£% United States
Environmental Protectio
^1 M^k. Agency
Office of Water
www.epa.gov June 2022
2nd 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-22-001
2nd Draft of Method 1633 - subject to further revision
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2nd Draft of Method 1633
Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in
Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS
June 2022
Notice
This document represents a draft of a PFAS method currently under development by the EPA Office
of Water, Engineering and Analysis Division (EAD), in conjunction with the Department of Defense
(DoD). This method is not required for Clean Water Act compliance monitoring until it has
been proposed and promulgated through rulemaking.
A single-laboratory validation of the procedure has been completed and the report on the results of
that study is being prepared. Historically, EAD posts draft methods on the Clean Water Act website
after the single-laboratory validation report is completed. However, due to a large number of public
and stakeholder requests, this method is being posted on the web before the single-laboratory
validation study report is finalized. A revision of this draft method with a later publication date may
be issued at that time. No procedural changes are expected as a result of the single-laboratory
validation, but some of the performance data (which are presented only as examples) may change
once the statistical analysis of the single-laboratory validation data is completed.
This draft method has been subjected to multiple levels of review across several EPA Program
Offices. DoD began a multi-laboratory validation study of the procedure in late 2021, in
collaboration with the Office of Water and the Office of Land and Emergency Management.
The Office of Water will use the results of the multi-laboratory validation study to finalize the method
and add formal performance criteria. The method validation process may eliminate some of the
parameters listed in this draft method.
In September 2021, the Office of Water released a draft of the method on its web site and encouraged
laboratories, regulatory authorities, and other interested parties to review the method, provide
feedback and comments to the Office of Water, and where appropriate, utilize it for their own
purposes, with the explicit understanding that method was a draft, subject to revision.
Partly as a result of such reviews and comments, as well as questions raised to DoD during the multi-
laboratory validation study, the Office of Water addressed some errors and less-than-clear aspects of
the method in an errata sheet that was posted on its web site. This 2nd draft of the method
incorporates the items from that errata sheet and a few other changes into one document.
The changes are indicated by the combination of red font and yellow highlighting.
2nd Draft of Method 1633 - subject to further revision
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Acknowledgements
This draft 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
draft 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
Ally son 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
Harrry 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
http s: //www. epa.gov/cwa-methods
http s: //www. epa.gov/cwa-methods/forms/contact-us -about-cwa-analytical-methods
2nd Draft of Method 1633 - subject to further revision
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Revision History
June 2022, 2nd Draft
This 2nd draft of Method 1633 incorporates the following changes presented in the errata sheet prepared
by the Office of Water in October 2021 and updated in February 2022, along with other changes in
response to questions raised during the early stages of the multi-laboratory validation study.
Location Rationale for the change
4.2.2 Some SPE manifold components may be too large to fit in commonly used ultrasonic baths.
Where size is not a concern, sonication is encouraged as an effective approach to cleaning
these parts.
6.3.4 The description of the blade and shaft of the originally listed device is incorrect, it has a
PTFE bearing that makes it inappropriate for use in a PFAS method. Use of the device itself
is never called out in the procedure after this section. Therefore, the description was deleted,
but the subsection number has been retained for the time being to avoid renumbering any
subsequent sections until the final version of the method is prepared.
6.9.3 Since the initial release of the draft method, the supplier has discontinued the snap cap that
was cited. The replacement shown is currently available and does not contain PTFE.
7.1.2 Erroneous description of the use of the solution was deleted.
7.1.9 Description of the use of the solution was added.
7.3.5 New "Note" regarding the standards was added.
7.4 The description of the mass calibration solution was made vendor agnostic in keeping with
changes to Section 10.1.
7.5 Naming this solution the bile salt interference check standard simplifies later discussions of
its use. The potential interference with PFOS from bile salts is affected by the mobile phase
used for the LC separation and laboratories choosing to use a mobile phase other than that
specified in the draft procedure need to make the adjustments described here. The
concentration of the solution also has been lowered so it does not overwhelm the PFOS peak.
8.2.3
The change addresses the discrepancy between Sections 8.2 and 8.5.
8.3.2
The change addresses the discrepancy between Sections 8.2 and 8.5.
9.1.2.2 (c) Section 1.6 does not exist in the draft method, and Section 1.5 is the correct citation.
9.2.2 Responds to questions about the example values in Table 6. Also see the discussion of Table
6 below.
10.1 A large portion of the description of the mass calibration process in Section 10.1 and all of its
subsections was revised in response to comments from some of the laboratories in the
validation study, relative to the existing mass calibration procedures from some instrument
manufacturers. The changes make the procedure more vendor-agnostic, while still
accomplishing the intended goal.
10.2.2.5 Naming this solution the bile salt interference check standard also simplifies discussions of its
use. The potential interference with PFOS from bile salts is affected by the mobile phase
used for the LC separation and laboratories choosing to use a mobile phase other than that
specified in the draft procedure need to make the adjustments described here. Also added the
requirement to run the bile salt interference check standard when initially establishing the
chromatography conditions, regardless of sample matrices.
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10.3.3.4 This new subsection was added to address questions about monitoring the areas of the non-
extracted internal standards in each sample.
10.3.5 Naming this solution the bile salt interference check standard simplifies these discussions of
its use. Added the requirement to run the bile salt interference check standard when initially
establishing the chromatography conditions, regardless of sample matrices.
11.2.5 Clarified that Section 11.2 addresses processing of aqueous samples, not extracts.
11.3.8 Based on questions raised by the laboratories in the DoD multi-laboratory validation study
and alternatives supported by data, the discussion of the dilution of solid sample extracts was
revised to clarify that flexibility in the specifics is allowed.
11.3.9 Additional discussion of the extract concentration procedure added in keeping with the
changes in Subsection 11.3.8.
13.1
Making the discussion more generic in the context of the bile salt interference check changes
noted above.
13.3, #9
Making the discussion more generic in the context of the bile salt interference check changes
noted above.
14.3.5
This new subsection was added to more accurately describe the calculation of ion abundance
ratios for the target analytes.
14.9
In conjunction with the addition of 10.3.3.4, additional material and an equation were added
here to address questions about monitoring the areas of the non-extracted internal standards in
each sample.
15.1
There is no subsection 15.1.5 in the draft method and subsection 15.1.4 is the correct citation.
15.1.3 This subsection was revised in conjunction with the addition of Section 14.3.5 to address ion
abundance ratios.
15.1.4 The requirements in subsection 15.1.1 were omitted from the original text.
15.3.1 The incorrect dilution solvent was called out in the first draft procedure. Also, the
explanation of how to deal with quantification of the analytes in diluted extracts was not clear
nor correct. Finally, the changes address samples where the diluted extract analysis does not
meet the requirements. This becomes two new paragraphs within Section 15.3.1.
15.4.2.4 Responds to questions from laboratories.
19.10 The placeholder for the report on the single-laboratory method validation study was replaced
with the actual citation.
Table 1
13C2-4:2FTS The full name of this labeled EIS compound was corrected.
Table 2
PFHxS
The quantitation and confirmation
ions for this analyte were reversed in the table.
13C3-PFHxS
The quantitation and confirmation
ions for this analyte were reversed in the table.
13c8-pfos
The quantitation and confirmation
ions for this analyte were reversed in the table.
13C3-HFPO-DA The parent ion mass was incorrect in the table.
Notes A new note was added to explain the ""NA" entries in this table
Table 6 Commenters noted that they could not reproduce the Minimum Level (ML) values in the
table from the details in the method. An explanatory footnote was added below the table.
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Table 7 The mass calibration verification citation was updated to reflect the changes to Section
10.1 described above.
Glossary Signal-to-noise ratio - The definition was corrected to refer to measuring the mean height
of the noise, not the width.
