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US EUUSLA ELLE - T-PFAS-WI20127 - Client Specific: Table 3 Compounds by Direct Injection Using LC/MS/MS, ver. 2
Always check on-line for validity.

Level:

Client Specific: Table 3 Compounds by Direct
Injection Using LC/MS/MS

Document number:Work Instruction

T-PFAS-WI20127
Old Reference:
Version:Organisation level:25-Sub-BU

Document users:
Approved by: UKL3
Effective Date 12-APR-2019 5_EUUSLA_PFAS_Manager, 6_EUUSLA_PFAS_Analyst,

Responsible:

5_EUUSLA_PFAS_Manager

6_EUUSLA_PFAS_Data_Reviewers,
6_EUUSLA_PFAS_Management_Team,
6_EUUSLA_PFAS_Sample_Prep

LIMS ID
14675
This documentation has been prepared by Eurofins Lancaster Laboratories Environmental LLC and its affiliates (“Eurofins”), solely for their own use. The
user of this document agrees by its acceptance to return it to Eurofins upon request and not to reproduce, copy, lend, or otherwise disclose its contents,
directly or indirectly, and not to use if for any other purpose other than that for which it was specifically provided. The user also agrees that where
consultants or other outside parties are involved in the evaluation process, access to these documents shall not be given to said parties unless those parties
also specifically agree to these conditions.

THIS DOCUMENT CONTAINS VALUABLE CONFIDENTIAL AND PROPRIETARY INFORMATION. DISCLOSURE, USE OR REPRODUCTION OF THESE
MATERIALS WITHOUT THE WRITTEN AUTHORIZATION OF EUROFINS IS STRICTLY PROHIBITED. THIS UNPUBLISHED WORK BY EUROFINS IS
PROTECTED BY STATE AND FEDERAL LAW OF THE UNITED STATES.

Revision Log
Reference
Cross Reference
Scope
Basic Principles
Interferences
Precaution to Minimize Method Interference
Safety Precautions and Waste Handling
Personnel Training and Qualifications
Sample Collection, Preservation, and HandlingApparatus and Equipment
Reagents and Standards
Calibration
ProcedureCalculations
Statistical Information/Method Performance
Quality Assurance/Quality Control

Revision Log
Revision
Section
Revision Log
Throughout
document

2

Revision
Section1

Effective Date:
Justification
Formatting requirement
Update requirements

Effective Date:
Justification

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This version
Changes
Removed revision logs up to the previous version
Add additional compounds requested by the client
throughout the document to reflect current practice.

27-SEP-2018
Changes
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Revision1

Effective Date:

27-SEP-2018
new sop

Reference

1. Determination of Table 3 Compounds by LC/MS/MS, Chemours Fluoroproducts Analytical Method
Draft, A. Petlick edit of J.Boyle, 1/8/2018.

2.

Chemical Hygiene Plan, current version.

Cross Reference
Document
T-PEST-WI9847
QA-SOP11892

Document Title
Common Equations Used During Chromatographic Analyses
Determining Method Detection Limits and Limits of Quantitation

Scope

This method is applicable for the determination of selected perfluoroethercarboxylic acids (PFECA)
in aqueous samples to include potable and non-potable waters. The compounds analyzed in thismethod are listed in the table below. The most current MDLs and LOQs are listed in the LIMS.
AnalyteAcronymCAS#

