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into both reference matrix aliquots. 
Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into one reference matrix aliquot.  This aliquot will serve as the
OPR (Section 15.5).  The other aliquot will serve as the Method blank.

11.5.5	Stir or tumble and equilibrate the aliquots for 1 to 2 hours.

11.5.6	Decant excess water.  If necessary to remove water, filter the
sample through a glass-fiber filter and discard the aqueous liquid.

11.5.7	If particles >1 mm are present in the sample (as determined in
Section 11.3.2), spread the sample on clean aluminum foil in a hood. 
After the sample is dry, grind to reduce the particle size (Section
11.7).

11.5.8	Extract the sample and QC aliquots using the SDS procedure in
Section 12.3.

11.6	Multi-phase samples

11.6.1	Using the percent solids determined in Section 11.2.1 or 11.2.2,
determine the volume of sample that will provide 10 g of solids, up to 1
L of sample.

11.6.2	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into the amount of sample determined in
Section 11.6.1, and into the OPR and blank.  Spike 1.0 mL of the Native
Toxics/LOC standard spiking solution (Section 7.11) into the OPR. 
Pressure filter the sample, blank, and OPR through Whatman GF/D
glass-fiber filter paper (Section 6.5.3).  If necessary to separate the
phases and/or settle the solids, centrifuge these aliquots prior to
filtration.

11.6.3	Discard any aqueous phase (if present).  Remove any non-aqueous
liquid present and reserve the maximum amount filtered from the sample
(Section 11.6.1) or 10 g, whichever is less, for combination with the
solid phase (Section 12.3.5).

11.6.4	If particles >1 mm are present in the sample (as determined in
Section 11.3.2) and the sample is capable of being dried, spread the
sample and QC aliquots on clean aluminum foil in a hood.  Observe the
precaution in Section 4.8.

11.6.5	After the aliquots are dry or if the sample cannot be dried,
reduce the particle size using the procedures in Section 11.7 and
extract the reduced-size particles using the SDS procedure in Section
12.3.  If particles >1 mm are not present, extract the particles and
filter in the sample and QC aliquots directly using the SDS procedure in
Section 12.3.

11.7	Sample grinding, homogenization, or blending – Samples with
particle sizes greater than 1 mm (as determined in Section 11.3.2) are
subjected to grinding, homogenization, or blending.  The method of
reducing particle size to less than 1 mm is matrix-dependent.  In
general, hard particles can be reduced by grinding with a mortar and
pestle.  Softer particles can be reduced by grinding in a Wiley mill or
meat grinder, by homogenization, or in a blender.

11.7.1	Each size-reducing preparation procedure on each matrix must be
verified by running the tests in Section 9.2 before the procedure is
employed routinely.

11.7.2	The grinding, homogenization, or blending procedures must be
carried out in a glove box or fume hood to prevent particles from
contaminating the work environment.

11.7.3	Grinding – Certain papers and pulps, slurries, and amorphous
solids can be ground in a Wiley mill or heavy duty meat grinder.  In
some cases, reducing the temperature of the sample to freezing or to dry
ice or liquid nitrogen temperatures can aid in the grinding process. 
Grind the sample aliquots from Sections 11.5.7 or 11.6.5 in a clean
grinder.  Do not allow the sample temperature to exceed 50 ºC.  Grind
the blank and reference matrix aliquots using a clean grinder.

11.7.4	Homogenization or blending – Particles that are not ground
effectively, or particles greater than 1 mm in size after grinding, can
often be reduced in size by high speed homogenization or blending. 
Homogenize and/or blend the particles or filter from Sections 11.5.7 or
11.6.5 for the sample, blank, and OPR aliquots.

11.7.5	Extract the aliquots using the SDS procedure in Section 12.3.

11.8	Fish and other tissues – Prior to processing tissue samples, the
laboratory must determine the exact tissue to be analyzed.  Common
requests for analysis of fish tissue include whole fish-skin on, whole
fish-skin removed, edible fish fillets (filleted in the field or by  the
laboratory), specific organs, and other portions.  Once the appropriate
tissue has been determined, the sample must be homogenized.

11.8.1	Homogenization

11.8.1.1	Samples are homogenized while still frozen, where practical. 
If the laboratory must dissect the whole fish to obtain the appropriate
tissue for analysis, the unused tissues may be rapidly refrozen and
stored in a clean glass jar for subsequent use.

11.8.1.2	Each analysis requires 10 g of tissue (wet weight).  Therefore,
the laboratory should homogenize at least 20 g of tissue to allow for
re-extraction of a second aliquot of the same homogenized sample, if
re-analysis is required.  When whole fish analysis is necessary, the
entire fish is homogenized.

11.8.1.3	Homogenize the sample in a tissue homogenizer (Section 6.3.3)
or grind in a meat grinder (Section 6.3.4).  Cut tissue that is too
large to feed into the grinder into smaller pieces.  To assure
homogeneity, grind three times.

11.8.1.4	Transfer approximately 10 g (wet weight) of homogenized tissue
to a clean, tared, 400- to 500-mL beaker.

11.8.1.5	Transfer the remaining homogenized tissue to a clean jar with a
fluoropolymer-lined lid.  Seal the jar and store the tissue at less than
-10 ºC.  Return any tissue that was not homogenized to its original
container and store at less than -10 ºC.

11.8.2	QC aliquots

11.8.2.1	Prepare a Method blank by adding approximately 1-2 g of the
oily liquid reference matrix (Section 7.6.4) to a 400- to 500-mL beaker.

11.8.2.2	Prepare a precision and recovery aliquot by adding 1-2 g of the
oily liquid reference matrix (Section 7.6.4) to a separate 400- to
500-mL beaker.  Record the weight to the nearest 10 mg.  If the initial
precision and recovery test is to be performed, use four aliquots; if
the ongoing precision and recovery test is to be performed, use a single
aliquot.

11.8.3	Spiking

11.8.3.1	Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard
spiking solution (Section 7.12) into the sample, blank, and OPR aliquot.

11.8.3.2	Spike 1.0 mL of the Native Toxics/LOC standard spiking solution
(Section 7.11) into the OPR aliquot.

11.8.4	Extract the aliquots using the procedures in Section 12.4.

12.0	Extraction and concentration

12.1	Extraction procedures include:  solid-phase (Section 12.2.1),
separatory funnel (Section 12.2.2), and continuous liquid/liquid
(Section 12.2.3) for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3)
for solids and filters; and Soxhlet extraction (Section 12.4) for
tissues.  Acid/base back-extraction (Section 12.5) is used for initial
cleanup of extracts.

	Macro-concentration procedures include:  rotary evaporation (Section
12.6.1), heating mantle (Section 12.6.2), and Kuderna-Danish (K-D)
evaporation (Section 12.6.3).  Micro-concentration uses nitrogen
evaporation (Section 12.7).

12.2	Extraction of aqueous liquids

12.2.1	Solid-phase extraction of samples containing less than one
percent solids

12.2.1.1	Disk preparation

12.2.1.1.1	Remove the test tube from the suction flask (Figure 4). 
Place an SPE disk on the base of the filter holder and  wet with
methylene chloride.  While holding a GMF 150 filter above the SPE disk
with tweezers, wet the filter with methylene chloride and lay the filter
on the SPE disk, making sure that air is not trapped between the filter
and disk.  Clamp the filter and SPE disk between the 1-L glass reservoir
and the vacuum filtration flask.

12.2.1.1.2	Rinse the sides of the reservoir with approx 15 mL of
methylene chloride using a squeeze bottle or pipet.  Apply vacuum
momentarily until a few drops appear at the drip tip.  Release the
vacuum and allow the filter/disk to soak for approx one minute.  Apply
vacuum and draw all of the methylene chloride through the filter/disk. 
Repeat the wash step with approx 15 mL of acetone and allow the
filter/disk to air dry.

12.2.1.2	Sample extraction

12.2.1.2.1	Pre-wet the disk by adding approx 20 mL of methanol to the
reservoir.  Pull most of the methanol through the filter/disk, retaining
a layer of methanol approx 2 mm thick on the filter.  Do not allow the
filter/disk to go dry from this point until the extraction is completed.

12.2.1.2.2	Add approx 20 mL of reagent water to the reservoir and pull
most through, leaving a layer approx 2 mm thick on the filter/disk.

12.2.1.2.3	Allow the sample (Section 11.4.2.6) to stand for 1-2 hours,
if necessary, to settle the suspended particles.  Decant the clear layer
of the sample, the blank (Section 11.4.2.4), or IPR/OPR aliquot (Section
11.4.2.5) into its respective reservoir and turn on the vacuum to begin
the extraction.  Adjust the vacuum to complete the  extraction in no
less than 10 minutes.  For samples containing a high concentration of
particles (suspended solids), the extraction time may be an hour or
longer.

12.2.1.2.4	Before all of the sample has been pulled through the
filter/disk, add approx 50 mL of reagent water to the sample bottle,
swirl to suspend the solids (if present), and pour into the reservoir. 
Pull through the filter/disk.  Use additional reagent water rinses until
all solids are removed.

12.2.1.2.5	Before all of the sample and rinses have been pulled through
the filter/disk, rinse the sides of the reservoir with small portions of
reagent water.

12.2.1.2.6	Partially dry the filter/disk under vacuum for approx 3
minutes.

12.2.1.3	Elution of the filter/disk

12.2.1.3.1	Release the vacuum, remove the entire filter/disk/reservoir
assembly from the vacuum flask, and empty the flask.  Insert a test tube
for eluant collection into the flask.  The test tube should have
sufficient capacity to contain the total volume of the elution solvent
(approx 50 mL) and should fit around the drip tip.  The drip tip should
protrude into the test tube to preclude loss of sample from spattering
when vacuum is applied.  Reassemble the filter/disk/reservoir assembly
on the vacuum flask.

12.2.1.3.2	Wet the filter/disk with 4-5 mL of acetone.  Allow the
acetone to spread evenly across the disk and soak for 15-20 seconds. 
Pull the acetone through the disk, releasing the vacuum when approx 1 mm
thickness remains on the filter.

12.2.1.3.3	Rinse the sample bottle with approx 20 mL of methylene
chloride and transfer to the reservoir.  Pull approx half  of the
solvent through the filter/disk and release the vacuum.  Allow the
filter/disk to soak for approx 1 minute.  Pull all of the solvent
through the disk.  Repeat the bottle rinsing and elution step with
another 20 mL of methylene chloride.  Pull all of the solvent through
the disk.

12.2.1.3.4	Release the vacuum, remove the filter/disk/reservoir
assembly, and remove the test tube containing the sample solution. 
Quantitatively transfer the solution to a 250-mL separatory funnel and
proceed to Section 12.5 for back-extraction.

12.2.2	Separatory funnel extraction

12.2.2.1	Pour the spiked sample (Section 11.4.2.2) into a 2-L separatory
funnel.  Rinse the bottle or flask twice with 5 mL of reagent water and
add these rinses to the separatory funnel.

12.2.2.2	Add 60 mL methylene chloride to the empty sample bottle.  Seal
the bottle and shake 60 seconds to rinse the inner surface.  Transfer
the solvent to the separatory funnel, and extract the sample by shaking
the funnel for 2 minutes with periodic venting.  Allow the organic layer
to separate from the aqueous phase for a minimum of 10 minutes.  If an
emulsion forms and is more than one-third the volume of the solvent
layer, employ mechanical techniques to complete the phase separation
(see note below).  Drain the  methylene chloride extract through a
solvent-rinsed glass funnel approximately one-half full of granular
anhydrous sodium sulfate (Section 7.2.1) supported on clean glass-fiber
paper into a solvent-rinsed concentration device (Section 12.6).

Note:	  If an emulsion forms, the laboratory must employ mechanical
techniques to complete the phase separation.  The optimum technique
depends upon the sample, but may include stirring, filtration through
glass wool, use of phase separation paper, centrifugation, use of an
ultrasonic bath with ice, addition of NaCl, or other physical methods. 
Alternatively, solid-phase (Section 12.2.1), CLLE (Section 12.2.3), or
other extraction techniques may be used to prevent emulsion formation. 
Any alternative technique is acceptable so long as the requirements in
Section 9.2 are met. ADVANCE \d7 

12.2.2.3	Extract the water sample two more times with 60-mL portions of
methylene chloride.  Drain each portion through the sodium sulfate into
the concentrator.  After the third extraction, rinse the separatory
funnel with at least 20 mL of methylene chloride, and drain this rinse
through the sodium sulfate into the concentrator.  Repeat this rinse at
least twice.

12.2.2.4	Concentrate the extract using one of the macro-concentration
procedures in Section 12.6 and proceed to back extraction in Section
12.5.  Set aside the concentration device for use after back extraction
or other cleanup.

12.2.3	Continuous liquid/liquid extraction

12.2.3.1	Place 100-150 mL methylene chloride in each continuous
extractor and 200-300 mL in each distilling flask.

12.2.3.2	Pour the sample(s), blank, and QC aliquots into the extractors.
 Rinse the sample containers with 50-100 mL methylene chloride and add
to the respective extractors.  Include all solids in the extraction
process.

12.2.3.3	Begin the extraction by heating the flask until the methylene
chloride is boiling.  When properly adjusted, 1-2 drops of methylene
chloride per second will fall from the condenser tip into the water. 
Extract for 16-24 hours.

12.2.3.4	Remove the distilling flask, estimate and record the volume of
extract (to the nearest 100 mL), and pour the contents through a drying
column containing 7 to 10 cm of granular anhydrous sodium sulfate into a
500-mL K-D evaporator flask equipped with a 10-mL concentrator tube. 
Rinse the distilling flask with 30-50 mL of methylene chloride and pour
through the drying column.  Concentrate and exchange to hexane per
Section 12.6 and back extract per Section 12.5.  Set aside the
concentration device for use after back extraction or other cleanup.

12.3	SDS extraction of samples containing particles

Note:	  SDS extraction with toluene may cause loss of some of the mono-
through tri- CB congeners.   If this loss is excessive, use Soxhlet
extraction with methylene chloride (Section 12.4) and increase the
amount of powdered, anhydrous sodium sulfate as necessary to provide a
free-flowing mixture. ADVANCE \d7 

12.3.1	Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of
100/200 mesh silica (Section 7.5.1.1) topped with 100 g of quartz sand
(Section 7.3.2).

Note:	  Do not disturb the silica layer throughout the extraction
process. ADVANCE \d7 

12.3.2	Place the thimble in a clean extractor.  Place 30 to 40 mL of
toluene in the receiver and 200 to 250 mL of toluene in the flask.

12.3.3	Pre-extract the glassware by heating the flask until the toluene
is boiling.  When properly adjusted, 1 to 2 drops of toluene will fall
per second from the condenser tip into the receiver.  Extract the
apparatus for a minimum of 3 hours.

12.3.4	After pre-extraction, cool and disassemble the apparatus.  Rinse
the thimble with toluene and allow to air dry.

12.3.5	Load the wet sample and/or filter from Sections 11.5.8, 11.6.5,
or 11.7.5 and any nonaqueous liquid from Section 11.6.3 into the thimble
and manually mix into the sand layer with a clean metal spatula,
carefully breaking up any large lumps  of sample.

12.3.6	Reassemble the pre-extracted SDS apparatus, and add a fresh
charge of toluene to the receiver and reflux flask.  Apply power to the
heating mantle to begin re-fluxing.  Adjust the reflux rate to match the
rate of percolation through the sand and silica beds until water removal
lessens the restriction to toluene flow.  Frequently check the apparatus
for foaming during the first 2 hours of extraction.  If foaming occurs,
reduce the reflux rate until foaming subsides.

12.3.7	Drain the water from the receiver at 1-2 hours and 8-9 hours, or
sooner if the receiver fills with water.  Reflux the sample for a total
of 16-24 hours.  Cool and disassemble the apparatus.  Record the total
volume of water collected.

12.3.8	Remove the distilling flask.  Drain the water from the Dean-Stark
receiver and add any toluene in the receiver to the extract in the
flask.

12.3.9	Concentrate the extracts from particles to approximately 10 mL
using the rotary evaporator (Section 12.6.1) or heating mantle (Section
12.6.2), transfer to a 250-mL separatory funnel, and proceed with
back-extraction (Section 12.5).  Set aside the concentration device for
use after back-extraction or other cleanup.

12.4	Soxhlet extraction of tissue (References 3 and 15)

Note:	  This procedure includes determination of the lipid content of
the sample (Sections 12.4.8 - 12.4.9), using the same sample extract
that is analyzed by GC/MS.  Alternatively, a separate sample aliquot may
be used for the lipid determination.  If a separate aliquot is used, use
nitrogen to evaporate the main portion of the sample extract only to the
extent necessary to effect the solvent exchange to n-hexane, so that
loss of low molecular weight CBs is avoided, i.e., it is not necessary
to dry the main portion of the sample to constant weight (Section
12.4.8). ADVANCE \d7 

12.4.1	Add 30 to 40 g of powdered anhydrous sodium sulfate (Section
7.2.2) to each of the beakers (Section 11.8.4) and mix thoroughly. 
Cover the beakers with aluminum foil and dry until the mixture becomes a
free-flowing powder (30 minutes minimum).  Remix prior to extraction to
prevent clumping.

12.4.2	Assemble and pre-extract the Soxhlet apparatus per Sections
12.3.1-12.3.4, except use methylene chloride for the pre-extraction and
rinsing and omit the quartz sand.

12.4.3	Reassemble the pre-extracted Soxhlet apparatus and add a fresh
charge of methylene chloride to the reflux flask.

12.4.4	Transfer the sample/sodium sulfate mixture (Section 12.4.1) to
the Soxhlet thimble, and install the thimble in the Soxhlet apparatus.

12.4.5	Rinse the beaker with several portions of solvent and add to the
thimble.  Fill the thimble/receiver with solvent.  Extract for 18-24
hours.

12.4.6	After extraction, cool and disassemble the apparatus.

12.4.7	Quantitatively transfer the extract to a macro-concentration
device (Section 12.6), and concentrate to near dryness.  Set aside the
concentration apparatus for re-use.

12.4.8	Complete the removal of the solvent using the nitrogen blow
evaporation procedure (Section 12.7) and a water bath temperature of 60
ºC.  Weigh the receiver, record the weight, and return the receiver to
the blowdown apparatus, concentrating the residue until a constant
weight is obtained.

12.4.9	Percent lipid determination

12.4.9.1	Redissolve the residue in the receiver in hexane and spike 1.0
mL of the Labeled cleanup standard spiking solution (Section 7.13) into
the solution.

12.4.9.2	Transfer the residue/hexane to the anthropogenic isolation
column (Section 13.6), retaining the boiling chips in the concentration
apparatus.  Use several rinses to assure that all material is
transferred.  If necessary, sonicate or heat the receiver slightly to
assure that all material is re-dissolved.  Allow the receiver to dry. 
Weigh the receiver and boiling chips.

12.4.9.3	Calculate the lipid content to the nearest three significant
figures as follows:

12.4.9.4	The laboratory should determine the lipid content of the blank,
IPR, and OPR to assure that the extraction system is working
effectively.

12.5	Back-extraction with base and acid

12.5.1	Back-extraction may not be necessary for some samples.  For some
samples, the presence of color in the extract may indicate that
back-extraction is necessary.  If back-extraction is not necessary,
spike 1.0 mL of the Labeled cleanup standard spiking solution (Section
7.13) into the extract and concentrate the extract for cleanup or
analysis (Sections 12.6 and 12.7).  If back-extraction is necessary,
spike 1.0 mL of the Labeled cleanup standard spiking solution (Section
7.13) into the  separatory funnels containing the sample and QC extracts
from Section 12.2.3.4 or 12.3.9.

12.5.2	Partition the extract against 50 mL of potassium hydroxide
solution (Section 7.1.1).  Shake for 2 minutes with periodic venting
into a hood.  Remove and discard the aqueous layer.  Repeat the base
washing until no color is visible in the aqueous layer, to a maximum of
four washings.  Minimize contact time between the extract and the base
to prevent degradation of the CBs.  Stronger potassium hydroxide
solutions may be employed for back-extraction, provided that the
laboratory meets the specifications for labeled compound recovery and
demonstrates acceptable performance using the procedure in Section 9.2.

12.5.3	Partition the extract against 50 mL of sodium chloride solution
(Section 7.1.4) in the same way as with base.  Discard the aqueous
layer.

12.5.4	Partition the extract against 50 mL of sulfuric acid (Section
7.1.2) in the same way as with base.  Repeat the acid washing until no
color is visible in the aqueous layer, to a maximum of four washings.

12.5.5	Repeat the partitioning against sodium chloride solution and
discard the aqueous layer.

12.5.6	Pour each extract through a drying column containing 7 to 10 cm
of granular anhydrous sodium sulfate (Section 7.2.1) into a
macro-concentration device (Section 12.6).  If a concentration device
was set aside from extraction, that concentration device may be re-used.
 Rinse the separatory funnel with 30 to 50 mL of solvent, and pour
through the drying column.  Re-concentrate the sample and QC aliquots
per Sections 12.6-12.7, and clean up the samples and QC aliquots per
Section 13.

12.6	Macro-concentration – Extracts in toluene are concentrated using
a rotary evaporator or a heating mantle; extracts in methylene chloride
or hexane are concentrated using a rotary evaporator, heating mantle, or
Kuderna-Danish apparatus.

Note:	  In the concentration procedures below, the extract must not be
allowed to concentrate to dryness because the mono- through
tri-chlorobiphenyls may be totally or partially lost.

12.6.1	Rotary evaporation – Concentrate the extracts in separate
round-bottom flasks. 

12.6.1.1	Assemble the rotary evaporator according to manufacturer's
instructions, and warm the water bath to 45 ºC.  On a daily basis,
pre-clean the rotary evaporator by concentrating 100 mL of clean
extraction solvent through the system.  Archive both the concentrated
solvent and the solvent in the catch flask for a contamination check if
necessary.  Between samples, three 2- to 3- mL aliquots of solvent
should be rinsed down the feed tube into a waste beaker.

12.6.1.2	Attach the round-bottom flask containing the sample extract to
the rotary evaporator.  Slowly apply vacuum to the system, and begin
rotating the sample flask.

12.6.1.3	Lower the flask into the water bath, and adjust the speed of
rotation and the temperature as required to complete concentration in 15
to 20 minutes.  At the proper rate of concentration, the flow of solvent
into the receiving flask will be steady, but no bumping or visible
boiling of the extract will occur.

Note:	  If the rate of concentration is too fast, analyte loss may
occur. ADVANCE \d7 

12.6.1.4	When the liquid in the concentration flask has reached an
apparent volume of approximately 2 mL, remove the flask from the water
bath and stop the rotation.  Slowly and carefully admit air into the
system.  Be sure not to open the valve so quickly that the sample is
blown out of the flask.  Rinse the feed tube with approximately 2 mL of
solvent.

12.6.1.5	Proceed to Section 12.6.4 for preparation for back-extraction
or micro-concentration and solvent exchange.

12.6.2	Heating mantle – Concentrate the extracts in separate
round-bottom flasks.

12.6.2.1	Add one or two clean boiling chips to the round-bottom flask,
and attach a three-ball macro Snyder column.  Prewet the column by
adding approximately 1 mL of solvent through the top.  Place the
round-bottom flask in a heating mantle, and apply heat as required to
complete the concentration in 15 to 20 minutes.  At the proper rate of
distillation, the balls of the column will actively chatter, but the
chambers will not flood.

12.6.2.2	When the liquid has reached an apparent volume of approximately
10 mL, remove the round-bottom flask from the heating mantle and allow
the solvent to drain and cool for at least 10 minutes.  Remove the
Snyder column and rinse the glass joint into the receiver with small
portions of solvent.

12.6.2.3	Proceed to Section 12.6.4 for preparation for back-extraction
or micro-concentration and solvent exchange.

12.6.3	Kuderna-Danish (K-D) – Concentrate the extracts in separate
500-mL K-D flasks equipped with 10-mL concentrator tubes.  The K-D
technique is used for solvents such as methylene chloride and hexane. 
Toluene is difficult to concentrate using the K-D technique unless a
water bath fed by a steam generator is used.

12.6.3.1	Add 1 to 2 clean boiling chips to the receiver.  Attach a
three-ball macro Snyder column.  Prewet the column by adding
approximately 1 mL of solvent through the top.  Place the K-D apparatus
in a hot water bath so that the entire lower rounded surface of the
flask is bathed with steam.

12.6.3.2	Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20
minutes.  At the proper rate of distillation, the balls of the column
will actively chatter but the chambers will not flood.

12.6.3.3	When the liquid has reached an apparent volume of 1 mL, remove
the K-D apparatus from the bath and allow the solvent to drain and cool
for at least 10 minutes.  Remove the Snyder column and rinse the flask
and its lower joint into the concentrator tube with 1 to 2 mL of
solvent.  A 5-mL syringe is recommended for this operation.

