Perfluoroalkyl Substances

(PFAS) Testing Guide

[Link]/PFAS
 PFAS Structure Complexity
Basic PFAS Structures

Anionic at environmental pH, water soluble

Perfluorosulfonic Acids
PFBS, PFHS, PFOS
PFAS Structures

Perfluorocarboxylic Acids
C6 acid - C12 acid
PFOA C8 acid

More volatile
Fluorotelomer Alcohols
6:2, 8:2 and 10:2

2
Complex PFAS Structures
Sulfonamides Telomer Acids
[Link]/PFAS

Phosphate Esters Phosphinic/phosphonic

Further Complex PFAS Structures

Perfluoroalkyl Sulfonates Fluorotelomer Sulfonamide Amines
PFBS n=4 Qn
PFPeS n=5 QI
PFHxS n=6 Qn 6:2 FtSaAM n = 6 Sq
PFHpS n=7 Qn 8:2 FtSaAm n = 8 QI
PFOS n=8 Qn
PFNS n=9 QI
PFDS n = 10 Qn

Perfluoroalkyl Carboxylates Fluorotelomer Betaines

5:1:2 FtB n=5 Sq

7:1:2 FtB n=7 Sq
9:1:2 FtB n=9 Sq

PFBA n=4 Qn
PFPeA n=5 Qn
PFHxA n=6 Qn 5:3 FtB n=5 Sq
PFHpA n=7 Qn 7:3 FtB n=7 Sq
PFOA n=8 Qn 9:3 FtB n=9 Sq
PFNA n=9 Qn
PFDA n = 10 Qn
PFUdA n = 11 Qn
PFDoA n = 12 Qn
Fluorotelomer Sulfonates
PFTrA n = 13 Qn
PFTeA n = 14 Qn
4:2 FtS n=4 Qr
6:2 FtS n=6 Qr
8:2 FtS n=8 Qr
 PFAS: The Forever Chemicals
PFAS have been around for 70 years, but are only now being found everywhere and
seemingly in everything. Increasing public concern about PFAS is driving unprecedented
growth in analytical technology and methodology. Now, explore HPLC column choices
and sample preparation options for diverse sample matrices, separation selectivities,
Table of Contents

and workflows. Here you will find the latest word on PFAS analysis - but certainly not the
final - on this rapidly expanding field of investigation.

3
[Link]/PFAS

Table of Contents

Introduction
1. The History and Future of PFAS Analytical Methodology 4-5

Drinking Water
2. EPA Method 537.1 7-9
3. EPA Method 533 10 - 14
4. Large-Volume Direct Injection 15 - 23

Wastewater, Sediment, and Soil

5. Strata PFAS SPE for DOD QSM 5.3. 25 - 26
6. Determination of PFAS in Sediments by QuEChERs, SPE and LC-MS/MS 27 - 29

Food and Food Packaging

7. New Concerns about PFAS in Food 31 - 37
8. PFAS in Food Contact Materials 38 - 41

New Analytical Frontiers

9. pH-Variable LC Mobile Phase Gradient 43 - 48
10. Column Chemistry Considerations 49 - 57

Product Guide and Ordering Information 58 - 64

 Introduction
1. The History and Future of PFAS Analytical Methodology
Introduction EPA Method 533
Analytical methods for PFAS have greatly evolved over the past In 2019 EPA published the PFAS Action Plan (3) which focused
decade. Beginning in 2009, USEPA published EPA Method 537 on proactive strategies and policies to address PFAS contami-
(1), their first standard method for PFAS in drinking water. EPA nation and protect public health and the environment. This co-
537, which targeted only 17 PFAS analytes, was developed to incided with the release of a new analytical method for PFAS
support the Third Unregulated Contaminant Monitoring Rule in drinking water – EPA Method 533 (4). This new method in-
(UCMR3) conducted from 2012-2017. From that time forward, creased the total number of analytes to 25 to include eleven
the scope an complexity of PFAS analysis has continued to short chain PFAS compounds that were more prevalent in the
Introduction

evolve. new manufacturing processes. However, four long chain PFAS

compounds that had been part of the EPA Method 537.1 were
EPA Method 537 removed from the panel due to 7 years of non-detects in the
The original EPA PFAS method, EPA Method 537, utilized Solid UCMR program. These additions and subtractions created the
Phase Extraction (SPE) to remove chemical interferences and to need for a new SPE sorbent in sample preparation step of the
concentrate the compounds of interest, followed by LC-MS/MS method because the hydrophobic SDVB sorbent used in EPA
to identify and quantitate the 14 analytes. The sample prepara- Method 537.1 was not able to retain the more hydrophilic short
tion step of the method specified a strongly hydrophobic SPE chain PFAS compounds. Consequently, EPA Method 533 speci-
sorbent composed of styrenedivinylbenzene polymer (SDVB) or fies the following requirements for the SPE sorbent used:
equivalent. The HPLC column used in method development and • Weak anion exchange, mixed-mode polymeric sorbent
validation was Phenomenex Gemini® 3u C18. The SPE equiva- (polymeric backbone with a diamino ligand)
4 lency specification allowed other SPE sorbents to be used, such
as Phenomenex Strata®-X (SDVB co-polymerized with polyvi- • Particle size approximately 33 µm
nyl-pyrrolidone).
[Link]/PFAS

• The SPE sorbent must have a pKa above 8, so it remains

positively charged during extraction
EPA Method 537.1
The original EPA Method 537 was updated to EPA Method EPA Method 533 was developed and validated around the use of
537.1 (2) in 2018. The new drinking water method recognized Phenomenex Strata-X-AW SPE sorbent which shows excellent
the major changes in the PFAS landscape that had been during recoveries for all 25 analytes. The analytical column identified in
the intervening nine years. PFOS and PFOA, the compounds EPA Method 533 is still Phenomenex Gemini 3 µm C18. How-
originally identified as being of greatest concern, had ceased ever, there was also the addition of an isotope dilution quality
to be manufactured and had been replaced in commerce by assurance scheme to account for any bias created in sampling,
the so-called “Gen-X” family of PFAS which were thought to be sample preparation or LC system variation. Method 533 will be
less toxic and more environment friendly. Four of the most com- employed in the UCMR5 cycle which will begin in 2022 (5). [See
monly encountered Gen-X compounds were therefore added to Chapter 3: EPA Method 533].
the original analyte list, giving EPA Method 537.1, a total of 18
analytes. The method also included a more restrictive SPE sor-
DOD QSM 5.3
bent specification in the sample preparation step. The authors
The DOD Quality Systems Manual 5.3 (6), is not a method per
of the method specified that only SDVB SPE Polymers could be
se, but rather a set of quality processes established by the De-
used, and included language that disallowed the use of other
partment of Defense (DOD) to ensure consistency across labo-
“co-polymers.” This language was updated in 2020 under Meth-
ratories that analyze non-drinking water samples (wastewater,
od 537.1, Revision 2.0, as indicated in Section 6.9.1, that “the
soils, sediment, etc.) from military installations. One of the few
sorbent may not be modified with monomers other than SDVB.”
method criteria specified in DOD QSM 5.3 (in Table B-15) is the
[See Chapter 2: EPA Method 527.1]
use of graphitized carbon black (GCB) media in the final step
of the sample preparation process following the SPE step. Ini-
tially, laboratories that follow the DOD guidelines used either a
separate GCB tube for this second extraction step, or added a
small amount of GCB media to the final extract as a dispersive
SPE step. However, both of these procedures require additional
time and resources, and can decrease the accuracy and preci-
sion of the overall analytical method owing to increased sample
manipulation.
 Introduction (continued)
In response to these procedural deficiencies, Phenomenex – in Pending Official PFAS Methods
partnership with a leading PFAS testing laboratory organization There are several new official method developments in progress.
- developed a new compliant SPE tube configuration, wherein FDA has developed a QuEChERs-based LC-MS/MS method for
Strata®-X-AW SPE media is packed on top of the GCB in the PFAS in food and feed products that has now undergone sin-
same tube. Using this new product - Strata PFAS - laborato- gle laboratory validation (7). In addition, USDA is developing a
ries have demonstrated comparable recoveries for all analytes UHPLC-MS/MS method for the analysis of PFAS in muscle
compared with the two step process, but with lower RSD val- tissue and plasma (8). Finally, several ASTM methods may be
ues. They have also demonstrated significant time savings in forthcoming soon, as well as the much-anticipated EPA Method
the sample preparation step, as well as reduction in the rate of 8327 (9)that should prove to be more applicable for ground wa-
samples re-runs due to QC batch failure. [See Chapter 5: Strata® ter, surface water and waste water matrices. However, discus-
Introduction

PFAS for DOD QSM 5.3] sion of these more recent developments will be deferred to the
next edition of the PFAS Guide when more operating experience
Other Multi-Media Methods and method validation data will be available. Please refer to the
All the validated EPA PFAS methods previously described were Product Guide for a complete summary of Phenomenex prod-
designed specifically for the analysis of PFAS in drinking wa- ucts that are referenced in current official methods or can be
ter. Analysts who wish to analyze PFAS in other matrices (such applied as equivalent.
as waste water, sediment, leachate, bio-solids, and tissues) are
generally free to develop and validate in-house methods for The Future of PFAS Method Development
these matrices. These customized “MOD Methods” are usually Clearly, the development of PFAS methodology is far from over.
based upon the analyte lists and LCMS operating conditions With over 9,000 PFAS compounds listed in the EPA PFAS Mas-
of EPA Method 537.1 or EPA Method 533, but with appropriate ter List (10), a great deal of advanced method development work
5 modifications to the sample preparation step. will be needed to fully establish the depth and breadth of the
PFAS contamination problem. Going forward, Phenomenex in-
tends to maintain its leading role as an analytical chemistry in-
[Link]/PFAS

novator in the field of PFAS analysis.

For example, waste water samples with high levels of particu-

lates cannot be reliably analyzed by strictly following the drink-
ing water methods because filtration is contra-indicated. There-
fore, these difficult samples are often analyzed by an improvised
method (based upon EPA method 537.1 or 533) wherein the To that end, we round out this PFAS Applications Guide with
official method specified SPE sorbent is replaced with a larger, two, forward-looking technical notes intended to stimulate fu-
100u particle sorbent such as Strata-XL, or Strata- XL-AW. In ture scientific thought and inquiry. [See Chapter 9: pH-Variable
addition, modified methods have been developed for the analy- LC Mobile Phase Gradient and also Chapter 10: Column Chem-
sis of PFAS in food products, sediments and other difficult ma- istry Considerations].
trices by incorporating the QuEChERS sample preparation pro-
cedure, either alone or in combination with weak ion exchange
Conclusion
SPE. [See Chapter 6: Determination of PFAS in Sediments, and
All of the above considerations point to significant challenges
also Chapter 7: New Concerns about PFAS in Food].
in developing new PFAS analytical methods. Phenomenex is
committed to lead the way with groundbreaking research
into the development of unique stationary phases that of-
References
1. [Link]
fer novel PFAS selectivity, as well as new SPE sorbents and
Id=198984&-simpleSearch=1&searchAll=EPA%2F600 %2FR-08 %2F092+ configurations for better sample cleanup and improved sen-
2. [Link] sitivity – especially for difficult matrices like food and biota.
3. EPA’s PFAS Action Plan | Per- and Polyfluoroalkyl Substances (PFAS) | US EPA While this new PFAS Testing Guide is a significant update to
4. Method 533: Determination of Per- and Polyfluoroalkyl Substances in Drinking Water by
Isotope Dilution Anion Exchange Solid Phase Extraction and Liquid Chromatography/
our original 2017 edition, we fully expect that new discoveries
Tandem Mass Spectrometry | Methods Approved to Analyze Drinking Water Samples to in both PFAS chemistry and analytical methodology will quickly
Ensure Compliance with Regulations | US EPA result in this new Guide becoming obsolete. We look forward to
5. Federal Register :: Revisions to the Unregulated Contaminant Monitoring Rule (UCMR 5) continuing to provide the tools and techniques to further under-
for Public Water Systems and Announcement of Public Meeting
6. [Link]
stand these uniquely challenging pollutants.
7. Determination of 16 Perfluoroalkyl and Polyfluoroalkyl Substances in Food using Liquid
Chromatography-Tandem Mass Spectrometry ([Link])
8. CLG-PFAS2.03 Screening, Determination and Confirmation of PFAS by UPLC-MS-MS
([Link])
9. Validated Test Method 8327: Per-and Polyfluoroalkyl Substances (PFAS) Using External
Standard Calibration and Multiple Reaction Monitoring (MRM) Liquid Chromatography/
Tandem Mass Spectrometry (LC/MS/MS) | Hazardous Waste Test Methods / SW-846 |
US EPA
10. CompTox Chemicals Dashboard | PFASMASTER Chemicals ([Link])
 Drinking Water
Drinking water has received the majority attention as a primary source of PFAS
exposure. Drinking water has also been the most widely studied, notably through the
USEPA Unregulated Contaminant Monitoring Rule (UCMR) program, initially using EPA
Method 537.1 and more recently using EPA Method 533 with its expanded analyte list
and attention to shorter chain PFAS and the GenX compounds. As analytical technology
Drinking Water

advances, method developers continue to create analytical methods for PFAS in drinking
water with expanded analyte lists and advanced techniques, such as large volume direct
injection and on-line Solid Phase Extraction (SPE). Although some of these modifications
are not considered “official methods” for regulatory purposes, they are widely used for
investigation and problem assessment.

6
[Link]/PFAS
 2. EPA Method 537.1

PFAS in Drinking Water Using Strata® SDB-L Solid Phase Extraction (SPE) and a
Luna® Omega 1.6 µm PS C18 UHPLC Column
David Kennedy1, Sam Lodge1, and Agustin Pierri2
1
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
Drinking Water

2
Weck Laboratories, Industry, CA 91745, USA

Overview Materials and Methods

This application demonstrates the suitability and advantage
of using Strata SDB-L SPE along with a Luna Omega 1.6 µm Solid Phase Extraction Protocol
PS C18 UHPLC column in the performance of EPA Method
537.1, the official SPE liquid chromatography/tandem mass Following the procedures of EPA Method 537.1, V2,
spectrometry (LC-MS/MS) method for the determination of Sections 6.9 - 6.11 and 11.3 - 11.4
selected per- and polyfluorinated alkyl substances (PFAS) in
drinking water in the United States. Cartridge: Strata SDB-L, 500 mg/6 mL
Part No.: 8B-S014-HCH
Introduction Load: 250 mL sample that has been
EPA Method 537 Version 1.1, was first published in 2009 for fortified with surrogates
Elution: 2x 3 mL Methanol
7 use in the Third Unregulated Contaminant Rule (UCMR3) na- Dry Down: With Nitrogen in a heated water bath
tionwide drinking water survey. This original PFAS method Reconstitute: Adjust final volume to 1 mL with
specified 14 target PFAS analytes, including Perfluorooctane- 96:4 Water:Methanol (v/v) and add
internal standards
sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), the
[Link]/PFAS

two PFAS compounds of largest historic use. However, both

the production and use of PFOS and PFOA were discontinued HPLC Conditions
between 2000 and 2015 and were replaced by shorter chain
PFAS compounds (informally referred to as the “GenX” com- Following the procedures of EPA Method 537.1, V2,
pounds), which were thought to be less persistent in the en- Sections 6.9 - 6.11 and 11.3 - 11.4
vironment and less toxic. Therefore, EPA Method 537.1 was
introduced in 2018 as an update to EPA Method 537 Version Column: Luna Omega 1.6 µm PS C18
1.1. It included the original 14 PFAS analytes and added 4 of Dimension: 100 x 2.1 mm
Part No.: 00D-4752-AN
the shorter chain PFAS compounds for a total of 18 analytes. Mobile Phase: A: 0.1 % Acetic acid in Water
As originally published, EPA Method 537.1 specified that only B: Methanol
SPE cartridges based upon SDVB (styrenedivinylbenzene) Gradient: Time (min) %B
0 20
polymers could be used in the extraction procedure, owing to 0.5 30
low recovery of the short chain PFAS compounds on non-SD- 7 90
VB polymers. This stipulation was continued in the most re- 7.5 100
9 100
cent update: Method 537.1 V2, published in March, 2020.1 Flow Rate: 0.7 mL/min
Injection Volume: 4 µL
In this technical note we present analytical results for the analy- LC System: Agilent® 1260 Series HPLC
Detection: Agilent Ultivo™ Triple Quadrupole MS
sis of drinking water by EPA Method 537.1 using Strata SDB-L
SPE (based upon a SDVB polymer) and a Luna Omega 1.6 µm
PS C18 UHPLC column. The data demonstrates excellent re- Data and Results
covery for all 18 PFAS analytes on Strata SDB-L. Likewise, Luna
Omega 1.6 µm PS C18 provides outstanding column efficiency
PFAS Target Analytes and UHPLC Retention Times
and analyte resolution for greater method sensitivity and shorter
run times.
Analyte RT (min) Internal Standard
PFBS 2.29 13C4 -PFOS
PFHxA 3.20 13C2 -PFOA
HFPO-DA 3.55 13C2 -PFOA
PFHpA 4.24 13C2 -PFOA
PFHxS 4.39 13C4 -PFOS
ADONA 4.41 13C2 -PFOA
PFOA 5.08 13C2 -PFOA
PFOS 5.72 13C4 -PFOS
PFNA 5.77 13C2 -PFOA
9Cl-PF3ONS 6.15 13C4 -PFOS
PFDA 6.35 13C2 -PFOA
NMeFOSAA 6.70 d3 -NMeFOSAA
PFUnA 6.83 13C2 -PFOA
NEtFOSAA 6.88 d3 -NMeFOSAA
11Cl-PF3OUdS 7.17 13C4 -PFOS
PFDoA 7.37 13C2 -PFOA
PFTrDA 7.80 13C2 -PFOA
PFTA 8.18 13C2 -PFOA
 2. EPA Method 537.1 (continued)

Full PFAS Target Analytes

PFAS Replacement Compounds

Drinking Water

2.2
2.15
2.1
2.05
2
1.95
1.9
1.85
1.8
1.75
1.7
1.65
1.6
1.55
1.5
1.45
1.4
1.35
1.3
1.25
1.2
1.15
1.1
1.05
1
0.95
8 0.9
0.85
0.8
0.75
0.7
0.65
0.6
[Link]/PFAS

0.55
0.5
0.45
0.4

App ID 25843
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 min
Counts vs. Acquisition Time

