vxEPA
United States
Environmental Protection
Agency
Method 1668B
Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, Biosolids, and Tissue by HRGC/HRMS
November 2008
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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-821-R-08-020
(November 2008) EPA revised Method 1668A with Method 1668B (the "Method") to replace single-lab quality
control (QC) acceptance criteria with interlaboratory criteria and other changes that are based on the results of
an interlaboratory validation study, and a peer review of that study. Method 1668B, and the validation study
report, Method 1668A Interlaboratory Validation Study Report (EPA-821 -08-021), are available at EPA's CWA
methods website at http://www.epa.gov/waterscience/methods/.
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Method 1668B Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, Biosolids, and Tissue by HRGC/HRMS November 2008
This revision of Method 1668 (Method 1668B; the "Method") revises EPA Method 1668Ato
replace single-lab quality control (QC) acceptance criteria with interlaboratory criteria, and make
other changes described below. Method 1668B was developed by the Office of Water's Office
of Science and Technology (OST) for use in Clean Water Act (CWA) programs. Method 1668B
is based on the results of an interlaboratory validation study, and a peer review of that study.
Method 1668B, and the validation study report, Methodl668A Interlaboratory Validation Study
Report (EPA-821-08-021), are available at EPA's CWA methods website at
www.epa.gov/waterscience/methods.
Method 1668B determines chlorinated biphenyl congeners in environmental samples by
isotope dilution and internal standard high resolution gas chromatography/high resolution mass
spectrometry (HRGC/HRMS). The Method was developed 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.
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
estimated minimum levels of quantitation in Table 2 are concentrations at which a congener can
be measured with laboratory contamination present. In water these values range from 10 to 500
parts per quadrillion (picograms per liter, pg/L). A laboratory may establish a lower reporting
level for a congener, see Sect. 17.6.1.4.
This Method was prepared by Interface, Inc. and CSC Environmental Systems and Solutions
under EPA Contract EP-C-06-085. Multi-lab (six labs for water and tissue, four for biosolids)
data in Table 6 of this Method were provided by laboratories that participated in EPA's inter-
laboratory validation of EPA Method 1668 A. Previously, single-lab data for 1668 A was
developed by Axys Analytical Services, Ltd., Sidney, BC, Canada.
Summary of changes between EPA Method 1668A (8-20-03) and 1668B
• The key changes (and goal of the validation study) are revised QC acceptance criteria in
Table 6. A new footnote 1 to Table 6 references the EPA interlaboratory study report, and
the other footnote numbers are incremented.
• Section 1.5, the performance-based discussion, reflects additional flexibility to modify
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.
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• 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 as
necessary to meet program or regulatory needs.
• 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, references the validation studies, and that performance data are in the interlab
validation study report.
• Reference 1, is updated to 2006 World Health Organization paper on toxicity equivalency
factors.
• References 4 and 18, adds titles to the papers in these references.
• Reference 22, references the Method 1668 A Interlab oratory Validation Study Report.
• Tables 2 and A-l, revise 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 1668 A Interlab oratory Validation Study Report.
• Table 7, adds footnote 2 to require meeting the 10:1 signal-to-noise specification at the CS-2
calibration level.
Disclaimer
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Contact
Please address questions, comments, or suggestions to:
The CWA Methods Team
Engineering and Analytical Support Branch
Engineering and Analysis Division (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460
E-mail: OSTCWAMethods@epa.gov
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Method 1668B November 2008
Method 1668B Chlorinated Biphenyl Congeners in Water, Soil, Sediment,
Biosolids and Tissue by HRGC/HRMS - November 2008
1.0 Scope and application
1.1 Method 1668B (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 poly chlorinated 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 This Method was developed 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 1668B is a revision of EPA Method 1668A. Both
methods are based on a compilation of methods from the technical literature (References 3-5),
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
estimated minimum levels of quantitation (EMLs) in Table 2 are the levels at which the CBs
can be determined with laboratory contamination present. The estimated method detection
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Method 1668B November 2008
limit (EMDL) for CB 126 in water is 5 pg/L (picograms-per-liter; parts-per-quadrillion) with no
interferences present.
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, or 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 othertissue—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
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Method 1668B November 2008
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 (iL. 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 (LOG) throughout a pre-determined retention time window.
2.4 An individual CB congener is identified by comparing the GC retention time and ion-
abundance ratio of two exact m/z's with the corresponding retention time of an authentic
standard and the theoretical or acquired ion-abundance ratio of the two exact m/z's. Isomer
specificity for certain of the CB congeners is achieved using GC columns that resolve these
congeners.
2.5 Quantitative analysis is performed in one of two ways using selected ion current profile (SICP)
areas:
2.5.1 For the Toxics and the LOC CBs, the GC/MS is multi-point calibrated and the
concentration is determined using the isotope dilution technique.
2.5.2 For all congeners other than the Toxics and LOC CBs, the GC/MS is calibrated at a
single concentration and the concentrations are determined using the internal standard
technique. The internal standards are the labeled congeners spiked into the sample,
thus affording recovery correction for all congeners.
2.5.3 For the labeled Toxics, labeled LOC CBs, and the cleanup standards, the GC/MS is
calibrated using replicates at a single concentration and the concentrations of these
labeled compounds in samples are determined using the internal standard technique.
The injection internal standards are labeled compounds spiked into the extract
immediately prior to injection of an aliquot of the extract into the HRGC/HRMS.
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, as well as toxic congeners 105, 114, 118, 123, 156, 157, and
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Method 1668B November 2008
167 have been shown to be very difficult to completely eliminate from the laboratory at levels
lower than the EMDLs in this Method (Table 2), and baking of glassware in a kiln or furnace at
450 - 500 °C may be necessary to remove these and other contaminants.
4.2 Proper cleaning of glassware is extremely important, because glassware may not only
contaminate the samples but may also remove the analytes of interest by adsorption on the glass
surface.
4.2.1 Glassware should be rinsed with solvent and washed with a detergent solution as soon
after use as is practical. Sonication of glassware containing a detergent solution for
approximately 30 seconds may aid in cleaning. Glassware with removable parts,
particularly separatory funnels with fluoropolymer stopcocks, must be disassembled
prior to detergent washing.
4.2.2 After detergent washing, glassware should be rinsed immediately, first with methanol,
then with hot tap water. The tap water rinse is followed by another methanol rinse,
then acetone, and then methylene chloride.
4.2.3 Baking of glassware in a kiln or other high temperature furnace (300 - 500 °C) 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.
4.3 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.
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Method 1668B November 2008
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, the 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 EMDLs and EMLs in Table 2 are the levels that
can be achieved with normal laboratory backgrounds present. Many of the EMLs are greater
than the equivalent concentrations of the calibration solutions. To prevent contamination of the
calibration solutions with the backgrounds allowed by the EMLs, the calibration solutions must
be prepared in an area free from CB contamination using glassware free from contamination. If
these requirements cannot be met or are difficult to meet in the laboratory, the laboratory
should prepare the calibration solutions in a contamination-free facility or have a vendor
prepare the calibration standards and guarantee freedom from contamination.
4.7 Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis of tissue
samples for the CBs. The lipid contents of different species and portions of tissue can vary
widely. Lipids are soluble to varying degrees in various organic solvents and may be present in
sufficient quantity to overwhelm the column chromatographic cleanup procedures used for
cleanup of sample extracts. Lipids must be removed by the anthropogenic isolation column
procedure in Section 13.6, followed by the gel permeation chromatography procedure in
Section 13.2. Florisil (Section 13.7) is recommended as an additional cleanup step.
4.8 If the laboratory air is a potential source of CB contamination, samples, reagents, glassware,
and other materials should be dried in a glove box or other area free from contamination.
5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical used in this Method has not been precisely
determined; however, each compound should be treated as a potential health hazard. Exposure
to these compounds should be reduced to the lowest possible level.
5.1.1 PCBs have been tentatively classified as known or suspected human or mammalian
carcinogens. On the basis of the available toxicological and physical properties of the
CBs, pure standards should be handled only by highly trained personnel thoroughly
familiar with handling and cautionary procedures and the associated risks.
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Method 1668B November 2008
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 6-9. The
references and bibliography at the end of Reference 8 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. We 11-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 10 for handling chlorinated dibenzo-/?-dioxins and dibenzofurans (CDDs/CDFs) are
also recommended for handling the CBs.
5.3.1 Facility—When finely divided samples (dusts, soils, dry chemicals) are handled, all
operations (including removal of samples from sample containers, weighing,
transferring, and mixing) should be performed in a glove box demonstrated to be leak
tight or in a fume hood demonstrated to have adequate air flow. Gross losses to the
laboratory ventilation system must not be allowed. Handling of the dilute solutions
normally used in analytical and animal work presents no inhalation hazards except in
the case of an accident.
5.3.2 Protective equipment—Disposable plastic gloves, apron or lab coat, safety glasses or
mask, and a glove box or fume hood adequate for radioactive work should be used.
During analytical operations that may give rise to aerosols or dusts, personnel should
wear respirators equipped with activated carbon filters. Eye protection (preferably full
face shields) must be worn while working with exposed samples or pure analytical
standards. Latex gloves are commonly used to reduce exposure of the hands. When
handling samples suspected or known to contain high concentrations of the CBs, an
additional set of gloves can also be worn beneath the latex gloves.
5.3.3 Training—Workers must be trained in the proper method of removing contaminated
gloves and clothing without contacting the exterior surfaces.
5.3.4 Personal hygiene—Hands and forearms should be washed thoroughly after each
manipulation and before breaks (coffee, lunch, and shift).
5.3.5 Confinement—Isolated work areas posted with signs, segregated glassware and tools,
and plastic absorbent paper on bench tops will aid in confining contamination.
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Method 1668B November 2008
5.3.6 Effluent vapors—The effluent of the sample splitter from the gas chromatograph (GC)
and from roughing pumps on the mass spectrometer (MS) should pass through either a
column of activated charcoal or be bubbled through a trap containing oil or high-
boiling alcohols to condense CB vapors.
5.3.7 Waste Handling—Good technique includes minimizing contaminated waste. Plastic
bag liners should be used in waste cans. Janitors and other personnel should be trained
in the safe handling of waste.
5.3.8 Decontamination
5.3.8.1 Decontamination of personnel—Use any mild soap with plenty of scrubbing
action.
5.3.8.2 Glassware, tools, and surfaces—Chlorothene NU Solvent is a less toxic
solvent that should be effective in removing CBs. Satisfactory cleaning may
be accomplished by rinsing with Chlorothene, then washing with any
detergent and water. If glassware is first rinsed with solvent, the wash water
may be disposed of in the sewer. Given the cost of disposal, it is prudent to
minimize solvent wastes.
5.3.9 Laundry—Clothing known to be contaminated should be collected in plastic bags.
Persons that convey the bags and launder the clothing should be advised of the hazard
and trained in proper handling. The clothing may be put into a washer without contact
if the launderer knows of the potential problem. The washer should be run through a
cycle before being used again for other clothing.
5.3.10 Wipe tests—A useful method of determining cleanliness of work surfaces and tools is
to perform a wipe test of the surface suspected of being contaminated.
5.3.10.1 Using a piece of filter paper moistened with Chlorothene or other solvent,
wipe an area approximately 10x10 cm.
5.3.10.2 Extract and analyze the wipe by GC with an electron capture detector (ECD)
or by this Method.
5.3.10.2 Using the area wiped (e.g., 10 x 10 cm = 0.01 m2), calculate the
concentration in (ig/m2. A concentration less than 1 (ig/m2 indicates
acceptable cleanliness; anything higher warrants further cleaning. More than
100 (ig/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.
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Method 1668B November 2008
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 tubing must be used. If the sampler uses a peristaltic pump, a minimum
length of compressible silicone rubber tubing may be used in the pump only. Before
use, the tubing must be thoroughly rinsed with methanol, followed by repeated rinsing
with reagent water to minimize sample contamination. An integrating flow meter is
used to collect proportional composite samples.
6.2 Equipment for glassware cleaning
Note: If blanks from bottles or other glassware or with fewer cleaning steps than required above
show no detectable CB contamination, unnecessary cleaning steps and equipment may be
eliminated.
6.2.1 Laboratory sink with overhead fume hood
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Method 1668B November 2008
6.2.2 Kiln—Capable of reaching 450 °C within 2 hours and maintaining 450 - 500 °C within
±10 °C, with temperature controller and safety switch (Cress Manufacturing Co, Santa
Fe Springs, CA, B31H, X3 ITS, 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 °C
6.3.5.2 Desiccator
6.3.6 Balances
6.3.6.1 Analytical—Capable of weighing 0.1 mg
6.3.6.2 Top loading—Capable of weighing 10 mg
6.4 Extraction apparatus
6.4.1 Water samples
6.4.1.1 pH meter, with combination glass electrode
6.4.1.2 pH paper, wide range (Hydrion Papers, or equivalent)
6.4.1.3 Graduated cylinder, 1-L capacity
6.4.1.4 Liquid/liquid extraction—Separatory funnels, 250-, 500-, and 2000-mL, with
fluoropolymer stopcocks
6.4.1.5 Solid-phase extraction
6.4.1.5.1 1-L filtration apparatus, including glass funnel, frit support,
clamp, adapter, stopper, filtration flask, and vacuum tubing
(Figure 4). For wastewater samples, the apparatus should accept
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Method 1668B November 2008
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 11) 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 x 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
10
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Method 1668B November 2008
6.5.3 Glass-fiber filter paper—Whatman GF/D (or equivalent), to fit glass runnel 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 FIW, 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 x 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-(iL 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
11
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Method 1668B November 2008
6.7.2.4 Column—Hypercarb, 100 x 4.6 mm, 5 (im particle size, Keystone Scientific,
or equivalent
6.7.2.5 Detector—Altex 110A (or equivalent) operated at 0.02 AUFS at 235 nm
6.7.2.6 Fraction collector—Isco Foxy II, or equivalent
6.7.3 Pipets
6.7.3.1 Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific 13-678-6A,
or equivalent)
6.7.3.2 Disposable, serological, 50-mL (8- to 10- mm ID)
6.7.4 Glass chromatographic columns
6.7.4.1 150-mm long x 8-mm ID, (Kontes K-420155, or equivalent) with coarse-
glass frit or glass-wool plug and 250-mL reservoir
6.7.4.2 200-mm long x 15-mm ID, with coarse-glass frit or glass-wool plug and 250-
mL reservoir
6.7.4.3 300-mm long x 22-mm ID, with coarse-glass frit, 300-mL reservoir, and
glass or fluoropolymer stopcock
6.7.5 Oven—For baking and storage of adsorbents, capable of maintaining a constant
temperature (± 5 °C) in the range of 105-250 °C
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.
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Method 1668B November 2008
6.8.2.2 Evaporation flask—500-mL (Kontes K-570001-0500, or equivalent),
attached to concentrator tube with springs (Kontes K-662750-0012 or
equivalent)
6.8.2.3 Snyder column—Three-ball macro (Kontes K-503000-0232, or equivalent)
6.8.2.4 Boiling chips
6.8.2.4.1 Glass or silicon carbide—Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450 °C 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 °C, installed in a fume hood
6.8.3 Nitrogen evaporation apparatus—Equipped with water bath controlled in the range of
30 - 60 °C (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
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Method 1668B November 2008
seconds at the peak maximum. Unique resolution means a valley
height less than 40 percent of the shorter of the two peaks that
result when the Diluted combined 209 congener solution
(Section 7.10.2.2) is analyzed (see Figures 6 and 7).
6.9.1.1.3 The column must be replaced when any of the criteria in
Sections 6.9.1 - 6.9.1.1.2 are not met.
6.9.1.2 If a column or column system alternate to the SPB-octyl column is used,
specifications similar to those for the SPB-octyl column (Sections 6.9.1 -
6.9.1.1.2) must be developed and be functionally equivalent to those
specifications.
6.9.1.3 Suggested column—30 ± 5-m long x 0.25 ± 0.02-mm ID; 0.25-(im 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)
14
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Method 1668B November 2008
performance should be computed and maintained, either on the instrument data system,
or on a separate computer system.
7.0 Reagents and standards
7.1 pH adjustment and back-extraction
7.1.1 Potassium hydroxide—Dissolve 20 g reagent grade KOH in 100 mL reagent water.
7.1.2 Sulfuric acid—Reagent grade (specific gravity 1.84)
7.1.3 Hydrochloric acid—Reagent grade, 6N
7.1.4 Sodium chloride—Reagent grade, prepare at 5% (w/v) solution in reagent water
7.2 Solution drying and evaporation
7.2.1 Solution drying—Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or
equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °C 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 °C 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.
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Method 1668B November 2008
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 °C 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 IN sodium hydroxide with 100 g
of activated silica gel in a clean container. Breakup aggregates with a
stirring rod until a uniform mixture is obtained. Store in a screw-capped
bottle with fluoropolymer-lined cap.
7.5.1.4 Potassium silicate
7.5.1.4.1 Dissolve 56 g of high purity potassium hydroxide (Aldrich, or
equivalent) in 300 mL of methanol in a 750- to 1000-mL flat-
bottom flask.
7.5.1.4.2 Add 100 g of activated silica gel (Section 7.5.1.1) and a stirring
bar, and stir on an explosion-proof hot plate at 60-70 °C 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 °C 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)
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Method 1668B November 2008
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 °C 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.
7.5.4.1.1 Fill a clean 1- to 2-L bottle !/2 to 2/3 full with Florisil and place
in an oven at 130-150 °C 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
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Method 1668B November 2008
7.6.2 High-solids reference matrix—Playground sand or similar material. Prepared by
extraction with methylene chloride and/or baking at 450 °C 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 given in Section 9.2. Ideally, the matrix should be free of the CBs, but in no
case must the background level of the CBs in the reference matrix exceed the minimum
levels in Table 2. If low background levels of the CBs are present in the reference
matrix, the spike level of the analytes used in Section 9.2 should be increased to
provide a spike-to-background ratio of approximately 5 (Reference 11).
7.7 Standard solutions—Prepare from materials of known purity and composition or purchase as
solutions or mixtures with certification to their purity, concentration, and authenticity. If the
chemical purity is 98 % or greater, the weight may be used without correction to calculate the
concentration of the standard. Observe the safety precautions in Section 5 and the
recommendation in Section 5.1.2.
Note: Native PCB standards are available from several suppliers. 13Cl2-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 1 to 2 mg of PCB 126 to
three significant figures in a 10-mL ground-glass-stoppered volumetric flask and fill to
the mark with nonane. After the compound is completely dissolved, transfer the
solution to a clean 15-mL vial with fluoropolymer-lined cap.
7.7.2 When not being used, store standard solutions in the dark at room temperature in
screw-capped vials with fluoropolymer-lined caps. Place a mark on the vial at the level
of the solution so that solvent loss by evaporation can be detected. Replace the
solution if solvent loss has occurred.
7.8 Native (unlabeled) stock solutions
Note: Some of the part numbers for solutions described below contain the identifier "1668A."
These part numbers remain valid for Method 1668B.
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.
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Method 1668B November 2008
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 the preparation of
calibration or performance test standards. Reference standards that can be used to
determine the accuracy of standard solutions are available from several vendors.
7.9 Labeled compound stock solutions (Table 3)
7.9.1 Labeled Toxics/LOC/window-defining stock solution—Prepare in isooctane or nonane
at the concentrations in Table 3 or purchase Cambridge Isotope Laboratories (CIL) EC-
4977, or equivalent. If additional CBs are to be determined by isotope dilution (e.g.,
170 and 180), include the additional labeled compounds in this stock solution.
7.9.2 Labeled cleanup standard stock solution—Prepare labeled CBs 28, 111, and 178 in iso-
octane or nonane at the concentration shown in Table 3 or purchase CIL EC-4978, or
equivalent.
7.9.3 Labeled injection internal standard stock solution—Prepare labeled CBs 9, 52, 101,
138, and 194 in nonane or isooctane at the concentrations shown in Table 3, or
purchase CIL EC-4979, or equivalent.
7.10 Calibration standards
7.10.1 Calibration standards—Combine and dilute the solutions in Sections 7.8.1 and 7.9 to
produce the calibration solutions in Table 5 or purchase CIL EC-4976, or equivalent,
for the CS-1 to CS-5 set of calibration solutions. If a 6-point calibration is used,
prepare the CS-0.2 solution or purchase CIL EC-4976-0.2, or equivalent. These
solutions permit the relative response (labeled to native) and response factor to be
measured as a function of concentration. The CS-3 standard (CIL EC-4976-3, or
equivalent) is used for calibration verification (VER).
7.10.2 Solutions of congener mixes
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Method 1668B November 2008
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-defming 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 (iL, the
concentration in the final volume will be 50 ng/mL (50 pg/(iL). Prepare only the amount
necessary for each reference matrix with each sample batch.
7.12 Labeled Toxics/LOC/window-defining standard spiking solution—This solution is spiked into
each sample (Section 9.3) and into the IPR (Section 9.2.1), OPR (Section 15.5), and blank
(Section 9.5) to measure recovery. Dilute the Labeled Toxics/LOC/window-defining stock
solution (Section 7.9.1) with acetone to produce a concentration of the labeled compounds at 2
ng/mL, as shown in Table 3. When 1 mL of this solution is spiked into an IPR, OPR, blank, or
sample and concentrated to a final extract volume of 20 (iL, the concentration in the final
extract volume will be 100 ng/mL (100 pg/(iL). Prepare only the amount necessary for each
reference matrix with each sample batch.