12.6.3.4	Remove the three-ball Snyder column, add a fresh boiling chip,
and attach a two ball micro Snyder column to the concentrator tube. 
Prewet the column by adding approximately 0.5 mL of solvent through the
top.  Place the apparatus in the hot water bath.

12.6.3.5	Adjust the vertical position and the water temperature as
required to complete the concentration in 5 to 10 minutes.  At the
proper rate of distillation, the balls of the column will actively
chatter but the chambers will not flood.

12.6.3.6	When the liquid reaches an apparent volume of 0.5 mL, remove
the apparatus from the water bath and allow to drain and cool for at
least 10 minutes.

12.6.3.7	Proceed to 12.6.4 for preparation for back-extraction or
micro-concentration and solvent exchange.

12.6.4	Preparation for back-extraction or micro-concentration and
solvent exchange

12.6.4.1	For back-extraction (Section 12.5), transfer the extract to a
250-mL separatory funnel.  Rinse the concentration vessel with small
portions of hexane, adjust the hexane volume in the separatory funnel to
10 to 20 mL, and proceed to back-extraction (Section 12.5).

12.6.4.2	For determination of the weight of residue in the extract, or
for clean-up procedures other than back-extraction, transfer the extract
 to a blowdown vial using 2-3 rinses of solvent.  Proceed with
micro-concentration and solvent exchange (Section 12.7).

12.7	Micro-concentration and solvent exchange

12.7.1	Extracts to be subjected to GPC cleanup are exchanged into
methylene chloride.  Extracts to be cleaned up using silica gel, carbon,
Florisil, and/or HPLC are exchanged into hexane.

12.7.2	Transfer the vial containing the sample extract to a nitrogen
evaporation device.  Adjust the flow of nitrogen so that the surface of
the solvent is just visibly disturbed.

Note:	  A large vortex in the solvent may cause analyte loss. ADVANCE
\d7 

12.7.3	Lower the vial into a 45 ºC water bath and continue
concentrating.

12.7.3.1	If the extract or an aliquot of the extract is to be
concentrated to dryness for weight determination (Sections 12.4.8 and
13.6.4), blow dry until a constant weight is obtained.

12.7.3.2	If the extract is to be concentrated for injection into the
GC/MS or  the solvent is to be exchanged for extract cleanup, proceed as
follows:

12.7.4	When the volume of the liquid is approximately 100 µL, add 2 to
3 mL of the desired solvent (methylene chloride for GPC and HPLC, or
hexane for the other cleanups) and continue concentration to
approximately 100 µL.  Repeat the addition of solvent and concentrate
once more.

12.7.5	If the extract is to be cleaned up by GPC, adjust the volume of
the extract to 5.0 mL with methylene chloride.  If the extract is to be
cleaned up by HPLC, concentrate the extract to 1.0 mL.  Proceed with GPC
or HPLC cleanup (Section 13.2 or 13.5, respectively).

12.7.6	If the extract is to be cleaned up by column chromatography
(silica gel, Carbopak/Celite, or Florisil), bring the final volume to
1.0 mL with hexane.  Proceed with column cleanup (Sections 13.3, 13.4,
or 13.7).

12.7.7	If the extract is to be concentrated for injection into the GC/MS
(Section 14), quantitatively transfer the extract to a 0.3-mL conical
vial for final concentration, rinsing the larger vial with hexane and
adding the rinse to the conical vial.  Reduce the volume to
approximately 100 µL.  Add 20 µL of nonane to the vial, and evaporate
the solvent to the level of the nonane.  Seal the vial and label with
the sample number.  Store in the dark at room temperature until ready
for GC/MS analysis.  If GC/MS analysis will not be performed on the same
day, store the vial at less than -10 ºC.

13.0	Extract cleanup

13.1	Cleanup may not be necessary for relatively clean samples (e.g.,
treated effluents, groundwater, drinking water).  If particular
circumstances require the use of a cleanup procedure, the laboratory may
use any or all of the procedures below or any other appropriate
procedure.  Before using a cleanup procedure, the laboratory must
demonstrate that the requirements of Section 9.2 can be met using the
cleanup procedure.

13.1.1	Gel permeation chromatography (Section 13.2) removes high
molecular weight interferences that cause GC column performance to
degrade.  It should be used for all soil and sediment extracts.  It may
be used for water extracts that are expected to contain high molecular
weight organic compounds (e.g., polymeric materials, humic acids).  It
should also be used for tissue extracts after initial cleanup on the
anthropogenic isolation column (Section 13.6).

13.1.2	Acid, neutral, and basic silica gel (Section 13.3) and Florisil
(Section 13.7) are used to remove non-polar and polar interferences.

13.1.3	Carbopak/Celite (Section 13.4) can be used to separate CBs 77,
126, and 169 from the mono- and di- ortho-substituted CBs, if desired.

13.1.4	HPLC (Section 13.5) is used to provide specificity for certain
congeners and congener groups.

13.1.5	The anthropogenic isolation column (Section 13.6) is used for
removal of lipids from tissue samples.

13.2	Gel permeation chromatography (GPC)

13.2.1	Column packing

13.2.1.1	Place 70 to 75 g of SX-3 Bio-beads (Section 6.7.1.1) in a 400-
to 500-mL beaker.

13.2.1.2	Cover the beads with methylene chloride and allow to swell
overnight (a minimum of 12 hours).

13.2.1.3	Transfer the swelled beads to the column (Section 6.7.1.1) and
pump solvent through the column, from bottom to top, at 4.5 to 5.5
mL/minute prior to connecting the column to the detector.

13.2.1.4	After purging the column with solvent for 1 to 2 hours, adjust
the column head pressure to 7 to 10 psig and purge for 4 to 5 hours to
remove air.  Maintain a head pressure of 7 to 10 psig.  Connect the
column to the detector (Section 6.7.1.4).

13.2.2	Column calibration

13.2.2.1	Load 5 mL of the GPC calibration solution (Section 7.4) into
the sample loop.

13.2.2.2	Inject the GPC calibration solution and record the signal from
the detector.  The elution pattern will be corn oil, BEHP, methoxychlor,
perylene, and sulfur.

13.2.2.3	Set the “dump time” to allow >85% removal of BEHP and >85%
collection of methoxychlor.

13.2.2.4	Set the “collect time” to the time of the sulfur peak
maximum.

13.2.2.5	Verify calibration with the GPC calibration solution after
every 20 extracts.  Calibration is verified if the recovery of the
methoxychlor is greater than 85%.  If calibration is not verified, the
system must be recalibrated using the GPC calibration solution, and the
previous sample batch must be re-extracted and cleaned up using the
calibrated GPC system.

13.2.3	Extract cleanup – GPC requires that the column not be
overloaded.  The column specified in this Method is designed to handle a
maximum of 0.5 g of material from an aqueous, soil, or mixed-phase
sample in a 5-mL extract, and has been shown to handle 1.5 g of lipid
from a tissue sample in a 5-mL extract.  If the extract is known or
expected to contain more than these amounts, the extract is split into
aliquots for GPC, and the aliquots are combined after elution from the
column.  The residue content of the extract may be obtained
gravimetrically by evaporating the solvent from a 50-µL aliquot.

13.2.3.1	Filter the extract or load through the filter holder (Section
6.7.1.3) to remove particles.  Load the 5.0-mL extract onto the column.

13.2.3.2	Elute the extract using the calibration data determined in
Section 13.2.2.  Collect the eluate in a clean 400- to 500-mL beaker. 
Allow the system to rinse for additional 10 minutes before injecting the
next sample.

13.2.3.3	Rinse the sample loading tube thoroughly with methylene
chloride between extracts to prepare for the next sample.

13.2.3.4	If an extract is encountered that could overload the GPC column
to the extent that carry-over could occur, a 5.0-mL methylene chloride
blank must be run through the system to check for carry-over.

13.2.3.5	Concentrate the eluate per Sections 12.6 and 12.7 for further
cleanup or injection into the GC/MS.

13.3	Silica gel cleanup

13.3.1	Place a glass-wool plug in a 15-mm ID chromatography column
(Section  6.7.4.2).  Pack the column bottom to top with: 1 g silica gel
(Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g silica
gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g
granular anhydrous sodium sulfate (Section 7.2.1).  Tap the column to
settle the adsorbents.

13.3.2	Pre-elute the column with 50 to 100 mL of hexane.  Close the
stopcock when the hexane is within 1 mm of the sodium sulfate.  Discard
the eluate.  Check the column for channeling.  If channeling is present,
discard the column and prepare another.

13.3.3	Apply the concentrated extract to the column.  Open the stopcock
until the extract is within 1 mm of the sodium sulfate.

13.3.4	Rinse the receiver twice with 1-mL portions of hexane, and apply
separately to the column.  Elute the CBs with 25 mL of hexane and
collect the eluate.

13.3.5	Concentrate the eluate per Section 12.6 and 12.7 for further
cleanup or injection into the HPLC or GC/MS.

13.3.6	For extracts of samples known to contain large quantities of
other organic compounds, it may be advisable to increase the capacity of
the silica gel column.  This may be accomplished by increasing the
strengths of the acid and basic silica gels.  The acid silica gel
(Section 7.5.1.2) may be increased in strength to as much as 40% w/w
(6.7 g sulfuric acid added to 10 g silica gel).  The basic silica gel
(Section 7.5.1.3) may be increased in strength to as much as 33% w/w (50
mL 1N NaOH added to 100 g silica gel), or the potassium silicate
(Section 7.5.1.4) may be used.

Note:	The use of stronger acid silica gel (44% w/w) may lead to charring
of organic compounds in some extracts.  The charred material may retain
some of the analytes and lead to lower recoveries of the CBs. 
Increasing the strengths of the acid and basic silica gel may also
require different volumes of hexane than those specified above to elute
the analytes from the column.  The performance of the Method after such
modifications must be verified by the procedure in Section 9.2.

 ADVANCE \d7 

13.4	Carbon column (Reference 16)

13.4.1	Cut both ends from a 50-mL disposable serological pipet (Section
6.7.3.2) to produce a 20-cm column.  Fire-polish both ends and flare
both ends if desired.  Insert a glass-wool plug at one end, and pack the
column with 3.6 g of Carbopak/Celite (Section 7.5.2.3) to form an
adsorbent bed 20 cm long.  Insert a glass-wool plug on top of the bed to
hold the adsorbent in place.

13.4.2	Pre-elute the column with 20 mL each in succession of toluene,
methylene chloride, and hexane.

13.4.3	When the solvent is within 1 mm of the column packing, apply the
n-hexane sample extract to the column.  Rinse the sample container twice
with 1-mL portions of hexane and apply separately to the column.  Apply
2 mL of hexane to complete the transfer.

13.4.4	Elute the column with 25 mL of n-hexane and collect the eluate. 
This fraction will contain the mono- and di-ortho CBs.  If carbon
particles are present in the eluate, filter through glass-fiber filter
paper.

13.4.5	Elute the column with 15 mL of methanol and discard the eluate. 
The fraction discarded will contain residual lipids and other potential
interferents, if present.

13.4.6	Elute the column with 15 mL of toluene and collect the eluate. 
This fraction will contain CBs 77, 126, and 169.  If carbon particles
are present in the eluate, filter through glass-fiber filter paper.

13.4.7	Concentrate the fractions per Section 12.6 and 12.7 for further
cleanup or injection into the HPLC or GC/MS.

13.5	HPLC (References 4 and 17)

13.5.1	Column calibration

13.5.1.1	Prepare a calibration standard containing the Toxics and other
congeners of interest at the concentrations of the stock solution in
Table 3, or at a concentration appropriate to the response of the
detector.

13.5.1.2	Inject the calibration standard into the HPLC and record the
signal from the detector.  Collect the eluant for reuse.  Elution will
be in the order of the di-ortho, mono-ortho, and non-ortho congeners.

13.5.1.3	Establish the collection time for the congeners of interest. 
Following calibration, flush the injection system with solvent to ensure
that residual CBs are removed from the system.

13.5.1.4	Verify the calibration with the calibration solution after
every 20 extracts.  Calibration is verified if the recovery of the CBs
is 75 to 125% compared to the calibration (Section 13.5.1.1).  If
calibration is not verified, the system must be recalibrated using the
calibration solution, and the previous 20 samples must be re-extracted
and cleaned up using the calibrated system.

13.5.2	Extract cleanup – HPLC requires that the column not be
overloaded.  The column specified in this Method is designed to handle a
maximum of 5-50 µg of a given CB, depending on the congener (Reference
17).  If the amount of material in the extract will overload the column,
split the extract into fractions  and combine the fractions after
elution from the column.

13.5.2.1	Rinse the sides of the vial containing the sample and adjust to
the volume required for the sample loop for injection.

13.5.2.2	Inject the sample extract into the HPLC.

13.5.2.3	Elute the extract using the calibration data determined in
Section 13.5.1.  Collect the fraction(s) in clean 20-mL concentrator
tubes.

13.5.2.4	If an extract containing greater than 500 µg of total CBs is
encountered, a blank must be run through the system to check for
carry-over.

13.5.2.5	Concentrate the eluate per Section 12.7 for injection into the
GC/MS.

13.6	Anthropogenic isolation column (Reference 3) – Used for removal
of lipids from tissue extracts

13.6.1	Prepare the column as given in Section 7.5.3.

13.6.2	Pre-elute the column with 100 mL of hexane.  Drain the hexane
layer to the top of the column, but do not expose the sodium sulfate.

13.6.3	Load the sample and rinses (Section 12.4.9.2) onto the column by
draining each portion to the top of the bed.  Elute the CBs from the
column into the apparatus used for concentration (Section 12.4.7) using
200 mL of hexane.

13.6.4	Remove a small portion (e.g., 50 µL) of the extract for
determination of residue content.  Estimate the percent of the total
that this portion represents.  Concentrate the small portion to constant
weight per Section 12.7.3.1.  Calculate the total amount of residue in
the extract.  If more than 500 mg of material remains, repeat the
cleanup using a fresh anthropogenic isolation column.

13.6.5	If necessary, exchange the extract to a solvent suitable for the
additional cleanups to be used (Section 13.2-13.5 and 13.7).

13.6.6	Clean up the extract using the procedures in Sections 13.2-13.5
and 13.7.  GPC (Section 13.2) and Florisil (Section 13.7) are
recommended as minimum additional cleanup steps.

13.6.7	Following cleanup, concentrate the extract to 20 µL as described
in Section 12.7 and proceed with the analysis in Section 14.

13.7	Florisil cleanup (Reference 18)

13.7.1	Begin to drain the n-hexane from the column (Section 7.5.4.1.2). 
Adjust the flow rate of eluant to 4.5-5.0 mL/min.

13.7.2	When the n-hexane is within 1 mm of the sodium sulfate, apply the
sample extract (in hexane) to the column.  Rinse the sample container
twice with 1-mL portions of hexane and apply to the column.

13.7.3	Elute the mono-ortho and di-ortho CBs with approx 165 mL of
n-hexane and collect the eluate.  Elute the non-ortho co-planar CBs with
approx 100 mL of 6% ether:hexane and collect the eluate.  The exact
volumes of solvents will need to be determined for each batch of
Florisil.  If the mono/di-ortho CBs are not to be separated from the
non-ortho co-planar CBs, elute all CBs with 6% ether:hexane.

13.7.4	Concentrate the eluate(s) per Sections 12.6-12.7 for further
cleanup or for injection into the HPLC or GC/MS.

14.0	HRGC/HRMS analysis

14.1	Establish the operating conditions given in Section 10.1.

14.2	Add 2 µL of the labeled injection internal standard spiking
solution (Section 7.14) to the 20 µL sample extract immediately prior
to injection to minimize the possibility of loss by evaporation,
adsorption, or reaction.  If an extract is to be reanalyzed and
evaporation has occurred, do not add more labeled injection internal
standard spiking solution.  Rather, bring the extract back to its
previous volume (e.g., 19 µL) with pure nonane (18 µL if 2 µL
injections are used).

14.3	Inject 1.0 or 2.0 µL of the concentrated extract containing the
Labeled injection internal standards using on-column or splitless
injection.  The volume injected must be identical to the volume used for
calibration (Section 10.3).

14.3.1	Start the GC column initial isothermal hold upon injection. 
Start MS data collection after the solvent peak elutes.

14.3.2	Monitor the exact m/z’s at each LOC throughout the LOC
retention time window.  Where warranted, monitor m/z’s associated with
congeners at higher levels of chlorination to assure that fragments are
not interfering with the m/z’s  for congeners at lower levels of
chlorination.  Also where warranted, monitor  m/z’s associated with
interferents expected to be present.

14.3.3	Stop data collection after 13C12-DeCB has eluted.  Return the
column to the initial temperature for analysis of the next extract or
standard.

15.0	System and laboratory performance

15.1	At the beginning of each 12-hour shift during which analyses are
performed, GC/MS system performance and calibration are verified for all
native CBs and labeled compounds.  For these tests, analyze the diluted
combined 209 congener solution (Section 7.10.2.2) to verify all
performance criteria.  Adjustment and/or recalibration (Section 10) must
be performed until all performance criteria are met.  Only after all
performance criteria are met may samples, blanks, IPRs, and OPRs be
analyzed.

15.2	MS resolution – Static resolving power checks must be performed
at the beginning and at the end of each shift per Section 10.2.1.  If
analyses are performed on successive shifts, only the beginning of shift
static resolving power check is required.  If the requirement in Section
10.2.1 cannot be met, the problem must be corrected before analyses can
proceed.  If any of the samples in the previous shift may be affected by
poor resolution, those samples must be re-analyzed.

15.3	Calibration verification

15.3.1	Inject and analyze the Diluted combined 209 congener solution
(Section 7.10.2.2.2) using the procedure in Section 14.

15.3.2	The m/z abundance ratios for each native CB and labeled compound
in the VER standard must be within the limits in Table 8; otherwise, the
mass spectrometer must be adjusted until the m/z abundance ratios fall
within the limits specified when the verification test is be repeated. 
If the adjustment alters the resolution of the mass spectrometer,
resolution must be verified (Section 10.2.1) prior to repeat of the
verification test.

15.3.3	The GC peak representing each native CB and labeled compound in
the VER standard must be present with a S/N of at least 10; otherwise,
the mass spectrometer must be adjusted and the verification test
repeated.

15.3.4	Compute the recovery of the Toxics/LOC CBs by isotope dilution
(Section 17.1) and the labeled compounds by internal standard (17.2). 
These recoveries are computed based on the calibration data in Section
10.

15.3.5	For each compound, compare the recovery with the calibration
verification limit in Table 6.  If all compounds meet the acceptance
criteria, calibration has been verified and analysis of standards and
sample extracts may proceed.  If, however, any compound fails its
respective limit, the measurement system is not performing properly.  In
this event, prepare a fresh calibration standard or correct the problem
and repeat the resolution (Section 15.2) and verification (Section 15.3)
tests, or recalibrate (Section 10).  If recalibration is required,
recalibration for the 209 congeners (Section 10.5) must also be
performed.

15.4	Retention times and GC resolution

15.4.1	Retention times

15.4.1.1	Absolute – The absolute retention times of the Labeled
Toxics/LOC/window defining standard congeners (Section 7.12) in the
verification test (Section 15.3) must be within ± 15 seconds of the
respective retention times in the calibration or, if an alternate column
or column system is employed, within ± 15 seconds of the respective
retention times in the calibration for the alternate column or column
system (Section 6.9.1.2).

15.4.1.2	Relative – The relative retention times of native CBs and
labeled compounds in the verification test (Section 15.3) must be within
their respective RRT limits in Table 2 or, if an alternate column or
column system is employed, within their respective RRT limits for the
alternate column or column system (Section 6.9.1.2).

15.4.1.3	If the absolute or relative retention time of any compound is
not within the limits specified, the GC is not performing properly.  In
this event, adjust the GC and repeat the verification test (Section
15.3) or recalibrate (Section 10), or replace the GC column and either
verify calibration or recalibrate.

15.4.2	GC resolution and minimum analysis time

15.4.2.1	As a final step in calibration verification, GC resolution and
minimum analysis time are verified and response factors for congeners
other than the Toxics and LOC CBs are updated.

15.4.2.2	The resolution and minimum analysis time specifications in
Sections 6.9.1.1.2 and 6.9.1.1.1, respectively, must be met for the
SPB-octyl column or, if an alternate column or column system is
employed, must be met as specified for the alternate column or column
system (Section 6.9.1.2).  If these specifications are not met, the GC
analysis conditions must be adjusted until the specifications are met,
or the column must be replaced and the calibration verification tests
repeated Sections 15.4.1 through 15.4.2.2), or the system must be
recalibrated (Section 10).

15.4.2.3	After the resolution and minimum analysis time specifications
are met, update the retention times and relative retention times for all
congeners, and response factors for all congeners except the Toxics and
LOC CBs.  For the Toxics and LOC CBs, the multi-point calibration data
must be used ( Section 10.4) and verified (Section 15.3.4).

15.5	Ongoing precision and recovery

15.5.1	Analyze the extract of the ongoing precision and recovery (OPR)
aliquot (Section 11.4.2.5, 11.5.4, 11.6.2, or 11.8.3.2) prior to
analysis of samples from the same batch.

15.5.2	Compute the percent recovery of the Toxics/LOC CBs by isotope
dilution  (Section 10.4).  Compute the percent recovery of each labeled
compound by the internal standard method (Section 10.5).

15.5.3	For the Toxics/LOC CBs and labeled compounds, compare the
recovery to the OPR limits given in Table 6.  If all compounds meet the
acceptance criteria, system performance is acceptable and analysis of
blanks and samples may proceed.  If, however, any individual
concentration falls outside of the range  given, the
extraction/concentration processes are not being performed properly for
that compound.  In this event, correct the problem, re-prepare, extract,
and clean up the sample batch and repeat the ongoing precision and
recovery test (Section 15.5).

15.5.4	If desired, add results that pass the specifications in Section
15.5.3 to initial and previous ongoing data for each compound in each
matrix.  Update QC charts to form a graphic representation of continued
laboratory performance.  Develop a statement of laboratory accuracy for
each congener in each matrix type by calculating the average percent
recovery (R) and the standard deviation of percent recovery (SR). 
Express the accuracy as a recovery interval from R - 2SR to R + 2SR. 
For example, if R = 95% and SR = 5%, the accuracy is 85 to 105%.

15.6	Blank – Analyze the Method blank extracted with each sample batch
immediately following analysis of the OPR aliquot to demonstrate freedom
from contamination and freedom from carryover from the OPR analysis. If
CBs will be carried from the OPR into the Method blank, analyze one or
more aliquots of solvent between the OPR and the Method blank.  The
results of the analysis of the blank must meet the specifications in
Section 9.5.2 before sample analyses may proceed.

16.0	Qualitative determination

A CB or labeled compound is identified in a standard, blank, or sample
when all of the criteria in Sections 16.1 through 16.4 are met.

16.1	The signals for the two exact m/z’s in Table 7 must be present
and must maximize within the same two scans.

16.2	The signal-to-noise ratio (S/N) for the GC peak at each exact m/z
must be greater than or equal to 2.5 for each CB detected in a sample
extract, and greater than or equal to 10 for all CBs in the calibration
and verification standards (Sections 10.3.3 and 15.3.3).

Note:	  An interference between DiCB m/z 223.9974 and PFK m/z 223.9872
may preclude meeting the S/N requirement for the DiCB congeners.  If
identification is ambiguous, an experienced spectrometrist (Section 1.4)
must determine the presence or absence of the congener. ADVANCE \d7 

16.3	The ratio of the integrated areas of the two exact m/z’s
specified in Table 7 must be within the limit in Table 8, or within ±
15 percent of the ratio in the midpoint (CS-3) calibration or
calibration verification (VER), whichever is most recent.

16.4	The relative retention time of the peak for a CB must be within the
RRT QC limits specified in Table 2 or within similar limits developed
from calibration data (Section 10.1.2).   If an alternate column or
column system is employed, the RRT for the CB must be within its
respective RRT QC limits for the alternate column or column system
(Section 6.9.1.2).

Note:	  For native CBs determined by internal standard quantitation, a
given CB congener may fall within more than one RT window and be
mis-identified unless the RRT windows are made very narrow, as in Table
2.  Therefore, consistency of the RT and RRT with other congeners and
the labeled compounds may be required for rigorous congener
identification.  Retention time regression analysis may aid in this
identification. ADVANCE \d7 

16.5	Because of congener overlap and the potential for interfering
substances, it is possible that all of the identification criteria
(Sections 16.1-16.4) may not be met.  It is also possible that loss of
one or more chlorines from a highly chlorinated congener may inflate or
produce a false concentration for a less-chlorinated congener that
elutes at the same retention time (see Section 18.5).  If identification
is ambiguous, an experienced spectrometrist (Section 1.4) must determine
the presence or absence of the congener.

16.6	If the criteria for identification in Sections 16.1-16.5 are not
met, the CB has not been identified and the result for that congener may
not be reported or used for permitting or regulatory compliance
purposes.  If interferences preclude identification, a new aliquot of
sample must be extracted, further cleaned up, and analyzed.