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Table of Contents
Acknowledgements i
Disclaimer i
Contact i
Revision History ii
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 3
6.0 Equipment and Supplies 5
7.0 Reagents and Standards 8
8.0 Sample Collection, Preservation, Storage, and Holding Times 13
9.0 Quality Control 14
10.0 Calibration and Standardization 19
II.0 Sample Preparation and Extraction 25
12.0 Extraction, Cleanup, and Concentration 30
13.0 Instrumental Analysis 32
14.0 Performance Tests during Routine Operations 33
15.0 Data Analysis and Calculations 36
16.0 Method Performance 39
17.0 Pollution Prevention 40
18.0 Waste Management 40
19.0 References 40
20.0 Tables, Diagrams, Flowcharts, and Validation Data 42
21.0 Glossary 56
Appendix A - Sample Pre-screening Instructions 60
Appendix B - Aqueous Sample Subsampling Instructions 61
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2nd DRAFT 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 analyte consisting of the sum of the linear and branched isomer concentrations.
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. Changes outside the scope
of 40 CFR Part 136.6 and Section 9.0 of this method may require prior review or approval.
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.
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.
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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 8). 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 Results for target analytes are recovery corrected by the method of quantification (i.e., either
isotope dilution or extracted internal standard quantification, see Section 10.3). 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
and may be used without cleaning if PFAS levels are 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.
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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 dichloromethane.
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 on a given analytical batch 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 but should
contain potential interferents in the concentrations expected to be found in the samples to
be analyzed.
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, when analyzing whole fish samples, bile salts (e.g.,
Taurodeoxycholic Acid [TDCA]) 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).
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.
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.
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5.1.1 PFOA has been described as likely to be carcinogenic to humans. Pure standards 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. It is also suggested that the laboratory perform personal
hygiene monitoring of each analyst who uses this method and that the results of this monitoring be
made available to the analyst. Additional information on laboratory safety can be found in
References 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 air flow. Gross losses to the laboratory ventilation
system must not be allowed. Handling of the dilute solutions normally used in analytical
and animal work presents no inhalation hazards except in the case of an accident.
5.3.2 Protective equipment - Disposable plastic gloves, apron or lab coat, safety glasses or mask,
and a glove box or fume hood adequate for radioactive work should be used. During
analytical operations that may give rise to aerosols or dusts, personnel should wear
respirators equipped with activated carbon filters. Eye protection (preferably full-face
shields) must be worn while working with exposed samples or pure analytical standards.
Latex gloves are commonly used to reduce exposure of the hands.
5.3.3 Training - Workers must be trained in the proper method of removing contaminated gloves
and clothing without contacting the exterior surfaces.
5.3.4 Personal hygiene - Hands and forearms should be washed thoroughly after each
manipulation and before breaks (coffee, lunch, and shift).
5.3.5 Confinement - Isolated work areas posted with signs, segregated glassware and tools, and
plastic absorbent paper on bench tops will aid in confining contamination.
5.3.6 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.
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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'.
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.
6.1.1.1 Liquid samples (waters, sludges, and similar materials containing < 50 mg
solids per sample) - Sample bottle, HDPE, with linerless HDPE or
polypropylene caps.
Note: At least two aliquots of aqueous samples are collected to allow sufficient volume
for the determination of percent solids and for pre-screening analysis. One
aliquot should be collected in a 500-mL container while the second aliquot may
be collected in a smaller sample container (e.g.. 250-mL or 125-mL).
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 Compositing equipment - Automatic or manual compositing system incorporating
containers cleaned per bottle cleaning procedure above. 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, the tubing must be thoroughly rinsed
with methanol, followed by repeated rinsing with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
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6.2 Equipment for glassware cleaning
Note: If blanks from bottles or 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 Text deleted, but Section number retained for the time being to avoid other numbering
changes
6.3.5 Meat grinder - Hobart, or equivalent, with 3- to 5-mm holes in inner plate
6.3.6 Equipment for determining percent moisture
6.3.6.1 Oven - Capable of maintaining a temperature of 110 ± 5 °C
6.3.6.2 Desiccator
6.3.7 Balances
6.3.7.1 Analytical - Capable of weighing 0.1 mg
6.3.7.2 Top loading - Capable of weighing 10 mg
6.3.8 Aluminum foil
6.3.9 Disposable spoons, 10 mg, polypropylene or stainless steel
6.3.10 Ultrasonic mixer (sonicator)
6.3.11 HDPE bottles, with linerless HDPE or polypropylene caps - 60 mL
6.3.12 pH Paper, range 0-14 - (Whatman® Panpeha™ or equivalent), 0.5-unit readability
6.3.13 Analog or digital vortex mixer, single or multi-tube (Fisher Scientific 02-215-452, or
equivalent)
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6.3.14 Volumetric flasks, Class A
6.3.15 Disposable polypropylene collection tubes (13 x 100 mm, 8 mL)
6.3.16 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-f.im Nylon membrane, PALL/Acrodisc or equivalent
6.4.3 Glass fiber filter, 47 mm, 1 |_im. PALL A/E or equivalent
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.
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)
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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 (mi 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)
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)
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.
7.1.1 Acetic acid - 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
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7.1.5 Ammonium hydroxide - certified ACS+ grade or equivalent, 30% in water, store at room
temperature
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%) - 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%) - 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%) - 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 - 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
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
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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 will be used for the multi-laboratory validation to
set statistically based method criteria. Once the method is multi-laboratory validated,
laboratories will have the flexibility to use carbon cartridges, as long as all method OC
criteria are met.
7.1.18 Toluene - HPLC grade, verified by lot number before use. Store at room temperature.
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. 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 4 °C unless the vendor
recommends otherwise in screw-capped vials with foiled-lined caps. Place a mark on the vial at the
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level of the solution so that solvent loss by evaporation can be detected. Replace 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.
lsO-mass labeledperfluoroalkyl sulfonates may undergo isotopic exchange with water under
certain conditions, which lowers the isotopic purity of the standards over time.
The laboratory must maintain records of the certificates for all 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.
7.3.1 Extracted Internal Standard (EIS) - (a.k.a. isotopically labeled compound) Prepare the EIS
solution containing the isotopically labeled compounds listed in Table 3 as extracted
internal standards in methanol from prime stocks. An aliquot of EIS solution, typically 50
l_iL. is added to each sample prior to extraction. Table 3 presents the nominal amounts of
EIS compounds added to each sample. The list of isotopically labeled compounds in Table
3 represents the compounds that were available at the time this method was validated.
Other isotopically labeled compounds may be used as they become 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.
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, NMeFOSAA, and NEtFOSAA.
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, methanolic
ammonium hydroxide (2%), water, acetic acid and the method analyte and isotopically
labeled compound standard solutions. After dilution, the final solution will match the
solvent mix of sample extracts, which contain methanol with 4% water, 1% ammonium
hydroxide and 0.625% acetic acid (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
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quantitation range (e.g., from the 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 quantitation range.
The lowest level calibration standard must meet a signal-to-noise ratio of 3:1 and be at a
concentration less than or equal to the Limit of Quantitation (LOQ). All initial calibration
requirements listed in Table 7 must be met. An instrument sensitivity check (ISC) standard
at the concentration of the lowest calibration standard within the quantitation range is
required to be analyzed at the beginning of the analytical run (Section 10.3.3.1 and Section
13.3). A mid-level calibration solution is analyzed at least every ten samples or less, on an
ongoing basis for the purpose of calibration verification. A mid-level calibration
verification (CV) standard must also be analyzed after all sample analyses in order to
bracket the analytical batch.
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/'N ratio criterion of 3:1, 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,
PFNA, PFOSA, NMeFOSA, NEtFOSA, NEtFOSE, and NMeFOSE.