Short Chain (“Gen X”) PFAS Analytes

1.95
1.9
1.85 ADONA
1.8
1.75
1.7
1.65
1.6
1.55
1.5
1.45 9CI-PF3ONS
1.4
1.35
1.3
1.25
1.2
1.15
1.1
1.05
1 11Cl-PF3OUdS
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1 HFPO-DA
0.05
0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 min
Counts vs. Acquisition Time
 2. EPA Method 537.1 (continued)

Method Precision and Accuracy from the Analysis of 50

Consecutive Laboratory Control Samples (LCS)

Mean % Standard Discussion

Drinking Water

Recovery Deviation These results fully demonstrate the suitability of the combina-
tion of Strata® SDB-L 500 mg/6 mL and Luna® Omega 1.6 µm
13C2-PFDA 106 15.8
PS C18 for use in EPA Method 537.1. Luna Omega 1.6 µm PS
13C2-PFHxA 108 18.2 C18 provides excellent separation of all analytes, including the
d5-EtFOSAA 104 19.8 shorter chain “Gen X” compounds. The accuracy and precision
13C2-HFPO-DA 104 17.1 of the data, as demonstrated by the analysis of 50 consecutive
LCS samples, are well within the requirements of the method.
11Cl-PF3OUdS 105 10.9
However, beyond meeting method requirements, the additional
9Cl-PF3OUNS 104 11.9 advantage of the Strata/Luna combination is its contribution to
ADONA 103 13.6 environmental laboratory productivity. In the published version
Et-FOSAA 111 13.8 of EPA Method 537.1, the run time for the 25 analytes (18 target
analytes and 7 internal standards) was 25 minutes. In the data
HFPO-DA 104 15.6
presented here, the equivalent run time is 8.5 minutes. Which
9 Me-FOSAA 113 18.5 represents a nearly 3-fold productivity increase in the chromato-
PFBS 104 14.7 graphic step compared to the method as originally published.
PFDA 106 12 This illustration of EPA Method 537.1 suitability and productivity
demonstrates why the combination of Strata SDB-L and Luna
[Link]/PFAS

PFDoA 105 17.4

Omega 1.6 µm PS C18 has become the environmental testing
PFHpA 111 14.8 industry’s go-to approach for PFAS drinking water analysis, re-
PFHxA 109 14.1 gardless of the instrumentation platform used.
PFHxS 108 15.1
PFNA 109 12.7 Conclusion
PFOA 109 12.5
EPA Method 537.1 is an official US regulatory method to be used
by environmental laboratories to quantitate PFAS in drinking wa-
PFOS 111 13 ter. Health advisory drinking water limits for PFOS and PFOA
PFTeDA 103 14.6 have been established at 70 µg/L and it is expected that official
PFTrDA 104 13.7 drinking water limits will be promulgated for these two com-
pounds, and for additional PFAS in the near future. As drinking
PFUnA 107 13.7
water utilities and water resource agencies opt (or are required)
to routinely test for PFAS in drinking water or water supplies,
EPA 537.1 is destined to transition from its prior status as an
exotic analytical method to a common environmental laboratory
test. The Strata/Luna combination is already widely used in EPA
Method 537.1 testing in the United States owing to the com-
bination of high accuracy and precision and reduced analysis
time. These properties will allow for higher sample throughput
at lower detection levels as PFAS water testing becomes ever
more prevalent.

Acknowledgments
We would like to acknowledge the invaluable assistance of
Weck Laboratories in demonstrating the suitability of Strata
SDB-L and Luna Omega 1.6 µm PS C18 for this essential envi-
ronmental application.

References
1. [Link]
tryId=348508&Lab=CESER&simpleSearch=0&showCrite-
ria=2&searchAll=537.1&TIMSType=&dateBeginPublishedPre-
sented=03 %2F24 %2F2018
 3. EPA Method 533

PFAS in Drinking Water

Sam Lodge1 and Agustin Pierri2
1
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
2
Weck Laboratories, Inc., 14859 Clark Avenue, Industry, CA 91745 USA
Drinking Water

Introduction Sample Preparation Protocol

The first official EPA method for Per- and Polyfluoroalkyl Sub-
Pre-treatment: 100-250 mL sample is fortified with isotopically labeled analogues
stances (PFAS) was EPA 537 developed in 2009, in part to sup- of the method analytes
port the UCMR3 study for drinking water systems in the US. This Cartridge: Strata-X-AW 500 mg/6 mL
method included 14 PFAS compounds, including both PFOS and Part No.: 8B-S038-HCH
Load: Pass pre-treated sample through the cartridge
PFOA, which were then considered to have potential impact on Wash 1: Aqueous Ammonium acetate followed by Methanol
human health. In 2018, EPA 537.1 was introduced to include 4 of Wash 2: Methanol
the “replacement” PFAS compounds which had replaced PFOA Elute: Ammonium hydroxide in Methanol
Dry Down: Under a gentle stream of Nitrogen in a heated water bath
and PFOS in many manufacturing processes in the interim. Reconstitute: Adjust the final volume to 1 mL with 20 % Water in Methanol (v/v)
before analyzing by LC-MS
In 2019, EPA released their PFAS Action Plan, which outlined
10 the steps that the EPA proposed to take to identify and regulate
PFAS in the environment. The PFAS Action Plan called for the LC Conditions
development and promulgation of new analytical methods that Column: Gemini 3 µm C18
Dimension: 50 x 2.0 mm
would allow scientists to effectively measure more PFAS com-
[Link]/PFAS

Part No.: 00B-4439-B0

pounds, with greater accuracy and precision. Published at the Mobile Phase: A: 20 mM Ammonium Acetate
end of 2019, EPA 533 is the first of these new PFAS analytical B: Methanol
Gradient: Time (min) %B
methods. 0 5
0.5 5
EPA 533 is complementary to EPA 537.1. It analyzes 14 of the 18 3 40
compounds from EPA 537.1, plus an additional 11 “short chain” 16 80
18 80
(C4-C12) PFAS compounds. Of the original EPA 537 and EPA 20 95
537.1 compounds, 4 were not included in EPA 533, since they 22 95
had been shown not to be present in drinking water during the 25 5
35 5
previous UCMR study. Of the new EPA 533 compounds, PFBA Flow Rate: 0.25 mL/min
and PFPeA, had been intentionally excluded from EPA 537.1 be- Injection Volume: 2 µL
cause they were too polar to be extracted by a styrene divin- MS Detection: Electrospray Ionization Tandem Mass Spectrometer (ESI-MS/MS)
ylbenzene (SDVB) solid phase extraction (SPE) sorbent during
the sample preparation step. However, EPA 533 was able to
include these 2 compounds, along with the other short chain Table 1.
analytes, because this new method employs a polymeric weak EPA Method Comparison
anion-exchange (WAX) sorbent in the SPE sample preparation
step which is very selective for the more polar/acidic PFAS an- EPA 537.1 EPA 533
alytes. An additional distinction of EPA 533 is that it uses the
isotope dilution technique to enhance method accuracy and ro- 18 analytes
25 analytes (including 14 from 537.1 and
bustness. 11 new short chain compounds)

SDVB SPE sorbent WAX SPE sorbent

Materials and Methods
Isotopic Internal Standards plus Isotope
The following is a summary of the prescribed experimental con- Isotopic Internal Standards
Dilution standards for each analyte
ditions taken from EPA 533. It should be noted that Strata®-X-AW
and Gemini® 3 μm C18 were the respective SPE sorbent and LC
column used in the development of EPA 533 and in its subse-
quent multi-laboratory validation.
 3. EPA Method 533 (continued)

Results Table 4.
Method Analytes, Retention Times, and Suggested Isotope
Table 2. Dilution Analogue References
Isotopically Labeled Isotope Performance Standards and
Drinking Water

Retention Times Isotope

Peak No.
Analyte RT (min) Dilution
(Figure 1)
Analogue
Isotopes Analytes RT (min)
PFBA 3 4.15 C4-PFBA
13

C3-PFBA
13
4.14
PFMPA 4 4.84 C4-PFBA
13

C2-PFOA
13
12.19
PFPeA 6 6.13 C5-PFPeA
13

C4-PFOS
13
13.73
PFBS 8 6.62 C3-PFBS
13

PFMBA 9 6.81 C5-PFPeA

13

PFEESA 10 7.53 C3-PFBS

13
Table 3.
Isotope Dilution Analogues: RTs and Suggested Isotope NFDHA 11 8.01 C5-PFHxA
13

11 Performance Standard References

4:2FTS 13 8.12 C2-4:2FTS
13

Isotopically Suggested Isotope PFHxA 15 8.36 C5-PFHxA

13
[Link]/PFAS

RT (min)
Labeled Analyte Performance Standard
PFPeS 16 8.69 C3-PFHxS
13

C4-PFBA
13
4.14 C3-PFBA
13

HFPO-DA 18 9.06 C3-HFPO-DA

13
C5-PFPeA
13
6.13 C3-PFBA
13

PFHpA 20 10.42 C4-PFHpA

13
C3-PFBS
13
6.62 C4-PFOS
13

PFHxS 22 10.62 C3-PFHxS

13
C2-4:2FTS
13
8.12 C4-PFOS
13

ADONA 23 10.73 C4-PFHpA

13
C5-PFHxA
13
8.35 C2-PFOA
13

6:2FTS 25 12.04 C2-6:2FTS

13
C3-HFPO-DA
13
9.06 C2-PFOA
13

PFOA 28 12.19 C8-PFOA

13
C4-PFHpA
13
10.34 C2-PFOA
13

PFHpS 29 12.28 C8-PFOS

13
C3-PFHxS
13
10.61 C4-PFOS
13

PFNA 31 13.70 C9-PFNA

13
C2-6:2FTS
13
12.05 C4-PFOS
13

PFOS 34 13.74 C8-PFOS

13
C8-PFOA
13
12.19 C2-PFOA
13

9Cl-PF3ONS 35 14.53 C8-PFOS

13
C9-PFNA
13
13.70 C2-PFOA
13

8:2 FTS 37 14.94 C2-8:2FTS

13
C8-PFOS
13
13.73 C -PFOS
13 4

PFDA 39 15.00 C6-PFDA

13
C2-8:2FTS
13
14.94 C4-PFOS
13

PFUnA 41 16.14 C7-PFUnA

13
C6-PFDA
13
15.00 C2-PFOA
13

11Cl-PF3OUdS 42 16.70 C8-PFOS

13
C7-PFUnA
13
16.14 C2-PFOA
13

PFDoA 44 17.13 C2-PFDoA

13
C2-PFDoA
13
17.13 C2-PFOA
13
 3. EPA Method 533 (continued)

Table 5.
Precision and Accuracy Data for Reagent Water
Drinking Water

Low High
Mean % Ra Mean % Ra
Analyte Fortification % RSDa Fortification % RSD
(n=7) (n=5)
(ng/L) (ng/L)

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

12 NFDHA 10 110 15 80 98.5 5.4

4:2FTS 10 94.4 14 80 100 5.7

[Link]/PFAS

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

9Cl-PF3ONS 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

11Cl-PF3OUdS 10 106 5.3 80 102 6.1

PFDoA 10 101 6.2 80 96.3 5.1

 3. EPA Method 533 (continued)

Table 6.
EPA 533 Precision and Accuracy Data from a Commercial
Laboratory
Drinking Water

Analyte MS % MSD % BS % BSD % Analyte MS % MSD % BS % BSD %

11Cl-PF3OUdS 85 84 95 86 % PFHpA 112 115 94 97 %

4-2FTS 113 104 109 100 PFHpS 119 117 119 114 %

6-2 FTS 94 96 108 102 PFHxA 113 107 91 95 %

8-2 FTS 97 100 89 101 PFHxS 96 101 108 110 %

9Cl-PF3OUdS 101 107 99 119 PFMBA 106 101 111 118 %

ADONA 118 116 111 99 PFMPA 99 100 108 117 %

HFPO-DA 100 97 110 101 PFNA 107 104 105 110 %

NFDHA 117 126 117 114 PFOA 101 104 101 100 %
13
PFBA 102 116 89 95 PFOS 117 115 108 108 %

PFBS 117 106 97 105 PFPeA 97 96 92 88 %

[Link]/PFAS

PFDA 102 99 112 104 PFPeS 86 99 103 104 %

PFDoA 104 107 108 109 PFUnA 105 103 115 113 %

PFEESA 116 109 119 115

Continued in next column

Figure 1.
Chromatogram from EPA Method 533
30,31,32,33,34
21,22,23

26,27,28,29
10

100
36,37,38,39
35
19,20
14,15
5,6
1,2,3
relative abundance (%)

11,12,13

43,44
42
40,41
17,18
7,8
9

24,25
PFHxS isomers

PFOS isomers
4

16

4 6 8 10 12 14 16 min
retention time (min)
 3. EPA Method 533 (continued)

Discussion Acknowledgement
In this application, the method is outlined for both the SPE The assistance of Dr. Agustin Pierri, Weck Laboratories, in pro-
method and the HPLC conditions. In Table 1, the EPA meth- viding the operational recovery data is gratefully acknowledged.
ods are compared to show where they differ. Tables 2-4 out-
line the specifics for the analytes in EPA Method 533 and then Reference
Drinking Water

the suggested isotopes in relation to each. Specified reten- EPA Method 533 ‘Determination of Per- and Polyfluoroalkyl
tion times (RT) are also mentioned for each of the analytes. In Substances in Drinking Water By Isotope Dilution Anion Ex-
Table 5, the acceptable precision and accuracy data is pre- change Solid Phase Extraction and Liquid Chromatography /
sented. In Table 6 the data is displayed from an actual lab- Tandem Mass Spectrometry’ (2019) [Link]
oratory example that displays the results of how a laborato- production/files/2019-12/documents/method-533-815b19020.
ry implements EPA 533 and in Figure 1 all necessary peaks pdf
from the specified method are shown in the example chromato-
gram. These data demonstrate that EPA Method 533 using
Strata®-X-AW SPE for clean-up and a Gemini® C18 column for
analysis provide accurate and sufficient results for a commer-
cial laboratory running this method.

Conclusion
14 EPA 533 is a significant improvement over EPA 537.1 for the
analysis of PFAS in drinking water. This new method elimi-
nates the 4 compounds from the EPA 537.1 analyte list that
[Link]/PFAS

were not detected over the 10 year period that EPA 537.1 was
being used to monitor these compounds. However, it also i.e.
features the addition of 11 new PFAS compounds that were
not included in EPA 537.1 which are believed to be of greater
environmental significance. These 11 compounds include many
of the “replacement” compounds that are currently being used
in the manufacturing of products that utilize PFAS chemistry.
This makes EPA 533 a much more relevant environmental meth-
od. Furthermore, EPA 533 is a more robust analytical method
owing to the use of the isotope dilution technique which pro-
vides a means to correct for the loss of analytes during sample
preparation step, as well as to offset the potential effects of ion
suppression or enhancement arising from matrix variation. Con-
sequently, EPA 533 will play a critical role in the UCMR5 cycle
beginning in 2021 to assess the safety of US public drinking
water systems. In this way, EPA 533 will play an essential role
in the EPA PFAS Action Plan, potentially leading to official PFAS
drinking water regulations.

However, there are a few specific requirements in this method

that the analyst must carefully follow. The SPE sorbent mass
(in mg) must be at least 2x the sample volume (in mL) to pre-
vent potential overloading of the sorbent. To illustrate, a 100 mL
sample must be extracted with an SPE mass of at least 200 mg,
a 250 mL sample must use an SPE sorbent mass of at least
500 mg and so forth. In addition, the SPE media must meet the
following specifications listed in the method:

• Approximately 33 μm particle size

• Employ a mixed-mode polymeric sorbent mechanism

(polymeric backbone and a diamino ligand functional
group)

• Display a pKa above 8 so that the SPE media remains

positively charged during extraction Now available
Strata-X-AW meets all these requirements and was found to
PFAS CRM Native Standard
show excellent performance in EPA 533 during routine labora-
Certified Reference Materials for EPA 533 and 537.1
tory operation as demonstrated by the performance data pre-
sented above. In addition, as has also been noted, both the • All analytes at the same concetration as acid form
Strata-X-AW SPE sorbent and the Gemini 3 μm C18 HPLC col- • Easy calculation and dilution
umn were used in the development of EPA 533 and its valida-
tion. Understandably, both products are now widely employed Visit [Link]
in environmental laboratories for the routine analysis of PFAS
by EPA 533.
 4. Large-Volume Direct Injection

Quantitation of PFAS in Water Samples using LC-MS/MS Large-Volume Direct

Injection and Solid Phase Extraction
Simon Roberts1, KC Hyland1, Craig Butt1, Scott Krepich2, Eric Redman3, and Christopher Borton1
Drinking Water

SCIEX, USA; 2Phenomenex, USA; 3TestAmerica Laboratories, Sacramento, USA

1

Introduction
PFASs are unique chemicals whose physiochemical properties
make them important for use in a variety of industrial and con-
sumer products including carpets, cookware, food packaging,
fire suppressants, and others (1). Chemically, PFASs are ali-
phatic structures containing one or more C atoms on which H
substituents have been replaced by F atoms. Classification and
naming is typically by the particular functional group present,
such as carboxylic acids, sulfonates, phosphonic acids, etc.,
as well as the length of the carbon chain. Desirable in various
industrial applications for their chemical stability and low reac-
15 tivity, these properties also make PFAS highly resistant to degra-
dation in aquatic environments. Typical concentrations of PFASs
found in various environmental water sources range from pg/L
[Link]/PFAS

to µg/L levels (2).