20
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Method 1668B November 2008
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 (iL, the concentration in the final volume will
be 100 ng/mL (100 pg/^L).
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 (iL of this solution is
spiked into a 20 (iL extract, the concentration of each injection internal standard will be
nominally 100 ng/mL (100 pg/(iL).
Note: The addition of 2 ^L of the Labeled injection internal standard spiking solution to a 20 ^L
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 13). Other sample collection techniques, or sample volumes may be used, if
documented.
8.2 Aqueous samples
8.2.1 Samples that flow freely are collected as grab samples or in refrigerated bottles using
automatic sampling equipment. Collect one liter (or a larger or smaller volume) of
sample sufficient to meet project needs.
8.2.2 If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. EPA
Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 14).
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Method 1668B November 2008
8.2.3 Maintain aqueous samples in the dark at <6 °C 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 <6 °C.
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 <6 °C 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 °C.
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 <6 °C 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 under dry ice.
8.4.3 Freeze tissue samples upon receipt at the laboratory and maintain in the dark at less
than -10 °C until prepared. Maintain unused sample in the dark at less than -10 °C.
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 <6
°C, aqueous samples may be stored for up to one year. Similarly, if stored in the dark
at less than -10 °C, 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 °C until analyzed. If stored in the
dark at less than -10 °C, 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 15). 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.
22
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Method 1668B November 2008
If the Method is to be applied to sample matrix other than water (e.g., soils, filter cake,
compost, tissue) the most appropriate alternate reference matrix (Sections 7.6.2 - 7.6.5 and
7.15) is substituted for the reagent water matrix (Section 7.6.1) in all performance tests.
9.1.1 The laboratory must make an initial demonstration of the ability to generate acceptable
precision and recovery with this Method. This demonstration is given in Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to overcome
matrix interferences, the laboratory is permitted certain options to improve separations
or lower the costs of measurements. These options include alternate extraction,
concentration, and cleanup procedures, and changes in sample volumes, columns and
detectors. Alternate determinative techniques, such as substitution of spectroscopic or
immunoassay techniques for HRGC/HRMS technology, and changes that degrade
Method performance, are not allowed without prior review and approval. If an
analytical technique other than the techniques specified in this Method is used, that
technique must have a specificity equal to or greater than the specificity of the
techniques in this Method for the analytes of interest. (Note: For additional flexibility
to make modifications without prior EPA review see 40 CFR Part 136.6.)
9.1.2.1 Each time a modification is made to this Method, the laboratory is required
to repeat the procedure in Section 9.2. If the detection limit of the Method
will 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 one-third the EMDLs 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 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).
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Method 1668B November 2008
b) Calibration verification (Section 15.3).
c) Initial precision and recovery (Section 9.2).
d) Labeled compound recovery (Section 9.3).
e) Analysis of blanks (Section 9.5).
f) Accuracy assessment (Section 9.4).
9.1.2.2.5 Data that will allow an independent reviewer to validate each
determination by tracing the instrument output (peak height,
area, or other signal) to the final result. These data are to
include:
a) Sample numbers and other identifiers.
b) Extraction dates.
c) Analysis dates and times.
d) Analysis sequence/run chronology.
e) Sample weight or volume (Section 11).
f) Extract volume prior to each cleanup step (Section 13).
g) Extract volume after each cleanup step (Section 13).
h) Final extract volume prior to injection (Section 14).
i) Injection volume (Section 14.3).
j) Dilution data, differentiating between dilution of a sample or
extract (Section 17.5).
k) Instrument and operating conditions.
1) 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
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Method 1668B November 2008
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-defming standard spiking solution
(Section 7.12), and the Labeled cleanup standard spiking solution (Section 7.13),
according to the procedures in Sections 11 through 18. For an alternative sample
matrix, four aliquots of the alternative reference matrix (Section 7.6) are used. All
sample processing steps that are to be used for processing samples, including
preparation (Section 11), extraction (Section 12), and cleanup (Section 13), must be
included in this test.
9.2.2 Using results of the set of four analyses, compute the average percent recovery (X) of
the extracts and the relative standard deviation (RSD) of the concentration for each
compound, by isotope dilution for CBs with a labeled analog, and by internal standard
for CBs without a labeled analog and for the labeled compounds.
9.2.3 For each CB and labeled compound, compare RSD and X with the corresponding limits
for initial precision and recovery in Table 6. If RSD and X for all compounds meet the
acceptance criteria, system performance is acceptable and analysis of blanks and
samples may begin. If, however, any individual RSD exceeds the precision limit or
any individual X falls outside the range for recovery, system performance is
unacceptable for that compound. Correct the problem and repeat the test (Section 9.2).
9.3 To assess Method performance on the sample matrix, the laboratory must spike all samples
with the Labeled Toxics/LOC/window-defming 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-defming 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
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Method 1668B November 2008
then be employed to attempt to bring the recovery within the normal range. If the
recovery cannot be brought within the normal range after all cleanup procedures have
been employed, water samples are diluted and smaller amounts of soils, sludges,
sediments, and other matrices are analyzed per Section 18.
9.4 It is suggested but not required that recovery of labeled compounds from samples be assessed
and records maintained.
9.4.1 After the analysis of 30 samples of a given matrix type (water, soil, sludge, pulp, etc.)
for which the labeled compounds pass the tests in Section 9.3, compute the average
percent recovery (R) and the standard deviation of the percent recovery (SR) for the
labeled compounds only. Express the assessment as a percent recovery interval from R
- 2SR to R + 2SR for each matrix. For example, if R = 90% and SR = 10% for five
analyses of pulp, the recovery interval is expressed as 70 to 110%.
9.4.2 Update the accuracy assessment for each labeled compound in each matrix on a regular
basis (e.g., after each five to ten new measurements).
9.5 Method blanks—A reference matrix Method blank is analyzed with each sample batch (Section
4.3) to demonstrate freedom from contamination. The matrix for the Method blank must be
similar to the sample matrix for the batch, e.g., a 1-L reagent water blank (Section 7.6.1), high-
solids reference matrix blank (Section 7.6.2), paper matrix blank (Section 7.6.3); tissue blank
(Section 7.6.4), or alternative reference matrix blank (Section 7.6.5).
9.5.1 Spike 1.0 mL each of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12), and the Labeled cleanup standard spiking solution (Section
7.13) into the Method blank, according to the procedures in Sections 11 through 18.
Prepare, extract, clean up, and concentrate the Method blank. Analyze the blank
immediately after analysis of the OPR (Section 15.5) to demonstrate freedom from
contamination.
9.5.2 If any CB (Table 1) is found in the blank at greater than 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
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Method 1668B November 2008
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.
10.1.1 Suggested GC operating conditions:
Injector temperature: 270 °C
Interface temperature: 290 °C
Initial temperature: 75 °C
Initial time: 2 minutes
Temperature program: 75-150 °C at 15 °C/minute
150-290 °C at 2.5 °C/minute
Final time: 1 minute
Note: All portions of the column that connect the GC to the ion source should remain at or
above the interface temperature specified above during analysis to preclude condensation of less
volatile compounds.
The GC conditions may be optimized for compound separation and sensitivity. Once
optimized, the same GC conditions must be used for the analysis of all standards,
blanks, IPR and OPR standards, and samples.
10.1.2 Retention time calibration for the CB congeners
10.1.2.1 Separately inject each of the diluted individual congener solutions (Section
7.10.2.1.2). Establish the beginning and ending retention times for the scan
descriptors in Table 7. Scan descriptors other than those listed in Table 7
may be used provided the MLs in Table 2 are met. Store the retention time
(RT) and relative retention time (RRT) for each congener in the data system.
10.1.2.2 The absolute retention time of CB 209 must exceed 55 minutes on the SPB-
octyl column; otherwise, the GC temperature program must be adjusted and
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Method 1668B November 2008
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 and
Table 5). 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 PFK (or other reference substance) and a molecular leak, tune the instrument to
meet the minimum required resolving power of 10,000 (10% valley) at m/z 330.9792
or any other significant PFK fragment in the range of 300 to 350. For each descriptor
(Table 7), monitor and record the resolution and exact m/z's of three to five reference
peaks covering the mass range of the descriptor. The level of PFK (or other reference
substance) metered into the HRMS during analyses should be adjusted so that the
amplitude of the most intense selected lock-mass m/z signal (regardless of the
descriptor number) does not exceed 10% of the full-scale deflection for a given set of
detector parameters. Under those conditions, sensitivity changes that might occur
during the analysis can be more effectively monitored.
Note: Different lots and types of PFK can contain varying levels of contamination, and
excessive PFK (or other reference substance) may cause noise problems and contamination of
the ion source necessitating increased frequency of source cleaning.
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 perfluorokerosene (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.
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Method 1668B November 2008
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 LOG 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 10.2.1.3) 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 (iL, consistent with the capability of the HRGC/HRMS instrument. Inject a 1
or 2 (iL 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 LOG window. The laboratory must also monitor exact m/z's for
congeners at higher levels of chlorination to determine if fragments will
compromise measurement of congeners at lower levels of chlorination.
10.3.1.2 The mass spectrometer must be operated in a mass-drift correction mode,
using PFK (or other reference substance) to provide lock m/z's. The lock
mass for each group of m/z's is shown in Table 7. Each lock mass must be
monitored and must not vary by more than ± 20% throughout its respective
retention time window. Variations of lock mass by more than 20% indicate
the presence of co-eluting interferences that raise the source pressure and
may significantly reduce the sensitivity of the mass spectrometer. Re-
injection of another aliquot of the sample extract may not resolve the
problem and additional cleanup of the extract may be required to remove the
interference. A lock mass interference or suppression in a retention time
region in which CBs and labeled compounds do not elute may be ignored.
10.3.2 All CBs and labeled compounds in the CS-1 standard must be within the QC limits in
Table 8 for their respective ion abundance ratios; otherwise, the mass spectrometer
must be adjusted and this test repeated until the m/z ratios fall within the limits
specified. If the adjustment alters the resolution of the mass spectrometer, resolution
must be verified (Section 10.2.1) prior to repeat of the test.
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Method 1668B November 2008
1 0.3.3 Verify that the HRGC/HRMS instrument meets the estimated minimum levels (EMLs)
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 EMDLs and EMLs 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 is employed. If lower levels are achievable, these 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:
RR -
(Al, + A2) Cn
Where:
Aln and A2n = The areas of the primary and secondary m/z's for the PCB.
Al1andA21 = The areas of the primary and secondary m/z's for the labeled
compound.
Q = The concentration of the labeled compound in the calibration
standard (Table 4).
Cn = The concentration of the native compound in the calibration
standard (Table 4).
10.4.3 To calibrate the analytical system by isotope dilution, inject calibration standards CS-1
through CS-5 (Section 7.10 and Table 5) for a less sensitive instrument or CS-0.2
through CS-5 for a more sensitive instrument. Use a volume identical to the volume
chosen in Section 10.3, the procedure in Section 14, and the conditions in Section
10.1.1. Compute and store the relative response (RR) for each Native Toxics/LOC CB
at each concentration. Compute the average (mean) RR and the RSD of the 5 (or 6)
RRs.
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Method 1668B November 2008
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, to determination of the Labeled
Toxics/LOC/window-defming congeners and Labeled cleanup congeners for performance tests
and intra-laboratory statistics (Sections 9.4 and 15.5.4), and to 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:
RF_ (Als + A2) Cis
(Alis + A2is> Cs
Where:
Als and A2S = The areas of the primary and secondary m/z's for the PCB.
AllsandA2ls = The areas of the primary and secondary m/z's for the internal
standard.
C1S = 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 (LOG) 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.
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Method 1668B November 2008
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
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Method 1668B November 2008
Note: This aliquot is used for determining the solids content of the sample, not for determination
ofCBs.
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 °C and cool in a desiccator.
11.2.1.4 Calculate percent solids as follows:
°/ solids = Wei8ht °f sample aliquot after drying (g) - weight of filter (g) 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 °C, and cool in a desiccator.
11.2.2.3 Calculate percent solids as follows:
% solids = wei%ht of sample aliquot after drying x 1QQ
weight of sample aliquot before drying
11.3 Estimation of particle size
11.3.1 Spread the dried sample from Section 11.2.2.2 onapiece 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.
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Method 1668B November 2008
11.4.2.2 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12) into the sample bottle. Cap the bottle and mix the
sample by careful shaking. Allow the sample to equilibrate for 1 to 2 hours,
with occasional shaking.
11.4.2.3 For each sample or sample batch (to a maximum of 20 samples) to be
extracted during the same 12-hour shift, place two 1.0-L aliquots of reagent
water in clean sample bottles or flasks.
11.4.2.4 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12) into both reagent water aliquots. One of these
aliquots will serve as the Method blank.
11.4.2.5 Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into the remaining reagent water aliquot. This aliquot will serve as the
OPR (Section 15.5).
11.4.2.6 For extraction using SPE, add 5 mL of methanol to the sample and QC
aliquots. Cap and shake the sample and QC aliquots to mix thoroughly, and
proceed to Section 12.2 for extraction.
11.5 Preparation of samples containing greater than one percent solids
11.5.1 Weigh a well-mixed aliquot of each sample (of the same matrix type) sufficient to
provide 10 g of dry solids (based on the solids determination in Section 11.2) into a
clean beaker or glass jar.
11.5.2 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking solution
(Section 7.12) into the sample.
11.5.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during
the same 12 hour shift, weigh two 10-g aliquots of the appropriate reference matrix
(Section 7.6) into clean beakers or glass jars.
11.5.4 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking solution
(Section 7.12) into both reference matrix aliquots. Spike 1.0 mL of the Native
Toxics/LOC standard spiking solution (Section 7.11) into one reference matrix aliquot.
This aliquot will serve as the OPR (Section 15.5). The other aliquot will serve as the
Method blank.
11.5.5 Stir or tumble and equilibrate the aliquots for 1 to 2 hours.
11.5.6 Decant excess water. If necessary to remove water, filter the sample through a glass-
fiber filter and discard the aqueous liquid.
11.5.7 If particles >1 mm are present in the sample (as determined in Section 11.3.2), spread
the sample on clean aluminum foil in a hood. After the sample is dry, grind to reduce
the particle size (Section 11.7).
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Method 1668B November 2008
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-defming standard spiking solution
(Section 7.12) into the amount of sample determined in Section 11.6.1, and into the
OPR and blank. Spike 1.0 mL of the Native Toxics/LOC standard spiking solution
(Section 7.11) into the OPR. Pressure filter the sample, blank, and OPR through
Whatman GF/D glass-fiber filter paper (Section 6.5.3). If necessary to separate the
phases and/or settle the solids, centrifuge these aliquots prior to filtration.
11.6.3 Discard any aqueous phase (if present). Remove any non-aqueous liquid present and
reserve the maximum amount filtered from the sample (Section 11.6.1) or 10 g,
whichever is less, for combination with the solid phase (Section 12.3.5).
11.6.4 If particles >1 mm are present in the sample (as determined in Section 11.3.2) and the
sample is capable of being dried, spread the sample and QC aliquots on clean
aluminum foil in a hood. Observe the precaution in Section 4.8.
11.6.5 After the aliquots are dry or if the sample cannot be dried, reduce the particle size using
the procedures in Section 11.7 and extract the reduced-size particles using the SDS
procedure in Section 12.3. If particles >1 mm are not present, extract the particles and
filter in the sample and QC aliquots directly using the SDS procedure in Section 12.3.
11.7 Sample grinding, homogenization, or blending—Samples with particle sizes greater than 1 mm
(as determined in Section 11.3.2) are subjected to grinding, homogenization, or blending. The
method of reducing particle size to less than 1 mm is matrix-dependent. In general, hard
particles can be reduced by grinding with a mortar and pestle. Softer particles can be reduced
by grinding in a Wiley mill or meat grinder, by homogenization, or in a blender.
11.7.1 Each size-reducing preparation procedure on each matrix must be verified by running
the tests in Section 9.2 before the procedure is employed routinely.
11.7.2 The grinding, homogenization, or blending procedures must be carried out in a glove
box or fume hood to prevent particles from contaminating the work environment.
11.7.3 Grinding—Certain papers and pulps, slurries, and amorphous solids can be ground in a
Wiley mill or heavy duty meat grinder. In some cases, reducing the temperature of the
sample to freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding
process. Grind the sample aliquots from Sections 11.5.7 or 11.6.5 in a clean grinder.
Do not allow the sample temperature to exceed 50 °C. 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
35
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Method 1668B November 2008
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 offish tissue include whole fish-
skin on, whole fish-skin removed, edible fish fillets (filleted in the field or by the laboratory),
specific organs, and other portions. Once the appropriate tissue has been determined, the
sample must be homogenized.
11.8.1 Homogenization
11.8.1.1 Samples are homogenized while still frozen, where practical. If the
laboratory must dissect the whole fish to obtain the appropriate tissue for
analysis, the unused tissues may be rapidly refrozen and stored in a clean
glass jar for subsequent use.
11.8.1.2 Each analysis requires 10 g of tissue (wet weight). Therefore, the laboratory
should homogenize at least 20 g of tissue to allow for re-extraction of a
second aliquot of the same homogenized sample, if re-analysis is required.
When whole fish analysis is necessary, the entire fish is homogenized.
11.8.1.3 Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind in a
meat grinder (Section 6.3.4). Cut tissue 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 °C.
Return any tissue that was not homogenized to its original container and
store at less than -10 °C.
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.
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Method 1668B November 2008
11.8.3 Spiking
11.8.3.1 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12) into the sample, blank, and OPR aliquot.
11.8.3.2 Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into the OPR aliquot.
11.8.4 Extract the aliquots using the procedures in Section 12.4.
12.0 Extraction and concentration
12.1 Extraction procedures include: solid-phase (Section 12.2.1), separatory funnel (Section 12.2.2),
and continuous liquid/liquid (Section 12.2.3) for aqueous liquids; Soxhlet/Dean-Stark (Section
12.3) for solids and filters; and Soxhlet extraction (Section 12.4) for tissues. Acid/base back-
extraction (Section 12.5) is used for initial cleanup of extracts.
Macro-concentration procedures include: rotary evaporation (Section 12.6.1), heating mantle
(Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-
concentration uses nitrogen evaporation (Section 12.7).
12.2 Extraction of aqueous liquids
12.2.1 Solid-phase extraction of samples containing less than one percent solids
12.2.1.1 Disk preparation
12.2.1.1.1 Remove the test tube from the suction flask (Figure 4). Place an
SPE disk on the base of the filter holder and wet with methylene
chloride. While holding a GMF 150 filter above the SPE disk
with tweezers, wet the filter with methylene chloride and lay the
filter on the SPE disk, making sure that air is not trapped
between the filter and disk. Clamp the filter and SPE disk
between the 1-L glass reservoir and the vacuum filtration flask.
12.2.1.1.2 Rinse the sides of the reservoir with approx 15 mL of methylene
chloride using a squeeze bottle or pipet. Apply vacuum
momentarily until a few drops appear at the drip tip. Release the
vacuum and allow the filter/disk to soak for approx one minute.
Apply vacuum and draw all of the methylene chloride through
the filter/disk. Repeat the wash step with approx 15 mL of
acetone and allow the filter/disk to air dry.
12.2.1.2 Sample extraction
12.2.1.2.1 Pre-wet the disk by adding approx 20 mL of methanol to the
reservoir. Pull most of the methanol through the filter/disk,
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Method 1668B November 2008
retaining a layer of methanol approx 2 mm thick on the filter.
Do not allow the filter/disk to go dry from this point until the
extraction is completed.
12.2.1.2.2 Add approx 20 mL of reagent water to the reservoir and pull
most through, leaving a layer approx 2 mm thick on the
filter/disk.
12.2.1.2.3 Allow the sample (Section 11.4.2.6) to stand for 1-2 hours, if
necessary, to settle the suspended particles. Decant the clear
layer of the sample, the blank (Section 11.4.2.4), or IPR/OPR
aliquot (Section 11.4.2.5) into its respective reservoir and turn on
the vacuum to begin the extraction. Adjust the vacuum to
complete the extraction in no less than 10 minutes. For samples
containing a high concentration of particles (suspended solids),
the extraction time may be an hour or longer.
12.2.1.2.4 Before all of the sample has been pulled through the filter/disk,
add approx 50 mL of reagent water to the sample bottle, swirl to
suspend the solids (if present), and pour into the reservoir. Pull
through the filter/disk. Use additional reagent water rinses until
all solids are removed.
12.2.1.2.5 Before all of the sample and rinses have been pulled through the
filter/disk, rinse the sides of the reservoir with small portions of
reagent water.