17.0	Quantitative determination

17.1	Isotope dilution quantitation

17.1.1	By adding a known amount of the Labeled
Toxics/LOC/window-defining compounds to every sample prior to
extraction, correction for recovery of the CBs can be made because the
native compound and its labeled analog exhibit similar effects upon
extraction, concentration, and gas chromatography.  Relative responses
(RRs) are used in conjunction with the calibration data in Section 10.4
to determine concentrations in the final extract, so long as labeled
compound spiking levels are constant.

17.1.2	Compute the concentrations in the extract of the Native
Toxics/LOC CBs using the RRs from the calibration data (Section 10.4)
and following equation:

 

	

	where:

	Cex	=	concentration of the PCB in the extract (ng/mL) and the other
terms are as defined in Section 10.5.1

17.2	Internal standard quantitation and labeled compound recovery

17.2.1	Compute the concentrations in the extract of the labeled
compounds (except labeled CB 178) and of the native compounds other than
those in the Native Toxics/LOC standard using the response factors
determined from calibration (Section 10.5)  or calibration verification
(Section 15.4.2.3) and the following equation:

	where:

	Cex	=	concentration of the native or labeled compound in the extract
(ng/mL) and the other terms are as defined in Section 10.5.1

17.2.2	Using the concentration in the extract determined above, compute
the percent recovery of the Labeled Toxics/LOC/window-defining CBs and
the Labeled cleanup standard CBs using the following equation:

17.3	The concentration of a native CB in the solid phase of the sample
is computed using the concentration of the compound in the extract and
the weight of the solids (Section 11.2.2.3), as follows:

where:

Cex	=	The concentration of the compound in the extract (ng/mL).

Vex	=	The extract volume in mL.

Ws	=	The sample weight (dry weight) in kg.

17.4	The concentration of a native CB in the aqueous phase of the sample
is computed using the concentration of the compound in the extract and
the volume of water extracted (Section 11.4.2.1), as follows:

where:

Cex	=	The concentration of the compound in the extract (pg/mL).

Vex	=	The extract volume in mL.

Vs	=	The sample volume in liters.

17.5	If the SICP area at either quantitation m/z for any congener
exceeds the calibration range of the system, dilute the sample extract
by the factor necessary to bring the concentration within the
calibration range, adjust the concentration of the Labeled injection
internal standard to 100 pg/µL in the extract, and analyze an aliquot
of this diluted extract.  If the CBs cannot be measured reliably by
isotope dilution, dilute and analyze an aqueous sample or analyze a
smaller portion of a soil, tissue, or mixed-phase sample.  Adjust the CB
congener concentrations, detection limits, and minimum levels to account
for the dilution.

17.6	Reporting of results – Results are reported to three significant
figures for the CBs and labeled compounds found in all standards,
blanks, and samples.

17.6.1	 Reporting units and levels

17.6.1.1	Aqueous samples – Report results in pg/L
(parts-per-quadrillion).

17.6.1.2	Samples containing greater than 1% solids (soils, sediments,
filter cake, compost) – Report results in ng/kg based on the dry
weight of the sample.  Report the percent solids so that the result may
be converted to aqueous units.

17.6.1.3	Tissues – Report results in ng/kg of wet tissue, not on the
basis of the lipid content of the tissue.  Report the percent lipid
content, so that the data user can calculate the concentration on a
lipid basis if desired.

17.6.1.4	Reporting level

17.6.1.4.1	Report the result for each congener at or above the minimum
level of quantitation (ML; Table 2) for analyses of blanks, standards,
and samples.  The MLs in Table 2 are the levels that can be achieved in
the presence of common laboratory contamination.  A laboratory may
establish an ML for a CB congener lower than the MLs in Table 2.  MLs
may be established as low as the lowest calibration point (Table 5)
provided that the concentration of the congener in a minimum of 10
blanks for a sample medium (e.g., water, soil, sludge, tissue) is
significantly below the ML in Table 2.  “Significant” means that the
ML for the congener is no less than 2 standard deviations above the mean
(average) level in the minimum of 10 blanks (Reference 19).  The blanks
must be analyzed during the same period that samples are analyzed,
ideally over an approximately 1-month period.

17.6.1.4.2	Standards (VER, IPR, OPR) and samples – Report the result
for each congener at or above the ML (Table 2) to 3 significant figures.
 Report results below the ML as <ML (where ML is the concentration at
the ML) or as required by the regulatory authority or permit.

17.6.1.4.3	Blanks – Report the result for each congener above the ML
to 3 significant figures.  Report a result below the ML but above the
MDL to 2 significant figures.  Report a result below the MDL as <MDL
(where MDL is the concentration at the MDL) or as required by the
regulatory authority or permit.

17.6.1.4.4	Blank correction – Blank-corrected results may be reported
in addition to reporting of separate results for samples (Section
17.6.1.4.1) and blanks (Section 17.6.1.4.2).  The recommended procedure
for blank correction (Reference 19) is that a result is significantly
above the blank level, and the level in the blank may be subtracted, if
the result is 2 standard deviations above the mean (average) of results
of analyses of 10 or more blanks for a sample medium.

17.6.2	Results for a CB in a sample that has been diluted are reported
at the least dilute level at which the area at the quantitation m/z is
within the calibration range (Section 17.5).

17.6.3	For a CB having a labeled analog, report results at the least
dilute level at which the area at the quantitation m/z is within the
calibration range (Section 17.5) and the labeled compound recovery is
within the normal range for the Method  (Section 9.3 and Table 6).

17.6.4	If requested, the total concentration of all congeners at a given
level of  chlorination (homolog; i.e., total TrCB, total PeCB, total
HxCB) may be reported by summing the concentrations of all congeners
identified at that LOC, including both the Toxics and other congeners. 
Also if requested, total CBs may be reported by summing all congeners
identified at all LOCs.

17.6.5	Reporting of coeluting PCB congeners–Optionally, Delaware River
Basin Commission (DRBC) data qualifier flags and conventions for
reporting coeluting congeners (see
http://www.state.nj.us/drbc/PCB_info.htm), or other reporting convention
agreed upon between the laboratory and the discharger/permittee or
regulatory/control authority, may be used.

18.0	Analysis of complex samples

18.1	Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the
compounds of interest, interfering compounds, and/or polymeric
materials.  Some extracts may not concentrate to 20 µL (Section
12.7.7); others may overload the GC column and/or mass spectrometer. 
Fragment ions from congeners at higher levels of chlorination may
interfere with determination of congeners at lower levels of
chlorination.

18.2	Analyze a smaller aliquot of the sample (Section 17.5) when the
extract will not concentrate to 20 µL after all cleanup procedures have
been exhausted.  If a smaller aliquot of soils or mixed-phase samples is
analyzed, attempt to assure that the sample is representative.

18.3	Perform integration of peak areas and calculate concentrations
manually when interferences preclude computerized calculations.

18.4	Several laboratories have reported that backgrounds of many of the
CB congeners are difficult to eliminate, and that these backgrounds can
interfere with the determination of the CBs in environmental samples. 
Backgrounds of Toxics with congener numbers 105, 114, 118, 123, 156,
157, and 167 are common.  The effects of contamination on results for
these congeners should be understood in order to make a reliable
determination.

18.5	Interferences may pose a problem in the determination of congeners
81, 123, 126, and 169 in  some environmental samples.  Loss of one or
more chlorines from a highly chlorinated congener may inflate or produce
a false concentration for a less-chlorinated congener that elutes at the
same retention time.  If, upon inspection of the chromatogram, the
possibility of interferences is evident (e.g., high concentrations of
fragments from loss of one or two chlorines from higher chlorinated
closely eluting congeners), carbon column fractionation (Section 13.4)
and analysis is recommended.

18.6	Recovery of labeled compounds – In most samples, recoveries of
the labeled compounds will be similar to those from reagent water or
from the alternate matrix (Section 7.6).

18.6.1	If the recovery of any of the labeled compounds is outside of the
normal range (Table 6), a diluted sample must be analyzed (Section
17.5).

18.6.2	If the recovery of any of the labeled compounds in the diluted
sample is outside of normal range, the Diluted combined 209 congener
solution (Section 7.10.2.2.2) must be analyzed and calibration verified
(Section 15.3).

18.6.3	If the calibration cannot be verified, a new calibration must be
performed and the original sample extract reanalyzed.

18.6.4	If calibration is verified and the diluted sample does not meet
the limits for labeled compound recovery, the Method does not apply to
the sample being analyzed and the result may not be reported or used for
permitting or regulatory compliance purposes.  In this case, alternate
extraction and cleanup procedures in this Method or an alternate GC
column must be employed to resolve the interference.  If all cleanup
procedures in this Method and an alternate GC column have been employed
and labeled compound recovery remains outside of the normal range,
extraction and/or cleanup procedures that are beyond this scope of this
Method will be required to analyze the sample.

19.0	Pollution prevention

19.1	Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of generation.
 Many opportunities for pollution  prevention exist in laboratory
operation.  EPA has established a preferred hierarchy of environmental
management techniques that places pollution prevention as the management
option of first choice.  Whenever feasible, laboratory personnel should
use pollution prevention techniques to address waste generation.  When
wastes cannot be reduced feasibly at the source, the Agency recommends
recycling as the next best option.

19.2	The CBs in this Method are used in extremely small amounts and pose
little threat to the environment when managed properly.  Standards
should be prepared in volumes  consistent with laboratory use to
minimize the disposal of excess volumes of expired standards.

19.3	For information about pollution prevention that may be applied to
laboratories and research institutions, consult Less is Better:
Laboratory Chemical Management for Waste Reduction, available from the
American Chemical Society's Department of Governmental Relations and
Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-4477.

20.0	Waste management

20.1	The laboratory is responsible for complying with all Federal,
State, and local regulations governing waste management, particularly
the hazardous waste identification rules and land disposal restrictions,
and to protect the air, water, and land by minimizing and controlling
all releases from fume hoods and bench operations.  Compliance is also
required with any sewage discharge permits and regulations.  An overview
of requirements can be found in Environmental Management Guide for Small
Laboratories (EPA 233-B-98-001).

20.2	Samples containing HCl or H2SO4 to pH <2 are hazardous and must be
neutralized before being poured down a drain or must be handled as
hazardous waste.

20.3	The CBs decompose above 800 ºC.  Low-level waste such as absorbent
paper, tissues, animal remains, and plastic gloves may be burned in an
appropriate incinerator.  Gross quantities (milligrams) should be
packaged securely and disposed of through commercial or governmental
channels that are capable of handling extremely toxic wastes.

20.4	Liquid or soluble waste should be dissolved in methanol or ethanol
and irradiated with ultraviolet light with a wavelength shorter than 290
nm for several days.  Use F40 BL or equivalent lamps.  Analyze liquid
wastes, and dispose of the solutions when the CBs can no longer be
detected.

20.5	For further information on waste management, consult The Waste
Management Manual for Laboratory Personnel and Less is Better-Laboratory
Chemical Management for Waste Reduction, available from the American
Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street NW, Washington, DC 20036.

21.0	Method performance

The original version of Method 1668 was validated in single-laboratory
studies at Pacific Analytical, Inc., Carlsbad, California and AXYS
Analytical Services, Ltd., Sidney, British Columbia, Canada.  The next
version, Method 1668A, was validated and data were collected at AXYS
Analytical (Reference 20).  Method 1668A was subjected to peer review in
1999, and published in 2000.  In 2003-2004, EPA conducted an
interlaboratory method validation study of Method 1668A (Reference 21),
subjected the study to a peer review, and subsequently published
interlaboratory performance data in Method 1668B.  

After release of Method 1668B, it was reported to EPA that some of the
QC acceptance criteria in Method 1668B did not allow excursions above
100 percent.  As a result, the QC acceptance criteria were re-developed
using data from the interlaboratory study and data from AXYS Analytical
and TestAmerica-Knoxville, Tennessee.  The revised QC acceptance
criteria were published in addendum to the Interlaboratory Study Report
(Reference 22).  

Subsequent to development of the revised QC acceptance criteria, AXYS
Analytical, TestAmerica-Knoxville, and Battelle-Columbus provided method
detection limit (MDL) data to EPA.  These data were combined to produced
pooled MDLs and MLs (Reference 23).  Method 1668B was revised to Method
1668C to incorporate the revised QC acceptance criteria and revised MDLs
and MLs.  

Figure 8 is a chromatogram showing method performance at each level of
chlorination.

22.0	References												

1.	Van den Berg, Martin, Linda S. Birnbaum, Michael Denison, Mike De
Vito, William Farland, Mark Feeley, Heidelore Fiedler, Helen Hakansson,
Annika Hanberg, Laurie Haws, Martin Rose, Stephen Safe, Dieter Schrenk,
Chiharu Tohyama, Angelika Tritscher, Jouko Tuomisto, Mats Tysklind,
Nigel Walker, and Richard E. Peterson, 2006, “The 2005 World Health
Organization Reevaluation of Human and Mammalian Toxic Equivalency
Factors for Dioxins and Dioxin-like Compounds,”  Toxicological
Sciences 93(2): 223-241.

2.	“Sampling and Analytical Methods of the National Status and Trends
Program Mussel Watch Project: 1993-1996 Update,” NOAA Technical
Memorandum NOS ORCS 130, Coastal Monitoring and Bioeffects Assessment
Division, Office of Ocean Resources Conservation and Assessment,
National Ocean Service, National Oceanic and Atmospheric Administration,
U.S. Department of Commerce, N/ORCA2, SSMC4, 1305 East-West Highway,
Silver Spring, MD 20910, p. 3, 1998.

3.	Kuehl, D.W., B.C. Butterworth, J. Libal, and P. Marquis, “An
Isotope Dilution High Resolution Gas Chromatography-High Resolution Mass
Spectrometric Method for the Determination of Coplanar Polychlorinated
Biphenyls: Application to Fish and Marine Mammals,” Chemosphere
22:9-10, 849-858, 1991.

4.	Echols, Kathy, Robert Gale, Donald E. Tillitt, Ted Schwartz, and
Jerome O'Laughlin, “An Automated HPLC Method for the Fractionation of
Polychlorinated Biphenyls, Polychlorinated Dibenzo-p-dioxins, and
Polychlorinated Dibenzofurans in Fish Tissue on a Porous Graphitic
Carbon Column,” Environmental Toxicology and Chemistry 16:8 1590-1597,
1997.

5.	“Working with Carcinogens,” Department of Health, Education, &
Welfare, Public Health Service, Centers for Disease Control, NIOSH,
Publication 77-206, August 1977, NTIS PB-277256.

6.	“OSHA Safety and Health Standards, General Industry,” OSHA 2206,
29 CFR 1910.

7.	“Safety in Academic Chemistry Laboratories,” ACS Committee on
Chemical Safety, 1979.

8.	“Standard Methods for the Examination of Water and Wastewater,”
18th edition and later revisions, American Public Health Association,
1015 15th St, NW, Washington, DC 20005, 1-35: Section 1090 (Safety),
1992.

9.	“Method 613 – 2,3,7,8-Tetrachlorodibenzo-p-dioxin,” 40 CFR 136,
Appendix A, Section 4.1.

10.	Lamparski, L.L., and Nestrick, T.J., “Novel Extraction Device for
the Determination of Chlorinated Dibenzo-p-dioxins (PCDDs) and
Dibenzofurans (PCDFs) in Matrices Containing Water,” Chemosphere,
19:27-31, 1989.

11.	Provost, L.P., and Elder, R.S., “Interpretation of Percent
Recovery Data,” American Laboratory, 15: 56-83, 1983.

12.	“Standard Practice for Sampling Water,” ASTM Annual Book of
Standards, ASTM, 1916 Race Street, Philadelphia, PA 19103-1187, 1980.

13.	“Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA,
EMSL, Cincinnati, OH 45268, EPA 600/4-70-020, April 1979.

14.	“Handbook of Analytical Quality Control in Water and Wastewater
Laboratories,” USEPA EMSL, Cincinnati, OH 45268, EPA-600/4-79-019,
April 1979.

15.	“Analytical Procedures and Quality Assurance Plan for the
Determination of PCDD/PCDF in Fish”, U.S. Environmental Protection
Agency, Environmental Research Laboratory, Duluth, MN 55804,
EPA/600/3-90/022, April 1990.

16.	Storr-Hansen, E. and T. Cederberg, “Determination of Coplanar
Polychlorinated Biphenyl (CB) Congeners in Seal Tissues by
Chromatography on Active Carbon, Dual-Column High Resolution GC/ECD and
High Resolution GC/High Resolution MS,” Chemosphere 24:9, 1181-1196,
1992.

17.	Echols, Kathy R., Robert W. Gale, Kevin Feltz, Jerome O'Laughlin,
Donald E. Tillitt, and Ted R. Schwartz, “Loading capacity and
chromatographic behavior of a porous graphitic carbon column for
polychlorinated biphenyls,” J. Chromatog. A 811: 135-144, 1998.

18.	Tessari, J.D., Personal communication with Dale Rushneck, available
from U.S. Environmental Protection Agency, Engineering and Analysis
Division (4303T), 1200 Pennsylvania Avenue NW, Washington, DC 20460.

19.	Ferrario, J.C., C. Byrne, A.E. Dupuy, Jr., “Background
Contamination by Coplanar Polychlorinated Biphenyls (PCBs) in Trace
Level High Resolution Gas Chromatography/High Resolution Mass
Spectrometry (HRGC/HRMS) Analytical Procedures” Chemosphere 34:11,
2451-2465, 1997.

20.	“Development of a Full Congener Version of Method 1668 and
Application to the Analysis of 209 PCB Congeners in Aroclors,” AXYS
Analytical Services, available from U.S. Environmental Protection
Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania
Avenue NW, Washington, DC 20460.

21.	“Method 1668A Interlaboratory Validation Study Report,” March
2010, EPA-820-R-10-004, U.S. Environmental Protection Agency,
Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.

22.	“Method 1668A Interlaboratory Study Report Addendum,” March
2010, EPA-820-R-10-003, U.S. Environmental Protection Agency,
Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.

23.	“Development of Pooled Method Detection Limits (MDLs) and Minimum
Levels of Quantitation (MLs) for EPA Method 1668C,” Brian Englert, 
USEPA, May 18 2010; available from U.S. Environmental Protection Agency,
Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.

23.0	Tables and Figures													

Table 1.	Names, Congener Numbers, and CAS Registry Numbers for Native
and Labeled Chlorinated Biphenyl (CB) Congeners Determined by Isotope
Dilution and Internal Standard HRGC/HRMS

CB congener name1	Congener number	CAS Registry number	Labeled analog
name	Labeled analog congener number	CAS Registry number

