£EPA
United States
Environmental Protection
Agency
METHOD 533: DETERMINATION OF PER- AND
POLYFLUOROALKYL SUBSTANCES IN DRINKING WATER BY
ISOTOPE DILUTION ANION EXCHANGE SOLID PHASE
EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM
MASS SPECTROMETRY
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Technical questions concerning this analytical method should be addressed to:
Steven C. Wendelken, Ph.D.
U.S. EPA, Office of Ground Water and Drinking Water, Standards and Risk Management Division,
Technical Support Center, 26 W. Martin Luther King Dr. Cincinnati, OH 45268
Phone:(513)569-7491
wendelken.steve@epa.gov
Questions concerning this document or policy should be addressed to: safewater@epa.gov
Office of Water (MS-140)
EPA Document No. 815-B-19-020
EPA contract EP-C-17-014
November 2019
Laura Rosenblum, Ph.D., APTIM (Cincinnati, OH)
Contractor's role did not include establishing Agency policy.
Steven C. Wendelken, Ph.D., U.S. EPA (Cincinnati, OH)
Ackno- I in in^
Alan Zaffiro, APTIM (Cincinnati, OH)
The following organizations completed a validation study in their laboratories using this method,
provided valuable feedback on the method procedures and reviewed the draft method manuscript:
Babcock Laboratories, Inc. (Riverside, CA)
Eurofins Eaton Analytical, LLC (South Bend, IN)
Eurofins TestAmerica (Sacramento, CA)
Merit Laboratories, Inc. (East Lansing, Ml)
Shimadzu Scientific Instruments (Columbia, MD)
Thermo Fisher Scientific (Sunnyvale, CA)
Week Laboratories, Inc. (City of Industry, CA)
Vogon Laboratory Services Ltd. (Cochrane, Alberta, Canada)
It •iv'.tmifi'T
This analytical method may support a variety of monitoring applications, which include the analysis of
multiple short-chain per- and polyfluoroalkyl substances (PFAS) that cannot be measured by Method
537.1. This publication meets an agency commitment identified within the 2019 EPA PFAS Action Plan.
Publication of the method, in and of itself, does not establish a requirement, although the use of this
method may be specified by the EPA or a state through independent actions. Terms such as "must" or
"required," as used in this document, refer to procedures that are to be followed to conform with the
method. References to specific brands and catalog numbers are included only as examples and do not
imply endorsement of the products. Such reference does not preclude the use of equivalent products
from other vendors or suppliers.
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Table of Contents
1 Scope and Application 1
2 Method Summary 2
3 Definitions 3
4 Interferences 5
5 Safety 7
6 Equipment and Supplies 8
7 Reagents and Standards 10
8 Sample Collection, Preservation, and Storage 16
9 Quality Control 17
10 Calibration and Standardization 22
11 Procedure 26
12 Data Analysis and Calculations 29
13 Method Performance 31
14 Pollution Prevention 31
15 Waste Management 31
16 References 31
17 Tables, Figures and Method Performance Data 32
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Tables
Table 1. HPLC Method Conditions 32
Table 2. ESI-MS Method Conditions 32
Table 3. Isotopically Labeled Isotope Performance Standards and Retention Times 33
Table 4. Isotope Dilution Analogues: RTs and Suggested Isotope Performance Standard References 33
Table 5. Method Analytes, Retention Times and Suggested Isotope Dilution Analogue References 34
Table 6. MS/MS Method Conditions 35
Table 7. LCMRL Results 37
Table 8. Precision and Accuracy Data for Reagent Water 38
Table 9. P&A in Reagent Water: Isotope Dilution Analogue Recovery Data 39
Table 10. Precision and Accuracy Data for Finished Ground Water 40
Table 11. P&A in Finished Ground Water: Isotope Dilution Analogue Recovery Data 41
Table 12. Precision and Accuracy Data for a Surface Water Matrix 42
Table 13. P&A in Surface Water Matrix: Isotope Dilution Analogue Recovery Data 43
Table 14. Aqueous Sample Holding Time Data 44
Table 15. Extract Holding Time Data 45
Table 16. Initial Demonstration of Capability (IDC) Quality Control Requirements 46
Table 17. Ongoing Quality Control Requirements 46
Figures
Figure 1. Example Chromatogram for Reagent Water Fortified with Method Analytes at 80 ng/L 48
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1 Scope and Application
This is a solid phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS)
method for the determination of select per- and polyfluoroalkyl substances (PFAS) in drinking water.
Method 533 requires the use of MS/MS in Multiple Reaction Monitoring (MRM) mode to enhance
selectivity. Accuracy and precision data have been generated in reagent water and drinking water for
the compounds included in the Analyte List.
This method is intended for use by analysts skilled in the performance of solid phase extractions, the
operation of LC-MS/MS instrumentation, and the interpretation of the associated data.
Analyte List
Analyte3
Abbreviation
CASRN
ll-Chloroeicosafluoro-3-oxaundecane-l-sulfonic acid
HCI-PF30UdS
763051-92-9
9-Chlorohexadecafluoro-3-oxanonane-l-sulfonic acd
9CI-PF30NS
756426-58-1
4,8-Dioxa-3H-perfluorononanoic acid
ADONA
919005-14-4
Hexafiuoropropyiene oxide dimer acid
HFPO-DA
13252-13-6
Nonafluoro-3,6-dioxaheptanoic acid
NFDHA
151772-58-6
Perfluorobutanoic acid
PFBA
375-22-4
Perfluorobutanesulfonic acid
PFBS
375-73-5
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid
8:2FTS
39108-34-4
Perfluorodecanoic acid
PFDA
335-76-2
Perfluorododecanoic acid
PFDoA
307-55-1
Perfluoro(2-ethoxyethane)sulfonic acid
PFEESA
113507-82-7
Perfluoroheptanesulfonic acid
PFHpS
375-92-8
Perfluoroheptanoic acid
PFHpA
375-85-9
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid
4:2FTS
757124-72-4
Perfluorohexanesulfonic acid
PFHxS
355-46-4
Perfluorohexanoic acid
PFHxA
307-24-4
Perfluoro-3-methoxypropanoic acid
PFMPA
377-73-1
Perfluoro-4-methoxybutanoic acid
PFMBA
863090-89-5
Perfluorononanoic acid
PFNA
375-95-1
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid
6:2FTS
27619-97-2
Perfluorooctanesulfonic acid
PFOS
1763-23-1
Perfluorooctanoic acid
PFOA
335-67-1
Perfluoropentanoic acid
PFPeA
2706-90-3
Perfluoropentanesulfonic acid
PFPeS
2706-91-4
Perfluoroundecanoic acid
PFUnA
2058-94-8
Some PFAS are commercially available as ammonium, sodium, and potassium salts. This method measures all
forms of the analytes as anions while the identity of the counterion is inconsequential. Analytes may be
purchased as acids or as any of the corresponding salts.
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1.1 Detection of PFAS Isomers
Both branched and linear PFAS isomers may be found in the environment. This method includes
procedures for summing the contribution of multiple isomers to the final reported concentration. In
those cases where standard materials containing multiple isomers are commercially available,
laboratories should obtain such standards for the method analytes.
1.2 Lowest Concentration Minimum Reporting Limits
The lowest concentration minimum reporting level (LCMRL) is the lowest concentration for which the
future recovery is predicted to fall between 50 and 150% with high confidence (99%). Single-laboratory
LCMRLs determined for the method analytes during method development are reported in Table 7. It
should be noted that most of the LCMRL values determined during the second laboratory evaluation
were lower than the values listed in Table 7. The values that a laboratory can obtain are dependent on
the design and capability of the instrumentation used. The procedure used to determine the LCMRL is
described elsewhere. ^ Laboratories using this method are not required to determine LCMRLs, but they
must demonstrate that they are able to meet the minimum reporting level (MRL) (Sect. 3.15) for each
analyte per the procedure described in Section 9.1.4.
1.3 Method Flexibility
The laboratory may select LC columns, LC conditions, and MS conditions different from those used to
develop the method. At a minimum, the isotope dilution standards and the isotope performance
standards specified in the method must be used, if available. The laboratory may select the aqueous
sample volume within the range of 100-250 mLthat meets their objectives. During method
development, 250 mL aqueous samples were extracted using a 500 mg solid phase extraction (SPE)
sorbent bed volume. The ratio of sorbent mass to aqueous sample volume may not be decreased. If a
laboratory uses 100 mL aqueous samples, the sorbent mass must be at least 200 mg. Changes may not
be made to sample preservation, the quality control (QC) requirements, or the extraction procedure.
The chromatographic separation should minimize the number of compounds eluting within a retention
window to obtain a sufficient number of scans across each peak. Instrumental sensitivity (or signal-to-
noise) will decrease if too many compounds are permitted to elute within a retention time window.
Method modifications should be considered only to improve method performance. In all cases where
method modifications are proposed, the analyst must perform the procedures outlined in the Initial
Demonstration of Capability (IDC, Sect. 9.1). verify that all QC acceptance criteria in this method
(Sect. 9.2) are met, and verify method performance in a representative sample matrix (Sect. 9.3.2).
2 Method Summary
A 100-250 mL sample is fortified with isotopically labeled analogues of the method analytes that
function as isotope dilution standards. The sample is passed through an SPE cartridge containing
polystyrene divinylbenzene with a positively charged diamino ligand to extract the method analytes and
isotope dilution analogues. The cartridge is rinsed with sequential washes of aqueous ammonium
acetate followed by methanol, then the compounds are eluted from the solid phase sorbent with
methanol containing ammonium hydroxide. The extract is concentrated to dryness with nitrogen in a
heated water bath. The extract volume is adjusted to 1.0 mL with 20% water in methanol (v/v), and
three isotopically labeled isotope performance standards are added. Extracts are analyzed by LC-MS/MS
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in the MRM detection mode. The concentration of each analyte is calculated using the isotope dilution
technique. For QC purposes, the percent recoveries of the isotope dilution analogues are calculated
using the integrated peak areas of isotope performance standards, which are added to the final extract
and function as traditional internal standards, exclusively applied to the isotope dilution analogues.
3 Definitions
3.1 Analysis Batch
A set of samples that are analyzed on the same instrument during a 24-hour period that begins and ends
with the analysis of the appropriate Continuing Calibration Check (CCC) standards. Additional CCCs may
be required depending on the length of the Analysis Batch and the number of field samples.
3.2 Calibration Standard
A solution of the method analytes, isotope dilution analogues, and isotope performance standards
prepared from the Primary Dilution Standards and stock standards. The calibration standards are used
to calibrate the instrument response with respect to analyte concentration.
3.3 Continuing Calibration Check (CCC)
A calibration standard that is analyzed periodically to verify the accuracy of the existing calibration.
3.4 Extraction Batch
A set of up to 20 field samples (not including QC samples) extracted together using the same lot of solid
phase extraction devices, solvents, and fortifying solutions.
3.5 Field Duplicates (FD)
Separate samples collected at the same time and sampling location, shipped and stored under identical
conditions. Method precision, including the contribution from sample collection procedures, is
estimated from the analysis of Field Duplicates. Field Duplicates are used to prepare Laboratory Fortified
Sample Matrix and Laboratory Fortified Sample Matrix Duplicate QC samples. For the purposes of this
method, Field Duplicates are collected to support potential repeat analyses (if the original field sample is
lost or if there are QC failures associated with the analysis of the original field sample).
3.6 Field Reagent Blank (FRB)
An aliquot of reagent water that is placed in a sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling site, exposure to sampling site conditions,
storage, and all analytical procedures. The purpose of the FRB is to determine if method analytes or
other interferences are introduced into the sample from shipping, storage, and the field environment.
3.7 Isotope Dilution Analogues
Isotopically labeled analogues of the method analytes that are added to the sample prior to extraction in
a known amount. Note: Not all target PFAS currently have an isotopically labelled analogue. In these
cases, an alternate isotopically labelled analogue is used as recommended in Table 5.
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3.8 Isotope Dilution Technique
An analytical technique for measuring analyte concentration using the ratio of the peak area of the
native analyte to that of an isotopically labeled analogue, added to the original sample in a known
amount and carried through the entire analytical procedure.
3.9 Isotope Performance Standards
Quality control compounds that are added to all standard solutions and extracts in a known amount and
used to measure the relative response of the isotopically labelled analogues that are components of the
same solution. For this method, the isotope performance standards are three isotopically labeled
analogues of the method analytes. The isotope performance standards are indicators of instrument
performance and are used to calculate the recovery of the isotope dilution analogues through the
extraction procedure. In this method, the isotope performance standards are not used in the calculation
of the recovery of the native analytes.
3.10 Laboratory Fortified Blank (LFB)
An aliquot of reagent water to which known quantities of the method analytes and isotope dilution
analogues are added. The results of the LFB verify method performance in the absence of sample
matrix.
3.11 Laboratory Fortified Sample Matrix (LFSM)
An aliquot of a field sample to which known quantities of the method analytes and isotope dilution
analogues are added. The purpose of the LSFM is to determine whether the sample matrix contributes
bias to the analytical results. Separate field samples are required for preparing fortified matrix so that
sampling error is included in the accuracy estimate.
3.12 Laboratory Fortified Sample Matrix Duplicate (LFSMD)
A Field Duplicate of the sample used to prepare the LFSM that is fortified and analyzed identically to the
LFSM. The LFSMD is used instead of the Field Duplicate to assess method precision when the method
analytes are rarely found at concentrations greater than the MRL.
