Ł% United States
Environmental Protectio
^1 M^k. Agency
Office of Water
www.epa.gov December 2022
3rd Draft Method 1633
Analysis of Per- and Polyfluoroalkyl Substances
(PFAS) in Aqueous, Solid, Biosolids, and Tissue
Samples by LC-MS/MS
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U.S. Environmental Protection Agency
Office of Water (4303T)
Office of Science and Technology
Engineering and Analysis Division
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA 821-D-22-003
3rd Draft Method 1633
December 2022
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Method 1633
Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in
Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS
December 2022
Notice
This document represents the third draft of Method 1633 for PFAS currently under development by
the EPA Office of Water, Engineering and Analysis Division (EAD), in collaboration with the
Department of Defense (DoD), and includes the wastewater results of the multi-laboratory validation
study. Overall, the method demonstrated good recovery for all the spiked wastewaters. The multi-
laboratory validation study of the method is still underway, and the Office of Water will use the final
results of the multi-laboratory validation study to finalize the method and add formal performance
criteria for all of the matrices.
Issuing this third draft version of the method does not require its use for Clean Water Act compliance
monitoring at the Federal level; that will only occur after it has been proposed and promulgated
through rulemaking (e.g., added to 40 CFR Part 136). However, EPA recommends the use of this
method, and it is currently the only PFAS method that has been validated for wastewater by 8
laboratories in 6 diverse and challenging wastewater matrices.
EPA anticipates issuing two more versions of the method in the next year:
1. 4th Draft: Once all the groundwater and surface water data are reviewed and analyzed, the QC
acceptance criteria in the method generated from the wastewater data will be reexamined and the
method may be revised to apply those criteria to all aqueous matrices. Alternatively, EPA may
develop separate QC acceptance criteria for groundwater and surface water samples.
(Wastewater is generally a more difficult matrix to analyze than groundwater or surface water,
and the wastewater data often drives the statistical determinations of the upper and lower limits of
QC criteria. Preliminary review of the surface water and groundwater data indicates this may be
the case for Method 1633 as well.)
2. Final: When the data for all the solid matrices and landfill leachate are reviewed and analyzed,
final QC criteria for the solid matrices (soil, sediment, biosolids, and tissue) and landfill leachate
will be added to the method to produce the version of the method that EPA expects to propose
through rulemaking.
Those future versions are unlikely to involve substantive changes to the procedure. They will update
the tables that dictate the required performance criteria for the relevant matrices. EPA decided to
release multiple draft of the method in response to stakeholder requests to update the method with the
best data as soon as practical.
3rd Draft Method 1633
December 2022
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Acknowledgements
This method was prepared under the direction of Adrian Hanley of the Engineering and Analysis
Division, Office of Science and Technology, within EPA's Office of Water, in collaboration with the
Department of Defense.
EPA acknowledges the support of a number of organizations in the development and validation of this
method, including the developers of the original procedure, the Department of Defense, the members of
EPA's workgroup, and EPA's support contractor staff at General Dynamics Information Technology,
including:
Adrian Hanley
EPA Office of Water, Office of Science and Technology, Engineering and Analysis Division
S. Bekah Burket
EPA Office of Water, Office of Science and Technology, Engineering and Analysis Division
Troy Strock
EPA Office of Land and Emergency Management
Marc Mills
EPA Office of Research and Development
Diane Reese
EPA Region 4
Steve Reimer
EPA Region 10
Janice Willey
Department of Defense, Naval Sea Systems Command
Richard H. Anderson
Department of Defense, Air Force Civil Engineer Center
Allyson Buytendyk
Institute for Defense Analysis
Coreen Hamilton
SGS-AXYS Analytical
Ivona Zysk
SGS-AXYS Analytical
Ting Chen
SGS-AXYS Analytical
Henry Huang
SGS-AXYS Analytical
Mirna Alpizar
General Dynamics Information Technology
Harry McCarty
General Dynamics Information Technology
Disclaimer
See the notice on the title page regarding the status of this method.
Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.
Contact
Please address questions, comments, or suggestions to:
CWA Methods Team, Engineering and Analysis Division (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460
https://www.epa.gov/cwa-methods
https://www.epa.gov/cwa-methods/forms/contact-us-about-cwa-analytical-methods
3rd Draft Method 1633
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Table of Contents
Acknowledgements i
Disclaimer i
Contact i
I.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Contamination and Interferences 2
5.0 Safety 4
6.0 Equipment and Supplies 5
7.0 Reagents and Standards 9
8.0 Sample Collection, Preservation, Storage, and Holding Times 14
9.0 Quality Control 16
10.0 Calibration and Standardization 20
II.0 Sample Preparation and Extraction 27
12.0 Extraction, Cleanup, and Concentration 34
13.0 Instrumental Analysis 36
14.0 Performance Tests during Routine Operations 37
15.0 Data Analysis and Calculations 40
16.0 Method Performance 44
17.0 Pollution Prevention 44
18.0 Waste Management 44
19.0 References 45
20.0 Tables, Diagrams, Flowcharts, and Validation Data 47
21.0 Glossary 64
Appendix A - Sample Pre-screening Instructions 68
Appendix B - Aqueous Sample Subsampling Instructions 69
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Method 1633 - Analysis of Per- and Polyfluoroalkyl Substances (PFAS)
in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS
1.0 Scope and Application
1.1 Method 1633 is for use in the Clean Water Act (CWA) for the determination of the per- and
polyfluoroalkyl substances (PFAS) in Table 1 in aqueous, solid (soil, biosolids, sediment) and tissue
samples by liquid chromatography/mass spectrometry (LC-MS/MS).
1.2 The method calibrates and quantifies PFAS analytes using isotopically labeled standards. Where
linear and branched isomers are present in the sample and either qualitative or quantitative standards
containing branched and linear isomers are commercially available, the PFAS analyte is reported as
a single result calculated from the combined responses of the linear and branched isomers.
1.3 The instrumental portion of this method is for use only by analysts experienced with LC-MS/MS 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.
1.4 By their very nature, many components of PFAS present analytical challenges unique to this class
of analytes. For example, PFAS analytes readily adhere to the walls of the sample containers and
may also stratify in the container. EPA has included procedures in the method that must be
employed to address such challenges (see Section 11.0 and Appendices A and B).
1.5 This method is "performance-based," which means that modifications may be made without
additional EPA review to improve performance (e.g., overcome interferences, or improve the
sensitivity, accuracy, or precision of the results) provided that all performance criteria in this
method are met. Requirements for establishing equivalency are in Section 9.1.2 and include
9.1.2.2c. For CWA uses, additional flexibility is described at 40 CFR 136.6. Changes in
performance, sensitivity, selectivity, precision, recovery, etc., that result from modifications within
the scope of 40 CFR Part 136.6, and Section 9.0 of this method must be documented, as well as
how these modifications compare to the specifications in this method. After promulgation, changes
outside the scope of 40 CFR Part 136.6 and Section 9.0 of this method may require prior review or
approval by EPA under the Clean Water Act Alternate Test Procedure program described at 40
CFR 136.4 and 136.5.
1.6 The target analytes in Table 1 were included in this method based in part on the availability of
standards for both unlabeled and isotopically labeled PFAS compounds at the time that the method
was first developed. Data from the single-laboratory and multi-laboratory validation studies
suggest that the method does not perform as well for some of the PFAS listed in Table 1 as for
others, which is not surprising given the wide range of structures across the nine classes of
compounds in that table. EPA has identified the analyte classes that are poor performers in Table 1
and data users and laboratories should take that information into account during project planning.
2.0 Summary of Method
Environmental samples are prepared and extracted using method-specific procedures. Sample extracts
are subjected to cleanup procedures designed to remove interferences. Analyses of the sample extracts
are conducted by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Sample concentrations
are determined by isotope dilution or extracted internal standard quantification (see Section 10.3) using
isotopically labeled compounds added to the samples before extraction.
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2.1 Extraction
2.1.1 Aqueous samples are spiked with isotopically labeled standards, extracted using
solid-phase extraction (SPE) cartridges and undergo cleanup using carbon before
analysis.
2.1.2 Solid samples are spiked with isotopically labeled standards, extracted into basic
methanol, and cleaned up by carbon and SPE cartridges before analysis.
2.1.3 Tissue samples are spiked with isotopically labeled standards, extracted in potassium
hydroxide and acetonitrile followed by basic methanol, and cleaned up by carbon and SPE
cartridges before analysis.
2.2 This method measures the analytes as either their anions or neutral forms. The default approach for
Clean Water Act uses of the method is to report the analytes in their acid or neutral forms, using the
equations in Section 15.2, although the differences between the anion and acid form concentrations
are minimal (See Table 2). Other project-specific reporting schemes may be used where required.
2.3 Individual PFAS analytes are identified through peak analysis of the quantification and
confirmation ions, where applicable.
2.4 Quantitative determination of target analyte concentrations is made with respect to an isotopically
labeled PFAS standard; the concentrations are then used to convert raw peak areas in sample
chromatograms to final concentrations.
2.5 By virtue of the use of isotope dilution and extracted internal standard quantification (see Section
10.3), the results for the target analytes are corrected for any losses that may occur during sample
extraction, extract cleanup, and concentration. Isotope dilution calibration also may address matrix
effects that lead to signal suppression or enhancement in the LC-MS/MS system and would
otherwise lead to measurement bias. Isotopically labeled compound recoveries are determined by
comparison to the responses of one of seven non-extracted internal standards (a.k.a., the "recovery"
standards) and are used as general indicators of overall analytical quality.
2.6 The quality of the analysis is assured through reproducible calibration and testing of the extraction,
cleanup, and LC-MS/MS systems.
3.0 Definitions
Definitions are provided 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 and
elevated baselines causing misinterpretation of chromatograms. Specific selection of reagents and
solvents may be required.
4.2 Clean all equipment prior to, and after each use to avoid PFAS cross-contamination. Typical
cleaning solvents used include water, methanol, and methanolic ammonium hydroxide. The
residual PFAS content of disposable plasticware and filters must be verified by batch/lot number
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and may be used without cleaning if the mass of any PFAS analyte found in a nominal 500-mL
aqueous sample is less than half the Minimum Level (ML, see Table 6).
4.2.1 All glass equipment that is used in the preparation or storage of reagents is cleaned by
washing with detergent and baking in a kiln or furnace (Section 6.2.2). After detergent
washing, glassware should be rinsed immediately with reagent water. Prior to use, baked
glassware must be solvent rinsed and then air dried. A solvent rinse procedure using
methanolic ammonium hydroxide (1%), toluene, and methanol is recommended.
4.2.2 All parts of the SPE manifold must be cleaned between samples with methanolic
ammonium hydroxide (1%) and air dried prior to use. Sonication with methanolic
ammonium hydroxide (1%) may be used for components that will fit in an ultrasonic bath.
Smaller parts, like the needles, adapters, reservoirs, and stopcocks associated with the
manifold, require rinsing with tap water prior to manual cleaning or sonicating with
methanolic ammonium hydroxide (1%) and air drying. When in use, after loading the
samples but prior to elution procedures, the chamber must be rinsed with methanolic
ammonium hydroxide (1%).
4.2.3 All equipment used in the filleting, dissecting, shucking, compositing, and homogenization
of tissue must be cleaned with detergent and hot water, then rinsed with ultra-pure water
followed by a series of solvent rinses. A typical solvent rinse procedure would be acetone,
followed by toluene, and then methanol.
4.3 All materials used in the analysis must be demonstrated to be free from interferences by running
method blanks (Section 9.5) at the beginning and with each sample batch (samples started through
the extraction process in a given batch during the same work shift, to a maximum of 20 field
samples).
4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix being tested.
Ideally, the reference matrix should not contain PFAS in detectable amounts (i.e., above the
laboratory's method detection limits (MDLs).
4.3.2 For tissue, chicken breast or other similar animal tissue (see Section 7.2.3) may be used as
the reference matrix. The laboratory must verify that the source product used does not
contain PFAS in detectable amounts.
4.3.3 When a reference matrix that simulates the sample matrix under test is not available,
reagent water (Section 7.2.1) can be used to simulate water samples and Ottawa sand
and/or reagent-grade sand (Section 7.2.2) can be used to simulate soils.
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 native PFAS. Because low levels of PFAS are
measured by this method, elimination of interferences is essential. The cleanup steps given in
Section 12.0 can be used to reduce or eliminate these interferences and thereby permit reliable
determination of the PFAS at the levels shown in Table 6. The most frequently encountered
interferences are fluoropolymers; however, bile salts (e.g., Taurodeoxycholic Acid [TDCA]) may
be present in various matrices, including fish and wastewaters, and can interfere in the
chromatography. For this reason, analysis of a standard containing TDCA is required as part of
establishing the initial chromatographic conditions (see Sections 10.2.2.5 and 10.3.5).
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4.5 Each piece of reusable glassware may be numbered to associate that glassware with the processing
of a particular sample. This may 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. If that approach is
used, the numbered glassware should be assigned to field samples, QC samples, and method blanks
in a random manner (e.g., do not use the same glassware for method blanks in every batch).
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 Several PFAS, including PFOA, have been described as likely to be carcinogenic to
humans. Pure standards and materials known or suspected to contain PFAS should be
handled by trained personnel, with suitable protection to skin and eyes, and care should be
taken not to breathe the vapors or ingest the materials.
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,
following universal safety measures.
5.2 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified
in this method. A reference file of safety data sheets (SDS) should also be made available to all
personnel involved in these analyses. Additional information on laboratory safety can be found in
References 1-4. The references and bibliography at the end of Reference 3 are particularly
comprehensive in dealing with the general subject of laboratory safety.
5.3 Samples suspected to contain these compounds are handled using essentially the same techniques
employed in handling radioactive or infectious materials. Well-ventilated, controlled access
laboratories are required. Assistance in evaluating the health hazards of particular laboratory
conditions may be obtained from certain consulting laboratories and from State Departments of
Health or Labor, many of which have an industrial health service. Each laboratory must develop a
strict safety program for handling these compounds.
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 face velocity. Gross losses to the laboratory
ventilation system must not be allowed. Handling of the dilute solutions normally used in
analytical 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 with adequate face velocity 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.
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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) using soaps or detergents that are
free of PFAS. Before starting work, staff should avoid the use of personal-care products on
exposed skin, because such products may be a source of some PFAS.
5.3.5 Confinement - Isolated work areas posted with signs, segregated glassware and tools, and
plastic absorbent paper on bench tops will aid in confining contamination.
5.3.6 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.
5.3.7 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.
5.4 Biosolids samples may contain high concentrations of biohazards and must be handled with gloves
and opened in a fume hood or biological safety cabinet to prevent exposure. Laboratory staff
should know and observe the safety procedures required in a microbiology laboratory that handles
pathogenic organisms when handling biosolids samples.
6.0 Equipment and Supplies
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. All equipment described below must be constructed of materials that will not
react with or sorb PFAS constituents and before use must be demonstrated to be free of PFAS at
levels that would be detectable in blanks or samples. Where available, certification of the PFAS
levels of the materials provided by the supplier will suffice. However, in the absence of such
certification from the supplier, and in the event ofpersistent problems with method blanks and
other QC samples, the laboratory is responsible for independent testing of all equipment and
supplies.
6.1 Sampling equipment for discrete or composite sampling.
6.1.1 Sample bottles and caps
Note: Do not use PTFE-lined caps on sample containers. All containers must be demonstrated to
be PFAS-free at the laboratory's MDLs for the target analytes by testing one or more
representative containers from each lot.
6.1.1.1 Liquid samples (waters, sludges, and similar materials containing < 50 mg
solids per sample) - Sample bottle, HDPE, 500-mL, 250-mL, and 125-mL, with
linerless HDPE or polypropylene caps.
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6.1.1.2 Solid samples (soils, sediments, and biosolids that contain more than 50 mg
solids) - Sample bottle or jar, wide-mouth, HDPE, 500-mL, with linerless
HDPE or polypropylene caps.
6.1.1.3 Tissue samples - Sample jar, wide-mouth HDPE, 100-mL, with linerless HDPE
or polypropylene caps.
6.1.2 Grab sampling equipment - Sample containers may be attached to a metal or wooden pole
with stainless steel hose clamps or cable ties in order to reach into flowing waters.
Stainless steel scoops or spoons may be used to collect samples of soils, sediments, and
biosolids.
6.1.3 Compositing equipment -Because some PFAS are known surfactants, EPA does not
recommend composite sampling for compliance monitoring (see Section 8.2), but if
composite sampling is approved for given project, the equipment described below may be
used.
Automatic or manual compositing system incorporating properly cleaned containers . An
integrating flow meter is used to collect proportional composite samples. Only HDPE
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, each lot of
tubing must be thoroughly rinsed with methanol, followed by repeated rinsing with reagent
water to minimize sample contamination. The final reagent water rinse should be collected
and analyzed for PFAS to confirm that the tubing is suitable for use.
6.2 Equipment for glassware cleaning
Note: If blanks from other glassware show no detectable PFAS contamination when using fewer
cleaning steps than required above, unnecessary cleaning steps and equipment may be
eliminated.
6.2.1 Laboratory sink with overhead fume hood
6.2.2 Kiln - Capable of reaching 450 °C within 2 hours and maintaining 450 - 500 °C ± 10 °C,
with temperature controller and safety switch (Cress Manufacturing Co., Santa Fe Springs,
CA, B31H, X3 ITS, or equivalent). For safety, the kiln or furnace should be vented outside
the laboratory, or to a trapping system.
6.3 Equipment for sample preparation
6.3.1 Polyethylene gloves
6.3.2 Laboratory fume hood (of sufficient size to contain the sample preparation equipment listed
below)
6.3.3 Glove box (optional)
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 105 ± 5 °C
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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
6.3.7 Aluminum foil
6.3.8 Disposable spoons, 10 mg, polypropylene or stainless steel
6.3.9 Ultrasonic mixer (sonicator) - Immersion style, for use with tissue samples
6.3.10 HDPE bottles, with linerless HDPE or polypropylene caps - 60 mL
6.3.11 pH Paper, range 0-14 - (Whatman® Panpeha™ or equivalent), 0.5-unit readability (papers
with other pH ranges may be suitable as well)
6.3.12 Analog or digital vortex mixer, single or multi-tube (Fisher Scientific 02-215-452, or
equivalent)
6.3.13 Volumetric flasks, Class A
6.3.14 Disposable polypropylene collection tubes (13 x 100 mm, 8 mL)
6.3.15 Variable speed mixing table (Fisherbrand™ Nutating mixer or equivalent)
6.4 Filtration
6.4.1 Silanized glass wool (Sigma-Aldrich, Cat # 20411 or equivalent) - store in a clean glass jar
and rinsed with methanol (2 times) prior to use.
6.4.2 Disposable syringe filter, 25-mm, 0.2-(.un Nylon membrane, PALL/Acrodisc or equivalent
6.4.3 Glass fiber filter, 47-mm, PALL A/E or equivalent, for use in determining total suspended
solids
6.5 Centrifuge apparatus
6.5.1 Centrifuge (Thermo Scientific Legend RT+, 16-cm rotor, or equivalent), capable of
reaching at least 3000 rpm
6.5.2 Centrifuge tubes - Disposable polypropylene centrifuge tubes (50 mL)
6.6 Pipettes
6.6.1 Norm-Ject® syringe (or equivalent), polypropylene/HDPE, 5 mL
6.6.2 Variable volume pipettes with disposable HDPE or polypropylene tips (10 (iL to 5 mL) -
used for preparation of calibration standards and spiked samples.
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6.6.3 Disposable glass pipets
6.6.4 Calibrated mechanical pipettes or Hamilton graduated syringes
6.7 Solid-phase extraction
6.7.1 Solid-phase extraction (SPE) cartridges (Waters Oasis WAX 150 mg, Cat # 186002493 or
equivalent). The SPE sorbent must have a pKa above 8 so that it remains positively
charged during the extraction.
Note: SPE cartridges with a different bed volume (e.g., 500 mg) may be used; however, the
laboratory must demonstrate that the bed volume does not negatively affect analyte
absorption and elution, by performing the initial demonstration of capability analyses
described in Section 9.2.
6.7.2 Vacuum manifold for SPE Cartridges (Waters™ extraction manifold #WAT200607 or
equivalent)
6.8 Evaporation
6.8.1 Automatic or manual solvent evaporation system (TurboVap® LV or
equivalent)
6.8.2 Evaporation/concentrator tubes: 60 mL clear glass vial, 30 x 125 mm, without
caps (Wheaton Cat # W226060 or equivalent). Cover with foil if required.
