ASTM D8421-22

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation:D8421 −22
Standard Test Method for
Determination of Per- and Polyfluoroalkyl Substances
(PFAS) in Aqueous Matrices by Co-solvation followed by
Liquid Chromatography Tandem Mass Spectrometry (LC/
MS/MS)
This standard is issued under the fixed designation D8421; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.2.2 Depending on data usage, you may modify this test
method but limit to modifications that improve performance
1.1 This test method covers the determination of per- and
whilestillmeetingorexceedingthemethodqualityacceptance
polyfluoroalkyl substances (PFASs) in aqueous matrices using
criteria. Modifications to the solvents, ratio of solvent to
liquid chromatography (LC) and detection with tandem mass
sample, or shortening the chromatographic run simply to save
spectrometry (MS/MS). These analytes are co-solvated by a
time are not allowed. Use Practice E2935 or similar statistical
1+1 ratio of sample and methanol then qualitatively and
tests to confirm that modifications produce equivalent results
quantitatively determined by this test method. Quantitation is
on non-interfering samples. In addition, use Guide E2857 or
by selected reaction monitoring (SRM) or sometimes referred
equivalent statistics to re-validate the modified test.
to as multiple reaction monitoring (MRM).
1.2.3 Analytedetectionsbetweenthemethoddetectionlimit
1.2 The method detection limit (MDL) (see Note 1) and
and the reporting limit are estimated concentrations. The
reportingrange(seeNote2)forthetargetanalytesarelistedin
reporting limit is based upon the concentration of the Level 1
Table1.Thetargetconcentrationforthereportinglimitforthis
calibration standard as shown in Table 5.
test method is an integer value that is calculated from the
1.3 The values stated in SI units are to be regarded as
concentration from the lowest standard from the final volume
standard. No other units of measurement are included in this
of the prepared sample. This value may be lower than the
standard.
calculated MDL due to sporadic PFAS hits due to PFAS
1.4 This standard does not purport to address all of the
contamination in consumables/collection tools used during
safety concerns, if any, associated with its use. It is the
samplecollectionandpreparation.Allsamplesshouldbetaken
responsibility of the user of this standard to establish appro-
at a minimal as duplicates in order to compare the precision
priate safety, health, and environmental practices and deter-
between the two prepared samples to help ensure the
mine the applicability of regulatory limitations prior to use.
concentration/positive result is reliable.
1.5 This international standard was developed in accor-
NOTE 1—The MDL is determined following the Code of Federal
dance with internationally recognized principles on standard-
Regulations (CFR), 40 CFR Part 136, Appendix B utilizing dilution and
ization established in the Decision on Principles for the
filtration. A detailed process determining the MDL is explained in the
Development of International Standards, Guides and Recom-
reference and is beyond the scope of this test method.
mendations issued by the World Trade Organization Technical
NOTE 2—Injection volume variations, and sensitivity of the instrument
used will change the reporting limit and ranges.
Barriers to Trade (TBT) Committee.
1.2.1 Recognizingcontinualadvancementsinthesensitivity
2. Referenced Documents
of instrumentation, advancements in column chromatography
and other processes not recognized here, the reporting limit 2.1 ASTM Standards:
may be lowered assuming the minimum performance require- D1129Terminology Relating to Water
ments of this test method at the lower concentrations are met. D1193Specification for Reagent Water
D2777Practice for Determination of Precision and Bias of
Applicable Test Methods of Committee D19 on Water
This test method is under the jurisdiction ofASTM Committee D19 on Water
andisthedirectresponsibilityofSubcommitteeD19.06onMethodsforAnalysisfor
Organic Substances in Water. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved May 1, 2022. Published June 2022. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2021. Last previous edition approved in 2021 as D8421–21. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D8421-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D8421−22
D3856Guide for Management Systems in Laboratories 3.2.6 precursor ion, n—ion that reacts to form product ions
Engaged in Analysis of Water or undergoes specified neutral losses.
D4841Practice for Estimation of Holding Time for Water
3.2.7 production,n—ionformedastheproductofareaction
Samples Containing Organic and Inorganic Constituents
involving a precursor ion.
D5847Practice for Writing Quality Control Specifications
3.2.8 single (or selected) reaction monitoring (SRM),
for Standard Test Methods for Water Analysis
n—data acquired from one or more specific product ions
D8272Guide for Development and Optimization of D19
corresponding to m/z selected precursor ions recorded via two
Chemical Analysis Methods Intended for EPA Compli-
or more stages of mass spectrometry.
ance Reporting
3.2.9 tandem mass spectrometer, n—mass spectrometer de-
E694Specification for Laboratory Glass Volumetric Appa-
signed for mass spectrometry/mass spectrometry.
ratus
E2554Practice for Estimating and Monitoring the Uncer- 3.2.10 triple quadrupole mass spectrometer (triple quad or
tainty of Test Results of a Test Method Using Control
QQQ), n—tandem mass spectrometer comprising two trans-
Chart Techniques mission quadrupole mass spectrometers in series, with a
E2857Guide for Validating Analytical Methods (non-selecting) RF-only quadrupole (or other multipole) be-
E2935Practice for Evaluating Equivalence of Two Testing
tween them to act as a collision cell.