3.13 Laboratory Reagent Blank (LRB)
An aliquot of reagent water fortified with the isotope dilution analogues and processed identically to a
field sample. An LRB is included in each Extraction Batch to determine if the method analytes or other
interferences are introduced from the laboratory environment, the reagents, glassware, or extraction
apparatus.
3.14 Lowest Concentration Minimum Reporting Level (LCMRL)
The single-laboratory LCMRL is the lowest spiking concentration such that the probability of spike
recovery in the 50% to 150% range is at least 99%.
3.15 Minimum Reporting Level (MRL)
The minimum concentration that may be reported by a laboratory as a quantified value for a method
analyte. For each method analyte, the concentration of the lowest calibration standard must be at or
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below the MRL and the laboratory must demonstrate its ability to meet the MRL per the criteria defined
in Section 9.1.4.
3.16 Precursor Ion
The gas-phase species corresponding to the method analyte that is produced in the electrospray
ionization interface. During tandem mass spectrometry, or MS/MS, the precursor ion is mass selected
and fragmented by collision-activated dissociation to produce distinctive product ions of smaller mass to
charge (m/z) ratio. For this method, the precursor ion is usually the deprotonated molecule ([M - H]~) of
the method analyte, except for HFPO-DA. For this analyte, the precursor ion is formed by
decarboxylation of HFPO-DA.
3.17 Primary Dilution Standard (PDS)
A solution that contains method analytes (or QC analytes) prepared from stock standards. PDS solutions
are used to fortify QC samples and diluted to prepare calibration standards.
3.18 Product Ion
One of the fragment ions that is produced in MS/MS by collision-activated dissociation of the precursor
ion.
3.19 Quality Control Standard (QCS)
A calibration standard prepared independently from the primary calibration solutions. For this method,
the QCS is a repeat of the entire dilution scheme starting with the same stock materials (neat
compounds or purchased stock solutions) used to prepare the primary calibration solutions.
Independent sources and separate lots of the starting materials are not required, provided the
laboratory has obtained the purest form of the starting materials commercially available. The purpose of
the QCS is to verify the integrity of the primary calibration standards.
3.20 Quantitative Standard
A quantitative standard of assayed concentration and purity traceable to a Certificate of Analysis.
3.21 Stock Standard Solution
A concentrated standard that is prepared in the laboratory using assayed reference materials or that is
purchased from a commercial source with a Certificate of Analysis.
3.22 Technical-Grade Standard
As defined for this method, a technical-grade standard includes a mixture of the branched and linear
isomers of a method analyte. For the purposes of this method, technical-grade standards are used to
identify retention times of branched and linear isomers of method analytes.
4 Interferences
4.1 Labware, Reagents and Equipment
Method interferences may be caused by contaminants in solvents, reagents (including reagent water),
sample bottles and caps, and other sample processing hardware that lead to discrete artifacts or
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elevated baselines in the chromatograms. The analytes in this method can also be found in many
common laboratory supplies and equipment, such as PTFE (polytetrafluoroethylene) products, LC
solvent lines, methanol, aluminum foil, deactivated syringes, SPE sample transfer lines, etc.-
Laboratories must demonstrate that these items are not contributing to interference by analyzing LRBs
as described in Section 9.2.1.
4.2 Sample Contact with Glass
Aqueous samples should not come in contact with any glass containers or pipettes as PFAS analytes can
potentially adsorb to glass surfaces. Standards dissolved in organic solvent may be purchased in glass
ampoules. These standards in organic solvent are acceptable and subsequent transfers may be
performed using glass syringes and pipets. Following extraction, the eluate must be collected in a
polypropylene tube prior to concentration to dryness. Concentration to dryness in glass tubes may cause
poor recovery.
4.3 Matrix Interferences
Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent
of matrix interferences will vary considerably from source to source, depending upon the nature of the
water. Humic and fulvic material may be co-extracted during SPE and high levels may cause
enhancement or suppression in the electrospray ionization source.- Inorganic salts may cause low
recoveries during the anion-exchange SPE procedure.
4.3.1 Co-extracted Organic Material
Under the LC conditions used during method development, matrix effects due to co-extracted organic
material enhanced the ionization of 4:2 FTS appreciably. Total organic carbon (TOC) is a good indicator
of humic content of the sample.
4.3.2 Inorganic Salts
The authors confirmed acceptable method performance for matrix ion concentrations up to 250 mg/L
chloride, 250 mg/L sulfate, and 340 mg/L hardness measured as CaC03. Acceptable performance was
defined as recovery of the isotope dilution analogues between 50-200%.
4.3.3 Ammonium Acetate
Relatively large quantities of ammonium acetate are used as a preservative. The potential exists for
trace-level organic contaminants in this reagent. Interferences from this source should be monitored by
analysis of LRBs, particularly when new lots of this reagent are acquired.
4.3.4 SPE Cartridges
Solid phase extraction cartridges may be a source of interferences. The analysis of LRBs provides
important information regarding the presence or absence of such interferences. Each brand and lot of
SPE devices must be monitored to ensure that contamination does not preclude analyte identification
and quantitation. SPE cartridges should be sealed while in storage to prevent ambient contamination of
the SPE sorbent.
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4.4 Bias Caused by IsotopicaIly Labeled Standards
During method development, no isotopically labeled standard solution yielded any signal that gave the
same mass and retention time as any native analyte. However, due to isotopic impurity, the 13C3-PFBA
isotope performance standard contained a small amount of 13C4-PFBA, slightly contributing to the signal
of the isotope dilution analogue. Further, due to natural abundance of 34S, the native telomer sulfonates
produced a small contribution to the 13C2 labeled telomer sulfonate isotope dilution analogues. The
effects on quantitation are insignificant. However, these cases are described below in Sections 4.4.2 and
4.4.3 to alert the user that these situations could occur.
4.4.1 Method Analytes
At the concentrations used to collect method performance data, the authors could not detect any
contribution from the isotope dilution analogues or isotope performance standards to the
corresponding native analyte response. However, the user should evaluate each source of isotopically
labeled analogues and isotope performance standards to verify that they do not contain any native
analyte at concentrations greater than 1/3 of the MRL.
4.4.2 Isotopic purity of 13C3-PFBA
In this method, 13C3-PFBA is used as an isotope performance standard and 13GrPFBA is used as an
isotope dilution analogue. Both share the same product ion, m/z 172. Ten nanograms per liter of 13C4-
PFBA is added to the sample prior to extraction (10 ng/mL extract concentration assuming 100%
recovery), and 10 ng/mL of 13C3-PFBA is added to the final extract. Because the natural abundance of 13C
is 1.1%, there is a 1.1% contribution to the 13C4-PFBA area from the lone, unlabeled 12C atom in 13C3-
PFBA. The authors confirmed this contribution empirically. Users of this method may consider this bias
to the area of the PFBA isotope dilution analogue insignificant.
4.4.3 Isotopic purity of 13C4-PFBA
A trace amount of 13C3-PFBA was detected in the 13C4-PFBA. The contribution was no greater than 1%.
The contribution of the isotope performance standard to the isotope dilution analogue is insignificant.
4.4.4 Telomer Sulfonates
Each of the three telomer sulfonates in the analyte list (4:2FTS, 6:2FTS, and 8:2FTS) are referenced to
their 13C2 isotope dilution analogue. The mass difference between the telomer sulfonates and the
isotope dilution analogues is 2 mass units. The single sulfur atom in each of the unlabeled molecules has
a naturally occurring M+2 isotope (34S) at 4.25%. Thus, the precursor ions of the 13C2 isotopically labeled
analogues and the naturally occuring 34S analogues present in the native analytes have the same
nominal masses. The product ions of the telomer sulfonate isotope dilution analogues listed in Table 6
would contain a small contribution from the 34S analogue of the native telomer sulfonates. At the
concentrations used in this study, the contribution of the 34S analogue to the isotope dilution analogue
was not greater than 2.7%. Alternate product ions may be used if there is sufficient abundance.
5 Safety
Each chemical should be treated as a potential health hazard and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining an awareness of OSHA regulations regarding
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safe handling of chemicals used in this method. A reference file of safety data sheets should be made
available to all personnel involved in the chemical analysis.
6 Equipment and Supplies
References to specific brands and catalog numbers are included as examples only and do not imply
endorsement of the products. Such reference does not preclude the use of equivalent products from
other vendors or suppliers. Due to potential adsorption of analytes onto glass, polypropylene containers
were used for sample preparation and extraction steps. Other plastic materials (e.g., polyethylene) that
meet the QC requirements of Section 9 may be substituted.
6.1 Sample Containers
Polypropylene bottles with polypropylene screw caps (for example, 250 mL bottles, Fisher Scientific, Cat.
No. 02-896-D or equivalent).
6.2 Polypropylene Vials
These vials are used to store stock standards and PDS solutions (4 mL, VWR Cat. No. 16066-960 or
equivalent).
6.3 Centrifuge Tubes
Conical polypropylene centrifuge tubes (15 mL) with polypropylene screw caps for storing standard
solutions and for collection of the eluate during the extraction procedure (Thomas Scientific Cat. No.
2602A10 or equivalent).
6.4 Autosampler Vials
Polypropylene autosampler vials (ThermoFisher, Cat. No. C4000-14) with polypropylene caps
(ThermoFisher, Cat. No. C5000-50 or equivalent). Note: Polypropylene vials and caps are necessary to
prevent contamination of the sample from PTFE coated septa. However, polypropylene caps do not
reseal, creating the potential for evaporation to occur after injection. Multiple injections from the same
vial are not permissible unless the cap is replaced immediately after injection.
6.5 Micro Syringes
Suggested sizes include 10, 25, 50, 100, 250, 500 and 1000 piL.
6.6 Pi pets
Polypropylene or glass pipets may be used for methanolic solutions.
6.7 Analytical Balance
Capable of weighing to the nearest 0.0001 g.
6.8 Solid Phase Extraction (SPE) Apparatus
6.8.1 SPE Cartridges
SPE cartridges containing weak anion exchange, mixed-mode polymeric sorbent (polymeric backbone
and a diamino ligand), particle size approximately 33 pim. The SPE sorbent must have a pKa above 8 so
that it remains positively charged during extraction. SPE cartridges containing 500 mg sorbent
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(Phenomenex Cat. No. 8B-S038-HCH) were used during method development. Use of 200 mg cartridges
is acceptable for the extraction of 100 mL samples.
6.8.2 Vacuum Extraction Manifold
Equipped with flow and vacuum control [Supelco Cat. No. 57030-U, UCT Cat. No. VMF016GL (the latter
requires UCT Cat. No. VMF02116 control valves), or equivalent systems]. Automated devices designed
for use with SPE cartridges may be used; however, all extraction and elution steps must be the same as
in the manual procedure. Care must be taken with automated SPE systems to ensure that Teflon tubing
and other PTFE components commonly used in these systems, do not contribute to unacceptable
analyte concentrations in LRBs.
6.8.3 Sample Delivery System
Use of large volume sampling lines, constructed with polyethylene tubing, are recommended, but not
mandatory. Large volume sample transfer lines, constructed with PTFE tubing, are commercially
available for standard extraction manifolds (Supelco Cat. No. 57275 or equivalent). The PTFE tubing can
be replaced with 1/8" o.d. x 1/16" i.d. polyethylene tubing [Freelin-Wade (McMinnville, Oregon) LLDPE
or equivalent] cut to an appropriate length. This prevents potential contamination from PTFE transfer
lines. Other types of non-PTFE tubing may be used provided it meets the LRB and LFB QC requirements.
PTFE tubing may be used, but an LRB must be run on each individual transfer line and the QC
requirements in Section 9.2.1 must be met. In the case of automated SPE, the removal of PTFE lines may
not be feasible; therefore, acceptable performance for the LRB must be met for each port during the IDC
(Sect 9.1.1). LRBs must be rotated among the ports during routine analyses thereafter. Plastic reservoirs
are difficult to rinse during elution and their use may lead to lower recovery.
6.9 Extract Concentration System
Extracts are concentrated by evaporation with high-purity nitrogen using a water bath set no higher
than 60 °C [N-Evap, Model 11155, Organomation Associates (Berlin, MA), Inc., or equivalent],
6.10 Laboratory Vacuum System
Sufficient capacity to maintain a vacuum of approximately 15 to 20 inches of mercury for extraction
cartridges.
6.11 pH Meter
Used to verify the pH of the phosphate buffer and to measure the pH of the aqueous sample prior to
anion exchange SPE.
6.12 LC-MS/MS System
6.12.1 LC System
The LC system must provide consistent sample injection volumes and be capable of performing binary
linear gradients at a constant flow rate. On some LC systems, PFAS may build up in PTFE transfer lines
when the system is idle for more than one day. To prevent long delays in purging high levels of PFAS
from the LC solvent lines, it may be useful to replace PTFE tubing with PEEK™ tubing and the PTFE
solvent frits with stainless steel frits. These modifications were not used on the LC system used for
method development. However, a delay column, HLB Direct Connect 2.1 x 30 mm (Waters 186005231),
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was placed in the mobile phase flow path immediately before the injection valve. This direct connect
column may have reduced the co-elution of PFAS originating from sources prior to the sample loop from
the PFAS injected in the sample. It may not be possible to remove all PFAS background contamination.
6.12.2 Analytical Column
C18 liquid chromatography column (2 x 50 mm) packed with 3 pim C18 solid phase particles
(Phenomenex Part Number 00B-4439-B0 or equivalent).