Note: During the multi-laboratory validation study, laboratories reported that NMeFOSA was an
impurity in the branched isomer qualitative standard for NMeFOSE and NEtFOSA was an
impurity in the branched isomer qualitative standard for NEtFOSE supplied for the study.
Those impurities did not preclude the use of these standards, but laboratories should be
aware of the possibility.
7.3.6 Instrument Blank - During the analysis of a batch of samples, a solvent blank is analyzed
after samples containing high level of target compounds (e.g., calibration, CV) to monitor
carryover from the previous injection. The injection 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 that will attest to the
authenticity and 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 tissue
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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 and analyzing tissues, it will be necessary to evaluate taurochenodeoxycholic acid (TCDCA)
and tauroursodeoxycholic acid (TUDCA) as well.
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 Samples that flow freely are collected as grab samples or in refrigerated bottles using
automatic sampling equipment. Collect 500 mL of sample (other than leachates) in an
HDPE bottle. Do not fill the bottle past the shoulder, to allow room for expansion during
frozen storage.
Note: Collect at least two aliquots of all aqueous samples to allow sufficient volume for the
determination of percent solids and for pre-screening analysis. That second aliquot may be
collected in a smaller sample container (e.g., 250-mL or 125-mL).
Because the target analvtes 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.
Leachate samples from landfills can present significant challenges and therefore only 100
mL of sample is collected for the analysis. Collect two 100-mL leachate sample aliquots in
a similar manner as described in Section 8.2.1, using appropriately sized containers.
Maintain all aqueous samples protected from light at 0 - 6 °C from the time of collection
until shipped to the laboratory. Samples must be shipped as soon as practical with
sufficient ice to maintain the sample temperature below 6 °C during transport and be
received by the laboratory within 48 hours of collection. 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 < -20 °C, or at 0 - 6 °C, until sample preparation. However, the
allowable holding time for samples depends on the storage temperature, as described in
Section 8.5.
(soil, sediment, biosolids), excluding tissue
Collect samples as grab samples using wide-mouth jars and fill no more than % full (see
Section 6.1.1.2 for container size and type).
Maintain solid samples protected from light (in HDPE containers) at 0 - 6 °C from the time
of collection until receipt at the laboratory. 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 < -20 °C or at 0 - 6 °C, until sample preparation. However, the allowable holding
time for samples depends on the storage temperature, as described in Section 8.5.
8.2.2
8.2.3
8.3 Solid
8.3.1
8.3.2
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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 90 days from collection, when stored at
< -20 °C and protected from the light. When stored at 0 - 6 °C and protected from the light,
aqueous samples may be held for up to 28 days, with the caveat that issues were 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.
8.5.2 Solid samples (soils and sediments) and tissue 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.
8.5.3 Biosolids samples may be held for up to 90 days, if stored by the laboratory in the dark at
0 - 6 °C or at -20 °C. Because microbiological activity in 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.
8.5.4 Store sample extracts in the dark at less than 0 - 4 °C until analyzed. If stored in the dark at
less than 0 - 4 °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. 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
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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., soils, biosolids, tissue), 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.
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 equivalent or superior to results produced by this
method as written, that modification may be used routinely thereafter, so long as
the other requirements in this method are met (e.g., isotopically labeled
compound recovery).
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:
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)
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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. Final extract volume prior to injection (Section 12)
ix. Injection volume (Section 13.3)
x. Dilution data, differentiating between dilution of a sample or an extract
(Section 15.3)
xi. Instrument
xii. Column (dimensions, liquid phase, solid support, film thickness, etc.)
xiii. Operating conditions (temperatures, temperature program, flow rates)
xiv. Detector (type, operating conditions, etc.)
xv. Chromatograms, printer tapes, and other recordings of raw data
xvi. 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 elation 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
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 14.1 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.
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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 ofNIS
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. If RSD and R for all compounds meet the acceptance criteria, system
performance is acceptable, and analysis of blanks and samples may begin. If,
however, any individual RSD exceeds the precision limit or any individual 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
analytes using the MDL procedure at 40 CFR Part 136, Appendix B. An MDL
determination must be performed for all compounds. 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.
9.3 To assess method performance on the sample matrix, the laboratory must spike all samples with the
isotopically labeled compound 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 isotopically labeled compound 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 isotopically labeled compound must be within the limits in Tables 9
and 10 (once the tables are finalized). If the recovery of any compound falls outside of
these limits, method performance is unacceptable for that compound in that sample.
Additional cleanup procedures must then be employed to attempt to bring the recovery
within the normal range. If the recovery cannot be brought within the normal range after
all cleanup procedures have been employed, water samples are diluted, and smaller
amounts of soils, biosolids, sediments, and other matrices are prepared and analyzed, per
Section 15.3.
9.4 Recovery of isotopically labeled compounds from samples must also be assessed and records
maintained.
9.4.1 After the analysis of 30 samples of a given matrix type (water, soil, biosolids, tissues, etc.)
for which the isotopically labeled compounds pass the tests in Section 9.3, compute the R
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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 five 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 measurements).
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 matrix 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
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 (Sections 14.2 and 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 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and spiked samples may be required to determine the accuracy
of the analysis when the extracted internal standard method is used.
9.8 Matrix spikes generally are not required for isotope dilution methods because any deleterious
effects of the matrix should be evident in the recoveries of the isotopically labeled compounds
spiked into every sample. However, because some of the compounds are quantified by a non-
analogous isotopically labeled compounds (e.g., PFPeS is quantified by 13C3-PFHxS), the analysis
of matrix spike samples may help diagnose matrix interferences for specific compounds.
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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 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 single-laboratory validation of this method are listed in Table 2 for each
native analyte, EIS, and NIS.
10.1.1 During the development of this method, instrumental parameters were optimized for the
precursor and product ions listed on Table 2. Product ions other than those listed may be
selected; however, the use of ions with lower mass 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
MS/MS
Conditions
(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/li) ~70
Desolvation gas (L/h) -800
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.
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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 2-5 (ig/mL
of 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.
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 CAL standard under LC-MS/MS conditions to obtain the
retention times of each method analyte. Divide the chromatogram into retention
time windows, 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 2, 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,
then the instrument will need to be recalibrated following the manufacturer's
instructions.
10.2 Chromatographic conditions
10.2.1 The chromatographic conditions should be optimized for compound separation and
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.
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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 inL/min
Initial
0.2
2% eluent A, 98% eluent B
0.35 inL/min
2
4.0
30% eluent A, 70% eluent B
0.40 inL/min
7
7.0
55% eluent A, 45% eluent B
0.40 inL/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
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 each method analyte,
EIS analyte, and NIS analyte 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 Method 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. All branched isomer peaks identified in either the calibration
standard or the qualitative (technical grade) standard must fall within in the
retention time window for that analyte.
10.2.2.4 For all method analytes with exact corresponding isotopically labeled analogs,
method analytes must elute within 0.1 minutes of the associated EIS.
10.2.2.5 When establishing the chromatographic conditions, it is important to consider the
potential interference of bile salts during analyses of tissue samples. Inject the
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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 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. This
evaluation is required when establishing the chromatographic conditions for the
method, 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. (If a second-order calibration model is used,
then one additional concentration is required.) The initial calibration solutions contain the entire
suite of isotopically labeled compounds, NISs, and target compounds. Calibration is verified with a
calibration verification (CV) standard at least once every ten field samples or less, 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 PFAS 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 corrective
action that 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 OC samples.
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10.3.3 Initial calibration calculations
10.3.3.1 Instrument sensitivity
Sufficient instrument sensitivity is established if a signal-to-noise ratio >3:1 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 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 2. RR is used for the 24 compounds quantified by true isotope dilution.