6.9 Vials
6.9.1 Snap cap/crimp top vials, 300 |_iL. polypropylene (12x32 mm) - used in sample
pre-screening (DWK Life Sciences Cat # 225180 or equivalent)
6.9.2 Polypropylene crimp/snap vials, 1 mL (Agilent Cat # 5182-0567 or equivalent)
6.9.3 Clear snap cap, polyethylene, 11 mm (Fisher Scientific # 03-375-24E, or
equivalent)
6.9.4 Single step filter vials (Restek Thomson SINGLE StEP® Standard Filter Vials,
0.2-|im Nylon membrane, with Black Preslit caps Cat # 25891 or equivalent) -
used in sample pre-screening.
6.10 Instrument
6.10.1 Ultra high-performance liquid chromatograph (UPLC, also called UHPLC) or high-
performance liquid chromatograph (HPLC) equipped with tandem quadrupole mass
spectrometer (Waters Xevo TQ-S Micro or equivalent) capable of collecting at least 10
scans across a chromatographic peak
6.10.2 C18 column, 1.7 |_im. 50x2.1 mm (Waters Acquity UPLC® BEH or equivalent)
6.10.3 Guard column (Phenomenex Kinetex® Evo C18 or equivalent)
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6.10.4 Trap/delay column (Purospher Star RP-18 endcapped [3 |im | Hibar® RT 50-4 or
equivalent)
6.11 Bottles, HDPE or glass, with linerless HDPE or polypropylene caps. Various sizes. To store
prepared reagents.
7.0 Reagents and Standards
7.1 Reagents
Reagents prepared by the laboratory may be stored in either glass or HDPE containers. Proper
cleaning procedures (Section 4.2) must be followed prior to using the containers. Before use, all
reagents described below must be demonstrated to be free of PFAS at levels that would be
detectable in blanks or samples Where available, certification of the PFAS levels of the reagents
provided by the supplier will suffice. However, in the absence of such certification from the
supplier, and in the event of persistent problems with method blanks and other QC samples, the
laboratory is responsible for independent testing of each lot.
7.1.1 Acetic acid (concentrated) - ACS grade or equivalent, store at room temperature
7.1.2 Acetic acid (0.1%) - dissolve acetic acid (1 mL) in reagent water (1 L), store at room
temperature, replace after 3 months.
7.1.3 Acetonitrile - UPLC grade or equivalent, verified before use, store at room temperature
7.1.4 Ammonium acetate - (Caledon Ultra LC/MS grade, or equivalent), store at 2-8 °C, replace
2 years after opening date
7.1.5 Ammonium hydroxide - certified ACS+ grade or equivalent, 30% in water, store at room
temperature, and replace 2 years after opening date
7.1.6 Aqueous ammonium hydroxide (3%) - add ammonium hydroxide (10 mL, 30%) to reagent
water (90 mL), store at room temperature, replace after 3 months
7.1.7 Methanolic ammonium hydroxide
7.1.7.1 Methanolic ammonium hydroxide (0.3% v/v) - add ammonium hydroxide (1 mL,
30%) to methanol (99 mL), store at room temperature, replace after 1 month
7.1.7.2 Methanolic ammonium hydroxide (1% v/v) - add ammonium hydroxide (3.3 mL,
30%) to methanol (97 mL), store at room temperature, replace after 1 month
7.1.7.3 Methanolic ammonium hydroxide (2% v/v) - add ammonium hydroxide (6.6 mL,
30%) to methanol (93.4 mL), store at room temperature, replace after 1 month
7.1.8 Methanolic potassium hydroxide (0.05 M) - add 3.3 g of potassium hydroxide to 1 L of
methanol, store at room temperature, replace after 3 months
7.1.9 Methanol with 4% water, 1% ammonium hydroxide and 0.625% acetic acid (v/v) - add
ammonium hydroxide (3.3 mL, 30%), reagent water (1.7 mL) and acetic acid (0.625 mL) to
methanol (92 mL), store at room temperature, replace after 1 month. This solution is used
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to prepare the instrument blank (Section 7.3.6) and is used to dilute the extracts of samples
that exceed the calibration range (see Section 15.3).
7.1.10 Eluent A - Acetonitrile, Caledon Ultra LCMS grade or equivalent
7.1.11 Eluent B - 2 mM ammonium acetate in 95:5 water/acetonitrile. Dissolve 0.154 g of
ammonium acetate (Section 7.1.4) in 950 mL of water and 50 mL of acetonitrile (Caledon
Ultra LCMS grade, or equivalent). Store at room temperature, shelf life 2 months.
7.1.12 Formic acid - (greater than 96% purity or equivalent), verified by lot number before use,
store at room temperature
7.1.13 Formic acid
7.1.13.1 Formic acid (aqueous, 0.1 M) - dissolve formic acid (4.6 g) in reagent water (1
L), store at room temperature, replace after 2 years
7.1.13.2 Formic acid (aqueous, 0.3 M) - dissolve formic acid (13.8 g) in reagent water (1
L), store at room temperature, replace after 2 years
7.1.13.3 Formic acid (aqueous, 5% v/v) - mix 5 mL formic acid with 95 mL reagent
water, store at room temperature, replace after 2 years
7.1.13.4 Formic acid (aqueous, 50% v/v) - mix 50 mL formic acid with 50 mL reagent
water, store at room temperature, replace after 2 years
7.1.13.5 Formic acid (methanolic 1:1, 0.1 M formic acid/methanol) - mix equal volumes
of methanol and 0.1 M formic acid, store at room temperature, replace after 2
years
7.1.14 Methanol - (HPLC grade or better, 99.9 % purity), verified by lot number before use, store
at room temperature
7.1.15 Potassium hydroxide - certified ACS or equivalent, store at room temperature, replace after
2 years
7.1.16 Reagent water - Laboratory reagent water, test by lot/batch number for residual PFAS
content
7.1.17 Carbon - EnviCarb® 1-M-USP or equivalent, verified by lot number before use, store at
room temperature. Loose carbon allows for better adsorption of interferent organics.
Note: The single-laboratory validation laboratory achieved better performance with loose carbon
than carbon cartridges. Loose carbon was used for the multi-laboratory validation to
establish statistically based method performance criteria. Now that the method has been
validated for wastewater matrices, laboratories have the flexibility to implement the use
carbon cartridges for wastewater samples, as long as all method QC criteria applicable to
wastewater analyses are met (see 40 CFR 136.6). (This flexibility may be extended to other
matrices in subsequent revisions of this method.)
7.1.18 Toluene - HPLC grade, verified by lot number before use. Store at room temperature.
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7.1.19 Acetone - Pesticide grade, verified by lot number before use in rinsing tissue dissection
and processing equipment.
7.1.20 Dichloromethane (methylene chloride) - Pesticide grade, verified by lot number before use
in rinsing tissue dissection and processing equipment.
7.2 Reference matrices - Matrices in which PFAS and interfering compounds are not detected by this
method. These matrices are to be used to prepare the batch QC samples (e.g., method blank, and
ongoing precision and recovery sample).
7.2.1 Reagent water - purified water, Type I
7.2.2 Solids reference matrix - Ottawa or reagent-grade sand
7.2.3 Tissue reference matrix - chicken breast or similar animal tissue
7.3 Standard solutions - Prepare from materials of known purity and composition or purchase as
solutions or mixtures with certification to their purity, concentration, and authenticity. Observe the
safety precautions in Section 5.
Purchase of commercial standard solutions or mixtures is highly recommended for this method;
however, when these are not available, preparation of stock solutions from neat materials may be
necessary. Some PFAS, notably the fluorinated carboxylic acids, will esterify in anhydrous acidic
methanol. To such prevent esterification, standards must be stored under basic conditions. If base
is not already present, this may be accomplished by the addition of sodium hydroxide
(approximately 4 mole equivalents) when standards are diluted in methanol. If the chemical purity
is 98% or greater, the weight may be used without correction to calculate the concentration of the
standard. Dissolve an appropriate amount of assayed reference material in the required solvent.
For example, weigh 10 to 20 mg of an individual compound to three significant figures in a 10-mL
ground-glass-stoppered volumetric flask and fill to the mark with the required solvent. Once the
compound is completely dissolved, transfer the solution to a clean vial and cap.
When not being used, store standard solutions in the dark at less than 6 °C, but not frozen, unless
the vendor recommends otherwise, in screw-capped vials with foiled-lined caps. Place a mark on
the vial at the level of the solution so that solvent loss by evaporation can be detected. Discard the
solution if solvent loss has occurred.
Note: Native PFAS standards are available from several suppliers. Isotopically labeled compounds are
available from Cambridge Isotope Laboratories and Wellington Laboratories, but may also be
available from other suppliers. Listing of these suppliers does not constitute a recommendation
or endorsement for use. All diluted solutions must be stored in glass or HDPE containers that
have been thoroughly rinsed with methanol.
180-mass labeledperfluoroalkyl sulfonates may undergo isotopic exchange with water under
certain conditions, which lowers the isotopic purity of the standards over time. Similarly, some of
the deuterated standards may undergo isotopic exchange in protic solvents such as methanol.
The laboratory must maintain records of the certificates for all standards, as well as records for the
preparation of intermediate and working standards, for traceability purposes. Copies of the
certificates must be provided as part of the data packages in order to check that proper calculations
were performed.
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7.3.1 Extracted Internal Standard (EIS) - (a.k.a. isotopically labeled compounds) Prepare the EIS
solution containing the isotopically labeled compounds listed in Table 4 as extracted
internal standards in methanol from prime stocks. An aliquot of EIS solution, typically
50 |_iL. is added to each sample prior to extraction. Table 3 presents the nominal amounts
of the EIS compounds added to each sample.
Note: Larger EIS amounts may be added to samples for which pre-screening results (see Section
11.0) indicate that the sample extract will require dilution, provided that the extract
dilution will result in approximately the same masses of the EISs as are found in the
calibration standards (assuming 100% recovery).
The list of EIS compounds in Table 3 represents the compounds that were available at the
time this method was validated. Additional isotopically labeled PFAS compounds may be
included as EISs as soon as practical, once they become commercially available.
7.3.2 Non-Extracted Internal Standard (NIS) - The NIS solution containing the isotopically
labeled compounds listed in Table 3 as non-extracted internal standards is prepared in
methanol from prime stock. An aliquot of NIS solution, typically 50 |_iL. is added to each
sample prior to instrumental analysis. Table 3 presents the nominal amounts of NIS
compounds added to each sample. As with the EIS solution above, larger amounts of the
NIS compounds may be used for samples known to require extract dilution.
7.3.3 Native Standards Solution - Prepare a spiking solution, containing the method analytes
listed in Table 4, in methanol from prime stocks. The solution is used to prepare the
calibration standards and to spike the known reference QC samples that are analyzed with
every batch. Quantitative standards containing a mixture of branched and linear isomers
must be used for method analytes if they are commercially available. Currently, these
include PFOS, PFHxS, PFOSA, NMeFOSAA, NEtFOSAA, NMeFOSA, NEtFOSA,
NMeFOSE, and NEtFOSE. Additional mixtures of branched and linear isomers must be
included as soon as practical, once they become commercially available
7.3.4 Calibration standard solutions - A series of calibration solutions containing the target
analytes and the 13C-, 180-, and deuterium-labeled extracted internal standards (EIS) and
non-extracted internal standards (NIS) is used to establish the initial calibration of the
analytical instrument. The concentration of the method analytes in the solutions varies to
encompass the working range of the instrument, while the concentrations of the EIS and
NIS remain constant. The calibration solutions are prepared using methanol, 2%
methanolic ammonium hydroxide, reagent water, acetic acid, and the target analyte and
isotopically labeled compound standard solutions. After dilution, the solvent composition
of the final calibration solutions will approximate the solvent composition of the sample
extracts, which contain methanol with roughly 4% water (due to the solubility of water
from the sample in the methanolic extraction fluid), 1% ammonium hydroxide and about
0.6% acetic acid (also see Section 7.1.9). Calibration standard solutions do not undergo
solid-phase extraction/cleanup.
Concentrations for seven calibration solutions are presented in Table 4. A minimum of six
contiguous calibrations standards are required for a valid analysis when using a linear
calibration model, with at least five of the six calibration standards being within the
quantitation range (e.g., from the Limit of Quantitation [LOQ] to the highest calibration
standard). If a second-order calibration model is used, then a minimum of seven calibration
standards are required, with at least six of the seven calibration standards within the
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quantitation range. The lowest level calibration standard must meet a signal-to-noise ratio
of 3:1 for the quantitation ions and confirmation ions, and 10:1 for quantitation ions that
have no confirmation ion and be at a concentration less than or equal to the LOQ. The
calibration is verified with a standard in the middle of the laboratory's calibration range,
i.e., the CS4 standard in Table 4 if using the default calibration range in that table.
Note: Additional calibration standards, at levels lower than the lowest calibration standard listed
in the method, may be added to accommodate a lower limit of quantitation if the instrument
sensitivity allows. Calibration standards at the high end of the calibration may be
eliminated if the linearity of the instrument is exceeded or at the low end if those
calibration standards do not meet the S/Nratio criterion of 3:1, or 10:1 for analytes
without a confirmation ion, as long as the required number of calibration points is met. All
analytes with commercially available stable isotope analogues must be quantified using
isotope dilution.
7.3.5 Qualitative Standards - Standards that contain mixtures of the branched and linear isomers
of the method analytes and that are used for comparison against suspected branched isomer
peaks in field samples. These qualitative standards are not required for those analytes
where the quantitative standards in Section 7.3.3 already contain the branched and linear
isomers. Qualitative standards that are currently commercially available include PFOA and
PFNA. Additional qualitative standards must be included as soon as practical, once they
become commercially available.
7.3.6 Instrument Blank - During the analysis of a batch of samples, a solvent blank is analyzed
after standards (e.g., calibration, CV) and based on screening results or prior knowledge of
the source, after samples containing high levels of target compounds to monitor carryover
from the previous injection. The instrument blank consists of the solution in Section 7.1.9
fortified with the EIS and NIS for quantitation purposes.
7.3.7 Stability of solutions - Standard solutions used for quantitative purposes (Sections 7.3.1
through 7.3.5) should be assayed periodically (e.g., every 6 months) against certified
standard reference materials (SRMs) from the National Institute of Science and Technology
(NIST), if available, or certified reference materials from a source accredited under ISO
Guide 17034 that attests to the concentration, to assure that the composition and
concentrations have not changed.
7.4 Mass calibration solution - Use the mass calibration solution specified by the instrument
manufacturer.
7.5 Bile salt interference check standard containing Taurodeoxycholic Acid (TDCA) or Sodium
taurodeoxychloate hydrate - (Sigma Aldrich 580221-5GM, or equivalent). This standard is used to
evaluate the chromatographic program relative to the risk of an interference from bile salts in
samples when using acetonitrile as the mobile phase in the instrument. Prepare solution at a
concentration of 1 (ig/mL in the same solvent as the calibration standards. If using other mobile
phases, it will be necessary to evaluate taurochenodeoxycholic acid (TCDCA) (Sigma Aldrich
T6260-1G, or equivalent) and tauroursodeoxycholic acid (TUDCA) (Sigma Aldrich 580549-1GM,
or equivalent) as well.
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8.0 Sample Collection, Preservation, Storage, and Holding Times
8.1 Collect samples in HDPE containers following conventional sampling practices (Reference 5). All
sample containers must have linerless HDPE or polypropylene caps. Other sample collection
techniques, or sample volumes may be used, if documented.
8.2 Aqueous samples
8.2.1 Because some PFAS are known surfactants, EPA does not recommend composite sampling
for compliance monitoring. Therefore, samples that flow freely are collected as grab
samples. Collect multiple sample aliquots in HDPE bottles that have been lot-certified to
be PFAS-free. Do not fill the bottle past the shoulder, to allow room for expansion during
frozen storage.
For aqueous sources other than leachates that have not been analyzed previously, the
nominal sample size is 500-mL. For sources that are known or expected to contain levels
of any target analytes above the calibration range, samples may be collected in smaller size
containers, provided that the volume analyzed is sufficient to meet any regulatory limits.
Because the target analytes are known to bind to the interior surface of the sample
container, the entire aqueous sample that is collected must be prepared and analyzed and
subsampling avoided whenever possible. Therefore, if a sample volume smaller than 500
mL is to be used for analysis, collect the sample in an appropriately sized HDPE container.
Note: In the absence of source-specific information (e.g., historical data) on the levels of PFAS or
project-specific requirements, collect at least three aliquots of all aqueous samples to allow
sufficient volume for an original whole-volume analysis, a re-extraction and second
analysis, and for the determination ofpercent solids and for pre-screening analysis. That
third aliquot may be collected in a smaller sample container (e.g., 250-mL or 125-mL).
If composite sampling is approved for given project, the equipment described in Section
6.1.2 may be used to collect samples in refrigerated bottles using automated sampling
equipment.
8.2.2 Leachate samples from landfills can present significant challenges and therefore only
100 mL of sample is collected for the analysis. Collect three 100-mL leachate sample
aliquots in a similar manner as described in Section 8.2.1, using appropriately sized
containers that have been lot-certified to be PFAS-free.
8.2.3 Maintain all aqueous samples protected from light and at 0 - 6 °C from the time of
collection until shipped to the laboratory. Samples must be shipped with sufficient ice to
maintain the sample temperature below 6 °C during transport for a period of at least 48
hours to allow for shipping delays. The laboratory must confirm that the sample
temperature is 0 - 6 °C upon receipt. Once received by the laboratory, the samples may be
stored at 0 - 6 °C or at < -20 °C, until sample preparation. However, the allowable holding
time for samples depends on the storage temperature, as described in Section 8.5, so
samples should be shipped to the laboratory as soon as practical.
8.3 Solid (soil, sediment, biosolids), excluding tissue
8.3.1 Collect samples using wide-mouth HDPE jars that have been lot-certified to be PFAS-free,
and fill no more than % full (see Section 6.1.1.2 for container size and type).
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8.3.2 Maintain solid samples protected from light (in HDPE containers) from the time of
collection until receipt at the laboratory. Samples must be shipped with sufficient ice to
maintain the sample temperature below 6 °C for a period of at least 48 hours to allow for
shipping delays. The laboratory must confirm that the sample temperature is 0 - 6 °C upon
receipt. Once received by the laboratory, the samples may be stored at 0 - 6 °C or at
< -20 °C, until sample preparation. However, the allowable holding times for samples
depend on the storage temperature, as described in Section 8.5, so samples should be
shipped to the laboratory as soon as practical.
8.4 Fish and other tissue samples
The nature of the tissues of interest may vary by project. Field sampling plans and protocols should
explicitly state the samples to be collected and if any processing will be conducted in the field (e.g.,
filleting of whole fish or removal of organs). All field procedures must involve materials and
equipment that have been shown to be free of PFAS.
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 If whole fish are collected, wrap the fish in aluminum foil or food-grade polyethylene
tubing, and maintain at 0 - 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
before shipping. Ideally, fish should be frozen upon collection and shipped to the
laboratory on dry ice.
8.4.3 Once received by the laboratory, the samples must be maintained protected from light at
< -20 °C until prepared. Store unused samples in HDPE containers or wrapped in
aluminum foil at < -20 °C.
8.5 Holding times
8.5.1 Aqueous samples (including leachates) should be analyzed as soon as possible; however,
samples may be held in the laboratory for up to 28 days from collection, when stored at
0 - 6 °C and protected from the light, with the caveat that issues have been observed with
certain perfluorooctane sulfonamide ethanols and perfluorooctane sulfonamidoacetic acids
after 7 days. These issues are more likely to elevate the observed concentrations of other
PFAS compounds via the transformation of these precursors if they are present in the
sample (see Reference 10).
When stored at < -20 °C and protected from the light, aqueous samples may be held for up
to 90 days.
8.5.2 Soil and sediment samples may be held for up to 90 days, if stored by the laboratory in the
dark at either 0 - 6 °C or < -20 °C, with the caveat that samples may need to be extracted as
soon as possible if NFDHA is an important analyte for a given project (see Reference 10).
However, some soils and sediments may exhibit microbial growth when stored at 0 - 6 °C.
8.5.3 Tissue samples may be held for up to 90 days, if stored by the laboratory in the dark at
< -20 °C, with the same caveat regarding NFDHA.
8.5.4 Biosolids samples may be held for up to 90 days, if stored by the laboratory in the dark at
0 - 6 °C, but preferably at < -20 °C (see Reference 10). Because microbiological activity in
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biosolids samples at 0 - 6 °C may lead to production of gases which may cause the sample
to be expelled from the container when it is opened, as well as producing noxious odors,
EPA recommends that samples be frozen if they need to be stored for more than a few days
before extraction.
8.5.5 Store sample extracts in the dark at less than 0 - 6 °C until analyzed. If stored in the dark at
< 0 °C, sample extracts may be stored for up to 90 days, with the caveat that issues were
observed for some ether sulfonates after 28 days (see Reference 10). These issues may
elevate the observed concentrations of the ether sulfonates in the extract over time.