6.12.3 Electrospray Ionization Tandem Mass Spectrometer (ESI-MS/MS)
The mass spectrometer must be capable of electrospray ionization in the negative ion mode. The system
must be capable of performing MS/MS to produce unique product ions for the method analytes within
specified retention time segments. A minimum of 10 scans across the chromatographic peak is needed
to ensure adequate precision. Some ESI-MS/MS instruments may not be suitable for PFAS analysis. See
the procedures in Section 10.1.2.1 to ensure that the selected MS/MS platform is capable of monitoring
all the required MS/MS transitions for the method analytes.
6.12.4 MS/MS Data System
An interfaced data system is required to acquire, store, and output MS data. The computer software
must have the capability of processing stored data by recognizing a chromatographic peak within a given
retention time window. The software must allow integration of the abundance of any specific ion
between specified time or scan number limits. The software must be able to construct a linear
regression or quadratic regression calibration curve and calculate analyte concentrations using the
internal standard technique.
7 Reagents and Standards
Reagent grade or better chemicals must be used. Unless otherwise indicated, all reagents must conform
to the specifications of the Committee on Analytical Reagents of the American Chemical Society (ACS),
where such specifications are available. Other grades may be used if the reagent is demonstrated to be
free of analytes and interferences and all requirements of the IDC are met when using these reagents.
7.1 Reagent Water
Purified water which does not contain any measurable quantities of any method analytes or interfering
compounds greater than one-third of the MRLfor each method analyte. It may be necessary to flush the
water purification unit to rinse out any build-up of PFAS in the system prior to collection of reagent
water.
7.2 Methanol
CH3OH, CASRN 67-56-1, LC grade (Fisher Scientific, Cat. No. A456 or equivalent).
7.3 Ammonium Acetate
NH4C2H3O2, CASRN 631-61-8, HPLC grade, molecular weight equals 77.08 g/mole.
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7.3.1 20 mM Ammonium Acetate
Chromatographic mobile phase. To prepare 1 L, add 1.54 g ammonium acetate to 1 L of reagent water.
This solution is volatile and must be replaced at least once per week. More frequent replacement may
be necessary if unexplained losses in sensitivity or retention time shifts are encountered.
7.3.2 1 g/L Ammonium Acetate
Used to rinse SPE cartridges after loading the aqueous sample and prior to the methanol rinse. Prepare
in reagent water.
7.4 Concentrated Ammonium Hydroxide Reagent
NH4OH, CASRN 1336-21-6, approximately 56.6% in water as ammonium hydroxide (w/w), approximately
28% in water as ammonia, approximately 14.5 N (Fisher Scientific, Cat. No. A669, Certified ACS Plus
grade, or equivalent).
7.5 Solution of Ammonium Hydroxide in Methanol
Used for elution of SPE cartridges. Dilute 2 mL of concentrated ammonium hydroxide (56.6% w/w) in
100 mL methanol. This solution should be made fresh on the day of extraction.
7.6 Sodium Phosphate Dibasic (Na2HP04)
Used for creating the aqueous buffer for conditioning the SPE cartridges. Dibasic sodium phosphate may
be purchased in either the anhydrous or any hydrated form. The formula weight will vary based on
degree of hydration.
7.7 Sodium Phosphate Monobasic (NaH2P04)
Used for creating the aqueous buffer for conditioning the SPE cartridges. Monobasic sodium phosphate
may be purchased in either the anhydrous or any hydrated form. The formula weight will vary based on
degree of hydration.
7.8 0.1 M Phosphate Buffer pH 7.0
Mix 500 mL of 0.1 M dibasic sodium phosphate with approximately 275 mL of 0.1 M monobasic sodium
phosphate. Verify that the solution pH is approximately 7.0.
7.9 Nitrogen
7.9.1 Nitrogen Nebulizer Gas
Nitrogen used as a nebulizer gas in the ESI interface and as collision gas in some MS/MS platforms
should meet or exceed the instrument manufacturer's specifications.
7.9.2 Nitrogen used for Concentrating Extracts
Ultra-high-purity-grade nitrogen should be used to concentrate sample extracts.
7.10 Argon
Used as collision gas in MS/MS instruments. Argon should meet or exceed instrument manufacturer's
specifications. Nitrogen may be used as the collision gas if recommended by the instrument
manufacturer.
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7.11 Sodium Hydroxide
May be purchased as pellets or as aqueous solution of known concentration. Added to methanolic
solutions of PFASto prevent esterification.
7.12 Acetic Acid (glacial)
May be necessary to adjust pH of aqueous samples. The pH of the aqueous sample containing 1 g/L
ammonium acetate must be between 6 and 8.
7.13 Standard Solutions
7.13.1 Stability of Methanolic Solutions
Fluorinated carboxylic acids will esterify in anhydrous acidic methanol. To prevent esterification,
standards must be stored under basic conditions. If base is not already present, this may be
accomplished by the addition of sodium hydroxide (approximately 4 mole equivalents) when standards
are diluted in methanol. When calculating molarity for solutions containing multiple PFAS, the molecular
weight can be estimated as 250 atomic mass units (amu). It is necessary to include sodium hydroxide in
solutions of both isotopically labeled and native analytes. The amount of sodium hydroxide needed may
be calculated using the following equation:
Total PFAS mass (g) x 160(-^-r)
—— = Mass of NaOH Required (g)
250
7.13.2 Preparation of Standards
When a compound purity is assayed to be 96% or greater, the weight can be used without correction to
calculate the concentration of the stock standard. Sorption of PFAS analytes in methanol solution to
glass surfaces after prolonged storage has not been evaluated. PFAS analyte and isotopically labeled
analogues commercially purchased in glass ampoules are acceptable; however, all subsequent transfers
or dilutions performed by the analyst must be stored in polypropylene containers.
Solution concentrations listed in this section were used to develop this method and are included as
examples. Alternate concentrations may be used as necessary depending on instrument sensitivity and
the calibration range used. Standards for sample fortification generally should be prepared in the
smallest volume that can be accurately measured to minimize the addition of excess organic solvent to
aqueous samples. Laboratories should use standard QC practices to determine when standards need to
be replaced. The analyte supplier's guidelines may be helpful when making this determination.
7.14 Storage Temperatures for Standards Solutions
Store stock standards at less than 4 °C unless the vendor recommends otherwise. The Primary Dilution
Standards may be stored at any temperature, but cold storage is recommended to prevent solvent
evaporation. During method development, the PDS was stored at -20 °C and no change in analyte
concentrations was observed over a period of 6 months.
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7.15 Isotope Performance Standards
This method requires three isotope performance standards listed in the table below. These isotopically
labeled compounds were chosen during method development to include the analogues of three method
analytes: two carboxylates with different chain lengths and a sulfonate.
Obtain the isotope performance standards as certified standard solutions, if available, or as the neat
compounds. During method development, the isotope performance standards were obtained from
Wellington Laboratories (Guelph, ON, Canada) as certified stocks in basic methanol. Note that Chemical
Abstracts Registry Numbers are not currently available for these compounds. The concentrations of the
stocks supplied by Wellington are listed in the table below.
Isotope Performance Standards
Abbreviation
Wellington
Stock, ng/mL
PDS, ng/nL
Perfluoro-/7-[2,3,4-13C3]butanoic acid
13c3-pfba
50
1.0
Perfluoro-[l,2-13C2]octanoic acid
13c2-pfoa
50
1.0
Sodium perfluoro-l-[l,2,3,4-13C4]octanesulfonate
13c4-pfos
50a
3.0
a 47.8 ng/mL as the anion.
All the isotope performance standards listed in this section must be used, if available. Additional isotope
performance standards may be used provided they are isotopically labeled analytes or labeled analytes
with similar functional groups as the method analytes. Linear isomers are recommended to simplify
peak integration. Method modification QC requirements must be met (Sect. 9.3) whenever additional
isotope performance standards are used.
7.15.1 Isotope Performance Standard PDS
Prepare the isotope performance standard PDS in methanol and add sodium hydroxide if not already
present to prevent esterification as described in Section 7.13.1. The PDS concentrations used to develop
the method are listed in the table above (Sect. 7.15). During collection of method performance data, the
final extracts were fortified with 10 piL of the PDS to yield a concentration of 10 ng/mL for 13C3-PFBA and
13C2-PFOA, and 30 ng/mL for 13C4-PFOS (28.7 ng/mL as the anion).
7.16 Isotope Dilution Analogues
Obtain the isotopically labeled analogues listed in the table in this section as individual certified
standard solutions or as certified standard mixes. All listed isotope dilution analogues must be used, if
available. Linear isomers are recommended to simplify peak integration. During method development,
the isotope dilution analogues were obtained from Wellington Laboratories (Guelph, ON, Canada) as
certified stocks in basic methanol. These analogues were chosen during method development because
they encompass most of the functional groups, as well as the molecular weight range of the method
analytes. Note that Chemical Abstracts Registry Numbers are not currently available for these
isotopically labeled analogues.
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Isotope Dilution Standards
Abbreviation
PDS,
ng/nLa
Perfluoro-n-[l,2,3,4-13C4]butanoic acid
13c4-pfba
0.50
Perfluoro-n-[l,2,3,4,5-13C5]pentanoic acid
13Cs-PFPeA
0.50
Sodium perfluoro-l-[2,3,4-13Cs]butanesulfonate
13Cs-PFBS
0.50
Sodium lH,lH,2H,2H-perfluoro-l-[l,2-13C2]hexane sulfonate
13C2-4:2FTS
2.0
Perfluoro-n-[l,2,3,4,6-13C5]hexanoic acid
13C5-PFHxA
0.50
2,3,3,3-Tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy-13C3-propanoic acid
13Cs-HFPO-DA
0.50
Perfluoro-n-[l,2,3,4-13C4]heptanoic acid
13C4-PFHpA
0.50
Sodium perfluoro-l-[l,2,3-13Cs]hexanesulfonate
13Cs-PFHxS
0.50
Sodium lH,lH,2H,2H-perfluoro-l-[l,2-13C2]-octane sulfonate
13C2-6:2FTS
2.0
Perfluoro-f7-[13Cs]octanoic acid
13Cs-PFOA
0.50
Perfluoro-n-[13C9]nonanoic acid
13C9-PFNA
0.50
Sodium perfluoro-[13Cs]octanesulfonate
13Cs-PFOS
0.50
Sodium lH,lH,2H,2H-perfluoro-l-[l,2-13C2]-decane sulfonate
13C2-8:2FTS
2.0
Perfluoro-n-[l,2,3,4,5,6-13C6]decanoic acid
13c6-pfda
0.50
Perfluoro-n-[l,2,3,4,5,6,7-13C7]undecanoic acid
13C7-PFUnA
0.50
Perfluoro-n-[l,2-13C2]dodecanoic acid
13C2-PFDoA
0.50
a Concentrations used during method development.
As additional isotopically labelled PFAS analogues become commercially available they may be
integrated into the method provided they have similar functional groups as the method analytes or are
isotopically labeled analogues of the method analytes. Method modification QC requirements must be
met (Sect. 9.3) whenever new analogues are proposed.
7.16.1 Isotope Dilution Analogue PDS
Prepare the isotope dilution analogue PDS in methanol and add sodium hydroxide if not already present
to prevent esterification as described in Section 7.13.1. The PDS concentrations used during method
development are listed in the table above. Method performance data were collected using 20 piL of this
PDS to yield concentrations of 40-160 ng/L in the 250 mL aqueous samples. Note that the
concentrations of sulfonates in the isotope dilution analogue PDS is based on the weight of the salt. It is
not necessary to account for difference in the formula weight of the salt compared to the free acid for
sample quantitation.
7.17 Analyte Standard Materials
Analyte standards may be purchased as certified standard solutions or prepared from neat materials of
assayed purity. If available, the method analytes should be purchased as technical-grade (as defined in
Sect. 3.22) to ensure that linear and branched isomers are represented. Standards or neat materials that
contain only the linear isomer can be substituted if technical-grade analytes are not available as
quantitative standards.
During method development, analyte standards were obtained from AccuStandard, Inc. (New Haven,
CT), Absolute Standards (Hamden, CT), Wellington Laboratories (Guelph, Ontario, Canada), Santa Cruz
Biotechnology (Dallas, TX), and Synquest Laboratories, Inc. (Alachua, FL). Stock standards are made by
dilution in methanol containing 4 mole equivalents of sodium hydroxide as described in Section 7.13.1
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7.17.1 PFOA
A quantitative standard for PFOA is currently available only for the linear isomer; however, a technical-
grade standard (Sect. 3.22) is available for PFOA that contains the linear and branched isomers
(Wellington Labs, Cat. No. T-PFOA, or equivalent). This product or a similar technical-grade PFOA
standard must be used to identify the retention times of the branched and linear PFOA isomers.
However, the linear-only PFOA standard must be used for quantitation until a quantitative PFOA
standard containing the branched and linear isomers becomes commercially available.
7177 PFHxS and PFOS
/ » / »Z. ii I I /\ d I I U r i
Technical grade, quantitative PFHxS and PFOS standards containing branched and linear isomers must
be used when available.
7.17.3 Correction for Analytes Obtained in the Salt Form
This method measures all forms of the analytes as anions while the identity of the counterion is
inconsequential. Analytes may be commercially available as neat materials or as certified stock
standards as their corresponding ammonium, sodium, or potassium salts. These salts are acceptable
standards provided the measured mass, or concentration, is corrected for the salt content. The equation
for this correction is provided below.