Processes
4. Summary of Test Method
2.2 Other Standards:
Code of Federal Regulations 40 CFR Part 136,Appendix B
4.1 The operating conditions presented in this test method
have been validated for use in the determination of PFASs in
3. Terminology
aqueous samples. Alternative instrument operating conditions
3.1 Definitions:
may be used provided data quality objectives are met. Follow
3.1.1 For definitions of terms used in this standard, refer to
the manufacturer’s instructions. The preparation process, as
Terminology D1129.
summarized in 4.2 and described in Section 14 may be
automated, but cannot be modified.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 collision cell, n—chamberintheionpathbetweenm/z
4.2 Samplesareshippedtothelabatatemperaturebetween
separation elements, or between ion source and the first
0°C and 6°C and analyzed within 28 days of collection. A
analyzer,intandemmassspectrometryinspaceconfigurations.
sample (5 mL) is collected and processed in the same collec-
tion tube in order to limit analyte loss; extra samples must be
3.2.2 continuing calibration verification (CCV), n—a mid-
collected for duplicates/triplicates and matrix spikes. All
range calibration standard which checks the continued validity
samples and associated QC samples are spiked with labeled
of the initial calibration of the instrument.
surrogates (QC samples such as laboratory control and matrix
3.2.3 mass spectrometry/mass spectrometry (MS/MS),
spike samples are additionally spiked with target PFASs) and
n—acquisition and study of the spectra of the product ions or
shaken for 2 minutes after adding 5 mL of methanol. The
precursor ions of m/z selected ions, or of precursor ions of a
samples are then filtered through a polypropylene filter.Acetic
selected neutral mass loss.
acid(~10µL)isaddedtoallthesamplestoadjusttopH~4and
3.2.3.1 Discussion—MS/MS can be accomplished using
analyzed by LC/MS/MS. If samples contain more than about
instruments incorporating more than one analyzer (tandem
1.0 g/L suspended or settled solids, (for example, sludge,
mass spectrometry in space) or in trap instruments (tandem
pretreatment, or wastewater influent) adjust to pH ~9 (adding
mass spectrometry in time).
~20 µL of ammonium hydroxide), shake for 2 minutes, filter,
3.2.4 multiple reaction monitoring (MRM), n—application
acidify to pH ~4 (~50 µL acetic acid), and then analyze by
of selected reaction monitoring to multiple product ions from
LC/MS/MS.
one or more precursor ions.
NOTE 3—Sludge in this test method is defined as sewage sample
containing between 0.1 and 2 % solids based upon a sample by weight.
3.2.5 per- and polyfluoroalkyl substances (PFAS),
NOTE 4—Since contact with surfaces may bias data, collect a 5.0-mL
n—synthetic organofluorine chemical compounds with mul-
sample in a graduated 15-mL polypropylene tube in the field so that the
tiple fluorine atoms that includes PFOA, PFOS, GenX, and
whole sample is processed in the lab. Once this 5.0-mL sample is spiked
many other chemicals.
accordingtothistestmethodandmethanolisadded,thesampleisfiltered
3.2.5.1 Discussion—PFAS have a hydrophobic and oleop- into another 15 mL polypropylene tube without analyte loss.
NOTE 5—For accurate volume, the weight of the 15-mLpolypropylene
hobic fluorinated “tail” and a hydrophilic “head” making them
tube may be taken before and after sampling. The density of water is
surfactants. They include the perfluoro sulfonic acids such as
assumed to be 1.0 g/mL unless the exact density of the water sample is
the perfluorooctanesulfonic acid (PFOS) and the perfluoro
known, then that conversion should be used.
carboxylic acids, such as the perfluorooctanoic acid (PFOA).
4.3 Most analytes are identified by comparing the SRM
PFOS and PFOAare persistent organic pollutants. The defini-
transitionanditsconfirmatorySRMtransitioncorrelatedtothe
tion does not include the mass labeled surrogates or internal
known standard SRM transition (Table 3) and quantitated
standards.
utilizing an external calibration. The retention times and ion
ratiosareshowninTable4foreachnativeanalyteandisotope.
Available from National Technical Information Service (NTIS), U.S. Depart-
The surrogates and some analytes only have one SRM transi-
ment of Commerce, 5285 Port Royal Road, Springfield, VA, 22161 or at http://
www.epa.gov/epawaste/hazard/testmethods/index.htm tion due to a less sensitive or non-existent secondary SRM
D8421−22
transition. As an additional quality-control measure, isotopi- qualified,backgroundsubtractionofblankcontaminationisnot
cally labeled surrogate (Table 1, Section 13.3) recoveries are allowed. It has become difficult to ensure consumables are
monitored. With external standard calibrations, there is no PFAS free at the lower concentrations (approximately at less
correction to the data based upon surrogate recoveries. than 30 ng/L) for the entire lot by testing only a very small
Alternatively, extract an isotopically labelled analog of each sub-sample. At a minimum duplicates/triplicates should be
analyte(isotopedilution),ifavailable,andcorrectforrecovery. taken of each sample to evaluate precision between the set.
Only exact isotopes of the native analytes may be used for
6.5 The LC system used should consist, as much as
isotope dilution correction. If a structurally different isotope is
practical, of sample solution or eluent contacting components
used to correct a native analyte this is called surrogate
free of PFAS of interest.
correctionandeithermustbeclearlystatedasperformedinthe
6.6 Polyethylene LC vial caps or any other target analyte
accompanying data report or not allowed. For isotope dilution,
free vial caps should be used.
the analog and the native compound concentrations (areas)
should be within 30% of each other to obtain more accurate
6.7 Polyethylene disposable pipettes or target analyte free
results.The final report issued lists the concentration of PFAS, pipettes should be used. All disposable pipettes should be
ifdetected,orasanon-detectattheRL,ifnotdetected,inng/L checked for release of target analytes of interest.
and the surrogate recoveries.
6.8 DegassersareimportanttocontinuousLCoperationand
NOTE 6—For greater accuracy in the isotope dilution method, add the
most commonly are made of fluorinated polymers. To enable
isotopes at the time sampling or allow the sample and isotope to
use,anisolatorcolumnshouldbeplacedafterthedegasserand
equilibrate for at least 48 hours prior to addition of methanol.
prior to the sample injection valve to separate the PFAS in the
5. Significance and Use
sample from the PFAS in the LC system.