Area,, M,
RR =
Areai Mn
where:
Arean = The measured area of the Q1 m/z for the native (unlabeled) PFAS
Areai = The measured area at the Q1 m/z for the corresponding isotopically
labeled PFAS added to the sample before extraction
Mi = The mass of the isotopically labeled compound in the calibration
standard
Mn = The mass of the native compound in the calibration standard
Similarly, the response factor (RF) for each unlabeled compound calibrated by
extracted internal standard is calculated according to the equation below. RF is
used for the 16 compounds quantified by extracted internal standard.
Rp = Areas MEIS
AreaEIS Ms
where:
Areas = The measured area of the Q1 m/z for the target (unlabeled) PFAS
Areanis = The measured area at the Q1 m/z for the isotopically labeled PFAS
used as the extracted internal standard (EIS)
Meis = The mass of the isotopically labeled PFAS used as the extracted
internal standard (EIS) in the calibration standard
Ms = The mass of the target (unlabeled) PFAS in the calibration standard
A response factor (RFS) is calculated for each isotopically labeled compound in
the calibration standard using the equation below. RFS is used for the 24
isotopically labeled compounds quantified by non-extracted internal standard.
Are^ Mnis
RFS =
AreaNIS M,
where:
Areai = The measured area of the Q1 m/z for the isotopically labeled PFAS
standard added to the sample before extraction
AreaNis = The measured area at the Q1 m/z for the isotopically labeled PFAS
used as the non-extracted internal standard (NIS)
Mnis = The mass of the isotopically labeled compound used as the
non-extracted internal standard (NIS) in the calibration standard
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Mi = The mass of the isotopically labeled PFAS standard added to the
sample before extraction
Note: Other calculation approaches may be used, such as linear regression or non-linear
regression, based on the capability of the data system used by the laboratory'.
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 of the six initial calibration standards for each native
compound and isotopically labeled compound. The RSD must be
< 20% to establish instrument linearity.
Option 2: Calculate the relative standard error (RSE) of the six initial calibration
standards for each native compound and isotopically labeled
compound. The RSE for all method analytes must be < 20% to
establish instrument linearity.
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.
2AreaNist
Mean AreaNIS. =
where:
AreaNis; = Area counts for the ith NIS, where i ranges from 1 to 7, for the seven
NIS compounds listed in Table 1
n = 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. All initial
calibration criteria must be met before any samples or required blanks are analyzed.
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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, even if tissue samples
are not going to be run. If an interference is present, the chromatographic conditions must
be modified to eliminate the interference from the bile salts (e.g., changing the retention
time of the bile salts such that they 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. If tissue sample analyses are not being
conducted, this check may be skipped.
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 (Section 11.2), solid (soil, sediment or biosolid) samples
(Section 11.3) and tissue samples (Section 11.4).
Note: It is highly recommended that the laboratory pre-screen all samples prior to performing the
analysis (see Appendix A). For aqueous samples, use the secondary container provided for
percent solids to perform the pre-screening. If high levels ofPFAS are present in the sample, a
lower volume is required for analysis.
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. 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 fhioropolymer articles or task wipes in these extraction procedures. Use only
HDPE or polypropylene wash bottles and centri fuge tubes. Reagents and solvents for cleaning
syringes may be kept in glass containers.
11.1 Determination of percent solids
11.1.1 Determination of percent suspended solids - Aqueous liquids and multi-phase samples
consisting of mainly an aqueous phase
11.1.1.1 Desiccate and weigh a glass fiber filter (Section 6.4.3) to three significant
figures.
11.1.1.2 Filter 10.0 ± 0.02 mL of well-mixed sample through the filter.
11.1.1.3 Dry the filter a minimum of 12 hours at 110 ± 5 °C and cool in a desiccator.
11.1.1.4 Calculate percent solids as follows:
weight of sample aliquot after drying (g) — weight of filter (g)
% solids = x 100
10g
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11.1.2 Solids (excluding tissues)
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.
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 is applicable to aqueous samples containing up to 50 mg of suspended solids per
sample. The procedure requires the preparation of the entire sample. Smaller sample volumes may
be analyzed for samples containing solids greater than specified for this method, or when
unavoidable due to high level of PFAS; however, subsampling should be avoided whenever
possible. Typical sample size is 500 mL; however, sample size may be up to 1000 mL. The
sample is to be analyzed in its entirety and should not be filtered. Leachate samples are analyzed
using a 100-mL sample volume. Therefore, they must not be included in the same sample
preparation batch as aqueous samples analyzed which 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.
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. 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 a
concentration 3 to 5 times the background concentration determined during screening of
the unspiked sample. 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.
11.2.5 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).
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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 method blanks (10% of sample weight or less) can
be added to unusually dry samples. This is an option, not a requirement.
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
included in the same sample preparation batch as solid samples analyzed with 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 for MS/'MSD samples with native standard solution (Section 7.3.3) at the
concentration 3 to 5 times the background concentration determined during screening of
the unspiked sample. 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.
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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.
Using a 10-mg scoop, add 10 mg of carbon (Section 7.1.17) to the combined extract, mix
by occasional hand shaking for no more than five minutes and then centrifuge at 2800 rpm
for 10 minutes. Immediately decant the extract into a 60-mL glass evaporation or
concentrator tube.
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 (a) x Moisture (%)
Water Content in Sample =
p 100
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
< 5 g 7 mL
5 - 8 g 8 mL
8 - 9 g 9 mL
9 - 10 g 10 mL
* 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 highly volatile compounds. The extract must be concentrated to remove the methanol
as excess methanol present during SPE clean-up results in poor recovery of Cn and Cu
carboxylic acids and Cw and Cn sulfonates.
11.3.6
11.3.7
11.3.8
+ any water added in 11.3.2 and 11.3.8
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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 7.1.6.2]). 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 (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 stainless-steel bowl during
grinding, then mixed using a stainless-steel spoon. Homogenized samples must be stored in clean HDPE
containers and stored frozen for subsequent use.
If using a 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.
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 the
concentration 3 to 5 times the background concentration determined during screening of
the unspiked sample. 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 to extract for at least 16 hours.
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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.
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 no more than five minutes and 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 win-
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 sample analysis
(by comparing results when skipping the carbon cleanup during reanalysis), then the carbon
cleanup may be skipped for that speci fic sample.
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 of0.3M formic acid (Section 7.1.13.2) (do
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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.
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 and concentration steps based on the matrix
(see Section 12.2 - Aqueous, Section 12.3 - Solids, and Section 12.4 - Tissue).
12.2 Elution and extract concentration of aqueous samples
Note: If two cartridges were used, perform Sections 12.2.1 through 12.2.3 with each cartridge.
Filter the eluate s through a 25-mm, 0.2-/um syringe filter. Combine both sets of filtered
eluates into a clean tube, add the NIS solution, and vortex to mix. Transfer 350 jj.L of the
filtered extract into a 1-mL polypropylene microvial and mark the level. Add another
350-juL portion and using a gentle stream of nitrogen (water bath at 40 °C), concentrate to
the 350-juL 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 no more than 5 minutes. 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.
12.2.4 Add NIS solution (Section 7.3.2) to a clean collection tube. Place a syringe filter (25-mm
filter, 0.2-f.im 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
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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 - 4 °C.
12.3 Elution and extract concentration 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-f.im 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 microvial
for LC-MS/MS analysis. Cap the collection tube containing the remaining extract and store
at 0 - 4 °C.
12.4 Elution and extract concentration 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-f.im 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 - 4 °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) prior to analyzing samples. If tissue samples are to be
analyzed during the analytical shift, repeat the analysis of the bile salt interference check standard
in Section 10.3.5 before analyzing any tissue samples.
13.2 Only after all performance criteria are met may blanks, MDLs, IPRs/OPRs, and samples be
analyzed.
