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

EPA Method 1668C ii April 2010

 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 inter­
ferences 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.
EPA Method 1668C iii April 2010
• 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.

EPA Method 1668C iv April 2010

• 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.

EPA Method 1668C v April 2010

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.

EPA Method 1668C vi April 2010

• 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.

EPA Method 1668C vii April 2010

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: [email protected]

EPA Method 1668C viii April 2010

 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).

EPA Method 1668C 1 April 2010

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.

EPA Method 1668C 2 April 2010

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.

EPA Method 1668C 3 April 2010

 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

EPA Method 1668C 4 April 2010

 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

EPA Method 1668C 5 April 2010

 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.

EPA Method 1668C 6 April 2010

 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.

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 tub-
EPA Method 1668C 7 April 2010
 ing 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.

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

EPA Method 1668C 8 April 2010

 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

EPA Method 1668C 9 April 2010

 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

EPA Method 1668C 10 April 2010

 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)

EPA Method 1668C 11 April 2010

 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 -

EPA Method 1668C 12 April 2010

 6.9.1.1.2) must be developed and be functionally equivalent to those
specifications.

6.9.1.3 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.

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

EPA Method 1668C 13 April 2010

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.

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.

EPA Method 1668C 14 April 2010

 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.

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.

EPA Method 1668C 15 April 2010

 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 solu­
tions 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.

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.
EPA Method 1668C 16 April 2010
 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.

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.

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.

EPA Method 1668C 17 April 2010

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.

EPA Method 1668C 18 April 2010

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.

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.

EPA Method 1668C 19 April 2010

 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 sub­
stituted for the reagent water matrix (Section 7.6.1) in all performance tests.

EPA Method 1668C 20 April 2010

 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).

EPA Method 1668C 21 April 2010

 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),

EPA Method 1668C 22 April 2010

 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

EPA Method 1668C 23 April 2010

 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

10.1 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

EPA Method 1668C 24 April 2010

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

EPA Method 1668C 25 April 2010

 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

EPA Method 1668C 26 April 2010

 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.

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.

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:

(A1n + A2 n ) C l
RR =
(A1l + A2 l ) C n
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)

EPA Method 1668C 27 April 2010

 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:

(A1s + A2 s ) C is
RF =
(A1is + A2 is ) C s

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.

EPA Method 1668C 28 April 2010

 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.

EPA Method 1668C 29 April 2010

 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:

weight of sample aliquot after drying (g) - weight of filter (g)

% solids = x 100
10 g
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:

weight of sample aliquot after drying (g)

% solids = x 100
weight of sample aliquot before drying (g)

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.

EPA Method 1668C 30 April 2010

 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

EPA Method 1668C 31 April 2010

 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.

EPA Method 1668C 32 April 2010

 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.

EPA Method 1668C 33 April 2010

 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.

EPA Method 1668C 34 April 2010

 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 separa­
tory 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).

EPA Method 1668C 35 April 2010

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.

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.

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.

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.
EPA Method 1668C 36 April 2010
 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 non-
aqueous 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).

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.
EPA Method 1668C 37 April 2010
 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:

weight of residue (g)

% lipid = x 100
weight of tissue (g)

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 degrada­
tion 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.
EPA Method 1668C 38 April 2010
 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.

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.

EPA Method 1668C 39 April 2010

 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.

EPA Method 1668C 40 April 2010

 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.

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 temper­
ature 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.

EPA Method 1668C 41 April 2010

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.

EPA Method 1668C 42 April 2010

 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 gravimetri­
cally 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.

EPA Method 1668C 43 April 2010

 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.

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.

EPA Method 1668C 44 April 2010

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.

EPA Method 1668C 45 April 2010

 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.

EPA Method 1668C 46 April 2010

 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.

EPA Method 1668C 47 April 2010

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).

EPA Method 1668C 48 April 2010

 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.

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).

EPA Method 1668C 49 April 2010

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.

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:

(A1n + A2 n ) C l
C ex (ng/mL) =
(A1l + A2 l ) RR

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:

EPA Method 1668C 50 April 2010

 ( A1s + A2 s ) C is
C ex (ng/mL) =
(A1is + A2 is ) RF
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:

Concentration found (ng/mL)

Recovery(%) = x 100
Concentration spiked (ng/mL)

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:
C V
Concentrat ion in solid sample (ng/kg) = ex ex
Ws
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:
C V
Concentration in aqueous sample (ng/L) = ex ex x 1000
Vs
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).

EPA Method 1668C 51 April 2010

 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).

EPA Method 1668C 52 April 2010

 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).

EPA Method 1668C 53 April 2010

 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.

EPA Method 1668C 54 April 2010

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

EPA Method 1668C 55 April 2010

 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.

EPA Method 1668C 56 April 2010

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.

EPA Method 1668C 57 April 2010

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
Labeled
analog
Congener CAS Registry congener CAS Registry
CB congener name1 number number Labeled analog name number number
13
2-MoCB 1 2051-60-7 C12-2-MoCB2 1L 234432-85-0
3-MoCB 2 2051-61-8
13
4-MoCB 3 2051-62-9 C12-4-MoCB2 3L 208263-77-8
13
2,2'-DiCB 4 13029-08-8 C12-2,2'-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
13
2,5-DiCB 9 34883-39-1 C12-2,5-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
13
4,4'-DiCB 15 2050-68-2 C12-4,4'-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
13
2,2',6-TrCB 19 38444-73-4 C12-2,2',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 13
C12-2,4,4'-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
13
3,4,4'-TrCB 37 38444-90-5 C12-3,4,4'-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

EPA Method 1668C 58 April 2010

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
Labeled
analog
Congener CAS Registry congener CAS Registry
CB congener name1 number number Labeled analog name number number
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 13
C12-2,2',5,5'-TeCB4 52L 208263-80-3
2,2',5,6'-TeCB 53 41464-41-9
13
2,2',6,6'-TeCB 54 15968-05-5 C12-2,2',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 13
C12-3,3',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 13
C12-3,4,4',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

EPA Method 1668C 59 April 2010

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
Labeled
analog
Congener CAS Registry congener CAS Registry
CB congener name1 number number Labeled analog name number number
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 13
C12-2,2',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
13
2,2',4,6,6'-PeCB 104 56558-16-8 C12-2,2',4,6,6'-PeCB2 104L 234432-89-4
2,3,3',4,4'-PeCB3,6 105 32598-14-4 13
C12-2,3,3',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
13
2,3,3',5,5'-PeCB 111 39635-32-0 C12-2,3,3',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 13
C12-2,3,4,4',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 13
C12-2,3',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 13
C12-2',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 13
C12-3,3',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

EPA Method 1668C 60 April 2010

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
Labeled
analog
Congener CAS Registry congener CAS Registry
CB congener name1 number number Labeled analog name number number
2,2',3,4,4',5-HxCB 137 35694-06-5
2,2',3,4,4',5'-HxCB3 138 35065-28-2 13
C12-2,2',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
13
2,2',4,4',6,6'-HxCB 155 33979-03-2 C12-2,2',4,4',6,6'-HxCB2 155L 234432-90-7
2,3,3',4,4',5-HxCB6 156 38380-08-4 13
C12-2,3,3',4,4',5-HxCB7 156L 208263-68-7
2,3,3',4,4',5'-HxCB6 157 69782-90-7 13
C12-2,3,3',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 13
C12-2,3',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 13
C12-3,3',4,4',5,5'-HxCB2,7 169L 208263-70-1
2,2',3,3',4,4',5-HpCB3 170 35065-30-6 13
C12-2,2',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
13
2,2',3,3',5,5',6-HpCB 178 52663-67-9 C12-2,2',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 13
C12-2,2',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

EPA Method 1668C 61 April 2010

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
Labeled
analog
Congener CAS Registry congener CAS Registry
CB congener name1 number number Labeled analog name number number
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
13
2,2',3,4',5,6,6'-HpCB 188 74487-85-7 C12-2,2',3,4',5,6,6'-HpCB2 188L 234432-91-8
2,3,3',4,4',5,5'-HpCB6 189 39635-31-9 13
C12-2,3,3',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
13
2,2',3,3',4,4',5,5'-OcCB 194 35694-08-7 C12-2,2',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
13
2,2',3,3',5,5',6,6'-OcCB 202 2136-99-4 C12-2,2',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
13
2,3,3',4,4',5,5',6-OcCB 205 74472-53-0 C12-2,3,3',4,4',5,5',6-OcCB2 205L 234446-64-1
2,2',3,3',4,4',5,5',6-NoCB3 206 40186-72-9 13
C12-2,2',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
13
2,2',3,3',4,5,5',6,6'-NoCB 208 52663-77-1 C12-2,2',3,3',4,5,5',6,6'-NoCB2 208L 234432-92-9
DeCB3 209 2051-24-3 13
C12-DeCB2 209L 105600-27-9

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

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

EPA Method 1668C 62 April 2010

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.