12.2.1.2.6 Partially dry the filter/disk under vacuum for approx 3 minutes.
12.2.1.3 Elution of the filter/disk
12.2.1.3.1 Release the vacuum, remove the entire filter/disk/reservoir
assembly from the vacuum flask, and empty the flask. Insert a
test tube for eluant collection into the flask. The test tube should
have sufficient capacity to contain the total volume of the elution
solvent (approx 50 mL) and should fit around the drip tip. The
drip tip should protrude into the test tube to preclude loss of
sample from spattering when vacuum is applied. Reassemble the
filter/disk/reservoir assembly on the vacuum flask.
12.2.1.3.2 Wet the filter/disk with 4-5 mL of acetone. Allow the acetone to
spread evenly across the disk and soak for 15-20 seconds. Pull
the acetone through the disk, releasing the vacuum when approx
1 mm thickness remains on the filter.
12.2.1.3.3 Rinse the sample bottle with approx 20 mL of methylene
chloride and transfer to the reservoir. Pull approx half of the
solvent through the filter/disk and release the vacuum. Allow
38
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Method 1668B November 2008
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 a2-L separatory funnel.
Rinse the bottle or flask twice with 5 mL of reagent water and add these
rinses to the separatory funnel.
12.2.2.2 Add 60 mL methylene chloride to the empty sample bottle. Seal the bottle
and shake 60 seconds to rinse the inner surface. Transfer the solvent to the
separatory funnel, and extract the sample by shaking the funnel for 2
minutes with periodic venting. Allow the organic layer to separate from the
aqueous phase for a minimum of 10 minutes. If an emulsion forms and is
more than one-third the volume of the solvent layer, employ mechanical
techniques to complete the phase separation (see note below). Drain the
methylene chloride extract through a solvent-rinsed glass funnel
approximately one-half full of granular anhydrous sodium sulfate (Section
7.2.1) supported on clean glass-fiber paper into a solvent-rinsed
concentration device (Section 12.6).
Note: If an emulsion forms, the laboratory must employ mechanical techniques to complete the
phase separation. The optimum technique depends upon the sample, but may include stirring,
filtration through glass wool, use of phase separation paper, centrifugation, use of an ultrasonic
bath with ice, addition ofNaCl, 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
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Method 1668B November 2008
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.
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.
40
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Method 1668B November 2008
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
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.
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.
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Method 1668B November 2008
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 blowdown procedure (Section
12.7) and a water bath temperature of 60 °C. 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:
Percent lipid = Weight of residue (g) x m
Weight of tissue (g)
12.4.9.4 The laboratory should determine the lipid content of the blank, IPR, and
OPRto 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 (Section 12.7). If back-extraction is necessary, spike 1.0 mL of the Labeled
cleanup standard spiking solution (Section 7.13) into the separatory funnels containing
the sample and QC extracts from Section 12.2.3.4 or 12.3.9.
12.5.2 Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1).
Shake for 2 minutes with periodic venting into a hood. Remove and discard the
aqueous layer. Repeat the base washing until no color is visible in the aqueous layer,
to a maximum of four washings. Minimize contact time between the extract and the
base to prevent degradation of the CBs. Stronger potassium hydroxide solutions may
be employed for back-extraction, provided that the laboratory meets the specifications
for labeled compound recovery and demonstrates acceptable performance using the
procedure in Section 9.2.
12.5.3 Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the
same way as with base. Discard the aqueous layer.
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Method 1668B November 2008
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 °C. 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.
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Method 1668B November 2008
12.6.1.5 Proceed to Section 12.6.4 for preparation for back-extraction or micro-
concentration and solvent exchange.
12.6.2 Heating mantle—Concentrate the extracts in separate round-bottom flasks.
12.6.2.1 Add one or two clean boiling chips to the round-bottom flask, and attach a
three-ball macro Snyder column. Prewet the column by adding
approximately 1 mL of solvent through the top. Place the round-bottom
flask in a heating mantle, and apply heat as required to complete the
concentration in 15 to 20 minutes. At the proper rate of distillation, the balls
of the column will actively chatter, but the chambers will not flood.
12.6.2.2 When the liquid has reached an apparent volume of approximately 10 mL,
remove the round-bottom flask from the heating mantle and allow the solvent
to drain and cool for at least 10 minutes. Remove the Snyder column and
rinse the glass joint into the receiver with small portions of solvent.
12.6.2.3 Proceed to Section 12.6.4 for preparation for back-extraction or micro-
concentration and solvent exchange.
12.6.3 Kuderna-Danish (K-D)—Concentrate the extracts in separate 500-mL K-D flasks
equipped with 10-mL concentrator tubes. The K-D technique is used for solvents such
as methylene chloride and hexane. Toluene is difficult to concentrate using the K-D
technique unless a water bath fed by a steam generator is used.
12.6.3.1 Add 1 to 2 clean boiling chips to the receiver. Attach a three-ball macro
Snyder column. Prewet the column by adding approximately 1 mL of
solvent through the top. Place the K-D apparatus in a hot water bath so that
the entire lower rounded surface of the flask is bathed with steam.
12.6.3.2 Adjust the vertical position of the apparatus and the water temperature as
required to complete the concentration in 15 to 20 minutes. At the proper
rate of distillation, the balls of the column will actively chatter but the
chambers will not flood.
12.6.3.3 When the liquid has reached an apparent volume of 1 mL, remove the K-D
apparatus from the bath and allow the solvent to drain and cool for at least 10
minutes. Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 mL of solvent. A 5-mL syringe is
recommended for this operation.
12.6.3.4 Remove the three-ball Snyder column, add a fresh boiling chip, and attach a
two ball micro Snyder column to the concentrator tube. Prewet the column
by adding approximately 0.5 mL of solvent through the top. Place the
apparatus in the hot water bath.
12.6.3.5 Adjust the vertical position and the water temperature as required to
complete the concentration in 5 to 10 minutes. At the proper rate of
44
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Method 1668B November 2008
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 appara-
tus from the water bath and allow to drain and cool for at least 10 minutes.
12.6.3.7 Proceed to 12.6.4 for preparation for back-extraction or micro-concentration
and solvent exchange.
12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange
12.6.4.1 For back-extraction (Section 12.5), transfer the extract to a 250-mL
separatory funnel. Rinse the concentration vessel with small portions of
hexane, adjust the hexane volume in the separatory funnel to 10 to 20 mL,
and proceed to back-extraction (Section 12.5).
12.6.4.2 For determination of the weight of residue in the extract, or for clean-up
procedures other than back-extraction, transfer the extract to a blowdown
vial using 2-3 rinses of solvent. Proceed with micro-concentration and
solvent exchange (Section 12.7).
12.7 Micro-concentration and solvent exchange
12.7.1 Extracts to be subjected to GPC cleanup are exchanged into methylene chloride.
Extracts to be cleaned up using silica gel, carbon, Florisil, and/or HPLC are exchanged
into hexane.
12.7.2 Transfer the vial containing the sample extract to a nitrogen evaporation device. Adjust
the flow of nitrogen so that the surface of the solvent is just visibly disturbed.
Note: A large vortex in the solvent may cause analyte loss.
12.7.3 Lower the vial into a 45 °C 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 \\L, 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 \\L. 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
45
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Method 1668B November 2008
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 (iL. Add 20 (iL of nonane to the vial, and evaporate the
solvent to the level of the nonane. Seal the vial and label with the sample number.
Store in the dark at room temperature until ready for GC/MS analysis. If GC/MS
analysis will not be performed on the same day, store the vial at less than -10 °C.
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
46
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Method 1668B November 2008
13.2.1.1 Place 70to 75 g of SX-3 Bio-beads (Section 6.7.1.1) in a400-to 500-mL
beaker.
13.2.1.2 Cover the beads with methylene chloride and allow to swell overnight (a
minimum of 12 hours).
13.2.1.3 Transfer the swelled beads to the column (Section 6.7.1.1) and pump solvent
through the column, from bottom to top, at 4.5 to 5.5 mL/minute prior to
connecting the column to the detector.
13.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig and purge for 4 to 5 hours to remove air.
Maintain a head pressure of 7 to 10 psig. Connect the column to the detector
(Section 6.7.1.4).
13.2.2 Column calibration
13.2.2.1 Load 5 mL of the GPC calibration solution (Section 7.4) into the sample
loop.
13.2.2.2 Inject the GPC calibration solution and record the signal from the detector.
The elution pattern will be corn oil, BEHP, methoxychlor, perylene, and
sulfur.
13.2.2.3 Set the "dump time" to allow >85% removal of BEHP and >85% collection
of methoxychlor.
13.2.2.4 Set the "collect time" to the time of the sulfur peak maximum.
13.2.2.5 Verify calibration with the GPC calibration solution after every 20 extracts.
Calibration is verified if the recovery of the methoxychlor is greater than
85%. If calibration is not verified, the system must be recalibrated using the
GPC calibration solution, and the previous sample batch must be re-extracted
and cleaned up using the calibrated GPC system.
13.2.3 Extract cleanup—GPC requires that the column not be overloaded. The column
specified in this Method is designed to handle a maximum of 0.5 g of material from an
aqueous, soil, or mixed-phase sample in a 5-mL extract, and has been shown to handle
1.5 g of lipid from a tissue sample in a 5-mL extract. If the extract is known or
expected to contain more than these amounts, the extract is split into aliquots for GPC,
and the aliquots are combined after elution from the column. The residue content of
the extract may be obtained gravimetrically by evaporating the solvent from a 50-(iL
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.
47
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Method 1668B November 2008
13.2.3.2 Elute the extract using the calibration data determined in Section 13.2.2.
Collect the eluate in a clean 400- to 500-mL beaker. Allow the system to
rinse for additional 10 minutes before injecting the next sample.
13.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
13.2.3.4 If an extract is encountered that could overload the GPC column to the extent
that carry-over could occur, a 5.0-mL methylene chloride blank must be run
through the system to check for carry-over.
13.2.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or
injection into the GC/MS.
13.3 Silica gel cleanup
13.3.1 Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.4.2).
Pack the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel
(Section 7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4
g granular anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the
adsorbents.
13.3.2 Pre-elute the column with 50 to 100 mL of hexane. Close the stopcock when the
hexane is within 1 mm of the sodium sulfate. Discard the eluate. Check the column
for channeling. If channeling is present, discard the column and prepare another.
13.3.3 Apply the concentrated extract to the column. Open the stopcock until the extract is
within 1 mm of the sodium sulfate.
13.3.4 Rinse the receiver twice with 1-mL portions of hexane, and apply separately to the
column. Elute the CBs with 25 mL of hexane and collect the eluate.
13.3.5 Concentrate the eluate per Section 12.6 and 12.7 for further cleanup or injection into
the HPLC or GC/MS.
13.3.6 For extracts of samples known to contain large quantities of other organic compounds,
it may be advisable to increase the capacity of the silica gel column. This may be
accomplished by increasing the strengths of the acid and basic silica gels. The acid
silica gel (Section 7.5.1.2) may be increased in strength to as much as 40% w/w (6.7 g
sulfuric acid added to 10 g silica gel). The basic silica gel (Section 7.5.1.3) may be
increased in strength to as much as 33% w/w (50 mL IN NaOH added to 100 g silica
gel), or the potassium silicate (Section 7.5.1.4) may be used.
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Method 1668B November 2008
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 ofhexane 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 17)
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 ofhexane
and apply separately to the column. Apply 2 mL ofhexane 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
theHPLCorGC/MS.
13.5 HPLC (References 4 and 18)
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.
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Method 1668B November 2008
1 3.5.1 .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.
1 3.5.1 .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.
1 3.5.2 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 (ig of a given CB,
depending on the congener (Reference 18). 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.
1 3.5.2.1 Rinse the sides of the vial containing the sample and adjust to the volume
required for the sample loop for injection.
1 3.5.2.2 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 (ig of total CBs is encountered, a
blank must be run through the system to check for carry-over.
1 3.5.2.5 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
1 3.6.1 Prepare the column as given in Section 7.5.3.
1 3.6.2 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.
1 3.6.3 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.
1 3.6.4 Remove a small portion (e.g, 50 (iL) 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.
1 3.6.5 If necessary, exchange the extract to a solvent suitable for the additional cleanups to be
used (Section 13.2-13.5 and 13.7).
50
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Method 1668B November 2008
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 (iL as described in Section 12.7 and
proceed with the analysis in Section 14.
13.7 Florisil cleanup (Reference 19)
13.7.1 Begin to drain the n-hexane from the column (Section 7.5.4.1.2). Adjust the flow rate
of eluantto 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% etherhexane
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
theHPLCorGC/MS.
14.0 HRGC/HRMS analysis
14.1 Establish the operating conditions given in Section 10.1.
14.2 Add 2 (iL of the labeled injection internal standard spiking solution (Section 7.14) to the 20 (iL
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 (iL) with pure nonane (18 (iL if 2 (iL injections are used).
14.3 Inject 1.0 or 2.0 (iL of the concentrated extract containing the Labeled injection internal
standards using on-column or splitless injection. The volume injected must be identical to the
volume used for calibration (Section 10.3).
14.3.1 Start the GC column initial isothermal hold upon injection. Start MS data collection
after the solvent peak elutes.
14.3.2 Monitor the exact m/z's at each LOC throughout the LOC retention time window.
Where warranted, monitor m/z's associated with congeners at higher levels of
chlorination to assure that fragments are not interfering with the m/z's for congeners at
lower levels of chlorination. Also where warranted, monitor m/z's associated with
interferents expected to be present.
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Method 1668B November 2008
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, analysis of the CS-3 calibration verification (VER) standard (Section 7.10.1 and Table 5)
and the diluted combined 209 congener solution (Section 7.10.2.2 and Table 5) must be used 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 Sections 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 the VER (CS-3) standard using the procedure in Section 14.
15.3.2 The m/z abundance ratios for all CBs 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 concentration of the Toxics/LOC CBs by isotope dilution (Section 17.1).
These concentrations are computed based on the calibration data in Section 10.
15.3.5 For each compound, compare the concentration with the calibration verification limit in
Table 6. If all compounds meet the acceptance criteria, calibration has been verified
and analysis of standards and sample extracts may proceed. If, however, any
compound fails its respective limit, the measurement system is not performing
properly. In this event, prepare a fresh calibration standard or correct the problem and
repeat the resolution (Section 15.2) and verification (Section 15.3) tests, or recalibrate
(Section 10). If recalibration is required, recalibration for the 209 congeners (Section
10.5) must also be performed.
15.4 Retention times and GC resolution
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Method 1668B November 2008
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, inject the Diluted combined 209
congener solution (Section 7.10.2.2 and Table 5).
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, relative retention times, and response factors for
the all congeners except the Toxics and LOG CBs. For the Toxics and LOG
CBs, the multi-point calibration data must be used (see Section 10.4 and
15.3).
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) priorto 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).
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Method 1668B November 2008
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 - 2SRto 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 PFKm/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).
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Method 1668B November 2008
Note: For native CBs determined by internal standard quantitation, a given CB congener may
fall within more than 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-defming compounds
to every sample prior to extraction, correction for recovery of the CB 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:
(Al + A2J C,
C (ng/mL) = —-? 2—^
" (Al, + A2) RR
Where:
Cex = The concentration of the PCB in the extract, 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 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) using the response factors deter-
mined from the calibration data (Section 10.5) and the following equation:
55
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Method 1668B November 2008
(Al, H- A2) Cis
C (ng/mL) =
(Alis + A2J RF
Where:
Cex = The concentration of the labeled compound in the extract, and the other terms
are as defined in Section 10.6.1.
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:
Recovery (%) = Concentration found (ng/mL) x
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:
Concentration in solid (ng/kg) = ——
Where:
Cex = The concentration of the compound in the extract.
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),
as follows:
(C x VJ
Concentration in aqueous phase (pg/L) = 1000 x ——
Where:
Cex = The concentration of the compound in the extract.
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/(iL 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.
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Method 1668B November 2008
17.6 Reporting of results—Results are reported to three significant figures for the CBs and labeled
compounds found in all standards, blanks, and samples.
17.6.1 Reporting units and levels
17.6.1.1 Aqueous samples—Report results in pg/L (parts-per-quadrillion).
17.6.1.2 Samples containing greater than 1% solids (soils, sediments, filter cake,
compost)—Report results in ng/kg based on the dry weight of the sample.
Report the percent solids so that the result may be converted to aqueous
units.
17.6.1.3 Tissues—Report results in ng/kg of wet tissue, not on the basis of the lipid
content of the tissue. Report the percent lipid content, so that the data user
can calculate the concentration on a lipid basis if desired.
17.6.1.4 Reporting level
17.6.1.4.1 Report results above the minimum level of quantitation (ML) for
analyses of blanks, standards, and samples. The estimated
minimum levels (EMLs) in Table 2 are based on common
laboratory contamination levels. A laboratory may establish an
ML for a CB lower than the EMLs 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 EML in Table 2. Significant means
that the ML for the congener is no less than 2 standard
deviations above the average (mean) level in the minimum of 10
blanks (Reference 20). 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 (or EML Table 2) to 3
significant figures. Report results below the ML (or EML) as
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Method 1668B November 2008
procedure for blank correction (Reference 20) is that a result is
significantly above the blank level, and the level in the blank
may be subtracted, if the result is 2 standard deviations above the
mean (average) of results of analyses of 10 or more blanks for a
sample medium.
17.6.2 Results for a CB in a sample that has been diluted are reported at the least dilute level
at which the area at the quantitation m/z is within the calibration range (Section 17.5).
17.6.3 For a CB having a labeled analog, report results at the least dilute level at which the
area at the quantitation m/z is within the calibration range (Section 17.5) and the
labeled compound recovery is within the normal range for the Method (Section 9.3 and
Table 6).
17.6.4 If requested, the total concentration of all congeners at a given level of chlorination
(homolog; i.e., total TrCB, total PeCB, total HxCB) may be reported by summing the
concentrations of all congeners identified at that LOG, including both the Toxics and
other congeners. Also if requested, total CBs may be reported by summing all
congeners identified at all LOCs.
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
(iL (Section 12.7); 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 (iL 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
58
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Method 1668B November 2008
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 calibration verification standard (Section 7.10.1 and Table 5) must be
analyzed and calibration verified (Section 15.3).
18.6.3 If the calibration cannot be verified, a new calibration must be performed and the
original sample extract reanalyzed.
18.6.4 If calibration is verified and the diluted sample does not meet the limits for labeled
compound recovery, the Method does not apply to the sample being analyzed and the
result may not be reported or used for permitting or regulatory compliance purposes.
In this case, alternate extraction and cleanup procedures in this Method or an alternate
GC column must be employed to resolve the interference. If all cleanup procedures in
this Method and an alternate GC column have been employed and labeled compound
recovery remains outside of the normal range, extraction and/or cleanup procedures
that are beyond this scope of this Method will be required to analyze the sample.
19.0 Pollution prevention
19.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or
toxicity of waste at the point of generation. Many opportunities for pollution prevention exist
in laboratory operation. EPA has established a preferred hierarchy of environmental
management techniques that places pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to
address waste generation. When wastes cannot be reduced feasiblely 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.
59
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Method 1668B November 2008
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 HC1 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 °C. Low-level waste such as absorbent paper, tissues, animal
remains, and plastic gloves may be burned in an appropriate incinerator. Gross quantities
(milligrams) should be packaged securely and disposed of through commercial or governmental
channels that are capable of handling extremely toxic wastes.
20.4 Liquid or soluble waste should be dissolved in methanol or ethanol and irradiated with
ultraviolet light with a wavelength shorter than 290 nm for several days. Use F40 BL or
equivalent lamps. Analyze liquid wastes, and dispose of the solutions when the CBs can no
longer be detected.
20.5 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste
Reduction, available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
21.0 Method performance
The original version of Method 1668 was validated in two separate single-laboratory studies.
The next version, 1668A, was validated and data were collected in a single laboratory
(Reference 21), and published in 1999. In 2003 - 2004, EPA conducted and peer-reviewed an
interlaboratory method validation study of 1668A (Reference 22), and subsequently published
interlaboratory performance data in this Method, 1668B. 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
60
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Method 1668B November 2008
Monitoring and Bioeffects Assessment Division, Office of Ocean Resources Conservation and
Assessment, National Ocean Service, National Oceanic and Atmospheric Administration, U.S.
Department of Commerce, N/ORCA2, SSMC4, 1305 East-West Highway, Silver Spring, MD
20910,p 3, 1998.
3 Kuehl, D.W., B.C. Butterworth, J. Libal, and P. Marquis, "An Isotope Dilution High Resolution
Gas Chromatography-High Resolution Mass Spectrometric Method for the Determination of
Coplanar Polychlorinated Biphenyls: Application to Fish and Marine Mammals," Chemosphere
22:9-10,849-858, 1991.
4 Echols, Kathy, Robert Gale, Donald E. Tillitt, Ted Schwartz, and Jerome O'Laughlin, "An
Automated HPLC Method for the Fractionation of Polychlorinated Biphenyls, Polychlorinated
Dibenzo-p-dioxins, and Polychlorinated Dibenzofurans in Fish Tissue on a Porous Graphitic
Carbon Column," Environmental Toxicology and Chemistry 16:8 1590-1597, 1997.
5 "Analysis of Coplanar CBs," Axys Environmental Systems Ltd., Fax from Mary McFarland to
Dale Rushneck dated November 25, 1994, available from U.S. Environmental Protection
Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.