TAF n=4 C7HF13O7 (aka PFO5DoA)TAFN439492-91-6

PFESA Byproduct 1 C7HF13O5S

PFESA-B1

29311-67-9

PFO4DA C6HF11O6

PFO4DA

39492-90-5

PFO2HxA C4HF7O4

PFO2HXA

39492-88-1

PFESA Byproduct 2 C7H2F14O5S

PFESA-B2

749836-20-2

PFECA-G C7HF13O3PFECA-G

801212-59-9

PFO3OA C5HF9O5

PFO3OA

39492-89-2

PFMOAA C3HF5O3PFMOAA

674-13-5

FRD903 HFPODA C6HF11O3

HFPODA

13252-13-6

PEPA

PEPA

267239-61-2

PMPA C4HF7O3

PMPA

13140-29-9

DFSA C2H2F2O5S

DFSA

422-67-3

MMF C3H2F2O4

MMF

1514-85-8

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MTP C4H4F4O3

MTP

93449-21-9

PPF Acid C3HF5O2

PPF Acid

422-64-0

R-EVE C8H2F12O5

R-EVE

NA

Byproduct 4 C7H2F12O6S

Byproduct 4

NA

Byproduct 5 C7H3F11O7S

Byproduct 5

NA

NVHOS C4H2F8O4S

NVHOS

1132933-86-8

PFECA B C5HF9O4PFECA B

151772-58-6

PES C4HF9O4S

PES113507-82-7

Hydro-EVE Acid C8H2F14O4

Hydro-EVE
Acid773804-62-9

EVE Acid C8HF13O4EVE Acid

69087-46-3

Byproduct 6 C6H2F12O4S

Byproduct 6

NA

Basic Principles

A 5-mL aqueous sample is diluted 1:1 with methanol followed by analysis by LC/MS/MS operated innegative electrospray ionization (ESI) mode for detection and quantification of the analytes. Quantitative
analysis is performed using external standard calibration.

Interferences

Compounds which have similar structures to the compounds of interest and similar molecular weights
would potentially interfere. Method interferences may be caused by contaminants in solvents, reagents(including reagent water), sample bottles and caps, and other sample processing hardware that lead todiscrete artifacts and/or 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, etc. A laboratory blank is performed with eachbatch of samples to demonstrate that the extraction system is free of contaminants.
Precaution to Minimize Method Interference

1. LC system components contain many of the target analytes. To minimize the background PFAS
peaks, PTFE solvent frits and tubing are replaced by PEEK™ solvent frits and tubing where possible.
2. A precolumn, Phenomenex Luna, 30 x 2 mm, 5 µm C18 column, is installed before the injection
valve to separate PFAS in standards/samples from those from the LC system and mobile phases.
3. PFAS standards, extracts and samples should not come in contact with any glass containers asthese analytes can potentially adsorb to glass surfaces. PFAS analytes and internal standards
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commercially purchased in glass ampules are acceptable; however, all subsequent transfers or dilutions
performed by the analyst must be prepared and stored in polypropylene containers.
Safety Precautions and Waste Handling

See Chemical Hygiene Plan for general information regarding employee safety, waste management, andpollution prevention.
The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined.
PFOA has been described as “likely to be carcinogenic to humans”. Each chemical should be treated asa potential health hazard and exposure to these chemicals should be minimized.
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 solvents andchemicals and getting them on the skin. Wear gloves when handling neat materials. When working withacids and bases, take care not to come in contact and to wipe any spills. Always add acid 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, andsubsequently 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, and agreeing tofollow 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 of time until they
can independently perform the extraction. Also, several batches of sample extractions must be performed under
the direct observation of another experienced chemist to assure the trainee is capable of independent
preparation. Proficiency is measured through a documented 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 maintenance procedures.
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 for the DOC)
that is carried through all steps of the extraction and meets the defined acceptance criteria. The criteria include
the calculation of mean accuracy and standard deviation.
Sample Collection, Preservation, and HandlingA.

Sample Collection

The samples are collected in polypropylene bottles as per the client's sample collection protocols for
submission to the laboratory for analysis.
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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 willaid 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 first 48
hours after collection. Sample temperature must be confirmed to be at or below 10°C when the samples
are received at the laboratory.
2.
extraction.
3.

Samples stored in the lab must be held at a temperature of 0° to 6°C, not frozen, until
Water samples must be analyzed within 14 days.

Apparatus and Equipment

A.

Apparatus
1.

250ml HDPE bottles: Scientific Specialties; # 334008-blk-1, or equivalent.

2. Centrifuge tubes – 15-mL conical polypropylene with polypropylene screw caps; Fisher Scientific,
Cat. No. 05-539-5 or equivalent
3. 10-mL polypropylene volumetric flask, class B – BrandTech Scientific, Inc., Cat. No. V677941 orequivalent.
4.

Polypropylene bottles for reagent storage: 1000ml, Fisher; Cat. No. 02896F.

5.

Analytical Balance – Capable of weighing to 0.0001 g

6.