Human exposure to PFAS residues has been implicated in the

incidence of cancer, obesity, endocrine system disruption, and
other adverse health effects (3-4). In recognition of these poten-
tial risks, sources of human exposure to these chemicals (e.g., Key features of PFAS methods
via drinking water) are receiving public and scientific attention. • LC-MS detection using a Shimadzu® LC-20ADXR coupled
to a SCIEX® Triple Quad 5500 System
PFASs exhibit relatively high aqueous solubility and can be
transported and bioaccumulated from contaminated water • Special modifications to the pumps and autosampler are
sources. The US EPA maintains health advisory limits for select described to mitigate laboratory-based contamination of
PFASs (e.g., perfluorooctanoic acid (PFOA) at a limit of 70 ng/L) PFASs.
in water, but these levels have been exceeded in some areas • Use of a delay column for separation of a contamination
experiencing extreme point source inputs of these chemicals (5). PFAS peak from the analytical peak
Given the tremendous persistence of PFASs in the environment • The first method presented here utilizes a weak-anion ex-
and their known presence in human populations exposed via change solid phase extraction (SPE) method to concentrate
drinking water and other environmental routes, demonstration of water samples for analysis using a 7.5 minute HPLC gra-
the capability for accurate and precise low-level quantitation is dient.
paramount for research and testing laboratories. Robust quanti-
tative analytical methods utilize the specificity and sensitivity of • The second method utilizes dilution of a water sample in
LC-MS/MS with MRM monitoring. However, a primary analytical methanol and direct injection of 950 µL of the diluted sam-
challenge to this assay is the prevention and reduction of back- ple using a 17.5 minute HPLC gradient.
ground PFASs originating from the LC system and contamina-
tion during sample collection and preparation. • Large volume injection of an aqueous sample is intended
to achieve method sensitivity while reducing accumulated
This application note presents two methods for the quantitation background during sample concentration steps.
of per- and polyfluorinated alkyl substances (PFASs) in water
samples. While the MS/MS detection method using the SCIEX • Both methods achieved accurate quantitation at levels of
Triple Quad™ 5500 System is similar between the two methods, approximately 1-10 ng/L for more than 17 PFASs.
the sample preparation and injection volume differ significantly.
 4. Large-Volume Direct Injection (continued)

Methods Table 2. LC gradient for method 1 at a flow rate of

Standards and internal standards (IS): The PFAS standards 0.6 mL/min.
and internal standards were obtained from Wellington Laborato-
ries (Guelph, Ontario) and were prepared in Baker HPLC-grade Step Time (min) A (%) B (%)
methanol. Standard stock solutions were prepared by dilution
Drinking Water

with 96 % methanol and 4 % water (purified using a Millipore® 0 0.00 90 10

water purification system). 1 0.10 45 55
Sampling and sample preparation: Water samples were ob- 2 4.50 1 99
tained anonymously from various sources in the United States.
Samples were stored in the dark at 4 °C in 250 mL high density 3 4.95 1 99
polyethlyene bottles until analysis.
4 5.00 90 10
Chromatography: Shimadzu® LC-20ADXR binary pumps with
End 6.50
a Shimadzu DGU-20A5 degasser was used for separations. All
fluoroethylene polymer (FEP) tubing on the Shimadzu pumps
and degasser was replaced with PEEK tubing with similar inter-
nal and external dimensions. A Phenomenex Luna® C18(2) col- Method 2: Dilution and large volume injection: A 1 mL aliquot
umn (dimensions shown in Table 1) was installed between the of a water sample was added to a 2 mL clear glass autosam-
16 pump mixing chamber and the column, outside of a Shimadzu pler vial with a polyethylene septum cap containing 0.65 mL of
CTO-20AC column oven. This column served as a delay or hold- methanol and a mix of surrogate standards at a final concentra-
up column to isolate PFAS contamination originating from the tion of 50 ng/L. The final concentration of methanol in the diluted
[Link]/PFAS

pumps and eluents. A longer and/or larger diameter Luna C18(2) sample was 40 %, and standards, blanks, and quality control
column must be installed on heavily contaminated systems to samples were all prepared at the same concentration. A PAL
prevent breakthrough of contamination. HTC-xt autosampler was modified to inject 950 µL of the diluted
samples and standards.
Chromatographic separation was performed using a Phenome-
nex Gemini® C18 HPLC column at 0.6 mL/min (Table 1). The For Method 2, samples were analyzed using an extended 15.5
Gemini C18 column was heated to 40 °C in the column oven. min gradient method (Table 3) with a 17.5 min total runtime, in-
A PAL-HTC-xt autosampler with dynamic load-wash (DLW) cluding the 2 min autosampler injection cycle. Water with 20 mM
was modified by replacing all FEP tubing from the rinse solvent ammonium acetate was used as the “A” solvent, and methanol
lines, the needle seal, and the sample holding loop with PEEK was the “B” solvent.
or stainless steel. The autosampler syringe and sample holding
loop was rinsed with methanol and 1:1 methanol:acetonitrile be- MS/MS detection: A SCIEX® Triple Quad™ 5500 System with a
tween samples. Turbo V™ Ion Source and ESI probe was used for analysis in neg-
ative polarity. The ion source parameters were optimized for the
LC conditions using the Compound Optimization (FIA) function
Table 1. LC columns for methods 1 and 2. in Analyst® Software (Table 4).

Method Column Dimensions

Table 3. LC gradient for method 2 at a Flow Rate of
Delay column Phenomenex Luna C18 (2), 5 µm 30 x 2 mm 0.6 mL/min.
Method 1 HPLC Column Phenomenex Gemini C18, 3 µm 50 x 2 mm
Step Time (min) A (%) B (%)
Method 2 HPLC Column Phenomenex Gemini C18, 3 µm 100 x 3 mm
0 0.0 90 10

1 1.5 35 65
Method 1: Solid phase extraction and 10 µL injection: A mix-
ture of surrogate standards (25 ng) was added to 250 mL water 2 8.0 5 95
samples in the sampling bottle, and the entire volume was ex-
3 8.1 1 99
tracted using weak anion exchange SPE as recommended by
ISO standard 251016. The empty sample container was rinsed 4 12.0 1 99
with 10 mL of methanol with 0.3 % NH4OH, which was then add-
ed to the SPE tube to elute the PFASs. The extract was evapo- 5 12.5 90 10
rated to dryness, reconstituted in 500 µL of 80 % methanol/20 %
End 15.5
water, and transferred to a polypropylene vial for analysis. All
standards and blanks were also prepared at a final methanol
concentration of 80 %.
One characteristic MRM transition was monitored for each an-
For Method 1, 10 µL injections of the standards and samples alyte and internal standard (Appendix Table 1). The Scheduled
were analyzed using a 6.5 min gradient method (Table 2) with a MRM™ algorithm was activated to monitor compounds only
7.5 min total runtime, including the 1 min autosampler injection during a 60 second expected retention time window to maxi-
cycle. Water with 20 mM ammonium acetate was used as the mize dwell times and optimize the cycle time of the method. As
“A” solvent and methanol was the “B” solvent. a result, all of the peaks in the calibration contained >12 points
per peak.
 4. Large-Volume Direct Injection (continued)

Table 4. Ion source parameters for methods 1 and 2. Figure 1.

Evaluating carryover
Parameter Value

Curtain Gas (CUR) 35 psi

Drinking Water

IonSpray voltage (IS) -4500 V

Temperature (TEM) 600 ˚C

Nebulizer Gas (GS1) 50 psi

Heater Gas (GS2) 50 psi

Calibration was performed using a 7-point curve at concen-

trations of 25, 50, 250, 1000, 2500, 10000, and 20000 ng/L for
Method 1 and 1, 2, 5, 20, 50, 100, and 200 ng/L for Method 2.
Quantitation was performed using MultiQuant™ Software 3.0.2
using 1.0 Gaussian smoothing and 1/x2 weighted linear regres-
17 sion. PFASs with matched isotopically labeled surrogate stan-
dards were quantified using isotope dilution, while PFASs with-
out matched surrogate standards were quantified using internal
standard calibration with structurally similar isotopically labeled
[Link]/PFAS

standards (full analyte and internal standard list shown in Ap-

pendix Figure 1). A concentration factor of 500 was applied to
samples analyzed using Method 1, and a dilution factor of 1.65 min
was applied to samples analyzed using Method 2. Time

Method 1 chromatography results

The Gemini® C18 column was selected for both methods based Overlaid MRM traces for PFHxS in the lowest calibration stan-
on its strong retention and predictable resolution of PFASs. All dard (black, 25 ng/L) and a blank injection (blue) that followed
of the other columns tested exhibited breakthrough of the short the highest concentration standard (20 µg/L). The delayed peak
chain acids in the column dead volume during optimization of in the calibration standard trace represents the ambient LC sys-
the 950 µL injection method. The Luna® C18(2) column was se- tem contamination retained by the delay column.
lected as the delay column for both methods after initial testing
indicated that it provided better separation of PFAS contamina-
tion than other columns. For PFASs, blank contamination is a
major concern for analysis due to potential contamination during
sample preparation or contamination originating from analytical
instrumentation. Figure 1 shows a small carryover peak at 2.5
min for PFHxS in a blank analyzed immediately following the
injection of the highest calibration standard of 20,000 ng/L. The
area of the carryover peak was only 0.078 % of the highest stan-
dard and 21 % of the lowest calibration standard for Method
1 (25 ng/L). The second peak at 3.2 min in Figure 1 is attribut-
ed to delayed PFHxS contamination originating from the HPLC
pumps. Without the delay column, this contamination would in-
stead focus on the analytical column and elute at 2.5 min along
with the standard and sample peak.

A 50 mm x 2 mm, 3 µm Gemini C18 column was selected for

Method 1, which utilized a 10 µL injection volume. The chro-
matographic separation of 25 PFASs is shown in Figure 2. The
average peak asymmetry factor for the first 2 eluting peaks
(PFBA and PFBS) in the initial calibration standards was calcu-
lated to be 1.3 in Method 1 using MultiQuant Software.
 4. Large-Volume Direct Injection (continued)

Figure 2.
Method 1 chromatography: Weak anion exchange SPE with 10 μL injection
Drinking Water

18
[Link]/PFAS

min
Time

Overlaid Chromatograms of a 1 µg/L Standard Injected using Method 1 calibration

Method 1. The initial 7-point calibration for Method 1 exhibited good ac-
curacy within +/- 30 % of the expected values for all points,
3.0.2. This is within the acceptance criteria set by EPA 537 of accuracy within +/- 10 % for the lowest calibrator, and R2 coef-
0.8-1.5 7. ficients of >0.990, as shown in Table 5. Based on the S/N ratio
Partial resolution of the branched and linear isotopes is neces- of the low calibrator and the linearity of the curve, the calibra-
sary for PFAS analysis to distinguish between samples contain- tion range could be extended on both the high and low levels
ing only linear isotopes or isotope mixtures. As shown in Figure to improve the dynamic range. A water sample analyzed using
2, the earlier eluting branched isotopes are clearly distinguish- Method 1 exhibited concentrations of several PFASs ranging
able from the major peak corresponding to the linear isotopes from 0.974 to 53.3 ng/L, as shown in Figure 3.
for the 2 compounds that contained both branched and linear
isotopes in the standards (PFHxS and PFOS). Most methods
recommend that these two peaks are summed for quantitation,
which was performed in this method using MultiQuant™ Soft-
ware 3.0.2.
 4. Large-Volume Direct Injection (continued)

Method 2 chromatography Figure 3.

Method 2 is a large-volume, direct aqueous injection method Overlaid chromatograms of PFASs quantified in a water
designed for drinking, surface, and ground water samples. After sample using method 1.
the addition of surrogate standards and a simple dilution with This method uses a solid-phase extraction and a 10 µL injection.
methanol, 950 µL of the sample was injected directly onto the
Drinking Water

Gemini® C18 column. In contrast to Method 1, a longer and

larger diameter column was used to improve retention of the
analytes in the large volume injection. This resulted in a longer
total runtime (17.5 minutes compared with 7.5 minutes), but pro-
vided robust results for the large volume injection and minimal
retention time shift (Figure 4). The only compound that exhibited
deteriorated peak shape due to the large injection volume was
PFBA. However, the broadened peak shape of PFBA did not
affect quantitation accuracy or precision.

19
[Link]/PFAS

Table 5.
Calibration curves for method 1 and 2.
Sensitivity and linearity from 25 to 20,000 ng/L and 1 to 200 ng/L min
(coefficient of regression, R2) using Method 1 and Method 2,
respectively. S/N calculated using MultiQuant™ Software 3.0.2. Time

Method 1 Method 2
Calibration Linear S/N of Accuracy Calibration Linear S/N of Accuracy
Compound range correlation 25 ng/L of 25 ng/L range correlation 1 ng/L of 1 ng/L
(ng/L) (R2) standard standard (ng/L) (R2) standard standard
PFCAs
PFBA 25-20,000 0.997 108 104 % 1-200 0.997 328 97 %
PFPeA 25-20,000 0.998 88 103 % 1-200 0.999 137 101 %
PFHxA 25-20,000 0.998 104 93 % 1-200 0.999 284 101 %
PFHpA 50-20,000 0.999 116 101 % 1-200 0.993 267 96 %
PFOA 25-20,000 0.999 117 106 % 1-200 0.999 113 99 %
PFNA 25-20,000 0.990 91 109 % 1-200 0.999 137 101 %
PFDA 25-20,000 0.998 103 105 % 1-200 0.997 176 96 %
PFUdA 25-20,000 0.995 84 101 % 1-200 0.998 168 99 %
PFDoA 25-20,000 0.998 60 101 % 1-200 0.994 127 94 %
PFTrDA 25-20,000 0.998 32 104 % 1-200 0.995 125 95 %
PFTeDA 25-20,000 0.994 15 107 % 1-200 0.998 56 98 %
PFHxDA 25-20,000 0.999 21 103 %
PFODA 25-20,000 0.999 33 102 %
PFSAs
PFBS 25-20,000 0.995 31 92 % 2-200 0.994 1178 100 %
PFHxS 25-20,000 0.999 604 103 % 1-200 0.998 229 96 %
PFHpS 25-20,000 0.997 103 105 % 1-200 0.999 327 99 %
PFOS 25-20,000 0.995 312 105 % 1-200 0.999 251 99 %
PFDS 25-20,000 0.998 88 102 % 1-200 0.999 516 98 %
Other PFASs
6:2 FTS 25-20,000 0.991 100 98 %
8:2 FTS 25-20,000 0.992 113 97 %
PFOSA 25-20,000 0.997 118 104 % 1-100 0.997 1012 96 %
MeFOSA 25-20,000 0.996 96 103 %
EtFOSA 25-20,000 0.994 90 101 %
N-MeFOSAA 25-20,000 0.996 109 100 %
N-EtFOSAA 25-20,000 0.994 61 103 %
 4. Large-Volume Direct Injection (continued)

Similar to Method 1, blank contamination from the instrument

was minimized by using a delay column in Method 2. Blank
contamination from sample preparation was also minimized in
Method 2 by reducing the number of pipetting steps and testing
all new batches of solvents prior to use. The integrated areas of
Drinking Water

the first blank after the highest concentration sample (200 ng/L)
were less than 50 % of the lowest calibrator. For example, the
area of the first blank analyzed after the 200 ng/L calibration
standard was 22 % of the area of the 1 ng/L standard for PFOA
as shown in Figure 5. The other blanks shown in Figure 5 exhib-
ited even lower response for PFOA, which could be contributed
to laboratory contamination for the method blank and solvent
contamination for the instrument blank.

To be compatible with common sampling practices, the Method

2 was not optimized for recovery of the longest chain PFASs,
PFHxDA and PFODA, from the sample container or from the
autosampler vial. Due to the stronger hydrophobicity of these
20 compounds compared with the shorter chain PFAS, PFHxDA
and PFODA appeared to bind to polypropylene containers when
the methanol concentration was <40 %. Modifications to this
method to improve the recovery and precision of PFHxDA and
[Link]/PFAS

PFODA analysis may include collecting smaller samples (10-

20 mL), diluting the entire sample with methanol in the sampling
container, and adding surrogate standard directly to the sam-
pling container.

Direct analysis of water samples is impaired by the presence of

5 g/L Trizma in samples, which is added to drinking water sam-
ples as a requirement by EPA method 537. Trizma suppresses
ionization of the PFASs and elutes slowly from the column for
minutes after the injection. Therefore, Trizma should not be add-
ed to samples that will be analyzed using direct aqueous injec-
tion, but is fully compatible with SPE as performed in Method 1.

Method 2 calibration
Similar to Method 1, the initial calibration results for Method 2
exhibited good accuracy within +/- 30 % of the expected values
for all points, accuracy within +/- 10 % for the lowest calibrator,
and R2 coefficients >0.990, as shown in Table 5. In the devel-
opment of Method 2, calibration standards for 6:2 and 8:2 FTS,
MeFOSA, EtFOSA, MeFOSAA, and EtFOSAA were not analyzed
in the full calibration curve.

Quotes, Methods, Tips... We're here to help

[Link]/chat
 4. Large-Volume Direct Injection (continued)

Figure 4.
Method 2 chromatography: Dilution of water sample in
methanol and 950 μL direct injection
Chromatogram of a 10 ng/L matrix spike into groundwater that
was diluted with methanol and injected according to Method 2.
Drinking Water

21
[Link]/PFAS

min
Time

Figure 6 Method 2 performance

Overlaid MRM traces of PFASs detected in a groundwa- Because large-volume injection methods are less common for
ter sample with the calculated concentrations of each PFASs compared with offline extraction methods, this applica-
PFAS. tion note reports the recovery and precision of continuing cali-
bration standards over 1 week of continuous water sample anal-
The sample was prepared and analyzed using Method 2. ysis to demonstrate the robustness and accuracy of Method 2.
The chromatogram and quantitated values for several PFASs in
one of these water samples are shown Figure 6.

As shown in Table 6, a continuing calibration standard at 20 ng/L

analyzed 1 week after the initial calibration exhibited quantita-
tive accuracy of 92-99 % for all compounds with the exception
of PFTrDA (81 %) and PFBS (84 %). Due to limited availability
of surrogate standards, PFBS was analyzed using 18O2 PFHxS
as an internal standard, and PFTrDA was analyzed using 13C2
PFDoA. The absence of an exact isotope-labelled surrogate for
these two compounds likely contributed to the decreased accu-
racy of the ongoing calibration standard.