5.1 PFAS are widely used in various industrial and com-
6.9 Electro Spray Ionization (ESI)—ESI should be heated
mercial products; they are persistent, bio-accumulative, and
andoptimizedforrecoveryofcomponentsanalyzedbythistest
ubiquitous in the environment. PFAS have been reported to
method. Using the suggested mobile phase, gradient, and
exhibit developmental toxicity, hepatotoxicity,
adequate column separation minimizes, or eliminates, quench-
immunotoxicity, and hormone disturbance. PFAS have been
ing and enhancing of signal. This method was validated using
detected in soils, sludges, surface, and drinking waters. This is
ESI, however other modes of ionization may be used provided
a quick, easy, and robust method to quantitatively determine
the detection limits and quality control acceptance criteria of
these compounds at trace levels in water matrices.
this method are met.
5.2 This test method has been validated using reagent water
and waters from sites that include landfill leachate, metal 7. Apparatus
finisher, POTW Effluent, Hospital, POTW Influent, Bus wash-
7.1 LC/MS/MS System:
ing station, Power Plant and Pulp and paper mill effluent for
7.1.1 Liquid Chromatography System—Acomplete LC sys-
selected PFAS, refer to the Precision and Bias (Section 17).
tem is required to analyze samples, this includes a sample
injection system, a solvent pumping system capable of mixing
6. Interferences
solvents, a sample compartment capable of maintaining re-
6.1 All glassware is washed in hot water (typically >45ºC)
quired temperature and a temperature-controlled column com-
with detergent and rinsed in hot water followed by distilled
partment. This test method was developed using a ternary
water. The glassware is then dried and heated in an oven
(Table2)pumpingsystem.AbinaryLCsystemmaybeusedby
(typically at 105ºC) for 15 to 30 minutes. All glassware is
adapting the ternary gradient to a binary system.ALC system
subsequently rinsed with methanol or acetonitrile.
that can perform at the flow rates, pressures, controlled
temperatures, sample volumes, and requirements of the stan-
6.2 All reagents and solvents should be pesticide residue
purity or higher to minimize interference. Avoid the use of dard shall be used.
7.1.2 Analytical Column—UHPLC CSH Phenyl-Hexyl, 2.1
PFAS containing caps.
×100mmand1.7µmparticlesizecolumn,oranycolumnthat
6.3 Matrix interferences may be caused by contaminants in
achieves adequate resolution may be used.The retention times
the sample. The extent of matrix interferences varies consid-
andorderofelutionmaychangedependingonthecolumnused
erably depending on variations of the sample matrices. Sepa-
and needs to be monitored.
ration of individual components by the LC is vital in minimi-
7.1.3 Isolator Column—A reverse phase C18 column is
zationofinterferences.Shorteningofruntimessimplytospeed
used to separate the target analytes in the LC system and
analysis should be avoided, unless interferences are known to
solventsfromthetargetanalytesintheanalyticalsample.Place
be absent.
the column between the solvent mixing chamber and the
6.4 Contaminants have been found in reagents, glassware,
injector sample loop.
tubing, glass disposable pipettes, filters, degassers, and other
7.2 Tandem Mass Spectrometer System—A MS/MS system
apparatus and consumables that release PFAS. All these
capable of multiple reaction monitoring (MRM) analysis or
materials and supplies must be routinely demonstrated to be
anysystemthatiscapableofperformingattherequirementsin
free from interferences by analyzing laboratory reagent blanks
this test method.
under the same conditions as the samples. If found, measures
should be taken to remove the contamination or data should be 7.3 Filtration Device:
D8421−22
TABLE 1 Analyte List with Method Detection Limit and Reporting Range
MDL Range
Analyte Name Acronym CAS Number
(ng/L) (ng/L)
Perfluorotetradecanoic acid PFTreA 376-06-7 8.2 10-400
Perfluorotridecanoic acid PFTriA 72629-94-8 17.2 10-400
Perfluorododecanoic acid PFDoA 307-55-1 6.6 10-400
Perfluoroundecanoic acid PFUnA 2058-94-8 3.9 10-400
Perfluorodecanoic acid PFDA 335-76-2 3.4 10-400
Perfluorononanoic acid PFNA 375-95-1 5.2 10-400
Perfluorooctanoic acid PFOA 335-67-1 2.5 10-400
Perfluoroheptanoic acid PFHpA 375-85-9 5.9 10-400
Perfluorohexanoic acid PFHxA 307-24-4 2.1 10-400
Perfluoropentanoic acid PFPeA 2706-90-3 13.0 50-1000
Perfluorobutanoic acid PFBA 375-22-4 17.1 50-1000
Perfluorodecanesulfonic acid PFDS 335-77-3 1.6 10-400