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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
QCs must be identical to the volume used for calibration (Section 10.2.3). 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) (only required during analytical sequences in
which tissue samples are being analyzed)
10. Samples (10 or fewer)
11. Calibration Verification Standard
12. Instrument Blank
13. Samples (10 or fewer)
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 sample, extracts are 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 that sample from the undiluted
analysis.
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.
14.1 MS resolution - A mass calibration must be performed prior to analysis of the calibration curve.
LC-MS/MS system performance is checked by performing an MS resolution verification after the
mass calibration. MS resolution must be verified prior to any samples or QC as per Section 10.1.
If the requirements in Section 10.1 cannot be met, the problem must be corrected before analyses
can proceed. If any of the samples in the previous shift may be affected by poor mass resolution,
the extracts of those samples must be re-analyzed.
14.2 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.
If the requirements cannot be met, the problem must be corrected before analyses can proceed.
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Note: An interim limit of 70-130% for 90% of the native and isotopically labeled compounds
should be used, with the other recoveries achieving 50-150%.
14.3 Calibration verification (CV)
After a passing MS resolution (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 and isotopically labeled compounds for the CVs must be within 70 -
130%.
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.
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 2, using the equation below. These ion
abundance ratios will be used a part of the qualitative identification criteria in Section 15.1.
Aream
IAR = —
AreaQ2
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: Six of the analytes in Table 2 do not produce confirmation ions, so the IAR does not
apply-
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.
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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 verification, analyze the extract of the OPR (Sections 12.2.4, 12.3.3, and 12.4.3) 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 isotopically labeled compounds, compare the recovery to the
OPR limits given in Table 5. If all compounds meet the acceptance criteria, system
performance is acceptable, and analysis of blanks and samples may proceed. If, however,
any individual concentration falls outside of the 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 and repeat the ongoing
precision and recovery test.
14.5.4 If desired, add results that pass the specifications in Section 14.5.3 to initial and previous
ongoing data for each compound in each matrix. Update QC charts to form a graphic
representation of continued laboratory performance. Develop a statement of laboratory
accuracy for each compound in each matrix type by calculating the average percent
recovery (R) and the standard deviation of percent recovery (SR). Express the accuracy as
a recovery interval from R - 2SR to R + 2SR. For example, if R = 95% and SR = 5%, the
accuracy is 85 to 105%.
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.
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 A qualitative identification standard (Section 7.3.5) containing all available isomers (branched and
linear) is analyzed 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 does not meet criteria. 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.
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Area Ratio
'NISj
= 100 x
where:
Area of NIS, in the Sample
Mean AreaNisi
/ =
Area ofNISi in the Sample
Mean AreaNIS.
Observed area counts for NIS, in the sample
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 should be within 50 - 200% of the mean area of
that NIS in the initial calibration standards. 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 Peak responses must be at least three times the background noise level (S/N 3:1). If the
S/N ratio is not met due to high background noise, the laboratory must correct the issue
(e.g., perform instrument troubleshooting to check and if needed, replace, the transfer line,
column, detector, liner, filament, etc.). 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.
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 total
quantification ion (Ql) response to the total confirmation ion (Q2) response ratio must fall
within ± 50% of the ratio observed in the mid-point initial calibration standard. If project-
specific requirements involve reporting sample concentrations below the LOQ or ML, the
response ratio must also fall within ± 50% of the ratio observed in the initial daily CV (see
Section 14.3.5).
The total response of all isomers (branched and linear) in the quantitative standards should
be used to define ratio. 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.
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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 2, 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 the
where:
Arean
Areai
Mi
M
RF
Ws
Note: For better accuracy, PFTrDA is quantitated using the average of the areas of labeled
compounds ISC2-PFTeDA and I3C2-PFDoA.
And for the EIS analytes:
Areai MNIS 1
Concentration (na/L orna/a) = =¦ x —
AreaNISRFs Ws
where:
Areai =
The measured area at the Q1 m/z for the isotopically labeled PFAS (EIS)
AreaNis =
The measured area of the Q1 m/z for the non-extracted internal standard (NIS)
Mnis =
The mass of the added non-extracted internal standard (NIS) compound (ng)
Ws
Sample volume (L) or weight (g)
RFS =
Average response factor used to quantify the isotopically labeled compound 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:
native analytes:
Arean M; 1
Concentration (nq/L ornq/q) = == =- x —
v y/ y/yj Area^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 isotopically labeled PFAS (EIS). See note
below.
= The mass of the isotopically labeled compound 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)
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c = r :: MW'*>**
*^Acid uAnion ^ MlAf
M vvAnion
where:
CAnion = The analyte concentration in anion form
MW'acki = 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 by a factor no greater than lOx 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 isotope dilution. 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 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 compound concentrations, detection limits, and minimum levels to account for the
dilution.
If a dilution greater than lOx is indicated, 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 isotopically labeled compound is outside of the acceptance limits
(Table 5), a diluted aqueous sample or smaller aliquot (for solids and tissue) must be
analyzed (Section 15.3.1). If the recovery of any isotopically labeled compound in the
diluted sample is outside of the normal range, 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. If all cleanup procedures in this method and an alternative column have been
employed and isotopically labeled compound recovery remains outside of the normal
range, extraction and/or cleanup procedures that are beyond this scope of this method will
be required to analyze the sample.
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
8 for the appropriate names and CAS Registry Numbers.
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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 "
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This method is being validated, and performance specifications will be developed using data from DoD's
interlaboratory validation study (Reference 10). A summary of the single-laboratory performance is
presented in Tables 5, 9, and 10.
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
operation. EPA has established a preferred hierarchy of environmental management techniques that
places pollution prevention as the management option of first choice. Whenever feasible,
laboratory personnel should use pollution prevention techniques to address waste generation. When
wastes cannot be reduced feasibly at the source, 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 the hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and controlling all releases from
fume hoods and bench operations. Compliance is also required with any sewage discharge permits
and regulations. An overview of requirements can be found in Environmental Management Guide
for Small Laboratories (Reference 8).
18.2 Samples at pH < 2 or pH >12, 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. It
is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions.
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.
2nd Draft of Method 1633 - subject to further revision
40
.Time 2022
-------
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 interlaborcttory study reference will be added here.
12. DoD QSM (US Department of Defense Quality Systems Manual for Environmental
Laboratories, version 5.3, 2019).
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.