During the 1 week period of full-time water sample analysis, 9

replicates of the 20 ng/L continuing calibration verification (CCV)
were analyzed as shown in Table 6. The precision (%CV) for all
of the PFASs was <5 %, which indicates excellent precision for
the large volume injections. The surrogate recovery, calculated
as the response of the surrogate standard in the 20 ng/L ongoing
min calibration standard divided by the response of the surrogate
standard during the initial calibration, was within 73-120 % for
all of the PFASs analyzed.
 4. Large-Volume Direct Injection (continued)

Summary Table 6.
The 2 methods reported here were designed for optimum ro- Method 2
bustness using the SCIEX® Triple Quad™ 5500 System as the
analytical platform. Both methods may be expanded to include Accuracy of a 20 ng/L CCV analyzed 1 week after the initial cal-
soil, sediment, and biological extracts. Minimum and maximum ibration and precision of 9 replicates of a 20 ng/L CCV analyzed
Drinking Water

reporting limits of approximately 1 ng/L to 400 µg/L could be between 5 and 7 days after the initial calibration using Method
achieved using both methods. These ranges could be expanded 2.
by increasing the extracted volume in Method 1 or by further di-
lutions in Method 2. The example chromatograms shown in this
Calculated Precision
application note also demonstrate that the lower calibration lev- conc of Accuracy of
Surrogate
of 20 ng/L
els than the levels analyzed here could be included in initial cal- Compound standard
20 ng/L 20 ng/L CCV CCVs
recovery
ibration curves to further improve the sensitivity of the method. CCV (%CV)

Method 1 has the advantage of compatibility with EPA Meth- PFCAs

od 537 (7) and allows sample concentration using solid phase PFBA 19.4 96 % 107 % 1.50
extraction. Method 2 has the advantages of minimal sample
PFPeA 19.7 98 % 107 % 1.40
preparation and fewer steps to introduce lab-based PFAS con-
tamination. With the growing need for PFAS analysis of environ- PFHxA 19.7 99 % 108 % 2.26
22 mental samples, these versatile methods will be useful for labs
aiming to evaluate growing lists of PFASs. PFHpA* 18.5 92 % 103 % 3.11

PFOA 19.2 96 % 105 % 2.07

[Link]/PFAS

PFNA 19.3 97 % 107 % 1.11

PFDA 19.4 97 % 107 % 2.62

PFUdA 18.8 94 % 109 % 2.90

PFDoA 18.7 94 % 99 % 1.90

PFTrDA 16.3 81 % 99 % 4.77

PFTeDA 18.9 95 % 73 % 1.43

PFSAs

PFBS 16.8 84 % 112 % 2.65

PFHxS 19.2 96 % 112 % 1.94

PFHpS 19.4 97 % 112 % 3.85

PFOS 18.8 94 % 120 % 2.62

PFDS 18.6 93 % 120 % 2.69

Other PFASs

PFOSA 19.0 95 % 112 % 0.98

 4. Large-Volume Direct Injection (continued)
Applications
Aknowledgements Appendix Table 1.
SCIEX® acknowledges TestAmerica (Sacramento, CA) for col- MRM masses for methods 1 and 2.
laborating with SCIEX by providing and conducting the analysis
of standards for this application note. SCIEX also acknowledges Analytes are shown in bold font, and internal standards are
Phenomenex (Torrance, CA) for providing HPLC columns and shown in italic font.
Drinking Water

expertise for this application note and other method develop-

ment efforts. Compound Q1 Q3 DP CE

References PFBA 212.9 169 -25 -12

1. Buck et al., Integr Environ Assess Manag. (2011), 7(4), 513-541) PFPeA 262.9 219 -20 -12

2. Rahman, M. F.; Peldszus, S.; Anderson, W. B. Water Research (2014), 50, PFHxA 313 269 -25 -12
318-340. PFHpA 363 319 -25 -12
3. Braun, J. M.; Chen, A.; Romano, M. E.; Calafat, A. M.; Webster, G. M.; PFOA 413 369 -25 -14
Yolton, K.; Lanphear, B. P. Obesity (2016), 24, 231−237.
PFNA 463 419 -25 -14
4. Barry, V.; Winquist, A.; Steenland, K. Environ. Health Perspect. (2013),
PFDA 513 469 -25 -16
121 (11−12), 1313− 1318.
PFUdA 563 519 -25 -18
23 5. Hu, X. C.; Andrews, D. Q.; Lindstrom, A. B.; Bruton, T. A.; Schaider, L. A.;
Grandjean, P.; Lohmann, R.; Carignan, C. C.; Blum, A.; Balan, S. A.; Hig- PFDoA 613 569 -25 -18
gins, C. P.; Sunderland, E. M. Environ. Sci. Technol. Lett. (2016). PFTrDA 663 619 -25 -20
[Link]/PFAS

6. ISO. 2009. Water quality – Determination of perfluorooctanesulfonate PFTeDA 713 669 -25 -22
(PFOS) and perfluorooctanoate (PFOA) – Method for unfiltered samples
PFHxDA 813 769 -25 -24
using solid phase extraction and liquid chromatography/mass spectrom-
etry. International Organization for Standardization, ISO 25101:2009 (TC PFODA 913 869 -25 -26
47/ SC 2). 19 pp. PFBS 298.9 80 -55 -58
7. EPA Method 537 ‘Determination of Selected Perfluorinated Alkyl Acids in PFHxS 399 80 -60 -74
Drinking Water by Solid Phase Extraction and Liquid Chromatography /
Tandem Mass Spectrometry LC/MS/MS)’ version 1.1 (2009) [Link] PFHpS 449 80 -65 -88
[Link]/microbes/documents/Method%2053 7_FI NAL_rev1.[Link] PFOS 499 80 -65 -108
PFDS 599 80 -85 -118
6:2 FTS 427 407 -50 -32
8:2 FTS 527 507 -50 -40
PFOSA 498 78 -60 -85
MeFOSA 512 169 -75 -37
EtFOSA 526 169 -75 -37
N-MeFOSAA 570 419 -40 -36
N-EtFOSAA 584 419 -50 -36
13C4_PFBA 217 172 -25 -12
13C5_PFPeA 268 223 -20 -12
13C2_PFHxA 315 270 -25 -12
13C4_PFHpA 367 322 -25 -12
13C2_PFOA 415 370 -25 -14
13C4_PFOA 417 372 -25 -14
13C5_PFNA 468 423 -25 -14
13C2_PFDA 515 470 -25 -16
13C2_PFUdA 565 520 -25 -18
13C2_PFDoA 615 570 -25 -18
13C2_PFTeDA 715 670 -25 -22
13C2_PFHxDA 815 770 -25 -24
18O2_PFHxS 403 84 -60 -74
13C4_PFOS 503 80 -65 -108
13C8_PFOSA 506 78 -60 -85
M2-6:2FTS 429 409 -50 -32
M2-8:2FTS 529 509 -50 -40
d3MeFOSA 515 169 -75 -37
d5EtFOSA 531 169 -75 -37
d3-MeFOSAA 573 419 -40 -36
d3-EtFOSAA 589 419 -50 -36
 Wastewater, Sediment, and Soil
Wastewater, Sediment, and Soil

As the PFAS story continues it is becoming more widely recognized that drinking water is
not the only environmental media of concern. From its primary sources in fire suppression
foams, industrial discharges and consumer products, PFAS is also widely found to occur
in soils, sediments, surface water, groundwater and wastewater discharges, illustrating
the widespread dispersion and persistence of this unique class of compounds. These
discoveries have required the development and application of more advanced sample
preparation, chromatography and mass spectrometry techniques to overcome the
challenges of matrix and spectral interferences. In this section, two recent applications
have been selected to illustrate the analytical challenges of these more difficult matrices.

24
[Link]/PFAS
[Link]
 5. SPE for DOD QSM 5.3
Wastewater, Sediment, and Soil

Per- and Polyfluoroalkyl Substances (PFAS) Extraction by LC-MS/MS Using

Strata PFAS for a Stacked Solid Phase Extraction (SPE) Solution
Andrew Patterson1 , Charles Neslund2 , Robert Brown2 , Sam Lodge3 , David Kennedy3 , and Brian Marshall3
1
Eurofins Environment Testing America, 800 Riverside Pkwy, Sacramento, CA 95605, USA
2
Eurofins Environment Testing America, 2425 New Holland Pike, Lancaster, PA 17601
3
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Overview
PFAS are a class of highly stable synthetic organic compounds Strata PFAS is a stacked single cartridge solution with polymer-
used in a wide variety of industrial and commercial applications. ic WAX and GCB sorbents that functions as a traditional Solid
They are also highly stable in the environment and strongly bio- Phase Extraction (SPE) cartridge with a built in polishing step
accumulate. As a result, they have become ubiquitous through- to meet the aforementioned DOD guidelines. This SPE product
out the global environment and are often referred to in popular increases lab productivity and reduces the need for multiple ex-
media as “Forever Chemicals”. Consequently, PFAS levels need traction tubes when compared to a traditional two tube method.
to be tested in drinking water and more recently methods have
been developed to measure PFAS in other environmental ma- Table 2 presents typical analyte recovery data from a routine
trices that require more complex clean-up solutions, such as Laboratory Control Sample (LCS) analyzed by a commercial
wastewater, soils and sediments. testing laboratory highly experienced with the performance of
25 DOD QSM 5.1/5.3. The LCS had been spiked with all 32 target
analytes at 25 μg/L and was analyzed with a batch of field sam-
ples to demonstrate method performance and data acceptabil-
[Link]/PFAS

ity. The recovery data show that all 32 analytes were well within
[Link]

method recovery limits with an average recovery of 98.8 % and

a mean recovery of 99.0 %, thereby demonstrating acceptability
of the use of Strata PFAS in the performance DOD QSM5.1/5.3.

The LCS sample was extracted with Strata PFAS under the
conditions shown below and analyzed on a LC-MS/MS system
using a Gemini® 3 µm C18 HPLC column under the conditions
described below.

SPE Conditions
Cartridge: Strata PFAS (200 mg WAX/50 mg GCB/ 6 mL)
Part No.: CS0-9207
Condition 1: 4 mL 0.3 % Ammonium hydroxide
Condition 2: 4 mL Methanol
Equilibrate: 5 mL Water
Load: Add sample at 4 mL/min
Wash: 2x 4 mL Water
The United States Department of Defense (DOD) is dealing with Elute: 2x 4 mL 0.3 % Ammonium hydroxide in Methanol
Evaporate: To dryness and reconstitute to 1 mL with
very extensive PFAS contamination owing to the widespread Methanol/Water (96:4)
use of PFAS based Aqueous Film Forming Foam (AFFF) used
as fire suppression foams at many military installations. As a LC-MS/MS Parameters
result, DOD has developed its own PFAS analytical guidelines
Column: Gemini® 3 µm C18
to deal with the unique environmental monitoring and clean-up Dimensions: 50 x 2.0 mm
challenges found on their installations. These guidelines, con- Part No.: 00B-4439-B0
tained within the DOD QSM 5.1/5.3 documentation (Department Mobile Phase: A: 20 mM Ammonium acetate in Water
B: Methanol
of Defense QSM ([Link]), feature a unique sample clean-up Gradient: Time (min) %B
and concentration approach not found in EPA Methods which 0 5
are designed only for drinking water application. 0.1 55
4.5 99
8.0 99
DOD QSM 5.1/5.3 specifies the use of a polymeric weak anion 8.5 5
exchange (WAX) SPE sorbent in combination with graphitized Flow Rate: 0.6 mL/min
carbon black (GCB) sorbent for the clean-up of solid samples, Delay column: Luna 5 µm C18(2) 30 x 3.0 mm
®

(00A-4252-Y0) installed between the

soils, biota, sediments, or non-drinking water samples. This autosampler and mobile phase pump mixer
can be performed by using two individual tubes of WAX and Injection Volume: 10 µL
GCB sorbent that are applied sequentially or the use of dis-
persive SPE (dSPE) utilizing GCB following the WAX SPE tube Mass Spec Parameters
extraction. Both methods add time to the clean-up procedure Mass Spec Detector: SCIEX® Triple Quad™ 4500
and present the opportunity for loss of analytes and introduction Ion Source Parameters: Samples were ionized using electrospray in
of imprecision. In this communication we describe a significant negative ion-mode
Parameter Value
improvement to the guidelines, Strata PFAS SPE, wherein the CAD 9
two sorbents are contained within a single tube, offering the op- CUR 30
portunity for decreased sample processing time and increased GS1 40
GS2 60
accuracy and precision. When comparing recoveries for a small IS Voltage -4500
subset of analytes for a WAX SPE and dSPE GCB method vs TEM 450
Strata® PFAS, the recovery is greatly improved for Strata PFAS
(Table 1). MRM Transitions for HFPO-DA
Compound Q1 Q3 RT DP CE
HFPO-DA (Quant) 329 185 3.7 -30 -32
HFPO-DA (Qual) 329 169 3.7 -30 -18
13C3 -HFPO-DA 332 185 3.7 -30 -32
 5. Strata® PFAS SPE for DOD QSM 5.3 (continued)
Wastewater, Sediment, and Soil

Table 1.
Recovery Comparisons of WAX SPE and dSPE using
GCB vs Strata PFAS Single Cartridge Method

Strata PFAS Stacked

WAX SPE + dSPE GCB
Analyte Cartridge
% Recovery
% Recovery

13C2-PFDoDA 77.0 84.5

13C2-PFTeDA 62.0 84.0
PFODA 38.0 78.3
PFHxDA 63.0 89.3

Table 2.
Recovery of QSM 5.3 Target Analytes from a Laboratory
Control Sample Using Strata PFAS SPE (WAX/GCB)
26 Analyte Actual Concentration Sample Result % Recovery Method Limits Pass/Fail

PFBA 25.600 22.640 88 84-135 Pass

[Link]/PFAS

PFPeA 25.600 22.157 87 75-138 Pass

[Link]

PFBS 22.640 22.300 99 81-133 Pass

4:2-FTS 23.920 22.078 92 64-134 Pass
PFHxA 25.600 24.644 96 80-137 Pass
PFPeS 24.000 21.699 90 82-132 Pass
HFPODA 25.600 26.336 103 0-130 Pass
PFHpA 25.600 27.018 106 80-140 Pass
PFHpA 25.600 27.018 106 80-140 Pass
PFHxS 24.200 24.713 102 71-131 Pass
DONA 24.120 26.083 108 70-130 Pass
6:2-FTS 24.280 24.217 100 51-155 Pass
PFHpS 24.360 23.015 94 80-129 Pass
PFOA 25.600 25.043 98 83-138 Pass
PFOS 24.480 22.492 92 54-139 Pass
PFNA 25.600 25.872 101 73-140 Pass
9Cl-PF3ONS 23.840 21.863 92 70-130 Pass
PFNS 24.560 21.993 90 71-121 Pass
PFNS 24.560 21.993 90 71-121 Pass
PFDA 25.600 25.047 98 78-137 Pass
8:2-FTS 24.520 22.231 91 62-133 Pass
PFOSA 25.600 25.714 100 73-121 Pass
NMEFOSAA 25.600 30.906 121 53-136 Pass
PFDS 24.640 22.873 93 69-124 Pass
PFUnDA 25.600 26.353 103 70-134 Pass
NEtFOSAA 25.600 28.765 112 59-145 Pass
11Cl-PF3OUdS 24.120 22.625 94 70-130 Pass
PFDoDA 25.600 27.710 108 75-139 Pass
10:2-FTS 24.680 26.626 108 50-124 Pass
PFDoS 24.800 21.509 87 39-121 Pass
PFTrDA 25.600 25.814 101 67-144 Pass
PFTeDA 25.600 25.446 99 79-134 Pass
PFODA 25.600 27.373 107 10-124 Pass

Recovery Range: 87 % - 116 %

Average Recovery: 98.8 %
Mean Recovery: 99.0 %
 6. Determination of PFAS in Sediments
Wastewater, Sediment, and Soil

Determination of Perfluoroalkyl Substances (PFAS) in Sediments

by QuEChERS Extraction and HPLC-MS/MS
Syljohn Estil1 , Eric Nelson1 and Scott Krepich2
1
Sanitation Districts of Los Angeles County-San Jose Creek Water Quality Laboratory, Whittier CA
2
Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501

Introduction Materials and Methods

Perfluoroalkyl substances (PFAS) are a class of highly stable Reagents/Chemicals
synthetic organic compounds used in a wide variety of industri- • QuEChERS Extraction – In a 50 mL plastic centrifuge
al and commercial applications including surface treatment for tube combine 2.0 g of Anhydrous Magnesium Sulfate,
textiles, packaging materials, and non-stick cookware. PFAS are and 1.5 g Sodium Acetate or use approximately 3.5 g of
characterized by a hydrophobic fully fluorinated alkyl chain and a AOAC 2007.01 roQ™ extraction packet
hydrophilic functional group. They are persistent in the environ- (part no. AH0-9043)
ment due to the exceptional stability of the C-F bond. Once re- • QuEChERS dSPE Clean-Up — roQ 15 mL dSPE Kit
leased in the aquatic environment, these chemicals will partition (part no. KS0-8926)
between the water phase and the sediment.

27 Sample Preparation
QuEChERS Extraction Protocol

1. Weigh 2.0 g of dried sediment into a polypropylene

[Link]/PFAS
[Link]

container and spike with isotopically-labeled internal standards.

PPCPs, Steroids, and Pyrethroids can be extracted concurrently
with this method by adding the appropriate internal standard and
spiking solutions to the samples and QCs 7,8,9.
2. Add 10 mL deionized water and vortex. Add 10 mL acidified
acetonitrile (1 % acetic acid) to the slurry and vortex.
3. Add the extraction salts (1.5 g Sodium Acetate and 2 g
MgSO4) to the sample and vortex for 1 minute.
4. Centrifuge the samples for 5 minutes at 4000 rpm.
5. Place the samples in a rack and freeze at -20º for 30-60
minutes. This freezing step allows for easier extraction of the
supernatant.

Currently, there are no federal regulatory limits controlling the 6. Transfer 8-9 mL of the acetonitrile supernatant into a roQ
discharge of PFAS compounds into the environment. Looking QuEChERS PSA/C18 dSPE clean-up tube
forward, it is possible that at some point EPA may establish reg- (Part no. KS0-8926) and vortex for one minute.
ulatory limits for the various PFAS compounds in drinking water, 7. Centrifuge the dSPE tubes for 10 minutes at 3000 rpm.
wastewater and solid waste. In anticipation of such future de- 8. Place an aliquot of the extract in a HPLC vial and dilute 1:1
velopments, it is prudent to develop robust analytical methods with deionized water. The sample is now ready for analysis.
and begin to better understand the fate and transport of these
compounds in both the solid and liquid environmental fractions.
HPLC-MS/MS Conditions
There are several methods available for the extraction and anal-
Column: Gemini® 3 μm C18
ysis of PFAS in aqueous samples, including the EPA Methods Dimensions: 100 x 3 mm
537.1 and 533 previously described in this Guide (5). Howev- Part No.: 00D-4439-Y0
er, very few procedures are available for extracting these com- Inline Filter: Phenomenex Krudkatcher™ Ultra
pounds in solid matrices such as sediments (1). Typical methods Delay Column: Luna® 5 µm C18 (2) 30 x 2.0 mm
used are mechanical shaker and ultrasonic-assisted Solid-Liq- Part No.: 00A-4252-B0
uid Extractions (SLE) 3, 4, 5). The extracts are then subjected to Mobile Phase: A: 20 mM Ammonium acetate in water
additional cleanup steps, usually by solid phase extraction, such B: Methanol
Gradient: Time (min) % B
as in the DoD QSM 5.3 approach previously described in this 0.0 10
Guide. These are generally solvent-intensive and time-consum- 1.5 65
ing processes. However, in 2003, an extraction procedure called 8.0 95
8.1 99
QuEChERS (Quick-Easy-Cheap-Effective-Rugged-and-Safe) 12.0 99
developed by researchers at the US Department of Agriculture 12.5 10
was introduced (6). It was originally developed to extract pesti- Injection: 90 µL
cide residues in food matrices but has since found many other Flow Rate: 0.6 mL/min
Temperature: 40 °C
applications in the field of environmental analytical chemistry.
Detector: SCIEX® 5500 QTRAP®
Detection: MS/MS ESI Negative (sMRM)
Our laboratory (LACSD) previously developed and validated a
Analytes: 1. PFBA
QuEChERS sediment extraction procedure for emerging con-
2. PFPeA
taminants including: pharmaceutical and personal care prod-
3. PFBS
ucts, steroids, alkylphenol ethoxylates, and pyrethroid pesti-
4. PFHxA
cides 7,8,9). We have successfully applied the same extraction 5. PFPS
method to determine perfluoroalkyl substances in marine and 6. PFHxS
freshwater sediments. 7. PFHpA
8. PFHpS
9. PFOA
 6. Determination of PFAS in Sediments (continued)
Wastewater, Sediment, and Soil

Mass Spectrometer Parameters Table 2.