Perfluorononanesulfonic acid PFNS 68259-12-1 1.2 10-400
Perfluorooctanesulfonic acid PFOS 1763-23-1 4.4 10-400
Perfluoroheptanesulfonic acid PFHpS 375-92-8 2.7 10-400
Perfluorohexanesulfonic acid PFHxS 355-46-4 2.3 10-400
Perfluoropentanesulfonic acid PFPeS 2706-91-4 2.7 10-400
Perfluorobutanesulfonic acid PFBS 375-73-5 3.3 10-400
Perfluorooctanesulfonamide PFOSA 754-91-6 2.2 10-400
8:2 Fluorotelomer sulfonic acid 8:2 FTS 39108-34-4 4.5 10-400
6:2 Fluorotelomer sulfonic acid 6:2 FTS 27619-97-2 2.7 10-400
4:2 Fluorotelomer sulfonic acid 4:2 FTS 757124-72-4 3.2 10-400
N-Ethylperfluorooctanesulfonamidoacetic acid NEtFOSAA 2991-50-6 2.6 10-400
N-Methylperfluorooctanesulfonamidoacetic acid NMeFOSAA 2355-31-9 1.3 10-400
Perfluorododecanesulfonic acid PFDoS 79780-39-5 2.2 10-400
N-Methylperfluorooctanesulfonamide NMeFOSA 31506-32-8 2.1 10-400
N-Ethylperfluorooctanesulfonamide NEtFOSA 4151-50-2 1.8 10-400
N-Methylperfluorooctanesulfonamidoethanol NMeFOSE 24448-09-7 3.1 10-400
N-Ethylperfluorooctanesulfonamidoethanol NEtFOSE 1691-99-2 2.7 10-400
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 3.7 10-400
4,8-dioxa-3H-perfluorononanoic acid ADONA 919005-14-4 2.1 10-400
9-chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 2.7 10-400
11-chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 2.2 10-400
Pentafluorpropanoic acid PFPrA 422-64-0 20.3 50-1000
Perfluoro-3,6-dioxaheptanoic acid NFDHA 151772-58-6 3.7 10-400
Perfluoro(2-ethoxyethane) sulfonic acid PFEESA 113507-82-7 2.2 10-400
Perfluoro-3-methoxypropanoic acid PFMPA 377-73-1 2.6 10-400
Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 2.2 10-400
2H,2H,3H,3H-Perfluorohexanoic Acid 3:3 FTCA 356-02-05 3.7 10-400
2H,2H,3H,3H-Perfluorooctanoic Acid 5:3 FTCA 914637-49-3 3.0 10-400
2H,2H,3H,3H-Perfluorodecanoic acid 7:3 FTCA 812-70-4 1.5 10-400
2H-perfluoro-2-octenoic acid FHUEA 70887-88-6 2.5 10-400
2H-perfluoro-2-decenoic acid FOUEA 70887-84-2 2.9 10-400
A
Lithium Bis(trifluoromethane)sulfonimide HQ-115 90076-65-6 9.0 10-400
Surrogates
Perfluoro-n-[ C ]butanoic acid MPFBA NA NA 10-400
Perfluor0-n-[ C ]pentanoic acid M5PFPeA NA NA 10-400
Perfluoro-n-[1,2,3,4,6- C ]hexanoic acid M5PFHxA NA NA 10-400
Perfluoro-n-[1,2,3,4- C ]heptanoic acid M4PFHpA NA NA 10-400
Perfluoro-n-[ C ]octanoic acid M8PFOA NA NA 10-400
Perfluoro-n-[ C ]nonanoic acid M9PFNA NA NA 10-400
Perfluoro-n-[1,2,3,4,5,6- C ]decanoic acid M6PFDA NA NA 10-400
Perfluoro-n-[1,2,3,4,5,6,7- C ]undecanoic acid M7PFUnA NA NA 10-400
Perfluoro-n-[1,2- C ]dodecanoic acid MPFDoA NA NA 10-400
Perfluoro-n-[1,2- C ]tetradecanoic acid M2PFTreA NA NA 10-400
Perfluoro-1-[ C ]octanesulfonamide M8FOSA NA NA 10-400
N-methyl-d -perfluoro-1-octanesulfonamidoacetic acid D3-N-MeFOSAA NA NA 10-400
N-ethyl-d -perfluoro-1-octanesulfonamidoacetic acid D5-N-EtFOSAA NA NA 10-400
N-methyl-d -perfluoro-1-octanesulfanamide d-N-MeFOSA NA NA 10-400
N-ethyl-d -perfluoro-1-octanesulfanamide d-N-EtFOSA NA NA 10-400
2-(N-methyl-d -perfluoro-1-octanesulfonamido)ethan-d4-ol d7-N-MeFOSE NA NA 10-400
2-(N-ethyl-d -perfluoro-1-octanesulfonamido)ethan-d4-ol D9-N-EtFOSE NA NA 10-400
2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy- C - MHFPO-DA NA NA 10-400
propanoic acid
1H,1H,2H,2H-perfluoro-1-[1,2- C ]hexane sulfonate M4:2FTS NA NA 10-400
1H,1H,2H,2H-perfluoro-1-[1,2- C ]-octane sulfonate M6:2FTS NA NA 10-400
1H,1H,2H,2H-perfluoro-1-[1,2- C ]-decane sulfonate M8:2FTS NA NA 10-400
Perfluoro-1-[ C ]octanesulfonate M8PFOS NA NA 10-400
Perfluoro-1-[2,3,4- C ]butanesulfonate MPFBS NA NA 10-400
Perfluoro-1-[1,2,3- C ]hexanesulfonate M3PFHxS NA NA 10-400
A
The Lithium is just the counter ion, report only Bis(trifluoromethane)sulfonimide.
D8421−22
TABLE 2 Gradient Conditions for a Ternary Pumping System
95 % Water:
Time Flow 95 % Water: 5 % Acetonitrile,
Acetonitrile %
(min) (mL/min) 5 % Acetonitrile % 400 mM
Ammonium Acetate %
00.3 95 0 5
1 0.3 75 20 5
6 0.3 50 45 5
13 0.3 15 80 5
14 0.4 0 95 5
17 0.4 0 95 5
18 0.4 95 0 5
21 0.4 95 0 5
7.3.1 Hypodermic Syringe—A luer-lock tip glass syringe 8.12 Acetonitrile (CAS #75-05-8).
capable of holding a syringe driven filter unit.
8.13 Methanol (CAS #67-56-1).
7.3.1.1 A 10-mL Lock Tip Glass Syringe size is recom-
8.14 Ammonium acetate (CAS #631-61-8).
mended in this test method.
7.3.2 Filter Unit—Polypropylene syringe-driven filter units 8.15 Acetic acid (CAS #64-19-7).
(0.2 µm) or equivalent, demonstrated contaminant free below
8.16 2-Propanol (isopropyl alcohol, CAS #67-63-0).
1/2 MRL.