Top-Loading Balance – Capable of weighing to 0.01 g

7. Centrifuge – “Q-Sep 3000”; Restek Corp. Cat. No. 26230, or equivalent, capable of a minimum
rotational speed of 3000 rpm.
8. Disposable polyethylene pipette – Fisher Scientific, Cat. No. S30467-1 or equivalent
9. Auto Pipettes – Eppendorf; capable of accurately dispensing 10ul – 1000ul.
10. Polypropylene pipette tips: 0-200ul. Fisher; Cat. No. 02-681-135
11. Polypropylene pipette tips: 101-1000ul. Fisher, Cat. No. 02-707-508
12. Pipettes – Disposable transfer. Fisher Scientific, Cat. No. 13-711-7M
13. Vortex mixer, variable speed, Fisher Scientific or equivalent
14. Reagent Water Purification System: Capable of producing ultrapure “Type 1/Milli-Q”-grade waterfrom in-house deionized water system. Millipore SAS; Cat. No. FTPF08831.
15. Thermo Target PP Polyspring inserts, catalog number C4010-630P
16. Waters 9mm vial kit pack with cap and PTFE/Sil Septa, catalog number 16005660CV, orequivalent
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17. Polypropylene bottles for standard storage - 4 mL; Fisher Scientific, Cat. No. 2006-9125
18. Syringes- Hamilton #80400 - 25 ul, #80500 - 50 ul, #80600 - 100 ul, #80700 - 250 ul, #80800 500ul.
19. Stainless steel spatula/scoop set. Bel-Art SP Scienceware; Product # 11-865-130.
20. Syringe filter - Acrodisc, Syringe Filter, GHP, 13 mm, 0.2 µm, Aqueous, 100/pkg ,Part #WAT097962.
21. EMD Millipore MColorpHast pH test strips and indicator paper, Fisher Scientific, Cat#M1095350001.

B.

Equipment
1.

AB Sciex Triple Quad 4500 Turbo V Ion Source
ExionLC Controller
ExionLC AC Pump
ExionLC AC Autosampler
Exion AC Column Oven
Data system –Analyst 1.6.3

2. AB Sciex API 4000 Turbo V Ion Source
ExionLC Controller
ExionLC AC Pump
ExionLC AC Autosampler
Exion AC Column Oven
Data system –Analyst 1.6.3
3.

HPLC columnsa.
equivalent

Analytical column-Gemini 3µm C18, 50 x 3 mm, Phenomenex Cat# 00B-4439-YO orb.
equivalent

Analytical column-Gemini 3µm C18, 100 x 3 mm, Phenomenex Cat# 00D-4439-YO orc.

Pre-column- Luna, 5 um C18, 30 x 2 mm, Phenomenex Cat# 00A-4252-B0, or equivalent

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:

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1.

Methanol (MeOH) – Honeywell Burdick and Jackson "Chromasolv LC-MS" grade or equivalent2.

Acetonitrile (ACN) – Fisher Scientific, Optima or equivalent

3.

Ammonium acetate – HPLC grade or equivalent

4. 20 mM ammonium acetate solution – Weigh 1.54 ± 0.01g ammonium acetate into a 1L glass
bottle. Add 1 L Milli Q water and mix well. The solution is prone to volatility losses and is replaced
weekly. Store at room temperature
5.

50:50 Methanol:water for preparing sample dilutions.

6.

Sulfuric acid(H2SO4), 1N(0.5M), Acros Organics(part number 124240010

7.

Potassium hydroxide(KOH) (8N): Acros Organics; 380625000

8. Working KOH solution for pH adjustment (0.1M): In a 100ml volumetric flask add approximately
75ml of MQ water. Add 1.25ml 8N KOH to the flask and bring to final volume of 100ml with MQ water.
Stable 6 months. Store at room temp.
9. Working H2SO4 solution for pH adjustment (0.1M): In a 100ml volumetric flask add approximately
50ml of MQ water. Add 20ml 0.5M H2SO4 to the flask and bring to final volume 100ml with MQ water.
Stable 6 months. Store at room temp.
B.

CalibrationStandards:

Standards are stored in accordance with the manufacturer recommended storage conditions andexpiration dates. Intermediate and calibration standards are stored at room temperature and expire
after 3 months.
1. Calibration and spiking standards are prepared from standard solutions received from the client(0.1% by weight in water) with the exception of HFPODA (Wellington Catalogue # HFPO-DA) which ispurchased as a 50,000 ppb stock solution.
ParentSolu on
ClientProvided/Commercial

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Analyte
DFSA
MMF
MTP
PPF Acid
PFMOAA
BP4
R-EVE
BP5
PMPA
NVHOS
PFO2HXA
PEPA
PES
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ParentSolu onAnalytePFECA-B
PFO3OA
Hydro-EVE Acid
BP6
PFESA-BP2
PFECA-G
PFO4DAPFESA-BP1
EVE Acid
TAFN4(PFO5DoA)HFPODA