Samples may need to be extracted as soon as possible if NFDHA is an important analyte.
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance program
(Reference 6). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, analysis of samples spiked with isotopically labeled compounds to evaluate
and document data quality, and analysis of standards and blanks as tests of continued performance.
Laboratory performance is compared to established performance criteria to determine if the results
of analyses meet the performance characteristics of the method.
If the method is to be applied to a sample matrix other than water (e.g., solids and tissues), the
appropriate alternative reference matrix (Sections 7.2.2 - 7.2.3) is substituted for the reagent water
matrix (Section 7.2.1) 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 alternative extraction,
concentration, and cleanup procedures, and changes in sample volumes, columns, and
detectors. Alternative determinative techniques and changes that degrade method
performance, are not allowed without prior review and approval (see 40 CFR 136.4 and
136.5).
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 calibration will be affected by the change,
the instrument must be recalibrated per Section 10. Once the modification is
demonstrated to produce results in a relevant reference matrix and are 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
(e.g., isotopically labeled compound recovery) are met in both the initial
demonstration in Section 9.2 and in field samples and other QC samples.
9.1.2.2 The laboratory is required to maintain records of any modifications made to this
method. These records include the following, at a minimum:
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a) The names, titles, business 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.
b) A listing of pollutant(s) measured, by name and CAS Registry number.
c) A narrative stating reason(s) for the modifications (see Section 1.5).
d) Results from all quality control (QC) tests comparing the modified method to
this method, including:
i. Calibration (Section 10)
ii. Calibration verification (Section 14.3)
iii. Initial precision and recovery (Section 9.2.1)
iv. Isotopically labeled compound recovery (Section 9.3)
v. Analysis of blanks (Section 9.5)
vi. Accuracy assessment (Section 9.4)
e) 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:
i. Sample numbers and other identifiers
ii. Extraction dates
iii. Analysis dates and times
iv. Analysis sequence/run chronology
v. Sample weight or volume (Section 11)
vi. Extract volume prior to each cleanup step (Section 12)
vii. Extract volume after each cleanup step (Section 12)
viii. Injection volume (Section 13.3)
ix. Dilution data, differentiating between dilution of a sample or an extract
(Section 15.3)
x. Instrument
xi. Column (dimensions, liquid phase, solid support, film thickness, etc.)
xii. Operating conditions (temperatures, temperature program, flow rates)
xiii. Detector (type, operating conditions, etc.)
xiv. Chromatograms, printer tapes, and other recordings of raw data
xv. Quantitation reports, data system outputs, and other data to link the raw
data to the results reported
9.1.2.3 Alternative columns and column systems - If a column or column system other
than those specified in this method is used, that column or column system must
meet all the requirements of this method.
Note: The use of alternative columns or programs will likely result in a different elution order.
9.1.3 Analyses of method blanks are required on an on-going basis to demonstrate the extent of
background contamination in any reagents or equipment used to prepare and analyze field
samples (Section 4.3). The procedures and criteria for analysis of a method blank are
described in Section 9.5.
9.1.4 The laboratory must spike all samples with isotopically labeled compounds to monitor
method performance. This test is described in Section 9.3. When results of these spikes
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indicate atypical method performance for samples, the samples are diluted to evaluate
whether the performance issue is caused by the sample matrix. Procedures for dilution are
given in Section 15.3.
9.1.5 The laboratory must, on an ongoing basis, demonstrate that the analytical system is in
control through calibration verification and the analysis of ongoing precision and recovery
standards (OPR), spiked at low (LLOPR) and mid-level, and blanks. These procedures are
given in Sections 14.3 through 14.7.
9.1.6 The laboratory must maintain records to define the quality of data generated. Development
of accuracy statements is described in Section 9.4.
9.2 Initial Demonstration of Capability
9.2.1 Initial precision and recovery (IPR) - To establish the ability to generate acceptable
precision and recovery, the laboratory must perform the following operations for each
sample matrix type to which the method will be applied by that laboratory.
9.2.1.1 Extract, concentrate, and analyze four aliquots of the matrix type to be tested
(Section 7.2.1 through 7.2.3), spiked with 200 |_iL of the native standard solution
(Section 7.3.3), 50 (iL of the EIS solution (Section 7.3.1), and 50 (iL of NIS
solution (Section 7.3.2). At least one method blank, matching the matrix being
analyzed, must be prepared with the IPR batch. In the event that more than one
MB was prepared and analyzed with the IPR batch, all blank results must be
reported. All sample processing steps that are to be used for processing samples,
including preparation and extraction (Sections 11.2 - 11.4), cleanup (Section
12.0) and concentration (Section 12.0), must be included in this test.
9.2.1.2 Using results of the set of four analyses, compute the average percent recovery
(R) of the extracts and the relative standard deviation (RSD) of the concentration
for each target and EIS compound.
9.2.1.3 For each native and isotopically labeled compound, compare RSD and %
recovery with the corresponding limits for initial precision and recovery in Table
5 and 5A. Table 5 includes the required QC acceptance limits for wastewater
samples that were derived from the multi-laboratory validation study. Table 5A
includes example performance data for solids and tissues from the single-
laboratory validation study and are provided for illustrative purposes (e.g., those
figures are not required acceptance criteria). For wastewater matrices, if RSD
and R for all compounds meet the acceptance criteria, system performance is
acceptable, and analysis of blanks and wastewater samples may begin. If,
however, any individual RSD exceeds the precision limit or any individual R
falls outside the range for recovery, system performance is unacceptable for that
compound. Correct the problem and repeat the test (Section 9.2).
9.2.2 Method detection limit (MDL) - Each laboratory must also establish MDLs for all the
target analytes using the MDL procedure at 40 CFR Part 136, Appendix B. The minimum
level of quantification (ML) can be calculated by multiplying the MDL by 3.18 and
rounding the result to the nearest 1, 2 or 5 x 10n, where n is zero or an integer (see the
Glossary for alternative derivations). Example matrix-specific method detection limits are
listed in Table 6.
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9.3 To assess method performance on the sample matrix, the laboratory must spike all samples with the
EIS standard solution (Section 7.3.1) and all sample extracts with the NIS spiking solution (Section
7.3.2).
9.3.1 Analyze each sample according to the procedures in Sections 11.0 through 16.0.
9.3.2 Compute the percent recovery of the EIS using the non-extracted internal standard method
(Section 15.2) and the equation in Section 14.5.2.
9.3.3 The recovery of each EIS in a wastewater sample must be within the limits in Table 8,
which are the required QC acceptance limits for wastewater samples that were derived from
the multi-laboratory validation study. If the recovery of any EIS falls outside of these
limits, method performance is unacceptable for that EIS in that wastewater sample.
Additional cleanup procedures or limited dilution of the sample extract may be employed to
attempt to bring the EIS recovery within the acceptance normal range. If the recovery
cannot be brought within the acceptance limits after extract dilution or additional cleanup
procedures have been employed, wastewater samples are diluted and prepared and
analyzed, per Section 15.3. Table 8A includes example performance data for solids and
tissues from the single-laboratory validation study and are provided for illustrative purposes
(e.g., those figures are not required acceptance criteria). EIS recoveries in solids and
tissues that fall well outside of the ranges in Table 8A are a potential cause for concern and
laboratories should take similar steps to those described for wastewater samples to improve
EIS recoveries.
9.4 Records of the recovery of EISs from samples must be maintained, and should be assessed
periodically.
9.4.1 After the analysis of 30 samples of a given matrix type (water, solids, tissues, etc.),
compute the recovery (R) and the standard deviation of the percent recovery (Sr) for the
isotopically 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 30
analyses of soil, the recovery interval is expressed as 70 to 110%.
9.4.2 Update the accuracy assessment for each isotopically labeled compound in each matrix on a
regular basis (e.g., after each five to ten new preparation batches).
9.5 Method blanks - A 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., reagent water blank [Section 7.2.1], solids blank [Section 7.2.2], or tissue
blank [Section 7.2.3]).
9.5.1 Analyze the cleaned extract (Section 12.0) of the method blank aliquot before the analysis
of the OPRs (Section 14.5).
9.5.2 If any PFAS is found in the blank at 1) at a concentration greater than the ML for the
analyte, 2) at a concentration greater than one-third the regulatory compliance limit, or 3) at
a concentration greater than one-tenth the concentration in a sample in the extraction batch,
whichever is greatest, analysis of samples must be halted, and the problem corrected. Other
project-specific requirements may apply; therefore, the laboratory may adopt more
stringent acceptance limits for the method blank at their discretion. If the contamination is
traceable to the extraction batch, samples affected by the blank must be re-extracted and
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analyzed, provided enough sample volume is available and the sample are still within
holding time.
If continued re-testing results in repeated blank contamination, the laboratory must
document and report the failures (e.g., as qualifiers on results), unless the failures are not
required to be reported as determined by the regulatory/control authority. Results
associated with blank contamination for an analyte regulated in a discharge cannot be used
to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee
of reporting timely results.
9.6 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 initial calibration (Section 10.3),
calibration verification (Section 14.3), and for initial (Section 9.2.1) and ongoing (Section 14.5)
precision and recovery may be prepared from the same source; however, the use of a secondary
source for calibration verification is highly recommended whenever available. If standards from a
different vendor are not available, a different lot number from the same vendor can be considered a
secondary source. A LC-MS/MS instrument will provide the most reproducible results if dedicated
to the settings and conditions required for determination of PFAS by this method.
9.7 Laboratory duplicates - A second aliquot of one sample is prepared and analyzed with each sample
batch to demonstrate within-laboratory precision for the analytes present in the sample. Use one of
the additional containers for a field sample. Do not divide the contents of a single bottle of an
aqueous sample into two smaller portions.
9.8 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique.
9.9 Matrix spikes generally are not required for methods that employ isotope dilution quantification
because any deleterious effects of the matrix should be evident in the recoveries of the EIS
compounds spiked into every sample. However, because some of the compounds are quantified by
a non-analogous EIS (e.g., PFPeS is quantified by 13C3-PFHxS), the analysis of matrix spike
samples can help determine the accuracy of the analysis for such compounds, and may help
diagnose matrix interferences for specific compounds.
10.0 Calibration and Standardization
10.1 Mass Calibration
The mass spectrometer must undergo mass calibration to ensure accurate assignments of m/z's by
the instrument. This mass calibration must be performed at least annually or as recommended by
the instrument manufacturer, whichever is more frequent, to maintain instrument sensitivity and
stability. Mass calibration must be repeated on an as-needed basis (e.g., QC failures, ion masses
fall outside of the required mass window, major instrument maintenance, or if the instrument is
moved). Mass calibration must be performed using the calibration compounds and procedures
prescribed by the manufacturer.
Multiple Reaction Monitoring (MRM) analysis is required to achieve better sensitivity than full-
scan analysis. The default parent ions, quantitation ions (Ql), and confirmation (Q2) ions that were
monitored during the validation of this method are listed in Table 7 for each native analyte, EIS,
and NIS.
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10.1.1 During the development of this method, instrumental parameters were optimized for the
precursor and product ions of the linear isomers of the target analytes listed on Table 7. If a
qualitative or quantitative standard containing an isomeric mixture (branched and linear
isomers) of an analyte is commercially available for an analyte, the quantification ion used
must be the quantification ion identified in Table 7, unless interferences render the product
ion unusable as the quantification ion. In cases where interferences render the product ion
unusable, consult the client before using the alternative product ion and document the
reason for the change when reporting results. However, the use of ions with lower masses
or common ions that may not provide sufficient discrimination between analytes of interest
and co-eluting interferences must be avoided.
10.1.2 Optimize the response of the precursor ion [M-H]" or [M-CO2]" for each method analyte
following the manufacturer's guidance. MS parameters (e.g., source voltages, source and
desolvation temperatures, gas flow, etc.) must be methodically changed until optimal
analyte responses are determined. Typically, carboxylic acids have similar MS/MS
conditions and sulfonic acids have similar MS/MS conditions. However, since analytes
may have different optimal parameters, some compromise on the final operating conditions
may be required.
10.1.3 Establish suitable operating conditions using the manufacturer's instructions and use the
table below of MS conditions used during the development of this method as guidance.
Operating Conditions for Waters Acquity UPLC, TQ-S Xevo MS/MS
Injection volume 2.0 |iL (This is the default volume, and may be
changed to improve performance)
Source Temp (°C) 140
Desolvation Temp (°C) 500
Capillary Voltage (kV) 0.70
Cone Gas (L/h) ~70
Desolvation gas (L/h) -800
MS/MS
Conditions
10.1.4 As noted above, perform the mass calibration following the instrument manufacturer's
instructions, using the calibrant prescribed by the manufacturer.
10.1.5 Regardless of the calibrant used, mass calibration is judged on the basis of the presence or
absence of the exact calibration masses (e.g., a limit on the number of masses that are
"missed"). If peaks are missing or not correctly identified, adjust the MS/MS, and repeat
the test. Only after the MS/MS is properly calibrated may standards, blanks, and samples
be analyzed.
10.1.6 Mass spectrometer optimization - Prior to measurements of a given analyte the mass
spectrometer must be separately optimized for that analyte.
10.1.6.1 Using the post-column pump, separately infuse a solution containing each
compound in methanol into the MS.
10.1.6.2 Optimize sensitivity for the product ion m/z for each compound. Precursor-
product ion m/z's other than those listed may be used provided requirements in
this method are met.
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10.1.6.3 After MS calibration and optimization and LC-MS/MS calibration, the same
LC-MS/MS conditions must be used for analysis of all standards, blanks, IPR
and OPR standards, and samples.
10.1.7 Mass Calibration Verification
The mass calibration must be verified prior to the analysis of any standards and samples
and after each subsequent mass calibration. Each laboratory must follow the instructions
for their instrument software to confirm the mass calibration, mass resolution, and peak
relative response.
10.1.7.1 Check the instrument mass resolution to ensure that it is at least unit resolution.
Inject a mid-level calibration standard under LC-MS/MS conditions to obtain the
retention times of each method analyte. Divide the chromatogram into segments
or retention time ranges, each of which contains one or more chromatographic
peaks. During MS/MS analysis, fragment a small number of selected precursor
ions ([M-H]") for the analytes in each window and choose the most abundant
product ion. The product ions (also the quantitation ions) chosen during method
development are listed in Table 7, although these will be instrument dependent.
Unit resolution must meet the manufacturer's criteria.
10.1.7.2 Check the mass calibration by measuring the amount of peak drift from the
expected masses. If the peak apex has shifted more than approximately 0.2 Da,
recalibrate the mass axis following the manufacturer's instructions.
10.2 Chromatographic conditions
10.2.1 The chromatographic conditions should be optimized for compound separation (including
analytes with both linear and branched isomers) and for sensitivity. The same optimized
operating conditions must be used for the analysis of all standards, blanks, IPR and OPR
standards, and samples. The following table gives the suggested chromatographic
conditions for this method using the specified instrument and column. Different
instruments may require slightly different operating conditions. Modification of the
solvent composition of the standard or extract by increasing the aqueous content to
prevent poor peak shape is not permitted. The peak shape of early eluting compounds
may be improved by increasing the volume of the injection loop or increasing the aqueous
content of the initial mobile phase composition.
General LC Conditions
Column Temp (°C) 40
Max Pressure (bar) 1100.0
LC Gradient Program
Time (min) Flow mixture 12 Flow Rate Program Gradient Curve
0.0
2% eluent A, 98% eluent B
0.35 mL/min
Initial
0.2
2% eluent A, 98% eluent B
0.35 mL/min
2
4.0
30% eluent A, 70% eluent B
0.40 mL/min
7
7.0
55% eluent A, 45% eluent B
0.40 mL/min
8
9.0
75% eluent A, 25% eluent B
0.40 mL/min
8
10.0
95% eluent A, 5% eluent B
0.40 mL/min
6
10.4
2% eluent A, 98% eluent B
0.40 mL/min
10
11.8
2% eluent A, 98% eluent B
0.40 mL/min
7
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General LC Conditions
12.0 2% eluent A, 98% eluent B 0.35 mL/min 1
1 Eluent A = Acetonitrile
2 Eluent B = 2 mM ammonium acetate in 95:5 water/acetonitrile
Note: LC system components, as well as the mobile phase constituents, may contain many of the
analytes in this method. Thus, these PFAS will build up on the head of the LC column
during mobile phase equilibration. To minimize the background PFAS peaks and to keep
baseline levels constant, the time the LC column sits at initial conditions must be kept
constant and as short as possible (while ensuring reproducible retention times). In
addition, priming the mobile phase and flushing the column with at least 90% methanol
before initiating a sequence may reduce background contamination.
10.2.2 Retention time calibration
10.2.2.1 Inject compound solution(s) to determine its retention time. The laboratory may
want to inject compounds separately the first time they perform the calibration.
All native compounds for which there is an isotopically labeled analog will elute
slightly before or with the labeled analog. Store the retention time (RT) for each
compound in the data system.
10.2.2.2 Once RT windows have been confirmed for each analyte, once per ICAL and at
the beginning of the analytical sequence, the position of all target analyte, EIS,
and NIS peaks shall be set using the midpoint standard of the ICAL curve when
ICAL is performed. When ICAL is not performed, the initial CV retention times
or the midpoint standard of the ICAL curve can be used to establish the RT
window position.
10.2.2.3 The RTs for the target analytes, EISs, and NISs must fall within 0.4 minutes of
the predicted retention times from the midpoint standard of the ICAL or initial
daily CV, whichever was used to establish the RT window position for the
analytical batch. All branched isomer peaks identified in either the calibration
standard or the qualitative (technical grade) standard also must fall within 0.4
minutes of the predicted retention times from the midpoint standard of the ICAL
or initial daily CV.
10.2.2.4 For all method analytes with exact corresponding isotopically labeled analogs,
target analytes must elute within 0.1 minutes of the associated EIS. (The
laboratory may use relative retention times (RRTs) of the target analytes and
their labeled analogs as an alternative, provided that they also develop
corresponding RRT acceptance criteria that are at least as stringent as those
described here.)
10.2.2.5 When establishing the chromatographic conditions, it is important to consider the
potential interference of bile salts during analyses of samples. Inject the bile salt
interference check standard containing TDCA (see Section 7.5 if the mobile
phase is not acetonitrile) during the retention time calibration process and adjust
the conditions to ensure that TDCA (or TDCA, TCDCA and TUDCA) does not
coelute with any of the target analytes, EIS, or NIS standards. Analytical
conditions must be set to allow a separation of at least 1 minute between the bile
salts and the retention time window of PFOS as described in Section 7.3.3. In
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order to ensure adequate chromatographic separation of the target analytes, the
method requires this evaluation when establishing the chromatographic
conditions, regardless of the sample matrices to be analyzed.
10.3 Initial calibration
Initial calibration is performed using a series of at least six solutions, with at least five of the six
calibration standards being within the quantification range, and with the lowest standard at or below
the LOQ. (If a second-order calibration model is used, then one additional concentration is
required, with at least six of the seven calibration standards within the quantitation range.) The
initial calibration solutions contain the entire suite of EISs, NISs, and target compounds.
Calibration is verified with a calibration verification (CV) standard at least once every ten
injections of a field sample extract, by analysis of a mid-level calibration solution. Calibration
verification uses the mean RRs or RFs determined from the initial calibration to calculate the
analyte concentrations in the verification standard.
Note: Six calibration standards is the minimum number that must be used in the initial
calibration; however, the laboratory may use more standards, as long as the criteria in
Section 10.3.3.3 can be met.
Prior to the analysis of samples, and after the mass calibration check has met all criteria in Section
10.1.4, each LC-MS/MS system must be calibrated at a minimum of 6 standard concentrations
(Section 7.3.4 and Table 4). This method procedure calibrates and quantifies 40 target analytes,
using the isotopically labeled compounds added to the sample prior to extraction, by one of two
approaches:
• True isotope dilution quantification (ID), whereby the response of the target compound is
compared to the response of its isotopically labeled analog. Twenty-four target compounds are
quantified in this way.
• Extracted internal standard quantification (EIS), whereby the response of the target compound
is compared to the response of the isotopically labeled analog of another compound with
chemical and retention time similarities. Sixteen target compounds are quantified in this way.
10.3.1 Initial calibration frequency
Each LC-MS/MS system must be calibrated whenever the laboratory takes an action that
changes the chromatographic conditions or might change or affect the initial calibration
criteria, or if either the CV or Instrument Sensitivity Check (ISC) acceptance criteria have
not been met.
10.3.2 Initial calibration procedure
Prepare calibration standards containing the native compounds, EISs, and NISs, at the
concentrations described in Table 4. Analyze each calibration standard by injecting 2.0 |_iL
(this volume may be changed to improve performance).