6 "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health
Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-
277256.
7 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
8 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
9 "Standard Methods for the Examination of Water and Wastewater," 18th edition and later
revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC 20005, 1-
35: Section 1090 (Safety), 1992.
10 "Method 613—2,3,7,8-Tetrachlorodibenzo-/?-dioxin," 40 CFR 136 (49 FR 43234), October 26,
1984, Section 4.1.
11 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.
12 Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American
Laboratory, 15: 56-83, 1983.
13 "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916 Race
Street, Philadelphia, PA 19103-1187, 1980.
14 "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL, Cincinnati, OH
45268, EPA 600/4-70-020, March 1979.
61
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Method 1668B November 2008
15 "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA
EMSL, Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.
16 "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, March 1990.
17 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.
18 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.
19 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.
20 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.
21 "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.
22 "Method 1668A Interlaboratory Validation Study Report," November 2008, EPA-821-R-08-
021, U.S. Environmental Protection Agency, Engineering and Analysis Division (4303T), 1200
Pennsylvania Avenue NW, Washington, DC 20460.
62
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Method 1668B November 2008
23.0 Tables and Figures
Table 1. Names, congener numbers, and CAS Registry numbers for native and labeled chlorinated
biphenyl (CB) congeners determined by isotope dilution and internal standard HRGC/HRMS.
CB congener1
2-MoCB
3-MoCB
4-MoCB
2,2'-DiCB
2,3-DiCB
2,3'-DiCB
2,4-DiCB
2,4'-DiCB3
2,5-DiCB
2,6-DiCB
3,3'-DiCB
3,4-DiCB
3,4'-DiCB
3,5-DiCB
4,4'-DiCB
2,2',3-TrCB
2,2',4-TrCB
2,2',5-TrCB3
2,2',6-TrCB
2,3,3'-TrCB
2,3,4-TrCB
2,3,4'-TrCB
2,3,5-TrCB
2,3,6-TrCB
2,3',4-TrCB
2,3',5-TrCB
2,3',6-TrCB
2,4,4'-TrCB3
2,4,5-TrCB
2,4,6-TrCB
2,4',5-TrCB
2,4',6-TrCB
2',3,4-TrCB
Congener
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CAS registry
number
2051-60-7
2051-61-8
2051-62-9
13029-08-8
16605-91-7
25569-80-6
33284-50-3
34883-43-7
34883-39-1
33146-45-1
2050-67-1
2974-92-7
2974-90-5
34883-41-5
2050-68-2
38444-78-9
37680-66-3
37680-65-2
38444-73-4
38444-84-7
55702-46-0
38444-85-8
55720-44-0
55702-45-9
55712-37-3
38444-81-4
38444-76-7
7012-37-5
15862-07-4
35693-92-6
16606-02-3
38444-77-8
38444-86-9
Labeled analog
13C12-2-MoCB2
13C12-4-MoCB2
13C12-2,2'-DiCB2
13C12-2,5-DiCB4
13C12-4,4'-DiCB2
13C12-2,2',6-TrCB2
13C12-2,4,4'-TriCB5
Congener
analog
1L
3L
4L
9L
15L
19L
28L
CAS registry
number
234432-85-0
208263-77-8
234432-86-1
250694-89-4
208263-67-6
234432-87-2
208263-76-7
63
-------
Method 1668B November 2008
CB congener1
2',3,5-TrCB
3,3',4-TrCB
3,3',5-TrCB
3,4,4'-TrCB
3,4,5-TrCB
3,4',5-TrCB
2,2',3,3'-TeCB
2,2',3,4-TeCB
2,2',3,4'-TeCB
2,2',3,5-TeCB
2,2',3,5'-TeCB3
2,2',3,6-TeCB
2,2',3,6'-TeCB
2,2',4,4'-TeCB
2,2',4,5-TeCB
2,2',4,5'-TeCB
2,2',4,6-TeCB
2,2',4,6'-TeCB
2,2',5,5'-TeCB3
2,2',5,6'-TeCB
2,2',6,6'-TeCB
2,3,3',4'-TeCB
2,3,3',4'-TeCB
2,3,3',5-TeCB
2,3,3',5'-TeCB
2,3,3',6-TeCB
2,3,4,4'-TeCB
2,3,4,5-TeCB
2,3,4,6-TeCB
2,3,4',5-TeCB
2,3,4',6-TeCB
2,3,5,6-TeCB
2,3',4,4'-TeCB3
2,3',4,5-TeCB
2,3',4,5'-TeCB
2,3',4,6-TeCB
2,3',4',5-TeCB
Congener
number
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
CAS registry
number
37680-68-5
37680-69-6
38444-87-0
38444-90-5
53555-66-1
38444-88-1
38444-93-8
52663-59-9
36559-22-5
70362-46-8
41464-39-5
70362-45-7
41464-47-5
2437-79-8
70362-47-9
41464-40-8
62796-65-0
68194-04-7
35693-99-3
41464-41-9
15968-05-5
74338-24-2
41464-43-1
70424-67-8
41464-49-7
74472-33-6
33025-41-1
33284-53-6
54230-22-7
74472-34-7
52663-58-8
33284-54-7
32598-10-0
73575-53-8
73575-52-7
60233-24-1
32598-11-1
Labeled analog
13C12-3,4,4'-TrCB2
13C12-2,2',5,5'-TeCB4
13C12-2,2',6,6'-TeCB2
Congener
analog
37L
52L
54L
CAS registry
number
208263-79-0
208263-80-3
234432-88-3
64
-------
Method 1668B November 2008
CB congener1
2,3',4',6-TeCB
2,3',5,5'-TeCB
2,3',5',6-TeCB
2,4,4',5-TeCB
2,4,4',6-TeCB
2',3,4,5-TeCB
3,3',4,4'-TeCB3'6
3,3',4,5-TeCB
3,3',4,5'-TeCB
3,3',5,5'-TeCB
3,4,4',5-TeCB6
2,2',3,3',4-PeCB
2,2',3,3',5-PeCB
2,2',3,3',6-PeCB
2,2',3,4,4'-PeCB
2,2',3,4,5-PeCB
2,2',3,4,5'-PeCB
2,2',3,4,6-PeCB
2,2',3,4,6'-PeCB
2,2',3,4',5-PeCB
2,2',3,4',6-PeCB
2,2',3,5,5'-PeCB
2,2',3,5,6-PeCB
2,2',3,5,6'-PeCB
2,2',3,5',6-PeCB
2,2',3,6,6'-PeCB
2,2',3',4,5-PeCB
2,2',3',4,6-PeCB
2,2',4,4',5-PeCB
2,2',4,4',6-PeCB
2,2',4,5,5'-PeCB3
2,2',4,5,6'-PeCB
2,2',4,5,'6-PeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB3'6
2,3,3',4,5-PeCB
2,3,3',4',5-PeCB
Congener
number
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
CAS registry
number
41464-46-4
41464-42-0
74338-23-1
32690-93-0
32598-12-2
70362-48-0
32598-13-3
70362-49-1
41464-48-6
33284-52-5
70362-50-4
52663-62-4
60145-20-2
52663-60-2
65510-45-4
55312-69-1
38380-02-8
55215-17-3
73575-57-2
68194-07-0
68194-05-8
52663-61-3
73575-56-1
73575-55-0
38379-99-6
73575-54-9
41464-51-1
60233-25-2
38380-01-7
39485-83-1
37680-73-2
68194-06-9
60145-21-3
56558-16-8
32598-14-4
70424-69-0
70424-68-9
Labeled analog
13C12-3,3',4,4'-TeCB2'7
13C12-3,4,4',5-TeCB7
13C12-2,2',4,5,5'-PeCB4
13C12-2,2',4,6,6'-PeCB2
13C12-2,3,3',4,4'-PeCB7
Congener
analog
77L
81L
101L
104L
105L
CAS registry
number
105600-23-5
208461-24-9
104130-39-4
234432-89-4
208263-62-1
65
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Method 1668B November 2008
CB congener1
2,3,3',4,5'-PeCB
2,3,3',4,6-PeCB
2,3,3',4',6-PeCB
2,3,3',5,5'-PeCB
2,3,3',5,6-PeCB
2,3,3',5',6-PeCB
2,3,4,4',5-PeCB6
2,3,4,4',6-PeCB
2,3,4,5,6-PeCB
2,3,4',5,6-PeCB
2,3',4,4',5-PeCB3'6
2,3',4,4',6-PeCB
2,3',4,5,5'-PeCB
2,3',4,5,'6-PeCB
2',3,3',4,5-PeCB
2',3,4,4',5-PeCB6
2',3,4,5,5'-PeCB
2',3,4,5,6'-PeCB
3,3',4,4',5-PeCB3'6
3,3',4,5,5'-PeCB
2,2',3,3',4,4'-HxCB3
2,2',3,3',4,5-HxCB
2,2',3,3',4,5'-HxCB
2,2',3,3',4,6-HxCB
2,2',3,3',4,6'-HxCB
2,2',3,3',5,5'-HxCB
2,2',3,3',5,6-HxCB
2,2',3,3',5,6'-HxCB
2,2',3,3',6,6'-HxCB
2,2',3,4,4',5-HxCB
2,2',3,4,4',5'-HxCB3
2,2',3,4,4',6-HxCB
2,2',3,4,4',6'-HxCB
2,2',3,4,5,5'-HxCB
2,2',3,4,5,6-HxCB
2,2',3,4,5,6'-HxCB
2,2',3,4,5',6-HxCB
Congener
number
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
CAS registry
number
70362-41-3
74472-35-8
38380-03-9
39635-32-0
74472-36-9
68194-10-5
74472-37-0
74472-38-1
18259-05-7
68194-11-6
31508-00-6
56558-17-9
68194-12-7
56558-18-0
76842-07-4
65510-44-3
70424-70-3
74472-39-2
57465-28-8
39635-33-1
38380-07-3
55215-18-4
52663-66-8
61798-70-7
38380-05-1
35694-04-3
52704-70-8
52744-13-5
38411-22-2
35694-06-5
35065-28-2
56030-56-9
59291-64-4
52712-04-6
41411-61-4
68194-15-0
68194-14-9
Labeled analog
13C12-2,3,3',5,5'-PeCB5
13C12-2,3,4,4',5-PeCB7
13C12-2,3',4,4',5-PeCB7
13C12-2',3,4,4',5-PeCB7
13C12-3,3',4,4',5-PeCB2'7
13C12-2,2',3,4,4',5'-HxCB4
Congener
analog
111L
114L
118L
123L
126L
138L
CAS registry
number
235416-29-2
208263-63-2
104130-40-7
208263-64-3
208263-65-4
208263-66-5
66
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Method 1668B November 2008
CB congener1
2,2',3,4,6,6'-HxCB
2,2',3,4',5,5'-HxCB
2,2',3,4',5,6-HxCB
2,2',3,4',5,6'-HxCB
2,2',3,4',5',6-HxCB
2,2',3,4',6,6'-HxCB
2,2',3,5,5',6-HxCB
2,2',3,5,6,6'-HxCB
2,2',4,4',5,5'-HxCB3
2,2',4,4',5',6-HxCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB6
2,3,3',4,4',5'-HxCB6
2,3,3',4,4',6-HxCB
2,3,3',4,5,5'-HxCB
2,3,3',4,5,6-HxCB
2,3,3',4,5',6-HxCB
2,3,3',4',5,5'-HxCB
2,3,3',4',5,6-HxCB
2,3,3',4',5',6-HxCB
2,3,3',5,5',6-HxCB
2,3,4,4',5,6-HxCB
2 3' 4 4' 5 5'-HxCB6
2,3',4,4',5',6-HxCB
3,3',4,4',5,5'-HxCB3'6
2,2',3,3',4,4',5-HpCB3
2,2'3,3',4,4',6-HpCB
2,2',3,3',4,5,5'-HpCB
2,2',3,3',4,5,6-HpCB
2,2',3,3',4,5,6'-HpCB
2 2' 3 3' 4 5' 6-HpCB
2,2',3,3',4,6,6'-HpCB
2,2',3,3',4',5,6-HpCB
2 2' 3 3' 5 5' 6-HpCB
2,2',3,3',5,6,6'-HpCB
2,2',3,4,4',5,5'-HpCB3
2,2',3,4,4',5,6-HpCB
Congener
number
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
CAS registry
number
74472-40-5
51908-16-8
68194-13-8
74472-41-6
38380-04-0
68194-08-1
52663-63-5
68194-09-2
35065-27-1
60145-22-4
33979-03-2
38380-08-4
69782-90-7
74472-42-7
39635-35-3
41411-62-5
74472-43-8
39635-34-2
74472-44-9
74472-45-0
74472-46-1
41411-63-6
52663-72-6
59291-65-5
32774-16-6
35065-30-6
52663-71-5
52663-74-8
68194-16-1
38411-25-5
40186-70-7
52663-65-7
52663-70-4
52663-67-9
52663-64-6
35065-29-3
74472-47-2
Labeled analog
13C12-2,2',4,4',6,6'-HxCB2
13C12-2,3,3',4,4',5-HxCB7
13C12-2,3,3',4,4',5'-HxCB7
13C12-2 3' 4 4' 5 5'-HxCB7
13C12-3,3',4,4',5,5'-HxCB2-7
13C12-2,2',3,3',4,4',5-HpCB
13C12-2 2' 3 3' 5 5' 6-HpCB5
13C12-2,2',3,4,4',5,5'-HpCB
Congener
analog
155L
156L
157L
167L
169L
170L
178L
180L
CAS registry
number
234432-90-7
208263-68-7
235416-30-5
208263-69-8
208263-70-1
160901-80-4
232919-67-4
160901-82-6
67
-------
Method 1668B November 2008
CB congener1
2,2',3,4,4',5,6'-HpCB
2,2',3,4,4',5',6-HpCB
2,2',3,4,4',6,6'-HpCB
2,2',3,4,5,5',6-HpCB
2,2',3,4,5,6,6'-HpCB
2,2',3,4',5,5',6-HpCB3
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB6
2,3,3',4,4',5,6-HpCB
2,3,3',4,4',5',6-HpCB
2,3,3',4,5,5',6-HpCB
2,3,3',4',5,5',6-HpCB
2,2',3,3',4,4',5,5'-OcCB
2,2',3,3',4,4',5,6-OcCB3
2,2',3,3',4,4',5,6'-OcCB
2,2',3,3',4,4',6,6'-OcCB
2,2',3,3',4,5,5',6-OcCB
2,2',3,3',4,5,5',6'-OcCB
2,2',3,3',4,5,6,6'-OcCB
2,2',3,3',4,5',6,6'-OcCB
2,2',3,3',5,5',6,6'-OcCB
2,2',3,4,4',5,5',6-OcCB
2,2',3,4,4',5,6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2 2' 3 3' 4 4' 5 5' 6-NoCB3
2,2',3,3',4,4',5,6,6'-NoCB
2,2',3,3',4,5,5',6,6'-NoCB
DeCB3
Congener
number
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
CAS registry
number
60145-23-5
52663-69-1
74472-48-3
52712-05-7
74472-49-4
52663-68-0
74487-85-7
39635-31-9
41411-64-7
74472-50-7
74472-51-8
69782-91-8
35694-08-7
52663-78-2
42740-50-1
33091-17-7
68194-17-2
52663-75-9
52663-73-7
40186-71-8
2136-99-4
52663-76-0
74472-52-9
74472-53-0
40186-72-9
52663-79-3
52663-77-1
2051-24-3
Labeled analog
13C12-2,2',3,4',5,6,6'-HpCB2
13C12-2,3,3',4,4',5,5'-HpCB2'7
13C12-2,2',3,3',4,4',5,5'-OcCB4
13C12-2,2',3,3',5,5',6,6'-OcCB2
13C12-2,3,3',4,4',5,5',6-OcCB2
13C,,-22'3 3' 4 4' 5 5' 6-
NoCB2
13C12-2,2',3,3',4,5,5',6,6'-
NoCB2
13C12-DeCB2
Congener
analog
188L
189L
194L
202L
205L
206L
208L
209L
CAS registry
number
234432-91-8
208263-73-4
208263-74-5
105600-26-8
234446-64-1
208263-75-6
234432-92-9
105600-27-9
Abbreviations for chlorination levels
MoCB monochlorobiphenyl
DiCB dichlorobiphenyl
TrCB trichlorobiphenyl
TeCB tetrachlorobiphenyl
PeCB pentachlorobiphenyl
HxCB hexachlorobiphenyl
HpCB heptachlorobiphenyl
OcCB octachlorobiphenyl
NoCB nonachlorobiphenyl
DeCB decachlorobiphenyl
Labeled level of chlorination (LOG) window-defining congener
68
-------
Method 1668B November 2008
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
69
-------
Method 1668B November 2008
Table 2. Retention times (RT), RT references, relative retention times (RRTs), estimated method detection limits (EMDLs), and estimated minimum
levels (EMLs) for the 209 CB congeners on SPB-octyl.