Detection limits and minimum levels -

Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
13
Compounds using 9L ( C12-2,5-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
2 12 15L 23:06 0.9865 0.9843-0.9886 6 4L/15L 19 50 1.9 5 2.5
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

EPA Method 1668C 63 April 2010

 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
3 26 19L 25:48 1.2709 1.2668-1.2750 10 19L/37L 12 50 1.2 5 2.5
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
3 20 37L 26:49 0.8604 0.8578-0.8631 10 19L/37L 22 50 2.2 5 2.5
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
3 33 37L 27:01 0.8668 0.8642-0.8695 10 19L/37L 21 50 2.1 5 2.5
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
EPA Method 1668C 64 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
4 53 54L 26:09 1.0972 1.0937-1.1007 10 54L/81L/77L 25 100 2.5 10 5
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
4 51 54L 26:58 1.1315 1.1280-1.1350 10 54L/81L/77L 22 50 2.2 5 2.5
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
4 49 54L 29:16 1.2280 1.2245-1.2315 10 54L/81L/77L 26 100 2.6 10 5
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
4 47 54L 29:50 1.2517 1.2483-1.2552 10 54L/81L/77L
40 100 4.0 10 5
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
4 75 54L 30:08 1.2643 1.2608-1.2678 10 54L/81L/77L
37 100 3.7 10 5
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
4 71 54L 30:58 1.2993 1.2958-1.3028 10 54L/81L/77L
42 100 4.2 10 5
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
EPA Method 1668C 65 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
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 59 200 5.9 20 10
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
13
Compounds using 101L ( C12-2,2',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
EPA Method 1668C 66 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
5 91 104L 33:55 1.1394 1.1366-1.1422 10 104L/123L/114L/118L/105L 22 50 2.2 5 2.5
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
5 90 123L 36:03 0.8789 0.8769-0.8809 10 104L/123L/114L/118L/105L
47 200 4.7 20 10
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
5 99 123L 36:41 0.8944 0.8923-0.8964 10 104L/123L/114L/118L/105L 29 100 2.9 10 5
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
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 74 200 7.4 20 10
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
5 116 123L 38:02 0.9273 0.9248-0.9297 12 104L/123L/114L/118L/105L
38 100 3.8 10 5
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
EPA Method 1668C 67 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
5 124 123L 40:40 0.9915 0.9894-0.9935 10 104L/123L/114L/118L/105L 29 100 2.9 10 5
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
13
Compounds using 138L ( C12-2,2',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
EPA Method 1668C 68 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
6 135 155L 39:17 1.0993 1.0970-1.1017 10 155L/156L/157L/167L/169L
46 100 4.6 10 5
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
6 149 155L 40:12 1.1250 1.1227-1.1273 10 155L/156L/157L/167L/169L 35 100 3.5 10 5
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
6 143 155L 40:30 1.1334 1.1311-1.1357 10 155L/156L/157L/167L/169L 33 100 3.3 10 5
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
6 140 155L 40:48 1.1418 1.1395-1.1441 10 155L/156L/157L/167L/169L 29 100 2.9 10 5
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
6 168 167L 43:21 0.9066 0.9048-0.9083 10 155L/156L/157L/167L/169L 30 100 3.0 10 5
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
6 163 167L 44:42 0.9348 0.9324-0.9373 14 155L/156L/157L/167L/169L 63 200 6.3 20 10
6 129 167L 44:47 0.9366 0.9341-0.9390 14 155L/156L/157L/167L/169L
EPA Method 1668C 69 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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
6 128 167L 46:09 0.9651 0.9634-0.9669 10 155L/156L/157L/167L/169L 29 100 2.9 10 5
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
6 157 156L/157L 49:09 1.0007 0.9990-1.0024 10 156L/157L 23 100 2.3 10 5
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
13
Compounds using 194L( C12-2,2',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
EPA Method 1668C 70 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
7 183 188L 46:42 1.1159 1.1147-1.1171 6 188L/189L
7 185 188L 46:53 1.1203 1.1191-1.1215 6 188L/189L 28 100 2.8 10 5
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
7 173 188L 48:11 1.1513 1.1501-1.1525 6 188L/189L 30 100 3.0 10 5
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
7 180 189L11 50:27 0.9156 0.9147-0.9165 6 188L/189L11 30 100 3.0 10 5
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
8 200 202L 49:40 1.0452 1.0442-1.0463 6 202L/205L 43 100 4.3 10 5
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
8 199 202L 52:32 1.1056 1.1045-1.1066 6 202L/205L 37 100 3.7 10 5
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
EPA Method 1668C 71 April 2010
 Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
Cl Window (pg/L) (ng/kg) (pg/µL)
No. 1 Congener No. 2,3 RT Ref4 RT5 RRT6 RRT limits7 (sec) 8 Quantitation reference9 MDL ML MDL ML ML
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.
EPA Method 1668C 72 April 2010
 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.

EPA Method 1668C 73 April 2010

 Table 3. Concentrations of Native and Labeled Chlorinated Biphenyls in Stock Solutions
Spiking Solutions
Solution Concentrations
CB Congener 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
EPA Method 1668C 74 April 2010
 Table 3. Concentrations of Native and Labeled Chlorinated Biphenyls in Stock Solutions
Spiking Solutions
Solution Concentrations
CB Congener Stock (µg/mL) Spiking (ng/mL) Extract (ng/mL)
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

EPA Method 1668C 75 April 2010

 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

EPA Method 1668C 76 April 2010

 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
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.

EPA Method 1668C 77 April 2010

Table 5. Concentration of Congeners in Calibration and Calibration Verification Standards
Congener Solution Concentration (ng/mL)
Congener Name No.1 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
13
C12-2-MoCB 1L 100 100 100 100 100 100
13
C12-4-MoCB 3L 100 100 100 100 100 100
13
C12-2,2'-DiCB 4L 100 100 100 100 100 100
13
C12-4,4'-DiCB 15L 100 100 100 100 100 100
13
C12-2,2',6'-TrCB 19L 100 100 100 100 100 100
13
C12-3,4,4'-TrCB 37L 100 100 100 100 100 100
13
C12-2,2',6,6'-TeCB 54L 100 100 100 100 100 100
13
C12-3,3',4,4'-TeCB 77L 100 100 100 100 100 100
13
C12-3,4,4',5-TeCB 81L 100 100 100 100 100 100
13
C12-2,2',4,6,6'-PeCB 104L 100 100 100 100 100 100
13
C12-2,3,3',4,4'-PeCB 105L 100 100 100 100 100 100
13
C12-2,3,4,4',5-PeCB 114L 100 100 100 100 100 100
13
C12-2,3',4,4',5-PeCB 118L 100 100 100 100 100 100
13
C12-2',3,4,4',5-PeCB 123L 100 100 100 100 100 100
13
C12-3,3',4,4',5-PeCB 126L 100 100 100 100 100 100
13
C12-2,2',4,4',6,6'-HxCB 155L 100 100 100 100 100 100
13
C12-2,3,3',4,4',5-HxCB 156L 100 100 100 100 100 100
13
C12-2,3,3',4,4',5'-HxCB 157L 100 100 100 100 100 100
13
C12-2,3',4,4',5,5'-HxCB 167L 100 100 100 100 100 100
13
C12-3,3',4,4',5,5'-HxCB 169L 100 100 100 100 100 100

EPA Method 1668C 78 April 2010

 Table 5. Concentration of Congeners in Calibration and Calibration Verification Standards
Congener Solution Concentration (ng/mL)
Congener Name No.1 CS-0.2 (Hi sens)2 CS-1 CS-2 CS-3 (VER) CS-4 CS-5
13
C12-2,2',3,4',5,6,6'-HpCB 188L 100 100 100 100 100 100
13
C12-2,3,3',4,4',5,5'-HpCB 189L 100 100 100 100 100 100
13
C12-2,2',3,3',5,5',6,6'-OcCB 202L 100 100 100 100 100 100
13
C12-2,3,3',4,4',5,5',6-OcCB 205L 100 100 100 100 100 100
13
C12-2,2',3,3',4,4',5,5',6-NoCB 206L 100 100 100 100 100 100
13
C12-2,2',3,3',4',5,5',6,6'-NoCB 208L 100 100 100 100 100 100
13
C12-DeCB 209L 100 100 100 100 100 100
Labeled clean-up
13
C12-2,4,4'-TrCB 28L 100 100 100 100 100 100
13
C12-2,3,3',5,5'-PeCB 111L 100 100 100 100 100 100
13
C12-2,2',3,3',5,5',6-HpCB 178L 100 100 100 100 100 100
Labeled injection internal
13
C12-2,5-DiCB 9L 100 100 100 100 100 100
13
C12-2,2',5,5'-TeCB 52L 100 100 100 100 100 100
13
C12-2,2',4',5,5'-PeCB 101L 100 100 100 100 100 100
13
C12-2,2',3',4,4',5'-HxCB 138L 100 100 100 100 100 100
13
C12-2,2',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.