2. Prepare 10,000 ppb intermediate A in water:

Compound
DFSA
MMF
MTP
PPF Acid
PFMOAA
BP4
R-EVE
BP5
PMPA
NVHOS
PFO2HXAPEPAPESPFECA-B
PFO3OA
Hydro-EVE Acid
BP6
PFESA-BP2
PFECA-G
PFO4DAPFESA-BP1
EVE Acid
TAFN4(PFO5DoA)

Conc. (ppb)
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000
1 000 000

Init. Vol.
(mL)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Final Vol.
(mL)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Intermediate
A Conc.
(ppb)
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 0001 000 000

0.1

10

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3. Prepare 100 ppb Intermediate B in water:

Compound
DFSA
MMF
MTP
PPF Acid
PFMOAA
BP4
R-EVE
BP5
PMPA
NVHOS
PFO2HXAPEPAPESPFECA-B
PFO3OAHFPODA
Hydro-EVE Acid
BP6
PFESA-BP2
PFECA-G
PFO4DAPFESA-BP1
EVE Acid
TAFN4(PFO5DoA)Intermediate
A/HFPODA stock
Conc. (ppb)
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000
10 00050 000
10 000
10 000
10 000
10 000
10 000
10 000
10 000Init. Vol.
(mL)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.02
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Final Vol.
(mL)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Intermediate
B Conc.
(ppb)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

10 000

0.1

10

100

4. Prepare 1 ppb Intermediate C in methanol:water(1:1):

Compound
DFSA
MMF
MTP
PPF Acid
PFMOAA
BP4
R-EVE

Intermediate B
Conc. (ppb)
100
100
100
100
100
100
100

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Init. Vol.
(mL)
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Final Vol.
(mL)
10
10
10
10
10
10
10

Intermediate
C Conc.
(ppb)
1
1
1
1
1
1
1
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Compound
BP5
PMPA
NVHOS
PFO2HXAPEPAPESPFECA-B
PFO3OAHFPODA
Hydro-EVE Acid
BP6
PFESA-BP2
PFECA-G
PFO4DAPFESA-BP1
EVE Acid
TAFN4(PFO5DoA)Intermediate B
Conc. (ppb)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

Init. Vol.
(mL)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Final Vol.
(mL)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Intermediate
C Conc.
(ppb)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

100

0.1

10

1

5. Prepare Calibration Standards in methanol:water (1:1)

CAL8
Ini alVolumeIntermediate
B (mL)
Final Volume(mL)
Intermediate
B Conc.
Compound
(ppb)
DFSA
100
MMF
100
MTP
100
PPF Acid100
PFMOAA
100
BP4
100
R-EVE
100
BP5
100
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CAL7 CAL6

CAL5

CAL4

CAL3

0.5

Ini alVolumeIntermediate
C (mL)

0.1

0.05

0.025

0.01

0.005 0.002 0.001

10

Final Volume
(mL)

10

10

10

10

10

CAL8
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5

Intermediate
C Conc.
(ppb)
1
1
1
1
1
1
1
1

CAL2

10

CAL1

10

CAL7 CAL6 CAL5 CAL4 CAL3 CAL2 CAL1
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
0.1 0.05 0.025 0.01 0.005 0.002 0.001
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PMPA
NVHOS
PFO2HXAPEPAPESPFECA-B
PFO3OAHFPODA
Hydro-EVE
Acid
BP6
PFESA-B2
PFECA-G
PFO4DAPFESA-B1

100
100
100
100
100
100
100
100

0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5

1
1
1
1
1
1
1
1

0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

0.025
0.025
0.025
0.025
0.025
0.025
0.025
0.025

0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01

0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.005

0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002

0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001

100
100
100
100
100
100

0.5
0.5
0.5
0.5
0.5
0.5

1
1
1
1
1
1

0.1
0.1
0.1
0.1
0.1
0.1

0.05
0.05
0.05
0.05
0.05
0.05

0.025
0.025
0.025
0.025
0.025
0.025

0.01
0.01
0.01
0.01
0.01
0.01

0.005
0.005
0.005
0.005
0.005
0.005

0.002
0.002
0.002
0.002
0.002
0.002

0.001
0.001
0.001
0.001
0.001
0.001

EVE Acid
TAFN4(PFO5DoA)

100

0.5

1

0.1

0.05

0.025

0.01

0.005 0.002 0.001

100

0.5

1

0.1

0.05

0.025

0.01

0.005 0.002 0.001

C. Native spiking solution
Spiking standards are stored at room temperature and expire after 3 months.
Working Native Spike Solution is prepared by placing 0.25 mL of Intermediate B from standard
preparation into a 10 mL volumetric flask and bringing to volume with Milli-Q water.