Note: The same injection volume must be used for all standards, samples, blanks, and QC samples.
10.3.3 Initial calibration calculations
10.3.3.1 Instrument sensitivity
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Sufficient instrument sensitivity is established if a signal-to-noise ratio > 3:1 for
the quantitation ions and the confirmation ions, or > 10:1 if the analyte only has
a quantitation ion, can be achieved when analyzing the lowest concentration
standard within the quantitation range that the laboratory includes in its
assessment of calibration linearity (Table 4).
10.3.3.2 Response Ratios (RR) and Response Factors (RF)
The response ratio (RR) for each native compound calibrated by isotope dilution
is calculated according to the equation below, separately for each of the
calibration standards, using the areas of the quantitation ions (Ql) with the m/z
shown in Table 7. RR is used for the 24 compounds measured by true isotope
dilution quantification.
Arean MEIS
t\t\ —
AreaEIS Mn
where:
Arean = The measured area of the Q1 m/z for the native (unlabeled) PFAS
Areasis = The measured area at the Ql m/z for the corresponding isotopically
labeled PFAS used as the EIS in the calibration standard
Meis = The mass of the isotopically labeled PFAS used as the EIS in the
calibration standard
Mn = The mass of the native compound in the calibration standard
Similarly, the response factor (RF) for each native compound calibrated by
extracted internal standard is calculated according to the equation below. RF is
used for the 16 compounds measured by extracted internal standard
quantification.
Rp = Areas MEIS
AreaEls Ms
where:
Areas = The measured area of the Q1 m/z for the native (unlabeled) PFAS
Areasis = The measured area at the Q1 m/z for the isotopically labeled PFAS
used as the EIS in the calibration standard
Meis = The mass of the isotopically labeled PFAS used as the EIS in the
calibration standard
Ms = The mass of the native (unlabeled) PFAS in the calibration standard
A response factor (RFS) is calculated for each isotopically labeled EIS
compounds in the calibration standard using the equation below. RFS is used for
the 24 isotopically labeled compounds measured by non-extracted internal
standard quantification.
AreaEISMNIS
RFs = n—
AreaNIS MEIS
where:
Areasis = The measured area of the Q1 m/z for the isotopically labeled EIS
added to the sample before extraction
Areams = The measured area at the Q1 m/z for the isotopically labeled PFAS
used as the NIS in the calibration standard
Mnis = The mass of the isotopically labeled compound used as the NIS in
the calibration standard
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Meis = The mass of the isotopically labeled EIS added to the sample before
extraction
Note: Other calculation approaches may be used, such as a weighted linear regression or
non-linear regression, based on the capability of the data system used by the
laboratory. If used, the regression must be weighted inversely proportional to
concentration and must not be forced through zero. Analysts should consult their
instrument vendor for details on regression calibration models. When using a
weighted regression calibration, linearity must be assessed using Option 2 below.
10.3.3.3 Instrument Linearity
One of the following two approaches must be used to evaluate the linearity of the
instrument calibration:
Option 1: Calculate the relative standard deviation (RSD) of the RR or RF
values for each native compound and isotopically labeled compound
for all the initial calibration standards that were analyzed. The RSD
must be < 20% to establish instrument linearity.
nn _ Z?=1(RRorRF)t
mean RR or RF =
n
SD =
Hf=i(RR or RFi ~ mean RR or RF)^
n
where:
RR or RF
n
SD
RSD = x 100
mean
RR or RF for calibration standard i
Number of calibration standards
Option 2: Calculate the relative standard error (RSE) for each native compound
and isotopically labeled compound for all the initial calibration
standards that were analyzed. The RSE for all method analytes must
be < 20% to establish instrument linearity.
where,
Xi =
x'i =
n =
P =
RSE = 100 X
IL
I'
( = 1
X,
n — p
Nominal concentration (true value) of each calibration standard
Measured concentration of each calibration standard
Number of standard levels in the curve
Type of curve (2 = linear, 3 = quadratic)
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In addition, although not required, it may be useful to compare the actual
responses for each standard to the calibration model. Differences outside of a
window of 70 - 130% of the modeled concentration may be cause for concern.
10.3.3.4 Non-extracted Internal Standard Area
Each time an initial calibration is performed, use the data from all the initial
calibration standards used to meet the linearity test in Section 10.3.3.3 to
calculate the mean area response for each of the NIS compounds, using the
equation below.
Y.Areams
MeanAreaNIS. =
I n
Area counts for the ith NIS, where i ranges from 1 to 7, for the seven
NIS compounds listed in Table 1
The number of ICAL standards (the default value is n = 6). If a
different number of standards is used for the ICAL, for example, to
increase the calibration range or by dropping a point at either end of
the range to meet the linearity criterion, change 6 to match the actual
number of standards used.
Record the mean areas for each NIS for use in evaluating results for sample
analyses (see Section 14.9). There is no acceptance criterion associated with the
mean NIS area data.
10.3.4 Initial calibration corrective actions
If the instrument sensitivity or the instrument linearity criteria for initial calibration are not
met, inspect the system for problems and take corrective actions to achieve the criteria.
This may require the preparation and analysis of fresh calibration standards or performing a
new initial calibration. All initial calibration criteria must be met before any samples or
required blanks are analyzed.
10.3.5 Bile salts interference check
The laboratory must analyze a bile salt interference check standard (see Section 7.5) after
the initial calibration as a check on the chromatographic conditions, regardless of the
sample matrix to be analyzed. If an interference is present, the chromatographic
conditions must be modified to eliminate the interference from the bile salts (e.g., changing
the conditions such that the retention time of the bile salts fall outside the retention time
window for any of the linear or branched PFOS isomers in the standard described in
Section 7.3.3 by at least one minute), and the initial calibration repeated.
11.0 Sample Preparation and Extraction
For aqueous samples that contain particles and solid samples, percent solids are determined using
the procedures in Section 11.1. This section describes the sample preparation procedures for
aqueous samples with < 50 mg solids in the sample volume to be extracted (Section 11.2), solid
(soil, sediment or biosolid) samples (Section 11.3) and tissue samples (Section 11.4).
where:
AreaNis; =
n =
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Absent of source-specific knowledge of the PFAS levels in samples or project-specific
requirements, the laboratory must pre-screen all samples prior to performing the quantitative
analysis (see Appendix A). For aqueous samples, use the secondary container provided for percent
solids to perform the pre-screening. If high levels of PFAS are present in the sample, a lower
volume may be required for analysis.
Note: The laboratory may subsample the aqueous samples as described in Appendix B; however,
subsampling must meet project-specific requirements. The laboratory must notify the client
before proceeding with subsampling, in the event that a more appropriate size sample can be
collected and sent to the laboratory. Once the laboratory becomes familiar with the levels of
PFAS in the samples for their clients, the samples should be collected in the appropriate sample
container size to avoid subsampling. The sample data report must state when subsampling has
been employed.
Do not use any fluoropolymer articles or task wipes in these extraction procedures. Use only
HDPE or polypropylene wash bottles and centrifuge tubes. Reagents and solvents for cleaning
syringes may be kept in glass containers.
11.1 Determination of solids contents of samples
Two types of solids determinations are described in this method. The first is the determination of
the total suspended solids (TSS) content of aqueous samples. Because aqueous samples are
processed with SPE cartridges that can be clogged by suspended solids in the sample, the method
recommends a limit of 50 mg of solids in the total volume of sample that is processed by SPE.
The second type of solids determination is the percent solids (% solids) of soil, sediment, and
biosolids samples. The percent solids is used to report results for these sample matrices as dry-
weight concentrations. (Tissue samples do not require any solids determination.)
Note: The earlier drafts of Method 1633 described the determination ofpercent solids for both aqueous
and solid matrices, in an attempt to "simplify" the procedures across matrix types. However, in
practice, the use of TSS for aqueous matrices is a more straightforward way to examine the risk
of clogging the SPE and the results do not need to be as accurate as the percent solids data used
for reporting dry-weight concentrations of the other matrices.
11.1.1 Determination of total suspended solids (TSS) in aqueous matrices
11.1.1.1 Desiccate and weigh a glass fiber filter (Section 6.4.3) in milligrams (mg) to two
significant figures.
11.1.1.2 Filter 10.0 ± 0.02 mL of well-mixed sample through the filter. This volume is
sufficient for the purposes of assessing the risk of clogging the SPE cartridge.
11.1.1.3 Dry the filter a minimum of 1 hour at 103 - 105 °C and cool in a desiccator.
11.1.1.4 Calculate TSS as follows:
weight of sample aliquot after drying (mg) — weight of filter (mg)
TSS (mg/L) = ——
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11.1.1.5 Multiply the TSS by the volume of the sample aliquot to be extracted, in liters,
to obtain the milligrams of solids in the sample. If the sample volume contains
more than 50 mg of TSS, at a minimum, the analyst should prepare a second
SPE cartridge (see the Note in Section 12.1.4) prior to start the extraction in the
event of clogging. Laboratories may develop other strategies for minimizing the
disruptions due to SPE clogging and slow extractions.
11.1.1.6 In the absence of client-specific requirements, an alternative to determining the
TSS may be to identify samples likely to contain more than 50 mg of solids by
visual comparison to examples maintained in the laboratory. More specifically,
a trained analyst should be able to distinguish samples with very low TSS and
focus the TSS determinations on only those samples that might present a risk of
clogging. However, given the translucent nature of HDPE containers, this may
require pouring a small volume of sample from the container designated for the
solids determination to a clear glass vessel. If this is done, that volume should
be discarded after the assessment.
11.1.1.7 Regardless of the approach used, the laboratory must maintain records of the
manner in which the solids content of each aqueous sample was assessed.
11.1.2 Determination of percent solids in soils, sediments, and biosolids
11.1.2.1 Weigh 5 to 10 g of sample to three significant figures in a tared beaker.
11.1.2.2 Dry a minimum of 12 hours at 110 ± 5 °C, and cool in a desiccator and weigh the
beaker.
11.1.2.3 Calculate percent solids as follows:
o weight of sample aliquot after drying (g)
weight of sample aliquot before drying (g)
11.2 Aqueous sample processing
This method was validated with aqueous samples containing no more than 50 mg of suspended
solids per sample. The procedure requires the preparation of the entire sample and samples
containing large amounts of suspended solids are likely to clog the SPE media, dramatically
slowing or precluding sample extraction. Smaller sample volumes may be analyzed for samples
containing solids greater than that specified for this method, or when unavoidable due to high levels
of PFAS; however, subsampling should be avoided whenever possible.
The nominal sample size for wastewater, surface water, and groundwater and their associated QC
samples is 500 mL; however, sample size may be increased up to 1,000 mL if required for a
specific project. The sample is to be analyzed in its entirety and must not be filtered. Leachate
samples and their associated QC samples are analyzed using a 100-mL sample volume. Therefore,
leachates must not be included in the same sample preparation batch as other aqueous samples that
are analyzed using 500-mL sample volumes.
11.2.1 Homogenize the sample by inverting the sample 3-4 times and allowing the sample to
settle. Do not filter the sample. The standard procedure is to analyze the entire sample,
plus a basic methanol rinse of the container.
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11.2.2 The volume of the aqueous sample analyzed is determined by weighing the full sample
bottle and then the empty sample bottle (see Section 12.2). Weigh each sample bottle (with
the lid) to 0.1 g.
11.2.3 Prepare a method blank and two OPRs using PFAS-free water in HDPE bottles. Select a
volume of water that is typical of the samples in the batch (nominally 500 mL). Spike one
OPR sample with native standard solution (Section 7.3.3) at 2x the LOQ (LLOPR). This
aliquot will serve to verify the LOQ. Spike the other OPR sample at the concentration of
the mid-level calibration point. This aliquot will serve as the traditional OPR.
Note: If matrix spikes are required for a specific project, spike the field sample bottles designated
for use as MS/MSD samples with native standard solution (Section 7.3.3) at concentrations
roughly 3 to 5 times the background concentration determined during screening of the
unspiked sample, but not to exceed the calibration range. This may require multiple
spiking solutions. If screening was not performed, then spike those samples at the
concentration of the mid-level calibration point.
11.2.4 Spike an aliquot of EIS solution (Section 7.3.1) directly into the sample in the original
bottle (or subsampled bottle) as well as to the bottles prepared for the QC samples. Mix by
swirling the sample container. If centrifugation is used to prevent samples with high TSS
from clogging the SPE, the EISs must be spiked into the original sample container prior to
centrifugation.
11.2.5 Using a PFAS-free pipette or other device, transfer a few drops of the sample to pH paper
and check that the pH is 6.5 ± 0.5. If necessary, adjust pH with 50% formic acid (Section
7.1.13.4) or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3%
aqueous ammonium hydroxide [Section 7.1.6.2]). The sample is now ready for solid-phase
extraction (SPE) and cleanup (Section 12.0).
11.3 Solid sample processing (excluding tissues)
Use a stainless spoon to mix the sample in its original jar. If it is impractical to mix the sample
within its container, transfer the sample to a larger container. Remove rocks, invertebrates, and
foreign objects. Vegetation can either be removed from the sample before homogenization or cut
into small pieces and included in the sample, based on project requirements. Mix the sample
thoroughly, stirring from the bottom to the top and in a circular motion along the sides of the jar,
breaking particles to less than 1 mm by pressing against the side of the container. The homogenized
sample should be even in colour and have no separate layers. Store the homogenized material in its
original container or in multiple smaller containers. Determine the percent solids as per Section
11.1.2.
Note: The maximum sample weight for sediment or soil is 5 g dry weight. The maximum sample weight
for biosolids is 0.5 g dry weight.
Small amounts of the reagent water used for aqueous method blanks (10% of sample weight or
less) can be added to unusually dry samples to facilitate extraction. This is an option, not a
requirement, and if used, the solid method blank associated with the samples must contain similar
amounts of added water.
11.3.1 Weigh out an aliquot of solid sample, not dried (aliquot should provide 5 g dry weight for
soil and sediment or 0.5 g dry-weight for biosolids) into a 50-mL polypropylene centrifuge
tube. Because biosolids samples are analyzed with a 0.5-g sample, they must not be
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included in the same sample preparation batch as solid samples analyzed with nominal 5-g
sample masses.
11.3.2 Prepare batch QC samples using 5 g of reference solid (Section 7.2.2) wetted with 2.5 g of
reagent water for the method blank and two OPRs (use 0.5 g of reference solid with 0.25 g
of reagent water for biosolid sample batches). The addition of reagent water to the sand
provides a matrix closer in composition to real-world samples. Spike one OPR sample with
native standard solution (Section 7.3.3) at 2x the LOQ (LLOPR). This aliquot will serve to
verify the LOQ. Spike the other OPR sample at the concentration of the mid-level
calibration point. This aliquot will serve as the traditional OPR.
Note: If matrix spikes are required for a specific project, spike the field sample aliquots
designated forMS/MSD samples with native standard solution (Section 7.3.3) at
concentrations roughly 3 to 5 times the background concentration determined during
screening of the unspiked sample, but not to exceed the calibration range. This may
require multiple spiking solutions. If screening was not performed, then spike those
samples at the concentration of the mid-level calibration point.
11.3.3 Spike an aliquot of EIS solution (Section 7.3.1) directly into each centrifuge tube
containing the aliquoted field and QC samples. Vortex the sample to disperse the standard
and allow to equilibrate for at least 30 minutes.
11.3.4 Add 10 mL of 0.3% methanolic ammonium hydroxide (Section 7.1.7.1) to each centrifuge
tube. Vortex to disperse, then shake for 30 minutes on a variable speed mixing table.
Centrifuge at 2800 rpm for 10 minutes and transfer the supernatant to a clean 50-mL
polypropylene centrifuge tube.
11.3.5 Add 15 mL of 0.3% methanolic ammonium hydroxide (Section 7.1.7.1) to the remaining
solid sample in each centrifuge tube. Vortex to disperse, then shake for 30 minutes on a
variable speed mixing table. Centrifuge at 2800 rpm for 10 minutes and decant the
supernatant from the second extraction into the centrifuge tube with the supernatant from
the first extraction.
11.3.6 Add another 5 mL of 0.3% methanolic ammonium hydroxide (Section 7.1.7.1) to the
remaining sample in each centrifuge tube. Shake by hand to disperse, centrifuge at 2800
rpm for 10 minutes and decant the supernatant from the third extraction into the centrifuge
tube with supernatant from the first and second extractions.
11.3.7 Using a 10-mg scoop, add 10 mg of carbon (Section 7.1.17) to the combined extract, mix
by occasional hand shaking for 5 minutes and no more, and then centrifuge at 2800 rpm for
10 minutes. Immediately decant the extract into a 60-mL glass evaporation or concentrator
tube.
11.3.8 The laboratory has the option to dilute the extract to approximately 35 mL with reagent
water. (Some laboratories may prefer not to add any additional water, therefore, this
dilution is optional.) A separate concentrator tube marked at the 35-mL level may be kept
for a visual reference to get the approximate volume. Samples containing more than 50%
water may yield extracts that are greater than 35 mL in volume; therefore, do not add water
to these. Determine the water content in the sample as follows (percent moisture is
determined from the % solids):
Sample Weight (g) x Moisture (%)
Water Content in Sample = —— 1- any water added in 11.3.2 and 11.3.8
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11.3.9 Concentrate each extract at approximately 55 °C with a N2 flow of approximately 1.2 L/min
to a final volume that is based on the water content of the sample (see table below). Allow
extracts to concentrate for 25 minutes, then mix (by vortex if the volume is < 20 mL or
using a glass pipette if the volume is > 20 mL). Continue concentrating and mixing every
10 minutes until the extract has been reduced to the required volume as specified in the
table below. If the extract volume appears to stop dropping, the concentration must be
stopped and the volume at which it was stopped recorded. The concentrated extract must
still contain some methanol, about 5-10 mL. The pre-cleanup extract in 11.3.10 should
contain no more than 20% methanol. The laboratory has flexibility to modify the volumes
used to achieve this goal. Some laboratories may prefer not to add water in Section 11.3.8.
The following table provides guidance to help determine the final extract volume, based on
the water content of the original solid sample.
Water Content in Sample* Concentrated Final Volume
* Based on the % solids result determined in Section 11.1.2.3, and
including any water added to the sample in Sections 11.3.2 or the
extract in Section 11.3.8.
A good rule of thumb is to make the "Concentrated Final Volume" 7-10 mL above the
"Water Content in Sample" value.
Note: Slowly concentrating extracts, in 1-mL increments, is necessary to prevent excessive
concentration and the loss of neutral compounds (methyl and ethyl FOSEs and FOSAs) and
other more volatile compounds. The extract must be concentrated to remove the methanol
as excess methanol present during SPE clean-up results in poor recovery of C13 and Cm
carboxylic acids and Cw and C12 sulfonates.
If all of the methanol is evaporated, the aforementioned neutral compounds are likely to
have poor recovery, if too much methanol is in the final concentrated extract, then the
aforementioned longer-chain compounds are likely to have poor recovery.
11.3.10 Add 40 - 50 mL of reagent water to the extract and vortex. Check that the pH is 6.5 ±0.5
and adjust as necessary with 50% formic acid (Section 7.1.13.4) or 30% ammonium
hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous ammonium
hydroxide [Section 7.1.6]). The extracts are ready for SPE and cleanup (Section 12.0).
11.4. Tissue sample processing
Prior to processing tissue samples, the laboratory must determine the exact tissue to be analyzed. Common
requests for analysis of fish tissue include whole fish with the skin on, whole fish with the 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 samples must be prepared and homogenized.
If the laboratory must dissect the whole fish to obtain the appropriate tissue for analysis, cover the benchtop
with clean aluminum foil and use clean processing equipment (e.g., knives, scalpels, tweezers) to dissect
each sample to prevent cross-contamination. Samples should be handled in a semi-thawed state for
compositing and/or homogenization. All tissue comprising a sample is collected in a tared stainless-steel
bowl during grinding or maceration, the total tissue mass weighed, and then mixed using a stainless-steel
<5 g
5-8g
8 - 9 g
9 - 10 g
7 mL
8 mL
9 mL
10 mL
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spoon. If not aliquoted immediately, homogenized samples must be stored in clean HDPE containers and
stored frozen for subsequent use.
If using a grinder, chilling the grinder briefly with a few pellets of dry ice may keep the tissue from sticking
to the equipment. Pellets of dry ice also may be added to the tissue as it enters the grinder. After the entire
sample has been processed, mix the ground tissue with a spoon, transfer back to the grinder, and repeat the
grinding at least two more times until the homogenize tissue has a consistent texture and color.
Between samples, disassemble the grinder or maceration device, remove any remaining tissue, and wash all
parts with PFAS-free detergents, rinse with tap water, then reagent water, and finally methanol. Do not
bake the grinder parts.