EPA Method 1668C 79 April 2010

 Table 6. QC Acceptance Criteria for VER, IPR, OPR, and Labeled Compounds in Samples1,2
Congener IPR Labeled Compound
Congener Name No.3 Test Conc. (ng/mL)4 VER (%)5 RSD (%) Mean Recovery (%) OPR Recovery (%) Recovery in Samples (%)
2-MoCB 1 50 75 - 125 25 70 - 130 60 - 135
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 NA
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
13
C12-2-MoCB 1L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-4-MoCB 3L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-2,2'-DiCB 4L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-4,4'-DiCB 15L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-2,2',6-TrCB 19L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-3,4,4'-TrCB 37L 100 50 - 145 70 20 - 135 15 - 145 5 - 145
13
C12-2,2',6,6'-TeCB 54L 100 50 - 145 70 20 - 135 15 - 145 5 - 145

EPA Method 1668C 80 April 2010

 Table 6. QC Acceptance Criteria for VER, IPR, OPR, and Labeled Compounds in Samples1,2
Congener IPR Labeled Compound
Congener Name No.3 Test Conc. (ng/mL)4 VER (%)5 RSD (%) Mean Recovery (%) OPR Recovery (%) Recovery in Samples (%)
13
C12-3,3',4,4'-TeCB 77L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-3,4,4',5-TeCB 81L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',4,6,6'-PeCB 104L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3,3',4,4'-PeCB 105L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3,4,4',5-PeCB 114L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3',4,4',5-PeCB 118L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2',3,4,4',5-PeCB 123L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-3,3',4,4',5-PeCB 126L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',4,4',6,6'-HxCB 155L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3,3',4,4',5 -HxCB 6 156L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3,3',4,4',5'-HxCB 6 157L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3',4,4',5,5'-HxCB 167L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-3,3',4,4',5,5'-HxCB 169L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',3,4',5,6,6'-HpCB 188L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2',3,3',4,4',5,5'-HpCB 189L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',3,3',5,5',6,6'-OcCB 202L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,3,3',4,4',5,5',6-OcCB 205L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',3,3',4,4',5,5',6-NoCB 206L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',3,3',4,5,5',6,6'-NoCB 208L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',3,3',4,4',5,5',6,6'-DeCB 209L 100 50 - 145 50 45 - 135 40 - 145 10 - 145
Cleanup standards
13
C12-2,4,4'-TrCB 28L 100 65 - 135 70 20 - 135 15 - 145 5 - 145
13
C12-2,3,3',5,5'-PeCB 111L 100 75 - 125 50 45 - 135 40 - 145 10 - 145
13
C12-2,2',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

EPA Method 1668C 81 April 2010

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
12
188.0393 M C12 H9 35Cl Cl-1 CB
12
190.0363 M+2 C12 H9 37Cl Cl-1 CB
13
Fn-1; Cl-1 200.0795 M C12 H9 35Cl 13
C12 Cl-1 CB
13
202.0766 M+2 C12 H9 37Cl 13
C12 Cl-1 CB
218.9856 lock C4 F9 PFK
12
222.0003 M C12 H8 35Cl2 Cl-2 PCB
223.9974 (2) M+2 12
C12 H8 35Cl 37 Cl Cl-2 PCB
12
225.9944 M+4 C12 H8 37Cl2 Cl-2 PCB
13
234.0406 M C12 H8 35Cl2 13
C12 Cl-2 PCB
13
236.0376 M+2 C12 H8 35Cl 37 Cl 13
C12 Cl-2 PCB
Fn-2; Cl-2, 3
242.9856 lock C6 F9 PFK
12
255.9613 M C12 H7 35Cl3 Cl-3 PCB
12
257.9584 M+2 C12 H7 35Cl2 37Cl Cl-3 PCB
13
268.0016 M C12 H7 35Cl3 13
C12 Cl-3 PCB
13
269.9986 M+2 C12 H7 35Cl2 37Cl 13
C12 Cl-3 PCB
12
255.9613 M C12 H7 35Cl3 Cl-3 PCB
12
257.9584 M+2 C12 H7 35Cl2 37Cl Cl-3 PCB
12
259.9554 M+4 C12 H7 35Cl 37Cl2 Cl-3 PCB
13
268.0016 M C12 H7 35Cl3 13
C12 Cl-3 PCB
13
269.9986 M+2 C12 H7 35Cl2 37Cl 13
C12 Cl-3 PCB
280.9825 lock C6 F11 PFK
12
289.9224 M C12 H6 35Cl4 Cl-4 PCB
12
291.9194 M+2 C12 H6 35Cl3 37Cl Cl-4 PCB
Fn-3; Cl-3, 4, 5 12
293.9165 M+4 C12 H6 35Cl2 37Cl2 Cl-4 PCB
13
301.9626 M C12 H6 35Cl4 13
C12 Cl-4 PCB
13
303.9597 M+2 C12 H6 35Cl3 37Cl 13
C12 Cl-4 PCB
13
323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB
12
289.9224 M C12 H6 35Cl4 Cl-4 PCB
12
291.9194 M+2 C12 H6 35Cl3 37Cl Cl-4 PCB
12
293.9165 M+4 C12 H6 35Cl2 37Cl2 Cl-4 PCB
13
301.9626 M C12 H6 35Cl3 37Cl 13
C12 Cl-4 PCB
13
303.9597 M+2 C12 H6 35Cl2 37Cl2 13
C12 Cl-4 PCB
12
Fn-4; Cl-4, 5, 6 323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
330.9792 lock C7 F15 PFK
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB

EPA Method 1668C 82 April 2010

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
12
359.8415 M+2 C12 H4 35Cl5 37Cl Cl-6 PCB
12
361.8385 M+4 C12 H4 35Cl4 37Cl2 Cl-6 PCB
12
Fn-4; Cl-4, 5, 6 363.8356 M+6 C12 H4 35Cl3 37Cl3 Cl-6 PCB
13
371.8817 M+2 C12 H4 35Cl5 37Cl 13
C12 Cl-6 PCB
13
373.8788 M+4 C12 H4 35Cl4 37Cl2 13
C12 Cl-6 PCB
12
323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB
354.9792 lock C9 F13 PFK
12
359.8415 M+2 C12 H4 35Cl5 37Cl Cl-6 PCB
12
361.8385 M+4 C12 H4 35Cl4 37Cl2 Cl-6 PCB
12
Fn-5; Cl-5, 6, 7 363.8356 M+6 C12 H4 35Cl3 37Cl3 Cl-6 PCB
13
371.8817 M+2 C12 H4 35Cl5 37Cl 13
C12 Cl-6 PCB
13
373.8788 M+4 C12 H4 35Cl4 37Cl2 13
C12 Cl-6 PCB
12
393.8025 M+2 C12 H3 35Cl6 37Cl Cl-7 PCB
12
395.7995 M+4 C12 H3 35Cl5 37Cl2 Cl-7 PCB
12
397.7966 M+6 C12 H3 35Cl4 37Cl3 Cl-7 PCB
13
405.8428 M+2 C12 H3 35Cl6 37Cl 13
C12 Cl-7 PCB
13
407.8398 M+4 C12 H3 35Cl5 37Cl2 13
C12 Cl-7 PCB
454.9728 QC C11 F17 PFK
12
393.8025 M+2 C12 H3 35Cl6 37Cl Cl-7 PCB
12
395.7995 M+4 C12 H3 35Cl5 37Cl2 Cl-7 PCB
12
397.7966 M+6 C12 H3 35Cl4 37Cl3 Cl-7 PCB
13
405.8428 M+2 C12 H3 35Cl6 37Cl 13
C12 Cl-7 PCB
13
407.8398 M+4 C12 H3 35Cl5 37Cl2 13
C12 Cl-7 PCB
12
427.7635 M+2 C12 H2 35Cl7 37Cl Cl-8 PCB
12
429.7606 M+4 C12 H2 35Cl6 37Cl2 Cl-8 PCB
12
431.7576 M+6 C12 H2 35Cl5 37Cl3 Cl-8 PCB
13
439.8038 M+2 C12 H2 35Cl7 37Cl 13
C12 Cl-8 PCB
13
441.8008 M+4 C12 H2 35Cl6 37Cl2 13
C12 Cl-8 PCB
Fn-6; Cl-7, 8, 9, 10
442.9728 QC C10 F13 PFK
454.9728 lock C11 F13 PFK
12
461.7246 M+2 C12 H1 35Cl8 37Cl Cl-9 PCB
12
463.7216 M+4 C12 H1 35Cl7 37Cl2 Cl-9 PCB
12
465.7187 M+6 C12 H1 35Cl6 37Cl3 Cl-9 PCB
13
473.7648 M+2 C12 H1 35Cl8 37Cl 13
C12 Cl-9 PCB
13
475.7619 M+4 C12 H1 35Cl7 37Cl2 13
C12 Cl-9 PCB
12
495.6856 M+2 C12 H4 35Cl9 37Cl Cl-10 PCB
12
497.6826 M+4 C12 35Cl8 37Cl2 Cl-10 PCB
12
499.6797 M+6 C12 35Cl7 37Cl3 Cl-10 PCB

EPA Method 1668C 83 April 2010

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
13
507.7258 M+2 C12 35Cl9 37Cl 13
C12 Cl-10 PCB
13
Fn-6; Cl-7, 8, 9, 10 509.7229 M+4 C12 35Cl8 37Cl2 13
C12 Cl-10 PCB
13
511.7199 M+6 C12 35Cl7 37Cl3 13
C12 Cl-10 PCB

1. Isotopic masses used for accurate mass calculation

1
H 1.0078
12
C 12.0000
13
C 13.0034
35
Cl 34.9689
37
Cl 36.9659
19
F 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.

