Certain sections are replaced by the words PROPRIETARY CONTENT to allow distribution of this document.

Always check on­line for validity.

Document number:

T­PFAS­WI14355
Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

Document users:

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Perfluoro-n-butanoic acidPFBA375-22-4

Perfluoro-n-pentanoic acid

PFPeA

2706-90-3

8:2 - Fluorotelomersulfonate

8:2FTS

39108-34-4

N-methylperfluoro-1-octanesulfonamidoacetic acidNMeFOSAA

2355-31-9N-ethylperfluoro-1-octanesulfonamidoacetic acid
Perfluoroundecanoic acid

NEtFOSAA

2991-50-6

4:2-Fluorotelomersulfonate

4:2-FTS

757124-72-4

Perfluoropentanesulfonate

PFPeS

2706-94-4

6:2-Fluorotelomersulfonate

6:2-FTS

27619-97-2

Perfluoroheptanesulfonate

PFHpS

375-92-8

Perfluorononanesulfonate

PFNS

68259-12-1

Perfluorodecanesulfonate

PFDS

335-77-3

10:2-Fluorotelomersulfonate

10:2-FTS

120226-60-0

Perfluorododecanesulfonate

PFDoDS

79780-39-5

Perfluorohexadecanoic acid

PFHxDA

67905-19-5

Perfluorooctadecanoic acid

PFODA

16517-17-6

Perfluorooctanesulfonamide

PFOSA

754-91-62-(N-methylperfluoro-1-octanesulfonamido)-ethanol NMePFOSAE

24448-09-7

N-methylperfluoro-1-octanesulfonamide

NMePFOSA

31506-32-82-(N-ethylperfluoro-1-octanesulfonamido)-ethanol

NEtPFOSAE

1691-99-2

N-ethylperfluoro-1-octanesulfonamide

NEtPFOSA

4151-50-2

Basic Principles
A 250-mL aqueous sample is fortified with isotopically-labeled extraction standards and is passed
through a solid phase extraction (SPE) cartridge to extract the analytes. The compounds are eluted
from the solid phase with a combination of solvents. The extract is concentrated to ~400-500ul withnitrogen in a heated water bath, and then reconstituted to 1ml with methanol. Isotopically-labeledinjection internal standards are added to the sample extract and it is analyzed by LC/MS/MS
operated in negative electrospray ionization (ESI) mode for detection and quantification of theanalytes. Quantitative analysis is performed using isotope dilution.

Reference Modifications
EPA Method 537 is written specifically for the analysis of drinking water samples. The following
modifications to the method have been made to accommodate all aqueous samples.
1. A labeled isotopic analog is spiked into samples for all compounds where an isotopic analog iscommercially available. These isotopic compounds are referred to as extraction standards. For
US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1
Modified Using LC/MS/MS
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Old Reference:

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Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

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Exposure to these chemicals must be reduced to the lowest possible level by whatever means
available, such as fume hoods, lab coats, safety glasses, and gloves. Gloves, lab coats, and safety
glasses should be worn when preparing standards and handling samples. Avoid inhaling solventsand chemicals and getting them on the skin. Wear gloves when handling neat materials. When
working with acids and bases, take care not to come in contact and to wipe any spills. Always addacid to water when preparing reagents containing concentrated acids.
All laboratory waste is accumulated, managed, and disposed of in accordance with all Federal, State,
and local laws and regulations. All solvent waste and extracts are collected in approved solvent
waste containers in the laboratory and subsequently emptied by personnel trained in hazardous
waste disposal into the lab-wide disposal facility. HPLC vials are disposed of in the lab container for
waste vials, and subsequently lab packed. Any solid waste material (disposable pipettes and broken
glassware, etc.) may be disposed of in the normal solid waste collection containers.

Personnel Training and Qualifications
All personnel performing this procedure must have documentation of reading, understanding, andagreeing to follow the current version of this SOP and an annual documented Demonstration of
Capability (DOC).

Each chemist performing the extraction must work with an experienced employee for a period oftime until they can independently perform the extraction. Also, several batches of sample
extractions must be performed under the direct observation of another experienced chemist toassure the trainee is capable of independent preparation. Proficiency is measured through adocumented Initial Demonstration of Capability (IDOC).
Each LC/MS/MS analyst must work with an experienced employee for a period of time until they can
independently calibrate the LC/MS/MS, review and process data, and perform maintenanceprocedures. Proficiency is measured through a documented Initial Demonstration of Capability(IDOC).
The IDOC and DOC consist of four laboratory control samples (or alternatively, one blind sample forthe DOC) that is carried through all steps of the extraction and meets the defined acceptancecriteria. The criteria include the calculation of mean accuracy and standard deviation.