Once during the preparation of each batch of tissue samples (up to 20), prepare an equipment blank by
pouring 500 mL of reagent water through the reassembled grinder and collecting the rinsate in a 500-mL
HDPE container. Process that rinsate as an aqueous sample, but record the result in nanograms (ng) of each
analyte. Barring other project-specific requirements, assess the levels of any PFAS in the rinsate by
assuming that the entire mass of the analyte in the rinsate was transferred to the smallest mass of any bulk
tissue sample that was collected during the grinding process (not the 2-g aliquot taken for analysis below).
For example, if the smallest fish sample in the batch yields 500 g of ground tissue, divide the mass of each
PFAS analyte in the rinsate by 500, and compare those amounts to the MDLs for tissue samples.
11.4.1 For each sample, weigh a 2-g aliquot of homogenized tissue into a 15-mL polypropylene
centrifuge tube. Reseal the container with the remaining homogenized portion of the
sample and return it to frozen storage in the event that it needs to be used for reanalysis.
Note: The default sample weight for tissue is 2 g wet weight; however, a 1-g sample may be used.
Higher sample weights are not recommended for this method.
11.4.2 Prepare the batch QC samples using 2 g of reference tissue matrix (Section 7.2.3) for the
method blank and two OPRs. Spike one OPR sample with native standard solution
(Section 7.3.3) at 2x the LOQ (LLOPR). This aliquot will serve to verify the LOQ. Spike
the other OPR sample at the concentration of the mid-level calibration point. This aliquot
will serve as the traditional OPR.
Note: If matrix spikes are required for a specific project, spike the field sample aliquots
designated as MS/MSD samples with native standard solution (Section 7.3.3) at
concentrations roughly 3 to 5 times the background concentration determined during
screening of the unspiked sample, but not to exceed the calibration range. This may
require multiple spiking solutions. If screening was not performed, then spike those
samples at the concentration of the mid-level calibration point.
11.4.3 Spike an aliquot of EIS solution (Section 7.3.1) directly into each field and QC sample.
Vortex and allow to equilibrate for at least 30 minutes.
11.4.4 Add 10 mL of 0.05M KOH in methanol (Section 7.1.8) to each sample. Vortex to disperse
the tissue then place tubes on a variable speed mixing table set at low speed to extract for at
least 16 hours. Avoid violent shaking of the samples. Centrifuge at 2800 rpm for 10
minutes and collect the supernatant in a 50-mL polypropylene centrifuge tube.
11.4.5 Add 10 mL of acetonitrile to remaining tissue in the 15-mL centrifuge tube, vortex to mix
and disperse the tissue. Sonicate for 30 minutes. Centrifuge at 2800 rpm for 10 minutes
and collect the supernatant, adding it to the 50-mL centrifuge tube containing the initial
extract.
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11.4.6 Add 5 mL of 0.05M KOH in methanol (Section 7.1.8) to the remaining sample in each
centrifuge tube. Vortex to disperse the tissue and hand mix briefly. Centrifuge at 2800
rpm for 10 minutes and collect the supernatant, adding it to the 50-mL centrifuge tube
containing the first two extracts.
11.4.7 Using a 10-mg scoop, add 10 mg of carbon (Section 7.1.17) to the combined extract, mix
by occasional hand shaking over a period of 5 minutes and no more, then centrifuge at
2800 rpm for 10 minutes. Immediately decant the extract into a 60-mL glass evaporation
or concentrator tube.
11.4.8 Add 1 mL of reagent water to each evaporation/concentrator tube, set the
evaporator/concentrator to 55 °C with a N2 flow of 1.2 L/min and concentrate the extract to
2.5 mL (only ~1 mL of the methanol should remain).
11.4.9 Add reagent water to each evaporation/concentrator tube to dilute the extracts to 50 mL.
Check that the pH = 6.5 ± 0.5 and adjust as needed with 50% formic acid (Section 7.1.13.4)
or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous
ammonium hydroxide [7.1.6.2]). The extracts are ready for SPE and cleanup (Section
12.0).
12.0 Extraction, Cleanup, and Concentration
Samples of all matrices (and the associated batch QC) must undergo SPE and carbon cleanup to remove
interferences (Section 12.1). Sample elution as well as any further extract treatment is matrix specific and
may be found in Sections 12.2 through 12.4.
Note: Carbon cleanup is required. Carbon cleanup may remove analytes if the sample has a very low
organic carbon content (this is unusual for non-drinking water environmental samples). This will
be apparent if the isotope dilution standard recoveries are significantly higher on the reanalysis.
If the laboratory can demonstrate that the carbon cleanup is detrimental to the analysis of
samples from a particular source (by comparing results when skipping the carbon cleanup during
reanalysis), then the carbon cleanup may be skipped for samples from that specific source, with
client approval.
12.1 All sample matrices
12.1.1 Pack clean silanized glass wool to half the height of the WAX SPE cartridge barrel (Section
6.7.1).
12.1.2 Set up the vacuum manifold with one WAX SPE cartridge plus a reservoir and reservoir
adaptor for each cartridge for each sample and QC aliquot.
12.1.3 Pre-condition the cartridges by washing them with 15 mL of 1% methanolic ammonium
hydroxide (Section 7.1.7.2) followed by 5 mL of 0.3M formic acid (Section 7.1.13.2) (do
not use the vacuum for this step). Do not allow the WAX SPE to go dry. Discard the wash
solvents.
12.1.4 Pour the sample into the reservoir (do not use a pipette), taking care to avoid splashing
while loading. Adjust the vacuum and pass the sample through the cartridge at 5 mL/min.
Retain the empty sample bottle and allow it to air dry for later rinsing (Section 12.2.2).
Discard eluate.
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Note: For aqueous samples, in the event the SPE cartridge clogs during sample loading, place a
second pre-conditioned cartridge and continue loading the remaining sample aliquot using
the same reservoir. Proceed to Section 12.1.5.
12.1.5 Rinse the walls of the reservoir with 5 mL reagent water (twice) followed by 5 mL of 1:1
0.1M formic acid/methanol (Section 7.1.13.5) and pass those rinses through the cartridge
using vacuum. Dry the cartridge by pulling air through for 15 seconds. Discard the rinse
solution. Continue to the elution steps based on the matrix (see Section 12.2 - Aqueous,
Section 12.3 - Solids, and Section 12.4 - Tissue).
12.2 Elution of aqueous samples
Note: If two cartridges were used, perform Sections 12.2.1 through 12.2.3 with each cartridge.
Filter the eluates through a 25-mm, 0.2-fim syringe filter. Combine both sets of filtered
eluates into a clean tube, add the NIS solution, and vortex to mix. Transfer 350 fiL of the
filtered extract into a 1-mL polypropylene microvial and mark the level. Add another
350-p,L portion and using a gentle stream of nitrogen (water bath at 40 °C), concentrate to
the 350-fiL mark and submit for LC-MS/MS analysis. This concentration step is only
applicable to situations where two SPE cartridges were eluted, each with 5 mL of elution
solvent.
12.2.1 Place clean collection tubes (13 x 100 mm polypropylene) inside the manifold, ensuring
that the extract delivery needles do not touch the walls of the tubes. DO NOT add NIS to
these collection tubes.
12.2.2 Rinse the inside of the sample bottle with 5 mL of 1% methanolic ammonium hydroxide
(Section 7.1.7.2), then, using a glass pipette, transfer the rinse to the SPE reservoir, washing
the walls of the reservoir. Use vacuum to pull the elution solvent through the cartridge and
into the collection tubes.
Note: Air dry the empty sample bottle after the rinse is transferred. Weigh the empty bottle with
the cap on and subtract from the weight with the sample determined in Section 11.2.2.
12.2.3 Add 25 (.iL of concentrated acetic acid to each sample eluted in the collection tubes and
vortex to mix. Add 10 mg of carbon (Section 7.1.17) to each sample and batch QC extract,
using a 10-mg scoop. Hand-shake occasionally for 5 minutes and no more. It is important
to minimize the time the sample extract is in contact with the carbon. Immediately vortex
(30 seconds) and centrifuge at 2800 rpm for 10 minutes (other rotational speeds may be
used for centrifuges other than the one described in Section 6.5.1).
12.2.4 Add NIS solution (Section 7.3.2) to a clean collection tube. Place a syringe filter (25-mm
filter, 0.2-(.un nylon membrane) on a 5-mL polypropylene syringe. Take the plunger out
and carefully decant the sample supernatant into the syringe barrel. Replace the plunger
and filter the entire extract into the new collection tube containing the NIS. Vortex to mix
and transfer a portion of the extract into a 1-mL polypropylene microvial for LC-MS/MS
analysis. Cap the collection tube containing the remaining extract and store at 0 - 6 °C.
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12.3 Elution of solid samples
12.3.1 Add NIS solution (Section 7.3.2) to a clean collection tube (13 x 100 mm polypropylene)
for each sample and QC aliquot and place them into the manifold rack, ensuring the extract
delivery needles are not touching the walls of the tubes.
12.3.2 Rinse the inside of the evaporation/concentrator tube using 5 mL of 1% methanolic
ammonium hydroxide (Section 7.1.7.2), then, using a glass pipette, transfer the rinse to the
reservoir, washing the walls of the reservoir. Use the vacuum to pull the elution solvent
through the cartridge and into the collection tubes.
12.3.3 Add 25 |_iL of concentrated acetic acid to each sample extract in its collection tube and
swirl to mix. Place a syringe filter (25-mm filter, 0.2-(.un nylon membrane) on a 5 mL
polypropylene syringe. Take the plunger out and carefully decant ~1 mL of sample extract
into the syringe barrel. Replace the plunger and filter into a 1-mL polypropylene micro vial
for LC-MS/MS analysis. Cap the collection tube containing the remaining extract and store
at 0 - 6 °C.
12.4 Elution of tissue samples
12.4.1 Add NIS solution (Section 7.3.2) to clean collection tubes (13 x 100 mm, polypropylene)
for each sample and QC aliquot. Place the tubes into the manifold rack and ensure the
extract delivery needles are not touching the walls of the tubes.
12.4.2 Rinse the inside of the evaporation/concentrator tube using 5 mL of 1% methanolic
ammonium hydroxide (Section 7.1.7.2), then, using a glass pipette, transfer the rinse to the
reservoir, washing the walls of the reservoir. Use the vacuum to pull the elution solvent
through the cartridge and into the collection tubes.
12.4.3 Add 25 |_iL of concentrated acetic acid to each sample extract. Place a syringe filter
(25-mm filter, 0.2-(.un nylon membrane) on a 5-mL polypropylene syringe. Take the
plunger out and carefully decant an aliquot (~1 mL) of the sample extract into the syringe
barrel. Replace the plunger and filter into a 1-mL polypropylene microvial for LC-MS/MS
analysis. Cap the collection tube containing the remaining extract and store at 0 - 6 °C.
13.0 Instrumental Analysis
Analysis of sample extracts for PFAS by LC-MS/MS is performed on an ultrahigh performance liquid
chromatograph coupled to a triple quadrupole mass spectrometer, running manufacturer's software. The
mass spectrometer is run with unit mass resolution in the multiple reaction monitoring (MRM) mode.
13.1 Perform mass calibration (Section 10.1), establish the operating conditions (Section 10.2), and
perform an initial calibration (Section 10.3) at the frequencies described in those sections prior to
analyzing samples.
13.2 Only after all performance criteria in Sections 10.1, 10.2, and 10.3 are met may blanks, MDLs,
IPRs/OPRs, and samples be analyzed.
13.3 After a successful initial calibration has been completed, the analytical sequence for a batch of
samples analyzed during the same time period is as follows. The volume injected for samples and
QC samples must be identical to the volume used for calibration (Section 10.2.3).
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Standards and sample extracts must be brought to room temperature and vortexed prior to
aliquoting into an instrument vial in order to ensure homogeneity of the extract.
1. Instrument Blank
2. Instrument Sensitivity Check (see Section 10.3.3.1)
3. Calibration Verification Standard
4. Qualitative Identification Standards
5. Instrument Blank
6. Method Blank
7. Low-level OPR (LLOPR)
8. OPR
9. Bile salt interference check standard (Section 7.5)
10. Injections of sample extracts, diluted extracts, and QC sample extracts (10 or fewer field sample
extracts)
11. Calibration Verification Standard
12. Instrument Blank
13. Injections (10 or fewer field sample extracts)
14. Calibration Verification Standard
15. Instrument Blank
If the results are acceptable, the closing calibration verification solution (#14 above) may be used as
the opening solution for the next analytical sequence.
13.4 If the response exceeds the calibration range for any analyte, the sample extract is diluted as per
Section 15.3 to bring all target responses within the calibration range.
Note: If the analytes that exceed the calibration range in the original analysis are known to not be of
concern for the specific project (e.g., are not listed in a discharge permit), then the laboratory
may consult with the client regarding the possibility of reporting sample results over the
calibration range from the undiluted analysis, provided that they are clearly identified as such
and appropriately qualified.
14.0 Performance Tests during Routine Operations
The following performance tests must be successfully completed as part of each routine
instrumental analysis shift described in Section 13.3 above (also see Table 9).
14.1 Instrument sensitivity check
The signal-to-noise ratio of the ISC standard (Section 7.3.4) must be greater than or equal to 3:1 for
the quantitation and confirmation ions that exist, and must meet the ion ratio requirements in
Section 15.1.3. If the analyte has no confirmation ions, then a 10:1 signal to noise ratio is required.
If the requirements cannot be met, the problem must be corrected before analyses can proceed. In
addition, the measured concentration of each native target analyte in the ISC must fall within ± 30%
of its nominal concentration. If that requirement cannot be met for any target analyte relevant to a
project, analysis must be halted and the sensitivity of the LC-MS/MS system adjusted before
analysis of field samples.
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14.2 Bile salt interference check
The retention time of the bile salts in the standard in Section 7.5 must fall at least one minute
outside the retention time window for any of the linear or branched PFOS isomers in the standard
described in Section 7.3.3. If this requirement is not met, the chromatographic conditions must be
adjusted to meet the requirement and the initial calibration must be repeated before any field sample
are analyzed.
14.3 Calibration verification (CV)
After a passing instrument sensitivity check (Section 14.1) and a successful initial calibration
(Section 10.3.3.3) is achieved, prior to the analysis of any samples, analyze a mid-level calibration
standard (Section 7.3.4).
14.3.1 The calibration is verified by analyzing a CV standard at the beginning of each analytical
sequence, every ten samples or less, and at the end of the analytical sequence.
14.3.2 Calculate concentration for each native and isotopically labeled compound in the CV using
the equation in Section 15.2.
14.3.3 The recovery of native compounds for the CVs must be within 70 - 130% unless the
analyte is not of concern for a given project.
14.3.4 If the CV criterion in Section 14.3.3 is not met, recalibrate the LC-MS/MS instrument
according to Section 10.3 and reanalyze any extracts that were analyzed between the last
passing CV and the one that failed with the following exception. If an analyte in the CV
failed because of high recovery, but that analyte was not detected in a sample extract, then
that sample extract need not be reanalyzed.
14.3.5 Ion abundance ratios
Using the data from the CV standard, compute the ion abundance ratio for each target
analyte listed with a confirmation ion mass in Table 7, using the equation below. These ion
abundance ratios will be used a part of the qualitative identification criteria in Section 15.1.
Arean-i
IAR = ^
Area,Q 2
where:
IAR = Ion abundance ratio
AreaQi = The measured area of the Q1 m/z for the analyte in the mid-point calibration
standard or daily CV standard, depending on the analyte concentration, as
described in Section 15.1.3
AreaQ2 = The measured area of the Q2 m/z for the analyte in the mid-point calibration
standard or daily CV standard, depending on the analyte concentration, as
described in Section 15.1.3
Note: Some of the native analytes in Table 7 do not produce confirmation ions, or
produce confirmation ions with very low relative abundances; therefore, for those
analytes, the IAR does not apply.
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Pending completion of the multi-laboratory validation study, construct an acceptance
window for the IAR of each target analyte as 50% to 150% of the IAR in the mid-point
calibration standard or daily CV standard as applicable per section 15.1.3.
14.4 Retention times and resolution
14.4.1 For all method analytes with exact corresponding isotopically labeled analogs, method
analytes must elute within ±0.1 minutes of the associated EIS.
14.4.2 The retention times of each native and isotopically labeled compound must be within ± 0.4
minutes of the ICAL or CV used to establish the RT windows for the samples and batch
QC.
14.5 Ongoing precision and recovery (OPR)
14.5.1 After completing the first 6 steps in the analytical sequence described in Section
13.3,analyze the extracts of the low-level OPR (LLOPR) and the mid-level OPR) (Sections
11.3.3, 11.3.2, and 11.4.2) prior to analysis of samples from the same batch to ensure the
analytical process is under control.
14.5.2 Compute the percent recovery of the native compounds by the appropriate quantification
method depending on the compound (Section 10.3). Compute the percent recovery of each
isotopically labeled compound by the non-extracted internal standard method (Sections 1.2
and 10.3).
Concentration found (nq/mL)
Recovery (%) = —— —— —- x 100
Concentration spiked (ng/mL)
14.5.3 For the native compounds and EISs, compare the recovery to the OPR and LLOPR limits
given in Table 5. Aqueous OPR and LLOPR results must meet the acceptance criteria in
that table. Pending completion on the multi-laboratory validation study and development
of formal acceptance criteria, OPR results for other matrices generally should fall within
the single-laboratory study ranges shown in Table 5A. Minor deviations (e.g., less than
10% lower or higher than the single-laboratory study range) are acceptable. Major
deviations for native PFAS analytes in solid and tissue matrices require corrective actions.
For wastewater matrices, if all compounds meet the acceptance criteria, system
performance is acceptable, and analysis of blanks and wastewater samples may proceed. If,
however, any individual concentration falls outside of the given range, 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,
including any QC samples, and repeat the ongoing precision and recovery test.
14.6 Instrument blank - At the beginning of the analytical sequence and after the analysis of high
concentration samples (e.g., highest calibration standard, CV), analyze an instrument blank to
ensure no instrument contamination has occurred. The instrument blank should not contain any
target analyte that would yield a response equivalent to the mass of the analyte that would be
present in a whole-volume sample at the analyte's MDL. If an analyte is present at such levels,
analyze one or more additional instrument blanks until the response of the analyte is no longer
detectable, or perform additional troubleshooting steps to identify and minimize other potential
sources of PFAS contamination.
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14.7 Method blank - After the analysis of the solvent blank and prior to the analysis of samples, analyze
a method blank (Section 9.5).
14.8 Analyze a qualitative identification standard (Section 7.3.5) containing all available isomers
(branched and linear) once daily, at the beginning of the analytical sequence, to confirm the
retention time of each linear and known branched isomer or isomer group.
14.9 Instrument sensitivity (optional)
This step is recommended as a follow-up step if the ISC 14.1 does not meet the criteria in Section
14.1. Calculate the ratio of the NIS peak areas from the QC and field samples relative to the mean
area of the corresponding NIS in the most recent initial calibration to check for possible bad
injections of NIS solution or loss of instrument sensitivity.
Area of NIS: in the Sample
Area RatioN,s.(°/o) = 100 x
1 Mean AreaNIS.
where:
Area of NIS; in the Sample = Observed area counts for NIS; in the sample
Mean AreaNisi = The mean area counts for the corresponding NIS from the most
recent initial calibration, calculated as described in Section 10.3.3.4
/' = Indicates each of the seven NIS compounds listed in Table 1
The NIS areas in the field samples and QC samples must be within 40 to 200% of the area of the
calibration verification standard run at the beginning of the analytical sequence. If the areas are
low for all the field samples and QC samples in the batch, it suggests a loss of instrument
sensitivity, while low areas in only some field or QC samples suggests a possible bad injection.
15.0 Data Analysis and Calculations
15.1 Qualitative determination and peak identification
A native or isotopically labeled compound is identified in a standard, blank, sample, or QC sample
when all of the criteria in Sections 15.1.1 through 15.1.4 are met.
15.1.1 For target analytes or EISs to be identified, peak responses of the quantitation and
confirmation ions must be at least three times the background noise level (S/N 3:1). The
quantitation ion must have a S/N > 10:1 if there is no confirmation ion . If the S/N ratio is
not met due to high background noise, the laboratory must correct the issue (e.g., perform
instrument troubleshooting and any necessary maintenance, such as cleaning the ion source,
replacing the LC column, or if needed, repeat the cleanup steps to remove background due
to the sample matrix). If the S/N ratio is not met but the background is low, then the
analyte is to be considered a non-detect.
15.1.2 Target analyte, EIS analyte, and NIS analyte RTs must fall within ± 0.4 minutes of the
predicted retention times from the midpoint standard of the ICAL or initial daily CV,
whichever was used to establish the RT window position for the analytical batch. The
retention time window used must be of sufficient width to detect earlier-eluting branched
isomers. For all method analytes with exact corresponding isotopically labeled analogs,
method analytes must elute within ±0.1 minutes of the associated EIS.