Cl
No.1
Congener No. 2'3
RTRef
RT5
RRT6
RRT limits7
Window
(sec)8
Quantitation reference9
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
EML
Other
(ng/kg)
EMDL
EML
Extract
(pg/nL)
EML
Compounds using 9L (13C12-2,5-DiCB) as Labeled injection internal standard
CB congener
Monochlorobiphenyls
1
1
1
1
2
o
J
1L
3L
3L
13:44
16:08
16:21
1.0012
0.9878
1.0010
0.9988-1.0036
0.9847-0.9908
0.9990-1.0031
-1+3
6
-1+3
1L
1L/3L
3L
82
4
88
200
10
200
8
0.4
9
20
1
20
10
0.5
10
Dichlorobiphenyls
2
2
2
2
2
2
2
2
2
2
2
2
2
4
10
9
7
6
5
8
14
11
13
12
13/12
15
4L
4L
4L
4L
4L
4L
4L
15L
15L
15L
15L
15L
15L
16:40
16:53
18:55
19:07
19:26
19:48
19:56
21:42
22:42
23:03
23:06
23:04
23:26
1.0010
1.0140
1.1361
1.1481
1.1672
1.1892
1.1972
0.9267
0.9694
0.9843
0.9865
0.9851
1.0007
0.9990-1.0030
1.0110-1.0170
1.1331-1.1391
1.1451-1.1512
1.1642-1.1702
1.1862-1.1922
1.1942-1.2002
0.9246-0.9288
0.9673-0.9715
0.9822-0.9865
0.9843-0.9886
0.9829-0.9872
0.9993-1.0021
-1+3
6
6
6
6
6
6
6
6
6
6
6
-1+3
4L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
15L
172
22
20
15
13
11
121
31
105
28
183
500
50
50
50
50
50
500
100
200
100
500
17
2
2
2
1
1
12
3
10
3
18
50
5
5
5
5
5
50
10
20
10
50
20
2
2
2
2
2
20
5
10
5
20
Trichlorobiphenyls
3
19
19L
20:19
1.0008
0.9992-1.0025
-1+3
19L
42
100
4
10
5
70
-------
Method 1668B November 2008
Cl
No.1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Congener No. 2'3
30
18
30/18
17
27
24
16
32
34
23
29
26
26/29
25
31
28
20
28/20
21
33
21/33
22
36
39
38
RTRef4
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
RT5
22:15
22:23
22:19
22:49
23:06
23:14
23:25
24:57
25:17
25:26
25:47
25:48
25:48
26:04
26:25
26:44
26:49
26:47
26:58
27:01
26:59
27:29
29:05
29:30
30:10
RRT6
1.0961
1.1026
1.0993
1.1240
1.1379
1.1445
1.1535
1.2291
1.2455
1.2529
1.2701
1.2709
1.2709
0.8364
0.8476
0.8578
0.8604
0.8594
0.8652
0.8668
0.8658
0.8818
0.9332
0.9465
0.9679
RRT limits7
1.0936-1.0985
1.1002-1.1051
1.0969-1.1018
1.1215-1.1264
1.1355-1.1404
1.1420-1.1470
1.1511-1.1560
1.2266-1.2315
1.2430-1.2479
1.2504-1.2553
1.2660-1.2742
1.2668-1.2750
1.2668-1.2750
0.8348-0.8380
0.8460-0.8492
0.8551-0.8604
0.8578-0.8631
0.8567-0.8620
0.8626-0.8679
0.8642-0.8695
0.8631-0.8684
0.8802-0.8834
0.9316-0.9348
0.9449-0.9481
0.9663-0.9695
Window
(sec)8
6
6
6
6
6
6
6
6
6
6
10
10
10
6
6
10
10
10
10
10
10
6
6
6
6
Quantitation reference9
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
175
86
59
53
35
84
74
50
83
55
152
192
51
90
79
85
83
EML
500
200
200
200
100
200
200
200
200
200
500
500
200
200
200
200
200
Other
(ng/kg)
EMDL
17
9
6
5
4
8
7
5
8
5
15
19
5
9
8
9
8
EML
50
20
20
20
10
20
20
20
20
20
50
50
20
20
20
20
20
Extract
(pg/nL)
EML
20
10
10
10
5
10
10
10
10
10
20
20
10
10
10
10
10
71
-------
Method 1668B November 2008
Cl
No.1
3
3
Congener No. 2'3
35
37
RTRef4
37L
37L
RT5
30:42
31:11
RRT6
0.9850
1.0005
RRT limits7
0.9834-0.9866
0.9995-1.0011
Window
(sec)8
6
-1+3
Quantitation reference9
19L/37L
37L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
77
132
EML
200
500
Other
(ng/kg)
EMDL
8
13
EML
20
50
Extract
(pg/nL)
EML
10
20
Labeled Compounds
1
1
2
2
3
3
1L
3L
4L
15L
19L
37L
9L
9L
9L
9L
9L
52L
13:43
16:20
16:39
23:25
20:18
31:10
0.7257
0.8642
0.8810
1.2390
1.0741
1.0841
0.7125-0.7390
0.8510-0.8774
0.8677-0.8942
1.2302-1.2478
1.0608-1.0873
1.0754-1.0928
30
30
30
20
30
30
9L
9L
9L
9L
9L
52L
Compounds using 52L (13C12-2,2',5,5'-TeCB) as Labeled injection internal standard
CB congener
Tetrachlorobiphenyls
4
4
4
4
4
4
4
4
4
4
4
4
54
50
53
50/53
45
51
45/51
46
52
73
43
69
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
23:51
26:07
26:09
26:08
26:55
26:58
26:57
27:18
28:45
28:52
28:58
29:08
1.0007
1.0958
1.0972
1.0965
1.1294
1.1315
1.1308
1.1455
1.2063
1.2112
1.2154
1.2224
0.9993-1.0021
1.0923-1.0993
1.0937-1.1007
1.0930-1.1000
1.1259-1.1329
1.1280-1.1350
1.1273-1.1343
1.1434-1.1476
1.2042-1.2084
1.2091-1.2133
1.2133-1.2175
1.2189-1.2259
-1+3
10
10
10
10
10
10
6
6
6
6
10
54L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
118
58
51
101
191
160
94
500
200
200
200
500
500
200
12
6
5
10
19
16
9
50
20
20
20
50
50
20
20
10
10
10
20
20
10
115
500
11
50
20
72
-------
Method 1668B November 2008
Cl
No.1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Congener No. 2'3
49
69/49
48
65
47
44
44/47/65
62
75
59
59/62/75
42
41
71
40
41/40/71
64
72
68
57
58
67
63
RTRef4
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
81L
81L
81L
81L
81L
81L
RT5
29:16
29:12
29:33
29:49
29:50
29:53
29:50
30:06
30:08
30:12
30:09
30:26
30:52
30:58
31:01
30:58
31:12
31:59
32:18
32:46
33:05
33:13
33:30
RRT6
1.2280
1.2252
1.2399
1.2510
1.2517
1.2538
1.2517
1.2629
1.2643
1.2671
1.2650
1.2769
1.2951
1.2993
1.3014
1.2993
1.3091
0.8336
0.8419
0.8540
0.8623
0.8658
0.8732
RRT limits7
1.2245-1.2315
1.2217-1.2287
1.2378-1.2420
1.2476-1.2545
1.2483-1.2552
1.2503-1.2573
1.2483-1.2552
1.2594-1.2664
1.2608-1.2678
1.2636-1.2706
1.2615-1.2685
1.2748-1.2790
1.2916-1.2986
1.2958-1.3028
1.2979-1.3049
1.2958-1.3028
1.3070-1.3112
0.8323-0.8349
0.8406-0.8432
0.8527-0.8553
0.8610-0.8636
0.8645-0.8671
0.8719-0.8745
Window
(sec)8
10
10
6
10
10
10
10
10
10
10
10
6
10
10
10
10
6
6
6
6
6
6
6
Quantitation reference9
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
76
195
57
61
119
70
158
149
125
127
147
138
EML
200
500
200
200
500
200
500
500
500
500
500
500
Other
(ng/kg)
EMDL
8
19
6
6
12
7
16
15
12
13
15
14
EML
20
50
20
20
50
20
50
50
50
50
50
50
Extract
(pg/nL)
EML
10
20
10
10
20
10
20
20
20
20
20
20
73
-------
Method 1668B November 2008
Cl
No.1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Congener No. 2'3
61
70
76
74
61/70/74/76
66
55
56
60
80
79
78
81
77
RTRef4
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
77L
RT5
33:46
33:53
33:55
33:57
33:55
34:15
34:28
35:03
35:16
35:32
37:16
37:52
38:23
39:02
RRT6
0.8801
0.8831
0.8840
0.8849
0.8840
0.8927
0.8983
0.9136
0.9192
0.9262
0.9713
0.9870
1.0004
1.0004
RRT limits7
0.8775-0.8827
0.8805-0.8858
0.8814-0.8866
0.8827-0.8871
0.8814-0.8866
0.8914-0.8940
0.8970-0.8997
0.9123-0.9149
0.9179-0.9205
0.9248-0.9275
0.9700-0.9726
0.9857-0.9883
0.9996-1.0013
0.9996-1.0013
Window
(sec)8
12
12
12
10
12
6
6
6
6
6
6
6
-1+3
-1+3
Quantitation reference9
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
81L
77L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
171
162
120
98
131
175
173
171
177
169
EML
500
500
500
200
500
500
500
500
500
500
Other
(ng/kg)
EMDL
17
16
12
10
13
18
17
17
18
17
EML
50
50
50
20
50
50
50
50
50
50
Extract
(pg/nL)
EML
20
20
20
10
20
20
20
20
20
20
Labeled compounds
4
4
4
54L
81L
77L
52L
52L
52L
23:50
38:22
39:01
0.8290
1.3345
1.3571
0.8232-0.8348
1.3287-1.3403
1.3513-1.3629
20
20
20
52L
52L
52L
Compounds using 101L (13C12-2,2',4,5,5'-PeCB) as Labeled injection internal standard
CB congener
Pentachlorobiphenyls
5
5
5
5
104
96
103
94
104L
104L
104L
104L
29:46
30:17
32:11
32:29
1.0000
1.0174
1.0812
1.0913
0.9994-1.0017
1.0146-1.0202
1.0795-1.0829
1.0896-1.0929
-1+3
10
6
6
104L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
228
210
225
121
500
500
500
500
23
21
23
12
50
50
50
50
20
20
20
20
74
-------
Method 1668B November 2008
Cl
No.1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Congener No. 2'3
95
100
93
102
98
95/100/93/102/98
88
91
88/91
84
89
121
92
113
90
101
113/90/101
83
99
83/99
112
119
109
86
97
RTRef4
104L
104L
104L
104L
104L
104L
104L
104L
104L
104L
104L
104L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
RT5
33:00
33:06
33:14
33:21
33:26
33:13
33:48
33:55
33:52
34:14
34:44
34:57
35:26
36:01
36:03
36:04
36:03
36:39
36:41
36:40
36:51
37:12
37:12
37:17
37:17
RRT6
1.1086
1.1120
1.1165
1.1204
1.1232
1.1159
1.1355
1.1394
1.1377
1.1501
1.1669
1.1741
0.8639
0.8781
0.8789
0.8793
0.8789
0.8935
0.8944
0.8939
0.8984
0.9069
0.9069
0.9090
0.9090
RRT limits7
1.1058-1.1114
1.1092-1.1148
1.1137-1.1193
1.1176-1.1232
1.1204-1.1260
1.1131-1.1187
1.1321-1.1389
1.1366-1.1422
1.1344-1.1411
1.1484-1.1517
1.1652-1.1685
1.1725-1.1758
0.8627-0.8651
0.8761-0.8801
0.8769-0.8809
0.8773-0.8813
0.8769-0.8809
0.8911-0.8960
0.8923-0.8964
0.8915-0.8964
0.8972-0.8996
0.9037-0.9102
0.9037-0.9102
0.9057-0.9122
0.9057-0.9122
Window
(sec)8
10
10
10
10
10
15
12
10
12
6
6
6
6
10
10
10
10
12
10
12
6
16
16
16
16
Quantitation reference9
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
221
118
124
195
209
115
241
217
245
149
EML
500
500
500
500
500
500
1000
500
1000
500
Other
(ng/kg)
EMDL
22
12
12
19
21
12
24
22
25
15
EML
50
50
50
50
50
50
100
50
100
50
Extract
(pg/nL)
EML
20
20
20
20
20
20
50
20
50
20
75
-------
Method 1668B November 2008
Cl
No.1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Congener No. 2'3
125
87
109/119/86/97/125/87
117
116
85
117/116/85
110
115
110/115
82
111
120
108
124
108/124
107
123
106
118
122
114
105
127
126
RTRef4
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
118L
118L
114L
105L
105L
126L
RT5
37:21
37:25
37:19
37:57
38:02
38:05
38:00
38:16
38:18
38:17
38:40
38:52
39:21
40:39
40:40
40:39
40:54
41:02
41:10
41:22
41:49
41:58
42:43
44:09
45:58
RRT6
0.9106
0.9122
0.9098
0.9252
0.9273
0.9285
0.9265
0.9330
0.9338
0.9334
0.9427
0.9476
0.9594
0.9911
0.9915
0.9911
0.9972
1.0004
1.0037
1.0004
1.0113
1.0004
0.9996
1.0332
1.0004
RRT limits7
0.9074-0.9139
0.9102-0.9143
0.9065-0.9130
0.9228-0.9277
0.9248-0.9297
0.9265-0.9305
0.9240-0.9289
0.9309-0.9350
0.9317-0.9358
0.9313-0.9354
0.9415-0.9439
0.9464-0.9488
0.9581-0.9606
0.9890-0.9931
0.9894-0.9935
0.9890-0.9931
0.9959-0.9984
0.9996-1.0012
1.0024-1.0049
0.9996-1.0012
1.0101-1.0125
0.9999-1.0012
0.9996-1.0012
1.0320-1.0343
0.9996-1.0011
Window
(sec)8
16
10
16
12
12
10
12
10
10
10
6
6
6
10
10
10
6
-1+3
6
-1+3
6
-1+3
-2+3
6
-1+3
Quantitation reference9
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
123L
104L/123L/1 14L/1 18L/105L
118L
104L/123L/1 14L/1 18L/105L
114L
105L
104L/123L/1 14L/1 18L/105L
126L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
104
243
133
243
147
271
103
150
143
193
117
120
109
278
136
EML
200
1000
500
1000
500
1000
200
500
500
500
500
500
200
1000
500
Other
(ng/kg)
EMDL
10
24
13
24
15
27
10
15
14
19
12
12
11
28
14
EML
20
100
50
100
50
100
20
50
50
50
50
50
20
100
50
Extract
(pg/nL)
EML
10
50
20
50
20
50
10
20
20
20
20
20
10
50
20
76
-------
Method 1668B November 2008
Cl
No.1
Congener No. 2'3
RTRef4
RT5
RRT6
RRT limits7
Window
(sec)8
Quantitation reference9
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
EML
Other
(ng/kg)
EMDL
EML
Extract
(pg/nL)
EML
Labeled compounds
5
5
5
5
5
5
104L
123L
118L
114L
105L
126L
101L
101L
101L
101L
101L
101L
29:46
41:01
41:21
41:57
42:44
45:57
0.8257
1.1378
1.1470
1.1637
1.1854
1.2746
0.8211-0.8303
1.1331-1.1424
1.1424-1.1516
1.1590-1.1683
1.1808-1.1900
1.2700-1.2792
20
20
20
20
20
20
101L
101L
101L
101L
101L
101L
Compounds using 138L (13C12-2,2',3,4,4',5'-HxCB) as Labeled injection internal standard
CB congener
Hexachlorobiphenyls
6
6
6
6
6
6
6
6
6
6
6
6
6
6
155
152
150
136
145
148
151
135
154
151/135/154
144
147
149
147/149
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
35:44
36:07
36:15
36:44
37:00
34:26
39:10
39:17
39:21
39:15
39:47
40:09
40:12
40:10
1.0000
1.0107
1.0145
1.0280
1.0354
1.0756
1.0961
1.0993
1.1012
1.0984
1.1133
1.1236
1.1250
1.1241
0.9995-1.0014
1.0093-1.0121
1.0131-1.0159
1.0266-1.0294
1.0340-1.0368
1.0742-1.0770
1.0938-1.0984
1.0970-1.1017
1.0989-1.1035
1.0961-1.1007
1.1119-1.1147
1.1213-1.1259
1.1227-1.1273
1.1217-1.1264
-1+3
6
6
6
6
6
10
10
10
10
6
10
10
10
155L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
339
238
328
91
317
324
112
167
179
1000
1000
1000
200
1000
1000
500
500
500
34
24
33
9
32
32
11
17
18
100
100
100
20
100
100
50
50
50
50
50
50
10
50
50
20
20
20
77
-------
Method 1668B November 2008
Cl
No.1
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Congener No. 2'3
134
143
134/143
139
140
139/140
131
142
132
133
165
146
161
153
168
153/168
141
130
137
164
138
163
129
160
138/163/129/160
RTRef4
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
RT5
40:27
40:30
40:29
40:47
40:48
40:47
41:03
41:13
41:36
41:57
42:23
42:38
42:47
43:17
43:21
43:19
43:34
44:01
44:14
44:22
44:42
44:42
44:47
44:53
44:47
RRT6
1.1320
1.1334
1.1329
1.1413
1.1418
1.1413
1.1488
1.1535
1.1642
1.1740
0.8864
0.8916
0.8947
0.9052
0.9066
0.9059
0.9111
0.9205
0.9251
0.9278
0.9348
0.9348
0.9366
0.9387
0.9366
RRT limits7
1.1297-1.1343
1.1311-1.1357
1.1306-1.1353
1.1390-1.1437
1.1395-1.1441
1.1390-1.1437
1.1474-1.1502
1.1521-1.1549
1.1618-1.1665
1.1726-1.1754
0.8853-0.8874
0.8906-0.8926
0.8937-0.8958
0.9035-0.9069
0.9048-0.9083
0.9041-0.9076
0.9101-0.9122
0.9195-0.9216
0.9240-0.9261
0.9268-0.9289
0.9324-0.9373
0.9324-0.9373
0.9341-0.9390
0.9369-0.9404
0.9341-0.9390
Window
(sec)8
10
10
10
10
10
10
6
6
10
6
6
6
6
10
10
10
6
6
6
6
14
14
14
10
14
Quantitation reference9
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
134
196
121
311
125
169
361
182
352
130
93
136
300
136
211
EML
500
500
500
1000
500
500
1000
500
1000
500
200
500
1000
500
500
Other
(ng/kg)
EMDL
13
20
12
31
12
17
36
18
35
13
9
14
30
14
21
EML
50
50
50
100
50
50
100
50
100
50
20
50
100
50
50
Extract
(pg/nL)
EML
20
20
20
50
20
20
50
20
50
20
10
20
50
20
20
78
-------
Method 1668B November 2008
Cl
No.1
6
6
6
6
6
6
6
6
6
6
6
Congener No. 2'3
158
166
128
128/166
159
162
167
156
157
156/157
169
RTRef4
167L
167L
167L
167L
167L
167L
167L
156L/157L
156L/157L
156L/157L
169L
RT5
45:05
45:59
46:09
46:04
46:59
47:18
47:49
49:05
49:09
45:07
52:31
RRT6
0.9428
0.9617
0.9651
0.9634
0.9826
0.9892
1.0000
0.9993
1.0007
1.0000
1.0003
RRT limits7
0.9418-0.9439
0.9599-0.9634
0.9634-0.9669
0.9617-0.9651
0.9815-0.9836
0.9881-0.9902
0.9997-1.0010
0.9983-1.0003
0.9990-1.0024
0.9990-1.0010
0.9997-1.0010
Window
(sec)8
6
10
10
10
6
6
-1+3
6
10
6
-1+3
Quantitation reference9
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
167L
156L/157L
156L/157L
156L/157L
169L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
96
124
348
355
115
132
161
EML
200
500
1000
1000
500
500
500
Other
(ng/kg)
EMDL
10
12
35
35
11
13
16
EML
20
50
100
100
50
50
50
Extract
(pg/nL)
EML
10
20
50
50
20
20
20
Labeled compounds
6
6
6
6
6
6
155L
167L
156L
157L
156L/157L
169L
138L
138L
138L
138L
138L
138L
35:44
47:49
49:05
49:08
49:07
52:30
0.7997
1.0701
1.0985
1.0996
1.0992
1.1749
0.7960-0.8034
1.0664-1.0739
1.0974-1.0996
1.0959-1.1033
1.0981-1.1003
1.1738-1.1761
20
20
20
20
20
20
138L
138L
138L
138L
138L
138L
Compounds using 194L(13C12-2,2',3,3',4,4',5,5'-OcCB) as Labeled injection internal standard
CB congener
Heptachlorobiphenyls
7
7
7
188
179
184
188L
188L
188L
41:51
42:19
42:45
1.0000
1.0112
1.0215
0.9996-1.0012
1.0100-1.0123
1.0203-1.0227
-1+3
6
6
188L
188L/189L
188L/189L
235
229
403
500
500
1000
23
23
40
50
50
100
20
20
50
79
-------
Method 1668B November 2008
Cl
No.1
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Congener No. 2'3
176
186
178
175
187
182
183
185
183/185
174
177
181
171
173
171/173
172
192
193
180
180/193
191
170
190
189
RTRef4
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
188L
189L
189L
189L
189L11
189L
189L
189L11
189L
189L
RT5
43:15
43:45
45:06
45:46
46:02
46:14
46:42
46:53
46:47
47:02
47:30
47:52
48:10
48:11
48:10
49:47
50:06
50:26
50:27
50:26
50:51
51:54
52:26
55:07
RRT6
1.0335
1.0454
1.0777
1.0936
1.1000
1.1047
1.1159
1.1203
1.1179
1.1239
1.1350
1.1438
1.1509
1.1513
1.1509
0.9035
0.9093
0.9153
0.9156
0.9153
0.9229
0.9419
0.9516
1.0003
RRT limits7
1.0323-1.0346
1.0442-1.0466
1.0765-1.0789
1.0924-1.0948
1.0988-1.1012
1.1035-1.1059
1.1147-1.1171
1.1191-1.1215
1.1167-1.1191
1.1227-1.1251
1.1338-1.1362
1.1426-1.1450
1.1489-1.1529
1.1501-1.1525
1.1489-1.1529
0.9026-0.9044
0.9083-0.9102
0.9144-0.9162
0.9147-0.9165
0.9144-0.9162
0.9220-0.9238
0.9410-0.9428
0.9507-0.9525
0.9997-1.0009
Window
(sec)8
6
6
6
6
6
6
6
6
6
6
6
6
10
6
10
6
6
6
6
6
6
6
6
-1+3
Quantitation reference9
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L11
188L/189L
188L/189L
188L/189L11
188L/189L
189L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
385
407
221
383
191
398
401
186
141
396
374
377
420
136
418
162
234
177
EML
1000
1000
500
1000
500
1000
1000
500
500
1000
1000
1000
1000
500
1000
500
500
500
Other
(ng/kg)
EMDL
39
41
22
38
19
40
40
19
14
40
37
38
42
14
42
16
23
18
EML
100
100
50
100
50
100
100
50
50
100
100
100
100
50
100
50
50
50
Extract
(pg/nL)
EML
50
50
20
50
20
50
50
20
20
50
50
50
50
20
50
20
20
20
Octachlorobiphenyls
80
-------
Method 1668B November 2008
Cl
No.1
8
8
8
8
8
8
8
8
8
8
8
8
8
8
Congener No. 2'3
202
201
204
197
200
197/200
198
199
198/199
196
203
195
194
205
RTRef4
202L
202L
202L
202L
202L
202L
202L
202L
202L
205L
205L
205L
205L
205L
RT5
47:32
48:31
49:11
49:27
49:40
49:33
52:30
52:32
52:31
53:13
53:26
54:55
57:19
57:49
RRT6
1.0004
1.0210
1.0351
1.0407
1.0452
1.0428
1.1049
1.1056
1.1052
0.9207
0.9245
0.9501
0.9916
1.0003
RRT limits7
0.9996-1.0011
1.0193-1.0228
1.0340-1.0361
1.0396-1.0417
1.0442-1.0463
1.0417-1.0438
1.1031-1.1066
1.1045-1.1066
1.1035-1.1070
0.9198-0.9216
0.9236-0.9253
0.9493-0.9510
0.9908-0.9925
0.9997-1.0009
Window
(sec)8
-1+3
10
6
6
6
6
10
6
10
6
6
6
6
-1+3
Quantitation reference9
202L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
205L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
442
440
447
245
203
429
444
427
170
449
EML
1000
1000
1000
1000
500
1000
1000
1000
500
1000
Other
(ng/kg)
EMDL
44
44
45
25
20
43
44
43
17
45
EML
100
100
100
100
50
100
100
100
50
100
Extract
(pg/nL)
EML
50
50
50
50
25
50
50
50
20
50
Nonachlorobiphenyls
9
9
9
208
207
206
208L
208L
206L
54:33
55:32
59:37
1.0003
1.0183
1.0003
0.9997-1.0009
1.0174-1.0193
0.9997-1.0008
-1+3
6
-1+3
208L
208L/206L
206L
455
453
451
1000
1000
1000
46
45
45
100
100
100
50
50
50
Decachlorobiphenyl
10
209
209L
61:15
1.0003
0.9997-1.0008
-1+3
209L
153
500
15
50
20
Labeled compounds
7
7
7
7
188L
180L
170L
189L
194L
194L
194L
194L
41:51
50:27
51:53
55:06
0.7304
0.8805
0.9055
0.9616
0.7275-0.7333
0.8775-0.8834
0.9026-0.9084
0.9587-0.9645
20
20
20
20
194L
194L
194L
194L
81
-------
Method 1668B November 2008
Cl
No.1
8
8
9
9
10
Congener No. 2'3
202L
205L
208L
206L
209L
RTRef4
194L
194L
194L
194L
194L
RT5
47:31
57:48
54:32
59:36
61:14
RRT6
0.8293
1.0087
0.9517
1.0401
1.0686
RRT limits7
0.8264-0.8322
1.0044-1.0131
0.9488-0.9546
1.0358-1.0445
1.0643-1.0730
Window
(sec)8
20
30
20
30
30
Quantitation reference9
194L
194L
194L
194L
194L
Detection limits and minimum levels -
Matrix and concentration10
Water
(pg/L)
EMDL
EML
Other
(ng/kg)
EMDL
EML
Extract
(pg/nL)
EML
Labeled clean-up standards
3
5
7
28L
111L
178L
52L
101L
138L
26:44
38:51
45:05
0.9266
1.0777
1.0090
0.9209-0.9324
1.0730-1.0823
1.0052-1.0127
20
20
20
52L
101L
138L
Labeled injection internal standards
2
4
5
6
8
9L
52L
101L
138L
194L
138L
138L
138L
138L
138L
18:54
28:45
36:03
44:41
57:18
0.4230
0.6434
0.8068
1.0000
1.2824
0.4183-0.4276
0.6388-0.6481
0.8021-0.8115
0.9996-1.0011
1.2777-1.2870
25
25
25
100
25
138L
138L
138L
138L
138L
1. Number of chlorines on congener.
2. Suffix "L" indicates labeled compound.
3. Multiple congeners in a box indicates a group of congeners that co-elute or may not be adequately resolved on a 30-m SPB-octyl column. Congeners
included in the group are listed as the last entry in the box.