MWacid
mass (acid form) = mass (salt form) x
7.17.4 Analyte PDS
The analyte PDS is used to prepare the calibration standards and to fortify the LFBs, LFSMs and LFSMDs
with the method analytes. Prepare the analyte PDS by combining and diluting the analyte stock
standards in 100% methanol and add sodium hydroxide if not already present to prevent esterification
as described in Section 7.13.1. Select nominal analyte concentrations for the PDS such that between 5
and 100 piL of the PDS is used to fortify samples and prepare standard solutions. More than one PDS
concentration may be necessary to meet this requirement. During method development, the analyte
PDS was prepared at an identical concentration for all analytes, 0.5 ng/piL. The user may modify the
concentrations of the individual analytes based on the confirmed MRLs and the desired monitoring
range. If the PDS is stored cold, warm the vials to room temperature and vortex prior to use.
7.17.5 Calibration Standards
Prepare a series of calibration standards of at least five levels by diluting the analyte PDS into methanol
containing 20% reagent water. The lowest calibration standard must be at or below the MRL for each
analyte. The calibration standards may also be used as Continuing Calibration Checks (CCCs). Using the
PDS solutions, add a constant amount of the isotope performance standards and the isotope dilution
analogues to each calibration standard. The concentration of the isotope dilution analogues should
match the concentration of the analogues in sample extracts, assuming 100% recovery through the
extraction process. During method development, the concentrations of the isotope dilution analogues
were 40 ng/mL extract concentration (160 ng/L in the aqueous sample) for 4:2FTS, 6:2FTS and 8:2FTS,
and 10 ng/mL (40 ng/L) for all others. The analyte calibration ranged from approximately 0.50 ng/mL to
25 ng/mL extract concentration.
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8 Sample Collection, Preservation and Storage
8.1 Sample Bottles
Samples must be collected in plastic bottles: polypropylene bottles fitted with polypropylene screw-
caps, or polyethylene bottles with polypropylene screw caps. Discard sample bottles after a single use.
The bottle volume should approximate the volume of the sample. Subsampling from a single bottle is
not permitted except as described in Section 12.5.
8.2 Sample Preservation
Based on sample volume, add ammonium acetate to each sample bottle as a solid (prior to shipment to
the field or immediately prior to sample collection) to achieve a lg/L concentration of ammonium
acetate. Ammonium acetate will sequester free chlorine to form chloramine.
8.3 Sample Collection
8.3.1 Precautions against Contamination
Workers must wash their hands before sampling and wear nitrile gloves while filling and sealing the
sample bottles. Users should seek to minimize accidental contamination of the samples.
8.3.2 Collection Procedure
Open the tap and allow the system to flush until the water temperature has stabilized. Collect samples
from the flowing system. Samples do not need to be collected headspace free. After collecting the
sample, cap the bottle and agitate by hand until the preservative is dissolved. Keep the sample sealed
from time of collection until extraction.
8.4 Field Reagent Blanks (FRB)
Each sample set must include an FRB. A sample set is defined as samples collected from the same site
and at the same time. The same lot of preservative must be used for the FRBs as for the field samples.
8.4.1 Analysis of Reagent Water used for FRBs
Reagent water used for the FRBs must be analyzed prior to shipment to ensure the water has minimal
residual PFAS. Extract an LRB prepared with reagent water using the same lot of sample bottles destined
for shipment to the sampling site and ensure that analyte concentrations are less than one-third the
MRL, as described in Section 9.2.1. This will ensure that any significant contamination detected in the
FRBs originated from exposure in the field.
8.4.2 Field Reagent Blank Procedure
In the laboratory, fill the FRB sample bottle with the analyzed reagent water (Sect. 8.4.1). then seal and
ship to the sampling site with the sample bottles. For each FRB shipped, a second FRB sample bottle
containing only preservative must also be shipped. At the sampling site, open the FRB bottle and pour
the reagent water into the second sample bottle containing preservative; seal and label this bottle as
the FRB with the date, time and location of the site.
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8.5 Sample Shipment and Storage
Samples must be shipped on ice. Samples are valid if any ice remains in the cooler when it is received at
the laboratory or bottles are received within 2 days of collection and below 10 °C. Once at the
laboratory, samples must be stored at or below 6 °C until extraction. Samples must not be frozen.
8.6 Sample and Extract Holding Times
Analyze samples as soon as possible. Samples must be extracted within 28 days of collection. Extracts
are generally stored at room temperature and must be analyzed within 28 days after extraction.
9 Quality Control
QC procedures include the IDC and ongoing QC requirements. This section describes each QC
parameter, its required frequency, and the performance criteria that must be met in order to satisfy
method objectives. The QC criteria discussed in the following sections are summarized in Table 16 and
Table 17. These QC requirements are considered the minimum for an acceptable QC program.
Laboratories are encouraged to institute additional QC practices to meet their specific needs.
9.1 Initial Demonstration of Capability
The IDC must be successfully performed prior to analyzing field samples. The IDC must be repeated if
changes are made to analytical parameters not previously validated during the IDC. This may include, for
example, changing the sample volume, selecting alternate quantitation ions, extending the calibration
range, adding additional isotope performance standards, or adding additional isotope dilution
analogues. Prior to conducting the IDC, the analyst must meet the calibration requirements outlined in
Section 10. The same calibration range used during the IDC must be used for the analysis of field
samples.
9.1.1 Demonstration of Low System Background
Analyze an LRB immediately after injecting the highest calibration standard in the selected calibration
range. Confirm that the blank is free from contamination as defined in Section 9.2.1. If an automated
extraction system is used, an LRB must be extracted on each port to fulfil this requirement.
9.1.2 Demonstration of Precision
Prepare, extract, and analyze seven replicate LFBs in a valid Extraction Batch (seven LFBs and an LRB).
Fortify the LFBs near the midpoint of the initial calibration curve. The percent relative standard deviation
(%RSD) of the concentrations of the replicate analyses must be less than 20% for all method analytes.
9.1.3 Demonstration of Accuracy
Using the same set of replicate data generated for Section 9.1.2, calculate the average percent recovery.
The average recovery for each analyte must be within a range of 70-130%.
9.1.4 Minimum Reporting Level (MRL) Confirmation
Establish a target concentration for the MRL (Sect. 3.15) based on the intended use of the method. If
there is a programmatic MRL requirement, the laboratory MRL must be set at or below this level. In
doing so, one should consider that establishing the MRL concentration too low may cause repeated
failure of ongoing QC requirements.
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Perform initial calibration following the procedures in Section 10.3. The lowest calibration standard used
to establish the initial calibration (as well as the low-level CCC) must be at, or below, the MRL. Confirm
the laboratory's ability to meet the MRL following the procedure outlined below.
9.1.4.1 Prepare and Analyze MRL Samples
Fortify, extract, and analyze seven replicate LFBs at, or below, the proposed MRL concentration.
9.1.4.2 Calculate MRL Statistics
Calculate the mean and standard deviation for each analyte in these replicates. Determine the Half
Range for the Prediction Interval of Results (HRpir) using the following equation:
HRpir = 3.963S
Where,
5 = the standard deviation and 3.963 is a constant value for seven replicates.-1
Calculate the Upper and Lower Limits for the Prediction Interval of Results (PIR = Mean ± HRpir) as shown
below. These equations are only defined for seven replicate samples.
Mean + HRP,P
Upper PIR Limit = —- x 100
Fortified Concentration
Mean — HRPir}
Lower PIR Limit = —- x 100
Fortified Concentration
9.1.4.3 MRL Acceptance Criteria
The laboratory's ability to meet the MRL is confirmed if the Upper PIR Limit is less than, or equal to,
150%; and the Lower PIR Limit is greater than, or equal to, 50%. If these criteria are not met, the MRL
has been set too low and must be confirmed again at a higher concentration.
9.1.5 Calibration Verification
Analyze a QCS (Sect. 9.2.9) to confirm the accuracy of the primary calibration standards.
9.2 Ongoing QC Requirements
This section describes the ongoing QC elements that must be included when processing and analyzing
field samples.
9.2.1 Laboratory Reagent Blank (LRB)
Analyze an LRB with each Extraction Batch. Background concentrations of method analytes must be less
than one-third the MRL. If method analytes are detected in the LRB at concentrations greater than or
equal to this level, then all positive field sample results (i.e., results at or above the MRL) for those
analytes are invalid for all samples in the Extraction Batch. Subtracting blank values from sample results
is not permitted.
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9.2.1.1 Estimating Background Concentrations
Although quantitative data below the MRL may not be accurate enough for data reporting, such data
are useful in determining the magnitude of background interference. Therefore, the analyte
concentrations in the LRB may be estimated by extrapolation when results are below the MRL.
9.2.1.2 Influence of Background on Selection of MRLs
Because background contamination can be a significant problem, some MRLs may be background
limited.
9.2.1.3 Evaluation of Background when Analytes Exceed the Calibration Range
After analysis of a sample in which method analytes exceed the calibration range, one or more LRBs
must be analyzed (to detect potential carryover) until the system meets the LRB acceptance criteria. If
this occurs during an automated sequence, examine the results of samples analyzed following the
sample that exceeded the calibration range. If the analytes that exceeded the calibration range in the
previous sample are detected at, or above, the MRL, these samples are invalid. If the affected analytes
do not exceed the MRL, these subsequent samples may be reported.
9.2.2 Continuing Calibration Check (CCC )
Analyze CCC standards at the beginning of each Analysis Batch, after every tenth field sample, and at the
end of the Analysis Batch. See Section 10.4 for concentration requirements and acceptance criteria for
CCCs.
9.2.3 Laboratory Fortified Blank
An LFB is required with each Extraction Batch. The concentration of the LFB must be rotated between
low, medium, and high concentrations from batch to batch.
9.2.3.1 LFB Concentration Requirements
Fortify the low concentration LFB near the MRL. The high concentration LFB must be near the high end
of the calibration range.
9.2.3.2 Evaluate Analyte Recovery
Results for analytes fortified at concentrations near or at the MRL (within a factor of two times the MRL
concentration) must be within 50-150% of the true value. Results for analytes fortified at all other
concentrations must be within 70-130% of the true value. If the LFB results do not meet these criteria,
then all data for the problem analytes must be considered invalid for all samples in the Extraction Batch.
9.2.4 Isotope Performance Standard Areas
The analyst must monitor the peak areas of the isotope performance standards in all injections of the
Analysis Batch. The isotope performance standard responses (as indicated by peak area) in any
chromatographic run must be within 50-150% of the average area measured during the initial
calibration. Random evaporation losses have been observed with the polypropylene caps causing high-
biased isotope performance standard areas. If an isotope performance standard area for a sample does
not meet these criteria, reanalyze the extract in a subsequent Analysis Batch. If the isotope performance
standard area fails to meet the acceptance criteria in the repeat analysis, extraction of the sample must
be repeated, provided the sample is still within holding time.
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9.2.5 Isotope Dilution Analogue Recovery
Calculate the concentration of each isotope dilution analogue in field and QC samples using the average
area in the initial calibration and the internal standard technique. Calculate the percent recovery (%R)
for each analogue as follows:
A
o/oR = - x 100
B
Where,
A = measured concentration of the isotope dilution analogue, and
B = fortification concentration of the isotope dilution analogue.
The percent recovery for each analogue must be within a range of 50-200%.
9.2.5.1 Corrective Action for Failed Analogue Recovery
If an isotope dilution analogue fails to meet the recovery criterion, evaluate the area of the isotope
performance standard to which the analogue is referenced and the recovery of the analogues in the
CCCs. If necessary, recalibrate and service the LC-MS/MS system. Take corrective action, then analyze
the failed extract in a subsequent Analysis Batch. If the repeat analysis meets the 50-200% recovery
criterion, report only data for the reanalyzed extract. If the repeat analysis fails the recovery criterion
after corrective action, extraction of the sample must be repeated provided a sample is available and
still within the holding time.
9.2.6 Laboratory Fortified Sample Matrix (LFSM)
Within each Extraction Batch, analyze a minimum of one LFSM. The native concentrations of the
analytes in the sample matrix must be determined in a separate field sample and subtracted from the
measured values in the LFSM. If various sample matrices are analyzed regularly, for example, drinking
water processed from ground water and surface water sources, collect performance data for each
source.
9.2.6.1 Prepare the LFSM
Prepare the LFSM by fortifying a Field Duplicate with an appropriate amount of the analyte PDS
(Sect. 7.17.4) and isotope dilution analogue PDS (Sect. 7.16.1). Generally, select a spiking concentration
that is greater than or equal to the native concentration for the analytes. Selecting a duplicate aliquot of
a sample that has already been analyzed aids in the selection of an appropriate spiking level. If this is not
possible, use historical data when selecting a fortifying concentration.
9.2.6.2 Calculate the Percent Recovery
Calculate the percent recovery (%R) using the equation:
04-5)
o/o R = x 100
Where,
A = measured concentration in the fortified sample,
B = measured concentration in the unfortified sample, and
C = fortification concentration.
In order to obtain meaningful percent recovery results, correct the measured values in the LFSM and
LFSMD for the native levels in the unfortified samples, even if the native values are less than the MRL.
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9,2,6,3 Evaluate Analyte Recovery in the LFSM
Results for analytes fortified at concentrations near or at the MRL (within a factor of two times the MRL
concentration) must be within 50-150% of the true value. Results for analytes fortified at all other
concentrations must be within 70-130% of the true value. If the accuracy for any analyte falls outside
the designated range, and the laboratory performance for that analyte is shown to be in control in the
CCCs and in the LFB, the recovery is judged matrix biased. Report the result for the corresponding
analyte in the unfortified sample as "suspect-matrix".
9.2.7 Laboratory Fortified Sample Matrix Duplicate (LFSMD) or Field Duplicate (FD)
Within each Extraction Batch, analyze a minimum of one Field Duplicate or one Laboratory Fortified
Sample Matrix Duplicate. If the method analytes are not routinely observed in field samples, analyze an
LFSMD rather than an FD.