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15.1.3 The laboratory must follow the identification requirements specified by the client for the
project. In the event there are no project-specific requirements, the following general
requirements apply. For concentrations at or above the method LOQ, the IAR must fall
within ± 50% of the IAR observed in the mid-point initial calibration standard. If project-
specific requirements involve reporting sample concentrations below the LOQ or ML, these
peaks should also meet the IAR criterion to be reported (see Section 14.3.5).
The total response of all isomers (branched and linear) in the quantitative standards should
be used to define the IAR. In samples, the total response should include only the branched
isomer peaks that have been identified in either the quantitative or qualitative standard (see
Section 7.3 regarding records of traceability of all standards). If standards (either
quantitative or qualitative) are not available for purchase, only the linear isomer can be
identified and quantitated in samples. The ratio requirement does not apply for PFBA,
PFPeA, NMeFOSE, NEtFOSE, PFMPA, and PFMBA because suitable (not detectable or
inadequate S/N) secondary transitions (Q2) are unavailable.
15.1.4 If the field sample result does not all meet the criteria stated in Sections 15.1.1 through
15.1.3, and all sample preparation avenues (e.g., extract cleanup, sample dilution, etc.) have
been exhausted, the result may only be reported with a data qualifier alerting the data user
that the result could not be confirmed because it did not meet the method-required criteria
and therefore should be considered an estimated value. If the criteria listed above are not
met for the standards, the laboratory must stop analysis of samples and correct the issue.
15.2 Quantitative determination
Concentrations of the target analytes are determined with respect to the extracted internal standard
(EIS) which is added to the sample prior to extraction. The EIS is quantitated with respect to a non-
extracted internal standard (NIS), as shown in Table 7, using the response ratios or response factors
from the most recent multi-level initial calibration (Section 10.3). Other equations may be used if
the laboratory demonstrates that those equations produce the same numerical result as produced by
the equations below.
For
where:
Arean
AreaEis
Meis
RR
RF
Ws
Note: For better accuracy, EPA recommends that PFTrDA be quantified using the average of the
areas of labeled compounds 13C2-PFTeDA and 13C2-PFDoA.
And for the EIS analytes:
AreaEls 1
Concentration (na/L orna/a) = ^=— x —
AreaNISRFs Ws
: native analytes:
Arean MEIS 1
Concentration (ng/L or ng/g) = - -== ==- x —
AreaEIS(RR or RF) Ws
The measured area of the Q1 m/z for the native (unlabeled) PFAS
The measured area at the Q1 m/z for the EIS. See note below.
The mass of the EIS added (ng)
Average response ratio used to quantify target compounds by the isotope dilution method
Average response factor used to quantify target compounds by the extracted internal
standard method
Sample volume (L) or weight (g)
3rd Draft Method 1633
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where:
A reap; is = The measured area at the Q1 m/z for the EIS
AreaNis = The measured area of the Q1 m/z for the NIS
Mms = The mass of the NIS added (ng)
Ws = Sample volume (L) or weight (g)
RFS = Average response factor used to quantify the EIS by the non-extracted internal standard
method
Results for native compounds are recovery corrected by the method of quantification. Extracted
internal standard (EIS) recoveries are determined similarly against the non-extracted internal
standard (NIS) and are used as general indicators of overall analytical quality.
The instrument measures the target analytes as either their anions or neutral forms. The default
approach for Clean Water Act uses of the method is to report the analytes in their acid or
neutral forms, using the following equation to convert the concentrations:
n — r v M^Acid
^Acid ~ Anion x '
M^Anton
where:
CAmon = The analyte concentration in anion form
MWacm = The molecular weight of the acid form
MWAmon = The molecular weight of the anion form
15.3 Sample dilutions
15.3.1 If the Q1 area for any compound exceeds the calibration range of the system, dilute a
subsample of the sample extract with the methanolic ammonium hydroxide and acetic acid
solution in Section 7.1.9 and analyze the diluted extract. If the responses for each EIS in
the diluted extract meet the S/N and retention time requirements in Sections 15.1.1 and
15.1.2, and the EIS recoveries from the analysis of the diluted extract are greater than 5%,
then the compounds associated with those EISs may be quantified using the EIS response.
Therefore, use the EIS recoveries from the original analysis to select the dilution factor,
with the objective of keeping the EIS recoveries in the dilution above that 5% lower limit
(i.e., if the EIS recovery of the affected analyte in the undiluted analysis is 50%, then the
sample cannot be diluted more than 10:1; if the EIS recovery of the affected analyte in the
undiluted analysis is 30%, then the sample cannot be diluted more than 6:1). Adjust the
compound concentrations, detection limits, and minimum levels to account for the dilution.
If the EIS responses in the diluted extract do not meet those S/N and retention time
requirements, then the compound cannot be measured reliably by isotope dilution in the
diluted extract. In such cases, the laboratory must take a smaller aliquot of any affected
aqueous sample and dilute it to 500 mL with reagent water and analyze the diluted aqueous
sample, or analyze a smaller aliquot of soil, biosolid, sediment, or tissue sample. Adjust
the calibration ranges, detection limits, and minimum levels to account for the dilution.
If a dilution results in a EIS recovery less than 5%, then the laboratory must prepare and
analyze a diluted aqueous sample or a smaller aliquot of a solid sample.
15.3.2 If the recovery of any EIS in a wastewater sample is outside of the acceptance limits in
Table 8, a diluted aqueous sample must be analyzed (Section 15.3.1). If the recovery of
any EIS in the diluted sample is below 5%, 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, an alternative column could be employed to resolve the interference.
3rd Draft Method 1633
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If all cleanup procedures in this method and an alternative column have been employed and
EIS recovery remains outside of the acceptance range, extraction and/or cleanup procedures
that are beyond this scope of this method may be needed to analyze the sample.
Table 8A includes example performance data for solids and tissues from the single-
laboratory validation study and are provided for illustrative purposes (e.g., those figures are
not required acceptance criteria). EIS recoveries in solids and tissues that fall well outside
of the ranges in Table 8A are a potential cause for concern and laboratories should take
similar steps to those described for wastewater samples to improve EIS recoveries,
including preparing and analyzing a smaller sample aliquot.
15.4 Reporting of analytical results (acid/neutral forms)
The data reporting practices described here are focused on NPDES monitoring needs and may not
be relevant to other uses of the method. For analytes reported in their acid form, use the equations
in Section 15.2 and the analyte names Table 1. For analytes reported in their anion form, see Table
2 for the appropriate names and CAS Registry Numbers.
15.4.1 Report results for aqueous samples in ng/L. Report results for solid samples in ng/g, on a
dry-weight basis, and report the percent solids for each sample separately. Report results
for tissue samples in ng/g, on a wet-weight basis. Other units may be used if required in a
permit or for a project. Report all QC data with the sample results.
15.4.2 Reporting level
Unless specified otherwise by a regulatory authority or in a discharge permit, results for
analytes that meet the identification criteria are reported down to the concentration of the
ML established by the laboratory through calibration of the instrument (see the glossary for
the derivation of the ML). EPA considers the terms "reporting limit," "quantitation limit,"
"limit of quantitation," and "minimum level" to be synonymous.
15.4.2.1 Report a result for each analyte in each field sample or QC standard at or above
the ML to 3 significant figures. Report a result for each analyte found in each
field sample or QC standard below the ML as "
-------
15.4.3 Results from tests performed with an analytical system that is not in control (i.e., that does
not meet acceptance criteria for any QC tests in this method) must be documented and
reported (e.g., as a qualifier on results), unless the failure is not required to be reported as
determined by the regulatory/control authority. Results associated with a QC failure cannot
be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or
permittee of reporting timely results. If the holding time would be exceeded for a
reanalysis of the sample, the regulatory/control authority should be consulted for
disposition.
16.0 Method Performance
Routine method performance is validated through analysis of matrix-specific reference samples, including
IPRs, MDLs, and certified reference materials. Ongoing method performance is monitored through QC
samples analyzed alongside samples. The parameters monitored include percent recovery of isotopically
labeled compounds, blank concentrations, and native compound recoveries.
This method is being validated, and performance specifications will be developed using data from DoD's
interlaboratory validation study (Reference 10). Wastewater data from that study were used to develop
the QC acceptance criteria in Table 5 (IPR/OPR/LLOPR) and Table 8 (EIS recoveries). Table 6 provides
the pooled MDL results from aqueous matrices portion of the multi-laboratory validation study.
For solid and tissue matrices, Table 5A and 8A summarize the results from the single-laboratory
validation study, which should be used as guidance in assessing the results for solid and tissue matrices
until EPA develops formal QC acceptance criteria. Table 6 provides examples of the MDL and ML
results from the single-laboratory validation study for solids and tissues.
17.0 Pollution Prevention
17.1 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
operations. 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 minimize waste generation.
When wastes cannot be reduced feasibly at the source, EPA recommends recycling as the next best
option.
17.2 The compounds 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.
17.3 For information about pollution prevention that may be applied to laboratories and research
institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction
(Reference 7).
18.0 Waste Management
18.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly regarding management of hazardous waste , and to protect the air,
water, and land by minimizing and controlling all releases from fume hoods and bench operations.
3rd Draft Method 1633
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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 (Reference
8).
18.2 Samples at pH < 2 or pH > 12.5, are hazardous and must be handled and disposed of as hazardous
waste or neutralized and disposed of in accordance with all federal, state, and local regulations
18.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction,
(Reference 9).
19.0 References
1. "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health
Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS
PB-277256.
2. "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
3. "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
4. "Standard Methods for the Examination of Water and Wastewater," 18th edition and later
revisions, American Public Health Association, 1015 15th St, NW, Washington, DC 20005,
1-35: Section 1090 (Safety), 1992.
5. "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916 Race
Street, Philadelphia, PA 19103-1187, 1980.
6. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA
EMSL, Cincinnati, OH 45268, EPA 600/4-79-019, April 1979.
7. "Less is Better: Laboratory Chemical Management for Waste Reduction," American Chemical
Society, 1993. Available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
8. "Environmental Management Guide for Small Laboratories," USEPA, Small Business
Division, Washington DC, EPA 233-B-00-001, May 2000.
9. "The Waste Management Manual for Laboratory Personnel," American Chemical Society,
1990. Available from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street NW, Washington, DC 20036.
10. Willey, J., R. Anderson, A. Hanley, M. Mills, C. Hamilton, T. Thompson, and A. Leeson. 2021.
"Report on the Single-Laboratory Validation of PFAS by Isotope Dilution LC-MS/MS,"
Strategic Environmental Research and Development Program (SERDP) Project ER19-1409.
11. DoD interlaboratory study reference will be added here.
12. DoD QSM (US Department of Defense Quality Systems Manual for Environmental
Laboratories, version 5.3, 2019).
3rd Draft Method 1633
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13. Woudneh, Million B., Bharat Chandramouli, Coreen Hamilton, Richard Grace, 2019, "Effects
of Sample Storage on the Quantitative Determination of 29 PFAS: Observation of Analyte
Interconversions during Storage," Environmental Science and Technology 53(21): 12576-
12585.
14. ISO 17034:2016, General requirements for the competence of reference material producers,
ISO, Geneva, Switzerland, 2016.
3rd Draft Method 1633
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20.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Names, Abbreviations, and CAS Registry Numbers for Target PFAS, Extracted Internal
Standards and Non-extracted Internal Standards1
Target Analyte Name
Abbreviation
( AS Number
l>eii°luoroiilk\ 1 carbowlic acids
Perfluorobutanoic acid
PFBA
375-22-4
Perfluoropentanoic acid
PFPeA
2706-90-3
Perfluorohexanoic acid
PFHxA
307-24-4
Perfluoroheptanoic acid
PFHpA
375-85-9
Perfluorooctanoic acid
PFOA
335-67-1
Perfluorononanoic acid
PFNA
375-95-1
Perfluorodecanoic acid
PFDA
335-76-2
Perfluoroundecanoic acid
PFUnA
2058-94-8
Perfluorododecanoic acid
PFDoA
307-55-1
Perfluorotridecanoic acid
PFTrDA
72629-94-8
Perfluorotetradecanoic acid
PFTeDA
376-06-7
Pcrfluoroalk\ 1 sulfonic acids
Acid Form
Perfluorobutanesulfonic acid
PFBS
375-73-5
Perfluoropentansulfonic acid
PFPeS
2706-91-4
Perfluorohexanesulfonic acid
PFHxS
355-46-4
Perfluoroheptanesulfonic acid
PFHpS
375-92-8
Perfluorooctanesulfonic acid
PFOS
1763-23-1
Perfluorononanesulfonic acid
PFNS
68259-12-1
Perfluorodecanesulfonic acid
PFDS
335-77-3
Pci°niii
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Table 1. Names, Abbreviations, and CAS Registry Numbers for Target PFAS, Extracted Internal
Standards and Non-extracted Internal Standards1
Target Analyte Name
Abbreviation
CAS Number
I-'.iIkt sulfonic acids
9-Chlorohexadecafluoro-3 -oxanonane-1 -sulfonic acid
9C1-PF30NS
756426-58-1
1 l-Chloroeicosafluoro-3-oxaundecane-l-sulfonic acid
llCl-PF30UdS
763051-92-9
Perfluoro(2-ethoxyethane)sulfonic acid
PFEESA
113507-82-7
l-'liioroiclomcr carbow lie acids
3-Perfluoropropyl propanoic acid
3:3FTCA
356-02-5
2 / /. 2 / /. 3 / /. 3 / /- Pc rfl uo ro o c t a no i c acid
5:3FTCA
914637-49-3
3-Perfluoroheptyl propanoic acid
7:3FTCA
812-70-4
EIS Compounds
Perfluoro-n-[13C4]butanoic acid
13c4-pfba
Perfluoro-n-[13C5]pentanoic acid
13C5-PFPeA
Perfluoro-n-[l,2,3,4,6-13C5]hexanoic acid
13C5-PFHxA
Perfluoro-n-[l,2,3,4-13C4]heptanoic acid
13C4-PFHpA
Perfluoro-n-[13C8]octanoic acid
13c8-pfoa
Perfluoro-n-[13C9]nonanoic acid
13c9-pfna
Perfluoro-n-[l,2,3,4,5,6-13C6]decanoic acid
13c6-pfda
Perfluoro-n-[l,2,3,4,5,6,7-13C7]undecanoic acid
13C7-PFUnA
Perfluoro-n- [1,2 -13C2]dodecanoic acid
13C2-PFDoA
Perfluoro-n- [1,2 -13C2]tetradecanoic acid
13C2-PFTeDA
Perfluoro-l-[2,3,4-13C3]butanesulfonic acid
13c3-pfbs
Perfluoro-l-[l,2,3-13C3]hexanesulfonic acid
13C3-PFHxS
NA
Perfluoro-l-[13C8]octanesulfonic acid
13c8-pfos
Perfluoro-l-[13C8]octanesulfonamide
13c8-pfosa
N-methyl-d3-perfluoro-1 -octanesulfonamidoacetic acid
D3-NMeFOSAA
N-ethyl-d5-perfluoro-1 -octanesulfonamidoacetic acid
Ds-NEtFOSAA
Ml. 1 //.2//.2//-Pcrfluoro-1 -| 1.2-l3C'2|lic\anc sulfonic acid
13C2-4:2FTS
MI. 1 //.2//.2//-Pcrfluoro-1 -| 1.2-l3C'2|octanc sulfonic acid
13C2-6:2FTS
MI. 1 //.2//.2//-Pcrfluoro-1 -| 1.2-' 3C2|dccanc sulfonic acid
13C2-8:2FTS
Tetrafluoro-2-heptafluoropropoxy-13C3-propanoic acid
13c3-hfpo-da
N-methyl-d7-perfluorooctanesulfonamidoethanol
Dv-NMeFOSE
N-ethyl-ds-perfluorooctanesulfonamidoethanol
D9-NEtFOSE
N-ethyl-d5-perfluoro-1 -octanesulfonamide
Ds-NEtFOSA
N-methyl-d3-perfluoro-1 -octanesulfonamide
D3-NMeFOSA
MS ( oiiipoulids
Perfluoro-n-[2,3,4-13C3]butanoic acid
13c3-pfba
Perfluoro-n-[l,2,3,4-13C4]octanoic acid
13c4-pfoa
Perfluoro-n-[l,2-13C2]decanoic acid
13c2-pfda
Perfluoro-n-[l,2,3,4-13C4]octanesulfonic acid
13c4-pfos
NA
Perfluoro-n-[l,2,3,4,5-13C5] nonanoic acid
13c5-pfna
Perfluoro-n-[l,2-13C2]hexanoic acid
13C2-PFHxA
Perfluoro-l-hexane[1802]sulfonic acid
1802-PFHxS
1 The target analyte names are for the acid and neutral forms of the analytes. See Table 2 for the names and CASRN of the
corresponding anion forms, where applicable.