4. Retention time (RT) reference used to locate target congener.
5. Retention time of target congener.
6. Relative retention time (RRT) between the RT for the congener and RT for the reference.
82
-------
Method 1668B November 2008
7. RRT limits based on RT window.
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. EMDLs and EMLs with common laboratory interferences present. Without interferences, EMDLs and EMLs will be, respectively, 5 and 10 pg/L for
aqueous samples, 0.5 and 1.0 ng/kg for soil, tissue, and mixed-phase samples, and EMLs for extracts will be 0.5 pg/uL. Reference 20 describes
calculation of an EMDL and EML.
11. If congeners 170L and 1 SOL are included in the calibration and spiking solutions, these congeners should be used as RT and quantitation references.
83
-------
Method 1668B November 2008
Table 3. Concentrations of native and labeled chlorinated biphenyls in stock solutions, spiking solutions,
and final extracts
CB congener
Solution concentrations
Stock (jig/mL)
Spiking (ng/mL)
Extract (ng/mL)
Native Toxics/LOC1
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Native congener mix stock solutions2
MoCB thru TrCB
TeCBthruHpCB
OcCB thru DeCB
2.5
5.0
7.5
84
-------
Method 1668B November 2008
CB congener
Solution concentrations
Stock (jig/mL)
Spiking (ng/mL)
Extract (ng/mL)
Labeled Toxics/LOC/window-defining3
1L
3L
4L
15L
19L
37L
54L
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
.2.0
2.0
2.0
2.0
2.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled clean-uo4
28L
111L
178L
1.0
1.0
1.0
2.0
2.0
2.0
100
100
100
Labeled injection internal5
9L
52L
101L
138L
5.0
5.0
5.0
5.0
1000
1000
1000
1000
100
100
100
100
85
-------
Method 1668B November 2008
CB congener
194L
Solution concentrations
Stock (jig/mL)
5.0
Spiking (ng/mL)
1000
Extract (ng/mL)
100
86
-------
Method 1668B November 2008
CB congener
Solution concentrations
Stock (jig/mL)
Spiking (ng/mL) Extract (ng/mL)
Diluted combined 209 congener6
Standard
Native congeners
MoCB thru TrCB
TeCBthruHpCB
OcCB thru DeCB
Labeled Toxics/LOC/window-defining
Labeled Cleanup
Labeled Injection internal
Solution concentration (ng/mL)
Native
25
50
75
Labeled
100
100
100
1. Stock solution: Section 7. 8.1; Spiking solution: Section 7. 11
2. Section 7. 8. 1.2
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
87
-------
Method 1668B November 2008
Table 4. Composition of individual native CB congener solutions1
Solution identifier
A2
B2
C2
D2
E2
Accu-Standard part number
M-1668A-1
2
10
9
6
8
14
11
30
27
32
34
26
31
33
36
38
35
50
45
52
49
75
41
72
57
63
66
79
78
81
96
103
95
M-1668A-2
7
5
12
18
24
23
28
22
39
53
51
73
48
62
71
68
58
61
55
60
94
100
91
121
90
99
109
117
111
108
118
114
150
M-1668A-3
13
17
29
20
46
65
59
40
67
76
80
93
84
101
112
86
116
107
154
147
140
146
141
164
158
182
174
173
193
M-1668A-4
25
21
69
47
42
64
70
102
97
115
123
134
131
163
180
M-1668A-5
1
3
4
15
19
16
37
54
43
44
74
56
77
104
98
125
110
126
155
138
169
188
189
202
205
208
206
209
-------
Method 1668B November 2008
Solution identifier
A2
B2
C2
D2
E2
Accu-Standard part number
M-1668A-1
88
89
92
113
83
119
87
85
82
120
124
106
122
105
127
152
136
148
151
144
143
142
133
161
153
130
129
166
159
167
156
179
176
178
175
M-1668A-2
145
135
149
139
132
165
168
137
160
128
162
157
184
186
187
185
181
192
197
199
203
M-1668A-3
M-1668A-4
M-1668A-5
89
-------
Method 1668B November 2008
Solution identifier
A2
B2
C2
D2
E2
Accu-Standard part number
M-1668A-1
183
177
171
172
191
170
190
200
204
201
198
196
195
194
207
Total number of
congeners
83
M-1668A-2
54
M-1668A-3
29
M-1668A-4
15
M-1668A-5
28
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 standard.
90
-------
Method 1668B November 2008
Table 5. Concentration of CB congeners in calibration and calibration verification standards
CB congener
Congener1
Solution concentration (ng/mL)
CS-0.2
(Hi sens)2
CS-1
CS-2
CS-3
(VER)
CS-4
CS-5
Native Toxics/LOC
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2',6'-TrCB
3,4,4'-TrCB
2,2',6,6'-TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB
2,3,3',4,4',5'-HxCB
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB
2,2',3,3',5,5',6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
2,2',3,3',4',5,5',6,6'-NoCB
DeCB
1
o
J
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
Labeled Toxics/LOC/window-defining
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
13C12-4,4'-DiCB
13C12-2,2',6'-TrCB
13C12-3,4,4'-TrCB
1L
3L
4L
15L
19L
37L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
91
-------
Method 1668B November 2008
CB congener
13C12-2,2',6,6'-TeCB
13C12-3,3',4,4'-TeCB
13C12-3,4,4',5-TeCB
13C12-2,2',4,6,6'-PeCB
13C12-2,3,3',4,4'-PeCB
13C12-2,3,4,4',5-PeCB
13C12-2,3',4,4',5-PeCB
13C12-2',3,4,4',5-PeCB
13C12-3,3',4,4',5-PeCB
13C12-2,2',4,4',6,6'-HxCB
13C12-2,3,3',4,4',5-HxCB
13C12-2,3,3',4,4',5'-HxCB
13C12-2,3',4,4',5,5'-HxCB
13C12-3,3',4,4',5,5'-HxCB
13C12-2,2',3,4',5,6,6'-HpCB
13C12-2,3,3',4,4',5,5'-HpCB
13C12-2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB
13C12-2,2',3,3',4',5,5',6,6'-NoCB
13C12-DeCB
Congener1
54L
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
Solution concentration (ng/mL)
CS-0.2
(Hi sens)2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CS-1
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CS-2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CS-3
(VER)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CS-4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CS-5
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled clean-up
13C12-2,4,4'-TrCB
13C12-2,3,3',5,5'-PeCB
13C12-2,2',3,3',5,5',6-HpCB
28L
111L
178L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled injection internal
13C12-2,5-DiCB
13C12-2,2',5,5'-TeCB
13C12-2,2',4',5,5'-PeCB
13C12-2,2',3',4,4',5'-HxCB
13C12-2,2',3,3',4,4',5,5'-OcCB
9L
52L
101L
138L
194L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1. Suffix "L" indicates labeled compound
2. Additional concentration used for calibration of high sensitivity HRGC/HRMS
abundance ratio (Table 8) cannot be achieved at this level (see Section 10.3.3),
0.5 ng/mL may be used.
systems. If the ion
a calibration point at 0.4 or
92
-------
Method 1668B November 2008
Table 6. QC acceptance criteria for VER, IPR, OPR, and labeled compounds in samples
1,2
Name
Congener
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2'6-TrCB
3,4,4'-TrCB
2,2'6,6'TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB 6
2,3,3',4,4',5'-HxCB 6
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB
2,2',3,3',5,5',6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
2,2',3,3,'4,5,5',6,6'-NoCB
DeCB
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
13C12-4,4'-DiCB
13C12-2,2',6-TrCB
13C12-3,4,4'-TrCB
Number 3
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
1L
3L
4L
15L
19L
37L
Test cone
(ng/mL)4
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
VER
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
70-
50-
50-
50-
50-
50-
50-
(%)5
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
150
150
150
150
150
150
RSD (%)
25
22
18
17
13
26
17
20
20
19
19
18
13
16
17
15
16
16
13
16
14
16
17
15
17
17
20
78
63
56
70
68
57
IPR
Recovery (%)
84-
83-
82-
85-
86-
77-
84-
81-
81-
83-
83-
83-
88-
82-
82-
86-
87-
87-
85-
80-
88-
85-
82-
87-
85-
86-
81-
21-
31-
35-
34-
32-
47-
119
112
105
107
103
109
106
106
106
107
107
105
105
102
104
105
108
108
101
100
106
106
104
107
106
108
106
100
100
100
100
100
104
OPR recovery (%)
71-
72-
73-
76-
79-
64-
76-
71-
70-
74-
73-
74-
81-
74-
74-
79-
78-
78-
79-
73-
81-
77-
74-
79-
76-
77-
71-
2-
13-
18-
10-
10-
24-
132
123
114
116
109
122
114
116
116
117
117
113
112
109
113
112
117
117
107
108
113
114
112
115
115
116
116
100
100
100
118
106
128
Labeled compound
recovery in samples (%)
N/A
4-100
11-106
14-107
19 - 107
1-108
25 - 123
93
-------
Method 1668B November 2008
13C12-2,2',6,6'-TeCB 54L
13C12-3,3',4,4'-TeCB 77L
13C12-3,4,4',5-TeCB 81L
13C12-2,2',4,6,6'-PeCB 104L
13C12-2,3,3',4,4'-PeCB 105L
13C12-2,3,4,4',5-PeCB 114L
13C12-2,3',4,4',5-PeCB 118L
13C12-2',3,4,4',5-PeCB 123L
13C12-3,3',4,4',5-PeCB 126L
13C12-2,2',4,4',6,6'-HxCB 155L
13C12-2,3,3',4,4',5-HxCB6 156L
13C12-2,3,3',4,4',5'-HxCB6 157L
13C12-2 3' 4 4' 5 5'-HxCB 167L
13C12-3,3',4,4',5,5'-HxCB 169L
13C12-2,2',3,4',5,6,6'-HpCB 188L
13C12-2' 3 3' 4 4' 5 5'-HpCB 189L
13C12-2,2',3,3',5,5',6,6'-OcCB 202L
13C12-2,3,3',4,4',5,5',6-OcCB 205L
13C12-2,2',3,3',4,4',5,5',6-NoCB 206L
13C12-2,2',3,3',4,5,5',6,6'-NoCB 208L
13C12-2 2' 3 3' 4 4' 5 5' 6 6'-DeCB 209L
Cleanup standards
13C12-2,4,4'-TrCB 28L
13C12-2,3,3',5,5'-PeCB 111L
13C12-2,2',3,3',5,5',6-HpCB 178L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1. Reference 22 describes how interlab results were pooled from
2. QC acceptance criteria for IPR, OPR, and samples based on a
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 two con
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50 - 150
60-130
60-130
60-130
62
35
33
48
31
41
33
32
29
42
35
35
24
33
47
28
50
21
29
32
30
63
23
30
37-100
57 - 100
57 - 100
49 - 100
66-101
57 - 100
65 - 102
66 - 103
67 - 100
58-103
61 - 100
61 - 100
74 - 103
66 - 103
53 - 102
68 - 100
56-113
70 - 100
64 - 100
62 - 100
65 - 100
43 - 106
75 - 102
78-117
analyses of wastewater, biosolids, and fish tissue
20 uL extract
eeners
final volume
16-111
43 - 105
44 - 102
30-115
52-116
39-117
51-117
52-118
54-113
40-121
46-115
46-115
63- 115
51-117
33-121
55- 112
33-136
61-103
51-107
48-111
52-111
18-131
64-113
62-133
samples.
13-105
31-109
14 - 127
36-115
50-111
41-121
49-111
49-116
50 - 106
25 - 124
40 - 120
40 - 120
45-118
37-117
23 - 125
47-116
31-134
46-115
38-122
31-126
43-115
14-131
57-112
57-125
94
-------
Method 1668B November 2008
Table 7. Scan descriptors, levels of chlorination, m/z information, and substances monitored by
HRGC/HRMS
Function and
chlorine level
Fn-1; Cl-1
Fn-2; Cl-2,3
Fn-3
Cl-3,4,5
m/z1
188.0393
190.0363
200.0795
202.0766
218.9856
222.0003
223. 9974 (2)
225.9944
234.0406
236.0376
242.9856
255.9613
257.9584
268.0016
269.9986
255.9613
257.9584
259.9554
268.0016
269.9986
280.9825
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
337.9207
339.9178
m/z type
M
M+2
M
M+2
lock
M
M+2
M+4
M
M+2
lock
M
M+2
M
M+2
M
M+2
M+4
M
M+2
lock
M
M+2
M+4
M
M+2
M
M+2
M+4
M+2
M+4
m/z formula
12C12 H9 35C1
12C12 H9 37C1
13C12 H9 35C1
C^2 ^9 ^1
C4F9
C12 H8 C12
12C12 H8 35C1 37 Cl
12C12 H8 37C12
13C12 H8 35C12
13C12 H8 35C1 37 Cl
QF9
12C12 H7 35C13
12C12 H7 35C12 37C1
13C12 H7 35C13
13C12 H7 35C12 37C1
12C12 H7 35C13
12C12 H7 35C12 37C1
12C12 H7 35C1 37C12
13C12 H7 35C13
13C12 H7 35C12 37C1
C6FU
12C12H635C14
12C12 H6 35C13 37C1
12C12 H6 35C12 37C12
13C12H635C14
13C12 H6 35C13 37C1
13C12 H5 35C15
12C12 H5 35C14 37C1
12C12 H5 35C13 37C12
13C12 H5 35C14 37C1
13C12 H5 35C13 37C12
Substance
Cl-1 CB
Cl-1 CB
13C12 Cl-1 CB
13C12 Cl-1 CB
PFK
Cl-2 PCB
Cl-2 PCB
Cl-2 PCB
13C12 Cl-2 PCB
13C12 Cl-2 PCB
PFK
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
PFK
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
95
-------
Method 1668B November 2008
Function and
chlorine level
Fn-4
Cl-4,5,6
Fn-5
Cl-5,6,7
m/z1
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
330.9792
337.9207
339.9178
359.8415
361.8385
363.8356
371.8817
373.8788
323.8834
325.8804
327.8775
337.9207
339.9178
354.9792
359.8415
361.8385
363.8356
371.8817
373.8788
393.8025
395.7995
397.7966
405.8428
407.8398
454.9728
m/z type
M
M+2
M+4
M
M+2
M
M+2
M+4
lock
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M
M+2
M+4
M+2
M+4
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
m/z formula
12C12H635C14
12C12 H6 35C13 37C1
12C12 H6 35C12 37C12
13C12 H6 35C13 37C1
13C12 H6 35C12 37C12
12C12 H5 35C15
12C12 H5 35C14 37C1
12C12 H5 35C13 37C12
C7F15
13C12 H5 35C14 37C1
13C12 H5 35C13 37C12
12C12 H4 35C15 37C1
12C12 H4 35C14 37C12
12C12 H4 35C13 37C13
13C12 H4 35C15 37C1
13C12H435C1437C12
12C12 H5 35C15
12C12 H5 35C14 37C1
12C12 H5 35C13 37C12
13C12 H5 35C14 37C1
13C12 H5 35C13 37C12
C9F13
12C12 H4 35C15 37C1
12C12H435C1437C12
12C12 H4 35C13 37C13
13C12 H4 35C15 37C1
13C12 H4 35C14 37C12
12C12 H3 35C16 37C1
12C12 H3 35C15 37C12
12C12 H3 35C14 37C13
13C12 H3 35C16 37C1
13C12 H3 35C15 37C12
CUF17
Substance
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
PFK
13C12 Cl-5 PCB
13C12 Cl-5 PCB
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
PFK
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
PFK
96
-------
Method 1668B November 2008
Function and
chlorine level
Fn-6
Cl-7,8,9,10
m/z1
393.8025
395.7995
397.7966
405.8428
407.8398
427.7635
429.7606
431.7576
439.8038
441.8008
442.9728
454.9728
461.7246
463.7216
465.7187
473.7648
475.7619
495.6856
497.6826
499.6797
507.7258
509.7229
511.7199
m/z type
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M+6
m/z formula
12C12 H3 35C16 37C1
12C12 H3 35C15 37C12
12C12 H3 35C14 37C13
13C12 H3 35Cl(i 37C1
13C12 H3 35C15 37C12
12C12 H2 35C17 37C1
12C12 H2 35C16 37C12
12C12 H2 35C15 37C13
13C12 H2 35C17 37C1
13C12 H2 35C16 37C12
r F
Mo r!3
Ml F13
12C12 H! 35C18 37C1
12C12 H! 35C17 37C12
12C12 H! 35C16 37C13
13C12 H! 35C18 37C1
13C12 H! 35C17 37C12
12C12 H4 35C19 37C1
12C12 35C18 37C12
12C12 35C17 37C13
13C12 35C19 37C1
13C12 35C18 37C12
13C12 35C17 37C13
Substance
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
PFK
Cl-9 PCB
Cl-9 PCB
Cl-9 PCB
13C12 Cl-9 PCB
13C12 Cl-9 PCB
Cl-10 PCB
Cl-10 PCB
Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
1. Isotopic masses used for accurate mass calculation
'H 1.0078
12C 12.0000
13C 13.0034
35C1 34.9689
37C1 36.9659
19F 18.9984
2. An interference with PFK m/z 223.9872 may preclude meeting 10:1 S/N for the DiCB congeners at the CS-0.2 and
CS-1 calibration levels (Section 10.3.3 and Table 5). If this interferences occurs, 10:1 S/N must be met at the CS-2
level
97
-------
Method 1668B November 2008
Table 8. Theoretical ion abundance ratios and QC limits
Chlorine atoms
1
2
3
4
5
6
7
8
9
10
m/zs forming ratio
m/m+2
m/(m+2)
m/(m+2)
m/(m+2)
(m+2)/(m+4)
(m+2)/(m+4)
(m+2)/(m+4)
(m+2)/(m+4)
(m+2)/(m+4)
(m+4)(m+6)]
Theoretical ratio
3.13
1.56
1.04
0.77
1.55
1.24
1.05
0.89
0.77
1.16
Lower QC limit
2.66
1.33
0.88
0.65
1.32
1.05
0.89
0.76
0.65
0.99
Upper QC limit
3.60
1.79
1.20
0.89
1.78
1.43
1.21
1.02
0.89
1.33
98
-------
Method 1668B November 2008
Table 9. Suggested Sample Quantities to be Extracted for Various Matrices1
Sample matrix2
Single-phase
Aqueous
Solid
Organic
Tissue
Multi-phase
Liquid/Solid
Aqueous/Solid
Organic/solid
Liquid/Liquid
Aqueous/organic
Aqueous/organic/solid
Example
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oil
Organic polymer
Fish
Human adipose
Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Industrial sludge
Oily waste
In-process effluent
Untreated effluent
Drum waste
Untreated effluent
Drum waste
Percent
solids
<1
>20
<1
1-30
1 1 f\f\
<1
^.1
Phase
3
Solid
Organic
Solid
T3r\fTi
J30tn
Organic
Organic & solid
Quantity
extracted
1000 mL
10 g
10 g
Ifl rr
1U g
10 g
1 ft rr
1U g
10 g
1 f\ rr
1U g
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.