2-MoCB	1	2051-60-7	13C12-2-MoCB2	1L	234432-85-0

3-MoCB	2	2051-61-8



	4-MoCB	3	2051-62-9	13C12-4-MoCB2	3L	208263-77-8

2,2'-DiCB	4	13029-08-8	13C12-2,2'-DiCB2	4L	234432-86-1

2,3-DiCB	5	16605-91-7



	2,3'-DiCB	6	25569-80-6



	2,4-DiCB	7	33284-50-3



	2,4'-DiCB3	8	34883-43-7



	2,5-DiCB	9	34883-39-1	13C12-2,5-DiCB4	9L	250694-89-4

2,6-DiCB	10	33146-45-1



	3,3'-DiCB	11	2050-67-1



	3,4-DiCB	12	2974-92-7



	3,4'-DiCB	13	2974-90-5



	3,5-DiCB	14	34883-41-5



	4,4'-DiCB	15	2050-68-2	13C12-4,4'-DiCB2	15L	208263-67-6

2,2',3-TrCB	16	38444-78-9



	2,2',4-TrCB	17	37680-66-3



	2,2',5-TrCB3	18	37680-65-2



	2,2',6-TrCB	19	38444-73-4	13C12-2,2',6-TrCB2	19L	234432-87-2

2,3,3'-TrCB	20	38444-84-7



	 2,3,4-TrCB	21	55702-46-0



	2,3,4'-TrCB	22	38444-85-8



	2,3,5-TrCB	23	55720-44-0



	2,3,6-TrCB	24	55702-45-9



	2,3',4-TrCB	25	55712-37-3



	2,3',5-TrCB	26	38444-81-4



	 2,3',6-TrCB	27	38444-76-7



	 2,4,4'-TrCB3	28	7012-37-5	13C12-2,4,4'-TriCB5	28L	208263-76-7

2,4,5-TrCB	29	15862-07-4



	 2,4,6-TrCB	30	35693-92-6



	2,4',5-TrCB	31	16606-02-3



	 2,4',6-TrCB	32	38444-77-8



	2',3,4-TrCB	33	38444-86-9



	2',3,5-TrCB	34	37680-68-5



	3,3',4-TrCB	35	37680-69-6



	3,3',5-TrCB	36	38444-87-0



	3,4,4'-TrCB	37	38444-90-5	13C12-3,4,4'-TrCB2	37L	208263-79-0

3,4,5-TrCB	38	53555-66-1



	3,4',5-TrCB	39	38444-88-1



	2,2',3,3'-TeCB	40	38444-93-8



	 2,2',3,4-TeCB	41	52663-59-9



	2,2',3,4'-TeCB	42	36559-22-5



	 2,2',3,5-TeCB	43	70362-46-8



	2,2',3,5'-TeCB3	44	41464-39-5



	2,2',3,6-TeCB	45	70362-45-7



	2,2',3,6'-TeCB	46	41464-47-5



	2,2',4,4'-TeCB	47	2437-79-8



	2,2',4,5-TeCB	48	70362-47-9



	2,2',4,5'-TeCB	49	41464-40-8



	2,2',4,6-TeCB	50	62796-65-0



	2,2',4,6'-TeCB	51	68194-04-7



	2,2',5,5'-TeCB3	52	35693-99-3	13C12-2,2',5,5'-TeCB4	52L	208263-80-3

2,2',5,6'-TeCB	53	41464-41-9



	2,2',6,6'-TeCB	54	15968-05-5	13C12-2,2',6,6'-TeCB2	54L	234432-88-3

2,3,3',4'-TeCB	55	74338-24-2



	2,3,3',4'-TeCB	56	41464-43-1



	2,3,3',5-TeCB	57	70424-67-8



	2,3,3',5'-TeCB	58	41464-49-7



	2,3,3',6-TeCB	59	74472-33-6



	 2,3,4,4'-TeCB	60	33025-41-1



	 2,3,4,5-TeCB	61	33284-53-6



	 2,3,4,6-TeCB	62	54230-22-7



	 2,3,4',5-TeCB	63	74472-34-7



	2,3,4',6-TeCB	64	52663-58-8



	 2,3,5,6-TeCB	65	33284-54-7



	2,3',4,4'-TeCB3	66	32598-10-0



	 2,3',4,5-TeCB	67	73575-53-8



	 2,3',4,5'-TeCB	68	73575-52-7



	 2,3',4,6-TeCB	69	60233-24-1



	2,3',4',5-TeCB	70	32598-11-1



	 2,3',4',6-TeCB	71	41464-46-4



	 2,3',5,5'-TeCB	72	41464-42-0



	 2,3',5',6-TeCB	73	74338-23-1



	2,4,4',5-TeCB	74	32690-93-0



	2,4,4',6-TeCB	75	32598-12-2



	 2',3,4,5-TeCB	76	70362-48-0



	3,3',4,4'-TeCB3,6	77	32598-13-3	13C12-3,3',4,4'-TeCB2,7	77L	105600-23-5

3,3',4,5-TeCB	78	70362-49-1



	3,3',4,5'-TeCB	79	41464-48-6



	3,3',5,5'-TeCB	80	33284-52-5



	3,4,4',5-TeCB6	81	70362-50-4	13C12-3,4,4',5-TeCB7	81L	208461-24-9

2,2',3,3',4-PeCB	82	52663-62-4



	2,2',3,3',5-PeCB	83	60145-20-2



	2,2',3,3',6-PeCB	84	52663-60-2



	2,2',3,4,4'-PeCB	85	65510-45-4



	2,2',3,4,5-PeCB	86	55312-69-1



	2,2',3,4,5'-PeCB	87	38380-02-8



	2,2',3,4,6-PeCB	88	55215-17-3



	  2,2',3,4,6'-PeCB	89	73575-57-2



	2,2',3,4',5-PeCB	90	68194-07-0



	2,2',3,4',6-PeCB	91	68194-05-8



	 2,2',3,5,5'-PeCB	92	52663-61-3



	 2,2',3,5,6-PeCB	93	73575-56-1



	 2,2',3,5,6'-PeCB	94	73575-55-0



	2,2',3,5',6-PeCB	95	38379-99-6



	2,2',3,6,6'-PeCB	96	73575-54-9



	2,2',3',4,5-PeCB	97	41464-51-1



	 2,2',3',4,6-PeCB	98	60233-25-2



	2,2',4,4',5-PeCB	99	38380-01-7



	2,2',4,4',6-PeCB	100	39485-83-1



	2,2',4,5,5'-PeCB3	101	37680-73-2	13C12-2,2',4,5,5'-PeCB4	101L
104130-39-4

2,2',4,5,6'-PeCB	102	68194-06-9



	2,2',4,5,'6-PeCB	103	60145-21-3



	2,2',4,6,6'-PeCB	104	56558-16-8	13C12-2,2',4,6,6'-PeCB2	104L
234432-89-4

2,3,3',4,4'-PeCB3,6	105	32598-14-4	13C12-2,3,3',4,4'-PeCB7	105L
208263-62-1

 2,3,3',4,5-PeCB	106	70424-69-0



	2,3,3',4',5-PeCB	107	70424-68-9



	2,3,3',4,5'-PeCB	108	70362-41-3



	2,3,3',4,6-PeCB	109	74472-35-8



	2,3,3',4',6-PeCB	110	38380-03-9



	 2,3,3',5,5'-PeCB	111	39635-32-0	13C12-2,3,3',5,5'-PeCB5	111 L
235416-29-2

 2,3,3',5,6-PeCB	112	74472-36-9



	2,3,3',5',6-PeCB	113	68194-10-5



	2,3,4,4',5-PeCB6	114	74472-37-0	13C12-2,3,4,4',5-PeCB7	114 L
208263-63-2

 2,3,4,4',6-PeCB	115	74472-38-1



	 2,3,4,5,6-PeCB	116	18259-05-7



	2,3,4',5,6-PeCB	117	68194-11-6



	2,3',4,4',5-PeCB3,6	118	31508-00-6	13C12-2,3',4,4',5-PeCB7	118 L
104130-40-7

2,3',4,4',6-PeCB	119	56558-17-9



	2,3',4,5,5'-PeCB	120	68194-12-7



	2,3',4,5,'6-PeCB	121	56558-18-0



	2',3,3',4,5-PeCB	122	76842-07-4



	2',3,4,4',5-PeCB6	123	65510-44-3	13C12-2',3,4,4',5-PeCB7	123L
208263-64-3

 2',3,4,5,5'-PeCB	124	70424-70-3



	 2',3,4,5,6'-PeCB	125	74472-39-2



	3,3',4,4',5-PeCB3,6	126	57465-28-8	13C12-3,3',4,4',5-PeCB2,7	126L
208263-65-4

3,3',4,5,5'-PeCB	127	39635-33-1



	2,2',3,3',4,4'-HxCB3	128	38380-07-3



	2,2',3,3',4,5-HxCB	129	55215-18-4



	2,2',3,3',4,5'-HxCB	130	52663-66-8



	2,2',3,3',4,6-HxCB	131	61798-70-7



	2,2',3,3',4,6'-HxCB	132	38380-05-1



	2,2',3,3',5,5'-HxCB	133	35694-04-3



	2,2',3,3',5,6-HxCB	134	52704-70-8



	2,2',3,3',5,6'-HxCB	135	52744-13-5



	2,2',3,3',6,6'-HxCB	136	38411-22-2



	2,2',3,4,4',5-HxCB	137	35694-06-5



	2,2',3,4,4',5'-HxCB3	138	35065-28-2	13C12-2,2',3,4,4',5'-HxCB4	138L
208263-66-5

 2,2',3,4,4',6-HxCB	139	56030-56-9



	 2,2',3,4,4',6'-HxCB	140	59291-64-4



	2,2',3,4,5,5'-HxCB	141	52712-04-6



	2,2',3,4,5,6-HxCB	142	41411-61-4



	 2,2',3,4,5,6'-HxCB	143	68194-15-0



	2,2',3,4,5',6-HxCB	144	68194-14-9



	2,2',3,4,6,6'-HxCB	145	74472-40-5



	2,2',3,4',5,5'-HxCB	146	51908-16-8



	2,2',3,4',5,6-HxCB	147	68194-13-8



	2,2',3,4',5,6'-HxCB	148	74472-41-6



	2,2',3,4',5',6-HxCB	149	38380-04-0



	2,2',3,4',6,6'-HxCB	150	68194-08-1



	2,2',3,5,5',6-HxCB	151	52663-63-5



	2,2',3,5,6,6'-HxCB	152	68194-09-2



	2,2',4,4',5,5'-HxCB3	153	35065-27-1



	2,2',4,4',5',6-HxCB	154	60145-22-4



	2,2',4,4',6,6'-HxCB	155	33979-03-2	13C12-2,2',4,4',6,6'-HxCB2	155L
234432-90-7

2,3,3',4,4',5-HxCB6	156	38380-08-4	13C12-2,3,3',4,4',5-HxCB7	156L
208263-68-7

2,3,3',4,4',5'-HxCB6	157	69782-90-7	13C12-2,3,3',4,4',5'-HxCB7	157L
235416-30-5

2,3,3',4,4',6-HxCB	158	74472-42-7



	2,3,3',4,5,5'-HxCB	159	39635-35-3



	 2,3,3',4,5,6-HxCB	160	41411-62-5



	2,3,3',4,5',6-HxCB	161	74472-43-8



	2,3,3',4',5,5'-HxCB	162	39635-34-2



	 2,3,3',4',5,6-HxCB	163	74472-44-9



	 2,3,3',4',5',6-HxCB	164	74472-45-0



	 2,3,3',5,5',6-HxCB	165	74472-46-1



	 2,3,4,4',5,6-HxCB	166	41411-63-6



	2,3',4,4',5,5'-HxCB6	167	52663-72-6	13C12-2,3',4,4',5,5'-HxCB7	167L
208263-69-8

2,3',4,4',5',6-HxCB	168	59291-65-5



	3,3',4,4',5,5'-HxCB3,6	169	32774-16-6	13C12-3,3',4,4',5,5'-HxCB2,7	169L
208263-70-1

2,2',3,3',4,4',5-HpCB3	170	35065-30-6	13C12-2,2',3,3',4,4',5-HpCB	170L
160901-80-4

2,2'3,3',4,4',6-HpCB	171	52663-71-5



	2,2',3,3',4,5,5'-HpCB	172	52663-74-8



	2,2',3,3',4,5,6-HpCB	173	68194-16-1



	2,2',3,3',4,5,6'-HpCB	174	38411-25-5



	2,2',3,3',4,5',6-HpCB	175	40186-70-7



	2,2',3,3',4,6,6'-HpCB	176	52663-65-7



	2,2',3,3',4',5,6-HpCB	177	52663-70-4



	2,2',3,3',5,5',6-HpCB	178	52663-67-9	13C12-2,2',3,3',5,5',6-HpCB5	178L
232919-67-4

2,2',3,3',5,6,6'-HpCB	179	52663-64-6



	2,2',3,4,4',5,5'-HpCB3	180	35065-29-3	13C12-2,2',3,4,4',5,5'-HpCB	180L
160901-82-6

 2,2',3,4,4',5,6-HpCB	181	74472-47-2



	 2,2',3,4,4',5,6'-HpCB	182	60145-23-5



	2,2',3,4,4',5',6-HpCB	183	52663-69-1



	2,2',3,4,4',6,6'-HpCB	184	74472-48-3



	2,2',3,4,5,5',6-HpCB	185	52712-05-7



	2,2',3,4,5,6,6'-HpCB	186	74472-49-4



	2,2',3,4',5,5',6-HpCB3	187	52663-68-0



	2,2',3,4',5,6,6'-HpCB	188	74487-85-7	13C12-2,2',3,4',5,6,6'-HpCB2	188L
234432-91-8

2,3,3',4,4',5,5'-HpCB6	189	39635-31-9	13C12-2,3,3',4,4',5,5'-HpCB2,7
189L	208263-73-4

2,3,3',4,4',5,6-HpCB	190	41411-64-7



	2,3,3',4,4',5',6-HpCB	191	74472-50-7



	2,3,3',4,5,5',6-HpCB	192	74472-51-8



	2,3,3',4',5,5',6-HpCB	193	69782-91-8



	2,2',3,3',4,4',5,5'-OcCB	194	35694-08-7	13C12-2,2',3,3',4,4',5,5'-OcCB4
194L	208263-74-5

2,2',3,3',4,4',5,6-OcCB3	195	52663-78-2



	2,2',3,3',4,4',5,6'-OcCB	196	42740-50-1



	2,2',3,3',4,4',6,6'-OcCB	197	33091-17-7



	2,2',3,3',4,5,5',6-OcCB	198	68194-17-2



	2,2',3,3',4,5,5',6'-OcCB	199	52663-75-9



	2,2',3,3',4,5,6,6'-OcCB	200	52663-73-7



	2,2',3,3',4,5',6,6'-OcCB	201	40186-71-8



	2,2',3,3',5,5',6,6'-OcCB	202	2136-99-4	13C12-2,2',3,3',5,5',6,6'-OcCB2
202L	105600-26-8

2,2',3,4,4',5,5',6-OcCB	203	52663-76-0



	2,2',3,4,4',5,6,6'-OcCB	204	74472-52-9



	2,3,3',4,4',5,5',6-OcCB	205	74472-53-0	13C12-2,3,3',4,4',5,5',6-OcCB2
205L	234446-64-1

2,2',3,3',4,4',5,5',6-NoCB3	206	40186-72-9
13C12-2,2',3,3',4,4',5,5',6-NoCB2	206L	208263-75-6

2,2',3,3',4,4',5,6,6'-NoCB	207	52663-79-3



	2,2',3,3',4,5,5',6,6'-NoCB	208	52663-77-1
13C12-2,2',3,3',4,5,5',6,6'-NoCB2	208L	234432-92-9

DeCB3	209	2051-24-3	13C12-DeCB2	209L	105600-27-9



.	Abbreviations for chlorination levels

MoCB	monochlorobiphenyl	HxCB	hexachlorobiphenyl

DiCB	dichlorobiphenyl	HpCB	heptachlorobiphenyl

TrCB	trichlorobiphenyl	OcCB	octachlorobiphenyl

TeCB	tetrachlorobiphenyl	NoCB	nonachlorobiphenyl

PeCB	pentachlorobiphenyl	DeCB	decachlorobiphenyl



2.	Labeled level of chlorination (LOC) window-defining congener

3.	National Oceanic and Atmospheric Administration (NOAA) congener of
interest

4.	Labeled injection internal standard

5.	Labeled clean-up standard

6.	World Health Organization (WHO) toxic congener

7.	Labeled analog of WHO toxic congener



Table 2.   Retention times (RT), RT references, relative retention times
(RRTs), method detection limits (MDLs), and minimum levels of
quantitation (MLs) for the 209 CB congeners on SPB-octyl.

Cl

No. 1	

Congener No. 2,3	

RT Ref4	

RT5	

RRT6	

RRT limits7	

Window

(sec) 8	

Quantitation reference9	Detection limits and minimum levels -

Matrix and concentration10









Water

 (pg/L)	Other

 (ng/kg)	Extract 

(pg/µL)









MDL	ML	MDL	ML	ML

Compounds using 9L (13C12-2,5-DiCB) as Labeled injection internal
standard

	CB congener

		Monochlorobiphenyls

1	1	1L	13:44	1.0012	0.9988-1.0036	-1+3	1L	10	20	1.0	2	1

1	2	3L	16:08	0.9878	0.9847-0.9908	6	1L/3L	7	20	0.7	2	1

1	3	3L	16:21	1.0010	0.9990-1.0031	-1+3	3L	11	50	1.1	5	2.5

		Dichlorobiphenyls

2	4	4L	16:40	1.0010	0.9990-1.0030	-1+3	4L	13	50	1.3	5	2.5

2	10	4L	16:53	1.0140	1.0110-1.0170	6	4L/15L	13	50	1.3	5	2.5

2	9	4L	18:55	1.1361	1.1331-1.1391	6	4L/15L	7	20	0.7	2	1

2	7	4L	19:07	1.1481	1.1451-1.1512	6	4L/15L	8	20	0.8	2	1

2	6	4L	19:26	1.1672	1.1642-1.1702	6	4L/15L	7	20	0.7	2	1

2	5	4L	19:48	1.1892	1.1862-1.1922	6	4L/15L	8	20	0.8	2	1

2	8	4L	19:56	1.1972	1.1942-1.2002	6	4L/15L	15	50	1.5	5	2.5

2	14	15L	21:42	0.9267	0.9246-0.9288	6	4L/15L	8	20	0.8	2	1

2	11	15L	22:42	0.9694	0.9673-0.9715	6	4L/15L	34	100	3.4	10	5

2	13	15L	23:03	0.9843	0.9822-0.9865	6	4L/15L	19	50	1.9	5	2.5

2	12	15L	23:06	0.9865	0.9843-0.9886	6	4L/15L





	2	13/12	15L	23:04	0.9851	0.9829-0.9872	6	4L/15L





	2	15	15L	23:26	1.0007	0.9993-1.0021	-1+3	15L	16	50	1.6	5	2.5

		Trichlorobiphenyls

3	19	19L	20:19	1.0008	0.9992-1.0025	-1+3	19L	8	20	0.8	2	1

3	30	19L	22:15	1.0961	1.0936-1.0985	6	19L/37L	16	50	1.6	5	2.5

3	18	19L	22:23	1.1026	1.1002-1.1051	6	19L/37L





	3	30/18	19L	22:19	1.0993	1.0969-1.1018	6	19L/37L





	3	17	19L	22:49	1.1240	1.1215-1.1264	6	19L/37L	9	20	0.9	2	1

3	27	19L	23:06	1.1379	1.1355-1.1404	6	19L/37L	8	20	0.8	2	1

3	24	19L	23:14	1.1445	1.1420-1.1470	6	19L/37L	10	20	1.0	2	1

3	16	19L	23:25	1.1535	1.1511-1.1560	6	19L/37L	9	20	0.9	2	1

3	32	19L	24:57	1.2291	1.2266-1.2315	6	19L/37L	8	20	0.8	2	1

3	34	19L	25:17	1.2455	1.2430-1.2479	6	19L/37L	7	20	0.7	2	1

3	23	19L	25:26	1.2529	1.2504-1.2553	6	19L/37L	7	20	0.7	2	1

3	29	19L	25:47	1.2701	1.2660-1.2742	10	19L/37L	12	50	1.2	5	2.5

3	26	19L	25:48	1.2709	1.2668-1.2750	10	19L/37L





	3	29/26	19L	25:48	1.2709	1.2668-1.2750	10	19L/37L





	3	25	37L	26:04	0.8364	0.8348-0.8380	6	19L/37L	8	20	0.8	2	1

3	31	37L	26:25	0.8476	0.8460-0.8492	6	19L/37L	18	50	1.8	5	2.5

3	28	37L	26:44	0.8578	0.8551-0.8604	10	19L/37L	22	50	2.2	5	2.5

3	20	37L	26:49	0.8604	0.8578-0.8631	10	19L/37L





	3	28/20	37L	26:47	0.8594	0.8567-0.8620	10	19L/37L





	3	21	37L	26:58	0.8652	0.8626-0.8679	10	19L/37L	21	50	2.1	5	2.5

3	33	37L	27:01	0.8668	0.8642-0.8695	10	19L/37L





	3	21/33	37L	26:59	0.8658	0.8631-0.8684	10	19L/37L





	3	22	37L	27:29	0.8818	0.8802-0.8834	6	19L/37L	9	20	0.9	2	1

3	36	37L	29:05	0.9332	0.9316-0.9348	6	19L/37L	8	20	0.8	2	1

3	39	37L	29:30	0.9465	0.9449-0.9481	6	19L/37L	8	20	0.8	2	1

3	38	37L	30:10	0.9679	0.9663-0.9695	6	19L/37L	7	20	0.7	2	1

3	35	37L	30:42	0.9850	0.9834-0.9866	6	19L/37L	9	20	0.9	2	1

3	37	37L	31:11	1.0005	0.9995-1.0011	-1+3	37L	10	20	1.0	2	1

	Labeled Compounds

1	1L	9L	13:43	0.7257	0.7125-0.7390	30	9L





	1	3L	9L	16:20	0.8642	0.8510-0.8774	30	9L





	2	4L	9L	16:39	0.8810	0.8677-0.8942	30	9L





	2	15L	9L	23:25	1.2390	1.2302-1.2478	20	9L





	3	19L	9L	20:18	1.0741	1.0608-1.0873	30	9L





	3	37L	52L	31:10	1.0841	1.0754-1.0928	30	52L





	Compounds using 52L (13C12-2,2',5,5'-TeCB) as Labeled injection
internal standard		

	CB congener

		Tetrachlorobiphenyls

4	54	54L	23:51	1.0007	0.9993-1.0021	-1+3	54L	14	50	1.4	5	2.5

4	50	54L	26:07	1.0958	1.0923-1.0993	10	54L/81L/77L	25	100	2.5	10	5

4	53	54L	26:09	1.0972	1.0937-1.1007	10	54L/81L/77L





	4	50/53	54L	26:08	1.0965	1.0930-1.1000	10	54L/81L/77L





	4	45	54L	26:55	1.1294	1.1259-1.1329	10	54L/81L/77L	22	50	2.2	5	2.5

4	51	54L	26:58	1.1315	1.1280-1.1350	10	54L/81L/77L





	4	45/51	54L	26:57	1.1308	1.1273-1.1343	10	54L/81L/77L





	4	46	54L	27:18	1.1455	1.1434-1.1476	6	54L/81L/77L	10	20	1.0	2	1

4	52	54L	28:45	1.2063	1.2042-1.2084	6	54L/81L/77L	15	50	1.5	5	2.5

4	73	54L	28:52	1.2112	1.2091-1.2133	6	54L/81L/77L	14	50	1.4	5	2.5

4	43	54L	28:58	1.2154	1.2133-1.2175	6	54L/81L/77L	14	50	1.4	5	2.5

4	69	54L	29:08	1.2224	1.2189-1.2259	10	54L/81L/77L	26	100	2.6	10	5

4	49	54L	29:16	1.2280	1.2245-1.2315	10	54L/81L/77L





	4	69/49	54L	29:12	1.2252	1.2217-1.2287	10	54L/81L/77L





	4	48	54L	29:33	1.2399	1.2378-1.2420	6	54L/81L/77L	14	50	1.4	5	2.5

4	65	54L	29:49	1.2510	1.2476-1.2545	10	54L/81L/77L	40	100	4.0	10	5

4	47	54L	29:50	1.2517	1.2483-1.2552	10	54L/81L/77L





	4	44	54L	29:53	1.2538	1.2503-1.2573	10	54L/81L/77L





	4	65/47/44	54L	29:50	1.2517	1.2483-1.2552	10	54L/81L/77L





	4	62	54L	30:06	1.2629	1.2594-1.2664	10	54L/81L/77L	37	100	3.7	10	5

4	75	54L	30:08	1.2643	1.2608-1.2678	10	54L/81L/77L





	4	59	54L	30:12	1.2671	1.2636-1.2706	10	54L/81L/77L





	4	62/75/59	54L	30:09	1.2650	1.2615-1.2685	10	54L/81L/77L





	4	42	54L	30:26	1.2769	1.2748-1.2790	6	54L/81L/77L	16	50	1.6	5	2.5

4	41	54L	30:52	1.2951	1.2916-1.2986	10	54L/81L/77L	42	100	4.2	10	5

4	71	54L	30:58	1.2993	1.2958-1.3028	10	54L/81L/77L





	4	40	54L	31:01	1.3014	1.2979-1.3049	10	54L/81L/77L





	4	41/71/40	54L	30:58	1.2993	1.2958-1.3028	10	54L/81L/77L





	4	64	54L	31:12	1.3091	1.3070-1.3112	6	54L/81L/77L	13	50	1.3	5	2.5

4	72	81L	31:59	0.8336	0.8323-0.8349	6	54L/81L/77L	13	50	1.3	5	2.5

4	68	81L	32:18	0.8419	0.8406-0.8432	6	54L/81L/77L	14	50	1.4	5	2.5

4	57	81L	32:46	0.8540	0.8527-0.8553	6	54L/81L/77L	11	50	1.1	5	2.5

4	58	81L	33:05	0.8623	0.8610-0.8636	6	54L/81L/77L	14	50	1.4	5	2.5

4	67	81L	33:13	0.8658	0.8645-0.8671	6	54L/81L/77L	12	50	1.2	5	2.5

4	63	81L	33:30	0.8732	0.8719-0.8745	6	54L/81L/77L	12	50	1.2	5	2.5

4	61	81L	33:46	0.8801	0.8775-0.8827	12	54L/81L/77L	59	200	5.9	20	10

4	70	81L	33:53	0.8831	0.8805-0.8858	12	54L/81L/77L





	4	76	81L	33:55	0.8840	0.8814-0.8866	12	54L/81L/77L





	4	74	81L	33:57	0.8849	0.8827-0.8871	10	54L/81L/77L





	4	61/70/76/74	81L	33:55	0.8840	0.8814-0.8866	12	54L/81L/77L





	4	66	81L	34:15	0.8927	0.8914-0.8940	6	54L/81L/77L	17	50	1.7	5	2.5

4	55	81L	34:28	0.8983	0.8970-0.8997	6	54L/81L/77L	12	50	1.2	5	2.5

4	56	81L	35:03	0.9136	0.9123-0.9149	6	54L/81L/77L	15	50	1.5	5	2.5

4	60	81L	35:16	0.9192	0.9179-0.9205	6	54L/81L/77L	14	50	1.4	5	2.5

4	80	81L	35:32	0.9262	0.9248-0.9275	6	54L/81L/77L	11	50	1.1	5	2.5

4	79	81L	37:16	0.9713	0.9700-0.9726	6	54L/81L/77L	13	50	1.3	5	2.5

4	78	81L	37:52	0.9870	0.9857-0.9883	6	54L/81L/77L	16	50	1.6	5	2.5

4	81	81L	38:23	1.0004	0.9996-1.0013	-1+3	81L	18	50	1.8	5	2.5

4	77	77L	39:02	1.0004	0.9996-1.0013	-1+3	77L	14	50	1.4	5	2.5

	Labeled compounds

4	54L	52L	23:50	0.8290	0.8232-0.8348	20	52L





	4	81L	52L	38:22	1.3345	1.3287-1.3403	20	52L





	4	77L	52L	39:01	1.3571	1.3513-1.3629	20	52L





	Compounds using 101L (13C12-2,2',4,5,5'-PeCB) as Labeled injection
internal standard		