9.2.7.1 Calculate the RPD for the LFSM and LFSMD
If an LFSMD is analyzed instead of a Field Duplicate, calculate the RPD using the equation:
| LFSMD - LFSM \
RPD = (LFSMD + LFSM)/2 * 10°
9.2.7.2 Acceptance Criterion for the RPD of the LFSM and LFSMD
RPDs for duplicate LFSMs must be less than, or equal to, 30% for each analyte. Greater variability may
be observed when the matrix is fortified at analyte concentrations near or at the MRL (within a factor of
two times the MRL concentration). LFSMs at these concentrations must have RPDs that are less than or
equal to 50%. If the RPD of an analyte falls outside the designated range, and the laboratory
performance for the analyte is shown to be in control in the CCCs and in the LFB, the precision is judged
matrix influenced. Report the result for the corresponding analyte in the unfortified sample as "suspect-
matrix".
9.2.7.3 Calculate the RPD for Field Duplicates
Calculate the relative percent difference (RPD) for duplicate measurements. (FD1 and FD2) using the
equation:
|FDi — FD2|
RPD = X 100
(FDi + FD2)/2
9.2.7.4 Acceptance Criterion for Field Duplicates
RPDs for Field Duplicates must be less than, or equal to, 30% for each analyte. Greater variability may be
observed when Field Duplicates have analyte concentrations that are near or at the MRL (within a factor
of two times the MRL concentration). At these concentrations, Field Duplicates must have RPDs that are
less than or equal to 50%. If the RPD of an analyte falls outside the designated range, and the laboratory
performance for the analyte is shown to be in control in the CCC and in the LFB, the precision is judged
matrix influenced. Report the result for the corresponding analyte in the unfortified sample as "suspect-
matrix"
9.2.8 Field Reagent Blank (FRB)
The purpose of the FRB is to ensure that PFAS measured in the field samples were not inadvertently
introduced into the sample during sample collection and handling. The FRB is processed, extracted, and
analyzed in exactly the same manner as a field sample. Analysis of the FRB is required only if a field
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sample contains a method analyte or analytes at, or above, the MRL. If a method analyte found in the
field sample is present in the FRB at a concentration greater than one-third of the MRL, then the results
for that analyte are invalid for all samples associated with the failed FRB.
9.2.9 Calibration Verification using QCS
A QCS must be analyzed during the IDC, and then quarterly thereafter. For this method, the laboratory is
not required to obtain standards from a source independent of the primary calibration standards.
Instead, the laboratory should acquire the best available quantitative standards (Sect. 3.20) and use
these to prepare both the primary calibration standards and the QCS. The QCS must be an independent
dilution beginning with the common starting materials. Preparation by a second analyst is
recommended. The acceptance criterion for the QCS is 70-130% of the true value. If the accuracy for
any analyte fails the recovery criterion, prepare fresh standard dilutions and repeat the Calibration
Verification.
9.3 Method Modification QC Requirements
The analyst is permitted to modify the chromatographic and MS/MS conditions. Examples of permissible
method modifications include alternate LC columns, MRM transitions, and additional QC analytes
proposed for use with the method. Any method modifications must be within the scope of the
established method flexibility and must retain the basic chromatographic elements of this method
(Sect. 2). The following are required after a method modification.
9.3.1 Repeat the IDC
Establish an acceptable initial calibration (Sect. 10.3) using the modified conditions. Repeat the
procedures of the IDC (Sect. 9.1).
9.3.2 Document Performance in Representative Sample Matrices
The analyst is also required to evaluate and document method performance for the modifications in real
matrices that span the range of waters that the laboratory analyzes. This additional step is required
because modifications that perform acceptably in the IDC, which is conducted in reagent water, could
fail ongoing method QC requirements in real matrices. This is particularly important for methods subject
to matrix effects, such as LC-MS/MS-based methods. For example, a laboratory may routinely analyze
finished drinking water from municipal treatment plants that process ground water, surface water, or a
blend of surface and ground water. In this case, the method modification requirement could be
accomplished by assessing precision (Sect. 9.1.2) and accuracy (Sect. 9.1.3) in finished drinking waters
derived from a surface water with moderate to high total organic carbon (e.g., 2 mg/L or greater) and
from a hard ground water (e.g., 250 mg/L as calcium carbonate (CaC03) equivalent, or greater).
10 Calibration and Standardization
Demonstration and documentation of acceptable MS calibration and initial analyte calibration are
required before performing the IDC and prior to analyzing field samples. The initial calibration should be
repeated each time a major instrument modification or maintenance is performed.
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10.1 MS/MS Optimization
10.1.1 Mass Calibration
Calibrate the mass spectrometer with the calibration compounds and procedures specified by the
manufacturer.
10.1.2 MS Parameters
During the development of this method, instrumental parameters were optimized for the precursor and
product ions listed in Table 6. Product ions other than those listed may be selected; however, the
analyst should avoid using ions with lower mass or common ions that may not provide sufficient
discrimination between the analytes of interest and co-eluting interferences.
10.1.2.1 Requirement for Branched Isomers
There have been reports that not all product ions in the linear PFOS are produced in all branched PFOS
isomers.- (This phenomenon may exist for many of the PFAS.) For this method, the m/z 80 product ion
must be used for PFOS and PFHxS to minimize this problem and promote comparability between
laboratories. Some MS/MS instruments, may not be able to scan a product ion with such a wide mass
difference from the precursor ion. These instruments may not be used for this method if PFOS or PFHxS
analysis is to be conducted.
10.1.2.2 Precursor lort
Optimize the response of the precursor ion ([M - H]~ or [M - C02 - H]~) for each analyte following
manufacturer's guidance. Analyte concentrations of 1.0 ng/mL were used for this step during method
development. Vary the MS parameters (source voltages, source and desolvation temperatures, gas
flows, etc.) until optimal analyte responses are determined. The electrospray parameters used during
method development are listed in Table 2. The analytes may have different optimal parameters,
requiring some compromise on the final operating conditions. See Table 6 for ESI-MS conditions used to
collect method performance data.
10.1.2.3 Product Ion
Optimize the product ion for each analyte following the manufacturer's guidance. Typically, the
carboxylic acids have similar MS/MS conditions and the sulfonic acids have similar MS/MS conditions.
See Table 6 for MS/MS conditions used to collect method performance data.
10.2 Chromatographic Conditions
Establish LC operating parameters that optimize resolution and peak shape. Suggested LC conditions can
be found in Table 1. Modifying the solvent composition of the standard or extract by increasing the
aqueous content to better focus early eluting compounds on the column is not permitted. A decrease in
methanol concentration could lead to lower or imprecise recovery of the more hydrophobic method
analytes, while higher methanol concentration could lead to the precipitation of salts in some extracts.
The peak shape of the 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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10.2.1 Minimizing PFAS Background
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 Establishing Branched vs. Linear Isomer Profiles
Prepare and analyze the technical-grade standard of PFOA, discussed in Section 7.17.1. at a mid- to high-
level concentration. Identify the retention times of the branched isomers of PFOA present in the
technical-grade PFOA standard. When PFOA is chromatographed on a reversed-phase column, the
branched isomers elute prior to the linear isomer. Repeat the procedure in this section for PFHxS and
PFOS discussed in Sectioi , and any other analytes for which technical-grade standards have been
acquired. The branched isomer identification checks must be repeated anytime chromatographic
changes occur that alter analyte retention times.
10.2.3 Establish LC-MS/MS Retention Times and MRM Segments
Inject a mid- to high-level calibration standard under optimized LC-MS/MS conditions to obtain the
retention times of each method analyte. Divide the chromatogram into segments that contain one or
more chromatographic peaks. For maximum sensitivity, minimize the number of MRM transitions that
are simultaneously monitored within each segment. Ensure that the retention time window used to
collect data for each analyte is of sufficient width to detect earlier eluting branched isomers.
The retention times observed during collection of the method performance data are listed in Table 3.
Table 4. and Table 5.
10.3 Initial Calibration
This method has three isotope performance standards that are used as reference compounds for the
internal standard quantitation of the isotope dilution analogues. The suggested isotope performance
standard reference for each isotope dilution analogue is listed in Table 4. The sixteen isotope dilution
analogues are used as reference compounds to quantitate the native analyte concentrations. The
suggested isotope dilution analogue references for the native analytes are listed in Table 5.
10.3.1 Calibration Standards
Prepare a set of at least five calibration standards as described in Section 7.17.5. The analyte
concentrations in the lowest calibration standard must be at or below the MRL.
10.3.2 Calibration Curves of Native Analytes
Quantitate the native analytes using the internal standard calibration technique. The internal standard
technique calculates concentration based on the ratio of the peak area of the native analyte to that of
the isotope dilution analogue. Calibrate the LC-MS/MS and fit the calibration points with either a linear
or quadratic regression. Weighting may be used. Forcing the calibration curve through the origin is
mandatory for this method. Forcing zero allows for a better estimate of the background levels of
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method analytes. The MS/MS instrument used during method development was calibrated using
weighted (1/x) quadratic regression with forced zero.
10.3.3 Calibration of Isotope Dilution Analogues
The isotope dilution analogues are quantified using the internal standard calibration technique. Because
isotope dilution analogues are added at a single concentration level to the calibration standards,
calibrate for each of these using an average response factor.
10.3.4 Calibration of Isotope Performance Standards
Because Isotope performance standards are added at a single concentration level to the calibration
standards, calibrate for each of these using an average response factor.
10.3.5 Calibration Acceptance Criteria
Evaluate the initial calibration by calculating the concentration of each analyte as an unknown against its
regression equation. For calibration levels that are less than or equal to the MRL, the result for each
analyte should be within 50-150% of the true value. All other calibration points should be within 70-
130% of their true value. If these criteria cannot be met, the analyst could have difficulty meeting
ongoing QC criteria. In this case, corrective action is recommended such as reanalyzing the calibration
standards, restricting the range of calibration, or performing instrument maintenance. If the cause for
failure to meet the criteria is due to contamination or standard degradation, prepare fresh calibration
standards and repeat the initial calibration.
10.4 Continuing Calibration
Analyze a CCC to verify the initial calibration at the beginning of each Analysis Batch, after every tenth
field sample, and at the end of each Analysis Batch. The beginning CCC for each Analysis Batch must be
at, or below, the MRL for each analyte. This CCC verifies instrument sensitivity prior to the analysis of
samples. If standards have been prepared such that all low calibration levels are not in the same
solution, it may be necessary to analyze two standards to meet this requirement. Alternatively, the
nominal analyte concentrations in the analyte PDS may be customized to meet these criteria. Alternate
subsequent CCCs between the mid and high calibration levels. Verify that the CCC meets the criteria in
the following sections.
10.4.1 CCC Isotope Performance Standard Responses
The absolute area of the quantitation ion for each of the three isotope performance standards must be
within 50-150% of the average area measured during the initial calibration. If these limits are exceeded,
corrective action is necessary (Sect. 10.5).
10.4.2 CCC Isotope Dilution Analogue Recovery
Using the average response factor determined during the initial calibration and the internal standard
calibration technique, calculate the percent recovery of each isotope dilution analogue in the CCC. The
recovery for each analogue must be within a range of 70-130%. If these limits are exceeded, corrective
action is necessary (Sect. 10.5).
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10.4.3 CCC Analyte Responses
Calculate the concentration of each method analyte in the CCC. Each analyte fortified at a level less than
or equal to the MRL must be within 50-150% of the true value. The concentration of the analytes in
CCCs fortified at all other levels must be within 70-130%. If these limits are exceeded, then all data for
the failed analytes must be considered invalid. Any field samples analyzed since the last acceptable CCC
that are still within holding time must be reanalyzed after an acceptable calibration has been restored.
10.4.3.1 Exception for High Recovery
If the CCC fails because the calculated concentration is greater than 130% (150% for the low-level CCC)
for a method analyte, and field sample extracts show no concentrations above the MRL for that analyte,
non-detects may be reported without re-analysis.
10.5 Corrective Action
Failure to meet the CCC QC performance criteria requires corrective action. Following a minor remedial
action, such as servicing the autosampler or flushing the column, check the calibration with a mid-level
CCC and a CCC at the MRL, or recalibrate according to Section 10.3. If isotope performance standard and
calibration failures persist, maintenance may be required, such as servicing the LC-MS/MS system or
replacing the LC column. These latter measures constitute major maintenance and the analyst must
return to the initial calibration step (Sect. 10.3).
11 Procedure
This procedure may be performed manually or in an automated mode using a robotic or automatic
sample preparation device. The data published in this method (Sect. 17) demonstrate acceptable
performance using manual extraction. The authors did not evaluate automated extraction systems. If an
automated system is used to prepare samples, follow the manufacturer's operating instructions, but all
extraction and elution steps must be the same as in the manual procedure. Extraction and elution steps
may not be changed or omitted to accommodate the use of an automated system. If an automated
system is used, the LRBs should be rotated among the ports to ensure that all the valves and tubing
meet the LRB requirements (Sect. 9.2.1).
11.1 Sample Bottle Rinse
Some of the PFAS adsorb to surfaces, including polypropylene. During the elution step of the procedure,
sample bottles must be rinsed with the elution solvent whether extractions are performed manually or
by automation.
11.2 Reuse of Extraction Cartridges
The SPE cartridges described in this section are designed for a single use. They may not be reconditioned
for subsequent analyses.