Analyte
DFSA
MMF
MTP
PPF Acid
PFMOAA
BP4
R-EVE
BP5
PMPA
NVHOS
PFO2HXAPEPAPESPFECA-B
PFO3OA
Hydro-EVE Acid
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Na ve SpikeSolu onConcentra on
(ppb)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
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BP6
PFESA-BP2
PFECA-G
PFO4DAPFESA-BP1
EVE Acid
TAFN4(PFO5DoA)

2.5
2.5
2.5
2.5
2.5
2.5
2.5

Calibration

A.

Initial Calibration

1. Calibration standards are prepared at 0.001 ppb, 0.002 ppb, 0.005 ppb, 0.01 ppb, 0.025 ppb,
0.05 ppb, 0.1 ppb, and 0.5 ppb.
2.
Fit the curve with a linear through zero or linear with a concentration weighing factor of 1/x or
quadratic regression with a concentration weighing factor 1/x2.
3.

Initial calibration acceptance criteria

The R2 value for each calibration curve must be ≥ 0.99 for each analyte.

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 be reported with aqualifying comment.
4.

Initial Calibration Verification (ICV)

2nd source standards are not available. A separately prepared standard at a final concentration of 0.05
ppb is analyzed as the ICV. 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 theanalysis sequence.
a. The CCV run after the initial calibration must be at the 0.01 ppb level.

2.

Acceptance criteria
The calculated amount for each compound in the CCV standard must be within ±30% of the true
value. Samples that are not bracketed by acceptable CCV analyses must be reanalyzed.If CCV
fails, a new initial calibration will be analyzed.

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ProcedureA.

Sample Preparation
1. Check pH of the sample with pH paper to verify sample pH is 6-8. If necessary, use dilute KOH
or H2SO4 to adjust pH in the sample container.
2. Using auto pipette, pipet 5 ml of MQ water(blank) or water sample into 15 ml centrifuge tube.
3. Using auto pipette, add 5 ml of methanol to sample tube. Use vortex to mix. The resulting
solution is a 2X dilution of the sample.
4. Load 3 ml plastic syringe with about 2 ml of diluted sample. Attach GHP syringe filter. Placefilter over opening of vial insert and filter about 0.200 ml into insert.
5. Cap autosampler vial and submit for LC/MS/MS analysis.

Dilution of target analytes is required when the calculated concentration exceeds the calibration
range of the system. See example below.
100X dilution example:a) Using auto pipette, add 9.8 ml of 50:50 methanol water to a 15 ml centrifuge tube.
b) Add 0.200 ml of the 2X sample dilution.
c) Use vortex to mix. Td) The resulting solution is a 100X dilution of the sample. Complete sample preparation withsteps 4 and 5 above.

B. Matrix Spike/LCS Preparation.

1. Check pH of the sample with pH paper to verify sample pH is 6-8. If necessary, use dilute KOH
or H2SO4 to adjust pH in the sample container.
2. Using auto pipette, pipet 5 ml of MQ water(LCS) or water sample(MS) into 15 ml centrifuge
tube.
3. Add 0.05 ml of the native spiking solution.
4. Using auto pipette, add 5 ml of methanol to sample tube. Use vortex to mix. The resulting
solution is a 2X dilution of the sample.
5. Load 3 ml plastic syringe with about 2 ml of diluted sample. Attach GHP syringe filter. Placefilter over opening of vial insert and filter about 0.200 ml into insert.
6. Cap autosampler vial and submit for LC/MS/MS analysis.
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B.

1.

US EUUSLA ELLE - T-PFAS-WI20127 - Client Specific: Table 3 Compounds by Direct Injection Using LC/MS/MS, ver. 2

LC/MS/MS Analysis

Tuning and Chromatographic conditions for LC/MS/MS Analysis

Refer to the instrument manufacturer’s instructions for tuning and conditions. These values are stored in
the tune file for future reference and may not need to be changed unless loss of response is noted.

See the AB Sciex (4000 / 4500) Acquisition, Quantitation, Gradient, and detector condition files for themost up to date chromatographic conditions. Modifications to these conditions can be made at thediscretion of the analyst to improve resolution or the chromatographic process.