MS Source Parameters
Table 1. Source Parameters Settings
MRM Transitions and Compound Dependent
Parameters Temperature 400 ºC

Gas 1 50
Compound Name Q1 Q3 DP CE
Gas 2 50
Perfluorobutanoic acid (PFBA) 213 169 -71 -14
Curtain Gas 35
Perfluoropentanoic acid (PFPeA) 263 219 -71 -12 Ionization Energies -4500 V

Perfluorohexanoic acid (PFHxA) 313 269 -60 -14 Collision Gas High

Perfluoroheptanoic acid (PFHpA) 363 319 -62 -15

Table 3.
Perfluoroctanoic acid (PFOA) 413 369 -91 -12 Method Performance Data for Sediments Spiked at
1 ng/g of the Target Analytes (n=4)
Perfluorononanoic acid (PFNA) 463 419 -79 -15
28 Compound Average % % RSD
Perfluorodecanoic acid (PFDA) 513 469 -83 -17 Recovery

Perfluoroundecanoic acid (PFUdA) 563 519 -60 -17 PFBA 91.7 0.76
[Link]/PFAS

PFPeA 86.3 6
[Link]

Perfluorododecanoic acid (PFDoA) 613 569 -50 -21

PFHxA 89.4 1.2
Perfluorotridecanoic acid (PFTrDA) 663 619 -49 -18
PFHpA 93.1 2.9
Perfluorotetradecanoic acid (PFTeDA) 713 669 -63 -20 PFOA 98.3 1.5

Perfluorobutanesulfonate (PFBS) 299 80 -94 -70 PFNA 93 1.6

PFDA 87.7 4.5

Perfluoropentantesulfonate (PFPeS) 349 80 -96 -66
PFUdA 92.3 2.1
Perfluorohexanesulfonate (PFHxS) 399 80 -92 -75
PFDoA 92.5 4.1
Perfluoroheptanesulfonate (PFHpS) 449 80 -75 -84 PFTrDA 88.2 2.1

Perfluorooctanesulfonate (PFOS) 499 80 -78 -96 PFTeDA 87.6 2.1

Perflluorooctanesulfonamide (PFOSA) 498 78 -60 -84 PFBS 86.3 2.1

Perfluorononanesulfonate (PFNS) 549 80 -87 -100 PFPeS 96.2 3.2

PFHxS 81.3 5
Perfluorodecanesulfonate (PFDS) 599 80 -55 -100
PFHpS 92.3 2.6
Perfluoro-n-13C4-butanoic acid (M4PFBA) 217 171.9 -71 -13
PFOS 92.1 2.6
Perfluoro-n-13C5-pentanoic acid (M5PFPeA) 268 222.7 -71 -12
PFOSA 104.5 6.3
Perfluoro-n-13C5-hexanoic acid (M5PFHxA) 318 272.9 -60 -13 PFNS 89.8 6.8

Perfluoro-n- C4-heptanoic acid (M4PFHpA)

13
367 321.8 -62 -14 PFDS 87.3 6.7

Perfluoro-n-13C8-octanoic acid (M8PFOA) 421 376 -91 -12

Perfluoro-n-13C9-nonanoic acid (M9PFNA) 472 427 -79 -17

Perfluoro-n-13C6-decanoic acid (M6PFDA) 519 474 -83 -21

Perfluoro-n-13C7-undecanoic acid (M7PFUdA) 570 525 -60 -17

Perfluoro-n-13C2-dodecanoic acid (M2PFDoA) 615 570 -50 -24

Perfluor-n-13C2-tetradecanoic acid (M2PFTeDA) 715 670 -63 -25

Perfluro-13C3-butanesulfonate (M3PFBS) 302 80 -94 -55

Perfluoro-13C3-hexanesulfonate (M3PFHxS) 402 80 -92 -85

Perfluoro-13C8-octanesulfonate (M8PFOS) 507 80 -78 -100

Note: DP = Declustering Potential

CE = Collision Energy
 6. Determination of PFAS in Sediments (continued)
Wastewater, Sediment, and Soil

Figure 1.
Extracted ion chromatogram of sediments spiked with
1.0 ng/g of the target analytes
12
1.20e6
1.15e6 3
1.10e6
1.05e6
1.00e6 5
9.50e5
9.00e5
8.50e5
8.00e5 15
7.50e5 8
Intensity, cps

7.00e5
6.50e5 13
6.00e5 7
5.50e5 10,11
5.00e5 17 18
29 4.50e5
4.00e5 16 19
3.50e5 4 14
[Link]/PFAS

3.00e5
9
[Link]

2.50e5 2

App ID 24517
2.00e5
1.50e5 1
1.00e5 6
5.00e4
0.00
5.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 min

Results and Discussion References

QuEChERS is a vortex-assisted solid-liquid extraction proce- 1. Roberts S, et al. Quantitation of PFAS in Water Samples using LC/MS/MS:
dure that uses acetonitrile, salts, and buffering agents for ex- Large Volume Direct Injection and Solid Phase Extraction (2016) SCIEX
traction, phase-separation, and pH adjustment respectively. Ex- Application Note Publication Number: RUO-MKT-02-4707-A
tracts are subsequently transferred to a dispersive solid phase
extraction (dSPE) tube containing a drying agent (MgSO4) and 2. Berlioz-Barbier, A., et al., Multi-residue analysis of emerging pollutants in
SPE sorbents such as C18 or PSA for sample cleanup. sediment using QuECHERS-based extraction followed by LC-MS/MS
analysis. Analytical and Bioanalytical Chemistry (2014) 406:1259-1266
The modified QuEChERS method presented here is a simple, 3. Jahnke A, et al. Trace analysis of per- and polyfluorinated alkyl substances
efficient, and cost-effective method for determining PFAS levels in various matrices How do current methods perform? Journal of Chro-
in sediments. Accuracy and precision were assessed using four matography A (2009) 1216: 410-4213. Estil S, et al, A. Rapid Extraction
replicates of sediments spiked with the target analytes. Average and Analysis of Steroids and Pyrethroids
% recoveries are all within the 80-120 % range and % RSDs for
all analytes are below 10 % (Table 3). Reporting limits were set 4. Ahrens et. al. Partitioning behavior of per- and polyfluoroalkyl compounds
at 0.05 ng/g dry weight based on a 2.0 g initial sample weight. between pore water and sediment in two sediment cores from Tokyo Bay,
Japan. Environmental Science and Technology (2009) 43: 6969-6975
5. Long et. al. Quantification of Pharmaceuticals, Personal Care Products,
and Perfluoroalkyl Substances in the Marine Sediments of Puget Sound,
Washington, USA. Environmental Toxicology and Chemistry (2013) 32:
1701-1710
6. Lehotay, S.J., Mastovska, K.,Lightfield, A.R., Use of Buffering to Improve
Results of Problematic Pesticides in a Fast and Easy Method for Residue
Analysis of Fruits and Vegetables. Journal of Official Analytical Chemists
International. [Link]. 615-629.
7. Estil S, et al., A. Rapid Extraction and Analysis of PPCPs from Sediments
by QuEChERS and LC/MS/MS (2016) Phenomenex Application Note: TN-
0099 from Sediments by QuEChERS and LC/MS/MS (2016) Phenomenex
Application Note: TN-0096 & TN-0098
8. Estil, S., et. al., A. Rapid Extraction of Steroids and Pyrethroids from Sed-
iments by QuEChERS and LC/MS/MS (2016) Phenomenex Application
Note: TN-0098
9. Estil S, et al, A. Rapid Extraction and Analysis of Pyrethroids from Sedi-
ments by QuEChERS and LC/MS/MS (2016) Phenomenex Application
Note: TN-0098
Acknowledgements
Special thanks to Syljohn Estil and to the Sanitation Districts of
Converting Waste Into Resources Los Angeles County – San Jose Creek Water Quality Laborato-
ry for contributing this method.
 Food and Food Packaging
It was recognized fairly early that PFAS compounds used in food packaging materials
Food and Food Packaging

(such as pizza boxes and microwave popcorn bags) could migrate into consumable
food products and contribute to increased PFAS body burden. In addition, as PFAS
contamination has continued to spread throughout the environment it has been more
recently recognized that these materials can also enter the human food supply chain
through animal consumption of PFAS contaminated water and feed, thereby further
increasing our PFAS body burden. Regardless of source, the analysis of PFAS in food
and food packaging materials – and their myriad complex matrices - features additional
difficult analytical challenges.

30
[Link]/PFAS
[Link]
 7. New Concerns about PFAS in Food
Food and Food Packaging

The Convergence of Environmental Contamination and Food Safety

David C. Kennedy, PhD1
1
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Reprinted with the kind permission of International Labmate Ltd - first printed in International Labmate, February, 2021, Volume 46 Issue 1
[Link]

Abstract Convergence
Per- and Polyfluorinated Alkyl Substances (PFAS) are well known Initially, these three individual trains of concern seemed to be
environmental contaminants that have a newly recognized po- running on separate tracks. It was only more recently that they
tential to taint certain food products through agricultural con- were seen to be converging toward a much larger, more com-
sumption via environmental transport from contaminated indus- plex problem requiring multimedia, multi agency examination
trial sites [1]. The analysis of PFAS in food products requires and the use of more sophisticated analytical tools. The simpli-
more extensive analytical preparation techniques, compared to fied pathway model shown in Figure 1 illustrates the general
PFAS testing of simple matrices such as drinking water, in order scope of the problem. By the end of 2019, the FDA was fully
to reduce the impact of sample matrix interferences on the sub- on board with concerns about PFAS entering the general food
sequent instrumental analysis. An example is provided of a PFAS supply through environmental sources, potentially leading to
31 method applicable to milk, butter, cheese and fish. the contamination of dairy products, bottled water, seafood and
other consumables [7].
The Prequel
[Link]/PFAS

Per- and Polyfluorinated Alkyl Substances (PFAS) are an ex- Analytical Implications
tensive family of synthetic, fluorochemicals with a unique set This expanded concept of the PFAS problem is clearly a major
of physical and chemical properties. These properties have step forward, but it has presented some analytical challenges.
resulted in their widespread commercial use over the past 50 Much of the official PFAS methodology developed over the past
years in diverse applications ranging from fire fighting foams decade has been focused on the analysis of drinking water and
to stain resistant carpet to grease-proof pizza boxes. However, aimed at a very limited list of analytes. With little challenge from
these same unique physical and chemical properties also have matrix interference, easily surmountable chromatography issues
been found to bear serious environmental consequences: wide- and straight forward mass spectrometry, these official drink-
spread dispersion ability, extreme environmental persistence ing-water-only methods proved to be inadequate when applied
and a high degree of bioaccumulation [2]. Although PFAS do to the analysis of PFAS in soil, sediment, sludge and wastewa-
not exhibit acute toxic properties, researchers have found that ter. When applied to the analysis of foods - with a myriad of
PFAS can demonstrate a large number of subtle, chronic health complex matrices, they are quite ineffective, resulting in a surge
effects, primarily affecting the endochrine and reproductive sys- in PFAS analytical method development centered about com-
tems. Consequently, health experts have long been concerned plex matrices, with food testing occupying a prominent position.
that low-level, cumulative exposure to PFAS over an extend- The following section features one such application as an illus-
ed period of time could have serious health consequences [3]. tration of the approaches now being pursued in pursuit of the
Therefore, chronic lifetime PFAS exposure pathways - such as expanded PFAS challenge.
through food or drinking water – are of particular concern to
regulators and are receiving enhanced scrutiny.
Analysis of PFAS in Dairy Products, Eggs, and Fish by
LC-MS/MS
Initial Concerns
In the US, the initial US Food and Drug Administration (FDA)
concern about PFAS centered about the contamination of food Method Introduction
products through contact with PFAS containing food packaging The following work was performed through a collaboration be-
(and to a lesser extent with food processing equipment). The tween Weck Laboratories, Inc., City of Industry, CA, USA and
classic examples are those PFAS coated pizza boxes, fast-food Phenomenex, Inc.,Torrance, CA, USA, for the development of
hamburger wrappers and microwave popcorn bags that have new sample preparation and analysis procedures for determin-
done such a marvelous job of keeping grease off our clothes. ing low levels of PFAS in food products. This particular applica-
That problem was summarily solved in late 2016 when FDA re- tion was directed at achieving sub-ppb sensitivity for 23 PFAS
moved the approval for the use of PFAS in food packaging [4]. analytes in dairy products (milk, butter and cheese), eggs and
fish as representative of difficult to analyze fatty matrices. The
Likewise, the primary US Environmental Protection Agency following discussion is a synopsis of the full work [8].
(EPA) focus has been on drinking water as a primary source of
lifetime PFAS exposure. EPA is continuing to conduct extensive
Sample Preparation
nationwide testing for PFAS in drinking water under the Unregu-
One gram of homogenized sample was spiked with internal
lated Contaminant Monitoring Rule (UCMR) program [5]. These
standards and surrogates and an analyte mix of 23 PFAS com-
efforts will very likely result in specific regulatory limits for the
pounds (Table 1) at the 1 ng/g level, followed by the addition
allowable concentration of certain PFAS in drinking water.
of 10 mL acetonitrile and 10 mL water. Four replicates of each
Concurrently, other government agencies, such as the US De- matrix (milk, eggs, butter, cheese and fish) were prepared. The
partment of Defense (DOD) have been extensively studying the samples were processed by a modified QuEChERs procedure
widespread environmental contamination of military facilities using a commercial kit (Phenomenex roQ™ Extraction Kit). An
owing to the extensive historical use of PFAS firefighting foams, aliquot (500 µL) of the cleaned acetonitrile phase was transferred
principally at air bases [6]. to an LC vial for analysis. Figure 2 displays an extraction blank
and the five sample types following sample preparation.
 7. New Concerns about PFAS in Food (continued)
Food and Food Packaging

Optional Solid Phase Extraction The Sequel

A dispersive SPE cleanup was used to achieve a 10-fold lower The earlier discussion showed the use of current analytical tech-
level of quantitation. Four replicate samples of the egg matrix nology to address the challenge of environmental PFAS con-
were spiked with the PFAS analyte mix at the 0.1 ng/g level and tamination of the food supply. However, care should be taken
processed by the QuEChERs procedure. Following extraction, since experience with analytical chemistry teaches us that we
500 µL of the acetonitrile phase was diluted with 15 mL of wa- will inevitably be facing further analytical challenges from the
ter and loaded onto a preconditioned, weak-ion-exchange SPE realm of the “unknown-unknowns”.
tube (Phenomenex Strata®-X-AW 200 mg). The analytes of inter-
est were then eluted with 4 mL of 0.3 % [Link] In PFAS analysis, we are currently discussing a target analyte
eluate was evaporated to dryness, reconstituted with 500 µL of list of 20, 30 or 40 compounds? However, the number of com-
acetonitrile and transferred to an LC autosampler vial for anal- pounds in the PFAS universe has been estimated at 5000 - and
ysis. even as high as 8,000 - which doesn’t include potential deg-
radation products. Toxicity is largely a function of the unique
chemical and configurational state of a molecule that controls
Optional Solid Phase Extraction the biochemical interaction with the organism. So, there is much
A dispersive SPE cleanup was used to achieve a 10-fold lower more analytical work to identify the most important PFAS com-
level of quantitation. Four replicate samples of the egg matrix pounds from a toxicity perspective.
were spiked with the PFAS analyte mix at the 0.1 ng/g level and
processed by the QuEChERs procedure. Following extraction, Excellent work is being done with accurate mass and advanced
32 500 µL of the acetonitrile phase was diluted with 15 mL of wa- data analysis to give us a broader understanding of the chemical
ter and loaded onto a preconditioned, weak-ion-exchange SPE complexity of the PFAS universe. However, given the complexity
tube (Phenomenex Strata-X-AW 200 mg). The analytes of inter- and extent of the problem of environmental PFAS contamina-
[Link]/PFAS

est were then eluted with 4 mL of 0.3 % [Link] tion, it is clear that a lot of hard work has yet to be done.
eluate was evaporated to dryness, reconstituted with 500 µL of
acetonitrile and transferred to an LC autosampler vial for anal-
Acknowledgements
ysis.
The contribution of Dr. Agustin Pierri and his team at Weck Lab-
oratories, City of Industry, California, USA is gratefully acknowl-
LC-MS/MS Analysis edged.
The chromatography was performed on an Agilent® 1290 UH-
PLC system. The LC column employed was a Phenomenex
Luna® Omega 1.6 µL PS C18 operating at 40 degrees Celsius
with a flow rate of 0.55 mL/min and an injection volume of 20 µL.
The mass spectrometer used was an Agilent 6460 QQQ. Var-
ious LC-MS/MS conditions were explored and an ammonium
acetate/acetonitrile gradient (Table 2) proved to be optimum,
resulting in a run time of approximately 4 minutes.

Results and Discussion

System calibration showed a linear dynamic response from 0.05
ppb – 1000 ppb with a lower limit of quantization of 0.05 ppb as
shown in Figure 3 and a calibration chromatogram at the 0.05
ppb level is shown in Figure 4. Recovery data for the five ma-
trix types is summarized in Figures 5–9. Four replicates of each
matrix were spiked at the 1 ng/g level and prepared for analysis
as described above (but were not subjected to the solid phase
extraction process). Figure 10 presents the recovery data for
four replicates of the egg matrix spiked at 0.1 ng/g and prepared
as described above, but with the addition of the solid phase
extraction step to increase method sensitivity.