8.17 Ammonium hydroxide (CAS #1336-21-6).
8. Reagents and Materials
8.18 PFASs Standards —Refer to Table 1 for the complete
8.1 Purity of Reagents—High Performance Liquid Chroma- analyte list and CAS numbers. These may be purchased from
tography (HPLC) pesticide residue analysis and spectropho- a commercial supplier individually or some as a mixture.
tometry grade chemicals shall be used in all tests. Unless
9. Hazards
indicated otherwise, it is intended that all reagents shall
conform to the Committee on Analytical Reagents of the
9.1 Precaution—The toxicity or carcinogenicity of chemi-
American Chemical Society. Other reagent grades may be
cals used in this test method has not been precisely defined;
used provided they are first determined to be of sufficiently
each chemical should be treated as a potential health hazard,
highpuritytopermittheirusewithoutaffectingtheaccuracyof
and exposure to these chemicals should be minimized. Each
the measurements.
laboratory is responsible for maintaining awareness of OSHA
regulations regarding safe handling of chemicals used in this
8.2 Purity of Water—Unless otherwise indicated, references
test method.
towatershallbeunderstoodtomeanreagentwaterconforming
toType 1 of Specification D1193. It shall be demonstrated that
9.2 Warning—The compound analytes in this test method
this water does not contain contaminants at concentrations
have been classified as known or suspected human or mam-
sufficient to interfere with the analysis.
malian carcinogens. Pure standards and stock solutions should
be handled in a hood or glovebox.
8.3 Gases—Ultrapure nitrogen and argon.
8.4 Vials—Greater than 1.0 mLAmber glass or polypropyl-
10. Sampling
ene autosampler vials.
10.1 Sampling and Preservation—Avoid sample containers
8.5 Polyethylene autosampler vial caps, or equivalent.
and contact with surfaces of fluorinated polymers or PFAS
contaminateditems.Collectfieldblanksthatareexposedtothe
8.6 Syringe—10 or 25-mL filter-adaptable glass syringe
same field conditions as samples and analyze according to this
with luer lock.
test method to assess the potential for field contamination.
8.7 Polypropylene Tubes—15 and 50 mL conical with cali-
Collect 5 6 0.5 mL samples, duplicates/triplicates, matrix
bration lines.
spikes and field blanks in graduated 15 mL polypropylene
8.8 pH paper (pH range 1–14).
tubes. For greater accuracy, the tubes should be pre-weighed
andweighedaftersamplinginordertoachieveanexactweight
8.9 Class A Volumetric Glassware.
of the sample. This weight is then used to calculate volume
8.10 Pipette tips—Polypropylene pipette tips free of release
with the assumed density of the water sample as 1.0 g/mL.
agents or low retention coating of various sizes.
Conventional sampling practices should be followed with the
8.11 Polyethylene Disposable Pipettes.
caution that PFASs containing products may be present in
sampling equipment. All sampling equipment and supplies
shall be PFAS free to prevent contamination of the samples.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, PFASsstandardsmaybedifficulttofind,somesourcesofPFASsstandardsthat
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma- havebeenfoundsuitableforusewerefromAldrichChemicalCompany,Wellington
copeial Convention, Inc. (USPC), Rockville, MD. LaboratoriesInc.,andWakoLaboratory.Standardsfromothervendorsmaybeused.
D8421−22
EPA Publication SW-846, Guide D3856, and Practices E694 second source and samples shall be in a 50:50 methanol:water
may be used as guides. Ship samples on ice with a trip blank. solution containing 0.1 % acetic acid. In the case of extreme
The temperature of the samples upon receipt at the laboratory
concentration differences amongst samples, it is wise to ana-
shouldbelessthan6°C.Ifthereceivingtemperatureisgreater
lyze a blank after a concentrated sample and before a dilute
than6°C,thesampletemperatureisnotedinthecasenarrative
sample to eliminate carry-over of analytes from sample injec-
accompanying the data. Samples should be stored refrigerated
tiontosampleinjection.Ifaflowthroughneedledesignisused
between 0°C and 6°C from the time of collection until
carry-over should not be a problem. The gradient conditions
analysis. Analyze the sample within 28 days of collection.
for LC are shown in Table 2. To ensure chromatographic
Holdingtimemayvarydependingonthematrixandindividual
separation between the targeted analytes and any unknown
laboratoriesshoulddeterminetheholdingtimeintheirmatrix.
non-targeted potentially interfering compounds, avoid shorten-
ingtheanalysistimesimplytospeedtheanalysis.RefertoFig.
11. Preparation of LC/MS/MS
1 as an example chromatogram of 24 surrogates showing
11.1 LC Chromatograph Operating Conditions:
resolution with limited coelution.
11.1.1 Injections of all standards and samples are made at a
10–30-µL volume. Other injection volumes may be used to 11.2 LC Sample Manager Conditions:
optimize conditions. Calibration Standards, reagent blanks,
11.2.1 Needle Wash Solvent—60 % acetonitrile/40 %
2-propanol. Eight second wash time before and after injection.
Instrumentmanufacturer’sspecificationsshouldbefollowedin
Guides to help determine holding times can be found at: http://www.epa.gov/
order to eliminate sample carry-over.
esd/cmb/research/bs_033cmb06.pdf (2014) and Practice D4841.
FIG. 1Example Chromatogram of 24 Surrogates, at the Level 1 Calibration Concentration, Overlayed Showing Resolution with Limited
Coelution
D8421−22
11.2.2 Temperatures—Column, 35 °C; Sample 12.2.2 Aliquots of SolutionA(Calibration Level 9) are then
compartment, 15°C. dilutedwith50:50methanol:watercontaining0.1%aceticacid
11.2.3 Seal Wash—Solvent: 50% water⁄50 % methanol;
to prepare the desired calibration levels (Table 5) in polypro-
Time: 5 minutes. pylene LC vials. For best results, use the calibration standards
within 24 hours of preparation. Prepare the end CCV at a
11.3 Mass Spectrometer Parameters:
mid-level concentration in a separate LC vial. All calibration
11.3.1 To acquire the maximum number of data points per
standards should be used only once because the analyte
SRM channel while maintaining adequate sensitivity, optimize
concentration in the vial may change after the vial cap is
the tune parameters according to instrument manufacturer
pierced.Changingthecapsimmediatelyaftertheinjectionmay
instructions. Each peak requires a minimum of 10 scans per
alleviate this problem, however, this should be verified in each
peak for adequate quantitation. This test method containing
laboratory. Calibration standards do not need to be filtered.