EPA Method 1668C 84 April 2010

 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

EPA Method 1668C 85 April 2010

 Table 9. Suggested Sample Quantities to be Extracted for Various Matrices1
Sample Matrix2 Example Percent Solids Phase Quantity Extracted
Single-phase
Drinking water
Aqueous Groundwater <1 –3 1000 mL
Treated wastewater
Dry soil
Solid Compost >20 Solid 10 g
Ash
Waste solvent
Organic Waste oil <1 Organic 10 g
Organic polymer
Fish
Tissue – Organic 10 g
Human adipose
Multi-phase - Liquid/Solid
Wet soil
Untreated effluent
Aqueous/Solid Digested municipal sludge 1-30 Solid 10 g
Filter cake
Paper pulp
Industrial sludge
Organic/solid 1-100 Both 10 g
Oily waste
Multi-phase - Liquid/Liquid
In-process effluent
Aqueous/organic Untreated effluent <1 Organic 10 g
Drum waste
Multi-phase - Liquid/Liquid/Solid
Untreated effluent Organic
Aqueous/organic/solid >1 10 g
Drum waste and solid

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.

EPA Method 1668C 86 April 2010

EPA Method 1668C 87 April 2010
EPA Method 1668C 88 April 2010
EPA Method 1668C 89 April 2010
 Figure 4. Solid-phase Extraction Apparatus

EPA Method 1668C 90 April 2010

 Figure 5. Soxhlet/Dean Stark Extractor

EPA Method 1668C 91 April 2010

EPA Method 1668C 92 April 2010
EPA Method 1668C 93 April 2010
EPA Method 1668C 94 April 2010
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.

EPA Method 1668C 95 April 2010

 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
13
C12- labeled analogs of the chlorinated biphenyls. The 13C12-labeled CBs are spiked into each

EPA Method 1668C 96 April 2010

 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

EPA Method 1668C 97 April 2010

 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

EPA Method 1668C 98 April 2010

 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

EPA Method 1668C 99 April 2010

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.

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.

EPA Method 1668C 100 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
C12-2-MoCB4 1L 13
C12-4-MoCB4,5 3L 09:17 0.8855 0.8776-0.8935
13
2-MoCB 1 C12-2-MoCB4 1L 09:17 1.0000 0.9964-1.0072
13
3-MoCB 2 C12-4-MoCB4,5 3L 10:22 0.9889 0.9809-0.9968
13
C12-4-MoCB4,5 3L 13
C12-2,2',5,5'-TeCB7 52L 10:29 0.5561 0.5473-0.5650
13
4-MoCB 3 C12-4-MoCB4,5 3L 10:29 1.0000 0.9968-1.0064
13
C12-2,2'-DiCB4 4L 13
C12-4,4'-DiCB4,5 15L 11:08 0.7591 0.7477-0.7705
13
2,2'-DiCB 4 C12-2,2'-DiCB4 4L 11:08 1.0000 0.9925-1.0075
13
2,6-DiCB 10 C12-4,4'-DiCB4,5 15L 11:10 0.7614 0.7500-0.7727
13
2,5-DiCB 9 C12-4,4'-DiCB4,5 15L 12:08 0.8273 0.8216-0.8330
13
2,4-DiCB 7 C12-4,4'-DiCB4,5 15L 12:09 0.8284 0.8227-0.8341
13
2,3'-DiCB 6 C12-4,4'-DiCB4,5 15L 12:31 0.8534 0.8477-0.8591
2,4'-DiCB6 8 13
C12-4,4'-DiCB4,5 15L 12:43 0.8670 0.8614-0.8727
13
2,3-DiCB 5 C12-4,4'-DiCB4,5 15L 12:46 0.8705 0.8648-0.8761
13
C12-2,2',6-TrCB4 19L 13
C12-2,4,4'-TrCB5 28L 13:31 0.7990 0.7892-0.8089
13
3,5-DiCB 14 C12-4,4'-DiCB4,5 15L 13:36 0.9273 0.9216-0.9330
13
2,4,6-TrCB 30 C12-2,4,4'-TrCB5 28L 14:06 0.8335 0.8286-0.8384
13
3,3'-DiCB 11 C12-4,4'-DiCB4,5 15L 14:11 0.9670 0.9614-0.9727
13
3,4'-DiCB 13 C12-4,4'-DiCB4,5 15L 14:26 0.9841 0.9784-0.9898
13
3,4-DiCB 12 C12-4,4'-DiCB4,5 15L 14:27 0.9852 0.9795-0.9909
2,2',5-TrCB6 18 13
C12-2,4,4'-TrCB5 28L 14:36 0.8631 0.8581-0.8680
13
C12-4,4'-DiCB4,5 15L 13
C12-2,2',5,5'-TeCB7 52L 14:40 0.7781 0.7692-0.7869
13
4,4'-DiCB 15 C12-4,4'-DiCB4,5 15L 14:40 1.0000 0.9977-1.0043
13
2,2',4-TrCB 17 C12-2,4,4'-TrCB5 28L 14:43 0.8700 0.8650-0.8749
13
2,3',6-TrCB 27 C12-2,4,4'-TrCB5 28L 15:06 0.8926 0.8877-0.8975
13
2,3,6-TrCB 24 C12-2,4,4'-TrCB5 28L 15:06 0.8926 0.8877-0.8975
13
2,2',3-TrCB 16 C12-2,4,4'-TrCB5 28L 15:26 0.9123 0.9074-0.9172
13
2,4',6-TrCB 32 C12-2,4,4'-TrCB5 28L 15:29 0.9153 0.9103-0.9202
13
C12-2,2',6,6'-TeCB4 54L 13
C12-3,3',4,4'-TeCB4,5,9 77L 16:02 0.6139 0.6075-0.6203
13
2,2',6,6'-TeCB 54 C12-2,2',6,6'-TeCB4 54L 16:02 1.0000 0.9979-1.0042
13
2',3,5-TrCB 34 C12-2,4,4'-TrCB5 28L 16:03 0.9488 0.9438-0.9537
13
2,3,5-TrCB 23 C12-2,4,4'-TrCB5 28L 16:07 0.9527 0.9478-0.9576
13
2,4,5-TrCB 29 C12-2,4,4'-TrCB5 28L 16:18 0.9635 0.9586-0.9685

EPA Method 1668C 101 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
2,3',5-TrCB 26 C12-2,4,4'-TrCB5 28L 16:29 0.9744 0.9695-0.9793
13
2,3',4-TrCB 25 C12-2,4,4'-TrCB5 28L 16:36 0.9813 0.9764-0.9862
13
2,4',5-TrCB 31 C12-2,4,4'-TrCB5 28L 16:52 0.9970 0.9921-1.0020
13
C12-2,4,4'-TrCB5 28L 13
C12-2,2',5,5'-TeCB7 52L 16:55 0.8974 0.8930-0.9019
2,4,4'-TrCB6 28 13
C12-2,4,4'-TrCB5 28L 16:55 1.0000 0.9980-1.0039
13
2,2',4,6-TeCB 50 C12-3,3',4,4'-TeCB4,5,9 77L 16:55 0.6477 0.6414-0.6541
13
2,3,4-TrCB 21 C12-2,4,4'-TrCB5 28L 17:21 1.0256 1.0207-1.0305
13
2,2',5,6'-TeCB 53 C12-3,3',4,4'-TeCB4,5,9 77L 17:26 0.6675 0.6611-0.6739
13
2,3,3'-TrCB 20 C12-2,4,4'-TrCB5 28L 17:22 1.0266 1.0217-1.0315
13
2',3,4-TrCB 33 C12-2,4,4'-TrCB5 28L 17:24 1.0286 1.0236-1.0335
13
2,2',4,6'-TeCB 51 C12-3,3',4,4'-TeCB4,5,9 77L 17:42 0.6777 0.6713-0.6841
13
2,3,4'-TrCB 22 C12-2,4,4'-TrCB5 28L 17:43 1.0473 1.0424-1.0522
13
2,2',3,6-TeCB 45 C12-3,3',4,4'-TeCB4,5,9 77L 18:00 0.6892 0.6828-0.6956
13
3,3',5-TrCB 36 C12-2,4,4'-TrCB5 28L 18:16 1.0798 1.0749-1.0847
13
2,2',3,6'-TeCB 46 C12-3,3',4,4'-TeCB4,5,9 77L 18:24 0.7045 0.6981-0.7109
13
3,4',5-TrCB 39 C12-2,4,4'-TrCB5 28L 18:37 1.1005 1.0956-1.1054
13
C12-2,2',5,5'-TeCB7 52L 13
C12-2,2',5,5'-TeCB7 52L 18:51 1.0000 0.9956-1.0044
2,2',5,5'-TeCB6 52 13
C12-3,3',4,4'-TeCB4,5,9 77L 18:51 0.7218 0.7154-0.7281
13
2,3',4,6-TeCB 69 C12-3,3',4,4'-TeCB4,5,9 77L 18:52 0.7224 0.7160-0.7288
13
2,3',5',6-TeCB 73 C12-3,3',4,4'-TeCB4,5,9 77L 18:57 0.7256 0.7192-0.7320
13
2,2',4,5'-TeCB 49 C12-3,3',4,4'-TeCB4,5,9 77L 19:00 0.7275 0.7211-0.7339
13
2,2',3,5-TeCB 43 C12-3,3',4,4'-TeCB4,5,9 77L 19:04 0.7301 0.7237-0.7364
13
3,4,5-TrCB 38 C12-2,4,4'-TrCB5 28L 19:12 1.1350 1.1300-1.1399
13
2,2',4,4'-TeCB 47 C12-3,3',4,4'-TeCB4,5,9 77L 19:15 0.7371 0.7307-0.7435
13
2,4,4',6-TeCB 75 C12-3,3',4,4'-TeCB4,5,9 77L 19:20 0.7403 0.7339-0.7466
13
2,2',4,5-TeCB 48 C12-3,3',4,4'-TeCB4,5,9 77L 19:20 0.7403 0.7339-0.7466
13
2,3,5,6-TeCB 65 C12-3,3',4,4'-TeCB4,5,9 77L 19:31 0.7473 0.7409-0.7537
13
2,3,4,6-TeCB 62 C12-3,3',4,4'-TeCB4,5,9 77L 19:36 0.7505 0.7441-0.7569
13
3,3',4-TrCB 35 C12-2,4,4'-TrCB5 28L 19:41 1.1635 1.1586-1.1685
13
C12-2,2',4,6,6'-PeCB4 104L 13
C12-2,3',4,4',5-PeCB5,9 118L 19:45 0.7037 0.6977-0.7096
13
2,2',4,6,6'-PeCB 104 C12-2,2',4,6,6'-PeCB4 104L 19:45 1.0000 0.9983-1.0034
2,2',3,5'-TeCB6 44 13
C12-3,3',4,4'-TeCB4,5,9 77L 19:55 0.7626 0.7562-0.7690