99
-------
Method 1668B November 2008
Determine % solids
§11.2
Determine particle size
§11.3
Prep per § 11.5
Spike Labeled Toxics/LOC
window-definers per
§11.5.2.2
Particle
size > 1 mm?
(from §11.3)
Prep per § 11.4
Spike Labeled Toxics/LOC
window-definers per
§11.4.2.2
Spike Cleanup standard per
§12.5.1
Back extract per
§12.5
Transfer through
Na2S04 per §12.5.6
Extract per § 12.2.1,
§12.2.2, or §12.2.3
Concentrate per
§12.6-§12.7
Clean up per
§13.2-§13.5, or §13.7
Concentrate per
§12.6-§12.7
Spike injection internal
standard per § 14.2
Analyze per
§14-§18
Figure 1 Flow Chart for Analysis of Aqueous and Solid Samples
100
-------
Method 1668B November 2008
Determine % solids
per§11.2
Determine particle size
per §11.3
Spike Labeled Toxics/LOC
window-definers per §
11.6.2
Aqueous
Discard
Pressure filter aliquot per
§11.6.2
Spike Cleanup standard per
§12.5.1
Back extract
per §12.5
Transfer through
Na2S04 per 12.5.6
Non-aqueous (organic)
Figure 2 Flow Chart for Analysis of Multi-Phase Samples
Concentrate per
§12.6-§12.7
Clean up per
§13.2-§13.5, §13.7
Concentrate per
§12.6-§12.7
Spike injection internal
standard per § 14.2
Analyze per
§14-§18
101
-------
Method 1668B November 2008
Homogenize tissue
per §11.8.1
Remove 1 0
per§ 11.8.1
g
4
Spike Labeled Toxics/LOC
window-definers per § 11.8.3
Soxhlet extract
per §12.4
Concentrate to dryness
per §12.4.7-§12.4.8
Determine % lipids per
§12.4.9
Redissolve in n-C6 and spike
cleanup standard
per §12.4.9.1
Remove lipids per
§13.6
Concentrate per
§12.6-§12.7
Clean up per
§13.2-§13.5, §13.7
Concentrate per
§12.6-§12.7
Spike injection internal
standard per§ 14.2
Analyze per § 14-§18
Figure 3 Flow Chart for Analysis of Tissue Samples
102
-------
Method 1668B November 2008
GMF150 Filler
— Test Tube. 25-mm x 200-mm
1-Liter Suction Flask
Figure 4 Solid-phase Extraction Apparatus
103
-------
Method 1668B November 2008
Figure 5 Soxhlet/Dean-Stark Extractor
32-027-02
104
-------
Method 1668B November 2008
30 27
18
24
17 \\16 32
23
34
A
..!..
X
28/20
31 33
J21 32
Al
36 39 38 35 37
f
Figure 6 Octyl column resolution test #1: Separation of Cl-3 congeners 34 and 23 with valley <40% (i.e.
lOOx/y < 40%)
105
-------
Method 1668B November 2008
179 176
188 U4 IBS
174 17I/
175 182 185 177
178 l»7 183 1S1
A
fr*
193/180
I9i
in
!90
170
189
Jm
Figure 1 Octyl column resolution test #2: Separation of Cl-7 congeners 187 and 182 with valley < 40%
(i.e. 100x/y<40%)
106
-------
Method 1668B November 2008
C110
C19
CIS
C17
CT6
CIS
C14
C13
C12
Cll
yi
10 15 20 25 30 35
\
40
f
45
_U I 11 »
50 55
\
60
Figure 8. CB congeners at each level of chlorination on the SPB-octyl column
107
-------
Method 1668B November 2008
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
°C degrees Celsius
(iL microliter
(im 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 pounds-per-square inch gauge
v/v volume per unit volume
w/v weight per unit volume
108
-------
Method 1668B November 2008
24.2 Definitions and acronyms (in alphabetical order)
Analyte—A CB tested for by this Method. The analytes are listed in Table 1.
Calibration standard (CAL)—A solution prepared from a secondary standard and/or stock solutions
and used to calibrate the response of the HRGC/HRMS instrument.
Calibration verification standard (VER)—The mid-point calibration standard (CS-3) that is used to
verify calibration. See Table 5.
CB—chlorinated biphenyl congener. One of the 209 individual chlorinated biphenyl congeners
determined using this Method. The 209 CBs are listed in Table 1.
CS-0.2, CS-1, CS-2, CS-3, CS-4, CS-5—See Calibration standards and Table 5.
DeCB—decachlorobiphenyl (PCB 209).
DiCB—dichlorobiphenyl.
Estimated method detection limit (EMDL)—The lowest concentration at which a CB can be detected
with common laboratory interferences present. EMDLs are listed in Table 2. Reference 20 describes
calculation of an EMDL.
Estimated minimum level (EML)—The lowest concentration at which a CB can be measured reliably
with common laboratory interferences present. EMLs are listed in Table 2. Reference 20 describes
calculation of an EML.
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.
109
-------
Method 1668B November 2008
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 9, 52, 101, 138, and 194.
Internal standard—a labeled compound used as a reference for quantitation of other labeled
compounds and for quantitation of native CB congeners other than the congener of which it is a labeled
analog. See Internal standard quantitation.
Internal standard quantitation—A means of determining the concentration of (1) a naturally
occurring (native) compound by reference to a compound other than its labeled analog and (2) a labeled
compound by reference to another labeled compound.
IPR—Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes of
interest and labeled compounds and analyzed to establish the ability of the laboratory to generate
acceptable precision and recovery. An IPR is performed prior to the first time this Method is used and
any time the Method or instrumentation is modified.
Isotope dilution quantitation—A means of determining a naturally occurring (native) compound by
reference to the same compound in which one or more atoms has been isotopically enriched. In this
Method, all 12 carbon atoms in the biphenyl molecule are enriched with carbon-13 to produce 13C12-
labeled analogs of the chlorinated biphenyls. The 13C12-labeled CBs are spiked into each sample and
allow identification and correction of the concentration of the native compounds in the analytical
process.
K-D—Kuderna-Danish concentrator; a device used to concentrate the analytes in a solvent.
Laboratory blank—See Method blank.
Laboratory control sample (LCS)—See Ongoing precision and recovery standard (OPR).
Laboratory reagent blank—See Method blank.
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Method blank—An aliquot of reagent water that is treated exactly as a sample including exposure to
all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with
samples. The Method blank is used to determine if analytes or interferences are present in the
laboratory environment, the reagents, or the apparatus.
Minimum level of quantitation (ML)—The level at which the entire analytical system must give a
recognizable signal and acceptable calibration point for the analyte. It is equivalent to the concentration
of the lowest calibration standard, assuming that all Method-specified sample weights, volumes, and
cleanup procedures have been employed.
MoCB—monochlorobiphenyl.
110
-------
Method 1668B November 2008
MS—Mass spectrometer or mass spectrometry.
Must—This action, activity, or procedural step is required.
NoCB-nonachlorobiphenyl.
OcCB—octachlorobiphenyl.
OPR—Ongoing precision and recovery standard (OPR); a method blank spiked with known quantities
of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this Method for precision and recovery.
Perfluorokerosene (PFK)—A mixture of compounds used to calibrate the exact m/z scale in the
HRMS.
Preparation blank—See Method blank.
Quality control check sample (QCS)—A sample containing all or a subset of the analytes at known
concentrations. The QCS is obtained from a source external to the laboratory or is prepared from a
source of standards different from the source of calibration standards. It is used to check laboratory
performance with test materials prepared external to the normal preparation process.
PeCB—pentachlorobiphenyl.
PCB—poly chlorinated 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.
Ill
-------
Method 1668B November 2008
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-^-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.
Tr CB—trichlorobiphenyl.
Unique GC resolution or uniquely resolved—Two adjacent chromatographic peaks in which the
height of the valley is less than 40 percent of the height of the shorter peak (See section 6.9.1.1.2 and
Figures 6 and 7 for unique resolution specific to the SPB-octyl column).
VER—See Calibration verification.
112
-------
Method 1668B November 2008
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 (im film DB-1 (J&W, or equivalent).
1.2 Suggested GC operating conditions:
Injector temperature: 270 °C
Interface temperature: 290 °C
Initial temperature: 75 °C
Initial time: 2 minutes
Temperature program: 75-150 °C at 15 °C/minute
150-270 °C at 2.5 °C/minute
Final time: 7 minutes
Carrier gas velocity: 40 cm/sec at 200 °C
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 at the date of writing of these corrections
to Method 1668A, August 2003, EPA-821-R-07-004.
2.2 Elution order data—The congener mixes developed for the SPB-octyl column (Table 4 of Method
1668A) 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-l.
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 1668A.
2.4 Scan descriptors—The 6-function scan descriptors are shown in Appendix Table A-2.
113
-------
Method 1668B November 2008
Table A-l. Retention time (RT) references, quantitation references, and relative retention times (RRTs) for CB congeners using a DB-1 column.
Labeled or native CB1
13C12-2-MoCB4
2-MoCB
3-MoCB
13C12-4-MoCB4'5
4-MoCB
13C12-2,2'-DiCB4
2,2'-DiCB
2,6-DiCB
2,5-DiCB
2,4-DiCB
2,3'-DiCB
2,4'-DiCB6
2,3-DiCB
13C12-2,2',6-TrCB4
2,2',6-TrCB
3,5-DiCB
2,4,6-TrCB
3,3'-DiCB
3,4'-DiCB
3,4-DiCB
2,2',5-TrCB6
13C12-4,4'-DiCB4'5
4,4'-DiCB
2,2',4-TrCB
2,3',6-TrCB
Congener
number2
1L
1
2
3L
3
4L
4
10
9
7
6
8
5
19L
19
14
30
11
13
12
18
15L
15
17
27
Retention time and quantitation
references
13C12-4-MoCB4'5
13C12-2-MoCB4
13C12-4-MoCB4'5
13C12-2,2',5,5'-TeCB7
13C12-4-MoCB4'5
13C12-4,4'-DiCB4'5
13C12-2,2'-DiCB4
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-2,2',6-TrCB4
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
Congener
number
3L
1L
3L
52L
3L
15L
4L
15L
15L
15L
15L
15L
15L
28L
19L
15L
28L
15L
15L
15L
28L
52L
15L
28L
28L
RT
09:17
09:17
10:22
10:29
10:29
11:08
11:08
11:10
12:08
12:09
12:31
12:43
12:46
13:31
13:31
13:36
14:06
14:11
14:26
14:27
14:36
14:40
14:40
14:43
15:06
RRT
0.8855
1.0000
0.9889
0.5561
1.0000
0.7591
1.0000
0.7614
0.8273
0.8284
0.8534
0.8670
0.8705
0.7990
1.0000
0.9273
0.8335
0.9670
0.9841
0.9852
0.8631
0.7781
1.0000
0.8700
0.8926
RRT QC limits3
0.8776-0.8935
0.9964-1.0072
0.9809-0.9968
0.5473-0.5650
0.9968-1.0064
0.7477-0.7705
0.9925-1.0075
0.7500-0.7727
0.8216-0.8330
0.8227-0.8341
0.8477-0.8591
0.8614-0.8727
0.8648-0.8761
0.7892-0.8089
0.9975-1.0049
0.9216-0.9330
0.8286-0.8384
0.9614-0.9727
0.9784-0.9898
0.9795-0.9909
0.8581-0.8680
0.7692-0.7869
0.9977-1.0043
0.8650-0.8749
0.8877-0.8975
114
-------
Method 1668B November 2008
Labeled or native CB1
2,3,6-TrCB
2,2',3-TrCB
2,4',6-TrCB
13C12-2,2',6,6'-TeCB4
2,2',6,6'-TeCB
2',3,5-TrCB
2,3,5-TrCB
2,4,5-TrCB
2,3',5-TrCB
2,3',4-TrCB
2,4',5-TrCB
13C12-2,4,4'-TrCB5
2,4,4'-TrCB6
2,2',4,6-TeCB
2,3,4-TrCB
2,2',5,6'-TeCB
2,3,3'-TrCB
2',3,4-TrCB
2,2',4,6'-TeCB
2,3,4'-TrCB
2,2',3,6-TeCB
3,3',5-TrCB
2,2',3,6'-TeCB
3,4',5-TrCB
13C12-2,2',5,5'-TeCB7
2,2',5,5'-TeCB6
Congener
number2
24
16
32
54L
54
34
23
29
26
25
31
28L
28
50
21
53
20
33
51
22
45
36
46
39
52L
52
Retention time and quantitation
references
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,2',6,6'-TeCB4
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13C12-3,3',4,4'-TeCB4'5'9
Congener
number
28L
28L
28L
77L
54L
28L
28L
28L
28L
28L
28L
52L
28L
77L
28L
77L
28L
28L
77L
28L
77L
28L
77L
28L
52L
77L
RT
15:06
15:26
15:29
16:02
16:02
16:03
16:07
16:18
16:29
16:36
16:52
16:55
16:55
16:55
17:21
17:26
17:22
17:24
17:42
17:43
18:00
18:16
18:24
18:37
18:51
18:51
RRT
0.8926
0.9123
0.9153
0.6139
1.0000
0.9488
0.9527
0.9635
0.9744
0.9813
0.9970
0.8974
1.0000
0.6477
1.0256
0.6675
1.0266
1.0286
0.6777
1.0473
0.6892
1.0798
0.7045
1.1005
1.0000
0.7218
RRT QC limits3
0.8877-0.8975
0.9074-0.9172
0.9103-0.9202
0.6075-0.6203
0.9979-1.0042
0.9438-0.9537
0.9478-0.9576
0.9586-0.9685
0.9695-0.9793
0.9764-0.9862
0.9921-1.0020
0.8930-0.9019
0.9980-1.0039
0.6414-0.6541
1.0207-1.0305
0.6611-0.6739
1.0217-1.0315
1.0236-1.0335
0.6713-0.6841
1.0424-1.0522
0.6828-0.6956
1.0749-1.0847
0.6981-0.7109
1.0956-1.1054
0.9956-1.0044
0.7154-0.7281
115
-------
Method 1668B November 2008
Labeled or native CB1
2,3',4,6-TeCB
2,3',5',6-TeCB
2,2',4,5'-TeCB
2,2',3,5-TeCB
3,4,5-TrCB
2,2',4,4'-TeCB
2,4,4',6-TeCB
2,2',4,5-TeCB
2,3,5,6-TeCB
2,3,4,6-TeCB
3,3',4-TrCB
13C12-2,2',4,6,6'-PeCB4
2,2',4,6,6'-PeCB
2,2',3,5'-TeCB6
13C12-3,4,4'-TrCB4
3,4,4'-TrCB
2,3,3',6-TeCB
2,2',3,4'-TeCB
2,3',5,5'-TeCB
2,3',4',6-TeCB
2,3,4',6-TeCB
2,2',3,4-TeCB
2,2',3,6,6'-PeCB
2,3',4,5'-TeCB
2,2',3,3'-TeCB
2,3,3',5-TeCB
Congener
number2
69
73
49
43
38
47
75
48
65
62
35
104L
104
44
37L
37
59
42
72
71
64
41
96
68
40
57
Retention time and quantitation
references
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,6,6'-PeCB4
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,4,4'-TrCB4
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
Congener
number
77L
77L
77L
77L
28L
77L
77L
77L
77L
77L
28L
118L
104L
77L
28L
37L
77L
77L
77L
77L
77L
77L
118L
77L
77L
77L
RT
18:52
18:57
19:00
19:04
19:12
19:15
19:20
19:20
19:31
19:36
19:41
19:45
19:45
19:55
20:03
20:03
20:05
20:07
20:36
20:36
20:37
20:39
20:48
20:52
20:58
21:21
RRT
0.7224
0.7256
0.7275
0.7301
1.1350
0.7371
0.7403
0.7403
0.7473
0.7505
1.1635
0.7037
1.0000
0.7626
1.1852
1.0000
0.7690
0.7703
0.7888
0.7888
0.7894
0.7907
0.7411
0.7990
0.8028
0.8175
RRT QC limits3
0.7160-0.7288
0.7192-0.7320
0.7211-0.7339
0.7237-0.7364
1.1300-1.1399
0.7307-0.7435
0.7339-0.7466
0.7339-0.7466
0.7409-0.7537
0.7441-0.7569
1.1586-1.1685
0.6977-0.7096
0.9983-1.0034
0.7562-0.7690
1.1803-1.1901
0.9983-1.0033
0.7626-0.7754
0.7639-0.7766
0.7824-0.7951
0.7824-0.7951
0.7830-0.7958
0.7843-0.7971
0.7352-0.7470
0.7926-0.8054
0.7996-0.8060
0.8143-0.8207
116
-------
Method 1668B November 2008
Labeled or native CB1
2,2',4,5,'6-PeCB
2,3',4,5-TeCB
2,2',4,4',6-PeCB
2,3,3',5'-TeCB
2,3,4',5-TeCB
2,2',3,5,6'-PeCB
2,4,4',5-TeCB
2,3,4,5-TeCB
2,3',4',5-TeCB
2',3,4,5-TeCB
2,2',3',4,6-PeCB
2,3',4,4'-TeCB6
2,2',4,556'-PeCB
2,2',3,5',6-PeCB
2,2',3,5,6-PeCB
3,3',5,5'-TeCB
2,2',3,4,6-PeCB
2,2',3,4',6-PeCB
2,3,3',4'-TeCB
2,3',4,5,'6-PeCB
2,3,3',4'-TeCB
2,3,4,4'-TeCB
13C12-2,2',4,4',6,6'-HxCB4
2,2',4,4',6,6'-HxCB
2,2',3,3',6-PeCB
2,2',3,5,5'-PeCB
Congener
number2
103
67
100
58
63
94
74
61
70
76
98
66
102
95
93
80
88
91
55
121
56
60
155L
155
84
92
Retention time and quantitation
references
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,4',6,6'-HxCB4
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
Congener
number
118L
77L
118L
77L
77L
118L
77L
77L
77L
77L
118L
77L
118L
118L
118L
77L
118L
118L
77L
118L
77L
77L
167L
155L
118L
118L
RT
21:22
21:38
21:41
21:43
21:51
22:05
22:07
22:11
22:20
22:25
22:28
22:29
22:32
22:34
22:36
22:45
22:49
22:55
22:57
23:04
23:24
23:24
23:43
23:43
23:44
23:50
RRT
0.7613
0.8283
0.7726
0.8315
0.8366
0.7868
0.8468
0.8494
0.8551
0.8583
0.8005
0.8609
0.8029
0.8040
0.8052
0.8711
0.8129
0.8165
0.8787
0.8219
0.8960
0.8960
0.7104
1.0000
0.8456
0.8492
RRT QC limits3
0.7553-0.7672
0.8251-0.8315
0.7666-0.7785
0.8283-0.8347
0.8334-0.8398
0.7809-0.7928
0.8437-0.8500
0.8462-0.8526
0.8519-0.8583
0.8551-0.8615
0.7975-0.8034
0.8577-0.8641
0.7999-0.8058
0.8011-0.8070
0.8023-0.8082
0.8679-0.8743
0.8100-0.8159
0.8135-0.8195
0.8756-0.8819
0.8189-0.8248
0.8928-0.8992
0.8928-0.8992
0.7054-0.7154
0.9986-1.0028
0.8426-0.8486
0.8462-0.8521
117
-------
Method 1668B November 2008
Labeled or native CB1
2,2',3,4,6'-PeCB
2,2',3,4',5-PeCB
13C12-2,2',4,5,5'-PeCB7
2,2',4,5,5'-PeCB6
2,3,3',5',6-PeCB
3,3',4,5'-TeCB
2,2',4,4',5-PeCB
2,2',3,4',6,6'-HxCB
2,3',4,4',6-PeCB
2,3,3',5,6-PeCB
2,3,3',4,6-PeCB
2,2',3,5,6,6'-HxCB
2,2',3,3',5-PeCB
2,2',3',4,5-PeCB