2nd Draft of Method 1633 - subject to further revision
41
.Time 2022
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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
CAS Number
Perfluoroalkyl carboxylic 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
Perfluoroalkyl 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
Perfluorododecanesulfonic acid
PFDoS
79780-39-5
Fluorotelomer sulfonic acids
\I I. \I I. III. 2/f-Perfluorohexane sulfonic acid
4:2FTS
757124-72-4
\I I. \I I. III. 2/f-Perfluorooctane sulfonic acid
6:2FTS
27619-97-2
\II. \ II. III. 2 //- Pe rflnorodccanc sulfonic acid
8:2FTS
39108-34-4
Perfluorooctane sulfonamides
Perfluorooctanesulfonainide
PFOSA
754-91-6
N-methyl perfluorooctanesulfonainide
NMeFOSA
31506-32-8
N-ethyl perfluorooctanesulfonainide
NEtFOSA
4151-50-2
Perfluorooctane sulfonamidoacetic acids
N-methyl perfluorooctanesulfonamidoacetic acid
NMeFOSAA
2355-31-9
N-ethyl perfluorooctanesulfonamidoacetic acid
NEtFOSAA
2991-50-6
Perfluorooctane sulfonamide ethanols
N-methyl perfluorooctanesulfonamidoethanol
NMeFOSE
24448-09-7
N-ethyl perfluorooctanesulfonamidoethanol
NEtFOSE
1691-99-2
Per- and Polyfluoroether carboxylic acids
Hexafluoropropylene oxide dimer acid
HFPO-DA
13252-13-6
4.8-Dio\a-3//-perfluorononanoic acid
ADONA
919005-14-4
Perfluoro-3-methoxypropanoic acid
PFMPA
377-73-1
Perfluoro-4-methoxybutanoic acid
PFMBA
863090-89-5
Nonafluoro-3,6-dioxaheptanoic acid
NFDHA
151772-58-6
2nd Draft of Method 1633 - subject to further revision
42
.Time 2022
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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
Ether sulfonic acids
9-Chlorohexadecafluoro-3-oxanonane-l-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
Fluorotelomer carboxylic acids
3-Perfluoropropyl propanoic acid
3:3FTCA
356-02-5
2 //. 2//. 3 //. 3 //- Pe rfl uo rooc ta 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
13Cs-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-[l,2-13C2]dodecanoic acid
13C2-PFDoA
Perfluoro-n-[l,2-13C2]tetradecanoic acid
13C2-PFTeDA
Perfluoro-1 -|2.3.4-'3C3]butanesulfonic acid
13c3-pfbs
Perfluoro-1 -[ 1,2,3-13C3]hexanesulfonic acid
13C3-PFHxS
NA
Perfluoro-l-[13C8]octanesulfonic acid
13c8-pfos
Perfluoro-1 -|l3Cx| octanesulfonamide
13c8-pfosa
N-methyl-d3-perfluoro-1 -octanesulfonainidoacetic acid
Dj-NMeFOSAA
N-ethyl-ds-perfluoro-1 -octanesulfonainidoacetic acid
Ds-NEtFOSAA
\II. 1 //.2//.2//-Perriuoro-1 -| 1.2-l3C'2|lic\anc sulfonic acid
13C2-4:2FTS
ML 1 //.2//.2//-Pcrfluoro-1 -| 1.2-l3C'2|octanc sulfonic acid
13C2-6:2FTS
1//. l//.2//.2//-Pcrfluoro-l-| 1.2-l3C'2|dccanc sulfonic acid
13C2-8:2FTS
Tetrafluoro-2-heptafluoropropoxy-13C3-propanoic acid
13c3-hfpo-da
N-methyl-d7-perfluorooctanesulfonamidoethanol
D7-NMeFOSE
N-ethyl-ds-perfluorooctanesulfonamidoethanol
D9-NEtFOSE
N-ethyl-ds-perfluoro-1 -octanesulfonamide
Ds-NEtFOSA
N-methyl-d3-perfluoro-1 -octanesulfonamide
D3-NMeFOSA
NIS Compounds
Perfluoro-n-12.3.4-'3C3]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-1 -hexane|lxO:|sulfonic acid
1802-PFHxS
1 The target analyte names are for the acid and neutral forms of the analytes. See Table 8 for the names and CASRN of the
corresponding anion forms, where applicable.
NA Not assigned a CASRN
2nd Draft of Method 1633 - subject to further revision
43
.Time 2022
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Table 2. 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 Analytes
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
13Co-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
and13C 2-PFDoA
PFTeDA
8.96
713.1
669.0
168.9
6.0
13C 2-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
Ds-N-EtFOSAA
NMeFOSE
9.57
616.1
58.9
NA
NA
Dy-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
Extracted Internal Standards
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
2nd Draft of Method 1633 - subject to further revision
44
.Time 2022
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Table 2. 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
13c9-pfna
6.99
472.1
427.0
NA
13c5-pfna
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
Dy-NMeFOSE
9.56
623.2
58.9
NA
13c4-pfos
D9-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.
2nd Draft of Method 1633 - subject to further revision
45
.Time 2022
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Table 3. Nominal Masses of Spike Added to Samples or Extracts
Analyte
Amount Added (ng)
Extracted Internal Standards
13c4-pfba
40
13Cs-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
Dj-NMeFOSA
10
Ds-NEtFOSA
10
D3-NMeFOSAA
20
Ds-NEtFOSAA
20
D7-NMeFOSE
100
Dg-NEtFOSE
100
13c3-hfpo-da
40
Non-extracted Internal Standards
13c3-pfba
20
13C2-PFHxA
10
13c4-pfoa
10
13c5-pfna
5
13c2-pfda
5
1802-PFHxS
10
13c4-pfos
10
2nd Draft of Method 1633 - subject to further revision
46
.Time 2022
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Table 4. Calibration Solutions (ng/mL)
Compound
CS1 (LOQ)
CS2
CS3
CS4 (CV1)
CS5
CS6
CS72
Perfluoroalkyl carboxylic 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
Perfluoroalkyl 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
Fluorotelomer 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
Perfluorooctane sulfonamides
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
Perfluorooctane sulfonamidoacetic 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
Perfluorooctane sulfonamide ethanols
NMeFOSE
2
5
12.5
25
50
125
625
NEtFOSE
2
5
12.5
25
50
125
625
Per- and polyfluoroether carboxylic acids
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
2nd Draft of Method 1633 - subject to further revision
47
.Time 2022
-------
Table 4. Calibration Solutions (ng/mL)
Compound
CS1 (LOQ)
CS2
CS3
CS4 (CV1)
CS5
CS6
CS72
Fluorotelomer carboxylic 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) Analytes
13c4-pfba
10
10
10
10
10
10
10
13Cs-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