	CB congener

		Pentachlorobiphenyls

5	104	104L	29:46	1.0000	0.9994-1.0017	-1+3	104L	14	50	1.4	5	2.5

5	96	104L	30:17	1.0174	1.0146-1.0202	10	104L/123L/114L/118L/105L	15	50
1.5	5	2.5

5	103	104L	32:11	1.0812	1.0795-1.0829	6	104L/123L/114L/118L/105L	11	50
1.1	5	2.5

5	94	104L	32:29	1.0913	1.0896-1.0929	6	104L/123L/114L/118L/105L	13	50
1.3	5	2.5

5	95	104L	33:00	1.1086	1.1058-1.1114	10	104L/123L/114L/118L/105L	77	200
7.7	20	10

5	100	104L	33:06	1.1120	1.1092-1.1148	10	104L/123L/114L/118L/105L





	5	93	104L	33:14	1.1165	1.1137-1.1193	10	104L/123L/114L/118L/105L





	5	102	104L	33:21	1.1204	1.1176-1.1232	10	104L/123L/114L/118L/105L





	5	98	104L	33:26	1.1232	1.1204-1.1260	10	104L/123L/114L/118L/105L





	5	95/100/93/102/98	104L	33:13	1.1159	1.1131-1.1187	15
104L/123L/114L/118L/105L





	5	88	104L	33:48	1.1355	1.1321-1.1389	12	104L/123L/114L/118L/105L	22	50
2.2	5	2.5

5	91	104L	33:55	1.1394	1.1366-1.1422	10	104L/123L/114L/118L/105L





	5	88/91	104L	33:52	1.1377	1.1344-1.1411	12	104L/123L/114L/118L/105L





	5	84	104L	34:14	1.1501	1.1484-1.1517	6	104L/123L/114L/118L/105L	11	20
1.1	2	1

5	89	104L	34:44	1.1669	1.1652-1.1685	6	104L/123L/114L/118L/105L	13	50
1.3	5	2.5

5	121	104L	34:57	1.1741	1.1725-1.1758	6	104L/123L/114L/118L/105L	12	50
1.2	5	2.5

5	92	123L	35:26	0.8639	0.8627-0.8651	6	104L/123L/114L/118L/105L	13	50
1.3	5	2.5

5	113	123L	36:01	0.8781	0.8761-0.8801	10	104L/123L/114L/118L/105L	47	200
4.7	20	10

5	90	123L	36:03	0.8789	0.8769-0.8809	10	104L/123L/114L/118L/105L





	5	101	123L	36:04	0.8793	0.8773-0.8813	10	104L/123L/114L/118L/105L





	5	113/90/101	123L	36:03	0.8789	0.8769-0.8809	10
104L/123L/114L/118L/105L





	5	83	123L	36:39	0.8935	0.8911-0.8960	12	104L/123L/114L/118L/105L	29	100
2.9	10	5

5	99	123L	36:41	0.8944	0.8923-0.8964	10	104L/123L/114L/118L/105L





	5	83/99	123L	36:40	0.8939	0.8915-0.8964	12	104L/123L/114L/118L/105L





	5	112	123L	36:51	0.8984	0.8972-0.8996	6	104L/123L/114L/118L/105L	14	50
1.4	5	2.5

5	119	123L	37:12	0.9069	0.9037-0.9102	16	104L/123L/114L/118L/105L	74	200
7.4	20	10

5	109	123L	37:12	0.9069	0.9037-0.9102	16	104L/123L/114L/118L/105L





	5	86	123L	37:17	0.9090	0.9057-0.9122	16	104L/123L/114L/118L/105L





	5	97	123L	37:17	0.9090	0.9057-0.9122	16	104L/123L/114L/118L/105L





	5	125	123L	37:21	0.9106	0.9074-0.9139	16	104L/123L/114L/118L/105L





	5	87	123L	37:25	0.9122	0.9102-0.9143	10	104L/123L/114L/118L/105L





	5	119/109/86/97/125/87	123L	37:19	0.9098	0.9065-0.9130	16
104L/123L/114L/118L/105L





	5	117	123L	37:57	0.9252	0.9228-0.9277	12	104L/123L/114L/118L/105L	38
100	3.8	10	5

5	116	123L	38:02	0.9273	0.9248-0.9297	12	104L/123L/114L/118L/105L





	5	85	123L	38:05	0.9285	0.9265-0.9305	10	104L/123L/114L/118L/105L





	5	117/116/85	123L	38:00	0.9265	0.9240-0.9289	12
104L/123L/114L/118L/105L





	5	110	123L	38:16	0.9330	0.9309-0.9350	10	104L/123L/114L/118L/105L	39
100	3.9	10	5

5	115	123L	38:18	0.9338	0.9317-0.9358	10	104L/123L/114L/118L/105L





	5	110/115	123L	38:17	0.9334	0.9313-0.9354	10	104L/123L/114L/118L/105L





	5	82	123L	38:40	0.9427	0.9415-0.9439	6	104L/123L/114L/118L/105L	15	50
1.5	5	2.5

5	111	123L	38:52	0.9476	0.9464-0.9488	6	104L/123L/114L/118L/105L	14	50
1.4	5	2.5

5	120	123L	39:21	0.9594	0.9581-0.9606	6	104L/123L/114L/118L/105L	13	50
1.3	5	2.5

5	108	123L	40:39	0.9911	0.9890-0.9931	10	104L/123L/114L/118L/105L	29	100
2.9	10	5

5	124	123L	40:40	0.9915	0.9894-0.9935	10	104L/123L/114L/118L/105L





	5	108/124	123L	40:39	0.9911	0.9890-0.9931	10	104L/123L/114L/118L/105L





	5	107	123L	40:54	0.9972	0.9959-0.9984	6	104L/123L/114L/118L/105L	17	50
1.7	5	2.5

5	123	123L	41:02	1.0004	0.9996-1.0012	-1+3	123L	17	50	1.7	5	2.5

5	106	123L	41:10	1.0037	1.0024-1.0049	6	104L/123L/114L/118L/105L	17	50
1.7	5	2.5

5	118	118L	41:22	1.0004	0.9996-1.0012	-1+3	118L	30	100	3.0	10	5

5	122	118L	41:49	1.0113	1.0101-1.0125	6	104L/123L/114L/118L/105L	12	50
1.2	5	2.5

5	114	114L	41:58	1.0004	0.9999-1.0012	-1+3	114L	15	50	1.5	5	2.5

5	105	105L	42:43	0.9996	0.9996-1.0012	-2+3	105L	17	50	1.7	5	2.5

5	127	105L	44:09	1.0332	1.0320-1.0343	6	104L/123L/114L/118L/105L	14	50
1.4	5	2.5

5	126	126L	45:58	1.0004	0.9996-1.0011	-1+3	126L	16	50	1.6	5	2.5

	Labeled compounds

5	104L	101L	29:46	0.8257	0.8211-0.8303	20	101L





	5	123L	101L	41:01	1.1378	1.1331-1.1424	20	101L





	5	118L	101L	41:21	1.1470	1.1424-1.1516	20	101L





	5	114L	101L	41:57	1.1637	1.1590-1.1683	20	101L





	5	105L	101L	42:44	1.1854	1.1808-1.1900	20	101L





	5	126L	101L	45:57	1.2746	1.2700-1.2792	20	101L





	Compounds using 138L (13C12-2,2',3,4,4',5'-HxCB) as Labeled injection
internal standard

	CB congener

		Hexachlorobiphenyls

6	155	155L	35:44	1.0000	0.9995-1.0014	-1+3	155L	14	50	1.4	5	2.5

6	152	155L	36:07	1.0107	1.0093-1.0121	6	155L/156L/157L/167L/169L	14	50
1.4	5	2.5

6	150	155L	36:15	1.0145	1.0131-1.0159	6	155L/156L/157L/167L/169L	15	50
1.5	5	2.5

6	136	155L	36:44	1.0280	1.0266-1.0294	6	155L/156L/157L/167L/169L	16	50
1.6	5	2.5

6	145	155L	37:00	1.0354	1.0340-1.0368	6	155L/156L/157L/167L/169L	16	50
1.6	5	2.5

6	148	155L	34:26	1.0756	1.0742-1.0770	6	155L/156L/157L/167L/169L	14	50
1.4	5	2.5

6	151	155L	39:10	1.0961	1.0938-1.0984	10	155L/156L/157L/167L/169L	46	100
4.6	10	5

6	135	155L	39:17	1.0993	1.0970-1.1017	10	155L/156L/157L/167L/169L





	6	154	155L	39:21	1.1012	1.0989-1.1035	10	155L/156L/157L/167L/169L





	6	151/135/154	155L	39:15	1.0984	1.0961-1.1007	10
155L/156L/157L/167L/169L





	6	144	155L	39:47	1.1133	1.1119-1.1147	6	155L/156L/157L/167L/169L	15	50
1.5	5	2.5

6	147	155L	40:09	1.1236	1.1213-1.1259	10	155L/156L/157L/167L/169L	35	100
3.5	10	5

6	149	155L	40:12	1.1250	1.1227-1.1273	10	155L/156L/157L/167L/169L





	6	147/149	155L	40:10	1.1241	1.1217-1.1264	10	155L/156L/157L/167L/169L





	6	134	155L	40:27	1.1320	1.1297-1.1343	10	155L/156L/157L/167L/169L	33
100	3.3	10	5

6	143	155L	40:30	1.1334	1.1311-1.1357	10	155L/156L/157L/167L/169L





	6	134/143	155L	40:29	1.1329	1.1306-1.1353	10	155L/156L/157L/167L/169L





	6	139	155L	40:47	1.1413	1.1390-1.1437	10	155L/156L/157L/167L/169L	29
100	2.9	10	5

6	140	155L	40:48	1.1418	1.1395-1.1441	10	155L/156L/157L/167L/169L





	6	139/140	155L	40:47	1.1413	1.1390-1.1437	10	155L/156L/157L/167L/169L





	6	131	155L	41:03	1.1488	1.1474-1.1502	6	155L/156L/157L/167L/169L	17	50
1.7	5	2.5

6	142	155L	41:13	1.1535	1.1521-1.1549	6	155L/156L/157L/167L/169L	17	50
1.7	5	2.5

6	132	155L	41:36	1.1642	1.1618-1.1665	10	155L/156L/157L/167L/169L	16	50
1.6	5	2.5

6	133	155L	41:57	1.1740	1.1726-1.1754	6	155L/156L/157L/167L/169L	12	50
1.2	5	2.5

6	165	167L	42:23	0.8864	0.8853-0.8874	6	155L/156L/157L/167L/169L	13	50
1.3	5	2.5

6	146	167L	42:38	0.8916	0.8906-0.8926	6	155L/156L/157L/167L/169L	14	50
1.4	5	2.5

6	161	167L	42:47	0.8947	0.8937-0.8958	6	155L/156L/157L/167L/169L	13	50
1.3	5	2.5

6	153	167L	43:17	0.9052	0.9035-0.9069	10	155L/156L/157L/167L/169L	30	100
3.0	10	5

6	168	167L	43:21	0.9066	0.9048-0.9083	10	155L/156L/157L/167L/169L





	6	153/168	167L	43:19	0.9059	0.9041-0.9076	10	155L/156L/157L/167L/169L





	6	141	167L	43:34	0.9111	0.9101-0.9122	6	155L/156L/157L/167L/169L	17	50
1.7	5	2.5

6	130	167L	44:01	0.9205	0.9195-0.9216	6	155L/156L/157L/167L/169L	13	50
1.3	5	2.5

6	137	167L	44:14	0.9251	0.9240-0.9261	6	155L/156L/157L/167L/169L	15	50
1.5	5	2.5

6	164	167L	44:22	0.9278	0.9268-0.9289	6	155L/156L/157L/167L/169L	15	50
1.5	5	2.5

6	138	167L	44:42	0.9348	0.9324-0.9373	14	155L/156L/157L/167L/169L	63	200
6.3	20	10

6	163	167L	44:42	0.9348	0.9324-0.9373	14	155L/156L/157L/167L/169L





	6	129	167L	44:47	0.9366	0.9341-0.9390	14	155L/156L/157L/167L/169L





	6	160	167L	44:53	0.9387	0.9369-0.9404	10	155L/156L/157L/167L/169L





	6	138/163/129/160	167L	44:47	0.9366	0.9341-0.9390	14
155L/156L/157L/167L/169L





	6	158	167L	45:05	0.9428	0.9418-0.9439	6	155L/156L/157L/167L/169L	16	50
1.6	5	2.5

6	166	167L	45:59	0.9617	0.9599-0.9634	10	155L/156L/157L/167L/169L	29	100
2.9	10	5

6	128	167L	46:09	0.9651	0.9634-0.9669	10	155L/156L/157L/167L/169L





	6	128/166	167L	46:04	0.9634	0.9617-0.9651	10	155L/156L/157L/167L/169L





	6	159	167L	46:59	0.9826	0.9815-0.9836	6	155L/156L/157L/167L/169L	14	50
1.4	5	2.5

6	162	167L	47:18	0.9892	0.9881-0.9902	6	155L/156L/157L/167L/169L	13	50
1.3	5	2.5

6	167	167L	47:49	1.0000	0.9997-1.0010	-1+3	167L	13	50	1.3	5	2.5

6	156	156L/157L	49:05	0.9993	0.9983-1.0003	6	156L/157L	23	100	2.3	10	5

6	157	156L/157L	49:09	1.0007	0.9990-1.0024	10	156L/157L





	6	156/157	156L/157L	49:07	1.0000	0.9990-1.1010	6	156L/157L





	6	169	169L	52:31	1.0003	0.9997-1.0010	-1+3	169L	15	50	1.5	5	2.5

	Labeled compounds

6	155L	138L	35:44	0.7997	0.7960-0.8034	20	138L





	6	167L	138L	47:49	1.0701	1.0664-1.0739	20	138L





	6	156L	138L	49:05	1.0985	1.0974-1.0996	20	138L





	6	157L	138L	49:08	1.0996	1.0959-1.1033	20	138L





	6	156L/157L	138L	49:07	1.0992	1.0981-1.1003	20	138L





	6	169L	138L	52:30	1.1749	1.1738-1.1761	20	138L





	Compounds using 194L(13C12-2,2',3,3',4,4',5,5'-OcCB) as Labeled
injection internal standard		

	CB congener

		Heptachlorobiphenyls

7	188	188L	41:51	1.0000	0.9996-1.0012	-1+3	188L	15	50	1.5	5	2.5

7	179	188L	42:19	1.0112	1.0100-1.0123	6	188L/189L	14	50	1.4	5	2.5

7	184	188L	42:45	1.0215	1.0203-1.0227	6	188L/189L	14	50	1.4	5	2.5

7	176	188L	43:15	1.0335	1.0323-1.0346	6	188L/189L	12	50	1.2	5	2.5

7	186	188L	43:45	1.0454	1.0442-1.0466	6	188L/189L	15	50	1.5	5	2.5

7	178	188L	45:06	1.0777	1.0765-1.0789	6	188L/189L	14	50	1.4	5	2.5

7	175	188L	45:46	1.0936	1.0924-1.0948	6	188L/189L	14	50	1.4	5	2.5

7	187	188L	46:02	1.1000	1.0988-1.1012	6	188L/189L	17	50	1.7	5	2.5

7	182	188L	46:14	1.1047	1.1035-1.1059	6	188L/189L	13	50	1.3	5	2.5

7	183	188L	46:42	1.1159	1.1147-1.1171	6	188L/189L	28	100	2.8	10	5

7	185	188L	46:53	1.1203	1.1191-1.1215	6	188L/189L





	7	183/185	188L	46:47	1.1179	1.1167-1.1191	6	188L/189L





	7	174	188L	47:02	1.1239	1.1227-1.1251	6	188L/189L	15	50	1.5	5	2.5

7	177	188L	47:30	1.1350	1.1338-1.1362	6	188L/189L	11	50	1.1	5	2.5

7	181	188L	47:52	1.1438	1.1426-1.1450	6	188L/189L	13	50	1.3	5	2.5

7	171	188L	48:10	1.1509	1.1489-1.1529	10	188L/189L	30	100	3.0	10	5

7	173	188L	48:11	1.1513	1.1501-1.1525	6	188L/189L





	7	171/173	188L	48:10	1.1509	1.1489-1.1529	6	188L/189L





	7	172	189L	49:47	0.9035	0.9026-0.9044	6	188L/189L	13	50	1.3	5	2.5

7	192	189L	50:06	0.9093	0.9083-0.9102	6	188L/189L	13	50	1.3	5	2.5

7	193	189L	50:26	0.9153	0.9144-0.9162	6	188L/189L	30	100	3.0	10	5

7	180	189L11	50:27	0.9156	0.9147-0.9165	6	188L/189L11





	7	193/180	189L	50:26	0.9153	0.9144-0.9162	6	188L/189L





	7	191	189L	50:51	0.9229	0.9220-0.9238	6	188L/189L	13	50	1.3	5	2.5

7	170	189L11	51:54	0.9419	0.9410-0.9428	6	188L/189L11	12	50	1.2	5	2.5

7	190	189L	52:26	0.9516	0.9507-0.9525	6	188L/189L	14	50	1.4	5	2.5

7	189	189L	55:07	1.0003	0.9997-1.0009	-1+3	189L	13	50	1.3	5	2.5

		Octachlorobiphenyls

8	202	202L	47:32	1.0004	0.9996-1.0011	-1+3	202L	24	100	2.4	10	5

8	201	202L	48:31	1.0210	1.0193-1.0228	10	202L/205L	20	50	2.0	5	2.5

8	204	202L	49:11	1.0351	1.0340-1.0361	6	202L/205L	21	50	2.1	5	2.5

8	197	202L	49:27	1.0407	1.0396-1.0417	6	202L/205L	43	100	4.3	10	5

8	200	202L	49:40	1.0452	1.0442-1.0463	6	202L/205L





	8	197/200	202L	49:33	1.0428	1.0417-1.0438	6	202L/205L





	8	198	202L	52:30	1.1049	1.1031-1.1066	10	202L/205L	37	100	3.7	10	5

8	199	202L	52:32	1.1056	1.1045-1.1066	6	202L/205L





	8	198/199	202L	52:31	1.1052	1.1035-1.1070	10	202L/205L





	8	196	205L	53:13	0.9207	0.9198-0.9216	6	202L/205L	20	50	2.0	5	2.5

8	203	205L	53:26	0.9245	0.9236-0.9253	6	202L/205L	18	50	1.8	5	2.5

8	195	205L	54:55	0.9501	0.9493-0.9510	6	202L/205L	22	50	2.2	5	2.5

8	194	205L	57:19	0.9916	0.9908-0.9925	6	202L/205L	18	50	1.8	5	2.5

8	205	205L	57:49	1.0003	0.9997-1.0009	-1+3	205L	15	50	1.5	5	2.5

		Nonachlorobiphenyls

9	208	208L	54:33	1.0003	0.9997-1.0009	-1+3	208L	16	50	1.6	5	2.5

9	207	208L	55:32	1.0183	1.0174-1.0193	6	208L/206L	19	50	1.9	5	2.5

9	206	206L	59:37	1.0003	0.9997-1.0008	-1+3	206L	16	50	1.6	5	2.5

		Decachlorobiphenyl

10	209	209L	61:15	1.0003	0.9997-1.0008	-1+3	209L	16	50	1.6	5	2.5

	Labeled compounds

7	188L	194L	41:51	0.7304	0.7275-0.7333	20	194L





	7	180L	194L	50:27	0.8805	0.8775-0.8834	20	194L





	7	170L	194L	51:53	0.9055	0.9026-0.9084	20	194L





	7	189L	194L	55:06	0.9616	0.9587-0.9645	20	194L





	8	202L	194L	47:31	0.8293	0.8264-0.8322	20	194L





	8	205L	194L	57:48	1.0087	1.0044-1.0131	30	194L





	9	208L	194L	54:32	0.9517	0.9488-0.9546	20	194L





	9	206L	194L	59:36	1.0401	1.0358-1.0445	30	194L





	10	209L	194L	61:14	1.0686	1.0643-1.0730	30	194L





	Labeled clean-up standards

3	28L	52L	26:44	0.9266	0.9209-0.9324	20	52L





	5	111L	101L	38:51	1.0777	1.0730-1.0823	20	101L





	7	178L	138L	45:05	1.0090	1.0052-1.0127	20	138L





	Labeled injection internal standards

2	9L	138L	18:54	0.4230	0.4183-0.4276	25	138L





	4	52L	138L	28:45	0.6434	0.6388-0.6481	25	138L





	5	101L	138L	36:03	0.8068	0.8021-0.8115	25	138L





	6	138L	138L	44:41	1.0000	0.9996-1.0011	100	138L





	8	194L	138L	57:18	1.2824	1.2777-1.2870	25	138L





	

1.	Number of chlorines on congener.

2.	Suffix “L” indicates labeled compound.

3.	Multiple congeners in a box indicates congeners that co-elute or may
not be adequately resolved on a 30-m SPB-octyl column.

4.	Retention time (RT) reference used to locate target congener.

5.	Retention time of target congener.

6.	Relative retention time (RRT) between the RT for the congener and RT
for the reference.   

7.	RRT limits based on RT window.  RTs, RRTs, and RRT limits may differ
slightly from those in Table 2.

8.	RT window width necessary to attempt to unambiguously identify the
congener in the presence of other congeners.

9.	Labeled congeners that form the quantitation reference.  Areas from
the exact m/z’s of the congeners listed in the quantitation reference
are summed, and divided by the number of congeners in the quantitation
reference.  For example, for congener 10, the areas at the exact m/z’s
for 4L and 15L are summed and the sum is divided by 2 (because there are
2 congeners in the quantitation reference).

10.	MDLs for water pooled from data from AXYS Analytical,
TestAmerica-Knoxville, and Battelle-Columbus  (see Reference 24).  MLs
for water per ML procedure at 68 FR 11790.  MDLs and MLs for “Other”
and “Extract” calculated from sample amount and extract volume.

11.	If congeners 170L and 180L are included in the calibration and
spiking solutions, these congeners should be used as RT and quantitation
references.Table 3.	Concentrations of Native and Labeled Chlorinated
Biphenyls in Stock Solutions Spiking Solutions

CB Congener	Solution Concentrations

	Stock (µg/mL)	Spiking (ng/mL)	Extract (ng/mL)

  Native toxics/LOC1

1	20	1.0	50

3	20	1.0	50

4	20	1.0	50

15	20	1.0	50

19	20	1.0	50

37	20	1.0	50

54	20	1.0	50

77	20	1.0	50

81	20	1.0	50

104	20	1.0	50

105	20	1.0	50

114	20	1.0	50

118	20	1.0	50

123	20	1.0	50

126	20	1.0	50

155	20	1.0	50

156	20	1.0	50

157	20	1.0	50

167	20	1.0	50

169	20	1.0	50

188	20	1.0	50

189	20	1.0	50

202	20	1.0	50

205	20	1.0	50

206	20	1.0	50

208	20	1.0	50

209	20	1.0	50

  Native congener mix stock solutions2

	MoCB thru TrCB	2.5



	TeCB thru HpCB	5.0



	OcCB thru DeCB	7.5



  Labeled toxics/LOC/window-defining3

1L	1.0	2.0	100

3L	1.0	2.0	100

4L	1.0	2.0	100

15L	1.0	2.0	100

19L	1.0	2.0	100

37L	1.0	2.0	100

54L	1.0	2.0	100

77L	1.0	2.0	100

81L	1.0	2.0	100

104L	1.0	2.0	100

105L	1.0	2.0	100

114L	1.0	2.0	100

118L	1.0	2.0	100

123L	1.0	2.0	100

126L	1.0	2.0	100

155L	1.0	2.0	100

156L	1.0	2.0	100

157L	1.0	2.0	100

167L	1.0	2.0	100

169L	1.0	2.0	100

188L	1.0	2.0	100

189L	1.0	2.0	100

202L	1.0	.2.0	100

205L	1.0	2.0	100

206L	1.0	2.0	100

208L	1.0	2.0	100

209L	1.0	2.0	100

  Labeled clean-up4

28L	1.0	2.0	100

111L	1.0	2.0	100

178L	1.0	2.0	100

  Labeled injection internal5

9L	5.0	1000	100

52L	5.0	1000	100

101L	5.0	1000	100

138L	5.0	1000	100

194L	5.0	1000	100

 Diluted combined 209 congener6	Solution Concentration (ng/mL)

Standard	Native	Labeled

  Native congeners



      MoCB thru TrCB	25

	      TeCB thru HpCB	50

	      OcCB thru DeCB	75

	  Labeled toxics/LOC/window-defining

100

  Labeled cleanup

100

  Labeled injection internal

100



1.	Stock solution:  Section 7.8.1; Spiking solution: Section 7.11

2.	Section 7.8.2.1

3.	Stock solution:  Section 7.9.1; Spiking solution: Section 7.12

4.	Stock solution:  Section 7.9.2; Spiking solution: Section 7.13

5.	Stock solution:  Section 7.9.3; Spiking solution: Section 7.14

6.	Section 7.10.2.2.2

Table 4.	Composition of Individual Native CB Congener Solutions1

Solution Identifier

A2	B2	C2	D2	E2

Accu-Standard part number

M-1668A-1	M-1668A-2	M-1668A-3	M-1668A-4	M-1668A-5

2	7	13	25	1

10	5	17	21	3

9	12	29	69	4

6	18	20	47	15

8	24	46	42	19

14	23	65	a64	16

11	28	59	70	37

30	22	40	102	54

27	39	67	97	43

32	53	76	115	44

34	51	80	123	74

26	73	93	134	56

31	48	84	131	77

33	62	101	163	104

36	71	112	180	98

38	68	86

125

35	58	116

110

50	61	107

126

45	55	154

155

52	60	147

138

49	94	140

169

75	100	146

188

41	91	141

189

72	121	164

202

57	90	158

205

63	99	182

208

66	109	174

206

79	117	173

209

78	111	193



81	108



	96	118



	103	114



	95	150



	88	145



	89	135



	92	149



	113	139



	83	132



	119	165



	87	168



	85	137



	82	160



	120	128



	124	162



	106	157



	122	184



	105	186



	127	187



	152	185



	136	181



	148	192



	151	197



	144	199/201



	143	203



	142





133





161





153





130





129





166





159





167





156





179





176





178





175





183





177





171





172





191





170





190





201/200





204





200/199





198





196





195





194





207





Total number of congeners





83	54	29	15	28

1.	Congeners present in each standard solution are listed in elution
order for each level of chlorination.  Congener number (Table 1) listed
first; BZ number listed second, where ambiguous.  See Table 3 for
concentrations of congeners in stock solutions and Table 5 for
concentrations in calibration standards.