2.

Acquisition method: See attachment I

3. 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 and allow it to stabilize.
4. After the initial calibration, inject a solvent blank, followed by the CCV at 0.1 ppb, and samples.
Bracket each set of ten samples with a CCV standard. CAL3, CAL4 and CAL5 are alternated.
5. After injections are completed, check all CCV recoveries and absolute areas to make sure they
are within method control limits. See Calibration section B.2 for acceptance criteria. Process eachchromatogram and closely evaluate all integrations, baseline anomalies, and retention time differences.
If manual integrations are performed, they must be documented and a reason given for the 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.
6. Quantitate results for the method blank. No target analytes at or above the reporting limit may befound in the method blank for acceptable batch results. If a target analyte is detected in the method
blank but not detected in the sample, the data is reported. If a target analyte is detected in the method
blank at a concentration greater than the reporting limit and also in the sample, the sample must be
reprepared. If the target analyte in the sample is detected at a concentration greater than 10 times theamount detected in the method blank, the data is reported.
7. Calculate the recoveries of spiked analytes for the LCS, and matrix spike (MS) by comparing
concentrations observed to the true values. The QC acceptance limits for LCS and MS recovery are 70
to 130% for each analyte. The QC acceptance limit for the relative percent difference (%RPD)
between unspiked sample and the duplicate sample is ≤25% at mid and high range concentrations and≤50% at the minimum reporting limit . If LCS 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 QC acceptance limits, and there are no positive detectionsin the sample, the data may be reported. A comment must be added to the analytical report.
8. Compare the retention times of all of the analytes to the retention times of the calibration standards.
The relative retention times should not vary by more than 0.2 retention time units.
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9. The CAL1 standard is used when assessing the correctness of the computer generated peak
integrations. For results that have responses at or near the CAL1, the analysts will calculate 1/2 of thearea ratio of that compound in the CAL1 standard. If the area ratio for the compound in the sample
exceeds that 1/2 the area ratio from the CAL1 standard, the peak is reported as a positive detection.
10. If the calculated concentration exceeds the calibration range of the system, dilute the extract with
50:50 MeOH:water as described in the Sample Preparation section and reanalyze the diluted sample.

Calculations

A. Analyte Concentration using linear through zero curves (MQ Data processing system)
Concentration = (area ÷ slope) x Dilution Factor
B. Sample Concentration (used only for aqueous samples using the MultiQuant data processing systemon the AB Sciex LC/MS/MS)
Sample concentration (ug/l) = Calc conc x (Sample volume ÷ Sample weight) x DF
C. See T-PEST-WI9847 for additional calculations used to evaluate the calibrations and quality control
samples.

Statistical Information/Method Performance

The LCS should contain all compounds of interest. The limits for LCS and MS are defined by the method.
LCS, MS, and RPD are compared to the limits stored on the LIMS. Historical data for MSs, LCSs,
measurement of uncertainty, is reviewed at least annually. Reporting limits including method detection
limits (MDLs) and limits of quantitation (LOQs) are set according to EPA method requirements and are
evaluated annually. Refer to QA-SOP11892 for specific guidelines and procedures. Updates to the
LIMS are made as needed by the QA Department and only as directed by the supervisor.

Quality Assurance/Quality Control

For each batch of samples extracted, a method blank, an LCS (Milli Q water spiked with all compounds
to be determined carried through the entire procedure) must be extracted. For each sample an MS anda DUP must be extracted. A batch is defined as the samples to be extracted on any given day, but not
to exceed 20 field samples. If more than 20 samples are prepared in a day, an additional batch must be
prepared. If any client, state, or agency has more stringent QC or batching requirements, these must be
followed instead.

The QC acceptance criteria are specified in the method and are as follows:1.

Blank – Value less than the limit of quantitation

2.

LCS – 70% to 130% recovery

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3.

MS– 70% to 130% recovery

4.

Sample Duplicate RPD (relative percent difference)a. ≤25% at mid and high range concentrationsb. ≤50% at the minimum reporting limit

QA-SOP11892 Determining Method Detection Limits and Limits of Quantitation
T-PEST-WI9847 Common Equations Used During Chromatographic Analyses
Attachment:
Attachment 1 - Acquisition Parameters
End of document

Version history
VersionApproval
1
27.SEP.2018
2
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Revision information

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