surrogates, which are select isotopically labeled PFAS, and the
12.2.3 Incorporate a second source standard, if available.
targeted PFAS may be split into multiple MRM acquisition
The second source standard should be analyzed near the
functions to optimize sensitivity. Retention times, and primary
midpoint of the calibration range to verify that the standards
and confirmatory transitions are shown in Table 3. Retention
used are within 630 % of the expected concentration.
times will vary between columns and gradient used. Each
manufacturer may have different terminology to represent Currently, a second source from a different vendor may not be
various mass spectrometer settings, and different set values readily available for all target analytes. In this case, a second
depending on the manufacturer and instrument model. Please lot number from the same vendor may be used.
refer to the manufacturer’s instructions in optimizing detector
12.2.4 Inject each standard and obtain its chromatogram.
settings, including collision energies and cone voltages. Data
The instrument software collects the primary and confirmatory
for this method was collected using electrospray ionization
SRM transitions of each analyte at the specified retention
(ESI) operated in negative mode. In recognition of the ad-
times. Calibration software conducts the quantitation of the
vancement of LCMSMS instrumentation, other MS operating
target analytes and surrogates using the primary SRM transi-
conditions, including ionization techniques may be used pro-
tion. The ratios of the primary/confirmatory MRM transition
vided the quality control criteria of the method is met.
area counts will vary depending on the individual tuning
conditions. Refer to Table 4 for retentionTimes and Ion ratios.
12. Calibration and Standardization
For confirmation of analyte identity, the primary/confirmatory
12.1 The mass spectrometer is calibrated as in accordance
ratio shall be within 30 % of the individual ratios established
withmanufacturer’sspecificationspriortoanalysis.Prepareall
duringtheinitialcalibration.Theaverageionratioiscalculated
calibration solutions using Class A volumetric glassware
for each batch from the initial calibration levels.
(E694).
12.2.5 Depending on sensitivity and sample dependent ma-
trixinterference,theconfirmatorySRMtransitionmaybeused
12.2 Calibration and Standardization—Analyze up to nine
calibration standards containing the PFAS and surrogates prior astheprimarySRMtransitionforquantitationduringanalysis.
to analysis as shown in Table 5.The calibration stock standard
12.2.6 The calibration software manual or the instrument
solution is prepared from the target and surrogate spike
manufacturer should be consulted to ensure correct software
solutions. Stock standard SolutionAcontaining the PFAS and
use. The quantitation method is set using the peak areas in ppt
surrogatesispreparedatCalibrationLevel9concentrationand
(ng/L) units. Concentrations may be calculated using the data
aliquots of that solution are diluted to prepare Calibration
system software to generate linear regression or quadratic
Levels1through8.Thefollowingstepswillproducestandards
calibration curves. Forcing the calibration curve through the
with the concentration values shown in Table 5. The analyst is
origin (X = 0, Y = 0) is not recommended.
responsible for recording initial component weights carefully
12.2.7 Either of two procedures may be used to determine
when working with pure materials and correctly carrying the
calibration function acceptability for linear and non-linear
weights through the dilution calculations. At a minimum, five
curves. These include refitting the calibration data back to the
calibration levels are required when using a linear calibration
model. Both % Error and Relative Standard Error (RSE)
and six calibration levels are required when using a quadratic
evaluate the difference between the measured and the true
calibration curve.An initial nine-points may be used to enable
amounts or concentrations used to create the model.
dropping the lower calibration points if the instrument cannot
12.2.7.1 Calculation of % Error is shown as Eq 1. Percent
achieve low detection limits on certain PFAS. This will allow
error between the calculated and expected amounts should be
at least a five or six-point calibration curve per analyte to be
≤ 30% for all standards.
obtained.
'
x 2 x
12.2.1 Calibration Stock Standard Solution A (Calibration
i i
%Error 5 3100 (1)
Level 9, Table 5) is prepared from the target and surrogate x
i
spike solutions. Transfer 500 µL of the surrogate spike (20
where:
µg/L), 500 µL of PFAS Target Spike I and 500 µL of PFAS
’
x = measured amount of analyte at calibration level i,in
i
Target Spike II (refer to Table 7) to a 50-mL volumetric flask
mass or concentration units, and
and dilute to 50-mL volume with 50:50 methanol:water con-
x = true amount of analyte at calibration level i, in mass or
i
taining 0.1 % acetic acid. Ensure that the analytes are solubi-
concentration units.
lized in the Level 9 standard.