11.3 Sample Preparation
11.3.1 Sample Volume
Determine sample volume. An indirect measurement may be done in one of two ways: by marking the
level of the sample on the bottle or by weighing the sample and bottle to the nearest 1 gram. After
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extraction, proceed to Section 11.5 to complete the volume measurement. Some of the PFAS adsorb to
surfaces, thus the sample may not be transferred to a graduated cylinder for volume measurement. The
LRB, LFB and FRB must have the same volume as that of the field samples and may be prepared by
measuring reagent water with a graduated cylinder.
11.3.2 Verifying Sample pH
Verify that the sample containing 1 g/L ammonium acetate has a pH between 6.0 and 8.0. Acetic acid
may be added as needed to reduce the pH
11.3.3 Fortify QC Samples
Fortify LFBs, LFSMs, and LFSMDs, with an appropriate volume of Analyte PDS (Sect. ). Cap and
invert each sample several times to mix.
11.3.4 Addition of Isotope Dilution Analogues
Add an aliquot of the isotope dilution analogue PDS (Sect. 7.16.1) to each sample, then cap and invert to
mix. During method development, a 20 piL aliquot of the PDS (0.50-2.0 ng/piL) was added to achieve a
final concentration of 40 ng/Lof the isotopically labeled carboxylates and perfluorinated sulfonates, and
160 ng/L of the telomer sulfonates.
11.4 Extraction Procedure
11.4.1 Cartridge Cleaning and Conditioning
Do not allow cartridge packing material to go dry during any of the conditioning steps. If the cartridge
goes dry during the conditioning phase, the conditioning must be repeated. Rinse each cartridge with
10 mL of methanol. Next, rinse each cartridge with 10 mL of aqueous 0.1 M phosphate buffer (Sect. 7.8)
without allowing the water to drop below the top edge of the packing. Close the valve and add 2-3 mL
of phosphate buffer to the cartridge reservoir and fill the remaining volume with reagent water.
11.4.2 Cartridge Loading
Attach the sample transfer tubes (Sect. 6.8.3) and adjust the vacuum to approximately 5 inches Hg.
Begin adding sample to the cartridge. Adjust the vacuum and control valves so that the approximate
flow rate is 5 mL/min. Do not allow the cartridge to go dry before all the sample has passed through.
Flow rates above 5 mL/min during loading may cause low analyte recovery.
11.4.3 Sample Bottle Rinse and Cartridge Drying
After the entire sample has passed through the cartridge, rinse the sample bottle with a 10 mL aliquot of
1 g/L ammonium acetate in reagent water. Draw the rinsate through the sample transfer tubes and the
cartridges. Add 1 mL of methanol to the sample bottle and draw through the transfer tube and SPE
cartridge. This step is designed to remove most of the water from the transfer line and cartridge
resulting in the reduction of the salt and water present in the eluate. The methanol rinse may also
reduce interferences by removing weakly retained organic material prior to elution. If plastic reservoirs
are used instead of transfer lines, the reservoirs must be rinsed with the ammonium acetate solution
and the 1 mL aliquot of methanol.
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11.4.4 Cartridge Drying
Draw air or nitrogen through the cartridge for 5 min at high vacuum (15-20 in. Hg).
11.4.5 Sample Bottle and Cartridge Elution
After the drying step, release the vacuum on the extraction manifold and place a collection tube under
each sample position. Rinse the sample bottles with 5 mL of the elution solvent, methanol with 2%
ammonium hydroxide (v/v), then elute the analytes from the cartridges by pulling the elution solvent
through the sample transfer tubes and the cartridges. Use a low vacuum such that the solvent exits the
cartridge in a dropwise fashion. Repeat sample bottle rinse and cartridge elution with a second 5 mL
aliquot of elution solvent. If plastic reservoirs are used instead of transfer lines, attempt to rinse the
entire inner surface of the reservoir with the elution solvent.
11.4.6 Extract Concentration
Concentrate the extract to dryness under a gentle stream of nitrogen in a heated water bath (55-60 °C).
Reconstitute the extract with 1.0 mL of 20% reagent water in methanol (v/v). Add the isotope
performance standards to the extract and vortex.
11.4.7 Extract Transfer and Storage
Transfer the final extract to a polypropylene autosampler vial. Store extracts at room temperature.
Recap vials as soon as possible after injection to prevent evaporation losses; the polypropylene caps do
not reseal after puncture. Alternatively, extracts can be stored in the 15 mL collection tubes after
extraction. A small aliquot can be removed for analysis if the autosampler vial and injection system
accommodate small volumes.
11.5 Sample Volume Determination
Use a graduated cylinder to measure the volume of water required to fill the original sample bottle to
the mark made prior to extraction. If using weight to determine the volume, weigh the empty bottle to
the nearest 1 gram and subtract this value from the weight recorded prior to extraction. Assume a
sample density of 1.0 g/mL. Record the sample volumes for use in the final calculations of analyte
concentrations.
11.6 Sample Analysis
11.6.1 Establish LC-MS/MS Operating Conditions
Establish MS/MS operating conditions per the procedures in Section 10.1 and chromatographic
conditions per Section 10.2. Establish a valid initial calibration following the procedures in Section 10.3
or confirm that the existing calibration is still valid by analyzing a low-level CCC. If establishing an initial
calibration for the first time, complete the IDC prior to analyzing field samples. Analyze field and QC
samples in a properly sequenced Analysis Batch as described in Section 11.7.
11.6.2 Verify Retention Time Windows
The analyst must ensure that each method analyte elutes entirely within the assigned window during
each Analysis Batch. Make this observation by viewing the quantitation ion for each analyte in the CCCs
analyzed during an Analysis Batch. If an analyte peak drifts out of the assigned window, then data for
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that analyte is invalid in all injections acquired since the last valid CCC. In addition, all peaks representing
multiple isomers of an analyte must elute entirely within the same MRM window.
11.7 Analysis Batch Sequence
An Analysis Batch is a sequence of samples, analyzed within a 24-hour period, of no more than 20 field
samples and includes all required QC samples (LRB, CCCs, the LFSM and LFSMD (or FD)). The required QC
samples are not included in counting the maximum field sample total of 20. LC-MS/MS conditions for
the Analysis Batch must be the same as those used during calibration.
11.7.1 Analyze Initial CCC
After a valid calibration is established, begin every Analysis Batch by analyzing an initial low-level CCC at
or below the MRL. This initial CCC must be within 50-150% of the true value for each method analyte
and must pass both the isotope performance standard area response criterion (Sect. 10.4.1) and the
isotope dilution analogue recovery criterion (Sect. 10.4.2). The initial CCC confirms that the calibration is
still valid. Failure to meet the QC criteria may indicate that recalibration is required prior to analyzing
samples.
11.7.2 Analyze Field and QC Samples
After the initial CCC, continue the Analysis Batch by analyzing an LRB, followed by the field samples and
QC samples. Analyze a mid- or high-level CCC after every ten field samples and at the end each Analysis
Batch. Do not count QC samples (LRBs, FDs, LFSMs, LFSMDs) when calculating the required frequency of
CCCs.
11.7.3 Analyze Final CCC
The last injection of the Analysis Batch must be a mid- or high-level CCC. The acquisition start time of the
final CCC must be within 24 hours of the acquisition start time of the low-level CCC at the beginning of
the Analysis Batch. More than one Analysis Batch within a 24-hour period is permitted. An Analysis
Batch may contain field and QC samples from multiple extraction batches.
11.7.4 Initial Calibration Frequency
A full calibration curve is not required before starting a new Analysis Batch. A previous calibration can be
confirmed by running an initial, low-level CCC followed by an LRB. If a new calibration curve is analyzed,
an Analysis Batch run immediately thereafter must begin with a low-level CCC and an LRB.
12 Data Analysis and Calculations
Because environmental samples may contain both branched and linear isomers of the method analytes,
but quantitative standards that contain branched isomers do not exist for all method analytes,
integration and quantitation of the PFAS is dependent on the type of standard materials available.
12.1 Identify Peaks by Retention Times
At the conclusion of data acquisition, use the same software settings established during the calibration
procedure to identify analyte peaks in the predetermined retention time windows. Confirm the identity
of each analyte by comparison of its retention time with that of the corresponding analyte peak in an
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initial calibration standard or CCC. Proceed with quantitation based on the type of standard available for
each method analyte.
12.1.1 Method Analytes without Technical-Grade Standards
If standards containing the branched and linear isomers cannot be purchased (i.e., only the linear isomer
is available), only the linear isomer can be identified and quantitated in field samples and QC samples
because the retention time of the branched isomers cannot be confirmed.
12.1.2 PFHxS, PFOS, and other Analytes with Technical-Grade Standards
During method development, multiple chromatographic peaks, representing branched and linear
isomers, were observed for standards of PFHxS and PFOS using the LC conditions in Table 1. For PFHxS
and PFOS, all the chromatographic peaks observed in the standard must be integrated and the areas
summed. Chromatographic peaks in all field samples and QC samples must be integrated in the same
way as the calibration standard for analytes with quantitative standards containing the branched and
linear isomers.
12.1.3 PFOA
For PFOA, identify the branched and linear isomers by analyzing a technical-grade standard that includes
both linear and branched isomers as directed in Section 10.2.2 and ensure that all isomers elute within
the same acquisition segment. Quantitate field samples and fortified matrix samples by integrating the
total response, accounting for peaks that are identified as linear and branched isomers. Quantitate
based on the initial calibration with the quantitative PFOA standard containing just the linear isomer.
12.2 Calculate Analyte Concentrations
Calculate analyte concentrations using the multipoint calibration and the measured sample volume.
Report only those values that fall between the MRL and the highest calibration standard.
12.3 Calculate Isotope Dilution Analogue Recovery
Calculate the concentration of each isotope dilution analogue using the multipoint calibration and the
measured sample volume. Verify that the percent recovery is within 50-200% of the true value.
12.4 Significant Figures
Calculations must use all available digits of precision, but final reported concentrations should be
rounded to an appropriate number of significant figures (one digit of uncertainty), typically two, and not
more than three significant figures.
12.5 Exceeding the Calibration Range
The analyst must not extrapolate beyond the established calibration range. If an analyte result exceeds
the range of the initial calibration curve, a field duplicate of the sample must be extracted, if available.
Dilute an aliquot of the field duplicate with reagent water to a final volume equal to that used for the
IDC. Add ammonium acetate to a final concentration of 1 g/L and process the diluted sample. Report all
concentrations measured in the original sample that do not exceed the calibration range. Report
concentrations of analytes that exceeded the calibration range in the in the original sample based on
measurement in a diluted sample. Incorporate the dilution factor into final concentration calculations
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and the resulting data must be annotated as a dilution. This is the only circumstance when subsampling
is permitted.
13 Method Performance
13.1 Precision, Accuracy, and LCMRL Results
Tables for these data are presented in Section 17. LCMRLs are presented in Table 7. Single-laboratory
precision and accuracy data are presented for three water matrices: reagent water (Table 8). finished
ground water (Table 10). and a drinking water matrix from a surface water source (Table 12). The mean
isotope dilution analogue recoveries measured in the replicate samples used in these studies are
presented in Table 9 for reagent water, Table 11 for finished groundwater, and Table 13 for the surface
water matrix.
13.2 Analyte Stability Study
Chlorinated (finished) surface water samples were inoculated with microbial-rich water from an
impacted surface source and fortified with 40 ng/L of the PFAS method analytes. These samples were
stored as required in this method. The percent change from the initial analyzed concentration observed
after 7, 14, 21, and 28 days is presented in Section 17, Table 14.
13.3 Extract Storage Stability
Extract storage stability studies were conducted on extracts obtained from the analyte stability study
(Sect. 13.2). The percent change from the initial analyzed concentration observed after 14, 21, and 27
days storage is presented in Section 17, Table 15.
14 Pollution Prevention
For information about pollution prevention applicable to laboratory operations described in this
method, consult: Less is Better, Guide to Minimizing Waste in Laboratories, a publication available from
the American Chemical Society (accessed April 2019) at www.acs.org.
15 Waste Management
Laboratory waste management practices should be consistent with all applicable rules and regulations,
and that laboratories protect the air, water, and land by minimizing and controlling all releases from
fume hoods and bench operations. In addition, compliance is required with any sewage discharge
permits and regulations, particularly the hazardous waste identification rules and land disposal
restrictions.
16 References
1. US EPA. Statistical Protocol for the Determination of the Single-Laboratory Lowest Concentration
Minimum Reporting Level (LCMRL) and Validation of Laboratory Performance at or Below the
Minimum Reporting Level (MRL); EPA 815-R-05-006; Office of Water: Cincinnati, OH, November
2004.
2. US EPA. Technical Basis for the Lowest Concentration Minimum Reporting Level (LCMRL) Calculator;
EPA 815-R-11-001; Office of Water: Cincinnati, OH, December 2010.
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3. Martin, J.W., et al. Analytical Challenges Hamper Perfluoroalkyl Research. Environ. Sci. Technol.
2004, Vol. 38, 248A-255A.
4. Cahill, J.D., et al. Determination of Pharmaceutical Compounds in Surface- and Ground-Water
Samples by Solid-Phase Extraction and High-Performance Liquid Chromatography Electrospray
Ionization Mass Spectrometry. J. Chromatography A, 2004, 1041, 171-180.
5. Langlois, I. and Oehme, M. Structural Identification of Isomers Present in Technical Perfluorooctane
Sulfonate by Tandem Mass Spectrometry. Rapid Communication Mass Spectrometry. 2006, Vol. 20,
844-850.