Table 5.	Concentration of Congeners in Calibration and Calibration
Verification Standards

Congener Name	Congener

No.1	Solution Concentration (ng/mL)



CS-0.2 (Hi sens)2	CS-1	CS-2	CS-3 (VER)	CS-4	CS-5

Native toxics/LOC

2-MoCB	1	0.20	1.0	5.0	50	400	2000

4-MoCB	3	0.20	1.0	5.0	50	400	2000

2,2'-DiCB	4	0.20	1.0	5.0	50	400	2000

4,4'-DiCB	15	0.20	1.0	5.0	50	400	2000

2,2',6'-TrCB	19	0.20	1.0	5.0	50	400	2000

3,4,4'-TrCB	37	0.20	1.0	5.0	50	400	2000

2,2',6,6'-TeCB	54	0.20	1.0	5.0	50	400	2000

3,3',4,4'-TeCB	77	0.20	1.0	5.0	50	400	2000

3,4,4',5-TeCB	81	0.20	1.0	5.0	50	400	2000

2,2',4,6,6'-PeCB	104	0.20	1.0	5.0	50	400	2000

2,3,3',4,4'-PeCB	105	0.20	1.0	5.0	50	 400	2000

2,3,4,4',5-PeCB	114	0.20	1.0	5.0	50	 400	2000

2,3',4,4',5-PeCB	118	0.20	1.0	5.0	50	 400	2000

2',3,4,4',5-PeCB	123	0.20	1.0	5.0	50	 400	2000

3,3',4,4',5-PeCB	126	0.20	1.0	5.0	50	 400	2000

2,2',4,4',6,6'-HxCB	155	0.20	1.0	5.0	50	400	2000

2,3,3',4,4',5-HxCB	156	0.20	1.0	5.0	50	 400	2000

2,3,3',4,4',5'-HxCB	157	0.20	1.0	5.0	 50	 400	2000

2,3',4,4',5,5'-HxCB	167	0.20	1.0	5.0	50	400	2000

3,3',4,4',5,5'-HxCB	169	0.20	1.0	5.0	 50	 400	2000

2,2',3,4',5,6,6'-HpCB	188	0.20	1.0	5.0	50	400	2000

2,3,3',4,4',5,5'-HpCB	189	0.20	1.0	5.0	50	400	2000

2,2',3,3',5,5',6,6'-OcCB	202	0.20	1.0	5.0	50	400	2000

2,3,3',4,4',5,5',6-OcCB	205	0.20	1.0	5.0	50	400	2000

2,2',3,3',4,4',5,5',6-NoCB	206	0.20	1.0	5.0	50	400	2000

2,2',3,3',4',5,5',6,6'-NoCB	208	0.20	1.0	5.0	50	400	2000

DeCB	209	0.20	1.0	5.0	 50	 400	2000

Labeled toxics/LOC/window-defining

13C12-2-MoCB	1L	100	100	100	100	100	100

13C12-4-MoCB	3L	100	100	100	100	100	100

13C12-2,2'-DiCB	4L	100	100	100	100	100	100

13C12-4,4'-DiCB	15L	100	100	100	100	100	100

13C12-2,2',6'-TrCB	19L	100	100	100	100	100	100

13C12-3,4,4'-TrCB	37L	100	100	100	100	100	100

13C12-2,2',6,6'-TeCB	54L	100	100	100	100	100	100

13C12-3,3',4,4'-TeCB	77L	100	100	100	100	100	100

13C12-3,4,4',5-TeCB	81L	100	100	100	100	100	100

13C12-2,2',4,6,6'-PeCB	104L	100	100	100	100	100	100

13C12-2,3,3',4,4'-PeCB	105L	100	100	100	100	100	100

13C12-2,3,4,4',5-PeCB	114L	100	100	100	100	100	100

13C12-2,3',4,4',5-PeCB	118L	100	100	100	100	100	100

13C12-2',3,4,4',5-PeCB	123L	100	100	100	100	100	100

13C12-3,3',4,4',5-PeCB	126L	100	100	100	100	100	100

13C12-2,2',4,4',6,6'-HxCB	155L	100	100	100	100	100	100

13C12-2,3,3',4,4',5-HxCB	156L	100	100	100	100	100	100

13C12-2,3,3',4,4',5'-HxCB	157L	100	100	100	100	100	100

13C12-2,3',4,4',5,5'-HxCB	167L	100	100	100	100	100	100

13C12-3,3',4,4',5,5'-HxCB	169L	100	100	100	100	100	100

13C12-2,2',3,4',5,6,6'-HpCB	188L	100	100	100	100	100	100

13C12-2,3,3',4,4',5,5'-HpCB	189L	100	100	100	100	100	100

13C12-2,2',3,3',5,5',6,6'-OcCB	202L	100	100	100	100	100	100

13C12-2,3,3',4,4',5,5',6-OcCB	205L	100	100	100	100	100	100

13C12-2,2',3,3',4,4',5,5',6-NoCB	206L	100	100	100	100	100	100

13C12-2,2',3,3',4',5,5',6,6'-NoCB	208L	100	100	100	100	100	100

13C12-DeCB	209L	100	100	100	100	100	100

Labeled clean-up

13C12-2,4,4'-TrCB	28L	100	100	100	100	100	100

13C12-2,3,3',5,5'-PeCB	111L	100	100	100	100	 100	100

13C12-2,2',3,3',5,5',6-HpCB	178L	100	100	100	100	100	100

Labeled injection internal

13C12-2,5-DiCB	9L	100	100	100	100	100	100

13C12-2,2',5,5'-TeCB	52L	100	100	100	100	100	100

13C12-2,2',4',5,5'-PeCB	101L	100	100	100	100	100	100

13C12-2,2',3',4,4',5'-HxCB	138L	100	100	100	100	100	100

13C12-2,2',3,3',4,4',5,5'-OcCB	194L	100	100	100	100	100	100



1.	Suffix “L” indicates labeled compound.

2.	Additional concentration used for calibration of high sensitivity
HRGC/HRMS systems.  If the ion abundance ratio (Table 8) cannot be
achieved at this level (see Section 10.3.3), a calibration point at 0.4
or 0.5 ng/mL may be used.

Table 6.	QC Acceptance Criteria for VER, IPR, OPR, and Labeled
Compounds in Samples1,2

Congener Name	Congener No.3	Test Conc. (ng/mL)4	 

VER (%)5	IPR	 

OPR Recovery (%)	Labeled Compound Recovery in Samples (%)





RSD (%)	Mean Recovery (%)



2-MoCB	1	50	75 - 125	25	70 - 130	60 - 135	NA

4-MoCB	3	50	75 - 125	25	70 - 130	60 - 135

	2,2'-DiCB	4	50	75 - 125	25	70 - 130	60 - 135

	4,4'-DiCB	15	50	75 - 125	25	70 - 130	60 - 135

	2,2'6-TrCB	19	50	75 - 125	25	70 - 130	60 - 135

	3,4,4'-TrCB	37	50	75 - 125	25	70 - 130	60 - 135

	2,2'6,6'TeCB	54	50	75 - 125	25	70 - 130	60 - 135

	3,3',4,4'-TeCB	77	50	75 - 125	25	70 - 130	60 - 135

	3,4,4',5-TeCB	81	50	75 - 125	25	70 - 130	60 - 135

	2,2',4,6,6'-PeCB	104	50	75 - 125	25	70 - 130	60 - 135

	2,3,3',4,4'-PeCB	105	50	75 - 125	25	70 - 130	60 - 135

	2,3,4,4',5-PeCB	114	50	75 - 125	25	70 - 130	60 - 135

	2,3',4,4',5-PeCB	118	50	75 - 125	25	70 - 130	60 - 135

	2',3,4,4',5-PeCB	123	50	75 - 125	25	70 - 130	60 - 135

	3,3',4,4',5-PeCB	126	50	75 - 125	25	70 - 130	60 - 135

	2,2',4,4',6,6'-HxCB	155	50	75 - 125	25	70 - 130	60 - 135

	2,3,3',4,4',5-HxCB 6	156	50	75 - 125	25	70 - 130	60 - 135

	 2,3,3',4,4',5'-HxCB 6	157	50	75 - 125	25	70 - 130	60 - 135

	2,3',4,4',5,5'-HxCB	167	50	75 - 125	25	70 - 130	60 - 135

	3,3',4,4',5,5'-HxCB	169	50	75 - 125	25	70 - 130	60 - 135

	2,2',3,4',5,6,6'-HpCB	188	50	75 - 125	25	70 - 130	60 - 135

	2,3,3',4,4',5,5'-HpCB	189	50	75 - 125	25	70 - 130	60 - 135

	2,2',3,3',5,5',6,6'-OcCB	202	50	75 - 125	25	70 - 130	60 - 135

	2,3,3',4,4',5,5',6-OcCB	205	50	75 - 125	25	70 - 130	60 - 135

	2,2',3,3',4,4',5,5',6-NoCB	206	50	75 - 125	25	70 - 130	60 - 135

	2,2',3,3,'4,5,5',6,6'-NoCB	208	50	75 - 125	25	70 - 130	60 - 135

	DeCB	209	50	75 - 125	25	70 - 130	60 - 135

	13C12-2-MoCB	1L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-4-MoCB	3L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-2,2'-DiCB	4L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-4,4'-DiCB	15L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-2,2',6-TrCB	19L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-3,4,4'-TrCB	37L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-2,2',6,6'-TeCB	54L	100	50 - 145	70	20 - 135	15 - 145	5 - 145

13C12-3,3',4,4'-TeCB	77L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-3,4,4',5-TeCB	81L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2,2',4,6,6'-PeCB	104L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2,3,3',4,4'-PeCB	105L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2,3,4,4',5-PeCB	114L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2,3',4,4',5-PeCB	118L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2',3,4,4',5-PeCB	123L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-3,3',4,4',5-PeCB	126L	100	50 - 145	50	45 - 135	40 - 145	10 - 145

13C12-2,2',4,4',6,6'-HxCB	155L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2,3,3',4,4',5 -HxCB 6	156L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2,3,3',4,4',5'-HxCB 6	157L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2,3',4,4',5,5'-HxCB	167L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-3,3',4,4',5,5'-HxCB	169L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2,2',3,4',5,6,6'-HpCB	188L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2',3,3',4,4',5,5'-HpCB	189L	100	50 - 145	50	45 - 135	40 - 145	10 -
145

13C12-2,2',3,3',5,5',6,6'-OcCB	202L	100	50 - 145	50	45 - 135	40 - 145	10
- 145

13C12-2,3,3',4,4',5,5',6-OcCB	205L	100	50 - 145	50	45 - 135	40 - 145	10
- 145

13C12-2,2',3,3',4,4',5,5',6-NoCB	206L	100	50 - 145	50	45 - 135	40 - 145
10 - 145

13C12-2,2',3,3',4,5,5',6,6'-NoCB	208L	100	50 - 145	50	45 - 135	40 - 145
10 - 145

13C12-2,2',3,3',4,4',5,5',6,6'-DeCB	209L	100	50 - 145	50	45 - 135	40 -
145	10 - 145

Cleanup standards	 	 	 	 	 	 	 

13C12-2,4,4'-TrCB	28L	100	65 - 135	70	20 - 135	15 - 145	5 - 145

13C12-2,3,3',5,5'-PeCB	111L	100	75 - 125	50	45 - 135	40 - 145	10 - 145

13C12-2,2',3,3',5,5',6-HpCB	178L	100	75 - 125	50	45 - 135	40 - 145	10 -
145



1.	Reference 22 describes how interlaboratory results were pooled from
analyses of wastewater, biosolids, and fish tissue samples.

2.	QC acceptance criteria for IPR, OPR, and samples based on a 20-µL
extract final volume

3.	Suffix “L” indicates labeled compound.

4.	See Table 5.

5.	Section 15.3.

6.	CBs 156/157 and 156L/157L are tested as the sum of the two congeners

	NA = Not applicable

	

Table 7.	Scan Descriptors, Levels of Chlorination, m/z Information, and
Substances Monitored by HRGC/HRMS

Function and Chlorine Level	 m/z1	m/z Type	m/z Formula	Substance

Fn-1; Cl-1	188.0393	M	12C12 H9 35Cl	Cl-1 CB

	 190.0363	M+2	12C12 H9 37Cl	Cl-1 CB

	 200.0795	M	13C12 H9 35Cl	13C12  Cl-1 CB

	 202.0766	M+2	13C12 H9 37Cl	13C12  Cl-1 CB

	 218.9856	lock	C4 F9	PFK

Fn-2; Cl-2, 3	 222.0003	M	12C12 H8 35Cl2	Cl-2 PCB

	 223.9974 (2)	M+2	12C12 H8 35Cl 37 Cl	Cl-2 PCB

	 225.9944	M+4	12C12 H8 37Cl2	Cl-2 PCB

	 234.0406	M	13C12 H8 35Cl2	13C12  Cl-2 PCB

	 236.0376	M+2	13C12 H8 35Cl 37 Cl	13C12  Cl-2 PCB

	 242.9856	lock	C6 F9	PFK

	 255.9613	M	12C12 H7 35Cl3	Cl-3 PCB

	 257.9584	M+2	12C12 H7 35Cl2 37Cl	Cl-3 PCB

	 268.0016	M	13C12 H7 35Cl3	13C12  Cl-3 PCB

	 269.9986	M+2	13C12 H7 35Cl2 37Cl	13C12  Cl-3 PCB

Fn-3; Cl-3, 4, 5	 255.9613	M	12C12 H7 35Cl3	Cl-3 PCB

	 257.9584	M+2	12C12 H7 35Cl2 37Cl	Cl-3 PCB

	 259.9554	M+4	12C12 H7 35Cl 37Cl2	Cl-3 PCB

	 268.0016	M	13C12 H7 35Cl3	13C12  Cl-3 PCB

	 269.9986	M+2	13C12 H7 35Cl2 37Cl	13C12  Cl-3 PCB

	 280.9825	lock	C6 F11	PFK

	 289.9224	M	12C12 H6 35Cl4	Cl-4 PCB

	 291.9194	M+2	12C12 H6 35Cl3 37Cl	Cl-4 PCB

	 293.9165	M+4	12C12 H6 35Cl2 37Cl2	Cl-4 PCB

	 301.9626	M	13C12 H6 35Cl4	13C12  Cl-4 PCB

	 303.9597	M+2	13C12 H6 35Cl3 37Cl	13C12  Cl-4 PCB

	 323.8834	M	13C12 H5 35Cl5	Cl-5 PCB

	 325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	 327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	 337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12  Cl-5 PCB

	 339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12  Cl-5 PCB

Fn-4; Cl-4, 5, 6	 289.9224	M	12C12 H6 35Cl4	Cl-4 PCB

	 291.9194	M+2	12C12 H6 35Cl3 37Cl	Cl-4 PCB

	 293.9165	M+4	12C12 H6 35Cl2 37Cl2	Cl-4 PCB

	 301.9626	M	13C12 H6 35Cl3 37Cl	13C12  Cl-4 PCB

	 303.9597	M+2	13C12 H6 35Cl2 37Cl2	13C12  Cl-4 PCB

	 323.8834	M	12C12 H5 35Cl5	Cl-5 PCB

	 325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	 327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	 330.9792	lock	C7 F15	PFK

	 337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12  Cl-5 PCB

	 339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12  Cl-5 PCB

Fn-4; Cl-4, 5, 6	 359.8415	M+2	12C12 H4 35Cl5 37Cl	Cl-6 PCB

	 361.8385	M+4	12C12 H4 35Cl4 37Cl2	Cl-6 PCB

	 363.8356	M+6	12C12 H4 35Cl3 37Cl3	Cl-6 PCB

	 371.8817	M+2	13C12 H4 35Cl5 37Cl	13C12  Cl-6 PCB

	 373.8788	M+4	13C12 H4 35Cl4 37Cl2	13C12  Cl-6 PCB

Fn-5; Cl-5, 6, 7	 323.8834	M	12C12 H5 35Cl5	Cl-5 PCB

	 325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	 327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	 337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12  Cl-5 PCB

	 339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12  Cl-5 PCB

	 354.9792	lock	C9 F13	PFK

	 359.8415	M+2	12C12 H4 35Cl5 37Cl	Cl-6 PCB

	 361.8385	M+4	12C12 H4 35Cl4 37Cl2	Cl-6 PCB

	 363.8356	M+6	12C12 H4 35Cl3 37Cl3	Cl-6 PCB

	 371.8817	M+2	13C12 H4 35Cl5 37Cl	13C12  Cl-6 PCB

	 373.8788	M+4	13C12 H4 35Cl4 37Cl2	13C12  Cl-6 PCB

	 393.8025	M+2	12C12 H3 35Cl6 37Cl	Cl-7 PCB

	 395.7995	M+4	12C12 H3 35Cl5 37Cl2	Cl-7 PCB

	 397.7966	M+6	12C12 H3 35Cl4 37Cl3	Cl-7 PCB

	 405.8428	M+2	13C12 H3 35Cl6 37Cl	13C12  Cl-7 PCB

	 407.8398	M+4	13C12 H3 35Cl5 37Cl2	13C12  Cl-7 PCB

	 454.9728	QC	C11 F17	PFK

Fn-6; Cl-7, 8, 9, 10	 393.8025	M+2	12C12 H3 35Cl6 37Cl	Cl-7 PCB

	 395.7995	M+4	12C12 H3 35Cl5 37Cl2	Cl-7 PCB

	 397.7966	M+6	12C12 H3 35Cl4 37Cl3	Cl-7 PCB

	 405.8428	M+2	13C12 H3 35Cl6 37Cl	13C12  Cl-7 PCB

	 407.8398	M+4	13C12 H3 35Cl5 37Cl2	13C12  Cl-7 PCB

	 427.7635	M+2	12C12 H2 35Cl7 37Cl	Cl-8 PCB

	 429.7606	M+4	12C12 H2 35Cl6 37Cl2	Cl-8 PCB

	 431.7576	M+6	12C12 H2 35Cl5 37Cl3	Cl-8 PCB

	 439.8038	M+2	13C12 H2 35Cl7 37Cl	13C12  Cl-8 PCB

	 441.8008	M+4	13C12 H2 35Cl6 37Cl2	13C12  Cl-8 PCB

	 442.9728	QC	C10 F13	PFK

	 454.9728	lock	C11 F13	PFK

	 461.7246	M+2	12C12 H1 35Cl8 37Cl	Cl-9 PCB

	 463.7216	M+4	12C12 H1 35Cl7 37Cl2	Cl-9 PCB

	 465.7187	M+6	12C12 H1 35Cl6 37Cl3	Cl-9 PCB

	 473.7648	M+2	13C12 H1 35Cl8 37Cl	13C12  Cl-9 PCB

	 475.7619	M+4	13C12 H1 35Cl7 37Cl2	13C12  Cl-9 PCB

	 495.6856	M+2	12C12 H4 35Cl9 37Cl	Cl-10 PCB

	 497.6826	M+4	12C12 35Cl8 37Cl2	Cl-10 PCB

	 499.6797	M+6	12C12 35Cl7 37Cl3	Cl-10 PCB

Fn-6; Cl-7, 8, 9, 10	 507.7258	M+2	13C12 35Cl9 37Cl	13C12  Cl-10 PCB

	 509.7229	M+4	13C12 35Cl8 37Cl2	13C12  Cl-10 PCB

	 511.7199	M+6	13C12 35Cl7 37Cl3	13C12  Cl-10 PCB



1.	Isotopic masses used for accurate mass calculation

	1H 	  1.0078

	12C	12.0000

	13C	13.0034

	35Cl	34.9689

	37Cl	36.9659

	19F	18.9984

2.	An interference with PFK m/z 223.9872 may preclude meeting 10:1 S/N
for the DiCB congeners at the CS-0.2 and CS-1 calibration levels
(Section 10.3.3 and Table 5).  If this interferences occurs, 10:1 S/N
must be met at the CS-2 level.  See the note at Section 10.2.1 for
information on how to minimize this interference.



Table 8.	Theoretical Ion Abundance Ratios and QC Limits

Chlorine Atoms	m/z’s Forming Ratio	Theoretical Ratio	Lower QC Limit
Upper QC Limit

1	M/(M+2)	3.13	2.66	3.60

2	M/(M+2)	1.56	1.33	1.79

3	M/(M+2)	1.04	0.88	1.20

4	M/(M+2)	0.77	0.65	0.89

5	(M+2)/(M+4)	1.55	1.32	1.78

6	(M+2)/(M+4)	1.24	1.05	1.43

7	(M+2)/(M+4)	1.05	0.89	1.21

8	(M+2)/(M+4)	0.89	0.76	1.02

9	(M+2)/(M+4)	0.77	0.65	0.89

10	(M+4)(M+6)	1.16	0.99	1.33



Table 9.	Suggested Sample Quantities to be Extracted for Various
Matrices1

Sample Matrix2	Example	Percent Solids	Phase	Quantity Extracted

Single-phase

Aqueous	Drinking water	<1	 – 3	1000 mL

	Groundwater





Treated wastewater



	Solid	Dry soil	>20	Solid	10 g

	Compost





Ash



	Organic	Waste solvent	<1	Organic	10 g

	Waste oil





Organic polymer



	Tissue	Fish	 – 	Organic	10 g

	Human adipose



	Multi-phase - Liquid/Solid

Aqueous/Solid	Wet soil	1-30	Solid	10 g

	Untreated effluent





Digested municipal sludge





Filter cake





Paper pulp



	Organic/solid	Industrial sludge	1-100	Both	10 g

	Oily waste



	Multi-phase - Liquid/Liquid

Aqueous/organic	In-process effluent	<1	Organic	10 g

	Untreated effluent





Drum waste



	Multi-phase - Liquid/Liquid/Solid

Aqueous/organic/solid	Untreated effluent	>1	Organic and solid	10 g

	Drum waste



	

1.	The quantity of sample to be extracted is adjusted to provide 10 g of
solids (dry weight).  One liter of aqueous samples containing one
percent solids will contain 10 grams of solids.  For aqueous samples
containing greater than one percent solids, a lesser volume is used so
that 10 grams of solids (dry weight) will be extracted.  Other sample
volumes may be used to meet project needs.

2.	The sample matrix may be amorphous for some samples.  In general,
when the CBs are in contact with a multi-phase system in which one of
the phases is water, they will be preferentially dispersed in or
adsorbed on the alternate phase because of their low solubility in
water.

3.	Aqueous samples are filtered after spiking with the labeled
compounds.  The filtrate and the materials trapped on the filter are
extracted separately, and the extracts are combined for cleanup and
analysis.



 

 

 

Figure 4.  Solid-phase 

Extraction Apparatus



Figure 5.  Soxhlet/Dean Stark Extractor

 

24.0	Glossary

These definitions and purposes are specific to this method, but have
been conformed to common usage to the extent possible.

24.1	Units of weight and measure and their abbreviations

	24.1.1	Symbols

ºC	degrees Celsius

µL	microliter

µm	micrometer

<	less than

>	greater than

%	percent

24.1.2	Alphabetical abbreviations

cm	centimeter

g	gram

h	hour

ID	inside diameter

in.	inch

L	liter

M	molecular ion

m	meter

mg	milligram

min	minute

mL	milliliter

mm	millimeter

m/z	mass-to-charge ratio

N	normal; gram molecular weight of solute divided by hydrogen equivalent
of solute, per liter of solution

OD	outside diameter

pg	picogram

ppb	part-per-billion

ppm	part-per-million

ppq	part-per-quadrillion

ppt	part-per-trillion

psig	pound-per-square-inch gauge

v/v	volume per unit volume

w/v	weight per unit volume

24.2	Definitions and acronyms (in alphabetical order)

Analyte – A CB tested for by this method.  The analytes are listed in
Table 1.

Calibration standard (CAL) – A solution prepared from a secondary
standard and/or stock solutions and used to calibrate the response of
the HRGC/HRMS instrument.

Calibration verification standard (VER) – The mid-point calibration
standard (CS-3) that is used to verify calibration.  See Table 5.

CB – Chlorinated biphenyl congener.  One of the 209 individual
chlorinated biphenyl congeners determined using this method.  The 209
CBs are listed in Table 1.

CS-0.2, CS-1, CS-2, CS-3, CS-4, CS-5 – See Calibration standards and
Table 5

DeCB – Decachlorobiphenyl (PCB 209)

DiCB – Dichlorobiphenyl

Field blank – An aliquot of reagent water or other reference matrix
that is placed in a sample container in the laboratory or the field, and
treated as a sample in all respects, including exposure to sampling site
conditions, storage, preservation, and all analytical procedures.  The
purpose of the field blank is to determine if the field or sample
transporting procedures and environments have contaminated the sample.

GC – Gas chromatograph or gas chromatography

GPC – Gel permeation chromatograph or gel permeation chromatography

HpCB – Heptachlorobiphenyl

HPLC – High performance liquid chromatograph or high performance
liquid chromatography

HRGC – High resolution GC

HRMS – High resolution MS

HxCB – Hexachlorobiphenyl

Labeled injection internal standard – All five, or any one of the
five, 13C12-labeled CB congeners spiked into the concentrated extract
immediately prior to injection of an aliquot of the extract into the
HRGC/HRMS.  The five Labeled injection internal standards in this method
are CBs with congener numbers 9L, 52L, 101L, 138L, and 194L.

Internal standard – a labeled compound used as a reference for
quantitation of other labeled compounds and for quantitation of native
CB congeners other than the congener of which it is a labeled analog. 
See Internal standard quantitation.

Internal standard quantitation – A means of determining the
concentration of (1) a naturally occurring (native) compound by
reference to a compound other than its labeled analog and (2) a labeled
compound by reference to another labeled compound

IPR – Initial precision and recovery; four aliquots of a reference
matrix spiked with the analytes of interest and labeled compounds and
analyzed to establish the ability of the laboratory to generate
acceptable precision and recovery.  An IPR is performed prior to the
first time this method is used and any time the method or
instrumentation is modified.