2,2',3,4,5-PeCB
13C12-3,4,4',5-TeCB9
3,4,4',5-TeCB10
2',3,4,5,6'-PeCB
2,3,4',5,6-PeCB
2,2',3,4,5'-PeCB
3,3',4,5-TeCB
2,2',3,4,6,6'-HxCB
2,3,4,4',6-PeCB
13C12-2,3,3',5,5'-PeCB8
2,3,3',5,5'-PeCB
2,2',3,4,4'-PeCB
Congener
number2
89
90
101L
101
113
79
99
150
119
112
109
152
83
97
86
81L
81
125
117
87
78
145
115
111L
111
85
Retention time and quantitation
references
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',5,5'-TeCB7
13C12-3,4,4',5-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
Congener
number
118L
118L
101L
118L
118L
77L
118L
167L
118L
118L
118L
167L
118L
118L
118L
52L
77L
118L
118L
118L
77L
167L
118L
101L
118L
118L
RT
23:53
24:07
24:11
24:11
24:23
24:27
24:28
24:52
24:54
25:00
25:09
25:17
25:20
25:22
25:27
25:32
25:32
25:36
25:37
25:38
25:40
25:42
25:44
25:51
25:51
25:51
RRT
0.8510
0.8593
1.0000
0.8616
0.8688
0.9362
0.8717
0.7449
0.8872
0.8907
0.8961
0.7574
0.8919
0.9038
0.9068
1.3546
1.0000
0.9121
0.9127
0.9133
0.9598
0.7698
0.9169
1.0689
0.9210
0.9210
RRT QC limits3
0.8480-0.8539
0.8563-0.8622
0.9966-1.0034
0.8587-0.8646
0.8658-0.8717
0.9330-0.9394
0.8688-0.8747
0.7399-0.7499
0.8842-0.8901
0.8878-0.8937
0.8931-0.8990
0.7524-0.7624
0.8890-0.8949
0.9008-0.9068
0.9038-0.9097
1.3457-1.3634
0.9987-1.0026
0.9091-0.9151
0.9097-0.9157
0.9103-0.9163
0.9566-0.9630
0.7649-0.7748
0.9139-0.9198
1.0655-1.0724
0.9181-0.9240
0.9181-0.9240
118
-------
Method 1668B November 2008
Labeled or native CB1
2,3,4,5,6-PeCB
13C12-3,3',4,4'-TeCB4'5'9
3,3',4,4'-TeCB6'10
2,2',3,3',6,6'-HxCB
2,3',4,555'-PeCB
2,2',3,4',5,6'-HxCB
2,3,3',4',6-PeCB
2,2',4,4',5,6'-HxCB
2,2',3,3',4-PeCB
2,2',3,5,5',6-HxCB
2,2',3,3',5,6'-HxCB
2',3,4,5,5'-PeCB
2,2',3,4,5',6-HxCB
2,3,3',4,5'-PeCB
2,2',3,4',5,6-HxCB
2,3,3',4',5-PeCB
2,2',3,4',5',6-HxCB
2,2',3,3',5,6-HxCB
2,2',3,4,5,6'-HxCB
13C12-2',3,454',5-PeCB9
2',3,4,4',5-PeCB10
2,2',3,4,4',6-HxCB
2,3,3',4,5-PeCB
13C12-2,3',4,4',5-PeCB5'9
2,3',4,4',5-PeCB6'10
2,2',3,4,4',6'-HxCB
Congener
number2
116
77L
77
136
120
148
110
154
82
151
135
124
144
108
147
107
149
134
143
123L
123
139
106
118L
118
140
Retention time and quantitation
references
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',5,5'-TeCB7
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2',3,4,4',5-PeCB9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
Congener
number
118L
52L
77L
167L
118L
167L
118L
167L
118L
167L
167L
118L
167L
118L
167L
118L
167L
167L
167L
101L
123L
167L
118L
101L
118L
167L
RT
25:48
26:07
26:07
26:10
26:12
26:14
26:16
26:44
26:48
27:18
27:31
27:36
27:38
27:40
27:44
27:45
28:01
28:35
28:34
27:53
27:53
28:01
28:04
28:04
28:04
28:12
RRT
0.9192
1.3855
1.0000
0.7793
0.9335
0.7858
0.9359
0.8008
0.9549
0.8178
0.8243
0.9834
0.8278
0.9857
0.8308
0.9887
0.8392
0.8562
0.8557
1.1530
1.0000
0.8392
1.0000
1.1606
1.0000
0.8447
RRT QC limits3
0.9163-0.9222
1.3767-1.3943
0.9987-1.0026
0.7743-0.7843
0.9305-0.9365
0.7808-0.7908
0.9329-0.9388
0.7983-0.8033
0.9519-0.9578
0.8153-0.8203
0.8218-0.8268
0.9804-0.9863
0.8253-0.8303
0.9828-0.9887
0.8283-0.8333
0.9857-0.9917
0.8367-0.8417
0.8537-0.8587
0.8532-0.8582
1.1496-1.1564
0.9988-1.0024
0.8367-0.8417
0.9970-1.0030
1.1571-1.1640
0.9988-1.0024
0.8422-0.8472
119
-------
Method 1668B November 2008
Labeled or native CB1
13C12-2,3,4,4',5-PeCB9
2,3,4,4',5-PeCB10
2',3,3',4,5-PeCB
2,2',3,3',4,6-HxCB
2,2',3,4,5,6-HxCB
2,2',3,3',5,5'-HxCB
2,2',3,3',4,6'-HxCB
2,3,3',5,5',6-HxCB
13C12-2,2',3,4',5,6,6'-HpCB4
2,2',3,4',5,6,6'-HpCB
2,2',3,4',5,5'-HxCB
13C12-2,3,3',4,4'-PeCB9
2,3,3',4,4'-PeCB6'10
2,3,3',4,5',6-HxCB
2,2',4,4',5,5'-HxCB6
2,2',3,4,4',6,6'-HpCB
3,3',4,555'-PeCB
2,3',4,4',5',6-HxCB
2,2',3,4,5,5'-HxCB
2,2',3,3',5,6,6'-HpCB
2,2',3,4,4',5-HxCB
2,2',3,3',4,5'-HxCB
2,2',3,3',4,6,6'-HpCB
13C12-2,2',3,454',5'-HxCB7
2,2',354,4',5'-HxCB6
2,3,3',4',5',6-HxCB
Congener
number2
114L
114
122
131
142
133
132
165
188L
188
146
105L
105
161
153
184
127
168
141
179
137
130
176
138L
138
164
Retention time and quantitation
references
13C12-2,2',4,5,5'-PeCB7
13C12-2,3,4,4',5-PeCB9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4',5,6,6'-HpCB4
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3,3',4,4'-PeCB9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
Congener
number
101L
114L
118L
167L
167L
167L
167L
167L
189L
188L
167L
101L
105L
167L
167L
189L
118L
167L
167L
189L
167L
167L
189L
138L
167L
167L
RT
28:38
28:38
28:48
28:52
28:59
28:59
29:32
29:21
29:22
29:22
29:24
29:30
29:30
29:32
29:48
29:49
29:57
29:59
30:31
30:33
30:51
30:57
31:01
31:20
31:20
31:22
RRT
1.1840
1.0000
1.0261
0.8647
0.8682
0.8682
0.8847
0.8792
0.9511
1.0000
0.8807
1.2198
1.0000
0.8847
0.8927
0.7482
1.0671
0.8982
0.9141
0.7666
0.9241
0.9271
0.7783
1.0000
0.9386
0.9396
RRT QC limits3
1.1806-1.1875
0.9988-1.0023
1.0232-1.0291
0.8622-0.8672
0.8657-0.8707
0.8657-0.8707
0.8822-0.8872
0.8767-0.8817
0.7327-0.7411
0.9989-1.0023
0.8782-0.8832
1.2130-1.2267
0.9989-1.0023
0.8822-0.8872
0.8902-0.8952
0.7440-0.7524
1.0641-1.0701
0.8957-0.9006
0.9116-0.9166
0.7624-0.7708
0.9216-0.9266
0.9246-0.9296
0.7742-0.7825
0.9973-1.0027
0.9361-0.9411
0.9371-0.9421
120
-------
Method 1668B November 2008
Labeled or native CB1
2,3,3',4',5,6-HxCB
2,3,3',4,5,6-HxCB
2,3,3',4,4',6-HxCB
2,2',3,4,5,6,6'-HpCB
2,2',3,3',4,5-HxCB
13C12-3,3',4,4',5-PeCB4'9
3,3',4,4',5-PeCB6'10
2,3,4,4',5,6-HxCB
13C12-2,2',3,3',5,5',6-HpCB7
2,2',3,3',5,5',6-HpCB
2,2',3,3',4,5',6-HpCB
2,3,3',4,5,5'-HxCB
2,2',3,4',5,5',6-HpCB6
2,2',3,4,4',5,6'-HpCB
2,2',3,3',4,4'-HxCB6
2,3,3',4',5,5'-HxCB
2,2',3,4,4',5',6-HpCB
13C12-2,3',4,4',5,5'-HxCB5'9
2,3',4,4',5,5'-HxCB10
2,2',3,4,5,5',6-HpCB
2,2',3,3',4,5,6'-HpCB
2,2',3,4,4',5,6-HpCB
2,2',3,3',4',5,6-HpCB
2,2'3,3',4,4',6-HpCB
13C12-2,3,3',4,4',5 -HxCB9
2,3,3',4,4',5-HxCB10
Congener
number2
163
160
158
186
129
126L
126
166
178L
178
175
159
187
182
128
162
183
167L
167
185
174
181
177
171
156L
156
Retention time and quantitation
references
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-3,3',4,4',5-PeCB4'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',3,3',5,5',6-HpCB7
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4'55'-HxCB7
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3,3',4,4',5 -HxCB9
Congener
number
167L
167L
167L
189L
167L
101L
126L
167L
178L
189L
189L
167L
189L
189L
167L
167L
189L
138L
167L
189L
189L
189L
189L
189L
138L
156L
RT
31:28
31:33
31:35
31:36
31:48
31:49
31:49
32:13
32:14
32:14
32:33
32:43
32:46
32:47
32:52
33:00
33:06
33:23
33:23
33:43
34:07
34:11
34:22
34:40
34:40
34:40
RRT
0.9426
0.9451
0.9461
0.7930
0.9526
1.3156
1.0000
0.9651
1.0000
0.8089
0.8168
0.9800
0.8223
0.8227
0.9845
0.9885
0.8306
1.0654
1.0000
0.8461
0.8561
0.8578
0.8624
0.8699
1.1064
1.0000
RRT QC limits3
0.9401-0.9451
0.9426-0.9476
0.9436-0.9486
0.7888-0.7972
0.9501-0.9551
1.3088-1.3225
0.9990-1.0021
0.9626-0.9675
0.9974-1.0026
0.8068-0.8110
0.8147-0.8189
0.9775-0.9825
0.8202-0.8243
0.8206-0.8248
0.9820-0.9870
0.9860-0.9910
0.8285-0.8327
1.0628-1.0681
0.9990-1.0020
0.8440-0.8482
0.8540-0.8582
0.8557-0.8599
0.8603-0.8645
0.8678-0.8720
1.1037-1.1090
0.9990-1.0019
121
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Method 1668B November 2008
Labeled or native CB1
13C12-2,2',3,3',5,5',6,6'-OcCB4
2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5'-HxCB9
2,3,3',4,4',5'-HxCB10
2,2',3,3',4,5,6-HpCB
2,2',3,3',4,5',6,6'-OcCB
2,2',3,4,4',5,6,6'-OcCB
2,2',3,3',4,5,5'-HpCB
2,3,3',4,5,5',6-HpCB
2,2',3,3',4,4',6,6'-OcCB
2,2',3,4,4',5,5'-HpCB6
2,3,3',4',5,5',6-HpCB
2,3,3',4,4',5',6-HpCB
2,2',3,3',4,5,6,6'-OcCB
13C12-3,3',4,4',5,5'-HxCB4'9
3,3',4,4',5,5'-HxCB6'10
2,2',3,3',4,4',5-HpCB6
2,3,3',4,4',5,6-HpCB
2,2',3,3',4,5,5',6-OcCB
2,2',3,3',4,5,5',6'-OcCB
2,2',3,3',4,4',5,6'-OcCB
2,2',3,4,4',5,5',6-OcCB
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
2,3,3',4,4',5,5'-HpCB10
2,2',3,3',4,4',5,6-OcCB6
13C12-2,2',3,3',4,555',6,6'-NoCB4
Congener
number2
202L
202
157L
157
173
201
204
172
192
197
180
193
191
200
169L
169
170
190
198
199
196
203
189L
189
195
208L
Retention time and quantitation
references
13C12-C18-PCB-1945
13C12-2,2',3,3',555',6,6'-OcCB4
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3,3',4,4',5'-HxCB9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-2,2',3,4,4',5'-HxCB7
13C12-3,3',4,4',5,5'-HxCB4'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-2,2',3,3',5,5',6-HpCB7
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-C19-PCB-2064'5
Congener
number
194L
202L
138L
157L
189L
194L
194L
189L
189L
194L
189L
189L
189L
194L
138L
169L
189L
189L
194L
194L
194L
194L
178L
189L
194L
206L
RT
34:56
34:56
34:57
34:57
35:04
35:25
35:36
35:41
35:51
35:55
36:07
36:20
36:34
36:49
37:19
37:19
37:44
37:56
38:34
38:43
39:05
39:05
39:51
39:51
40:45
41:03
RRT
0.8265
1.0000
1.1154
1.0000
0.8800
0.8379
0.8423
0.8954
0.8996
0.8498
0.9063
0.9118
0.9176
0.8711
1.1910
1.0000
0.9469
0.9519
0.9125
0.9160
0.9247
0.9247
1.2363
1.0000
0.9641
0.9149
RRT QC limits3
0.8245-0.8285
0.9990-1.0019
1.1128-1.1181
0.9990-1.0019
0.8779-0.8821
0.8360-0.8399
0.8403-0.8442
0.8934-0.8975
0.8975-0.9017
0.8478-0.8517
0.9042-0.9084
0.9097-0.9138
0.9155-0.9197
0.8691-0.8730
1.1883-1.1936
0.9991-1.0018
0.9448-0.9490
0.9498-0.9540
0.9105-0.9144
0.9140-0.9180
0.9227-0.9267
0.9227-0.9267
1.2311-1.2415
0.9992-1.0017
0.9621-0.9661
0.9131-0.9168
122
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Method 1668B November 2008
Labeled or native CB1
2,2',3,3',4,5,5',6,6'-NoCB
2,2',3,3',4,4',5,6,6'-NoCB
13C12-2,2',3,3',4,4',5,5'-OcCB5
2,2',3,3',4,4',5,5'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB4
2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB4'5
2,2',3,3',4,4',5,5',6-NoCB6
13C12-2,2',3,3',4,4',5,5',6,6'-DeCB4'5
2,2',3,3',4,4',5,5',6,6'-DeCB6
Congener
number2
208
207
194L
194
205L
205
206L
206
209L
209
Retention time and quantitation
references
13C12-2,2',3,3',4,5,5',6,6'-NoCB4
13C12-C19-PCB-2064'5
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-2,3,3',4,4',5,5',6-OcCB4
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C19-PCB-2064'5
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C110-PCB-2094'5
Congener
number
208L
206L
178L
194L
194L
205L
178L
206L
178L
209L
RT
41:03
41:32
42:16
42:16
42:44
42:44
44:52
44:52
46:55
46:55
RRT
1.0000
0.9257
1.3113
1.0000
1.0110
1.0000
1.3919
1.0000
1.4555
1.0000
RRT QC limits3
0.9992-1.0016
0.9238-0.9276
1.3061-1.3164
0.9992-1.0016
1.0091-1.0130
0.9992-1.0016
1.3868-1.3971
0.9993-1.0015
1.4504-1.4607
0.9993-1.0014
1. Abbreviations for chlorination levels
MoCB monochlorobiphenyl HxCB
DiCB dichlorobiphenyl HpCB
TrCB trichlorobiphenyl OcCB
TeCB tetrachlorobiphenyl NoCB
PeCB pentachlorobiphenyl DeCB
hexachlorobiphenyl
heptachlorobiphenyl
octachlorobiphenyl
nonachlorobiphenyl
decachlorobiphenyl
123
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Method 1668B November 2008
2. Suffix "L" indicates labeled compound.
3. For native CBs determined by isotope dilution quantitation, RRT QC limits were constructed using -2 to +4 seconds around the retention
time for the labeled analog. For native CBs determined by internal standard quantitation, RRT QC limits were constructed using a ± 2
percent window around the retention time for retention times in the range of 0.8-1.2 and a ± 4 percent window around the retention time
for retention times <0.8 and >1.2. These windows may not be adequate for analyte identification (See the note in Section 16.4)
4. Labeled level of chlorination (LOC) window-defining congener
5. Labeled level of chlorination (LOC) quantitation congener
6. National Oceanic and Atmospheric Administration (NOAA) congener of interest
7. Instrument internal standard
8. Clean-up standard
9. Labeled internal standard for World Health Organization (WHO) toxic congener
10. WHO toxic congener
Table A-2. Scan descriptors, levels of chlorination, m/z information, and substances monitored by HRGC/HRMS
Function and chlorine level
Fn-1
Cl-1
Fn-2
m/z
188.0393
190.0363
200.0795
202.0766
218.9856
222.0003
m/z type
M
M+2
M
M+2
lock
M
m/z formula
12C12 H9 35C1
C]2 Hg Cl
C]2 Hg Cl
13C12 H9 37C1
C4F9
C12 H8 C12
Substance
Cl-1 PCB
C1-1P CB
13C12 Cl-1 PCB
13C12 Cl-1 PCB
PFK
Cl-2 PCB
124
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Method 1668B November 2008
Function and chlorine level
Cl-2,3
Fn-3
Cl-3,4,5
m/z
223.9974
225.9944
234.0406
236.0376
242.9856
255.9613
257.9584
255.9613
257.9584
259.9554
268.0016
269.9986
280.9825
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
337.9207
339.9178
m/z type
M+2
M+4
M
M+2
lock
M
M+2
M
M+2
M+4
M
M+2
lock
M
M+2
M+4
M
M+2
M
M+2
M+4
M+2
M+4
m/z formula
12C12H835C137C1
12C12H837C12
13C12H835C12
13C12 H8 35C1 37 Cl
C6F9
Cj2 H7 03
12C12H735C1237C1
12C12H735C13
12C12H735C1237C1
12C12 H7 35C1 37C12
13C12H735C13
13C12 H7 35C12 37C1
C6Fn
C12 H6 C14
12C12H635C1337C1
12C12H635C1237C12
13C12H635C14
13C12H635C1337C1
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
13C12 H5 35C14 37C1
13C12 H5 35C13 37C12
Substance
Cl-2 PCB
Cl-2 PCB
13C12 Cl-2 PCB
13C12 Cl-2 PCB
PFK
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
PFK
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
125
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Method 1668B November 2008
Function and chlorine level
Fn-4
Cl-4,5,6
Fn-5
Cl-5,6,7,8
m/z
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
330.9792
337.9207
339.9178
359.8415
361.8385
363.8356
371.8817
373.8788
323.8834
325.8804
327.8775
337.9207
339.9178
354.9792
359.8415
m/z type
M
M+2
M+4
M+2
M+4
M
M+2
M+4
lock
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M
M+2
M+4
M+2
M+4
lock
M+2
m/z formula
12C12H635C14
12C12H635C1337C1
12C12H635C1237C12
13C12 H6 35C13 37C1
13C12H635C1237C12
Cj2 FL; 05
12C12H535C1437C1
12C12H535C1337C12
C7F15
13C12 H5 35C14 37C1
13C12H535C1337C12
13C12H435Cl537ClCl-6
13C12H435C1437C12
13C12 H4 35C13 37C12
13C12 H4 35C15 37C1
13C12H435C1437C12
12C12H535C15
12C12 H5 35C14 37C1
12C12H535C1337C12
13C12 H5 35C14 37C1
13C12H535C1337C12
C9F13
12C12H435C1537C1
Substance
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
PFK
13C12 Cl-5 PCB
13C12 Cl-5 PCB
PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
PFK
Cl-6 PCB
126
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Method 1668B November 2008
Function and chlorine level
Fn-6
Cl-8,9,10
m/z
361.8385
363.8356
371.8817
373.8788
393.8025
395.7995
397.7966
405.8428
407.8398
427.7635
429.7606
431.7576
439.8038
441.8008
454.9728
427.7635
429.7606
431.7576
439.8038
441.8008
442.9728
454.9728
461.7246
m/z type
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
M+2
M+4
M+6
M+2
M+4
QC
lock
M+2
m/z formula
12C12H435C1437C12
12C12H435C1337C13
13C12H435C1537C1
13C12 H4 35C14 37C12
12C12H335C1637C1
12C12H335C1537C12
12C12H335C1437C13
13C12H335C1637C1
13C12 H3 35C15 37C12
12C12 H2 35C17 37C1
12C12H235C1637C12
12C12H235C1537C13
13C12H235C1737C1
13C12 H2 35C16 37C12
^11 M7
12C12H235C1737C1
12C12H235C1637C12
12C12H235C1537C13
13C12H235C1737C1
13C12 H2 35C16 37C12
CIO-TIS
CnF13
^H^Cl^Cl
Substance
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
PFK
Cl-9 PCB
127
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Method 1668B November 2008
Function and chlorine level
m/z
463.7216
465.7187
473.7648
475.7619
495.6856
499.6797
501.6767
507.7258
509.7229
511.7199
m/z type
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M+6
m/z formula
^C^H^CV'Ci,
'^H^d^Cls
"CuH^CV'Cl
13C12 Hj 35C17 37C12
C]2rl4 Clg Cl
12C1235C1737C13
12C1235C1637C14
C]2rl4 Clg Cl
Cj2rl4 C18 C12
C12H4 Clg C14
Substance
Cl-9 PCB
Cl-9 PCB
13C12 Cl-9 PCB
13C12 Cl-9 PCB
Cl-10 PCB
Cl-10 PCB
Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
1. Isotopic masses used for accurate mass calculation
^1.0078
12.0000
13.0034
34.9689
12C
13C
35C1
37
C1
36.9659
18.9984
128
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