Sample Collection, Preservation, and HandlingA.

Sample Collection

The samples are collected in 250 mL polyethylene bottles containing 1.25 grams of Trizma, resulting
in a Trizma concentration in the sample of 5 g/L. Trizma functions as a free chlorine scavenger;
therefore, any chlorinated water supplies require the preservative. Water samples from nonUS Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1
Modified Using LC/MS/MS
Printed by: Jeremy Beckley, d. 2019/01/14 15:34 CETPage 6 of 17

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T­PFAS­WI14355
Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

Document users:

Responsible:

Effective Date 11­SEP­2018

6_EUUSLA_PFAS_Analyst,
6_EUUSLA_PFAS_Data_Reviewers,
6_EUUSLA_PFAS_Sample_Prep

5_EUUSLA_PFAS_Manager

chlorinated water sources would not necessarily require the Trizma preservative. Keep the sample
sealed from time of collection until extraction.

NOTE: PFAS contamination during sampling can occur from a number of common sources, such as
food packaging and certain foods and beverages. Proper hand washing and wearing nitrile gloves
will aid in minimizing this type of accidental contamination of the samples.
B.

Sample Storage and Shipment

1.
Samples must be chilled during shipment and must not exceed 10°C during the first48 hours after collection. Sample temperature must be confirmed to be at or below 10°C when thesamples are received at the laboratory.
2.
Samples stored in the lab must be held at a temperature of 0° to 6°C, not frozen,
until extraction.
3.
Water samples must be extracted within 14 days. Extracts must be analyzed within
28 days after extraction. Extracts are stored at room temperature.

Apparatus and Equipment

PROPRIETARY CONTENT

US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1
Modified Using LC/MS/MS
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Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

Document users:

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US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1
Modified Using LC/MS/MS
Printed by: Jeremy Beckley, d. 2019/01/14 15:34 CETPage 8 of 17

Always check on­line for validity.

Document number:

T­PFAS­WI14355
Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

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Reagents and Standards
All solvents, acids, and bases are stored in glass bottles in flammable proof cabinets or pressure
resistant steel drums. Solvents, acids, and bases are stored at ambient temperature for up to 1
year. All non-solvents are stored according to manufacturer’s storage conditions.

A.

Reagents: PROPRIETARY CONTENT

B.

Standards: See SOP T-PFAS-WI13881

US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1
Modified Using LC/MS/MS
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Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

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If the criteria are not met, the source of the problem must be determined and corrected.
Situations may exist where the initial calibration can be used. In those cases, the data will bereported with a qualifying comment.
7. Initial Calibration Verification (ICV)A check standard prepared from a second source (ICV) is injected to confirm the validity of thecalibration curve/standard. The calculated amount for each analyte should be ± 30% of the true
value.
B. Continuing calibration
1. Once the calibration curve has been established, the continuing accuracy must be verified byanalysis of a continuing calibration verification (CCV) standard every ten samples and at the end of
the analysis sequence.
a. The CCV run after the initial calibration must be at the CAL3 level.
b. Subsequent CCV standards should alternate between the low, mid and high levels of thecalibration curve.
2.

Acceptance criteria

a. The calculated amount for each compound (native and extraction standard) in the CCV
standard must be within ±30% of the true value. Samples that are not bracketed by acceptable CCV
analyses must be reanalyzed. The exception to this would be if the CCV recoveries are high,indicating increased sensitivity, and there are no positive detections in the associated samples, thedata may be reported with a qualifying comment. If two consecutive CCVs fail criteria for target
analytes, two passing CCVs must be analyzed or the source of the problem determined and thesystem recalibrated before continuing sample analysis.
b. The absolute areas of the injection internal standards should be within 50-150% of theaverage areas measured during the initial calibration.

ProcedureA.

Sample Preparation

1. Weigh full sample container on a calibrated top loading balance and record the first reading in
the automated prep entry system.
2. If required, add 1.25 grams of Trizma to a 250 ml HDPE bottle for the method blank and thelaboratory control sample (LCS) and LCSD if needed. Fill each bottle with 250 ml of Milli-Q water.
Record 250 ml as the volume for the batch QC samples on the batchlog.
3. If sample has dissolved and/or settleable solid content; i.e., is cloudy or has a layer of
sediment/solids at the bottom of the bottle, an aliquot should be taken from the original bottle anddiluted with reagent water in order to minimize difficulty passing through the SPE sorbent bed. If

US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1 Page 11 of 17
Modified Using LC/MS/MS
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T­PFAS­WI14355
Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

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3.