Isotope dilution quantitation – A means of determining a naturally
occurring (native) compound by reference to the same compound in which
one or more atoms has been isotopically enriched.  In this method, all
12 carbon atoms in the biphenyl molecule are enriched with carbon-13 to
produce 13C12- labeled analogs of the chlorinated biphenyls.  The
13C12-labeled CBs are spiked into each sample and allow identification
and correction of the concentration of the native compounds in the
analytical process.

K-D – Kuderna-Danish concentrator; a device used to concentrate the
analytes in a solvent

Laboratory blank – See Method blank

Laboratory control sample (LCS) – See Ongoing precision and recovery
standard (OPR)

Laboratory reagent blank – See Method blank

May – This action, activity, or procedural step is neither required
nor prohibited.

May not – This action, activity, or procedural step is prohibited.

Method blank – An aliquot of reagent water that is treated exactly as
a sample including exposure to all glassware, equipment, solvents,
reagents, internal standards, and surrogates that are used with samples.
 The method blank is used to determine if analytes or interferences are
present in the laboratory environment, the reagents, or the apparatus.

Method Detection Limit – The minimum concentration of a substance that
can be measured and reported with 99% confidence that the analyte
concentration is greater than zero (40 CFR 136, Appendix B)

Minimum level of quantitation (ML) – The lowest level at which the
entire analytical system must give a recognizable signal and acceptable
calibration point for the analyte.  The ML represents the lowest
concentration at which an analyte can be measured with a known level of
confidence.  It may be equivalent to the concentration of the lowest
calibration standard, assuming that all method-specified sample weights,
volumes, and cleanup procedures have been employed.  The ML is
calculated by multiplying the MDL (pooled or unpooled, as appropriate)
by 3.18 and rounding the result to the number nearest to 1, 2, or 5 x 10
n, where n is zero or an integer (see 68 FR 11790).

MoCB – Monochlorobiphenyl

MS – Mass spectrometer or mass spectrometry

Must – This action, activity, or procedural step is required.

NoCB – Nonachlorobiphenyl

OcCB – Octachlorobiphenyl

OPR – Ongoing precision and recovery standard (OPR); a method blank
spiked with known quantities of analytes.  The OPR is analyzed exactly
like a sample.  Its purpose is to assure that the results produced by
the laboratory remain within the limits specified in this method for
precision and recovery.

Perfluorokerosene (PFK) – A mixture of compounds used to calibrate the
exact m/z scale in the HRMS

Preparation blank – See Method blank

Quality control check sample (QCS) – A sample containing all or a
subset of the analytes at known concentrations.  The QCS is obtained
from a source external to the laboratory or is prepared from a source of
standards different from the source of calibration standards.  It is
used to check laboratory performance with test materials prepared
external to the normal preparation process.

PeCB – Pentachlorobiphenyl

PCB – Polychlorinated biphenyl

Reagent water – Water demonstrated to be free from the analytes of
interest and potentially interfering substances at the method detection
limit for the analyte.

Relative standard deviation (RSD) – The standard deviation times 100
divided by the mean.  Also termed “coefficient of variation.”

RF – Response factor.  See Section 10.5.

RR – Relative response.  See Section 10.4.

SDS – Soxhlet/Dean-Stark extractor; an extraction device applied to
the extraction of solid and semi-solid materials (Reference 11 and
Figure 5)

Signal-to-noise ratio (S/N) – The height of the signal as measured
from the mean (average) of the noise to the peak maximum divided by the
width of the noise

Should – This action, activity, or procedural step is suggested but
not required.

SICP – Selected ion current profile; the line described by the signal
at an exact m/z

SPE – Solid-phase extraction; an extraction technique in which an
analyte is extracted from an aqueous sample by passage over or through a
material capable of reversibly adsorbing the analyte.  Also termed
liquid-solid extraction.

Stock solution – A solution containing an analyte that is prepared
using a reference material traceable to EPA, the National Institute of
Science and Technology (NIST), or a source that will attest to the
purity and authenticity of the reference material.

TeCB – Tetrachlorobiphenyl

TEF – Toxicity equivalency factor; an estimate of the toxicity of a
specific congener relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin

TEQ – The toxicity equivalent concentration in an environmental
sample.  It is the sum of the concentrations of each individual toxic
PCB and each individual 2,3,7,8-substituted, tetra-through
octa-chlorinated, dibenzo-p-dioxin and dibenzofuran multiplied by their
respective TEFs (Reference 1).

TEQPCB – The portion of the TEQ attributable to the toxic PCBs

TrCB – Trichlorobiphenyl

Unique GC resolution or uniquely resolved – Two adjacent
chromatographic peaks in which the height of the valley is less than 40
percent of the height of the shorter peak. See Section 6.9.1.1.2 and
Figures 6 and 7 for unique resolution specific to the SPB-octyl column.

VER – See Calibration verification

Appendix A   -	Preliminary Information for Determination of 209 CBs on
the DB-1 Column

1.0	Column and Conditions

1.1	Column – 30 ± 5-m long x 0.25 ± 0.02-mm ID; 0.25 µm film DB-1
(J&W, or equivalent).

1.2	Suggested GC operating conditions:

Injector temperature:	270 ºC

Interface temperature:	290 ºC

Initial temperature:	75 ºC

Initial time:	2 minutes

Temperature program:	75-150 ºC at 15 ºC/minute

	150-270 ºC at 2.5 ºC/minute

Final time:	7 minutes

Carrier gas velocity:	40 cm/sec at 200 ºC



Note:	The GC conditions may be optimized for compound separation and
sensitivity.  Once optimized, the same GC conditions must be used for
the analysis of all standards, blanks, IPR and OPR aliquots, and
samples. ADVANCE \d7 

2.0	Operating Information

2.1	Congener solutions – Mixes of individual congeners that will allow
separation of all 209 congeners on the DB-1 column had not been
developed when writing Method 1668C.

2.2	Elution order data – The congener mixes developed for the
SPB-octyl column (Table 4 of Method 1668C) were run on the DB-1 column. 
Although some congeners in these mixes co-elute, the mixes allow
determination of retention times for many congeners on the DB-1 column. 
These retention times are shown in Appendix Table A-1.

2.3	Window-defining congeners – The beginning and ending congeners at
each level of chlorination are the same as for the SPB-octyl column. 
See Table 2 in Method 1668C.

2.4	Scan descriptors – The 6-function scan descriptors are shown in
Appendix Table A-2.

Table A-1.	Retention time (RT) References, Quantitation References, and
Relative Retention Times (RRTs) for CB Congeners using a 

DB-1 Column

Labeled or Native CB1	Congener No.2	Retention Time and Quantitation
References	Congener No.2	RT	RRT	RRT QC Limits3

13C12-2-MoCB4	1L	13C12-4-MoCB4,5	3L	09:17	0.8855	0.8776-0.8935

2-MoCB	1	13C12-2-MoCB4	1L	09:17	1.0000	0.9964-1.0072

3-MoCB	2	13C12-4-MoCB4,5	3L	10:22	0.9889	0.9809-0.9968

13C12-4-MoCB4,5	3L	13C12-2,2',5,5'-TeCB7	52L	10:29	0.5561	0.5473-0.5650

4-MoCB	3	13C12-4-MoCB4,5	3L	10:29	1.0000	0.9968-1.0064

13C12-2,2'-DiCB4	4L	13C12-4,4'-DiCB4,5	15L	11:08	0.7591	0.7477-0.7705

2,2'-DiCB	4	13C12-2,2'-DiCB4	4L	11:08	1.0000	0.9925-1.0075

2,6-DiCB	10	13C12-4,4'-DiCB4,5	15L	11:10	0.7614	0.7500-0.7727

2,5-DiCB	9	13C12-4,4'-DiCB4,5	15L	12:08	0.8273	0.8216-0.8330

2,4-DiCB	7	13C12-4,4'-DiCB4,5	15L	12:09	0.8284	0.8227-0.8341

2,3'-DiCB	6	13C12-4,4'-DiCB4,5	15L	12:31	0.8534	0.8477-0.8591

2,4'-DiCB6	8	13C12-4,4'-DiCB4,5	15L	12:43	0.8670	0.8614-0.8727

2,3-DiCB	5	13C12-4,4'-DiCB4,5	15L	12:46	0.8705	0.8648-0.8761

13C12-2,2',6-TrCB4	19L	13C12-2,4,4'-TrCB5	28L	13:31	0.7990	0.7892-0.8089

3,5-DiCB	14	13C12-4,4'-DiCB4,5	15L	13:36	0.9273	0.9216-0.9330

 2,4,6-TrCB	30	13C12-2,4,4'-TrCB5	28L	14:06	0.8335	0.8286-0.8384

3,3'-DiCB	11	13C12-4,4'-DiCB4,5	15L	14:11	0.9670	0.9614-0.9727

3,4'-DiCB	13	13C12-4,4'-DiCB4,5	15L	14:26	0.9841	0.9784-0.9898

3,4-DiCB	12	13C12-4,4'-DiCB4,5	15L	14:27	0.9852	0.9795-0.9909

2,2',5-TrCB6	18	13C12-2,4,4'-TrCB5	28L	14:36	0.8631	0.8581-0.8680

13C12-4,4'-DiCB4,5	15L	13C12-2,2',5,5'-TeCB7	52L	14:40	0.7781
0.7692-0.7869

4,4'-DiCB	15	13C12-4,4'-DiCB4,5	15L	14:40	1.0000	0.9977-1.0043

2,2',4-TrCB	17	13C12-2,4,4'-TrCB5	28L	14:43	0.8700	0.8650-0.8749

 2,3',6-TrCB	27	13C12-2,4,4'-TrCB5	28L	15:06	0.8926	0.8877-0.8975

2,3,6-TrCB	24	13C12-2,4,4'-TrCB5	28L	15:06	0.8926	0.8877-0.8975

2,2',3-TrCB	16	13C12-2,4,4'-TrCB5	28L	15:26	0.9123	0.9074-0.9172

 2,4',6-TrCB	32	13C12-2,4,4'-TrCB5	28L	15:29	0.9153	0.9103-0.9202

13C12-2,2',6,6'-TeCB4	54L	13C12-3,3',4,4'-TeCB4,5,9	77L	16:02	0.6139
0.6075-0.6203

2,2',6,6'-TeCB	54	13C12-2,2',6,6'-TeCB4	54L	16:02	1.0000	0.9979-1.0042

2',3,5-TrCB	34	13C12-2,4,4'-TrCB5	28L	16:03	0.9488	0.9438-0.9537

2,3,5-TrCB	23	13C12-2,4,4'-TrCB5	28L	16:07	0.9527	0.9478-0.9576

2,4,5-TrCB	29	13C12-2,4,4'-TrCB5	28L	16:18	0.9635	0.9586-0.9685

2,3',5-TrCB	26	13C12-2,4,4'-TrCB5	28L	16:29	0.9744	0.9695-0.9793

2,3',4-TrCB	25	13C12-2,4,4'-TrCB5	28L	16:36	0.9813	0.9764-0.9862

2,4',5-TrCB	31	13C12-2,4,4'-TrCB5	28L	16:52	0.9970	0.9921-1.0020

13C12-2,4,4'-TrCB5	28L	13C12-2,2',5,5'-TeCB7	52L	16:55	0.8974
0.8930-0.9019

 2,4,4'-TrCB6	28	13C12-2,4,4'-TrCB5	28L	16:55	1.0000	0.9980-1.0039

2,2',4,6-TeCB	50	13C12-3,3',4,4'-TeCB4,5,9	77L	16:55	0.6477
0.6414-0.6541

 2,3,4-TrCB	21	13C12-2,4,4'-TrCB5	28L	17:21	1.0256	1.0207-1.0305

2,2',5,6'-TeCB	53	13C12-3,3',4,4'-TeCB4,5,9	77L	17:26	0.6675
0.6611-0.6739

2,3,3'-TrCB	20	13C12-2,4,4'-TrCB5	28L	17:22	1.0266	1.0217-1.0315

2',3,4-TrCB	33	13C12-2,4,4'-TrCB5	28L	17:24	1.0286	1.0236-1.0335

2,2',4,6'-TeCB	51	13C12-3,3',4,4'-TeCB4,5,9	77L	17:42	0.6777
0.6713-0.6841

2,3,4'-TrCB	22	13C12-2,4,4'-TrCB5	28L	17:43	1.0473	1.0424-1.0522

2,2',3,6-TeCB	45	13C12-3,3',4,4'-TeCB4,5,9	77L	18:00	0.6892
0.6828-0.6956

3,3',5-TrCB	36	13C12-2,4,4'-TrCB5	28L	18:16	1.0798	1.0749-1.0847

2,2',3,6'-TeCB	46	13C12-3,3',4,4'-TeCB4,5,9	77L	18:24	0.7045
0.6981-0.7109

3,4',5-TrCB	39	13C12-2,4,4'-TrCB5	28L	18:37	1.1005	1.0956-1.1054

13C12-2,2',5,5'-TeCB7	52L	13C12-2,2',5,5'-TeCB7	52L	18:51	1.0000
0.9956-1.0044

2,2',5,5'-TeCB6	52	13C12-3,3',4,4'-TeCB4,5,9	77L	18:51	0.7218
0.7154-0.7281

 2,3',4,6-TeCB	69	13C12-3,3',4,4'-TeCB4,5,9	77L	18:52	0.7224
0.7160-0.7288

 2,3',5',6-TeCB	73	13C12-3,3',4,4'-TeCB4,5,9	77L	18:57	0.7256
0.7192-0.7320

2,2',4,5'-TeCB	49	13C12-3,3',4,4'-TeCB4,5,9	77L	19:00	0.7275
0.7211-0.7339

 2,2',3,5-TeCB	43	13C12-3,3',4,4'-TeCB4,5,9	77L	19:04	0.7301
0.7237-0.7364

3,4,5-TrCB	38	13C12-2,4,4'-TrCB5	28L	19:12	1.1350	1.1300-1.1399

2,2',4,4'-TeCB	47	13C12-3,3',4,4'-TeCB4,5,9	77L	19:15	0.7371
0.7307-0.7435

2,4,4',6-TeCB	75	13C12-3,3',4,4'-TeCB4,5,9	77L	19:20	0.7403
0.7339-0.7466

2,2',4,5-TeCB	48	13C12-3,3',4,4'-TeCB4,5,9	77L	19:20	0.7403
0.7339-0.7466

2,3,5,6-TeCB	65	13C12-3,3',4,4'-TeCB4,5,9	77L	19:31	0.7473	0.7409-0.7537

 2,3,4,6-TeCB	62	13C12-3,3',4,4'-TeCB4,5,9	77L	19:36	0.7505
0.7441-0.7569

3,3',4-TrCB	35	13C12-2,4,4'-TrCB5	28L	19:41	1.1635	1.1586-1.1685

13C12-2,2',4,6,6'-PeCB4	104L	13C12-2,3',4,4',5-PeCB5,9	118L	19:45	0.7037
0.6977-0.7096

2,2',4,6,6'-PeCB	104	13C12-2,2',4,6,6'-PeCB4	104L	19:45	1.0000
0.9983-1.0034

2,2',3,5'-TeCB6	44	13C12-3,3',4,4'-TeCB4,5,9	77L	19:55	0.7626
0.7562-0.7690

13C12-3,4,4'-TrCB4	37L	13C12-2,4,4'-TrCB5	28L	20:03	1.1852	1.1803-1.1901

3,4,4'-TrCB	37	13C12-3,4,4'-TrCB4	37L	20:03	1.0000	0.9983-1.0033

2,3,3',6-TeCB	59	13C12-3,3',4,4'-TeCB4,5,9	77L	20:05	0.7690
0.7626-0.7754

2,2',3,4'-TeCB	42	13C12-3,3',4,4'-TeCB4,5,9	77L	20:07	0.7703
0.7639-0.7766

 2,3',5,5'-TeCB	72	13C12-3,3',4,4'-TeCB4,5,9	77L	20:36	0.7888
0.7824-0.7951

 2,3',4',6-TeCB	71	13C12-3,3',4,4'-TeCB4,5,9	77L	20:36	0.7888
0.7824-0.7951

2,3,4',6-TeCB	64	13C12-3,3',4,4'-TeCB4,5,9	77L	20:37	0.7894
0.7830-0.7958

 2,2',3,4-TeCB	41	13C12-3,3',4,4'-TeCB4,5,9	77L	20:39	0.7907
0.7843-0.7971

2,2',3,6,6'-PeCB	96	13C12-2,3',4,4',5-PeCB5,9	118L	20:48	0.7411
0.7352-0.7470

 2,3',4,5'-TeCB	68	13C12-3,3',4,4'-TeCB4,5,9	77L	20:52	0.7990
0.7926-0.8054

2,2',3,3'-TeCB	40	13C12-3,3',4,4'-TeCB4,5,9	77L	20:58	0.8028
0.7996-0.8060

2,3,3',5-TeCB	57	13C12-3,3',4,4'-TeCB4,5,9	77L	21:21	0.8175
0.8143-0.8207

2,2',4,5,'6-PeCB	103	13C12-2,3',4,4',5-PeCB5,9	118L	21:22	0.7613
0.7553-0.7672

 2,3',4,5-TeCB	67	13C12-3,3',4,4'-TeCB4,5,9	77L	21:38	0.8283
0.8251-0.8315

2,2',4,4',6-PeCB	100	13C12-2,3',4,4',5-PeCB5,9	118L	21:41	0.7726
0.7666-0.7785

2,3,3',5'-TeCB	58	13C12-3,3',4,4'-TeCB4,5,9	77L	21:43	0.8315
0.8283-0.8347

 2,3,4',5-TeCB	63	13C12-3,3',4,4'-TeCB4,5,9	77L	21:51	0.8366
0.8334-0.8398

 2,2',3,5,6'-PeCB	94	13C12-2,3',4,4',5-PeCB5,9	118L	22:05	0.7868
0.7809-0.7928

2,4,4',5-TeCB	74	13C12-3,3',4,4'-TeCB4,5,9	77L	22:07	0.8468
0.8437-0.8500

 2,3,4,5-TeCB	61	13C12-3,3',4,4'-TeCB4,5,9	77L	22:11	0.8494
0.8462-0.8526

2,3',4',5-TeCB	70	13C12-3,3',4,4'-TeCB4,5,9	77L	22:20	0.8551
0.8519-0.8583

 2',3,4,5-TeCB	76	13C12-3,3',4,4'-TeCB4,5,9	77L	22:25	0.8583
0.8551-0.8615

 2,2',3',4,6-PeCB	98	13C12-2,3',4,4',5-PeCB5,9	118L	22:28	0.8005
0.7975-0.8034

2,3',4,4'-TeCB6	66	13C12-3,3',4,4'-TeCB4,5,9	77L	22:29	0.8609
0.8577-0.8641

2,2',4,5,6'-PeCB	102	13C12-2,3',4,4',5-PeCB5,9	118L	22:32	0.8029
0.7999-0.8058

2,2',3,5',6-PeCB	95	13C12-2,3',4,4',5-PeCB5,9	118L	22:34	0.8040
0.8011-0.8070

 2,2',3,5,6-PeCB	93	13C12-2,3',4,4',5-PeCB5,9	118L	22:36	0.8052
0.8023-0.8082

3,3',5,5'-TeCB	80	13C12-3,3',4,4'-TeCB4,5,9	77L	22:45	0.8711
0.8679-0.8743

2,2',3,4,6-PeCB	88	13C12-2,3',4,4',5-PeCB5,9	118L	22:49	0.8129
0.8100-0.8159

2,2',3,4',6-PeCB	91	13C12-2,3',4,4',5-PeCB5,9	118L	22:55	0.8165
0.8135-0.8195

2,3,3',4'-TeCB	55	13C12-3,3',4,4'-TeCB4,5,9	77L	22:57	0.8787
0.8756-0.8819

2,3',4,5,'6-PeCB	121	13C12-2,3',4,4',5-PeCB5,9	118L	23:04	0.8219
0.8189-0.8248

2,3,3',4'-TeCB	56	13C12-3,3',4,4'-TeCB4,5,9	77L	23:24	0.8960
0.8928-0.8992

 2,3,4,4'-TeCB	60	13C12-3,3',4,4'-TeCB4,5,9	77L	23:24	0.8960
0.8928-0.8992

13C12-2,2',4,4',6,6'-HxCB4	155L	13C12-2,3',4,4',5,5'-HxCB5,9	167L	23:43
0.7104	0.7054-0.7154

2,2',4,4',6,6'-HxCB	155	13C12-2,2',4,4',6,6'-HxCB4	155L	23:43	1.0000
0.9986-1.0028

2,2',3,3',6-PeCB	84	13C12-2,3',4,4',5-PeCB5,9	118L	23:44	0.8456
0.8426-0.8486

 2,2',3,5,5'-PeCB	92	13C12-2,3',4,4',5-PeCB5,9	118L	23:50	0.8492
0.8462-0.8521

  2,2',3,4,6'-PeCB	89	13C12-2,3',4,4',5-PeCB5,9	118L	23:53	0.8510
0.8480-0.8539

2,2',3,4',5-PeCB	90	13C12-2,3',4,4',5-PeCB5,9	118L	24:07	0.8593
0.8563-0.8622

13C12-2,2',4,5,5'-PeCB7	101L	13C12-2,2',4,5,5'-PeCB7	101L	24:11	1.0000
0.9966-1.0034

2,2',4,5,5'-PeCB6	101	13C12-2,3',4,4',5-PeCB5,9	118L	24:11	0.8616
0.8587-0.8646

2,3,3',5',6-PeCB	113	13C12-2,3',4,4',5-PeCB5,9	118L	24:23	0.8688
0.8658-0.8717

3,3',4,5'-TeCB	79	13C12-3,3',4,4'-TeCB4,5,9	77L	24:27	0.9362
0.9330-0.9394

2,2',4,4',5-PeCB	99	13C12-2,3',4,4',5-PeCB5,9	118L	24:28	0.8717
0.8688-0.8747

2,2',3,4',6,6'-HxCB	150	13C12-2,3',4,4',5,5'-HxCB5,9	167L	24:52	0.7449
0.7399-0.7499

2,3',4,4',6-PeCB	119	13C12-2,3',4,4',5-PeCB5,9	118L	24:54	0.8872
0.8842-0.8901

 2,3,3',5,6-PeCB	112	13C12-2,3',4,4',5-PeCB5,9	118L	25:00	0.8907
0.8878-0.8937

2,3,3',4,6-PeCB	109	13C12-2,3',4,4',5-PeCB5,9	118L	25:09	0.8961
0.8931-0.8990

2,2',3,5,6,6'-HxCB	152	13C12-2,3',4,4',5,5'-HxCB5,9	167L	25:17	0.7574
0.7524-0.7624

2,2',3,3',5-PeCB	83	13C12-2,3',4,4',5-PeCB5,9	118L	25:20	0.8919
0.8890-0.8949

2,2',3',4,5-PeCB	97	13C12-2,3',4,4',5-PeCB5,9	118L	25:22	0.9038
0.9008-0.9068

2,2',3,4,5-PeCB	86	13C12-2,3',4,4',5-PeCB5,9	118L	25:27	0.9068
0.9038-0.9097

13C12-3,4,4',5-TeCB9	81L	13C12-2,2',5,5'-TeCB7	52L	25:32	1.3546
1.3457-1.3634

3,4,4',5-TeCB10	81	13C12-3,4,4',5-TeCB4,5,9	77L	25:32	1.0000
0.9987-1.0026

 2',3,4,5,6'-PeCB	125	13C12-2,3',4,4',5-PeCB5,9	118L	25:36	0.9121
0.9091-0.9151

2,3,4',5,6-PeCB	117	13C12-2,3',4,4',5-PeCB5,9	118L	25:37	0.9127
0.9097-0.9157

2,2',3,4,5'-PeCB	87	13C12-2,3',4,4',5-PeCB5,9	118L	25:38	0.9133
0.9103-0.9163

3,3',4,5-TeCB	78	13C12-3,3',4,4'-TeCB4,5,9	77L	25:40	0.9598
0.9566-0.9630

2,2',3,4,6,6'-HxCB	145	13C12-2,3',4,4',5,5'-HxCB5,9	167L	25:42	0.7698
0.7649-0.7748