The recovery data show good recovery for all five matrices
spiked at the 1 ng/g level, with most analytes falling into the
80 % - 120 % recovery range. Precision is generally somewhat
poorer for the higher fat dairy products than for the lower fat
matrices. The recoveries on tuna fish are particularly good, con-
sidering the complexity of the matrix. In comparing the analyte
recoveries from eggs at the 1 ng/g and 0.1 ng/g levels (Figure 9
and Figure 10), both show comparable recoveries although, as
expected, the higher spike level shows greater precision. Over-
all, the data suggest that the method has sufficient accuracy
and precision to potentially be used to assess environmental
PFAS contamination of food products. Clearly, this is preliminary
data and further development and multi-laboratory validation
would be required to demonstrate such a purpose. However, the
data clearly show that current sample preparation techniques,
coupled with the power of advanced chromatography and tri-
ple-quad mass spectrometry represent a suitable workflow.
 7. New Concerns about PFAS in Food (continued)
Food and Food Packaging

Figure 1.
Pathway Model for Environmental Transmission of
PFAS to Food and Consumer

33
[Link]/PFAS

Figure 2.
Samples after QuEChERs Cleanup:
From Left to Right: Blank, Butter, Cheese, Egg, Milk and Fish
 7. New Concerns about PFAS in Food (continued)
Applications
Food and Food Packaging

Figure 3.
System Calibration Dynamic Range (0.05 – 1000 ppb)

34
[Link]/PFAS

Figure 4.
Chromatogram of 0.05 ppb Lower Limit of
Quantization Standard

10

19
13 18
4
6 15
3
App ID 24662

2 7
17 20 22
9 1112 23
1 5 14 16
21

10
 7. New Concerns about PFAS in Food (continued)
Food and Food Packaging

Figure 5.
Milk Recoveries (QuEChERs: 1 ng/g, n=4)

35
[Link]/PFAS

Figure 6.
Butter Recoveries (QuEChERs: 1 ng/g, n=4)

Figure 7.
Tuna Recoveries (QuEChERs: 1 ng/g, n=4)
 7. New Concerns about PFAS in Food (continued)
Food and Food Packaging

Figure 8.
Cheese Recoveries (QuEChERs:1 ng/g, n=4)

36
[Link]/PFAS

Figure 9.
Egg Recoveries (QuEChERs: 1 ng/g, n=4)

Figure 10.
Egg Recoveries (QuEChERs + SPE: 0.1 ng/g, n=4)
 7. New Concerns about PFAS in Food (continued)
Food and Food Packaging

Table 1.
PFAS Analyte List References
1. Analytical Results of Testing Food for PFAS from Environmental Con-
Analytes: tamination. ([Link]
ing-pfas-environmental-contamination), US Food and Drug Administra-
1. PFBA 9. PFHpS 17. Et-FOSE
tion, October 20, 2020.
2. PFPeA 10. PFOS 18. Et-FOSA
2. Basic Information about PFAS ([Link]
3. PFBS 11. PFNA 19. PFDS tion-pfas), US Environmental Protection Agency, December 06, 2018.
4. PFHxA 12. FOSA 20. PFDS 3. Toxicological Profile for Perfluoroalkyls ([Link]
icprofiles/[Link]?id=1117&tid=237), Agency for Toxic Substances and
5. PFHpA 13. Me-FOSE 21. PFDoA
Disease Registry, Communicable Disease Center, September 30, 2020.
6. PFHxS 14. 8:2 FTS 22. PFTrDA
4. FDA Removes Approval for the Use of PFCs in Food Packaging Based on
7. 6:2 FTS 15. Me-FOSA 23. PFTeDA the Abandonment. ([Link]
fda-removes-approval-use-pfcs-food-packaging-based-abandonment),
8. PFOA 16. PFDA
US Food and Drug Administration, November 21, 2016.
5. Development of the Proposed Unregulated Contaminant Monitoring
37 Table 2.
Rule for the Fifth Monitoring Cycle (UCMR 5): Public Meeting and We-
binar ([Link]
LC-MS/MS Conditions
LC-MS/MS Conditions [Link]), US Environmental Protection
Column: Luna® Omega 1.6 µm PS C18 Agency, July 16, 2019
[Link]/PFAS

Dimensions: 100 x 2.1 mm

Part No.: 00D-4752-AN 6. Installations Conducting Assessments for PFAS use or Potential Release.
Mobile Phase: A: 5 mM Ammonium Acetate in Water ([Link]
B: Acetonitrile INSTALLATIONS_CONDUCTING_ASSESSMENTS_FOR_PFAS_USE_OR_
Gradient: Time (min) % B POTENTIAL_RELEASE.PDF), US Department of Defense, September 30,
0 40
0.5 40
2019.
3 90 7. Per and Polyfluoroalkyl Substances (PFAS) ([Link]
3.1 100
4 100 chemicals/and-polyfluoroalkyl-substances-pfas), US Food and Drug Ad-
Flow Rate: 0.55 mL/min ministration, December 20,2019.
Injection: 20 µL 8. Per- and Polyfluorinated Alkyl Substances (PFAS) from Milk, Eggs, Butter,
Temperature: 40 °C Cheese and Fish using QuEChERs, SPE and LC-MS/MS. Agustin Pierri
UHPLC System: Agilent® 1290 Weck Laboratories) and Scott Krepich (Phenomenex, Inc,). Phenomenex
Detection: Agilent 6460 QQQ Technical Note TN-0124 ([Link]
Analytes: 1. PFBA 9. PFHpS 17. Et-FOSE
documents/[Link]), Phenomen-
2. PFPeA 10. PFOS 18. Et-FOSA
ex, Inc., 2018.
3. PFBS 11. PFNA 19. PFDS
4. PFHxA 12. FOSA 20. PFUdA
5. PFHpA 13. Me-FOSE 21. PFDoA
6. PFHxS 14. 8:2 FTS 22. PFTrDA
7. 6:2 FTS 15. Me-FOSA 23. PFTeDA
8. PFOA 16. PFDA
 8. PFAS in Food Contact Materials
Food and Food Packaging

Identification and Quantification of PFAS in Food Contact Materials using

MRMHR Workflow on X500R QTOF System
Wang Sha, Liu Bingjie, Cheng Haiyan, Li Lijun, Jin Wenhai
SCIEX, China

Introduction
In comparison to other surfactants, perfluorinated alkyl substanc-
es (PFAS) have stable physiochemical structures with hydropho-
bic and oleophobic properties. They are widely used in industrial
and consumer products like plastic packaging materials for food
and as coating in non-stick pans. Due to their chemical stability
and low reactivity, PFAS are highly resistant to degradation even
in living organisms and can therefore be accumulated in the food
chain. Human exposure to PFAS residues has been implicated in
incidences of cancer, obesity, endocrine system disruption and
other adverse health effects. [1]

38 With the rapid growth in the food delivery industry in China

(and globally) in the past two years, one-time-use plastic pack-
aging materials are widely used by merchants due to their low
cost and high durability [2]. One-time-use plastic has become
[Link]/PFAS

a source of public concern and environmental pollution. Given

the tremendous persistence of PFAS in the environment and the
adverse effect on human health, monitoring of PFAS residue has
gained traction in China and elsewhere.

In China, the level of PFOS and PFOA in food contact materi- The X500R QTOF system has the industry’s fastest scanning
als and products is regulated according to the latest National speed, allowing for the implementation of the unique MRMHR
Food Safety Standard (GB 31604.35-2016). The detection limit acquisition mode to provide excellent quantitative performance
is set at 1.0 ng/g while the quantification limit is set at 2.0 ng/g. using high-resolution MS/MS data. This approach to quantita-
In 2006, the European Union (EU) has set a regulation that the tion with LC-QTOF-MS/MS minimizes matrix interferences and
level of PFOS in finished products should not exceed 0.005 % the patented Turbo V™ ion source with curtain gas interface,
of the product mass. twin sprayer technology and built-in automatic calibration sys-
tem help to improve and maintain instrument robustness and
maintain high mass accuracy results. The high resolution MS/
MS spectra can also be used for qualitative analysis by calcu-
lating the ion ratio for confirmation, thus reducing false positives
by taking advantage of the data acquired on the LC-QTOF plat-
form.

Key Workflow Advantages

• PFAS quantitation using an easily established method
and minimal method development
• 10-minute run time using a Phenomenex Kinetex® C18
column demonstrates separation of PFAS targets
• MRMHR workflow using MS/MS for selectivity vs high res-
olution TOF MS mode provides improved signal-to-noise
• QTOF technology can be utilized for quantitative analysis
of PFAS suite without compromising method perfor-
mance (excellent sensitivity, linearity demonstrated)

Figure 1.
Signal-to-Noise Comparison of PFHpA using TOF-MS
and MRMHR Data Using a Post Spiked 0.2 ppb Matrix
Blank
Monitoring the transition and the high resolution fragment ion
results in greater specificity and reduced baseline, so signal-
to-noise demonstrates marked improvement and method
sensitivity is maximized.
 8. PFAS in Food Contact Materials (continued)
Food and Food Packaging

Methods Table 1.
Scheduled MRMHR Method Setup in SCIEX OS
Sample Preparation Unique RTs can be defined for each transition for each analyte.
The food packaging material to be tested is cut into small piec-
es. For coating sample, scrape it with a small knife. The sample
preparation procedure was adapted from National Standard of
China (document number GB 31604.35-2016) which is imple-
mented on 19 April 2017 (Figure 2).

A total of eight samples were collected as test samples which

include disposable meal box, plastic bag, beverage bottle, coat-
ing of non-stick pan, etc. Packaging materials in the collected
samples were mainly polyethylene, polystyrene and polytetra-
fluoroethylene.

39 Establishing the Scheduled MRMHR Quantitative Method

The SCIEX OS software is fully automated with a user-friendly
interface, greatly reducing the time to establish the acquisition
method. The MRM parameters can be set up easily in two differ-
[Link]/PFAS

ent ways. For compounds which are in MS/MS spectral library,

fragment ions can be imported easily from the library to build
Blow dry with N2 to 0.5 mL, add 10 mL water the MRMHR method list. Up to 5 fragment ions can be imported
at the same time using a single click. For compounds not found
in the spectral library, spectra can be added easily to the library
using TOF MS-IDA-MS/MS data acquired for standards of the
desired targets.

MRM parameters like retention time, declustering potential (DP)

and collision energy (CE) from an existing triple quadrupole
method are fully transferrable.

Figure 2.
Extraction and Clean-up Process Flow Diagram

Chromatography
Using the SCIEX® ExionLC™ AD System with a Phenomenex Ki-
netex®, 2.6 μm C18, 100 X 2.0 mm, compounds were separated
using a gradient elution with mobile phase A of 5 mM NH4AC in
water and mobile phase B of 5 mM NH4AC in methanol (flow rate
of 0.3 mL/min, column temperature 40 ºC).

Mass Spectrometry
The SCIEX X500R QTOF System was used to analyse the com-
pounds operating in negative ion polarity using the Scheduled Figure 3.
MRMHR acquisition mode (Table 1). Source conditions were as TOF MS Extracted Ion Chromatogram of 17 PFAS
follows: CUR of 30psi; CAD of 7; IS of -4500V; Temp 500 ºC; Good separation was achieved for most of the 17 PFAS com-
GS1 of 50psi; GS2 of 55psi. pounds analysed.

Data Processing
All data was processed with SCIEX OS Software.
 8. PFAS in Food Contact Materials (continued)
Food and Food Packaging

MRMHR Quantitation of PFAS Detection of PFAS in Food Contact Materials

Chromatogram of 17 PFAS utilizing extracted precursor ion data SCIEX OS software combines both qualitative and quantitative
from TOF-MS scan are shown (Figure 3). results in one single interface (Figure 6). The result table show
the retention time, concentration, peak area, ion ratio confirma-
tion and the mass error of 0.9 ppm for a sample tested positive
High Selectivity Data
with PFOA.
Comparing 0.2 ppb post spiked in matrix blank, PFHpA show
higher selectivity in MRMHR mode as compared to TOF-MS Among the eight samples, eight types of PFAS were detect-
mode for quantification (Figure 1). Monitoring the high res- ed as shown in Table 2. Two out of eight samples have levels
olution fragment ion from the full scan MS/MS data collected which exceeded regulated level of 1 ng/g by national standard.
provides greater specificity and reduced baseline, so signal-to- Most of the detected PFAS are the acid derivatives of PFOA and
noise demonstrates marked improvement and method sensitiv- primarily found in non-stick pan coating and disposable meal
ity is maximized. boxes. The number of actual samples collected in this test is
rather small; hence statistically it does not imply that all related
Linearity and Accuracy products are unsafe for consumers.
The 17 monitored PFAS demonstrate good linearity and accura-
cy (Figure 4) with the correlation coefficients above 0.99. Accu-
racy values are within the permissible deviation range for LOD
40 and LOQ according to the national standards.

Ion Ratio Calculations

[Link]/PFAS

Ion ratios can be easily calculated using the SCIEX® OS soft-

ware. Ion ratio confirmation can be visually displayed in the
chromatogram and result table. Depending on the requirement,
the confirmation tolerance can be defined using either constant
tolerance or variable tolerance as shown in Figure 5.

Figure 6.
PFOA Results in Actual Sample

Figure 5.
Setting up Tolerance for Ion Ratios Confirmation
Constant tolerance (same percent difference from measured
standard ion ratio) or variable tolerance (varying percent differ-
ence dependant on concentration level) can be utilized when
determining whether an unknown same meets the criteria for
qualitative analyte identification by ion ratio confirmation. Differ-
ent levels of percent difference can be defined by the user to be
flagged as within “Acceptable,” “Marginal,” or “Unacceptable.”
 8. PFAS in Food Contact Materials (continued)
Food and Food Packaging

Summary References
The SCIEX® X500R QTOF system and SCIEX OS software 1. Xu R, Tan H, Yang H B, et al. (2014) Food and Fermentation Industries,
brings powerful performance capabilities for routine testing of 40(10): 205.
PFAS. The unique MRMHR quantification method enables high
selectivity even in real sample with matrix interference. This im- 2. Kannan K, Tao L, Sinclair E, et al. (2005) Arch Environ Con Tox, 48: 559.
proves the detection and quantification of PFAS which can meet 3. GB 31604.35-2016, National Food Safety Standard - Food contact ma-
the EU regulation and national standards in China. terials and products - Determination of perfluorooctane sulfonate (PFOS)
and perfluorooctanoic acid (PFOA).
Although the concentration of PFAS in most of the test samples
falls below the regulated level, the detection rate of perfluorinat-
ed alkyl substances is relatively high indicating that the quality
of food contact/packaging materials may pose potential risks to
consumer’s health.

Table 2.
PFAS Content in Different Food Contact Samples

Detected Amount (ng/g)

41
PFHxA PFHpA PFOA PFDA PFuDA PFDoA PFTrDA PFTeDA

Meal box 1 0.14 0.16 3.15 - - - - -

[Link]/PFAS

Meal box 2 - - 3.12 - - - - -

Plastic bag 1 - - - - - - - -
Plastic bag 2 - - - - - - - -
Drink bottle 1 - - - - - - - -
Drink bottle 2 - - - - - - - -
Non-stick pan 1 - - - 0.11 0.15 0.13 0.15 -
Non-stick pan 2 - - - - - - - 0.17
- Falls below the detection level of this method.

Figure 4.
Calibration Curve of 17 PFAS with Acceptable Accuracy
and Linear Response
 New Analytical Frontiers
The number of PFAS compounds found on current analyte lists represents less than 1 %
of the potential environmental contaminants that could be contributed by this large class
New Analytical Frontiers

of compounds. However, researchers continue to identify additional PFAS compounds

with potential human health and environmental impacts, thereby increasing the scope
of the problem. Consequently, it is inevitable that PFAS analyte lists will continue to
grow, and, future analytical challenges - sample preparation, chromatograpy and mass
spectrometry - will become more complex and difficult to overcome. Therefore, we
close this PFAS Guide with two visionary technical notes which propose new analytical
approaches that will help meet the evolving PFAS challenge.

42
[Link]/PFAS
 9. pH-Variable LC Mobile Phase Gradient
New Analytical Frontiers

PFAS Analysis Based Upon a pH-Variable LC Mobile Phase Gradient

David C. Kennedy, PhD1, Richard Jack, PhD1 , Sam Lodge1 and David Schiessel2
1
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
2
Babcock Laboratories, 6100 Quail Valley Ct, Riverside, CA 92507 USA

Introduction
Polyfluoroalkyl substances (PFAS) have been an environmental Recently introduced regulations in California (3) have significant-
concern ever since the 1970s when initial reports of potential ly expanded the PFAS target analyte list to include compounds
adverse health effects first came to light. While the analysis of such as PFBA, PFMBA, PFHxDA and PFOcDA, which have very
PFAS compounds has been ongoing for some time in academia, large differences in hydrophobicity. This presents a significant
they are a fairly recent addition to the suite of analyses common- analytical challenge because PFHxDA (C16) and PFOcDA (C18)
ly performed by commercial environmental laboratories. The are very hydrophobic with limited solubility in water. The pre-
only official methods for the analysis of PFAS in drinking water dicted solubility of PFOA (C8) and PFOcDA (C18) are 480,000
are EPA 537/537.1 and EPA 533 and there are currently no of- and 0.00047 ng/L respectively, using the WS-KOWIN from the
ficial methods for the analysis of PFAS in complex environmen- USEPA EPISuite Software (4).
tal matrices such as Wastewater, Sediment, and Soil. Although
ASTM has released methods for the analysis of PFAS in com- In addition, chromatographic analysis of PFBA in an extract that
plex matrices (ASTM D7979 and D7968), they have not gained is > 90 % organic results in poor peak shape for this early elut-
43 ing compound. Most methods that can successfully analyze for
widespread use within the environmental testing community.
As PFAS analyte lists continue to expand and matrices become PFBA are either direct injection (100 % water), a 1:1 water-meth-
ever more complex, we anticipate the need for a scalable ana- anol dilution or have at least 20 % water in the extract (EPA 533).
[Link]/PFAS

lytical framework that will enable the development of analytical Some methods (ASTM D7979, D7968 and EPA 8327) add acetic
methods for a wider range of PFAS compounds and matrices. In acid to the extract to help improve the peak shape of PFBA.
this Technical Note we present such a framework, based upon However, this results in poorer chromatographic performance
the use of a variable pH mobile phase gradient, which could for the longer chain PFHxDA (C16) and PFOcDA (C18).
facilitate the expansion of PFAS analyte lists beyond those in
common use today. A New Strategy
In recognition of these limitations, we have pursued a new chro-
matographic strategy using a 100 % organic system (for long
chain PFAS solubility) and variable mobile phase pH to provide
good chromatography for PFBA and other early eluting PFAS
compounds. By staying within the confines of the NH4OAc mo-
bile phase composition but employing pH as a variable, one can
realize the potential advantages mobile phase variation allowed
by EPA while avoiding the primary disadvantages. This ap-
proach could be useful in overcoming the difficulty of expanding
the analyte lists of the existing PFAS methods to incorporate
both the hydrophilic shorter chain compounds and the extreme-
ly hydrophobic longer chain compounds.