17 Tables, Figures and Method Performance Data
Table 1. HPLC Method Conditionsa
Time (min)
% 20 mM ammonium acetate
% Methanol
Initial
95.0
5.0
0.5
95.0
5.0
3.0
60.0
40.0
16.0
20.0
80.0
18.0
20.0
80.0
20.0
5.0
95.0
22.0
5.0
95.0
25.0
95.0
5.0
35.0
95.0
5.0
a Phenomenex Gemini C18, 2 x 50 mm, 3.0 pim silica with TMS end-capping. Flow rate of 0.25
mL/min; run time 35 minutes; 10 piL injection into a 50 piL loop. The chromatogram in Figure 1 was
obtained under these conditions.
ESI Conditions for Waters (Milford, MA) Xevo TQD
Polarity
Negative ion
Capillary needle voltage
-2.7 kV
Cone gas flow
40 L/hour
Nitrogen desolvation gas
800 L/hour
Desolvation gas temperature
300 °C
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Table 3, Isotopically Labeled Isotope Performance Standards and Retention Times
Isotope Performance Standard
Peak #
(Figure 1)
RT
(min)
13c3-pfba
1
4.14
13c2-pfoa
26
12.19
13c4-pfos
32
13.73
Table 4, Isotope Dilution Analogues: RTs and Suggested Isotope Performance Standard
References
Isotopically Labeled Analyte
Peak #
(Fig. 1)
RT
(min)
Suggested Isotope Performance Standard
13c4-pfba
2
4.14
13c3-pfba
13Cs-PFPeA
5
6.13
13c3-pfba
13c3-pfbs
7
6.62
13c4-pfos
13C2-4:2FTS
12
8.12
13c4-pfos
13C5-PFHxA
14
8.35
13c2-pfoa
13C3-HFPO-DA
17
9.06
13c2-pfoa
13C4-PFHpA
19
10.34
13c2-pfoa
13C3-PFHxS
21
10.61
13c4-pfos
13C2-6:2FTS
24
12.05
13c4-pfos
13c8-pfoa
27
12.19
13c2-pfoa
13c9-pfna
30
13.70
13c2-pfoa
13Cs-PFOS
33
13.73
13c4-pfos
13C2-8:2FTS
36
14.94
13c4-pfos
13CS-PFDA
38
15.00
13c2-pfoa
13C7-PFUnA
40
16.14
13c2-pfoa
13C2-PFDoA
43
17.13
13c2-pfoa
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Table 5, Method Analytes, Retention Times and Suggested Isotope Dilution Analogue
References
Analyte
Peak #
(Figure 1)
RT
(min)
Isotope Dilution Analogue
PFBA
3
4.15
13c4-pfba
PFMPA
4
4.84
13c4-pfba
PFPeA
6
6.13
13Cs-PFPeA
PFBS
8
6.62
13c3-pfbs
PFMBA
9
6.81
13Cs-PFPeA
PFEESA
10
7.53
13c3-pfbs
NFDHA
11
8.01
13C5-PFHxA
4:2FTS
13
8.12
13C2-4:2FTS
PFHxA
15
8.36
13C5-PFHxA
PFPeS
16
8.69
13C3-PFHxS
HFPO-DA
18
9.06
13c3-hfpo-da
PFHpA
20
10.42
13C4-PFHpA
PFHxS
22
10.62
13C3-PFHxS
ADONA
23
10.73
13C4-PFHpA
6:2FTS
25
12.04
13C2-6:2FTS
PFOA
28
12.19
13c8-pfoa
PFHpS
29
12.28
13Cs-PFOS
PFNA
31
13.70
13c9-pfna
PFOS
34
13.74
13Cs-PFOS
9CI-PF30NS
35
14.53
13Cs-PFOS
8:2 FTS
37
14.94
13C2-8:2FTS
PFDA
39
15.00
13CS-PFDA
PFUnA
41
16.14
13C7-PFUnA
HCI-PF30UdS
42
16.70
13Cs-PFOS
PFDoA
44
17.13
13C2-PFDoA
533-34
-------
Table 6, MS/MS Method Conditions0
Segment'5
Analyte
Precursor lonc
(m/z)
Product lonc
-------
Segment'5
Analyte
Precursor lonc
(m/z)
Product lonc 79.9 for PFOS). These precursor and
product ion masses (with at least one decimal place) should be used in the MS/MS method for all
analyses.
Ions used for quantitation purposes.
Argon used as collision gas.
HFPO-DA is not stable in the ESI source and the [M - H]~ yields a weak signal under typical ESI
conditions. The precursor ion used during method development was [M - C02 - H]~.
The isotope dilution analogue used during method development was composed of the linear isomer
exclusively.
h Analyte has multiple resolved chromatographic peaks due to linear and branched isomers. All peaks
summed for quantitation purposes. To reduce bias regarding detection of branched and linear
isomers, the m/z 80 product ion must be used for this analyte.
533-36
-------
Table 7. LCMRL Results
Analyte
LCMRL Fortification Levels (ng/L)
Calculated LCMRL (ng/L)
PFBA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
13
PFMPA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.8
PFPeA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.9
PFBS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.5
PFMBA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.7
PFEESA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
2.6
NFDHA
4.0, 6.0, 10, 14, 20, 41, 82
16
4:2FTS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
4.7
PFHxA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
5.3
PFPeS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
6.3
HFPO-DA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.7
PFHpA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
2.6
PFHxS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.7
ADONA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.4
6:2FTS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
14
PFOA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
3.4
PFHpS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
5.1
PFNA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
4.8
PFOS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
4.4
9CI-PF30NS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
1.4
8:2FTS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
9.1
PFDA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
2.3
PFUnA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
2.7
HCI-PF30UdS
1.0, 2.0, 4.0, 6.0, 10, 14, 20
1.6
PFDoA
1.0, 2.0, 4.0, 6.0, 10, 14, 20
2.2
533-37
-------
Table 3. Precision and Accuracy Data for Reagent Water
Analyte
Low Fortification
(ng/L)
Mean %Ra
(n=7)
%RSDa
High Fortification
(ng/L)
Mean %R
(n=5)
%RSD
PFBA
10
128
8.6
80
98.4
2.4
PFMPA
10
108
4.5
80
98.1
2.2
PFPeA
10
107
4.9
80
99.6
3.6
PFBS
10
102
9.1
80
96.2
2.9
PFMBA
10
111
6.8
80
101
3.4
PFEESA
10
107
10
80
98.8
4.0
NFDHA
10
110
15
80
98.5
5.4
4:2FTS
10
94.4
14
80
100
5.7
PFHxA
10
102
8.0
80
97
7.7
PFPeS
10
99.5
19
80
101
7.8
HFPO-DA
10
102
9.7
80
102
4.7
PFHpA
10
108
7.0
80
104
4.1
PFHxS
10
103
9.0
80
97.7
5.5
ADONA
10
96.3
3.1
80
96.8
5.6
6:2FTS
10
109
15
80
111
11
PFOA
10
108
7.4
80
98.5
6.9
PFHpS
10
98.8
8.9
80
102
7.0
PFNA
10
109
6.2
80
99.6
5.6
PFOS
10
104
8.7
80
98.0
4.3
9CI-PF30NS
10
99.7
4.6
80
103
6.8
8:2FTS
10
100
17
80
100
13
PFDA
10
100
4.2
80
100
1.8
PFUnA
10
102
10
80
97.3
8.1
HCI-PF30UdS
10
106
5.3
80
102
6.1
PFDoA
10
101
6.2
80
96.3
5.1
a %R = percent recovery; %RSD = percent relative standard deviation
533-38
-------
Analyte
Analogue Fortification
(ng/L)
Mean %Rbc
(n=7) P&A Low
%RSDbc
Mean %R
(n=5) P&A High
%RSD
13c4-pfba
40
95.6
11
92.5
3.4
13Cs-PFPeA
40
93.4
9.3
91.7
4.6
13c3-pfbs
40
98.6
9.6
107
6.6
13C2-4:2FTS
160
102
6.7
108
3.5
13C5-PFHxA
40
92.5
6.4
92.8
11
13C3-HFPO-DA
40
88.6
6.5
88.8
7.4
13C4-PFHpA
40
98.0
4.0
94.0
8.3
13C3-PFHxS
40
101
11
106
8.2
13C2-6:2FTS
160
109
9.5
99.8
4.7
13c8-pfoa
40
98.0
4.1
91.5
8.7
13c9-pfna
40
97.1
4.9
92.1
8.4
13Cs-PFOS
40
98.8
6.5
96.5
5.0
13C2-8:2FTS
160
106
13.9
108
8.7
13c6-pfda
40
104
7.7
104
6.1
13C7-PFUnA
40
107
6.0
98.8
7.5
13C2-PFDoA
40
100
5.7
94.0
6.7
a P&A = "precision and accuracy".
b %R = percent recovery; %RSD = percent relative standard deviation.
c Mean and %RSD of the isotope dilution analogue results for the fortified samples in the P&A study; number of replicates given in the header
row of the table.
533-39
-------
Analyte
Low Fortification
(ng/L)
Mean %Rb
(n=5)
%RSDb
High Fortification
(ng/L)
Mean %R
(n=5)
%RSD
PFBA
10
127
15
80
98.0
4.0
PFMPA
10
100
8.3
80
103
9.8
PFPeA
10
105
11
80
105
5.1
PFBS
10
111
12
80
101
10
PFMBA
10
99.0
4.6
80
100
2.3
PFEESA
10
101
3.5
80
107
8.8
NFDHA
10
95.1
17
80
98.5
18
4:2FTS
10
70.5
20
80
116
9.2
PFHxA
10
104
18
80
111
17
PFPeS
10
87.5
5.0
80
106
6.2
HFPO-DA
10
105
7.4
80
103
7.5
PFHpA
10
102
6.8
80
101
6.4
PFHxS
10
86.6
18
80
108
6.8
ADONA
10
97.6
8.1
80
94.2
6.9
6:2FTS
10
99.9
15
80
100
12
PFOA
10
95.8
8.1
80
104
9.8
PFHpS
10
94.0
6.3
80
113
6.0
PFNA
10
95.1
7.2
80
108
3.3
PFOS
10
C
C
80
109
5.8
9CI-PF30NS
10
92.7
7.2
80
111
7.9
8:2FTS
10
108
19
80
102
3.2
PFDA
10
90.8
9.8
80
104
7.1
PFUnA
10
98.3
8.8
80
105
3.0
HCI-PF30UdS
10
94.6
8.3
80
110
9.3
PFDoA
10
92.7
7.8
80
102
6.3
a Finished water from a ground water source. Hardness = 320 mg/L as CaC03. pH = 7.88 at 17 °C. Free Cl2 = 0.64 mg/L. Total Cl2 = 0.74 mg/L.
b %R = percent recovery, corrected for native concentration; %RSD = percent relative standard deviation.
c The spike level was below the ambient PFOS concentration of 25 ng/L.
533-40
-------
Table 11. P&A in Finished Ground Water: Isotope Dilution Analogue Recovery Dataa
Analyte
Analogue Fortification
(ng/L)
Mean %Rbc
(n=6) P&A Low
%RSDbc
Mean %R
(n=6) P&A High
%RSD
13c4-pfba
40
89.5
4.4
81.3
7.8
13Cs-PFPeA
40
94.0
4.2
84.6
7.7
13c3-pfbs
40
103
1.7
93.6
8.5
13C2-4:2FTS
160
107
6.1
105
2.6
13C5-PFHxA
40
93.8
9.8
75.8
16
13C3-HFPO-DA
40
77.8
8.5
72.0
9.8
13C4-PFHpA
40
90.5
8.4
83.3
10
13C3-PFHxS
40
101
7.8
94.7
6.4
13C2-6:2FTS
160
101
5.2
101
4.5
13c8-pfoa
40
89.5
5.7
82.8
10
13c9-pfna
40
103
6.6
78.0
11
13Cs-PFOS
40
101
7.6
89.7
4.5
13C2-8:2FTS
160
97.2
7.4
94.0
8.0
13c6-pfda
40
98.7
6.3
82.3
15
13C7-PFUnA
40
102
4.3
82.6
8.0
13C2-PFDoA
40
98.8
4.6
81.2
10
a P&A = "precision and accuracy".
b %R = percent recovery; %RSD = percent relative standard deviation.
c Mean and %RSD of the isotope dilution analogue results for the unfortified matrix sample and the fortified samples in the P&A study;
number of replicates given in the header row of the table.
533-41
-------
Table 12. Precision and Accuracy Data for a Surface Water Matrixa
Analyte
Low Fortification
(ng/L)
Mean %Rbc
(n=5)
%RSDb
High Fortification
(ng/L)
Mean %R
(n=5)
%RSD
PFBA
10
95.4
19
80
106
4.8
PFMPA
10
108
16
80
102
5.9
PFPeA
10
93
13
80
101
6.0
PFBS
10
111
17
80
98.3
2.7
PFMBA
10
93.0
12
80
103
3.0
PFEESA
10
95.6
15
80
99.1
2.4
NFDHA
10
102
14
80
101
2.5
4:2FTS
10
70.9
17
80
91.1
7.8
PFHxA
10
96.9
19
80
103
4.2
PFPeS
10
87.5
14
80
104
4.9
HFPO-DA
10
109
8.7
80
105
7.0
PFHpA
10
95.9
11
80
105
4.8
PFHxS
10
78.5
8.2
80
97.1
5.3
ADONA
10
94.3
7.9
80
95.8
6.0
6:2FTS
10
86.5
6.3
80
101
9.7
PFOA
10
91.9
9.8
80
98.7
4.9
PFHpS
10
88.4
14
80
106
3.4
PFNA
10
89.7
9.5
80
95.9
2.8
PFOS
10
95.1
11
80
105
8.0
9CI-PF30NS
10
82.4
5.0
80
94.1
3.9
8:2FTS
10
102
7.6
80
101
4.0
PFDA
10
87.3
12
80
98.5
8.0
PFUnA
10
96.9
5.4
80
95.2
2.7
HCI-PF30UdS
10
82.4
8.9
80
93.0
4.4
PFDoA
10
94.6
2.3
80
98.4
4.1
Surface water matrix was sampled after the clarifier and prior to granular activated carbon withi
chlorinated in our laboratory. pH = 8.1 at 20 °C. Free Cl2 = 0.98 mg/L. Total Cl2 = 1.31 mg/L. Total
%R = percent recovery; %RSD = percent relative standard deviation.