Dj-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
D7-NMeFOSE
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-extracted Internal Standard (NIS) Analytes
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.
2nd Draft of Method 1633 - subject to further revision
48
.Time 2022
-------
Table 5. Single-Laboratory Validation Performance Summary for Target Compounds and Extracted Internal
Standards
Compounds
Blank
(ng/mL)
Aqueous Matrices1
Solid Matrices1
Tissue Matrices1
IPRRec
(%)
RSD
(%)
OPR
Rec (%)
IPRRec
(%)
RSD
(%)
OPR Rec
(%)
IPRRec
(%)
RSD
(%)
OPR Rec
(%)
Target Compounds
PFBA
<0.4
89-107
4.8
89-113
95-99
1.0
92-108
89 -104
3.9
90-110
PFPeA
<0.2
85 -106
5.5
89-121
92 - 105
3.4
94-115
80-98
5.0
96-114
PFHxA
<0.1
75 -109
9.1
89-111
93-101
2.2
89-107
72-110
10.2
90-111
PFHpA
<0.1
87-102
4.1
90-110
94-102
2.2
89-107
87 - 102
4.0
87-118
PFOA
<0.1
88-98
2.8
87-112
92 - 100
2.0
90-106
78-85
2.4
82-114
PFNA
<0.1
88-104
4.1
90-111
91-102
2.7
88-112
85-110
6.3
87-119
PFDA
<0.1
82-115
8.3
92-115
97-103
1.5
89-118
76-115
10.2
84-112
PFUnA
<0.1
83-98
4.2
89-112
91-107
4.0
92-111
83 - 102
5.1
91-117
PFDoA
<0.1
58-111
15.7
84 -123
73 - 120
12.1
88-119
83-105
5.7
77-141
PFTrDA
<0.1
80-111
8.1
92-119
91-112
5.2
89-125
92-114
5.3
106-133
PFTeDA
<0.1
88-103
4.1
89-116
94-104
2.5
92-110
76 - 103
7.4
91-111
PFBS
<0.1
85-111
6.6
87-116
91-103
3.2
91-111
69-105
10.3
89-117
PFPeS
<0.1
87-115
6.9
87-115
87-103
4.3
89-112
77-96
5.4
89-112
PFHxS
<0.1
90-107
4.4
97-119
98-106
2.0
96-113
81 - 101
5.3
91-123
PFHpS
<0.1
84 -126
10.2
86-114
87-104
4.4
88-104
77-108
8.4
86-108
PFOS
<0.1
93 -122
6.7
91-120
95-108
3.4
94-115
98-112
3.2
97-124
PFNS
<0.1
64 -141
18.8
86-123
98-111
3.0
76-117
65-88
7.5
85-114
PFDS
<0.1
75-121
11.7
84-107
83 - 102
5.2
84-107
82-94
3.6
78-110
PFDoS
<0.1
74-114
10.6
78-102
76-99
6.5
77-100
73-96
6.9
29-108
4:2FTS
<0.4
76 -123
12.0
91-119
98-100
0.5
87-113
66 - 126
15.6
90-103
6:2FTS
<0.4
71 -148
17.5
81-129
94-123
6.5
60-166
77-105
7.8
92-119
8:2FTS
<0.4
85 -109
6.1
99 -124
109-128
3.8
104-127
66 - 148
19.3
102-136
PFOSA
<0.1
90-107
4.4
91 -122
92 - 106
3.4
94-114
92-116
5.7
96-121
NMeFOSA
<0.1
78-90
3.6
84-112
87-104
4.4
91-117
81 - 100
5.5
86-117
NEtFOSA
<0.1
79-97
5.0
83-108
98-102
1.0
96-115
74-114
10.7
90-127
NMeFOSAA
<0.1
82-115
8.2
81-120
91-107
4.0
90-113
89-136
10.4
93-117
NEtFOSAA
<0.1
79 -120
10.3
85 -124
102 - 108
1.6
87-117
53-115
18.3
90-117
NMeFOSE
<1
87-102
3.9
92-115
98-103
1.3
94-112
71-292
30.3
118-344
NEtFOSE
<1
87-104
4.7
91-118
97-104
1.9
96-115
97-133
8.0
61-159
HFPO-DA
<0.4
88-114
6.5
84-118
83-105
5.9
80-120
73 - 100
7.8
86-114
ADONA
<0.4
77-106
7.9
77-117
85-96
3.2
76 - 124
82-95
3.8
86-132
PFMPA
<0.2
86-106
6.6
83 -120
91-98
1.8
85-117
78-93
4.2
86-109
PFMBA
<0.2
62 -122
5.2
81-115
88-97
2.6
85-120
74 - 104
8.4
84-117
NFDHA
<0.2
44 -149
16.3
56-138
53-103
16.2
58-136
49-86
13.8
56-115
9C1-PF30NS
<0.4
84-101
27.4
80 -120
84-100
4.4
79-131
69-98
8.7
95-126
llCl-PF30UdS
<0.4
80-95
4.5
76-116
84-96
3.3
77-127
85 - 100
4.3
94-138
PFEESA
<0.2
80-104
4.4
85-115
80-93
3.8
89-109
68-99
9.3
88-107
3:3FTCA
<0.5
84-103
5.0
66 -127
86-98
3.3
76-116
66-94
9.0
41-126
5:3FTCA
<2.5
84-101
4.6
84-113
83-94
3.1
80-101
95-131
7.9
78-199
7:3FTCA
<2.5
78-103
7.0
82-116
90-106
4.1
75 - 104
84-111
6.7
99-139
2nd Draft of Method 1633 - subject to further revision
49
.Time 2022
-------
Table 5. Single-Laboratory Validation Performance Summary for Target Compounds and Extracted Internal
Standards
Compounds
Blank
(ng/mL)
Aqueous Matrices1
Solid Matrices1
Tissue Matrices1
IPRRec
(%)
RSD
(%)
OPR
Rec (%)
IPRRec
(%)
RSD
(%)
OPR Rec
(%)
IPRRec
(%)
RSD
(%)
OPR Rec
(%)
Extracted Internal Standard (EIS)
13c4-pfba
N/A
85-91
1.6
88-108
92-99
1.6
95-109
93-97
1.0
95-105
13C5-PFPeA
N/A
87-95
2.4
84-111
86-106
5.3
80-110
85-108
6.0
89-103
13C5-PFHxA
N/A
85-92
1.9
83-108
83-101
4.8
92 - 106
79-111
8.5
88-98
13C4-PFHpA
N/A
78-100
6.2
83-106
87-102
4.1
90-100
88-93
1.3
80-102
13Cs-PFOA
N/A
77-98
6.0
84-107
89-101
3.2
92 - 104
91-98
1.7
86-102
13C9-PFNA
N/A
82-96
3.8
84-107
86-101
4.1
90-106
91 - 104
3.3
89-101
13Co-PFDA
N/A
81-98
4.7
84-106
79-101
6.0
86-109
89 - 104
4.0
90-104
13C7-PFUnA
N/A
84-100
4.4
84-109
84-104
5.4
91-116
84-118
8.4
88-109
13C2-PFDoA
N/A
61 -103
12.9
73-101
70-93
7.1
73 - 106
95-125
6.8
70-108
13C2-PFTeDA
N/A
72-89
5.4
74-97
83-88
1.5
74-107
81 - 114
8.5
10-110
13C3-PFBS
N/A
87-94
2.0
88-110
97-105
1.8
96-109
87-114
6.5
95-106
13C3-PFHxS
N/A
83-89
1.9
85-103
92-97
1.4
92 - 106
92-97
1.4
91-103
13Cs-PFOS
N/A
78-92
3.9
86-110
87-107
4.9
95-109
87-93
1.6
95-103
13C2-4:2FTS
N/A
64-106
12.1
87-137
132-135
0.6
123-145
106-221
17.6
155-291
13C2-6:2FTS
N/A
93 -102
2.2
67-149
118-129
2.3
104-138
87-135
10.8
117-149
13C2-8:2FTS
N/A
99-109
2.5
71-137
96 - 122
6.1
93-123
179-299
12.5
79 - 304
13Cs-PFOSA
N/A
60-107
14.2
57-109
69-86
5.4
66-100
104-153
9.4
88-120
Ds-NMeFOSA
N/A
55-85
10.8
39-84
47-59
5.4
25-64
20-58
24.5
3-34
Ds-NEtFOSA
N/A
54-91
12.9
43-84
43-51
4.5
18-58
30-56
15.2
0-56*
Ds-NMeFOSAA
N/A
63-117
14.9
66-117
98-107
2.1
86-109
102-187
14.7
144-196
D5-NEIFOSAA
N/A
66-115
13.7
63-115
98-104
1.3
85-109
178-216
4.9
175-223
Dy-NMeFOSE
N/A
61 -106
13.6
42-99
50-61
5.1
35-76
3-5
11.6
0-8*
D9-NEIFOSE
N/A
63 -108
13.2
44-90
46-57
5.5
32-72
8-33
30.0
0-33*
13C3-HFPO-DA
N/A
89-106
4.5
88-121
98-108
2.4
83-125
87-106
4.9
81-106
1 Hie recovery limits are applied to all samples, method blanks, IPR, OPR samples for all matrix types.
* Ranges were determined at ± 2 standard deviations from the mean. Because of the low recoveries for these EIS, the calculated
lower limits were negative values. Therefore, the lower limits have been set to 0 for these analytes.
Data for this table are 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. Several sections of this method state that Table 5 criteria are required, this is standard
language that will be applicable when the method is finalized.