D8421−22
TABLE 3 Transitions for Target Analytes and Surrogates
Analyte Name Acronym CAS Number Primary Ion Transition Confirmation Ion Transition
Perfluorotetradecanoic acid PFTreA 376-06-7 712.9→ 668.9 712.9→ 168.9
Perfluorotridecanoic acid PFTriA 72629-94-8 662.9→ 618.9 662.9→ 168.9
Perfluorododecanoic acid PFDoA 307-55-1 612.9→ 568.9 612.9→ 168.9
Perfluoroundecanoic acid PFUnA 2058-94-8 562.9→ 519 562.9→ 269
Perfluorodecanoic acid PFDA 335-76-2 512.9→ 469 512.9→ 218.9
Perfluorononanoic acid PFNA 375-95-1 462.9→ 419 462.9→ 218.9
Perfluorooctanoic acid PFOA 335-67-1 412.9→ 369 412.9→ 168.9
Perfluoroheptanoic acid PFHpA 375-85-9 362.9→ 318.9 362.9→ 168.9
Perfluorohexanoic acid PFHxA 307-24-4 312.9→ 269 312.9→ 118.9
Perfluoropentanoic acid PFPeA 2706-90-3 262.9→ 218.9 NA
Perfluorobutanoic acid PFBA 375-22-4 212.9→168.9 NA
Perfluorodecanesulfonic acid PFDS 335-77-3 598.9→ 79.9 598.9→ 98.9
Perfluorononanesulfonic acid PFNS 68259-12-1 548.9→ 79.9 548.9→ 98.9
Perfluorooctanesulfonic acid PFOS 1763-23-1 498.9→ 79.9 498.9→ 98.9
Perfluoroheptanesulfonic acid PFHpS 375-92-8 448.9→ 79.9 448.9→ 98.9
Perfluorohexanesulfonic acid PFHxS 355-46-4 398.9→ 79.9 398.9→ 98.9
Perfluoropentanesulfonic acid PFPeS 2706-91-4 348.9→ 79.9 348.9→ 98.9
Perfluorobutanesulfonic acid PFBS 375-73-5 298.9→ 79.9 298.9→ 98.9
Perfluorooctanesulfonamide PFOSA 754-91-6 497.9→ 77.9 NA
8:2 Fluorotelomer sulfonic acid 8:2 FTS 39108-34-4 526.9→ 506.9 526.9→ 80.9
6:2 Fluorotelomer sulfonic acid 6:2 FTS 27619-97-2 427→ 407 427→ 80.9
4:2 Fluorotelomer sulfonic acid 4:2 FTS 757124-72-4 326.9→ 306.9 326.9→ 80.9
N-Ethylperfluorooctanesulfonamidoacetic acid NEtFOSAA 2991-50-6 584→ 419 584→ 482.9
N-Methylperfluorooctanesulfonamidoacetic NMeFOSAA 2355-31-9 569.9→ 419 569.9→ 482.9
acid
Perfluorododecanesulfonic acid PFDoS 79780-39-5 698.9→ 79.9 698.9→ 98.9
N-Methylperfluorooctanesulfonamide NMeFOSA 31506-32-8 511.9→ 168.9 511.9→ 218.9
N-Ethylperfluorooctanesulfonamide NEtFOSA 4151-50-2 525.9→ 168.9 525.9→ 218.9
N-Methylperfluorooctanesulfonamidoethanol NMeFOSE 24448-09-7 616→ 58.9 NA
N-Ethylperfluorooctanesulfonamidoethanol NEtFOSE 1691-99-2 630→ 58.9 NA
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 285→ 168.9 285→ 184.9
4,8-dioxa-3H-perfluorononanoic acid ADONA 919005-14-4 376.9→ 251 376.9→ 84.9
9-chlorohexadecafluoro-3-oxanonane-1- 9Cl-PF3ONS 756426-58-1 530.9→ 350.9 532.9→ 352.9
sulfonic acid
11-chloroeicosafluoro-3-oxaundecane-1- 11Cl-PF3OUdS 763051-92-9 630.8→ 450.9 632.8→ 452.9
sulfonic acid
Pentafluorpropanoic acid PFPrA 422-64-0 162.9→ 118.9 NA
Perfluoro-3,6-dioxaheptanoic acid NFDHA 151772-58-6 295→ 200.9 295→ 84.9
Perfluoro(2-ethoxyethane)sulfonic acid PFEESA 113507-82-7 314.9→ 134.9 314.9→ 82.9
Perfluoro-3-methoxypropanoic acid PFMPA 377-73-1 228.9→ 84.9 NA
Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 278.9→ 84.9 NA
2H,2H,3H,3H-Perfluorohexanoic Acid 3:3 FTCA 356-02-05 241→ 176.9 241→ 116.9
2H,2H,3H,3H-Perfluorooctanoic Acid 5:3 FTCA 914637-49-3 340.9→ 216.9 340.9→237
2H,2H,3H,3H-Perfluorodecanoic acid 7:3 FTCA 812-70-4 440.9→ 337 440.9→ 316.9
2H-perfluoro-2-octenoic acid FHUEA 70887-88-6 356.9→ 292.9 NA
2H-perfluoro-2-decenoic acid FOUEA 70887-70-4 456.9→ 393 NA
Lithium Bis(trifluoromethane)sulfonimide HQ-115 90076-65-6 279.9→146.9 279.9→210.9
Surrogates
Perfluoro-n-[ C ]butanoic acid MPFBA NA 216.9→ 171.9 NA
Perfluoro-n-[ C ]pentanoic acid M5PFPeA NA 267.9→ 222.9 NA
Perfluoro-n-[1,2,3,4,6- C ]hexanoic acid M5PFHxA NA 317.9→ 272.9 NA
Perfluoro-n-[1,2,3,4- C ]heptanoic acid M4PFHpA NA 366.9→ 321.9 NA
Perfluoro-n-[ C ]octanoic acid M8PFOA NA 421→ 376 NA
Perfluoro-n-[ C ]nonanoic acid M9PFNA NA 471.9→ 426.9 NA
Perfluoro-n-[1,2,3,4,5,6- C ]decanoic acid M6PFDA NA 518.9→ 473.9 NA
Perfluoro-n-[1,2,3,4,5,6,7- C7]undecanoic M7PFUnA NA 569.9→ 524.9 NA
acid
Perfluoro-n-[1,2- C ]dodecanoic acid MPFDoA NA 614.9→ 569.9 NA
Perfluoro-n-[1,2- C ]tetradecanoic acid M2PFTreA NA 714.9→ 669.9 NA
Perfluoro-1-[ C ]octanesulfonamide M8FOSA NA 505.9→ 77.9 NA
N-methyl-d -perfluoro-1- D3-N-MeFOSAA NA 572.9→ 418.9 NA
octanesulfonamidoacetic acid
N-ethyl-d -perfluoro-1- D5-N-EtFOSAA NA 589→ 418.9 NA
octanesulfonamidoacetic acid
N-methyl-d -perfluoro-1-octanesulfanamide d-N-MeFOSA NA 514.9→ 168.9 NA
N-ethyl-d -perfluoro-1-octanesulfanamide d-N-EtFOSA NA 531→168.9 NA
2-(N-ethyl-d -perfluoro-1- d7-N-MeFOSE NA 623→ 58.9 NA
octanesulfonamido)ethan-d4-ol
2-(N-methyl-d -perfluoro-1- D9-N-EtFOSE NA 639→ 58.9 NA
octanesulfonamido)ethan-d4-ol
2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3- MHFPO-DA NA 287→ 168.9 NA
heptafluoropropoxy- C -propanoic acid
1H,1H,2H,2H-perfluoro-1-[1,2- C ]hexane M4:2FTS NA 328.9→ 308.9 NA
A
sulfonate 328.9→ 80.9
D8421−22
TABLE3 Continued
Analyte Name Acronym CAS Number Primary Ion Transition Confirmation Ion Transition
1H,1H,2H,2H-perfluoro-1-[1,2- C ]-octane M6:2FTS NA 428.9→ 408.9 NA
A
sulfonate 428.9→ 80.9
1H,1H,2H,2H-perfluoro-1-[1,2- C ]-decane M8:2FTS NA 528.9→ 508.9 NA
A
sulfonate 528.9→ 80.9
Perfluoro-1-[ C ]octanesulfonate M8PFOS NA 506.9→ 79.9 NA
Perfluoro-1-[2,3,4- C ]butanesulfonate M3PFBS NA 301.9→ 79.9 NA
Perfluoro-1-[1,2,3- C ]hexanesulfonate M3PFHxS NA 401.9→ 79.9 NA
A
If high concentrations of the native FTS interfere with Isotope of the FTSs, this transition should be used. It is not as sensitive, but the interference/high bias is removed.