NA Not assigned a CASRN
* Analytes in this class may not perform as well as others (see Section 1.6)
3rd Draft Method 1633
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December 2022
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Table 2. Cross-reference of Abbreviations, Analyte Names, CAS Numbers for the Acid and Anion Forms
of the Perfluoroalkyl carboxylates and Perfluoroalkyl sulfonates
Pcrl'liioroalkt 1 carbnxjlic acids/anions
Abbreviation
Acid Name
CASRN
Anion Name
CASRN
PFBA
Perfluorobutanoic acid
375-22-4
Perfluorobutanoate
45048-62-2
PFPeA
Perfluoropentanoic acid
2706-90-3
Perfluoropentanoate
45167-47-3
PFHxA
Perfluorohexanoic acid
307-24-4
Perfluorohexanoate
92612-52-7
PFHpA
Perfluoroheptanoic acid
375-85-9
Perflluoroheptanoate
120885-29-2
PFOA
Perfluorooctanoic acid
335-67-1
Pefluorooctanoate
45285-51-6
PFNA
Perfluorononanoic acid
375-95-1
Perfluorononanoate
72007-68-2
PFDA
Perfluorodecanoic acid
335-76-2
Perfluorodecanoate
73829-36-4
PFUnA
Perfluoroundecanoic acid
2058-94-8
Perfluoroundecanoate
196859-54-8
PFDoA
Perfluorododecanoic acid
307-55-1
Perfluorododecanoate
171978-95-3
PFTrDA
Perfluorotridecanoic acid
72629-94-8
Perfluorotridecanoate
862374-87-6
PFTeDA
Perfluorotetradecanoic acid
376-06-7
Perfluorotetradecanoate
365971-87-5
Pcrl'liioroalkt 1 sulfonic acids/anions
PFBS
Perfluorobutanesulfonic acid
375-73-5
Perfluorobutane sulfonate
45187-15-3
PFPeS
Perfluoropentansulfonic acid
2706-91-4
Perfluoropentane sulfonate
175905-36-9
PFHxS
Perfluorohexanesulfonic acid
355-46-4
Perfluorohexane sulfonate
108427-53-8
PFHpS
Perfluoroheptanesulfonic acid
375-92-8
Perfluoroheptane sulfonate
146689-46-5
PFOS
Perfluorooctanesulfonic acid
1763-23-1
Perfluorooctane sulfonate
45298-90-6
PFNS
Perfluorononanesulfonic acid
68259-12-1
Perfluorononane sulfonate
474511-07-4
PFDS
Perfluorodecanesulfonic acid
335-77-3
Perfluorodecane sulfonate
126105-34-8
PFDoS
Perfluorododecanesulfonic acid
79780-39-5
Perfluorododecane sulfonate
343629-43-6
3rd Draft Method 1633
49
December 2022
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Table 3. Nominal Masses of Spike Added to Samples or Extracts
Analyte
Amount Added (ng)
l-Alraclcd Internal Standards
13c4-pfba
40
13C5-PFPeA
20
13C5-PFHxA
10
13C4-PFHpA
10
13c8-pfoa
10
13c9-pfna
5
13c6-pfda
5
13C7-PFUnA
5
13C2-PFDoA
5
13C2-PFTeDA
5
13c3-pfbs
10
13C3-PFHxS
10
13c8-pfos
10
13C2-4:2FTS
20
13C2-6:2FTS
20
13C2-8:2FTS
20
13c8-pfosa
10
D3-NMeFOSA
10
Ds-NEtFOSA
10
D3-NMeFOSAA
20
Ds-NEtFOSAA
20
D-NMcFOSE
100
Dg-NEtFOSE
100
13C3-HFPO-DA
40
Non-e\lracled Inlernal Siandards
13c3-pfba
20
13C2-PFHxA
10
13c4-pfoa
10
13c5-pfna
5
13c2-pfda
5
1802-PFHxS
10
13c4-pfos
10
3rd Draft Method 1633
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December 2022
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Table 4. Calibration Solutions (ng/mL) Used in the Method Validation Studies
Compound
CS1 (LOQ)
CS2
CS3
CS4 (CV1)
CS5
CS6
CS72
Perlluoroalkvl carboxvlic acids
PFBA
0.8
2
5
10
20
50
250
PFPeA
0.4
1
2.5
5
10
25
125
PFHxA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFHpA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFOA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFNA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFDA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFUnA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFDoA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFTrDA
0.2
0.5
1.25
2.5
5
12.5
62.5
PFTeDA
0.2
0.5
1.25
2.5
5
12.5
62.5
Pciiliioroalkv 1 sulfonic acids
PFBS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFPeS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFHxS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFHpS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFOS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFNS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFDS
0.2
0.5
1.25
2.5
5
12.5
62.5
PFDoS
0.2
0.5
1.25
2.5
5
12.5
62.5
l-'liioroiclomcr sulfonic acids
4:2FTS
0.8
2
5
10
20
50
NA
6:2FTS
0.8
2
5
10
20
50
NA
8:2FTS
0.8
2
5
10
20
50
NA
Pcrfliiorooclanc suHViiiamitlos
PFOSA
0.2
0.5
1.25
2.5
5
12.5
62.5
NMeFOSA
0.2
0.5
1.25
2.5
5
12.5
62.5
NEtFOSA
0.2
0.5
1.25
2.5
5
12.5
62.5
I'eii'liioi'ooclanc suiroiiamitloacclic acids
NMeFOSAA
0.2
0.5
1.25
2.5
5
12.5
62.5
NEtFOSAA
0.2
0.5
1.25
2.5
5
12.5
62.5
Pcrfliiorooclanc sulfonamide clhanols
Wlcl (>si:
5
i: 5
25
5(1
125
(.25
\iii osi:
Per- and poM'luoroclhcr car
>o\\lie acids
5
i: 5
25
5t>
125
(.25
HFPO-DA
0.8
2
5
10
20
50
250
ADONA
0.8
2
5
10
20
50
250
PFMPA
0.4
1
2.5
5
10
25
125
PFMBA
0.4
1
2.5
5
10
25
125
NFDHA
0.4
1
2.5
5
10
25
125
Ether sulfonic acids
9C1-PF30NS
0.8
2
5
10
20
50
250
llCl-PF30UdS
0.8
2
5
10
20
50
250
PFEESA
0.4
1
2.5
5
10
25
125
3rd Draft Method 1633
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Table 4. Calibration Solutions (ng/mL) Used in the Method Validation Studies
Compound
CS1 (LOQ)
CS2
CS3
CS4 (CV1)
CS5
CS6
CS72
Fluorotelomer carboxvlic acids
3:3FTCA
1.0
2.5
6.26
12.5
25
62.4
312
5:3FTCA
5.0
12.5
31.3
62.5
125
312
1560
7:3FTCA
5.0
12.5
31.3
62.5
125
312
1560
Extracted Internal Standard (EIS) Analvtcs
13c4-pfba
10
10
10
10
10
10
10
13C5-PFPeA
5
5
5
5
5
5
5
13C5-PFHxA
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13C4-PFHpA
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c8-pfoa
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c9-pfna
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13c6-pfda
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13C7-PFUnA
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13C2-PFDoA
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13C2-PFTeDA
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13c3-pfbs
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13C3-PFHxS
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c8-pfos
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13C2-4:2FTS
5
5
5
5
5
5
5
13C2-6:2FTS
5
5
5
5
5
5
5
13C2-8:2FTS
5
5
5
5
5
5
5
13c8-pfosa
2.5
2.5
2.5
2.5
2.5
2.5
2.5
D3-NMeFOSA
2.5
2.5
2.5
2.5
2.5
2.5
2.5
Ds-NEtFOSA
2.5
2.5
2.5
2.5
2.5
2.5
2.5
D3-NMeFOSAA
5
5
5
5
5
5
5
Ds-NEtFOSAA
5
5
5
5
5
5
5
D-NMcFOSE
25
25
25
25
25
25
25
D9-NEtFOSE
25
25
25
25
25
25
25
13C3-HFPO-DA
10
10
10
10
10
10
10
Non-e\lracled Inlernal Standard (MS) AnalMes
13c3-pfba
5
5
5
5
5
5
5
13C2-PFHxA
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c4-pfoa
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c5-pfna
1.25
1.25
1.25
1.25
1.25
1.25
1.25
13c2-pfda
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1802-PFHxS
2.5
2.5
2.5
2.5
2.5
2.5
2.5
13c4-pfos
2.5
2.5
2.5
2.5
2.5
2.5
2.5
1 This calibration point is used as the calibration verification (CV)
2 A minimum of six contiguous calibrations standards are required for linear models and a minimum of seven calibration
standards are required for second-order models.
3rd Draft Method 1633
52
December 2022
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Table 5. IPR/OPR/LLOPR Acceptance Limits for Wastewater Samples
Compounds
Aqueous Matrix 1,2
I PR
OPR Recovery (%)
LLOPR Recovery (%)
Recovery (%)
RSD (%)
PFBA
60 - 147
20
58-148
44 - 157
PFPeA
56 - 150
20
54-152
57 - 148
PFHxA
59 - 148
25
55-152
62 - 149
PFHpA
60 - 149
25
54-154
56 - 150
PFOA
55-158
25
52-161
57 - 161
PFNA
64 - 144
25
59-149
53 - 157
PFDA
57 - 142
25
52 - 147
43 - 158
PFUnA
54-153
30
48-159
50 - 155
PFDoA
73 - 133
25
64 - 142
60 - 141
PFTrDA
52 - 145
25
49-148
52 - 140
PFTeDA
49-158
25
47-161
52 - 156
PFBS
66-141
20
62 - 144
63 - 145
PFPeS
66 - 144
25
59-151
58 - 144
PFHxS
62-141
25
57 - 146
44 - 158
PFHpS
59 - 148
25
55-152
51 - 150
PFOS
61 - 145
20
58-149
43 - 162
PFNS
57 - 143
25
52-148
46 - 151
PFDS
56 - 142
25
51-147
50 - 144
PFDoS
41 - 140
30
36-145
30 - 138
4:2FTS
77-135
25
67 - 146
52 - 158
6:2FTS
75 - 137
30
61-151
48 - 158
8:2FTS
79-136
30
63 - 152
46 - 165
PFOSA
65 - 144
20
61-148
47 - 163
NMeFOSA
76 - 132
25
63 - 145
54 - 155
NEtFOSA
75 - 129
25
65-139
49 - 156
NMeFOSAA
69-134
25
58-144
32 - 160
NEtFOSAA
65 - 140
25
59-146
51 - 154
NMeFOSE
79 - 129
20
71-136
56 - 151
NEtFOSE
79 - 126
25
69-137
60 - 147
HFPO-DA
72-135
25
63 - 144
58 - 154
ADONA
75-138
20
68 - 146
61 - 148
PFMPA
55-141
25
51-145
48 - 150
PFMBA
59 - 145
20
55-148
49 - 154
NFDHA
63 - 146
35
48-161
47 - 160
9C1-PF30NS
72 - 140
30
56-156
44 - 167
llCl-PF30UdS
61 - 140
35
46-156
36 - 158
PFEESA
57 - 149
20
56-151
56 - 144
3:3FTCA
66 - 126
20
62 - 129
32 - 161
5:3FTCA
68-130
20
63-134
39 - 156
7:3FTCA
55-133
25
50-138
36 - 149
13c4-pfba
10-130
30
10-130
10-130
13C5-PFPeA
35-150
30
40-150
40-150
13C5-PFHxA
55 - 150
30
40-150
40-150
13C4-PFHpA
55 - 150
30
40-150
40-150
13c8-pfoa
60 - 140
30
30-140
30-140
13c9-pfna
55 - 140
30
30-140
30-140
13c6-pfda
50 - 140
30
20-140
20-140
3rd Draft Method 1633
53
December 2022
-------
Table 5. IPR/OPR/LLOPR Acceptance Limits for Wastewater Samples
Compounds
Aqueous Matrix 1,2
IPR
OPR Recovery (%)
LLOPR Recovery (%)
Recovery (%)
RSD (%)
13C7-PFUnA
30 - 140
30
20-140
20-140
13C2-PFDoA
10-150
30
10-150
10-150
13C2-PFTeDA
10-130
30
10-130
10-130
13c3-pfbs
55 - 150
30
25-150
25-150
13C3-PFHxS
55 - 150
30
25-150
25-150
13c8-pfos
45 - 140
30
20-140
20-140
13C2-4:2FTS
60 - 200
30
25-200
25-200
13C2-6:2FTS
60 - 200
30
25-200
25-200
13C2-8:2FTS
50 - 200
30
25-200
25-200
13c8-pfosa
30-130
30
10-130
10-130
D3-NMeFOSA
15-130
30
10-130
10-130
Ds-NEtFOSA
10-130
30
10-130
10-130
D3-NMeFOSAA
45 - 200
30
10-200
10-200
Ds-NEtFOSAA
10 - 200
30
10-200
10-200
D-NMcFOSE
10-150
30
10-150
10-150
D9-NEtFOSE
10-150
30
10-150
10-150
13C3-HFPO-DA
25 - 160
30
25-160
25-160
1 The recovery limits apply to the target analyte results for IPR, OPR, and LLOPR samples for wastewater matrices.
Data for this matrix type are derived from the multi-laboratory validation study and are therefore the limits required
for this method.
2 The recovery limits for the EIS compounds were derived by EPA from the wastewater sample data from multi-
laboratory validation study. To simplify laboratory operations, EPA has applied the same EIS recovery limits
used for field sample analyses to the EIS recoveries in the IPR, OPR, and LLOPR samples.
3rd Draft Method 1633
54
December 2022
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Table 5A Example Performance Data for Solids and Tissues
Compounds
Solid Matrix1
Tissue Matrix1
I PR
OPR
Recovery
(%)
I PR
OPR
Recovery
(%)
Recovery (%)
RSD2 (%)
Recovery (%)
RSD2 (%)
PFBA
95-99
5
92 - 108
89 - 104
5
90-110
PFPeA
92 - 105
5
94-115
80-98
5
96-114
PFHxA
93 - 101
5
89 - 107
72-110
10
90-111
PFHpA
94 - 102
5
89 - 107
87 - 102
5
87-118
PFOA
92 - 100
5
90 - 106
78-85
5
82-114
PFNA
91 - 102
5
88-112
85-110
6
87-119
PFDA
97 - 103
5
89-118
76-115
10
84-112
PFUnA
91 - 107
5
92-111
83 - 102
5
91-117
PFDoA
73 - 120
12
88-119
83 - 105
6
77-141
PFTrDA
91 - 112
5
89-125
92-114
5
106-133
PFTeDA
94 - 104
5
92-110
76 - 103
7
91-111
PFBS
91 - 103
5
91-111
69 - 105
10
89-117
PFPeS
87 - 103
5
89-112
77-96
5
89-112
PFHxS
98 - 106
5
96-113
81 - 101
5
91-123
PFHpS
87 - 104
5
88 - 104
77 - 108
8
86 - 108
PFOS
95 - 108
5
94-115
98-112
6
97 - 124
PFNS
98-111
5
76-117
65-88
8
85-114
PFDS
83 - 102
5
84 - 107
82-94
5
78-110
PFDoS
76-99
7
77 - 100
73-96
7
29-108
4:2FTS
98 - 100
5
87-113
66 - 126
16
90 - 103
6:2FTS
94 - 123
7
60 - 166
77 - 105
8
92-119
8:2FTS
109 - 128
5
104 - 127
66 - 148
19
102-136
PFOSA
92 - 106
5
94-114
92-116
6
96-121
NMeFOSA
87 - 104
5
91-117
81 - 100
6
86-117
NEtFOSA
98 - 102
5
96-115
74-114
11
90 - 127
NMeFOSAA
91 - 107
5
90-113
89-136
10
93-117
NEtFOSAA
102 - 108
5
87-117
53 - 115
18
90-117
NMeFOSE
98 - 103
5
94-112
71-292
30
118-344
NEtFOSE
97 - 104
5
96-115
97-133
8
61-159
HFPO-DA
83 - 105
6
80 - 120
73 - 100
8
86-114
ADONA
85-96
5
76 - 124
82-95
5
86-132
PFMPA
91-98
5
85-117
78-93
5
86 - 109
PFMBA
88-97
5
85 - 120
74 - 104
8
84-117
NFDHA
53 - 103
16
58-136
49-86
14
56-115
9C1-PF30NS
84 - 100
5
79-131
69-98
9
95 - 126
llCl-PF30UdS
84-96
5
77 - 127
85 - 100
5
94-138
PFEESA
80-93
5
89-109
68-99
9
88 - 107
3:3FTCA
86-98
5
76-116
66-94
9
41-126
5:3FTCA
83-94
5
80-101
95-131
8
78-199
7:3FTCA
90 - 106
5
75 - 104
84-111
7
99-139
13c4-pfba
92-99
5
95 - 109
93-97
5
95 - 105
13C5-PFPeA
86 - 106
5
80-110
85 - 108
6
89-103
13C5-PFHxA
83 - 101
5
92 - 106
79-111
9
88-98
13C4-PFHpA
87 - 102
5
90 - 100
88-93
5
80 - 102
13c8-pfoa
89-101
5
92 - 104
91-98
5
86 - 102
13c9-pfna
86-101
5
90 - 106
91 - 104
5
89-101
13c6-pfda
79-101
6
86 - 109
89 - 104
5
90 - 104
3rd Draft Method 1633
55
December 2022
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Table 5A Example Performance Data for Solids and Tissues
Compounds
Solid Matrix1
Tissue Matrix1
IPR
OPR
Recovery
(%)
IPR
OPR
Recovery
(%)
Recovery (%)
RSD2 (%)
Recovery (%)
RSD2 (%)
13C7-PFUnA
84 - 104
5
91-116
84-118
8
88-109
13C2-PFDoA
70-93
7
73 - 106
95 - 125
7
70 - 108
13C2-PFTeDA
83-88
5
74 - 107
81 - 114
9
10-110
13c3-pfbs
97 - 105
5
96 - 109
87-114
7
95 - 106
13C3-PFHxS
92-97
5
92 - 106
92-97
5
91-103
13c8-pfos
87 - 107
5
95 - 109
87-93
5
95 - 103
13C2-4:2FTS
132-135
5
123 - 145
106-221
18
155-291
13C2-6:2FTS
118-129
5
104-138
87-135
11
117-149
13C2-8:2FTS
96 - 122
6
93 - 123
179-299
13
79 - 304
13c8-pfosa
69-86
5
66 - 100
104-153
9
88 - 120
D3-NMeFOSA
47-59
5
25-64
20-58
25
3-34
Ds-NEtFOSA
43-51
5
18-58
30-56
15
0-56**
D3-NMeFOSAA
98 - 107
5
86 - 109
102 - 187
15
144-196
Ds-NEtFOSAA
98 - 104
5
85 - 109
178-216
5
175-223
D-NMcFOSE
50-61
5
35-76
3-5
12
0-8**
D9-NEtFOSE
46-57
5
32-72
8-33
30
0-33**
13C3-HFPO-DA
98 - 108
5
83 - 125
87 - 106
5
81-106
1 The data for these matrices were derived from the single-laboratory validation study, and are only provided as examples for
this draft method. The data will be updated to reflect the interlaboratory study results in a subsequent revision. Therefore,
these criteria will change after interlaboratory validation. Laboratories may use these data as guidance is assessing their IPR
and OPR results for solids and tissues.
2 RSD values from the single-laboratory validation study that were les than 5% have all been raised to 5% for the purposes of
this draft of the method.
** Statistically derived lower acceptance limits below 0% were set to 0% for the purposes of this table.
3rd Draft Method 1633
56
December 2022
-------
Table 6. Pooled MDL and ML Values for Aqueous Matrices and Example Solid and Tissue MDL and ML
Values*
Compound
Aqueous (ng/L)1
Solid (ng/g)2
Tissue (ng/g)2
Pooled MDLs
ML
MDLS
ML
MDLS
ML
PFBA
0.80
2.0
0.40
0.8
0.59
2.0
PFPeA
0.53
2.0
0.02
0.4
0.08
1.0
PFHxA
0.48
2.0
0.02
0.2
0.10
0.5
PFHpA
0.39
2.0
0.03
0.2
0.09
0.5
PFOA
0.55
2.0
0.04
0.2
0.09
0.5
PFNA
0.46
2.0
0.09
0.2
0.16
0.5
PFDA
0.53
2.0
0.03
0.2
0.12
0.5
PFUnA
0.44
2.0
0.03
0.2
0.15
0.5
PFDoA
0.37
2.0
0.06
0.2
0.13
0.5
PFTrDA
0.46
2.0
0.04
0.2
0.09
0.5
PFTeDA
0.51
2.0
0.03
0.2
0.19
0.5
PFBS
0.37
2.0
0.01
0.2
0.07
0.5
PFPeS
0.53
2.0
0.02
0.2
0.03
0.5
PFHxS
0.56
2.0
0.02
0.2
0.08
0.5
PFHpS
0.87
2.0
0.06
0.2
0.04
0.5
PFOS
0.64
2.0
0.07
0.2
0.29
0.5
PFNS
0.49
2.0
0.05
0.2
0.11
0.5
PFDS
0.90
2.0
0.04
0.2
0.10
0.5
PFDoS
0.64
2.0
0.04
0.2
0.18
0.5
4:2FTS
1.74
5.0
0.28
0.8
0.74
2.0
6:2FTS
2.52
10
0.12
0.8
1.15
2.0
8:2FTS
2.58
10
0.23
0.8
0.37
2.0
PFOSA
0.32
2.0
0.07
0.2
0.09
0.5
NMeFOSA
0.41
2.0
0.05
0.2
0.16
0.5
NEtFOSA
0.43
2.0
0.04
0.2
0.17
0.5
NMeFOSAA
1.04
2.0
0.03
0.2
0.09
0.5
NEtFOSAA
0.80
2.0
0.04
0.2
0.14
0.5
NMeFOSE
3.93
10
0.20
2.0
9.98
5.0
NEtFOSE
5.13
20
0.25
2.0
1.50
5.0
HFPO-DA
1.54
5.0
0.14
0.8
0.16
2.0
ADONA
1.47
5.0
0.06
0.8
0.08
2.0
PFEESA
0.79
2.0
0.02
0.4
0.05
1.0
PFMPA
0.54
2.0
0.03
0.4
0.07
1.0
PFMBA
0.53
2.0
0.03
0.4
0.07
1.0
NFDHA
1.92
5.0
0.08
0.4
0.29
1.0
9C1-PF30NS
1.42
5.0
0.04
0.8
0.15
2.0
llCl-PF30UdS
1.78
5.0
0.07
0.8
0.31
2.0
3:3FTCA
2.54
10
0.06
1.0
0.25
2.5
5:3FTCA
9.92
20
0.36
5.0
1.54
12.5
7:3FTCA
9.14
20
0.31
5.0
0.85
12.5
* A standard containing a mixture of branched and linear isomer of suitable quality to be used for quantitation is currently
available and required to be used for all calibration, calibration verifications, and QC samples. If more become commercially
available for other target analytes, they must be utilized in the same manner.
1 The pooled MDL and ML for aqueous matrices data are derived from the multi-laboratory validation study using data from
eight laboratories for a total of 24 individual MDL studies and are therefore the limits required for this method.
2 The MDL and ML values for solid and tissue matrices are example data from the single-laboratory validation study and are
only provided as examples for this draft method.