2nd Draft of Method 1633 - subject to further revision
50
.Time 2022
-------
Table 6. Pooled MDLS and ML values from the Single-laboratory Validation Study, by Matrix1
Compound
Aqueous (ng/L)
Solid (ng/g)
Tissue (ng/g)
MDLS
ML2
MDLS
ML
MDLs
ML
PFBA
0.330
6.4
0.401
0.8
0.593
2.0
PFPeA
0.196
3.2
0.021
0.4
0.083
1.0
PFHxA
0.318
1.6
0.020
0.2
0.096
0.5
PFHpA
0.221
1.6
0.029
0.2
0.088
0.5
PFOA
0.302
1.6
0.037
0.2
0.086
0.5
PFNA
0.221
1.6
0.086
0.2
0.160
0.5
PFDA
0.333
1.6
0.031
0.2
0.124
0.5
PFUnA
0.264
1.6
0.033
0.2
0.152
0.5
PFDoA
0.379
1.6
0.059
0.2
0.130
0.5
PFTrDA
0.238
1.6
0.038
0.2
0.086
0.5
PFTeDA
0.264
1.6
0.032
0.2
0.185
0.5
PFBS
0.245
1.6
0.014
0.2
0.070
0.5
PFPeS
0.204
1.6
0.015
0.2
0.032
0.5
PFHxS1
0.217
1.6
0.018
0.2
0.083
0.5
PFHpS
0.137
1.6
0.057
0.2
0.043
0.5
PFOS1
0.327
1.6
0.067
0.2
0.294
0.5
PFNS
0.303
1.6
0.046
0.2
0.114
0.5
PFDS
0.334
1.6
0.040
0.2
0.101
0.5
PFDoS
0.179
1.6
0.038
0.2
0.177
0.5
4:2FTS
2.281
6.4
0.282
0.8
0.740
2.0
6:2FTS
3.973
6.4
0.116
0.8
1.149
2.0
8:2FTS
1.566
6.4
0.225
0.8
0.373
2.0
PFOSA
0.227
1.6
0.068
0.2
0.094
0.5
NMeFOSA
0.196
1.6
0.049
0.2
0.161
0.5
NEtFOSA
0.585
1.6
0.038
0.2
0.169
0.5
NMeFOSAA1
0.586
1.6
0.030
0.2
0.093
0.5
NEtFOSAA1
0.324
1.6
0.044
0.2
0.138
0.5
NMeFOSE
1.191
16
0.203
2.0
9.978
5.0
NEtFOSE
1.022
16
0.247
2.0
1.501
5.0
HFPO-DA
0.406
6.4
0.136
0.8
0.161
2.0
ADONA
0.779
6.4
0.057
0.8
0.082
2.0
PFEESA
0.137
3.2
0.018
0.4
0.045
1.0
PFMPA
0.177
3.2
0.033
0.4
0.070
1.0
PFMBA
0.117
3.2
0.029
0.4
0.069
1.0
NFDHA
1.384
3.2
0.084
0.4
0.294
1.0
9CL-PF30NS
0.871
6.4
0.038
0.8
0.152
2.0
11CL-PF30UDS
0.819
6.4
0.071
0.8
0.312
2.0
3:3FTCA
0.721
8.0
0.060
1.0
0.247
2.5
5:3FTCA
5.066
40
0.363
5.0
1.537
12.5
7:3FTCA
5.942
40
0.308
5.0
0.845
12.5
1A 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.
2 The ML values in this table were derived from the concentrations of the lowest calibration standard in Table 4, based on the
alternative described in the Glossary, using the nominal sample volume (aqueous) or weight (all other matrices) described in the
method.
Data for this table are derived from the single-laboratory validation study, and are only provided
as examples for this draft method. The data will be updated with the pooled MDLs from the
interlaboratory study results in a subsequent revision.
2nd Draft of Method 1633 - subject to further revision
51
.Time 2022
-------
Table 7. 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 14.2
Calibration Verification (CV)
At the beginning and every 10 samples
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)
Prior to analyzing samples
Section 11.0
Matrix Spike (MS/MSD)
One per preparation batch (if required)
2nd Draft of Method 1633 - subject to further revision
52
.Time 2022
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Table 8. Cross-reference of Abbreviations, Analyte Names, CAS Numbers for the Acid and Anion Forms
of the Perfluoroalkyl carboxylates and Perfluoroalkyl sulfonates
Perfluoroalkyl carboxylic 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
Perfluoroalkyl 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
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Table 9. Range of Recoveries for Extracted Internal Standards (EIS) in the Single-laboratory Validation
Study, by Matrix
EIS Compounds
Aqueous
Solid
Tissue
% Recovery
RSD
(%)
% Recovery
RSD
(%)
% Recovery
RSD
(%)
Min
Max
Min
Max
Min
Max
13c4-pfba
9
97
15.9
3
113
37.4
84
99
8.0
13C5-PFPeA
39
103
13.3
28
112
17.2
86
107
11.1
13C5-PFHxA
73
97
2.7
79
110
5.5
92
95
1.6
13C4-PFHpA
77
95
2.4
73
111
6.0
80
93
8.2
13Cs-PFOA
87
95
0.8
86
115
4.4
90
95
2.8
13C9-PFNA
82
95
1.6
87
110
4.2
90
98
4.3
13Co-PFDA
71
93
3.3
87
112
4.9
83
97
7.7
13C7-PFUnA
56
94
6.5
66
124
11.6
71
91
12.9
13C2-PFDoA
34
87
13.7
26
109
24.3
54
96
29.2
13C2-PFTeDA
17
153
26.2
18
110
30.1
31
102
67.8
13C3-PFBS
72
100
4.7
89
120
5.4
89
98
5.1
13C3-PFHxS
79
95
1.6
87
110
4.4
98
99
0.1
13Cs-PFOS
67
96
3.6
79
113
5.7
92
103
6.0
13C2-4:2FTS
81
199
14.8
95
248
17.0
192
215
6.2
13C2-6:2FTS
64
183
16.4
76
127
9.4
145
230
27.2
13C2-8:2FTS
65
139
8.4
86
173
15.2
136
220
24.6
13Cs-PFOSA
27
93
15.4
61
123
10.0
87
96
4.5
Ds-NMeFOSA
14
74
16.4
28
86
22.7
8
38
61.9
D5-NEtFOSA
12
70
16.5
21
70
25.5
8
30
57.8
Ds-NMeFOSAA
21
113
7.3
52
142
14.8
106
139
13.1
D5-NEtFOSAA
12
106
8.2
68
151
16.9
79
151
31.8
Dy-NMeFOSE
11
77
18.6
13
107
27.9
5
30
81.1
Ds-NEtFOSE
8
73
19.6
16
97
30.4
0
29
103.1
13C3-HFPO-DA
92
113
2.0
70
119
10.4
93
102
5.1
Data for this table are derived from the single-laboratory validation study, and are only provided
as examples for this draft method. The data will be updated with the interlaboratory study results
in a subsequent revision.
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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
Data for this table are derived from the single-laboratory validation study, and are only provided
as examples for this draft method. The data will be updated with the interlaboratory study results
in a subsequent revision.
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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
±
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.
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) quantification - The response of the target compound is
compared to the response of the labeled analog of another compound in the same LOC.
LC - Liquid chromatograph or liquid chromatography
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.
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 quantitation - A means of determining the concentration of (1) a naturally
occurring (native) compound by reference to a compound other than its labeled analog and (2) a
labeled compound by reference to another labeled compound
IPR - Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes
of interest and labeled compounds and analyzed to establish the ability of the laboratory to
generate acceptable precision and recovery. An IPR is performed prior to the first time this
method is used and any time the method or instrumentation is modified.
Isotope dilution (ID) quantitation - A means of determining a naturally occurring (native)
compound by reference to the same compound in which one or more atoms has been isotopically
enriched. 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 quantitation or
other method-specific purposes.
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).
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.
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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
the results produced by the laboratory remain within the limits specified in this method for
precision and recovery.
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.
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.
Reagent water - Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.
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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.
Aqueous Samples
1. Weight out 10 (±0.1) g of sample into a 50-mL centrifuge tube.
2. Add 50 pL of EIS and NIS to the sample and vortex to mix.
3. Filter 1 mL of the sample through 0.2-pm 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 pL of EIS to a clean Single Step® filter vial (chamber).
b. Add 400 pL 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 pL of filtrate to a ~300-pL polypropylene micro-vial and dilute to 300 pL 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 ——-
1, U TYIL
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 Vi cm below the foam.
4. If no foam forms - pour or pipette based on volume required.
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