 2,3,4,4',6-PeCB	115	13C12-2,3',4,4',5-PeCB5,9	118L	25:44	0.9169
0.9139-0.9198

13C12-2,3,3',5,5'-PeCB8	111L	13C12-2,2',4,5,5'-PeCB7	101L	25:51	1.0689
1.0655-1.0724

 2,3,3',5,5'-PeCB	111	13C12-2,3',4,4',5-PeCB5,9	118L	25:51	0.9210
0.9181-0.9240

2,2',3,4,4'-PeCB	85	13C12-2,3',4,4',5-PeCB5,9	118L	25:51	0.9210
0.9181-0.9240

 2,3,4,5,6-PeCB	116	13C12-2,3',4,4',5-PeCB5,9	118L	25:48	0.9192
0.9163-0.9222

13C12-3,3',4,4'-TeCB4,5,9	77L	13C12-2,2',5,5'-TeCB7	52L	26:07	1.3855
1.3767-1.3943

3,3',4,4'-TeCB6,10	77	13C12-3,3',4,4'-TeCB4,5,9	77L	26:07	1.0000
0.9987-1.0026

2,2',3,3',6,6'-HxCB	136	13C12-2,3',4,4',5,5'-HxCB5,9	167L	26:10	0.7793
0.7743-0.7843

2,3',4,5,5'-PeCB	120	13C12-2,3',4,4',5-PeCB5,9	118L	26:12	0.9335
0.9305-0.9365

2,2',3,4',5,6'-HxCB	148	13C12-2,3',4,4',5,5'-HxCB5,9	167L	26:14	0.7858
0.7808-0.7908

2,3,3',4',6-PeCB	110	13C12-2,3',4,4',5-PeCB5,9	118L	26:16	0.9359
0.9329-0.9388

2,2',4,4',5,6'-HxCB	154	13C12-2,3',4,4',5,5'-HxCB5,9	167L	26:44	0.8008
0.7983-0.8033

2,2',3,3',4-PeCB	82	13C12-2,3',4,4',5-PeCB5,9	118L	26:48	0.9549
0.9519-0.9578

2,2',3,5,5',6-HxCB	151	13C12-2,3',4,4',5,5'-HxCB5,9	167L	27:18	0.8178
0.8153-0.8203

2,2',3,3',5,6'-HxCB	135	13C12-2,3',4,4',5,5'-HxCB5,9	167L	27:31	0.8243
0.8218-0.8268

 2',3,4,5,5'-PeCB	124	13C12-2,3',4,4',5-PeCB5,9	118L	27:36	0.9834
0.9804-0.9863

2,2',3,4,5',6-HxCB	144	13C12-2,3',4,4',5,5'-HxCB5,9	167L	27:38	0.8278
0.8253-0.8303

2,3,3',4,5'-PeCB	108	13C12-2,3',4,4',5-PeCB5,9	118L	27:40	0.9857
0.9828-0.9887

2,2',3,4',5,6-HxCB	147	13C12-2,3',4,4',5,5'-HxCB5,9	167L	27:44	0.8308
0.8283-0.8333

2,3,3',4',5-PeCB	107	13C12-2,3',4,4',5-PeCB5,9	118L	27:45	0.9887
0.9857-0.9917

2,2',3,4',5',6-HxCB	149	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:01	0.8392
0.8367-0.8417

2,2',3,3',5,6-HxCB	134	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:35	0.8562
0.8537-0.8587

 2,2',3,4,5,6'-HxCB	143	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:34	0.8557
0.8532-0.8582

13C12-2',3,4,4',5-PeCB9	123L	13C12-2,2',4,5,5'-PeCB7	101L	27:53	1.1530
1.1496-1.1564

2',3,4,4',5-PeCB10	123	13C12-2',3,4,4',5-PeCB9	123L	27:53	1.0000
0.9988-1.0024

 2,2',3,4,4',6-HxCB	139	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:01	0.8392
0.8367-0.8417

 2,3,3',4,5-PeCB	106	13C12-2,3',4,4',5-PeCB5,9	118L	28:04	1.0000
0.9970-1.0030

13C12-2,3',4,4',5-PeCB5,9	118L	13C12-2,2',4,5,5'-PeCB7	101L	28:04	1.1606
1.1571-1.1640

2,3',4,4',5-PeCB6,10	118	13C12-2,3',4,4',5-PeCB5,9	118L	28:04	1.0000
0.9988-1.0024

 2,2',3,4,4',6'-HxCB	140	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:12	0.8447
0.8422-0.8472

13C12-2,3,4,4',5-PeCB9	114L	13C12-2,2',4,5,5'-PeCB7	101L	28:38	1.1840
1.1806-1.1875

2,3,4,4',5-PeCB10	114	13C12-2,3,4,4',5-PeCB9	114L	28:38	1.0000
0.9988-1.0023

2',3,3',4,5-PeCB	122	13C12-2,3',4,4',5-PeCB5,9	118L	28:48	1.0261
1.0232-1.0291

2,2',3,3',4,6-HxCB	131	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:52	0.8647
0.8622-0.8672

2,2',3,4,5,6-HxCB	142	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:59	0.8682
0.8657-0.8707

2,2',3,3',5,5'-HxCB	133	13C12-2,3',4,4',5,5'-HxCB5,9	167L	28:59	0.8682
0.8657-0.8707

2,2',3,3',4,6'-HxCB	132	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:32	0.8847
0.8822-0.8872

 2,3,3',5,5',6-HxCB	165	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:21	0.8792
0.8767-0.8817

13C12-2,2',3,4',5,6,6'-HpCB4	188L	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L
29:22	0.9511	0.7327-0.7411

2,2',3,4',5,6,6'-HpCB	188	13C12-2,2',3,4',5,6,6'-HpCB4	188L	29:22	1.0000
0.9989-1.0023

2,2',3,4',5,5'-HxCB	146	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:24	0.8807
0.8782-0.8832

13C12-2,3,3',4,4'-PeCB9	105L	13C12-2,2',4,5,5'-PeCB7	101L	29:30	1.2198
1.2130-1.2267

2,3,3',4,4'-PeCB6,10	105	13C12-2,3,3',4,4'-PeCB9	105L	29:30	1.0000
0.9989-1.0023

2,3,3',4,5',6-HxCB	161	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:32	0.8847
0.8822-0.8872

2,2',4,4',5,5'-HxCB6	153	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:48	0.8927
0.8902-0.8952

2,2',3,4,4',6,6'-HpCB	184	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	29:49
0.7482	0.7440-0.7524

3,3',4,5,5'-PeCB	127	13C12-2,3',4,4',5-PeCB5,9	118L	29:57	1.0671
1.0641-1.0701

2,3',4,4',5',6-HxCB	168	13C12-2,3',4,4',5,5'-HxCB5,9	167L	29:59	0.8982
0.8957-0.9006

2,2',3,4,5,5'-HxCB	141	13C12-2,3',4,4',5,5'-HxCB5,9	167L	30:31	0.9141
0.9116-0.9166

2,2',3,3',5,6,6'-HpCB	179	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	30:33
0.7666	0.7624-0.7708

2,2',3,4,4',5-HxCB	137	13C12-2,3',4,4',5,5'-HxCB5,9	167L	30:51	0.9241
0.9216-0.9266

2,2',3,3',4,5'-HxCB	130	13C12-2,3',4,4',5,5'-HxCB5,9	167L	30:57	0.9271
0.9246-0.9296

2,2',3,3',4,6,6'-HpCB	176	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	31:01
0.7783	0.7742-0.7825

13C12-2,2',3,4,4',5'-HxCB7	138L	13C12-2,2',3,4,4',5'-HxCB7	138L	31:20
1.0000	0.9973-1.0027

2,2',3,4,4',5'-HxCB6	138	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:20	0.9386
0.9361-0.9411

 2,3,3',4',5',6-HxCB	164	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:22	0.9396
0.9371-0.9421

 2,3,3',4',5,6-HxCB	163	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:28	0.9426
0.9401-0.9451

 2,3,3',4,5,6-HxCB	160	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:33	0.9451
0.9426-0.9476

2,3,3',4,4',6-HxCB	158	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:35	0.9461
0.9436-0.9486

2,2',3,4,5,6,6'-HpCB	186	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	31:36
0.7930	0.7888-0.7972

2,2',3,3',4,5-HxCB	129	13C12-2,3',4,4',5,5'-HxCB5,9	167L	31:48	0.9526
0.9501-0.9551

13C12-3,3',4,4',5-PeCB4,9	126L	13C12-2,2',4,5,5'-PeCB7	101L	31:49	1.3156
1.3088-1.3225

3,3',4,4',5-PeCB6,10	126	13C12-3,3',4,4',5-PeCB4,9	126L	31:49	1.0000
0.9990-1.0021

 2,3,4,4',5,6-HxCB	166	13C12-2,3',4,4',5,5'-HxCB5,9	167L	32:13	0.9651
0.9626-0.9675

13C12-2,2',3,3',5,5',6-HpCB7	178L	13C12-2,2',3,3',5,5',6-HpCB7	178L
32:14	1.0000	0.9974-1.0026

2,2',3,3',5,5',6-HpCB	178	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	32:14
0.8089	0.8068-0.8110

2,2',3,3',4,5',6-HpCB	175	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	32:33
0.8168	0.8147-0.8189

2,3,3',4,5,5'-HxCB	159	13C12-2,3',4,4',5,5'-HxCB5,9	167L	32:43	0.9800
0.9775-0.9825

2,2',3,4',5,5',6-HpCB6	187	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	32:46
0.8223	0.8202-0.8243

 2,2',3,4,4',5,6'-HpCB	182	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	32:47
0.8227	0.8206-0.8248

2,2',3,3',4,4'-HxCB6	128	13C12-2,3',4,4',5,5'-HxCB5,9	167L	32:52	0.9845
0.9820-0.9870

2,3,3',4',5,5'-HxCB	162	13C12-2,3',4,4',5,5'-HxCB5,9	167L	33:00	0.9885
0.9860-0.9910

2,2',3,4,4',5',6-HpCB	183	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	33:06
0.8306	0.8285-0.8327

13C12-2,3',4,4',5,5'-HxCB5,9	167L	13C12-2,2',3,4,4',5'-HxCB7	138L	33:23
1.0654	1.0628-1.0681

2,3',4,4',5,5'-HxCB10	167	13C12-2,3',4,4',5,5'-HxCB5,9	167L	33:23	1.0000
0.9990-1.0020

2,2',3,4,5,5',6-HpCB	185	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	33:43
0.8461	0.8440-0.8482

2,2',3,3',4,5,6'-HpCB	174	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	34:07
0.8561	0.8540-0.8582

 2,2',3,4,4',5,6-HpCB	181	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	34:11
0.8578	0.8557-0.8599

2,2',3,3',4',5,6-HpCB	177	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	34:22
0.8624	0.8603-0.8645

2,2'3,3',4,4',6-HpCB	171	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	34:40
0.8699	0.8678-0.8720

13C12-2,3,3',4,4',5 -HxCB9	156L	13C12-2,2',3,4,4',5'-HxCB7	138L	34:40
1.1064	1.1037-1.1090

2,3,3',4,4',5-HxCB10	156	13C12-2,3,3',4,4',5 -HxCB9	156L	34:40	1.0000
0.9990-1.0019

13C12-2,2',3,3',5,5',6,6'-OcCB4	202L	13C12-Cl8-PCB-1945	194L	34:56
0.8265	0.8245-0.8285

2,2',3,3',5,5',6,6'-OcCB	202	13C12-2,2',3,3',5,5',6,6'-OcCB4	202L	34:56
1.0000	0.9990-1.0019

13C12-2,3,3',4,4',5'-HxCB9	157L	13C12-2,2',3,4,4',5'-HxCB7	138L	34:57
1.1154	1.1128-1.1181

2,3,3',4,4',5'-HxCB10	157	13C12-2,3,3',4,4',5'-HxCB9	157L	34:57	1.0000
0.9990-1.0019

2,2',3,3',4,5,6-HpCB	173	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	35:04
0.8800	0.8779-0.8821

2,2',3,3',4,5',6,6'-OcCB	201	13C12-Cl8-PCB-1945	194L	35:25	0.8379
0.8360-0.8399

2,2',3,4,4',5,6,6'-OcCB	204	13C12-Cl8-PCB-1945	194L	35:36	0.8423
0.8403-0.8442

2,2',3,3',4,5,5'-HpCB	172	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	35:41
0.8954	0.8934-0.8975

2,3,3',4,5,5',6-HpCB	192	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	35:51
0.8996	0.8975-0.9017

2,2',3,3',4,4',6,6'-OcCB	197	13C12-Cl8-PCB-1945	194L	35:55	0.8498
0.8478-0.8517

2,2',3,4,4',5,5'-HpCB6	180	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	36:07
0.9063	0.9042-0.9084

2,3,3',4',5,5',6-HpCB	193	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	36:20
0.9118	0.9097-0.9138

2,3,3',4,4',5',6-HpCB	191	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	36:34
0.9176	0.9155-0.9197

2,2',3,3',4,5,6,6'-OcCB	200	13C12-Cl8-PCB-1945	194L	36:49	0.8711
0.8691-0.8730

13C12-3,3',4,4',5,5'-HxCB4,9	169L	13C12-2,2',3,4,4',5'-HxCB7	138L	37:19
1.1910	1.1883-1.1936

3,3',4,4',5,5'-HxCB6,10	169	13C12-3,3',4,4',5,5'-HxCB4,9	169L	37:19
1.0000	0.9991-1.0018

2,2',3,3',4,4',5-HpCB6	170	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	37:44
0.9469	0.9448-0.9490

2,3,3',4,4',5,6-HpCB	190	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	37:56
0.9519	0.9498-0.9540

2,2',3,3',4,5,5',6-OcCB	198	13C12-Cl8-PCB-1945	194L	38:34	0.9125
0.9105-0.9144

2,2',3,3',4,5,5',6'-OcCB	199	13C12-Cl8-PCB-1945	194L	38:43	0.9160
0.9140-0.9180

2,2',3,3',4,4',5,6'-OcCB	196	13C12-Cl8-PCB-1945	194L	39:05	0.9247
0.9227-0.9267

2,2',3,4,4',5,5',6-OcCB	203	13C12-Cl8-PCB-1945	194L	39:05	0.9247
0.9227-0.9267

13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	13C12-2,2',3,3',5,5',6-HpCB7	178L
39:51	1.2363	1.2311-1.2415

2,3,3',4,4',5,5'-HpCB10	189	13C12-2',3,3',4,4',5,5'-HpCB4,5,9	189L	39:51
1.0000	0.9992-1.0017

2,2',3,3',4,4',5,6-OcCB6	195	13C12-Cl8-PCB-1945	194L	40:45	0.9641
0.9621-0.9661

13C12-2,2',3,3',4,5,5',6,6'-NoCB4	208L	13C12-Cl9-PCB-2064,5	206L	41:03
0.9149	0.9131-0.9168

2,2',3,3',4,5,5',6,6'-NoCB	208	13C12-2,2',3,3',4,5,5',6,6'-NoCB4	208L
41:03	1.0000	0.9992-1.0016

2,2',3,3',4,4',5,6,6'-NoCB	207	13C12-Cl9-PCB-2064,5	206L	41:32	0.9257
0.9238-0.9276

13C12-2,2',3,3',4,4',5,5'-OcCB5	194L	13C12-2,2',3,3',5,5',6-HpCB7	178L
42:16	1.3113	1.3061-1.3164

2,2',3,3',4,4',5,5'-OcCB	194	13C12-Cl8-PCB-1945	194L	42:16	1.0000
0.9992-1.0016

13C12-2,3,3',4,4',5,5',6-OcCB4	205L	13C12-Cl8-PCB-1945	194L	42:44	1.0110
1.0091-1.0130

2,3,3',4,4',5,5',6-OcCB	205	13C12-2,3,3',4,4',5,5',6-OcCB4	205L	42:44
1.0000	0.9992-1.0016

13C12-2,2',3,3',4,4',5,5',6-NoCB4,5	206L	13C12-2,2',3,3',5,5',6-HpCB7
178L	44:52	1.3919	1.3868-1.3971

2,2',3,3',4,4',5,5',6-NoCB6	206	13C12-Cl9-PCB-2064,5	206L	44:52	1.0000
0.9993-1.0015

13C12-2,2',3,3',4,4',5,5',6,6'-DeCB4,5	209L	13C12-2,2',3,3',5,5',6-HpCB7
178L	46:55	1.4555	1.4504-1.4607

2,2',3,3',4,4',5,5',6,6'-DeCB6	209	13C12-Cl10-PCB-2094,5	209L	46:55
1.0000	0.9993-1.0014



1.	Abbreviations for chlorination levels

MoCB

monochlorobiphenyl

HxCB

hexachlorobiphenyl

DiCB

dichlorobiphenyl

HpCB

heptachlorobiphenyl

TrCB

trichlorobiphenyl

OcCB

octachlorobiphenyl

TeCB

tetrachlorobiphenyl

NoCB

nonachlorobiphenyl

PeCB

pentachlorobiphenyl

DeCB

decachlorobiphenyl

	4.	Labeled level of chlorination (LOC) window-defining congener

5.	Labeled level of chlorination (LOC) quantitation congener

6.	National Oceanic and Atmospheric Administration (NOAA) congener of
interest

2.	Suffix “L” indicates labeled compound	7.	Instrument internal
standard

3.	For native CBs determined by isotope dilution quantitation, RRT QC
limits were constructed using -2 to +4 seconds around the retention time
for the labeled analog.  For native CBs determined by internal standard
quantitation, RRT QC limits were constructed using a ± 2 percent window
around the retention time for retention times in the range of 0.8-1.2
and a ± 4 percent window around the retention time for retention times
<0.8 and >1.2.  These windows may not be adequate for analyte
identification (See the note in Section 16.4)	8.	Clean-up standard

9.	Labeled internal standard for World Health Organization (WHO) toxic
congener

10.	WHO toxic congener



Table A-2.	Scan Descriptors, Levels of Chlorination, m/z Information,
and Substances Monitored by HRGC/HRMS

Function and Chlorine Level	 m/z	m/z Type	m/z Formula	Substance

Fn-1 Cl-1	188.0393	M	12C12 H9 35Cl	Cl-1 PCB

	190.0363	M+2	12C12 H9 37Cl	Cl-1P CB

	200.0795	M	13C12 H9 35Cl	13C12 Cl-1 PCB

	202.0766	M+2	13C12 H9 37Cl	13C12 Cl-1 PCB

	218.9856	lock	C4 F9	PFK

Fn-2 Cl-2,3	222.0003	M	12C12 H8 35Cl2	Cl-2 PCB

	223.9974	M+2	12C12 H8 35Cl 37 Cl	Cl-2 PCB

	225.9944	M+4	12C12 H8 37Cl2	Cl-2 PCB

	234.0406	M	13C12 H8 35Cl2	13C12 Cl-2 PCB

	236.0376	M+2	13C12 H8 35Cl 37 Cl	13C12 Cl-2 PCB

	242.9856	lock	C6 F9	PFK

	255.9613	M	12C12 H7 35Cl3	Cl-3 PCB

	257.9584	M+2	12C12 H7 35Cl2 37Cl	Cl-3 PCB

Fn-3 Cl-3,4,5	255.9613	M	12C12 H7 35Cl3	Cl-3 PCB

	257.9584	M+2	12C12 H7 35Cl2 37Cl	Cl-3 PCB

	259.9554	M+4	12C12 H7 35Cl 37Cl2	Cl-3 PCB

	268.0016	M	13C12 H7 35Cl3	13C12 Cl-3 PCB

	269.9986	M+2	13C12 H7 35Cl2 37Cl	13C12 Cl-3 PCB

	280.9825	lock	C6 F11	PFK

	289.9224	M	12C12 H6 35Cl4	Cl-4 PCB

	291.9194	M+2	12C12 H6 35Cl3 37Cl	Cl-4 PCB

	293.9165	M+4	12C12 H6 35Cl2 37Cl2	Cl-4 PCB

	301.9626	M	13C12 H6 35Cl4	13C12 Cl-4 PCB

	303.9597	M+2	13C12 H6 35Cl3 37Cl	13C12 Cl-4 PCB

	323.8834	M	12C12 H5 35Cl5	Cl-5 PCB

	325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12 Cl-5 PCB

	339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12 Cl-5 PCB

Fn-4 Cl-4,5,6	289.9224	M	12C12 H6 35Cl4	Cl-4 PCB

	291.9194	M+2	12C12 H6 35Cl3 37Cl	Cl-4 PCB

	293.9165	M+4	12C12 H6 35Cl2 37Cl2	Cl-4 PCB

	301.9626	M+2	13C12 H6 35Cl3 37Cl	13C12 Cl-4 PCB

	303.9597	M+4	13C12 H6 35Cl2 37Cl2	13C12 Cl-4 PCB

	323.8834	M	12C12 H5 35Cl5	Cl-5 PCB

	325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	330.9792	lock	C7 F15	PFK

	337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12 Cl-5 PCB

	339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12 Cl-5 PCB

	359.8415	M+2	13C12 H4 35Cl5 37Cl 	Cl-6 PCB

	361.8385	M+4	13C12 H4 35Cl4 37Cl2	Cl-6 PCB

	363.8356	M+6	13C12 H4 35Cl3 37Cl2	Cl-6 PCB

	371.8817	M+2	13C12 H4 35Cl5 37Cl	13C12 Cl-6 PCB

	373.8788	M+4	13C12 H4 35Cl4 37Cl2	13C12 Cl-6 PCB

Fn-5 Cl-5,6,7,8	323.8834	M	12C12 H5 35Cl5	Cl-5 PCB

	325.8804	M+2	12C12 H5 35Cl4 37Cl	Cl-5 PCB

	327.8775	M+4	12C12 H5 35Cl3 37Cl2	Cl-5 PCB

	337.9207	M+2	13C12 H5 35Cl4 37Cl	13C12 Cl-5 PCB

	339.9178	M+4	13C12 H5 35Cl3 37Cl2	13C12 Cl-5 PCB

	354.9792	lock	C9 F13	PFK

	359.8415	M+2	12C12 H4 35Cl5 37Cl	Cl-6 PCB

	361.8385	M+4	12C12 H4 35Cl4 37Cl2	Cl-6 PCB

	363.8356	M+6	12C12 H4 35Cl3 37Cl3	Cl-6 PCB

	371.8817	M+2	13C12 H4 35Cl5 37Cl	13C12 Cl-6 PCB

	373.8788	M+4	13C12 H4 35Cl4 37Cl2	13C12 Cl-6 PCB

	393.8025	M+2	12C12 H3 35Cl6 37Cl	Cl-7 PCB

	395.7995	M+4	12C12 H3 35Cl5 37Cl2	Cl-7 PCB

	397.7966	M+6	12C12 H3 35Cl4 37Cl3	Cl-7 PCB

	405.8428	M+2	13C12 H3 35Cl6 37Cl	13C12 Cl-7 PCB

	407.8398	M+4	13C12 H3 35Cl5 37Cl2	13C12 Cl-7 PCB

	427.7635	M+2	12C12 H2 35Cl7 37Cl	Cl-8 PCB

	429.7606	M+4	12C12 H2 35Cl6 37Cl2	Cl-8 PCB

	431.7576	M+6	12C12 H2 35Cl5 37Cl3	Cl-8 PCB

	439.8038	M+2	13C12 H2 35Cl7 37Cl	13C12 Cl-8 PCB

	441.8008	M+4	13C12 H2 35Cl6 37Cl2	13C12 Cl-8 PCB

	454.9728	QC	C11 F17	PFK

Fn-6 Cl-8,9,10	427.7635	M+2	12C12 H2 35Cl7 37Cl	Cl-8 PCB

	429.7606	M+4	12C12 H2 35Cl6 37Cl2	Cl-8 PCB

	431.7576	M+6	12C12 H2 35Cl5 37Cl3	Cl-8 PCB

	439.8038	M+2	13C12 H2 35Cl7 37Cl	13C12 Cl-8 PCB

	441.8008	M+4	13C12 H2 35Cl6 37Cl2	13C12 Cl-8 PCB

	442.9728	QC	C10 F13	PFK

	454.9728	lock	C11 F13	PFK

	461.7246	M+2	12C12 H1 35Cl8 37Cl	Cl-9 PCB

	463.7216	M+4	12C12 H1 35Cl7 37Cl2	Cl-9 PCB

	465.7187	M+6	12C12 H1 35Cl6 37Cl3	Cl-9 PCB

	473.7648	M+2	13C12 H1 35Cl8 37Cl	13C12 Cl-9 PCB

	475.7619	M+4	13C12 H1 35Cl7 37Cl2	13C12 Cl-9 PCB

	495.6856	M+2	13C12 H435Cl9 37Cl	Cl-10 PCB

	499.6797	M+4	12C12 35Cl7 37Cl3	Cl-10 PCB

	501.6767	M+6	12C12 35Cl6 37Cl4	Cl-10 PCB

	507.7258	M+2	13C12 H435Cl9 37Cl	13C12 Cl-10 PCB

	509.7229	M+4	13C12 H4 35Cl8 37Cl2	13C12 Cl-10 PCB

	511.7199	M+6	13C12 H4 35Cl8 37Cl4	13C12 Cl-10 PCB



Isotopic masses used for accurate mass calculation

1H 	1.0078	37Cl	36.9659

12C	12.0000	19F	18.9984

13C	13.0034	35Cl	34.9689

EPA Method 1668C	  PAGE  viii 	 April 2010

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