EPA Method 1668C 102 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
C12-3,4,4'-TrCB4 37L 13
C12-2,4,4'-TrCB5 28L 20:03 1.1852 1.1803-1.1901
13
3,4,4'-TrCB 37 C12-3,4,4'-TrCB4 37L 20:03 1.0000 0.9983-1.0033
13
2,3,3',6-TeCB 59 C12-3,3',4,4'-TeCB4,5,9 77L 20:05 0.7690 0.7626-0.7754
13
2,2',3,4'-TeCB 42 C12-3,3',4,4'-TeCB4,5,9 77L 20:07 0.7703 0.7639-0.7766
13
2,3',5,5'-TeCB 72 C12-3,3',4,4'-TeCB4,5,9 77L 20:36 0.7888 0.7824-0.7951
13
2,3',4',6-TeCB 71 C12-3,3',4,4'-TeCB4,5,9 77L 20:36 0.7888 0.7824-0.7951
13
2,3,4',6-TeCB 64 C12-3,3',4,4'-TeCB4,5,9 77L 20:37 0.7894 0.7830-0.7958
13
2,2',3,4-TeCB 41 C12-3,3',4,4'-TeCB4,5,9 77L 20:39 0.7907 0.7843-0.7971
13
2,2',3,6,6'-PeCB 96 C12-2,3',4,4',5-PeCB5,9 118L 20:48 0.7411 0.7352-0.7470
13
2,3',4,5'-TeCB 68 C12-3,3',4,4'-TeCB4,5,9 77L 20:52 0.7990 0.7926-0.8054
13
2,2',3,3'-TeCB 40 C12-3,3',4,4'-TeCB4,5,9 77L 20:58 0.8028 0.7996-0.8060
13
2,3,3',5-TeCB 57 C12-3,3',4,4'-TeCB4,5,9 77L 21:21 0.8175 0.8143-0.8207
13
2,2',4,5,'6-PeCB 103 C12-2,3',4,4',5-PeCB5,9 118L 21:22 0.7613 0.7553-0.7672
13
2,3',4,5-TeCB 67 C12-3,3',4,4'-TeCB4,5,9 77L 21:38 0.8283 0.8251-0.8315
13
2,2',4,4',6-PeCB 100 C12-2,3',4,4',5-PeCB5,9 118L 21:41 0.7726 0.7666-0.7785
13
2,3,3',5'-TeCB 58 C12-3,3',4,4'-TeCB4,5,9 77L 21:43 0.8315 0.8283-0.8347
13
2,3,4',5-TeCB 63 C12-3,3',4,4'-TeCB4,5,9 77L 21:51 0.8366 0.8334-0.8398
13
2,2',3,5,6'-PeCB 94 C12-2,3',4,4',5-PeCB5,9 118L 22:05 0.7868 0.7809-0.7928
13
2,4,4',5-TeCB 74 C12-3,3',4,4'-TeCB4,5,9 77L 22:07 0.8468 0.8437-0.8500
13
2,3,4,5-TeCB 61 C12-3,3',4,4'-TeCB4,5,9 77L 22:11 0.8494 0.8462-0.8526
13
2,3',4',5-TeCB 70 C12-3,3',4,4'-TeCB4,5,9 77L 22:20 0.8551 0.8519-0.8583
13
2 ',3,4,5-TeCB 76 C12-3,3',4,4'-TeCB4,5,9 77L 22:25 0.8583 0.8551-0.8615
13
2,2',3',4,6-PeCB 98 C12-2,3',4,4',5-PeCB5,9 118L 22:28 0.8005 0.7975-0.8034
2,3',4,4'-TeCB6 66 13
C12-3,3',4,4'-TeCB4,5,9 77L 22:29 0.8609 0.8577-0.8641
13
2,2',4,5,6'-PeCB 102 C12-2,3',4,4',5-PeCB5,9 118L 22:32 0.8029 0.7999-0.8058
13
2,2',3,5',6-PeCB 95 C12-2,3',4,4',5-PeCB5,9 118L 22:34 0.8040 0.8011-0.8070
13
2,2',3,5,6-PeCB 93 C12-2,3',4,4',5-PeCB5,9 118L 22:36 0.8052 0.8023-0.8082
13
3,3',5,5'-TeCB 80 C12-3,3',4,4'-TeCB4,5,9 77L 22:45 0.8711 0.8679-0.8743
13
2,2',3,4,6-PeCB 88 C12-2,3',4,4',5-PeCB5,9 118L 22:49 0.8129 0.8100-0.8159
13
2,2',3,4',6-PeCB 91 C12-2,3',4,4',5-PeCB5,9 118L 22:55 0.8165 0.8135-0.8195
13
2,3,3',4'-TeCB 55 C12-3,3',4,4'-TeCB4,5,9 77L 22:57 0.8787 0.8756-0.8819
13
2,3',4,5,'6-PeCB 121 C12-2,3',4,4',5-PeCB5,9 118L 23:04 0.8219 0.8189-0.8248

EPA Method 1668C 103 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
2,3,3',4'-TeCB 56 C12-3,3',4,4'-TeCB4,5,9 77L 23:24 0.8960 0.8928-0.8992
13
2,3,4,4'-TeCB 60 C12-3,3',4,4'-TeCB4,5,9 77L 23:24 0.8960 0.8928-0.8992
13
C12-2,2',4,4',6,6'-HxCB4 155L 13
C12-2,3',4,4',5,5'-HxCB5,9 167L 23:43 0.7104 0.7054-0.7154
13
2,2',4,4',6,6'-HxCB 155 C12-2,2',4,4',6,6'-HxCB4 155L 23:43 1.0000 0.9986-1.0028
13
2,2',3,3',6-PeCB 84 C12-2,3',4,4',5-PeCB5,9 118L 23:44 0.8456 0.8426-0.8486
13
2,2',3,5,5'-PeCB 92 C12-2,3',4,4',5-PeCB5,9 118L 23:50 0.8492 0.8462-0.8521
13
2,2',3,4,6'-PeCB 89 C12-2,3',4,4',5-PeCB5,9 118L 23:53 0.8510 0.8480-0.8539
13
2,2',3,4',5-PeCB 90 C12-2,3',4,4',5-PeCB5,9 118L 24:07 0.8593 0.8563-0.8622
13
C12-2,2',4,5,5'-PeCB7 101L 13
C12-2,2',4,5,5'-PeCB7 101L 24:11 1.0000 0.9966-1.0034
2,2',4,5,5'-PeCB6 101 13
C12-2,3',4,4',5-PeCB5,9 118L 24:11 0.8616 0.8587-0.8646
13
2,3,3',5',6-PeCB 113 C12-2,3',4,4',5-PeCB5,9 118L 24:23 0.8688 0.8658-0.8717
13
3,3',4,5'-TeCB 79 C12-3,3',4,4'-TeCB4,5,9 77L 24:27 0.9362 0.9330-0.9394
13
2,2',4,4',5-PeCB 99 C12-2,3',4,4',5-PeCB5,9 118L 24:28 0.8717 0.8688-0.8747
13
2,2',3,4',6,6'-HxCB 150 C12-2,3',4,4',5,5'-HxCB5,9 167L 24:52 0.7449 0.7399-0.7499
13
2,3',4,4',6-PeCB 119 C12-2,3',4,4',5-PeCB5,9 118L 24:54 0.8872 0.8842-0.8901
13
2,3,3',5,6-PeCB 112 C12-2,3',4,4',5-PeCB5,9 118L 25:00 0.8907 0.8878-0.8937
13
2,3,3',4,6-PeCB 109 C12-2,3',4,4',5-PeCB5,9 118L 25:09 0.8961 0.8931-0.8990
13
2,2',3,5,6,6'-HxCB 152 C12-2,3',4,4',5,5'-HxCB5,9 167L 25:17 0.7574 0.7524-0.7624
13
2,2',3,3',5-PeCB 83 C12-2,3',4,4',5-PeCB5,9 118L 25:20 0.8919 0.8890-0.8949
13
2,2',3',4,5-PeCB 97 C12-2,3',4,4',5-PeCB5,9 118L 25:22 0.9038 0.9008-0.9068
13
2,2',3,4,5-PeCB 86 C12-2,3',4,4',5-PeCB5,9 118L 25:27 0.9068 0.9038-0.9097
13
C12-3,4,4',5-TeCB9 81L 13
C12-2,2',5,5'-TeCB7 52L 25:32 1.3546 1.3457-1.3634
3,4,4',5-TeCB10 81 13
C12-3,4,4',5-TeCB4,5,9 77L 25:32 1.0000 0.9987-1.0026
13
2 ',3,4,5,6'-PeCB 125 C12-2,3',4,4',5-PeCB5,9 118L 25:36 0.9121 0.9091-0.9151
13
2,3,4',5,6-PeCB 117 C12-2,3',4,4',5-PeCB5,9 118L 25:37 0.9127 0.9097-0.9157
13
2,2',3,4,5'-PeCB 87 C12-2,3',4,4',5-PeCB5,9 118L 25:38 0.9133 0.9103-0.9163
13
3,3',4,5-TeCB 78 C12-3,3',4,4'-TeCB4,5,9 77L 25:40 0.9598 0.9566-0.9630
13
2,2',3,4,6,6'-HxCB 145 C12-2,3',4,4',5,5'-HxCB5,9 167L 25:42 0.7698 0.7649-0.7748
13
2,3,4,4',6-PeCB 115 C12-2,3',4,4',5-PeCB5,9 118L 25:44 0.9169 0.9139-0.9198
13
C12-2,3,3',5,5'-PeCB8 111L 13
C12-2,2',4,5,5'-PeCB7 101L 25:51 1.0689 1.0655-1.0724
13
2,3,3',5,5'-PeCB 111 C12-2,3',4,4',5-PeCB5,9 118L 25:51 0.9210 0.9181-0.9240
13
2,2',3,4,4'-PeCB 85 C12-2,3',4,4',5-PeCB5,9 118L 25:51 0.9210 0.9181-0.9240