Acquisition method: See attachment 3

4. Load sample vials containing standards, quality control samples, and sample extracts into
autosampler tray. Allow the instrument adequate time to equilibrate to ensure the mass spec and
LC have reached operating conditions (approximately 5 minutes) before the first injection. Analyze
several solvent blanks clean the instrument prior to sample acquisition.

5. After the initial calibration, inject a solvent blank, followed by the ICV, L/B standard, closing
Cal 3 level CCV , CCV, extraction batch QC, and samples. Bracket each set of ten samples with a
CCV standard, alternating between the Cal3, Cal4, and Cal5 levels.

6. After injections are completed, check all CCV recoveries and absolute areas to make surethey are within method control limits. See Calibration section B.2 for acceptance criteria. Processeach chromatogram and closely evaluate all integrations, baseline anomalies, and retention timedifferences. If manual integrations are performed, they must be documented and a reason given forthe change in integrations. The manual integrations are documented during data processing and all
original integrations are reported at the end of the sample PDF file with the reason for manual
integration clearly listed.
7. Quantitate results for the extraction blank.
a. No target analytes at or above the reporting limit may be found in the extraction blank for
acceptable batch results. If a target analyte is detected in the extraction blank but not detected in
the sample, the data is reported. If a target analyte is detected in the method blank at aconcentration greater than the reporting limit and also in the sample, the sample must be
reextracted. If the target analyte in the sample is detected at a concentration greater than 10 times
the amount detected in the method blank, the data is reported.

8. Calculate the recoveries of spiked analytes for the LCS, matrix spike and matrix spike
duplicate (MS/MSD) by comparing concentrations observed to the true values. The advisory QC
acceptance limits for LCS and MS/MSD recovery are 70 to 130% for each analyte. The advisory QC
acceptance limit for the relative percent difference (%RPD) between LCS/LCSD and MS/MSD is≤30%. The limits are advisory until sufficient data points are available to determine statistical QC
acceptance limits. If LCS and/or LCSD recoveries are acceptable, proceed to sample quantitation. If
the LCS recoveries are unacceptable, the samples associated with the LCS may need to bereanalyzed. If LCS recoveries are above the advisory QC acceptance limits, and there are no positive
detections in the sample, the data may be reported. If MS/MSD recoveries are outside QC
acceptance criteria, the associated data will be flagged or noted in the comments section of thereport.
9. Isotopically labeled extraction standards are added to all samples, extraction blank,
LCS/LCSD, and MS/MSD prior to extraction.

US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1 Page 15 of 17
Modified Using LC/MS/MS
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Document number:

T­PFAS­WI14355
Old Reference:

Level:

Polyfluorinated Alkyl
Substances (PFAS) in Aqueous Work Instruction
Samples by Method 537 Version
1.1 Modified Using LC/MS/MS

1­P­QM­WI­9039651 (1­P­QM­WI­9012802)
Version:Organisation level:

85­Sub­BU

Approved by: UDM6

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a. The recovery of the extraction standards should be within QC acceptance criteria. If theextraction standard recovery(ies) is(are) outside the QC limit(s), consult a supervisor to determine
the appropriate course of action based on batch and sample results.
10. Isotopically labeled injection standards are added to each QC and field sample extractprior to analysis.
a. The absolute areas of the injection standards should be within 50-150% of the average
areas measured during the initial calibration. If the internal standards are recovered outside 50150%, consult a supervisor to determine the appropriate course of action based on batch andsample results.

11. Compare the retention times of all of the analytes, surrogates and internals standards. The
relative retention times should not vary by more than 0.2 retention time units.

12. The MDL standard and the linear/branch chain standard are used when assessing thecorrectness of the computer generated peak integrations.
13. If the calculated concentration exceeds the calibration range of the system, dilute theextract with MeOH and add the appropriate amount of extraction standard to match the original
concentration. Add 10 ul of injection internal standard and analyze the dilution.
Dilution Example 1/10: Mix 0.877 mL of MEOH with 0.100 mL of sample extract and0.0225 mL of labeled extraction standard. Vortex to mix. Using an auto-pipette, transfer 200 uL ofthe mixed solution into a labeled auto-sampler vial containing a plastic insert. Using a syringe, add
10 uL of labeled injection std to the 200 uL aliquot. Cap and vortex thoroughly to mix.

Calculations
A.