Corrected for native concentration.
n the drinking water treatment plant and
Organic Carbon (TOC) = 3.8 mg/L C.
533-42
-------
Table 13. P&A in Surface Water Matrix: Isotope Dilution Analogue Recovery Dataa
Analyte
Analogue Fortification
(ng/L)
Mean %Rbc
(n=6) P&A Low
%RSDbc
Mean %R
(n=6) P&A High
%RSD
13c4-pfba
40
86.9
18
86.3
6.5
13Cs-PFPeA
40
105
15
102
5.7
13c3-pfbs
40
98.6
11
99.8
4.5
13C2-4:2FTS
160
136
13
138
6.3
13C5-PFHxA
40
88.8
16
84.8
4.5
13C3-HFPO-DA
40
78.4
14
75.4
13
13C4-PFHpA
40
91.6
12
89.3
6.0
13C3-PFHxS
40
98.2
6.5
96.0
9.6
13C2-6:2FTS
160
110
9.7
109
8.4
13c8-pfoa
40
90.1
14
86.6
4.5
13c9-pfna
40
91.0
14
87.2
6.0
13Cs-PFOS
40
98.8
15
95.6
5.0
13C2-8:2FTS
160
101
9.8
97.3
11
13c6-pfda
40
92.0
16
86.6
10
13C7-PFUnA
40
92.2
16
90.0
5.6
13C2-PFDoA
40
91.2
14
90.8
10
a P&A = "precision and accuracy".
b %R = percent recovery; %RSD = percent relative standard deviation.
c Mean and %RSD of the isotope dilution analogue results for the unfortified matrix sample and the fortified samples in the P&A study;
number of replicates given in the header row of the table.
533-43
-------
Table 14, Aqueous Sample Holding Time Data"
Analyte
Fortified
Cone. (ng/L)
Day Zero
Mean
(ng/L)
Day
Zero
%RSD
Day 7
%Changeb
Day 7
%RSD
Day 14
%Change
Day 14
%RSD
Day 21
%Change
Day 21
%RSD
Day 28
%Change
Day 28
%RSD
PFBA
40
42
4.6
9.1
2.3
3.1
7.2
5.1
5.4
4.2
5.0
PFMPA
40
41
5.2
5.5
2.2
-7.8
5.1
1.0
6.3
-10
3.1
PFPeA
40
43
4.1
1.2
1.9
-2.2
6.5
-0.29
2.5
-6.5
5.8
PFBS
40
43
9.7
-1.9
3.6
-6.1
1.8
-4.0
2.5
-7.6
8.9
PFMBA
40
40
3.0
-2.5
3.7
-5.7
4.3
0.20
5.0
-6.6
6.3
PFEESA
40
39
3.2
2.6
5.7
-1.8
6.7
-2.4
4.5
-1.7
2.6
NFDHA
40
39
6.5
-4.0
7.2
-11
6.9
-3.8
5.2
-2.9
8.0
4:2FTS
40
43
9.7
-1.7
3.8
-2.6
9.6
-2.0
6.1
-0.34
5.3
PFHxA
40
42
5.2
-0.37
4.6
-2.61
5.6
-1.7
5.8
-2.3
7.6
PFPeS
40
41
3.2
5.6
7.5
-3.1
2.6
6.0
9.2
-11
9.4
HFPO-DA
40
42
5.1
6.2
4.8
3.2
9.2
2.1
2.1
-3.5
4.2
PFHpA
40
41
4.6
-0.042
2.4
-4.7
1.7
-2.9
3.6
-3.0
5.4
PFHxS
40
41
4.3
1.8
3.0
-1.8
1.8
-1.8
9.0
-0.99
6.8
ADONA
40
39
4.2
-4.3
3.1
-12
5.7
-6.2
5.9
-2.3
3.1
6:2FTS
40
41
7.5
-4.3
4.4
-0.74
9.4
2.5
6.0
-1.5
6.0
PFOA
40
41
5.4
-1.5
6.7
1.6
5.1
-2.0
4.9
-6.5
7.2
PFHpS
40
41
4.7
-2.4
5.4
1.2
3.1
0.30
3.2
2.9
7.2
PFNA
40
42
4.1
2.05
0.57
-6.0
4.9
-6.1
3.4
-9.5
3.4
PFOS
40
41
7.0
-2.1
4.7
-1.8
5.2
1.0
5.8
-1.6
5.3
9CI-PF30NS
40
40
3.5
1.6
4.8
-0.34
1.8
4.0
4.8
-2.6
10
8:2FTS
40
44
7.9
-0.36
2.5
-1.4
6.7
0.026
3.8
-3.6
6.9
PFDA
40
41
5.0
0.12
3.1
-2.7
3.8
-1.4
3.8
-2.4
7.0
PFUnA
40
39
3.9
-1.3
4.7
-12
1.2
3.7
3.1
-6.7
3.5
HCI-PF30UdS
40
40
4.9
-1.1
4.5
-9.4
5.1
-11.0
4.7
-12
7.3
PFDoA
40
39
4.4
9.5
6.5
-4.8
6.0
-3.4
5.8
-16
6.1
a Finished water from a surface water source. pH = 8.84 at 18 °C; total organic carbon (TOC) = 0.75 mg/L C (mean of 2019 first quarter plant records); free
chlorine = 0.87 mg/L, total chlorine = 1.04 mg/L Day Zero: n=7. All other events: n=5.
b %Change = percent change from Day Zero calculated as follows: (Day X mean concentration - Day Zero mean concentration) / Day Zero mean
concentration * 100%, where X is the analysis day.
533-44
-------
Table 15. Extract Holding Time Dataa
Analyte
Fortified Cone. (ng/L)
Day Zero
Mean
(ng/L)
Day Zero %RSD
Day 14
%Changeb
Day 14 %RSD
Day 21
%Change
Day 21 %RSD
Day 27
%Change
Day 27 %RSD
PFBA
40
42
4.6
-8.0
4.2
-4.4
0.89
-12
6.4
PFMPA
40
41
5.2
-3.9
4.5
-0.10
5.1
-3.9
12
PFPeA
40
43
4.1
-6.0
6.0
-0.55
4.8
-5.4
1.1
PFBS
40
43
9.7
2.6
2.0
6.6
2.3
2.9
3.6
PFMBA
40
40
3.0
-10
7.1
-4.8
5.3
-8.8
2.7
PFEESA
40
39
3.2
1.3
8.9
-3.6
2.1
-4.9
3.6
NFDHA
40
39
6.5
-10
3.9
-13
6.8
-11
3.1
4:2FTS
40
43
9.7
-4.7
8.5
-6.2
8.8
-7.3
8.5
PFHxA
40
42
5.2
-4.6
6.3
-20
3.0
-14
4.7
PFPeS
40
41
3.2
-6.7
8.6
-11
5.2
-10
4.5
HFPO-DA
40
42
5.1
-4.9
4.9
-4.7
5.1
-4.4
7.7
PFHpA
40
41
4.6
-1.9
1.9
-6.1
4.8
-8.7
7.8
PFHxS
40
41
4.3
-19
9.9
-21
8.4
-22
11
ADONA
40
39
4.2
-1.2
1.9
-7.8
6.4
-7.5
5.0
6:2FTS
40
41
7.5
-5.3
13
-7.6
5.8
-8.4
14
PFOA
40
41
5.4
-5.7
6.3
-2.2
4.2
-2.4
3.3
PFHpS
40
41
4.7
-8.7
7.3
-6.0
5.2
-3.2
4.2
PFNA
40
42
4.1
-5.8
5.6
0.17
3.2
-2.0
6.0
PFOS
40
41
7.0
-3.8
10
-4.2
2.5
-3.7
4.4
9CI-PF30NS
40
40
3.5
-5.8
7.7
-9.3
4.0
-8.6
4.7
8:2FTS
40
44
7.9
-4.7
6.3
-1.3
5.8
-6.4
2.9
PFDA
40
41
5.0
-3.7
5.3
-1.8
5.6
-4.8
3.1
PFUnA
40
39
3.9
6.2
4.0
0.63
7.5
-2.8
5.2
llCI-PF30UdS
40
40
4.9
-12
5.9
-18
4.6
-10
6.3
PFDoA
40
39
4.4
1.9
5.5
1.0
6.4
-2.6
3.3
a Finished water from a surface water source. pH = 8.84 at 18 °C; total organic carbon (TOC) = approximately 0.75 mg/L C (2019 first quarter plant records);
free chlorine = 0.87 mg/L, total chlorine = 1.04 mg/L Day Zero: n=7. All other events: n=1.
b %Change = percent change from Day Zero calculated as follows: (Day X mean concentration - Day Zero mean concentration) / Day Zero mean
concentration * 100%, where X is the analysis day.
533-45
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Table 16, Initial Demonstration of Capability (IDC) Quality Control Requirements
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section 10,2,2
Establish retention times
for branched isomers
Each time chromatographic conditions change
All isomers of each analyte must
elute within the same MRM window.
Section 9,1,1
Demonstration of low
system background
Analyze a Laboratory Reagent Blank (LRB) after the highest
standard in the calibration range.
Demonstrate that the method
analytes are less than one-third of
the Minimum Reporting Level (MRL).
Section 9,1,2
Demonstration of precision
Extract and analyze 7 replicate Laboratory Fortified Blanks (LFBs)
near the mid-range concentration.
Percent relative standard deviation
must be <20%.
Section 9,1,3
Demonstration of accuracy
Calculate mean recovery for replicates used in Section 9,1,2.
Mean recovery within 70-130% of
the true value.
Section 9,1,4
MRL confirmation
Fortify and analyze 7 replicate LFBs at the proposed MRL
concentration. Confirm that the Upper Prediction Interval of
Results (PIR) and Lower PIR meet the recovery criteria.
Upper PIR <150%
Lower PIR >50%
Section 9,1,5
Calibration Verification
Analyze mid-level QCS.
Results must be within 70-130% of
the true value.
Table 17. Ongoing Quality Control Requirements
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section
10,3
Initial calibration
Use the isotope dilution calibration technique to
generate a linear or quadratic calibration curve. Use at
least 5 standard concentrations. Evaluate the
calibration curve as described in Section 10,3,5.
When each calibration standard is calculated as an
unknown using the calibration curve, analytes fortified at
or below the MRL should be within 50-150% of the true
value. Analytes fortified at all other levels should be
within 70-130% of the true value.
Section
9,2,1
Laboratory Reagent
Blank (LRB)
Include one LRB with each Extraction Batch. Analyze
one LRB with each Analysis Batch.
Demonstrate that all method analytes are below one-
third the Minimum Reporting Level (MRL), and that
possible interference from reagents and glassware do
not prevent identification and quantitation of method
analytes.
533-46
-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section
9.2.3
Laboratory Fortified
Blank
Include one LFB with each Extraction Batch.
For analytes fortified at concentrations <2 x the MRL,
the result must be within 50-150% of the true value; 70-
130% of the true value if fortified at concentrations
greater than 2 x the MRL.
Section
10.4
Continuing Calibration
Check (CCC)
Verify initial calibration by analyzing a low-level CCC
(concentrations at or below the MRL for each analyte)
at the beginning of each Analysis Batch. Subsequent
CCCs are required after every tenth field sample and
to complete the batch.
The lowest level CCC must be within 50-150% of the
true value. All other levels must be within 70-130% of
the true value.
Section
9.2.4
Isotope performance
standards
Isotope performance standards are added to all
standards and sample extracts.
Peak area counts for each isotope performance standard
must be within 50-150% of the average peak area in the
initial calibration.
Section
9.2.5
Isotope dilution
analogues
Isotope dilution analogues are added to all samples
prior to extraction.
50%-200% recovery for each analogue
Section
9.2.6
Laboratory Fortified
Sample Matrix (LFSM)
Include one LFSM per Extraction Batch. Fortify the
LFSM with method analytes at a concentration close
to but greater than the native concentrations (if
known).
For analytes fortified at concentrations <2 x the MRL,
the result must be within 50-150% of the true value; 70-
130% of the true value if fortified at concentrations
greater than 2 x the MRL.
Section
9 2 7
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD) or Field
Duplicate (FD)
Include at least one LFSMD or FD with each Extraction
Batch.
For LFSMDs or FDs, relative percent differences must be
<30% (<50% if analyte concentration <2 x the MRL).
Section
9.2.8
Field Reagent Blank
(FRB)
Analyze the FRB if any analyte is detected in the
associated field samples.
If an analyte detected in the field sample is present in
the associated FRB at greater than one-third the MRL,
the results for that analyte are invalid.
Section
9.2.9
Calibration Verification
using QCS
Perform a Calibration Verification at least quarterly.
Results must be within 70-130% of the true value.
533-47
-------
Figure 1.
Example Chromatogram for Reagent Water Fortified with Method Analytes at 80 ng/L°
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533-48
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