EPA Method 1668C 104 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
2,3,4,5,6-PeCB 116 C12-2,3',4,4',5-PeCB5,9 118L 25:48 0.9192 0.9163-0.9222
13
C12-3,3',4,4'-TeCB4,5,9 77L 13
C12-2,2',5,5'-TeCB7 52L 26:07 1.3855 1.3767-1.3943
3,3',4,4'-TeCB6,10 77 13
C12-3,3',4,4'-TeCB4,5,9 77L 26:07 1.0000 0.9987-1.0026
13
2,2',3,3',6,6'-HxCB 136 C12-2,3',4,4',5,5'-HxCB5,9 167L 26:10 0.7793 0.7743-0.7843
13
2,3',4,5,5'-PeCB 120 C12-2,3',4,4',5-PeCB5,9 118L 26:12 0.9335 0.9305-0.9365
13
2,2',3,4',5,6'-HxCB 148 C12-2,3',4,4',5,5'-HxCB5,9 167L 26:14 0.7858 0.7808-0.7908
13
2,3,3',4',6-PeCB 110 C12-2,3',4,4',5-PeCB5,9 118L 26:16 0.9359 0.9329-0.9388
13
2,2',4,4',5,6'-HxCB 154 C12-2,3',4,4',5,5'-HxCB5,9 167L 26:44 0.8008 0.7983-0.8033
13
2,2',3,3',4-PeCB 82 C12-2,3',4,4',5-PeCB5,9 118L 26:48 0.9549 0.9519-0.9578
13
2,2',3,5,5',6-HxCB 151 C12-2,3',4,4',5,5'-HxCB5,9 167L 27:18 0.8178 0.8153-0.8203
13
2,2',3,3',5,6'-HxCB 135 C12-2,3',4,4',5,5'-HxCB5,9 167L 27:31 0.8243 0.8218-0.8268
13
2 ',3,4,5,5'-PeCB 124 C12-2,3',4,4',5-PeCB5,9 118L 27:36 0.9834 0.9804-0.9863
13
2,2',3,4,5',6-HxCB 144 C12-2,3',4,4',5,5'-HxCB5,9 167L 27:38 0.8278 0.8253-0.8303
13
2,3,3',4,5'-PeCB 108 C12-2,3',4,4',5-PeCB5,9 118L 27:40 0.9857 0.9828-0.9887
13
2,2',3,4',5,6-HxCB 147 C12-2,3',4,4',5,5'-HxCB5,9 167L 27:44 0.8308 0.8283-0.8333
13
2,3,3',4',5-PeCB 107 C12-2,3',4,4',5-PeCB5,9 118L 27:45 0.9887 0.9857-0.9917
13
2,2',3,4',5',6-HxCB 149 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:01 0.8392 0.8367-0.8417
13
2,2',3,3',5,6-HxCB 134 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:35 0.8562 0.8537-0.8587
13
2,2',3,4,5,6'-HxCB 143 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:34 0.8557 0.8532-0.8582
13
C12-2',3,4,4',5-PeCB9 123L 13
C12-2,2',4,5,5'-PeCB7 101L 27:53 1.1530 1.1496-1.1564
2',3,4,4',5-PeCB10 123 13
C12-2',3,4,4',5-PeCB9 123L 27:53 1.0000 0.9988-1.0024
13
2,2',3,4,4',6-HxCB 139 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:01 0.8392 0.8367-0.8417
13
2,3,3',4,5-PeCB 106 C12-2,3',4,4',5-PeCB5,9 118L 28:04 1.0000 0.9970-1.0030
13
C12-2,3',4,4',5-PeCB5,9 118L 13
C12-2,2',4,5,5'-PeCB7 101L 28:04 1.1606 1.1571-1.1640
2,3',4,4',5-PeCB6,10 118 13
C12-2,3',4,4',5-PeCB5,9 118L 28:04 1.0000 0.9988-1.0024
13
2,2',3,4,4',6'-HxCB 140 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:12 0.8447 0.8422-0.8472
13
C12-2,3,4,4',5-PeCB9 114L 13
C12-2,2',4,5,5'-PeCB7 101L 28:38 1.1840 1.1806-1.1875
2,3,4,4',5-PeCB10 114 13
C12-2,3,4,4',5-PeCB9 114L 28:38 1.0000 0.9988-1.0023
13
2',3,3',4,5-PeCB 122 C12-2,3',4,4',5-PeCB5,9 118L 28:48 1.0261 1.0232-1.0291
13
2,2',3,3',4,6-HxCB 131 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:52 0.8647 0.8622-0.8672
13
2,2',3,4,5,6-HxCB 142 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:59 0.8682 0.8657-0.8707
13
2,2',3,3',5,5'-HxCB 133 C12-2,3',4,4',5,5'-HxCB5,9 167L 28:59 0.8682 0.8657-0.8707