12.2.7.2 Calculation of Relative Standard Error (RSE – 13.2 If a laboratory has not performed the test before or if
expressed as %) is shown in Eq 2. The RSE acceptance limit there has been a major change in the measurement system, for
criterionforthecalibrationmodelisthesameastheRSDlimit. example, new analyst, new instrument, etc., an instrument
qualification study including method detection limit (MDL),
n
' 2
x 2 x
i i
calibration range determination and precision and bias deter-
RSE 5100 3Œ ⁄ ~n 2 p! (2)
F G
(
x
i51
i
mination shall be performed to demonstrate laboratory capa-
bility.
where:
13.2.1 Analyze at least four replicates of a spiked water
x = true amount of analyte in calibration level i, in mass or
i
sample containing the analytes and surrogates at a prepared
concentration units,
’
sample concentration in the range of Calibration Levels 4–7.
x = measured amount of analyte in calibration level i,in
i
Calibration Level 6 was used to establish the QC acceptance
mass or concentration units,
criteria in this test method. Take each replicate through the
p = number of terms in the fitting equation (average = 1,
complete analytical test method including any sample manipu-
linear = 2, quadratic = 3, cubic = 4), and
lation and pretreatment steps.
n = number of calibration points.
13.2.2 Calculate the mean (average) percent recovery and
12.2.8 The retention time window of an unknown shall be
relative standard deviation (RSD) of the four values and
within5%ofthe retention time of the analyte in a midpoint
comparetotheacceptablerangesoftheQCacceptancecriteria
calibration standard. If this is not the case, re-analyze the
for the Initial Demonstration of Performance in Table 6.
calibration curve to determine if there was a shift in retention
13.2.3 Repeat until the single operator precision and mean
time during the analysis. If the retention time of the known
recovery are within the limits in Table 6. If a concentration
standard is correct, and the retention time of the peak in the
other than the recommended concentration is used, refer to
sampleisstillincorrectinthesample,refertotheanalyteasan
PracticeD5847forinformationonapplyingtheFtestandttest
unknown.
in evaluating the acceptability of the mean and standard
12.2.9 Analyze a CCV at the end of each batch of 20
deviation.
samples, 20 samples does not include QC samples. This
13.2.3.1 The QC acceptance criteria for the Initial Demon-
interval may be tightened according to the laboratory’s QA
stration of Performance in Table 6 were generated from the
programoraccreditationrequirements.ThisendCCV,inanew
combined single-laboratory data from reagent water as shown
never pierced sealed vial, should come from the same stock
inSection17.Laboratoriesshouldgeneratetheirownin-house
calibration standard solution that was used to generate the
QCacceptancecriteriawhichmeetorexceedthecriteriainthis
initial calibration curve.The concentration of each analyte and
test method. References on how to generate QC acceptance
surrogate in the end CCV standard shall be within 30 % of the
criteria are Practices D2777, D5847, and E2554.
expectedconcentration.Iftheconcentrationisnotwithin30%,
corrective action is performed and either all samples in the
13.3 Surrogate Spiking Solution:
batcharere-analyzedagainstanewcalibrationcurveorqualify
13.3.1 A surrogate spiking solution containing each isoto-
affected samples. If the analyst inspects the vial containing the
pically labeled PFAS are added to all samples; including
end CCV and notices a probable cause for the failure, a new
method blanks, duplicates, laboratory control samples, matrix
end CCVmay be prepared and analyzed. If this new end CCV
spikes, and reporting limit checks. A stock surrogate spiking
is within 30 % from the expected concentration for the target
solutionispreparedat20µg⁄Lin95%MeOH:5%water.Add
analytes and surrogates, the results do not need to be qualified.
40 µL of this spiking solution into a 5-mL water sample for a
concentration of 160 ng/L of the surrogate in the sample. The
13. Quality Control
results obtained for the surrogate recoveries shall fall within
13.1 Quality control (QC) requirements include the initial the limits of Table 6. If the limits are not met, the affected
resultsshallbequalifiedwithanindicationthattheydonotfall
demonstration of laboratory capability followed by routine
analysesoflaboratoryreagentblanks,fieldreagentblanks,and within the performance criteria of the test method.
laboratory fortified blanks, and matrix spikes. The laboratory 13.3.1.1 Preparethesurrogatespikingsolutionmixcontain-
must maintain records to document the quality of the data ing all twenty-four surrogates shown in Table 1 with 95 %
generated.The criteria in this section were used for, or derived MeOH: 5 % water. It should be replaced every year if not
from, the method validation. previously discarded for quality-control failure.
D8421−22
TABLE 4 Retention Times and Ion Ratios for Target An
...