3rd Draft Method 1633
57
December 2022
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Table 7. Analyte Ions Monitored, Extracted Internal Standard, and Non-extracted Internal Standard Used
for Quantification
Abbreviation
Example
Retention
Time 1
Parent Ion
Mass
Quantification
Ion Mass
Confirmation
Ion Mass
Typical Ion
Ratio
Quantification
Reference
Compound
Target Analvtes
PFBA
1.96
212.8
168.9
NA
NA
13c4-pfba
PFPeA
4.18
263.0
219.0
68.9
NA
13C5-PFPeA
PFHxA
4.81
313.0
269.0
118.9
13
13C5-PFHxA
PFHpA
5.32
363.1
319.0
169.0
3.5
13C4-PFHpA
PFOA
6.16
413.0
369.0
169.0
3.0
13Cs-PFOA
PFNA
6.99
463.0
419.0
219.0
4.9
13C9-PFNA
PFDA
7.47
512.9
469.0
219.0
5.5
13c6-pfda
PFUnA
7.81
563.1
519.0
269.1
6.9
13C7-PFUnA
PFDoA
8.13
613.1
569.0
319.0
10
13C2-PFDoA
PFTrDA2
8.53
663.0
619.0
168.9
6.7
avg.13C2-PFTeDA
and13C2-PFDoA
PFTeDA
8.96
713.1
669.0
168.9
6.0
13C2-PFTeDA
PFBS
4.79
298.7
79.9
98.8
2.1
13C3-PFBS
PFPeS
5.38
349.1
79.9
98.9
1.8
13C3-PFHxS
PFHxS
6.31
398.7
79.9
98.9
1.9
13C3-PFHxS
PFHpS
7.11
449.0
79.9
98.8
1.7
13Cs-PFOS
PFOS
7.59
498.9
79.9
98.8
2.3
13Cs-PFOS
PFNS
7.92
548.8
79.9
98.8
1.9
13Cs-PFOS
PFDS
8.28
599.0
79.9
98.8
1.9
13Cs-PFOS
PFDoS
9.14
699.1
79.9
98.8
1.9
13Cs-PFOS
4:2FTS
4.67
327.1
307.0
80.9
1.7
13C2-4:2FTS
6:2FTS
5.81
427.1
407.0
80.9
1.9
13C2-6:2FTS
8:2FTS
7.28
527.1
507.0
80.8
3.0
13C2-8:2FTS
PFOSA
8.41
498.1
77.9
478.0
47
13Cs-PFOSA
NMeFOSA
9.70
511.9
219.0
169.0
0.66
Ds-NMeFOSA
NEtFOSA
9.94
526.0
219.0
169.0
0.63
D5-NEtFOSA
NMeFOSAA
7.51
570.1
419.0
483.0
2.0
Ds-NMeFOSAA
NEtFOSAA
7.65
584.2
419.1
526.0
1.2
D5-N-EtFOSAA
NMeFOSE
9.57
616.1
58.9
NA
NA
D7-NMeFOSE
NEtFOSE
9.85
630.0
58.9
NA
NA
Ds-NEtFOSE
HFPO-DA
4.97
284.9
168.9
184.9
1.95
13C3-HFPO-DA
ADONA
5.79
376.9
250.9
84.8
2.8
13c3-hfpo-da
9C1-PF30NS
7.82
530.8
351.0
532.8^353.0
3.2
13c3-hfpo-da
llCl-PF30UdS
8.62
630.9
450.9
632.9^452.9
3.0
13c3-hfpo-da
3:3FTCA
3.89
241.0
177.0
117.0
1.70
13C5-PFPeA
5:3FTCA
5.14
341.0
237.1
217.0
1.16
13C5-PFHxA
7:3FTCA
6.76
441.0
316.9
336.9
0.69
13C5-PFHxA
PFEESA
5.08
314.8
134.9
82.9
9.22
13C5-PFHxA
PFMPA
3.21
229.0
84.9
NA
NA
13C5-PFPeA
PFMBA
4.53
279.0
85.1
NA
NA
13C5-PFPeA
NFDHA
4.84
295.0
201.0
84.9
1.46
13C5-PFHxA
I'.Mrailed Inlernal Slaiulanls
13c4-pfba
1.95
216.8
171.9
NA
13c3-pfba
13C5-PFPeA
4.18
268.3
223.0
NA
13C2-PFHxA
13C5-PFHxA
4.80
318.0
273.0
120.3
13C2-PFHxA
13C4-PFHpA
5.32
367.1
322.0
NA
13C2-PFHxA
13Cs-PFOA
6.16
421.1
376.0
NA
13c4-pfoa
13C9-PFNA
6.99
472.1
427.0
NA
13c5-pfna
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Table 7. Analyte Ions Monitored, Extracted Internal Standard, and Non-extracted Internal Standard Used
for Quantification
Abbreviation
Example
Retention
Time 1
Parent Ion
Mass
Quantification
Ion Mass
Confirmation
Ion Mass
Typical Ion
Ratio
Quantification
Reference
Compound
13c6-pfda
7.47
519.1
474.1
NA
13c2-pfda
13C7-PFUnA
7.81
570.0
525.1
NA
13c2-pfda
13C2-PFDoA
8.13
615.1
570.0
NA
13c2-pfda
13C2-PFTeDA
8.96
715.2
670.0
NA
13c2-pfda
13C3-PFBS
4.78
302.1
79.9
98.9
1802-PFHxS
13C3-PFHxS
6.30
402.1
79.9
98.9
1802-PFHxS
13Cs-PFOS
7.59
507.1
79.9
98.9
13c4-pfos
13C2-4:2FTS
4.67
329.1
80.9
309.0
1802-PFHxS
13C2-6:2FTS
5.82
429.1
80.9
409.0
1802-PFHxS
13C2-8:2FTS
7.28
529.1
80.9
509.0
1802-PFHxS
13Cs-PFOSA
8.41
506.1
77.8
NA
13c4-pfos
Ds-NMeFOSA
9.70
515.0
219.0
NA
13c4-pfos
D5-NEtFOSA
9.94
531.1
219.0
NA
13c4-pfos
Ds-NMeFOSAA
7.51
573.2
419.0
NA
13c4-pfos
D5-NEtFOSAA
7.65
589.2
419.0
NA
13c4-pfos
D7-NMeFOSE
9.56
623.2
58.9
NA
13c4-pfos
Dsi-NEtFOSE
9.83
639.2
58.9
NA
13c4-pfos
13C3-HFPO-DA
4.97
286.9
168.9
184.9
13C2-PFHxA
Non-Extracted Internal Standards
13c3-pfba
1.95
216.0
172.0
NA
13C2-PFHxA
4.80
315.1
270.0
119.4
13c4-pfoa
6.16
417.1
172.0
NA
13c5-pfna
6.99
468.0
423.0
NA
13c2-pfda
7.47
515.1
470.1
NA
1802-PFHxS
6.30
403.0
83.9
NA
13c4-pfos
7.59
502.8
79.9
98.9
1 Times shown are in decimal minute units. Example retention times are based on the instrument operating conditions and
column specified in Section 10.2.
2 For improved accuracy, PFTrDA is quantitated using the average areas of the labeled compounds 13C2-PFTeDA and
13C2-PFDoA.
NA= These analytes do not produce a confirmation ion mass.
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Table 8. QC Acceptance Limits for EIS
Recoveries in Wastewater Samples
EIS Compound
Recovery Range (%)
13c4-pfba
10-130 *
13C5-PFPeA
35 - 150
13C5-PFHxA
55 - 150
13C4-PFHpA
55 - 150
13c8-pfoa
60 - 140
13c9-pfna
55 - 140
13c6-pfda
50 - 140
13C7-PFUnA
30 - 140
13C2-PFDoA
10-150
13C2-PFTeDA
10-130 *
13c3-pfbs
55 - 150
13C3-PFHxS
55 - 150
13c8-pfos
45 - 140
13C2-4:2FTS
60 - 200 *
13C2-6:2FTS
60 - 200 *
13C2-8:2FTS
50 - 200 *
13c8-pfosa
30-130
D3-NMeFOSA
15-130
Ds-NEtFOSA
10-130
D3-NMeFOSAA
45 - 200 *
Ds-NEtFOSAA
10 - 200
D-NMcFOSE
10-150 *
D9-NEtFOSE
10-150 *
13C3-HFPO-DA
25 - 160
* In the multi-laboratory validation study data for
wastewater matrices, some laboratories had difficulties
achieving EIS recoveries in this range.
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Table 8A. Range of Recoveries for Extracted Internal
Standards (EIS) in the Single-laboratory
Validation Study for Solids and Tissues
EIS Compound
Solid Sample
Recovery (%)
Tissue Sample
Recovery (%)
Min
Max
Min
Max
13c4-pfba
3
113
84
99
13C5-PFPeA
28
112
86
107
13C5-PFHxA
79
110
92
95
13C4-PFHpA
73
111
80
93
13c8-pfoa
86
115
90
95
13c9-pfna
87
110
90
98
13c6-pfda
87
112
83
97
13C7-PFUnA
66
124
71
91
13C2-PFDoA
26
109
54
96
13C2-PFTeDA
18
110
31
102
13c3-pfbs
89
120
89
98
13C3-PFHxS
87
110
98
99
13c8-pfos
79
113
92
103
13C2-4:2FTS
95
248
192
215
13C2-6:2FTS
76
127
145
230
13C2-8:2FTS
86
173
136
220
13c8-pfosa
61
123
87
96
D3-NMeFOSA
28
86
8
38
Ds-NEtFOSA
21
70
8
30
D3-NMeFOSAA
52
142
106
139
Ds-NEtFOSAA
68
151
79
151
D-NMcFOSE
13
107
5
30
D9-NEtFOSE
16
97
0
29
13C3-HFPO-DA
70
119
93
102
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Table 9. Summary of Quality Control
Method Reference
Requirement
Specification and Frequency
Section 10.1
Mass Calibration
Annually and on as-needed basis
Section 10.1.7
Mass Calibration Verification
After mass calibration
Section 10.3
Initial Calibration (ICAL)
Minimum 6 calibration standards for linear model
and 7 calibration standards for non-linear models.
Sections 10.2.2, 14.4
Retention Time (RT) window
After ICAL and at the beginning of analytical
sequence
Sections 7.3.1, 9.4
Extracted Internal Standard (EIS)
Analytes
All CAL standards, batch QC and field samples
Sections 7.3.2
Non-extracted Internal Standards
(NIS)
All CAL standards, batch QC and field samples
Sections 7.3.4, 10.3.1,
13.3
Instrument Sensitivity Check (ISC)
Daily, prior to analysis
Section 14.3
Calibration Verification (CV)
At the beginning of the analytical sequence
(except for sample analyzed immediately after an
initial calibration) and every 10 field sample
injections
Section 14.6
Instrument Blank
Daily prior to analysis and after high standards
Sections 9.1.3, 9.5, 14.7
Method Blank (MB)
One per preparation batch
Section 14.5
Ongoing Precision Recovery
(OPR)
One per preparation batch
Section 11.0
Limit of Quantitation Verification
(LLOPR)
One per preparation batch
Section 11.0
Matrix Spike (MS/MSD)
One per preparation batch (if required)
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Table 10. Range of Recoveries for Non-Extracted Internal Standards in the Single-laboratory Validation
Study, by Matrix
NIS Compounds
Aqueous
Solid
Tissue
% Recovery
RSD
(%)
% Recovery
RSD
(%)
% Recovery
RSD
(%)
Min
Max
Min
Max
Min
Max
13c3-pfba
60
91
10.3
54
89
6.4
51
82
7.0
13C2-PFHxA
43
94
18.6
52
90
7.4
41
80
19.3
13c4-pfoa
59
87
9.7
54
89
6.4
51
82
9.5
13c5-pfna
64
87
7.5
59
94
7.1
52
88
11.2
13c2-pfda
57
86
10.0
55
91
8.6
47
85
19.4
1802-PFHxS
59
87
9.6
53
87
7.1
51
80
8.1
13c4-pfos
60
82
7.5
58
86
7.0
52
85
10.3
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21.0 Glossary
These definitions and purposes are specific to this method, but have been conformed to common usage to
the extent possible.
21.1 Units of weight and measure and their abbreviations
21.1.1 Symbols
°c
degrees Celsius
Da
Dalton (equivalent to "amu" below)
Mg
microgram
(iL
microliter
(im
micrometer
<
less than
<
less than or equal
>
greater than
>
greater than or equal
0/
/O
percent
lb
plus or minus
21.1.2 Alphabetical abbreviations
amu
atomic mass unit (equivalent to Dalton)
cm
centimeter
g
gram
h
hour
L
liter
M
molar
mg
milligram
min
minute
mL
milliliter
mm
millimeter
cm
centimeter
m/z
mass-to-charge ratio
ng
nanogram
Qi
quantitation ion
Q2
confirmation ion
rpm
revolutions per minute
v/v
percent volume per volume
21.2 Definitions and acronyms (in alphabetical order)
Analyte - A PFAS compound included in this method. The analytes are listed in Table 1.
Calibration standard (CS) - A solution prepared from a secondary standard and/or stock
solutions and used to calibrate the response of the LC-MS/MS instrument.
Calibration verification standard (CV) - The mid-point calibration standard (CS-4) that is used
to verify calibration. See Table 4.
CFR - Code of Federal Regulations
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Compound - One of many variants or configurations of a common chemical structure.
Individual compounds are identified by the number of carbon atoms and functional group
attached at the end of the chain.
Confirmation Ion - For the purpose of this method, the confirmation ion is produced by
collisionally activated dissociation of a precursor ion to produce distinctive ions of smaller m/z
than the precursor.
Class A glassware - Volumetric glassware that provides the highest accuracy. Class A
volumetric glassware complies with the Class A tolerances defined in ASTM E694, must be
permanently labeled as Class A, and is supplied with a serialized certificate of precision.
CWA - Clean Water Act
Extracted internal standard (EIS) - An isotopically labeled analog of a target analyte that is
structurally identical to a native (unlabeled) analyte. The EISs are added to the sample at the
beginning of the sample preparation process and are used to quantify the native target analytes.
Extracted internal standard (EIS) quantification - The process of determining the
concentration of the native target analyte by its comparing response to the response of a
structurally related isotopically labeled analog that was added to the sample at the beginning of
the sample preparation process.
LC - Liquid chromatograph or liquid chromatography
Instrument sensitivity check - solution used to check the sensitivity of the instrument. The
solution contains the native compounds at the concentration of the LOQ.
Internal standard - A labeled compound used as a reference for quantitation of other labeled
compounds and for quantitation of native PFAS compounds other than the compound of which it
is a labeled analog. See Internal standard quantitation.
Internal standard quantitation - A means of determining the concentration of (1) a 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 (ID) quantitation - A means of determining a native compound by reference to
the same compound in which one or more atoms has been isotopically enriched. The labeled
PFAS are spiked into each sample and allow identification and correction of the concentration of
the native compounds in the analytical process.
Isotopically labeled compound - An analog of a target analyte in the method which has been
synthesized with one or more atoms in the structure replaced by a stable (non-radioactive) isotope
of that atom. Common stable isotopes used are 13C (Carbon-13) or Deuterium (D or 2H). These
labeled compounds do not occur in nature, so they can be used for isotope dilution quantification
or other method-specific purposes.
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Limit of Quantitation (LOQ) - The smallest concentration that produces a quantitative result
with known and recorded precision and bias. The LOQ shall be set at or above the concentration
of the lowest initial calibration standard (the lowest calibration standard must fall within the
linear range).
Low-level OPR (LLOPR) - A version of the ongoing precision and recovery standard that is
spiked at twice the concentration of the laboratory's LOQ and used as a routine check of
instrument sensitivity.
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 labeled
compounds that are used with samples. The method blank is used to determine if analytes or
interferences are present in the laboratory environment, the reagents, or the apparatus.
Method Detection Limit (MDL) - The minimum measured concentration of a substance that
can be reported with 99% confidence that the measured analyte concentration is distinguishable
from method blank results (40 CFR 136, Appendix B).
MESA - Mining Enforcement and Safety Administration
Minimum level of quantitation (ML) - The lowest level at which the entire analytical system
must give a recognizable signal and acceptable calibration point for the analyte. The ML
represents the lowest concentration at which an analyte can be measured with a known level of
confidence. It may be equivalent to the concentration of the lowest calibration standard,
assuming that all method-specified sample weights, volumes, and cleanup procedures have been
employed. Alternatively, the ML may be established by multiplying the MDL (pooled or
unpooled, as appropriate) by 3.18 and rounding the result to the number nearest to 1, 2, or 5 x 10n,
where n is zero or an integer (see 68 FR 11770).
MS - Mass spectrometer or mass spectrometry
Matrix Spike/Matrix Spike Duplicate (MS/MSD) - Aliquots of field samples that have been
fortified with a known concentration of target compounds, prior to sample preparation and
extraction, and analyzed to measure the effect of matrix interferences. The use of MS/MSD
samples is generally not required in isotope dilution methods because the labeled compounds
added to every sample provide more performance data than spiking a single sample in each
preparation batch.
Multiple reaction monitoring (MRM) - Also known as selected reaction monitoring (SRM). A
type of mass spectrometry where a parent mass of the compound is fragmented through MS/MS
and then specifically monitored for a single fragment ion.
Must - This action, activity, or procedural step is required.
NIOSH - The National Institute of Occupational Safety and Health
Non-extracted internal standard (NIS) -Labeled PFAS compounds spiked into the
concentrated extract immediately prior to injection of an aliquot of the extract into the LC-
MS/MS.
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
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the results produced by the laboratory remain within the limits specified in this method for
precision and recovery.
PFAS - Per- and Polyfluoroalkyl substances -A group of man-made fluorinated compounds that
are hydrophobic and lipophobic, manufactured and used in a variety of industries globally. These
compounds are persistent in the environment as well as in the human body. This method
analyzes for the PFAS listed in Table 1.
Precursor Ion - For the purpose of this method, the precursor ion is the deprotonated molecule
([M-H]-) of the method analyte. In MS/MS, the precursor ion is mass selected and fragmented by
collisionally activated dissociation to produce distinctive product ions of smaller m/z. Also called
a parent ion.
Product Ion - For the purpose of this method, a product ion is a charged fragment ion that is
formed as the product of collisionally activated dissociation of a particular precursor ion. Also
called a transition or transition ion.
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 multiplied by 100 and divided by
the mean. Also termed "coefficient of variation."
Relative Standard Error (RSE) - The standard error of the mean divided by the mean and
multiplied by 100.
RF - Response factor. See Section 10.3.3.2.
RR- Relative response. See Section 10.3.3.2.
RT - Retention time; the time it takes for an analyte or labeled compound to elute off the
HPLC/UPLC column
Should - This action, activity, or procedural step is suggested but not required.
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 mean height of the noise.
SPE - Solid-phase extraction; a technique in which an analyte is extracted from an aqueous
solution or a solid/tissue extract 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, NIST, or a source that will attest to the purity and authenticity of the reference
material.
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Appendix A - Sample Pre-screening Instructions
Samples that are known or suspected to contain high levels of analytes may be pre-screened using the
following procedure. These are example procedures using smaller sample aliquots spiked with EIS and
NIS and no clean up procedures. Other pre-screening procedures may be used. The results of the pre-
screening should be used by the analyst to assess the need for sample or extract dilutions necessary to
keep the target analytes within the calibration range of the instrument. The results may also be used to
reduce the risk of prevent gross contamination of the instrument when dealing with unfamiliar sources of
samples.
Aqueous Samples
1. Weight out 10 (±0.1) g of sample into a 50-mL centrifuge tube.
2. Add 50 (iL of EIS and NIS to the sample and vortex to mix.
3. Filter 1 mL of the sample through 0.2-f.im membrane filter into a microvial. Sample is ready for
instrumental analysis.
Solid and Tissue Samples
1. Weigh 1.0 (±0.1) g sample into 50-mL polypropylene centrifuge tubes.
2. Add 20 mL of 0.3% methanolic ammonium hydroxide (Section 7.1.7.1). Vortex and mix on a shaker
table (or equivalent) for 10 min. Allow to settle and/or centrifuge to produce a clear extract.
3. Filter using a Single Step® filter vial:
a. Add 20 |_iL of EIS to a clean Single Step® filter vial (chamber).
b. Add 400 |_iL of clear extract from step 2 (e.g., by adding extract until it reaches the fill line),
carefully vortex to mix.
c. Use filter/plunger part and filter.
4. Transfer 30 |_iL of filtrate to a ~300-f.iL polypropylene micro-vial and dilute to 300 |_iL with 0.3%
methanolic ammonium hydroxide (Section 7.1.7.1). Add NIS to the filtrate.
5. The extract is now a lOx dilution.
6. Sample is ready for instrumental analysis.
Calculate results using the equivalent sample weight computed as follows:
0.4 mL
Equivalent Weight = Sample weight (g) x ——-
La U /TIL
Note that the EIS concentration in the diluted portion is 0.5x the level in the regular analysis of solid
samples.
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Appendix B - Aqueous Sample Subsampling Instructions
Warning: Because some target analytes may be stratified within the sample (e.g., AFFF-
contaminated media, surfactants), or adhere to the walls of the sample container,
subsampling may only be done on a project-specific basis. Subsampling has been shown
to increase uncertainty in PFAS analysis, especially on foaming samples.
If a reduced sample size is required, transfer a weighed subsample using the following subsampling
procedure to a 60-mL HDPE bottle and dilute to approximately 60 mL using reagent water. This
container is now considered the "sample bottle."
1. Gently invert sample 3-4 times being careful to avoid foam formation and subsample immediately (do
not let stand).
2. If foam forms and more than 5 mL is required - pour sample, avoiding any foam.
3. If foaming forms and a volume less than 5 mL is required - pipette from cm below the foam.
4. If no foam forms - pour or pipette based on volume required.
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