EPA Method 1668C 105 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
2,2',3,3',4,6'-HxCB 132 C12-2,3',4,4',5,5'-HxCB5,9 167L 29:32 0.8847 0.8822-0.8872
13
2,3,3',5,5',6-HxCB 165 C12-2,3',4,4',5,5'-HxCB5,9 167L 29:21 0.8792 0.8767-0.8817
13
C12-2,2',3,4',5,6,6'-HpCB4 188L 13
C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 29:22 0.9511 0.7327-0.7411
13
2,2',3,4',5,6,6'-HpCB 188 C12-2,2',3,4',5,6,6'-HpCB4 188L 29:22 1.0000 0.9989-1.0023
13
2,2',3,4',5,5'-HxCB 146 C12-2,3',4,4',5,5'-HxCB5,9 167L 29:24 0.8807 0.8782-0.8832
13
C12-2,3,3',4,4'-PeCB9 105L 13
C12-2,2',4,5,5'-PeCB7 101L 29:30 1.2198 1.2130-1.2267
2,3,3',4,4'-PeCB6,10 105 13
C12-2,3,3',4,4'-PeCB9 105L 29:30 1.0000 0.9989-1.0023
13
2,3,3',4,5',6-HxCB 161 C12-2,3',4,4',5,5'-HxCB5,9 167L 29:32 0.8847 0.8822-0.8872
2,2',4,4',5,5'-HxCB6 153 13
C12-2,3',4,4',5,5'-HxCB5,9 167L 29:48 0.8927 0.8902-0.8952
13
2,2',3,4,4',6,6'-HpCB 184 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 29:49 0.7482 0.7440-0.7524
13
3,3',4,5,5'-PeCB 127 C12-2,3',4,4',5-PeCB5,9 118L 29:57 1.0671 1.0641-1.0701
13
2,3',4,4',5',6-HxCB 168 C12-2,3',4,4',5,5'-HxCB5,9 167L 29:59 0.8982 0.8957-0.9006
13
2,2',3,4,5,5'-HxCB 141 C12-2,3',4,4',5,5'-HxCB5,9 167L 30:31 0.9141 0.9116-0.9166
13
2,2',3,3',5,6,6'-HpCB 179 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 30:33 0.7666 0.7624-0.7708
13
2,2',3,4,4',5-HxCB 137 C12-2,3',4,4',5,5'-HxCB5,9 167L 30:51 0.9241 0.9216-0.9266
13
2,2',3,3',4,5'-HxCB 130 C12-2,3',4,4',5,5'-HxCB5,9 167L 30:57 0.9271 0.9246-0.9296
13
2,2',3,3',4,6,6'-HpCB 176 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 31:01 0.7783 0.7742-0.7825
13
C12-2,2',3,4,4',5'-HxCB7 138L 13
C12-2,2',3,4,4',5'-HxCB7 138L 31:20 1.0000 0.9973-1.0027
2,2',3,4,4',5'-HxCB6 138 13
C12-2,3',4,4',5,5'-HxCB5,9 167L 31:20 0.9386 0.9361-0.9411
13
2,3,3',4',5',6-HxCB 164 C12-2,3',4,4',5,5'-HxCB5,9 167L 31:22 0.9396 0.9371-0.9421
13
2,3,3',4',5,6-HxCB 163 C12-2,3',4,4',5,5'-HxCB5,9 167L 31:28 0.9426 0.9401-0.9451
13
2,3,3',4,5,6-HxCB 160 C12-2,3',4,4',5,5'-HxCB5,9 167L 31:33 0.9451 0.9426-0.9476
13
2,3,3',4,4',6-HxCB 158 C12-2,3',4,4',5,5'-HxCB5,9 167L 31:35 0.9461 0.9436-0.9486
13
2,2',3,4,5,6,6'-HpCB 186 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 31:36 0.7930 0.7888-0.7972
13
2,2',3,3',4,5-HxCB 129 C12-2,3',4,4',5,5'-HxCB5,9 167L 31:48 0.9526 0.9501-0.9551
13
C12-3,3',4,4',5-PeCB4,9 126L 13
C12-2,2',4,5,5'-PeCB7 101L 31:49 1.3156 1.3088-1.3225
3,3',4,4',5-PeCB6,10 126 13
C12-3,3',4,4',5-PeCB4,9 126L 31:49 1.0000 0.9990-1.0021
13
2,3,4,4',5,6-HxCB 166 C12-2,3',4,4',5,5'-HxCB5,9 167L 32:13 0.9651 0.9626-0.9675
13
C12-2,2',3,3',5,5',6-HpCB7 178L 13
C12-2,2',3,3',5,5',6-HpCB7 178L 32:14 1.0000 0.9974-1.0026
13
2,2',3,3',5,5',6-HpCB 178 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 32:14 0.8089 0.8068-0.8110
13
2,2',3,3',4,5',6-HpCB 175 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 32:33 0.8168 0.8147-0.8189
13
2,3,3',4,5,5'-HxCB 159 C12-2,3',4,4',5,5'-HxCB5,9 167L 32:43 0.9800 0.9775-0.9825

EPA Method 1668C 106 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
2,2',3,4',5,5',6-HpCB6 187 13
C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 32:46 0.8223 0.8202-0.8243
13
2,2',3,4,4',5,6'-HpCB 182 C12-2',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 13
C12-2,3',4,4',5,5'-HxCB5,9 167L 32:52 0.9845 0.9820-0.9870
13
2,3,3',4',5,5'-HxCB 162 C12-2,3',4,4',5,5'-HxCB5,9 167L 33:00 0.9885 0.9860-0.9910
13
2,2',3,4,4',5',6-HpCB 183 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 33:06 0.8306 0.8285-0.8327
13
C12-2,3',4,4',5,5'-HxCB5,9 167L 13
C12-2,2',3,4,4',5'-HxCB7 138L 33:23 1.0654 1.0628-1.0681
2,3',4,4',5,5'-HxCB10 167 13
C12-2,3',4,4',5,5'-HxCB5,9 167L 33:23 1.0000 0.9990-1.0020
13
2,2',3,4,5,5',6-HpCB 185 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 33:43 0.8461 0.8440-0.8482
13
2,2',3,3',4,5,6'-HpCB 174 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 34:07 0.8561 0.8540-0.8582
13
2,2',3,4,4',5,6-HpCB 181 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 34:11 0.8578 0.8557-0.8599
13
2,2',3,3',4',5,6-HpCB 177 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 34:22 0.8624 0.8603-0.8645
13
2,2'3,3',4,4',6-HpCB 171 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 34:40 0.8699 0.8678-0.8720
13
C12-2,3,3',4,4',5 -HxCB9 156L 13
C12-2,2',3,4,4',5'-HxCB7 138L 34:40 1.1064 1.1037-1.1090
2,3,3',4,4',5-HxCB10 156 13
C12-2,3,3',4,4',5 -HxCB9 156L 34:40 1.0000 0.9990-1.0019
13
C12-2,2',3,3',5,5',6,6'-OcCB4 202L 13
C12-Cl8-PCB-1945 194L 34:56 0.8265 0.8245-0.8285
13
2,2',3,3',5,5',6,6'-OcCB 202 C12-2,2',3,3',5,5',6,6'-OcCB4 202L 34:56 1.0000 0.9990-1.0019
13
C12-2,3,3',4,4',5'-HxCB9 157L 13
C12-2,2',3,4,4',5'-HxCB7 138L 34:57 1.1154 1.1128-1.1181
2,3,3',4,4',5'-HxCB10 157 13
C12-2,3,3',4,4',5'-HxCB9 157L 34:57 1.0000 0.9990-1.0019
13
2,2',3,3',4,5,6-HpCB 173 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 35:04 0.8800 0.8779-0.8821
13
2,2',3,3',4,5',6,6'-OcCB 201 C12-Cl8-PCB-1945 194L 35:25 0.8379 0.8360-0.8399
13
2,2',3,4,4',5,6,6'-OcCB 204 C12-Cl8-PCB-1945 194L 35:36 0.8423 0.8403-0.8442
13
2,2',3,3',4,5,5'-HpCB 172 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 35:41 0.8954 0.8934-0.8975
13
2,3,3',4,5,5',6-HpCB 192 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 35:51 0.8996 0.8975-0.9017
13
2,2',3,3',4,4',6,6'-OcCB 197 C12-Cl8-PCB-1945 194L 35:55 0.8498 0.8478-0.8517
2,2',3,4,4',5,5'-HpCB6 180 13
C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 36:07 0.9063 0.9042-0.9084
13
2,3,3',4',5,5',6-HpCB 193 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 36:20 0.9118 0.9097-0.9138
13
2,3,3',4,4',5',6-HpCB 191 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 36:34 0.9176 0.9155-0.9197
13
2,2',3,3',4,5,6,6'-OcCB 200 C12-Cl8-PCB-1945 194L 36:49 0.8711 0.8691-0.8730
13
C12-3,3',4,4',5,5'-HxCB4,9 169L 13
C12-2,2',3,4,4',5'-HxCB7 138L 37:19 1.1910 1.1883-1.1936
3,3',4,4',5,5'-HxCB6,10 169 13
C12-3,3',4,4',5,5'-HxCB4,9 169L 37:19 1.0000 0.9991-1.0018
2,2',3,3',4,4',5-HpCB6 170 13
C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 37:44 0.9469 0.9448-0.9490
13
2,3,3',4,4',5,6-HpCB 190 C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 37:56 0.9519 0.9498-0.9540

EPA Method 1668C 107 April 2010

 Table A-1. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Congener Retention Time and Quantitation Congener
Labeled or Native CB1 No.2 References No.2 RT RRT RRT QC Limits3
13
2,2',3,3',4,5,5',6-OcCB 198 C12-Cl8-PCB-1945 194L 38:34 0.9125 0.9105-0.9144
13
2,2',3,3',4,5,5',6'-OcCB 199 C12-Cl8-PCB-1945 194L 38:43 0.9160 0.9140-0.9180
13
2,2',3,3',4,4',5,6'-OcCB 196 C12-Cl8-PCB-1945 194L 39:05 0.9247 0.9227-0.9267
13
2,2',3,4,4',5,5',6-OcCB 203 C12-Cl8-PCB-1945 194L 39:05 0.9247 0.9227-0.9267
13
C12-2',3,3',4,4',5,5'-HpCB4,5,9 189L 13
C12-2,2',3,3',5,5',6-HpCB7 178L 39:51 1.2363 1.2311-1.2415
2,3,3',4,4',5,5'-HpCB10 189 13
C12-2',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 13
C12-Cl8-PCB-1945 194L 40:45 0.9641 0.9621-0.9661
13
C12-2,2',3,3',4,5,5',6,6'-NoCB4 208L 13
C12-Cl9-PCB-2064,5 206L 41:03 0.9149 0.9131-0.9168
13
2,2',3,3',4,5,5',6,6'-NoCB 208 C12-2,2',3,3',4,5,5',6,6'-NoCB4 208L 41:03 1.0000 0.9992-1.0016
13
2,2',3,3',4,4',5,6,6'-NoCB 207 C12-Cl9-PCB-2064,5 206L 41:32 0.9257 0.9238-0.9276
13
C12-2,2',3,3',4,4',5,5'-OcCB5 194L 13
C12-2,2',3,3',5,5',6-HpCB7 178L 42:16 1.3113 1.3061-1.3164
13
2,2',3,3',4,4',5,5'-OcCB 194 C12-Cl8-PCB-1945 194L 42:16 1.0000 0.9992-1.0016
13
C12-2,3,3',4,4',5,5',6-OcCB4 205L 13
C12-Cl8-PCB-1945 194L 42:44 1.0110 1.0091-1.0130
13
2,3,3',4,4',5,5',6-OcCB 205 C12-2,3,3',4,4',5,5',6-OcCB4 205L 42:44 1.0000 0.9992-1.0016
13
C12-2,2',3,3',4,4',5,5',6-NoCB4,5 206L 13
C12-2,2',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 13
C12-Cl9-PCB-2064,5 206L 44:52 1.0000 0.9993-1.0015
13
C12-2,2',3,3',4,4',5,5',6,6'-DeCB4,5 209L 13
C12-2,2',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 13
C12-Cl10-PCB-2094,5 209L 46:55 1.0000 0.9993-1.0014