Technical Approach
This work specifically focused on a secondary chemical char-
acteristic of most PFAS compounds: the hydrophilic or polar
functional head of the molecule which are either carboxylic or
sulfonic acids which can be charged or neutral, depending on
Method Limitations the pH of the eluent. Chromatographers can take advantage of
Most PFAS methods in use today employ an ammonium acetate secondary interactions by employing a mobile phase in which
(NH4OAc) mobile phase at a pH of 7 and with a concentration a pH gradient is performed, i.e. changing the pH of the mobile
between 2 and 20 mM. Although EPA methods 537.1 and 533 phase over time. Mobile phase pH becomes important when an-
both specify 20 mM NH4OAc, EPA’s method flexibility criteria alytes contain acidic, basic or both functional groups. The mo-
allow for the use of alternative mobile phases (1, 2). This allow- bile phase pH determines the charge state (protonation state) of
ance is useful in pursuing potentially better eluent systems and the analyte and thereby influences its interactions with the mo-
allowing the analyst to run various PFAS methods on the same bile and stationary phase. This technique allows for more control
instrument using the same column and similar mobile phase. The of the ionic interactions between the PFAS analytes within a col-
benefit of changing the eluent system is the ability to change an- umn’s stationary phase and the mobile phase. This is analogous
alyte selectivity and potentially analyte resolution. Selectivity dif- to the WAX SPE technique used in EPA method 533, wherein
ferences can also be useful when trying to discriminate analytes the ion exchange mechanism allows for stronger interaction with
from matrix interferences. However, the drawback to changing the shorter-chain PFAS compounds than does the styrenedivin-
eluent systems is that it takes time and can create other issues ylbenzene (SVDB) SPE sorbent used in method 537.1 which op-
associated with differing mobile phase composition. erates primarily in a reversed phase mode. Shorter chain PFAS
compounds have a lower degree of binding ability due to their
shorter chain length and thus often pass through, owing to bind-
ing mechanisms that rely exclusively or primarily on a reversed
phase interaction.
 9. pH-Variable LC Mobile Phase Gradient (continued)
New Analytical Frontiers

In this new technique, the mobile phase at the beginning of the Table 1.
run has a low pH (~ pH 3.9) and changes over time to a higher pH LC Conditions (neutral, pH=7)
(~ pH 9.3). This protonates or deprotonates the functional heads
of the various PFAS compounds over time, depending upon 20 mM NH4OAc MeOH
the pKa of the functional group. This correspondingly changes
the elution profile for the separation, in terms of both relative Time %A %B
and absolute retention times. In principle, the protonation of
0.00 95 5
short-chain, anionic PFAS will lead to greater retention, while
the deprotonation of the later-eluting, long-chain PFAS may 1.20 55 45
lead to less retention, thereby compressing the chromatogram.
3.60 35 65
This will lead to less suppression from non-retained interferenc-
es, and shorter run times, allowing greater sample throughput. 11.00 10 90
Separating interferences from early eluting analytes is particu- 13.00 10 90
larly important when there is only one sensitive MRM transition
available, as in the case of PFBA and PFPeA. It is reasonable 13.01 95 5
to think that these orthogonal retention mechanisms (hydropho- 17.00 95 5
bicity vs. ionizability or pKa) could offer greater opportunity to
resolve complex PFAS mixtures. This Technical Note provides
44 an illustration of the potential power of this approach. Table 2.
LC Conditions (gradient pH)
Experimental Conditions
20 mM HOAc 25 mM NH4OH in MeOH
[Link]/PFAS

Instrumentation and Consumables. All PFAS analyses were per-

formed on an Agilent® 1100 HPLC with a Thermo Scientific® TSQ Time %A %B
Vantage triple quadrupole mass spectrometer. All samples were
prepared using a Phenomenex Strata®-X-AW 200 mg 33 μm in 0.00 95 5
a 6cc format (pn: 8B-S038-FCH). The LC column employed 1.20 55
was a Phenomenex Kinetex® C18 EVO 5 µm 100 x 2.1 mm (pn:
00B-4633-AN). 3.60 35 65

11.00 10
Reagent Preparation. Eluents: (1A) Ammonium Acetate
(NH4OAc) was prepared at 20 mM by dissolving 1.54 g NH4OAc 13.00 10 90
into 1.0 L of water. LC-MS methanol (MeOH) was used for (1B).
13.01 95 5
Acetic acid (HOAc) was prepared at 20 mM by diluting 1.22 mL
of glacial acetic acid into 1.0 L of water (2A). Basic methanol 17.00 95 5
was prepared by diluting 1.46 mL of concentrated. Ammonium
Hydroxide (NH4OH) into 1.0 L of LC-MS methanol. Reference
materials were purchased from Wellington Labs (Guelph, Cana-
da) and diluted into LC-MS methanol for analysis.

Mass Spectrometer Operating Conditions: The capillary and va-

porizer temperature were 250 ˚C and 300 ˚C respectively. The
sheath and aux gas were held at 40 arb and 50 arb respectively. Table 3.
The ESI voltages for positive and negative mode were +3.0/- Measured pH of a Binary Mixture of Eluents
2.5add spacekV. See Appendix 1 for MS/MS Parameters.
20 mM HOAc 25 mM NH4OH in MeOH
LC Operating Conditions: A moderate organic gradient profile
was used in both analyses being compared. The only difference %A %B Actual pH
between the two LC systems was the pH modifiers that were
used in the aqueous and organic eluents. To illustrate the effect 100 0 3.62
of improved peak shape and selectivity differences solely due 95 5 3.86
to the pH modifiers, the times used to change from aqueous to
high organic were identical. 90 10 4.17

80 20 4.55

70 30 5.14
Results and Discussion
Although it is difficult to determine the actual pH in any eluent 60 40 5.77
system especially in the presence of methanol and a particular
50 50 6.45
stationary phase, this was estimated in an offline experiment. In
order to ascertain the pH change as 20 mM HOAc mixes with 40 60 7.13
the 25 mM NH4OH, the pH was measured offline for different
35 65 8.15
mixture ratios of this binary system. The measured pH values
are shown in Table 3. Based on this data, it is estimated that the 30 70 8.52
gradient pH elution profile has a pH no wider than 3.9 and 9.3
20 80 8.98
from start to finish respectively.
10 90 9.33

0 99.5 10.25
 9. pH-Variable LC Mobile Phase Gradient (continued)
New Analytical Frontiers

One of the first notable improvements using the new gradient Additionally, the latest eluters (PFTrA, PFTeDA, PFHxDA and
pH upon injecting an extract containing PFAS in 100 % metha- PFODA) not only elute early, but the peak height is noticeably
nol is that the peak shape for PFBA is drastically improved due higher. The increase in height would improve detection limit
to shifting the equilibrium of unprotonated PFBA to a protonated with a greater s/n. This indicates that NH4OH, which increases
form. Protonated PFBA will interact with the nonpolar stationary in concentration as the organic (methanol) gradient progresses,
phase much more than the mobile phase causing increased re- is affecting analyte retention by shifting their equilibrium to a
tention and a better focused peak. This is illustrated in Figures deprotonated anion since the anions favor interactions with the
1 and 2; PFBA (light blue). Under the commonly used eluent sys- mobile phase and the neutral analyte favors interaction with the
tem of 20 mM NH4OAc, PFBA and PFMPA exhibit severe front- stationary phase. In fact, the NH4OH must be present in slightly
ing in 100 % methanol (required for PFODA solubility). However, higher molar concentration than the HOAc in order to move the
using the gradient pH profile, these peaks are focused much pH into the slightly basic range.
better on the column.

Figure 1.
Chromatogram of 48 PFAS using 20 mM NH4OAc (pH=7)
Total Ion Chromatogram
5 ppb Std in 100 % MeOH

45
[Link]/PFAS

Figure 2.
Chromatogram of 48 PFAS using 20 mM HOAc and
25 mM NH4OH (varied pH from 3.9 to 9.3)
Total Ion Chromatogram
5 ppb Std in 100 % MeOH
 9. pH-Variable LC Mobile Phase Gradient (continued)
New Analytical Frontiers

The selectivity of these two mobile phase systems was further

investigated to see how they affect different PFAS compounds
varying in chain length.

Upon close examination of the ΔRT data there were certain To validate the regression model and the prediction interval of
analytes (e.g PFOSA) that indicated possible differences in se- significance at 95 %, a Global Validation of Linear Models As-
lectivity. In order to evaluate significant selectivity differences sumptions (GVLMA) was used (5) The plots in Figure 3 high-
between the two eluent systems that were not obvious, a sta- light the most important aspects of the advantages of this
tistical approach was used. This is necessary because not ev- new system. These are increased retention for early eluters
ery slight change in RT or resolution may be significant. First, a (3a), decreased elution for late eluters (3b), and significant se-
least squares regression was performed on the ΔRT as a func- lectivity differences (3c). To evaluate significant differences,
tion of RT of the new method. The equation that was used to the x-axis shows the retention time (RT) for the new mobile
model the change in the two systems is listed in equation (1) phase and the y-axis shows the ΔRT relative to the neutral
where a, b, c are the coefficients for the intercept, linear term, ammonium acetate mobile phase.
and inverse term respectively:

Figure 3.
46 Notable Mobile Phase Elution Changes
a) PFAS Analytes with Increased Retention
[Link]/PFAS

dRT > +0.25

b) PFAS Analytes with Decreased Retention

Delta-RT of pH Gradient vs. 20 mM NH4OAc (pH = 7) dRT < -0.25

5 ppb Std in 100 % MeOH
 9. pH-Variable LC Mobile Phase Gradient (continued)
New Analytical Frontiers

It is also worth noting that this new eluent system also has
an effect on sensitivity for certain compounds. Specifical-
ly, N-TAmP-FHxSA, N-CMAmP-62FOSA, and N-AP-FHxSA2
(which are detected in ESI+) had an increase in response more
than 2x in the new pH gradient eluent system (Figure 1-2).

Lastly, the robustness of the stationary phase was examined

by evaluating a “well used” LC column versus a brand new
column. The “well used” column had been used to analyze
thousands of samples over approximately six months. This in-
cluded drinking water extracts as well as non-potable aqueous
and soil extracts. The Kinetex® EVO C18 showed reasonable
robustness and, although some retention is lost over time,
there was no significant (P<0.05) selectivity difference ob-
served. Again, the GVLMA cross-validation was used (Figure
4) to detect significant elution order changes (ie: all analytes
had statistically the same elution order) although “absolute”
elution order was different in some cases.
47
Figure 4.
Retention Difference of New vs Used column Under
[Link]/PFAS

Varied pH conditions
Delta-RT Plot using the Varied-pH Gradient
Kinetex C18 EVO - New vs. 6 mo used

Conclusion
The objective of using a pH gradient mobile phase for PFAS Moving forward, this promising mobile phase gradient ap-
analysis is that it allows the analyst to widen the scope of an- proach could be combined with work investigating alternative
alyte chemistry to properly chromatograph short-chain and HPLC stationary phases to determine optimal conditions for
long-chain PFAS in 100 % organic extracts as well as change PFAS panels that are much broader in scope and chemistry. In
the selectivity of the method. This holds true for any analyte principle, this approach should allow the separation of an even
panel outside the scope of method EPA 537.1 and EPA 533, wider class of PFAS including non-volatile short-chain PFAS.
in that the absolute and relative retention of some analytes are Preliminary data suggest that the use of Formic acid (ie: 25 mM
different than when using a standard organic gradient with am- HOFo) instead of 25 mM HOAc can drop the pH slightly lower;
monium acetate (NH4OAc). closer to pH = 3. This has the benefit of increased retention for
TFA, TFMS, and PFPrA in extracts that are 100 % methanol.
Additionally, this solution may provide the ability to move cer-
tain peaks away from interferences and high ion suppression
zones at the beginning of the chromatographic run. It may also
allow for the inclusion of other PFAS analytes with a minimal
redevelopment and optimization. The pH gradient method
shows excellent robustness and reproducibility, with stable
PFAS analyte retention times, even when using different col-
umns, systems, and analysts. The changes in retention times
(both absolute and relative) offer another tool for more complex
PFAS mixtures - either those with more PFAS analytes or from
working with dirtier matrices.
 9. pH-Variable LC Mobile Phase Gradient (continued)
New Analytical Frontiers

Appendix 1.
Instrumental Conditions for MS/MS Analysis and RT Data
Retention Time Data

Analyte Precursor Product CE S-Lens Polarity Gradient pH Constant pH=7

PFBA 213 169 9 35 - 4.18 3.22

PFMPA 229 85 12 35 - 4.51 3.98
PFPeA 263 219 9 38 - 4.93 4.68
3:3-FTCA 241 177 8 37 - 5.30 4.83
PFEESA 315 135 23 90 - 5.32 4.88
PFBS 299 80 36 100 - 5.04 4.89
PFMBA 279 85 12 40 - 5.15 5.13
NFHDA 295 201 10 33 - 5.50 5.28
4:2-FTS 327 307 20 110 - 5.48 5.35
PFHxA 313 269 9 47 - 5.57 5.42
PFPeS 349 80 41 100 - 5.64 5.53
HFPO-DA 285 169 8 37 - 5.74 5.60
48
PFHpA 363 319 9 56 - 6.15 6.04
PFHxS 399 80 44 120 - 6.18 6.10
ADONA 377 251 11 60 - 6.22 6.12
[Link]/PFAS

5:3-FTCA 341 237 13 57 - 6.41 6.28

6:2-FTS 427 407 22 130 - 6.64 6.57
PFOA 413 369 9 62 - 6.69 6.63
N-TAmP-FHxSA 499.1 60 37 140 + 6.66 6.66
PFHpS 449 80 46 110 - 6.71 6.66
N-CMamP-
571.1 440 31 140 + 6.92 6.98
6:2FOSA
N-AP-FHxSA 485.1 85 34 130 + 7.01 7.05
PFNA 463 419 10 65 - 7.24 7.27
PFOS 499 80 46 105 - 7.25 7.29
9Cl-PF3ONS 530.9 351 28 120 - 7.56 7.67
7:3FTCA 441 337 11 70 - 7.44 7.68
8:2-FTS 527 507 27 130 - 7.65 7.93
PFDA 513 469 10 75 - 7.81 7.97
PFNS 549 80 48 130 - 7.80 8.01
N-MeFOSAA 570 419 20 120 - 8.13 8.40
PFUnDA 563 519 10 85 - 8.42 8.69
PFDS 599 80 49 110 - 8.40 8.71
PFOSA 498 78 34 110 - 7.84 8.75
N-EtFOSAA 584 419 20 120 - 8.45 8.76
11Cl-PF3OUdS 630.9 451 30 120 - 8.74 9.05
PFDoDA 613 569 12 92 - 9.04 9.38
10:2-FTS 627 607 31 150 - 9.03 9.40
MeFOSE 616 59 15 90 - 9.95 9.96
MeFOSA 512 169 30 110 - 9.48 10.00
PFTrA 663 619 12 101 - 9.67 10.05
EtFOSE 630 59 15 91 - 10.45 10.46
EtFOSA 526 169 30 120 - 10.10 10.51
PFTeDA 713 669 12 108 - 10.27 10.64
PFHxDA 813 769 12 120 - 11.29 11.72
PFOcDA 913 869 13 140 - 12.13 12.54

References
1) U.S. Environmental Protection Agency, Method 537.1—Determination of 4) U.S. Environmental Protection Agency, Estimation Program Interface Suite
Selected Per- And Polyfluorinated Alkyl Substances In Drinking Water By (EPISuite) Software, v4.11, 2000-2017. [Link]
Solid Phase Extraction And Liquid Chromatography/Tandem Mass Spec- ing-tools/download-epi-suitetm-estimation-program-interface-v411
trometry (LC/MS/MS). November 2018. 5) Peña, Edsel A., and Elizabeth H. Slate. “Global Validation of Linear Model
2) U.S. Environmental Protection Agency, Method 533— Determination of Assumptions.” Journal of the American Statistical Association, vol. 101,
Per- And Polyfluoroalkyl Substances In Drinking Water By Isotope Dilution no. 473, 2006, pp. 341–54, doi:10.1198/016214505000000637.
Anion Exchange Solid Phase Extraction And Liquid Chromatography/Tan-
dem Mass Spectrometry (LC/MS/MS). November 2019.
3) Analytical Reporting Limits for PFAS compliance with DoD Table B-15 of
QSM, Version 5.1 or later (updated 07/22/20) [Link]
[Link]/pfas/docs/reporting_limits_dod_qsm_v5_1_or_later_july_22_2020.
pdf
 10. Column Chemistry Considerations
New Analytical Frontiers

Column Chemistry Considerations for Full Coverage of PFAS Analyte Ranges

Dr. J Preston, Zara Jalali, Scott Krepich, Dr. David Kennedy, Sam Lodge, Laura Snow, Dr. Richard Jack, and Dr. Bryan Tackett
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Introduction
Per- and polyfluorinated alkyl substances (PFAS) are man-made Common Chromatographic Approaches
chemicals, that have been widely used since the 1940s. They PFAS compounds are typically determined by LC-MS/MS and
have been employed in a large variety of consumer products, LC-HRMS instrumentation. The use of mass spectrometry de-
such as nonstick cookware, food containers, stain and water re- tection has played a significant role in the quantitation of specif-
pellent fabrics, polishes, waxes, paints, and cleaning products ic compounds where standards are available. Where standards
and are now widely distributed in the global environment. A sig- are not available, the use of time of flight (TOF) and Orbitrap™
nificant source of PFAS environmental contamination has been MS detectors are used to semi-quantify unknown PFAS com-
the widespread use of PFAS-containing aqueous firefighting pounds. The chromatographic separation of PFAS compounds
foams (AFFF), which are known to migrate into groundwaters in currently validated methods typically involves a reversed
at airports and military bases. Further environmental exposure phase mechanism using a C18 or Phenyl column in an acid-
to PFAS comes from industrial production facilities (e.g. chrome ic-methanol eluent. For example, EPA method 537.1 uses a C18
49 plating, electronics, manufacturing, or oil recovery). Living or- column (5 µm, 2.1 x 150 mm C18) and EPA Method 533 was
ganisms, including plants, animals, and humans, can accumu- validated using a C18 Phenomenex Gemini® column (3 µm, 2 x
late PFAS compounds in their tissue, which can build up over 50 mm). Conversely, ASTM D7979 and EPA 8327 were validated
time and impact their health.1-3 A total of 9,252 PFAS are listed using a Phenyl-Hexyl column (1.7 µm, 2.1 × 100 mm), ISO 21675
[Link]/PFAS

in EPA’s most recent list of PFAS substances. 4 However, only used a C18 column (5 μm, 2 × 50 mm) and the Department of
a handful of these, such as perfluorooctanoic acid (PFOA) and Agriculture CLG-PFAS 2.01 method used a C8 column, Phe-
perfluorooctanesulfonic acid (PFOS), have been widely moni- nomenex Luna® C8(2) (3 µm, 2 x 50 mm).
tored in the environment or have been thoroughly studied for
their toxicological effects.
PFAS Chromatographic Challenges
While these methods are generally adequate for a limited list of
analytes, the large number of potential PFAS analytes that could
potentially be present in a sample will inevitably challenge sim-
ple chromatographic separation approaches. This phenomenon
was seen early in the development of the EPA drinking water
methods. EPA 537.1 when validated, identified several overlap-
ping peaks which can be seen in Figure 1 as demonstrated by
peaks, 2,3; 4,5; 7,8; 9,10; 11,12,13; 15,16; 17, 18; 19, 20, 21.
 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

Figure 1.
Example chromatogram for reagent water fortified with
method 537.1 analytes at 80 ng/L

50
[Link]/PFAS

min

Likewise, when EPA 533 was developed and validated with an expanded list of PFAS compounds, it also shows several overlapping
peaks, as seen in Figure 2.