Peak Area Ratio

B. Analyte Concentration using linear through zero curves (MQ Data processing system)
Concentration = (area ratio ÷ slope) x Dilution Factor x Internal Standard concentration
C. Sample Concentration (used only for aqueous samples using the MultiQuant data processing
system on the AB Sciex LC/MS/MS)
Sample concentration (ng/l) = Calc conc x (Sample volume ÷ Sample weight) x DF
US Eurofins US Lancaster Laboratories Environmental ­ Polyfluorinated Alkyl Substances (PFAS) in Aqueous Samples by Method 537 Version 1.1 Page 16 of 17
Modified Using LC/MS/MS
Printed by: Jeremy Beckley, d. 2019/01/14 15:34 CET

PFOAPFOA (2)
13C4-PFOS
13C8-PFOSPFOS
PFOS (2)
13C9-PFNA
PFNA
PFNA (2)
13C8-PFOSA
PFOSA
PFNS
PFNS (2)13C2-PFDA
13C6-PFDA
PFDA
PFDA (2)13C2-8:2-FTS
8:2-FTS
8:2-FTS (2)d7-NMePFOSAE
NMePFOSAE
d3-NMePFOSANMEPFOSA
d3-NMeFOSAA
NMeFOSAA
NMeFOSAA (2)d9-NEtPFOSAE
NEtPFOSAE
d5-NETPFOSA
NEtPFOSAPFDSPFDS (2)
13C7-PFUnDA

413
413
503
507
499
413
472
463
463
506
498
549
549
515
519
513
513
529
527
527
623
616
515
512
573
570
570
639
630
531
526
599
599
570

369
169
80
80
80
169
427
419
169
78
78
80
99
470
474
469
169
81
507
81
59
59
169
169
419
419
483
59
59
169
169
80
99
525

PFUnDAPFUnDA (2)d5-NEtFOSAA
NEtFOSAA
NEtFOSAA (2)13C-PFDoDA
PFDoDA
PFDoDA (2)
10:2-FTS
10:2-FTS (2)PFDoS
PFTrDA
PFTrDA (2)13C2-PFTeDA
PFTeDA
PFTeDA (2)PFHxDAPFHxDA (2)PFODA
PFODA (2)

563
563
589
584
584
615
613
613
627
627
699
663
663
715
713
713
813
813
913
913

519
169
419
419
526
570
569
169
607
81
80
619
169
670
669
169
769
169
869
169

Attachment 2

PFAS Injection Standards/Extraction Standards/Native Compounds
Injection Standards
Inj Std

I13C3-PFBA
I13C2-PFOA
I13C4-PFOS
I13C2-PFDA

Internal
Standard/Injection
Standard
13C3-PFBA
13C2-PFOA
13C4-PFOS
13C2-PFDA

Extraction Standards
Extraction Standard
E13C4-PFBA
E13C5-PFPeA
E13C3-PFBS
E13C2-4:2-FTS
E13C5-PFHxA
E13C3-PFHxS
E13C4-PFHpA
E13C2-6:2-FTS
E13C8-PFOA
E13C8-PFOS
E13C9-PFNA
E13C8-PFOSA
E13C6-PFDA
E13C2-8:2-FTS
Ed7-NMePFOSAE
Ed3-NMePFOSA
Ed3-NMeFOSAA
Ed9-NEtPFOSAE
Ed5-NEtPFOSA
E13C7-PFUnDA
Ed5-NEtFOSAA
E13C2-PFDoDA
E13C2-PFTeDA

Internal Standard
13C3-PFBA

13C2-PFOA

13C4-PFOS

13C2-PFDA

Page 1 of 2

Native PFAS Compounds
Native
PFBA
PFPeA
PFBS
PFPeS
4:2-FTS
PFHxA
PFHxS
PFHpS
PFHpA
6:2-FTS
PFOA
PFOS
PFNS
PFDS
PFDoS
PFNA
PFOSA
PFDA
8:2-FTS
10:2-FTS
NMePFOSAE
NMePFOSANMeFOSAA
NEtPFOSAE
NEtPFOSAPFUnDA
NEtFOSAA
PFDoDA
PFTrDA
PFTeDAPFHxDA
PFODA

Extraction Standard
13C4-PFBA
13C5-PFPeA
13C3-PFBS
13C2-4:2-FTS
13C5-PFHxA
13C3-PFHxS
13C4-PFHpA
13C2-6:2-FTS
13C8-PFOA
13C8-PFOS
13C9-PFNA
13C8-PFOSA
13C6-PFDA
13C2-8:2-FTS
d7-NMePFOSAE
d3-NMePFOSA
d3-NMeFOSAA
d9-NEtPFOSAE
d5-NEtPFOSA
13C7-PFUnDA
d5-NEtFOSAA
13C2-PFDoDA
13C2-PFTeDA

Page 2 of 2