1. Abbreviations for chlorination levels 4. Labeled level of chlorination (LOC) window-

MoCB monochlorobiphenyl HxCB hexachlorobiphenyl defining congener
DiCB dichlorobiphenyl HpCB heptachlorobiphenyl 5. Labeled level of chlorination (LOC) quantitation
TrCB trichlorobiphenyl OcCB octachlorobiphenyl congener
TeCB tetrachlorobiphenyl NoCB nonachlorobiphenyl 6. National Oceanic and Atmospheric Administration
PeCB pentachlorobiphenyl DeCB decachlorobiphenyl (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 8. Clean-up standard
seconds around the retention time for the labeled analog. For native CBs determined by internal standard 9. Labeled internal standard for World Health
quantitation, RRT QC limits were constructed using a ± 2 percent window around the retention time for Organization (WHO) toxic congener
retention times in the range of 0.8-1.2 and a ± 4 percent window around the retention time for retention times 10. WHO toxic congener
<0.8 and >1.2. These windows may not be adequate for analyte identification (See the note in Section 16.4)

EPA Method 1668C 108 April 2010

 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
12
188.0393 M C12 H9 35Cl Cl-1 PCB
12
190.0363 M+2 C12 H9 37Cl Cl-1P CB
13
Fn-1 Cl-1 200.0795 M C12 H9 35Cl 13
C12 Cl-1 PCB
13
202.0766 M+2 C12 H9 37Cl 13
C12 Cl-1 PCB
218.9856 lock C4 F9 PFK
12
222.0003 M C12 H8 35Cl2 Cl-2 PCB
12
223.9974 M+2 C12 H8 35Cl 37 Cl Cl-2 PCB
12
225.9944 M+4 C12 H8 37Cl2 Cl-2 PCB
13
234.0406 M C12 H8 35Cl2 13
C12 Cl-2 PCB
Fn-2 Cl-2,3 13
236.0376 M+2 C12 H8 35Cl 37 Cl 13
C12 Cl-2 PCB
242.9856 lock C6 F9 PFK
12
255.9613 M C12 H7 35Cl3 Cl-3 PCB
12
257.9584 M+2 C12 H7 35Cl2 37Cl Cl-3 PCB
12
255.9613 M C12 H7 35Cl3 Cl-3 PCB
12
257.9584 M+2 C12 H7 35Cl2 37Cl Cl-3 PCB
12
259.9554 M+4 C12 H7 35Cl 37Cl2 Cl-3 PCB
13
268.0016 M C12 H7 35Cl3 13
C12 Cl-3 PCB
13
269.9986 M+2 C12 H7 35Cl2 37Cl 13
C12 Cl-3 PCB
280.9825 lock C6 F11 PFK
12
289.9224 M C12 H6 35Cl4 Cl-4 PCB
12
291.9194 M+2 C12 H6 35Cl3 37Cl Cl-4 PCB
Fn-3 Cl-3,4,5 12
293.9165 M+4 C12 H6 35Cl2 37Cl2 Cl-4 PCB
13
301.9626 M C12 H6 35Cl4 13
C12 Cl-4 PCB
13
303.9597 M+2 C12 H6 35Cl3 37Cl 13
C12 Cl-4 PCB
12
323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB
12
289.9224 M C12 H6 35Cl4 Cl-4 PCB
12
291.9194 M+2 C12 H6 35Cl3 37Cl Cl-4 PCB
12
293.9165 M+4 C12 H6 35Cl2 37Cl2 Cl-4 PCB
13
301.9626 M+2 C12 H6 35Cl3 37Cl 13
C12 Cl-4 PCB
13
303.9597 M+4 C12 H6 35Cl2 37Cl2 13
C12 Cl-4 PCB
12
323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
Fn-4 Cl-4,5,6
330.9792 lock C7 F15 PFK
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB
13
359.8415 M+2 C12 H4 35Cl5 37Cl Cl-6 PCB
13
361.8385 M+4 C12 H4 35Cl4 37Cl2 Cl-6 PCB
13
363.8356 M+6 C12 H4 35Cl3 37Cl2 Cl-6 PCB
13
371.8817 M+2 C12 H4 35Cl5 37Cl 13
C12 Cl-6 PCB
13
373.8788 M+4 C12 H4 35Cl4 37Cl2 13
C12 Cl-6 PCB
EPA Method 1668C 109 April 2010
 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
12
323.8834 M C12 H5 35Cl5 Cl-5 PCB
12
325.8804 M+2 C12 H5 35Cl4 37Cl Cl-5 PCB
12
327.8775 M+4 C12 H5 35Cl3 37Cl2 Cl-5 PCB
13
337.9207 M+2 C12 H5 35Cl4 37Cl 13
C12 Cl-5 PCB
13
339.9178 M+4 C12 H5 35Cl3 37Cl2 13
C12 Cl-5 PCB
354.9792 lock C9 F13 PFK
12
359.8415 M+2 C12 H4 35Cl5 37Cl Cl-6 PCB
12
361.8385 M+4 C12 H4 35Cl4 37Cl2 Cl-6 PCB
12
363.8356 M+6 C12 H4 35Cl3 37Cl3 Cl-6 PCB
13
371.8817 M+2 C12 H4 35Cl5 37Cl 13
C12 Cl-6 PCB
13
373.8788 M+4 C12 H4 35Cl4 37Cl2 13
C12 Cl-6 PCB
Fn-5 Cl-5,6,7,8 12
393.8025 M+2 C12 H3 35Cl6 37Cl Cl-7 PCB
12
395.7995 M+4 C12 H3 35Cl5 37Cl2 Cl-7 PCB
12
397.7966 M+6 C12 H3 35Cl4 37Cl3 Cl-7 PCB
13
405.8428 M+2 C12 H3 35Cl6 37Cl 13
C12 Cl-7 PCB
13
407.8398 M+4 C12 H3 35Cl5 37Cl2 13
C12 Cl-7 PCB
12
427.7635 M+2 C12 H2 35Cl7 37Cl Cl-8 PCB
12
429.7606 M+4 C12 H2 35Cl6 37Cl2 Cl-8 PCB
12
431.7576 M+6 C12 H2 35Cl5 37Cl3 Cl-8 PCB
13
439.8038 M+2 C12 H2 35Cl7 37Cl 13
C12 Cl-8 PCB
13
441.8008 M+4 C12 H2 35Cl6 37Cl2 13
C12 Cl-8 PCB
454.9728 QC C11 F17 PFK
12
427.7635 M+2 C12 H2 35Cl7 37Cl Cl-8 PCB
12
429.7606 M+4 C12 H2 35Cl6 37Cl2 Cl-8 PCB
12
431.7576 M+6 C12 H2 35Cl5 37Cl3 Cl-8 PCB
13
439.8038 M+2 C12 H2 35Cl7 37Cl 13
C12 Cl-8 PCB
13
441.8008 M+4 C12 H2 35Cl6 37Cl2 13
C12 Cl-8 PCB
442.9728 QC C10 F13 PFK
454.9728 lock C11 F13 PFK
12
461.7246 M+2 C12 H1 35Cl8 37Cl Cl-9 PCB
12
463.7216 M+4 C12 H1 35Cl7 37Cl2 Cl-9 PCB
Fn-6 Cl-8,9,10 12
465.7187 M+6 C12 H1 35Cl6 37Cl3 Cl-9 PCB
13
473.7648 M+2 C12 H1 35Cl8 37Cl 13
C12 Cl-9 PCB
13
475.7619 M+4 C12 H1 35Cl7 37Cl2 13
C12 Cl-9 PCB
13
495.6856 M+2 C12 H435Cl9 37Cl Cl-10 PCB
12
499.6797 M+4 C12 35Cl7 37Cl3 Cl-10 PCB
12
501.6767 M+6 C12 35Cl6 37Cl4 Cl-10 PCB
13
507.7258 M+2 C12 H435Cl9 37Cl 13
C12 Cl-10 PCB
13
509.7229 M+4 C12 H4 35Cl8 37Cl2 13
C12 Cl-10 PCB
13
511.7199 M+6 C12 H4 35Cl8 37Cl4 13
C12 Cl-10 PCB

Isotopic masses used for accurate mass calculation

1 37
H 1.0078 Cl 36.9659
12 19
C 12.0000 F 18.9984
13 35
C 13.0034 Cl 34.9689

EPA Method 1668C 110 April 2010