Figure 2.
Example chromatogram for reagent water fortified with
method 533 analytes at 80 ng/L

min

.
 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

Whereas many of these overlapping peaks can be successfully Table 1.

resolved by the mass analyzer, the potential presence of isobar-
ic homologues and unresolved matrix interferences point to the Chemical Name Abbreviation
continuing need for good chromatographic separation to assure Perfluoroalkyl carboxylic acids (PFCAs)
reliable identification and quantitation. Although the problem
Perfluorohexanoic acid PFHxA
may be manageable for today’s small analyte lists, the challenge
will inevitably grow as new PFAS compounds are added for in- Perfluoroheptanoic acid PFHpA
vestigational or regulatory purposes. perfluoro-n-octanoic acid PFOA

perfluoro-n-nonanoic acid PFNA

Looking to the Future
Current PFAS methods primarily rely upon C18 solid phase perfluoro-n-decanoic acid PFDA
chemistry and simple methanol-ammonium acetate mobile perfluoro-n-undecanoic acid PFUdA
phase gradients. These methods do not make full use of all
perfluoro-n-dodecanoic acid PFDoA
the tools in the chromatographer’s toolbox, nor need they, giv-
en today’s limited analyte lists. However, this simple situation perfluoro-n-tridecanoic acid PFTrDA
will inevitably change and there will be a need to develop more
perfluoro-n-tetradecanoic acid PFTeDA
sophisticated chromatographic methods to tease out the sub-
tle chemical and structural differences between closely related Perfluoroalkyl ether carboxylic acids (PFECA)
51 PFAS compounds. Chief among these will be the application hexafluoropropylene oxide-dimer acid HFPO-DA
of novel stationary phases and mobile phases to exploit the
different interactions between closely related PFAS molecules. dodecafluoro-3H-4,8-diosanonanoate NaDONA
[Link]/PFAS

This Tech Note was designed to provide a vision of the potential Perfluorooctane sulfonamides and derivatives
power of such new chromatographic approaches.
N-methylperfluoro-1-octanesulfonamidoacetic acid N-MeFOSAA

N-ethylperfluoro-1-octanesulfonamidoacetic acid N-EtFOSAA

Scope
In this Tech Note we will present data for a select list of PFAS Perfluorinated sulfonic acids (PFSAs)
compounds (Table 1) that were selected to reflect some of the Perfluorobutanesulfonic acid L-PFBS
chemical diversity of the PFAS universe. This color-coded group-
ing will be used to illustrate the differences in chromatograph- perfluoro-1-hexanesulfonate L-PFHxS

ic retention time and elution order between various stationary perfluoro-1-octanesulfonate L-PFOS
phases including C8, C18, Phenyl-Hexyl, Biphenyl and F5 which
Chlorinated polyfluoroalkyl ether sulfonic acids (Cl-PFESAs)
can have significantly different sorptive properties. We will also
examine how differing mobile phase polarity (e.g., methanol vs. 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate 9Cl-PF3ONS
acetonitrile) influences chromatographic performance for these
11-chloroeicosalfluoro-3-oxaundecane-1-sulfonate 11Cl-PF3OUdS
various phases. Ideally, this information can be used to enhance
chromatographic resolution as the list of PFAS compounds con-
tinues to increase. The goal is to provide insights that will allow
method developers to identify useful separation strategies.

Method Variables
PFAS Chemistries
There are established, validated methods set forth by the EPA and ISO for chromatographic separation of PFAS compounds using
specific types of columns and packing materials. Unfortunately, not all PFAS compounds can be separated with sufficient accuracy
using these methods because of the different types of functional groups that are on different PFAS compounds. In the select list
that was used, there are 5 categories of PFAS compounds as shown in Figure 3, with an example of each. Owing to the variety of
functional groups that can potentially be found on PFAS compounds, there are a variety of HPLC column chemistries that could aid
enhanced separation.
Figure 3.

Perfluoroalkyl carboxylic acids (PFCAs) Perfluoroalkyl ether carboxylic acids (PFECAs) Perfluoro sulfomamide carborylic acids (PFSACAs)

Perfluoro-n-octanoic acid Hexafluoropropylene oxide-dimcer acid N-ethylperfluoro-1-octanesulfonamidoacetic acid

Perfluorinated sulfonic acids Chlorinated polyfluoroalkyl ether sulfoninic acids

Perfluorobutanesulfonic acid 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate

 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

Solid Phase Chemistries

A representation of the different solid phase chemistries that are Different combinations of these variables serve to enhance the
available in Phenomenex HPLC columns that could be used in separation of polar compounds, increase surface areas, add pH
PFAS separations is presented in Figure 4. This wide variety of stability, decrease system backpressures, etc. These, and addi-
ligand chemistries – combined with differences in porosity and tional column properties, provide chromatographers with a high
other morphological variations – was developed to offer a wide degree of flexibility with which to tackle challenging separations.
range of variables for method development.

Figure 4.
Available column chemistries appropriate for PFAS
compound separation

Kinetex® Core-Shell 1.3, 1.7, 2.6, and 5 μm

Kinetex PS C18 Kinetex Polar C18 Kinetex EVO C18 Kinetex XB-C18 Kinetex C18 Kinetex C8

52

TMS TMS TMS TMS TMS TMS TMS TMS TMS TMS
Si
[Link]/PFAS

TMS TMS
+ + Polar Polar

PS C18 Polar EVO XB- C18 C8

C18 C18 C18

Kinetex Biphenyl Kinetex Phenyl-Hexyl Kinetex F5 Kinetex HILIC Kinetex PAH

ACN H20 ACN ACN ACN

F ACN ACN ACN ACN

ACN ACN ACN ACN

F F H20
Si Si
ACN H 0 ACN H20 ACN H 0 ACN
2 2

O O
F F
H20 HO H20 OH H20
Si Si
H 20 H20
TMS TMS TMS TMS TMS TMS HO HO O O
H20 O Si O H20
HO Si OH
Si

Biphenyl Phenyl-
Hexyl F5 HILIC PAH

Luna® Omega Fully Porous 1.6, 3, and 5 μm

Luna Omega C18 Luna Omega Polar C18 Luna Omega PS C18

TMS TMS TMS TMS TMS TMS

Polar Polar + +
 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

Mobile Phase Chemistries

However, in addition to column selection, chromatographers In the second elution regime, acetonitrile was added to the mo-
can also make changes in mobile phase polarity to further en- bile phase at a ratio of 80:20 methanol:acetonitrile to increase
hance selectivity. For example, EPA method 533 was altered in mobile phase polarity (but with all other factors remaining the
several ways to enhance separation of the selected PFAS com- same as in the “533 Similar” elution regime). This second elution
pounds used in the present study. In the first elution regime, the regime will be referred to as “533 Acetonitrile Altered” (Table 3).
percentage of methanol was increased at run initiation and then The results from these two elution regimes will be addressed
further increased to a higher percentage than had been previ- separately. Clearly, there are many other potential mobile phase
ously used in the published method. This decreased the overall variations that could be investigated. However, the two varia-
run time but kept the percentage increase of methanol roughly tions presented here will suffice to demonstrate the power of
the same. This elution regime will be referred to later in this Tech mobile phase polarity combined with solid phase chemistry vari-
Note as “533 Similar” (Table 2). ation to effect PFAS chromatographic behavior.

Table 2.

EPA 533 - As Published 533 Similar

53
% 20 mM % 20 mM
Time (min) % Methanol Time (min) % Methanol
Ammonium Acetate Ammonium Acetate
[Link]/PFAS

Initial 95 5 Initial 55 45

0.5 95 5 15 10 90

3 60 40 21 10 90

16 20 80 21.5 55 45

18 20 80 40 → 90 in 15 min 3.0 % per min

20 5 95

22 5 95

25 95 5

35 95 5

40 → 80 in 13 min 3.08 % per min

Table 3.

533 Similar 533 Acetonitrile Altered

% 20 mM % 20 mM % 80-20
Time (min) % Methanol Time (min)
Ammonium Acetate Ammonium Acetate Methanol Acetonitrile

Initial 55 45 Initial 55 45

15 10 90 15 10 90

21 10 90 21 10 90

21.5 55 45 21.5 55 45

Results and Discussion

For ease of comparison, all chromatographic data will be pre-
sented in tabular format with the chromatography columns on
the left, the PFAS compounds across the top, and the specific
analyte retention times under the PFAS compounds. The high-
lighted boxes identify two compounds that have overlapping
retention times (∆RT ≤ 5 seconds) and the arrows at the bottom
indicate when two compounds have changed elution order. The
different PFAS compound classes are represented by the colors
referenced in Table 1. This representation is a more insightful
way to present the data because overlaying or stacking indi-
vidual chromatograms makes it very difficult to compare results
across columns. The two mobile phase chemistry regimes iden-
tified above will now be discussed separately.
 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

1. EPA 533 Similar

In order to determine how the selected PFAS compounds would However, the C8 phases also demonstrated two compound
elute and separate, seven different chromatography columns functionality pairs with a reverse order elution from the C18
with different solid phase chemistries were examined. Figure 5 phases: L-PFOS (a perfluoronated sulfonic acid) vis-á-vis PFNA
displays columns that have C18-functionality or PAH-function- (a perfluroalkyl carboxylic acid) and (again) PFUdA vis-á-vis
ality. The PFAS elution order was generally consistent for most N-EtFOSSA, presumably is response to the lower hydrophobic-
of the C18 phases, although specific elution times varied. The ity of the C8 phase functionality. Interestingly, both C8 phases
Kinetex® PAH column demonstrated two compound function- and the PAH phase had fewer overlapping peaks compared to
al pairs with a reverse elution order: NaDONA (a perfluoroether the C18 phases, but in different parts of the elution order spec-
carboxylic acid) vis-á-vis L-PFHxS (a perfluronated sulfon- trum. This likely represents the greater contribution of pi-electron
ic acid) and PFUdA (a perfluoroalkyl carboxylic acid) vis-á-vis interaction with the PAH phase in contrast with more consistent
N-EtFOSSA (a perfluorooctane sulfonamide). In addition, there hydrophobic interaction characteristic of the C18 phases. These
were slight differences in overlapping peaks amongst the vari- variations are subtle rather than dramatic, but they offer insights
ous C18 phases, whereas the Kinetex PAH phase had only one into interactions between solid phase chemistry and PFAS com-
overlapping pair. When compared to two C8 phases (Figure 6), pound class that could be useful for better separating adjacent
the elution order was similar to the C18 phases, and the reten- compound pairs or shifting analytes away from mass spectral
tion times were similar, but there were fewer overlapping peak interferences.
pairs (one pair vs. 3 pairs).
54
[Link]/PFAS

Figure 5.
C18 and PAH summary

11Cl-PF3OUdS
9-Cl-PF3ONS

N-MeFOSAA

N-EtFOSAA
HFPO-DA

NaDONA
L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFUdA
PFHpA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Gemini® C18 5.28 7.37 8.23 9.53 10.02 10.09 11.41 13.12 13.12 13.58 14.28 15.03 15.35 15.36 16.05 16.34 17.23 18.11

Luna Omega
®
Polar C18 4.15 6.18 7.10 8.31 8.38 8.47 10.16 11.43 11.43 12.34 12.58 13.30 14.04 14.05 14.38 14.59 15.47 16.29

Luna Omega Ps C18 4.29 6.34 7.21 8.46 8.55 9.05 10.35 12.06 12.03 12.53 13.24 14.01 14.31 14.36 14.58 15.23 16.08 16.44

Kinetex C18 3.36 6.38 7.38 9.44 10.04 10.07 12.03 13.48 13.52 14.4 15.1 15.41 16.17 16.15 16.5 17.15 18.06 18.48

Kinetex XB-C18 3.27 5.30 6.18 7.56 8.10 8.13 9.54 11.31 11.34 12.24 12.51 13.28 13.58 14.02 14.31 14.56 15.47 16.31

Kinetex Polar C18 3.05 4.59 5.49 7.18 7.33 7.37 9.16 10.54 10.57 11.52 12.17 12.51 13.25 13.27 14.03 14.25 15.18 16.05
11Cl-PF3OUdS
9-Cl-PF3ONS

N-MeFOSAA

N-EtFOSAA
HFPO-DA

NaDONA

L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFUdA
PFHpA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Kinetex PAH 1.24 2.07 2.60 3.99 4.27 4.31 5.81 7.59 7.76 8.61 9.16 9.73 10.32 10.52 11.14 11.69 12.75 13.71

Elution Order Shifts from C18 Elution Order Shifts from C18
Overlaps are Eliminated

Figure 6.
C8 summary
11Cl-PF3OUdS
9-Cl-PF3ONS

N-MeFOSAA

N-EtFOSAA
HFPO-DA

NaDONA
L-PFHxS

L-PFOS

PFTeDA
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Luna C8 5.41 7.51 8.38 9.51 10.04 10.15 11.33 12.47 12.49 13.28 13.53 14.12 14.40 14.46 15.13 15.33 16.14 16.48

Kinetex C8 5.30 7.54 8.47 10.14 10.26 10.33 11.57 13.18 13.19 14.03 14.26 14.45 15.15 15.24 15.56 16.14 16.56 17.33

Elution Order Shifts from C18

 10. Column Chemistry Considerations (continued)
Finally, additional differences are seen when comparing Kine- The elution order in the Kinetex Biphenyl and Phenyl-Hexyl
tex® Biphenyl, Phenyl-Hexyl, and F5 columns. These phases columns are consistent, but markedly different from the Kinetex
New Analytical Frontiers

were designed with different chemistries having varying polar- F5 column. The Biphenyl and F5 phases showed only one set
ities to provide better selectivity for aromatic compounds. How- of overlapping peaks, but the Phenyl-Hexyl column had 3 sets
ever, these polarity differences and greater pi-electron interac- of overlapping peaks. Interestingly, the compound classes
tability also come into play with the different PFAS chemistries, that overlapped were different between the Phenyl-Hexyl and
as evidenced by the various reverse order elution pairs from the Biphenyl columns (Figure 7).
C18 phases.

Figure 7.
Phenyl Stationary Phase Summary

11Cl-PF3OUdS

11Cl-PF3OUdS
9-Cl-PF3ONS

9-Cl-PF3ONS

N-MeFOSAA

N-EtFOSAA
HFPO-DA

NaDONA

NaDONA
L-PFHxS

L-PFHxS

L-PFOS

PFTeDA
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFDoA
PFHxA

PFOA

PFNA

PFDA

PFDA
Kinetex Biphenyl 1.47 2.06 2.19 3.67 4.00 4.43 5.53 7.02 7.49 8.64 8.21 9.23 9.24 9.88 10.16 10.56 10.96 11.67
Phenyl-
Kinetex 2.53 3.89 4.57 5.97 6.39 6.42 7.69 9.10 9.35 10.30 10.37 11.13 11.33 11.69 12.21 12.31 13.01 13.67
Hexyl

55 Elution Order Shifts from C18

11Cl-PF3OUdS
9-Cl-PF3ONS

N-MeFOSAA

N-EtFOSAA
HFPO-DA

NaDONA

L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFHxA
[Link]/PFAS

PFOA

PFNA

PFDA
Kinetex F5 3.37 5.12 5.84 7.49 7.85 7.98 9.45 11.10 11.35 11.88 12.47 13.37 13.63 13.84 14.09 14.61 15.47 16.18

Elution Order Shifts from C18

2. EPA 533 Acetonitrile Altered

Acetonitrile is a highly polar molecule and is often added to the The addition of acetonitrile to the mobile phase increased the
mobile phase to alter how analytes interact with the solid phase. number of overlapping peaks for the Gemini® C18, Luna® Ome-
The previously discussed experimental sequence was repeated ga Polar C18, and Kinetex Polar C18 columns, but it conversely
using a 80:20 methanol:acetonitrile mobile phase with the same decreased the number of overlapping peaks for the Luna Ome-
PFAS compounds and HPLC columns. The C18 columns all still ga PS-C18 and Kinetex C18 columns. In the Kinetex PAH col-
had a consistent elution order as compared to 533 Similar but umn, the methanol:acetonitrile mobile phase also significantly
displayed earlier retention times (Figure 8). However, compared changed the elution order as compared to methanol-only mobile
to 533 Similar, the conditions of 533 Acetonitrile Altered resulted phase, but with some differences in the effected compounds
in a much larger number of retention time elution order shifts. (Figure 8). However, with Kinetex PAH there were also more
overlapping peaks, resulting in compromised separation for ear-
ly eluters.
Figure 8.
C18 and PAH Summary
11Cl-PF3OUdS
11Cl-PF3OUdS
9-Cl-PF3ONS
9-Cl-PF3ONS

N-MeFOSAA

N-MeFOSAA
N-EtFOSAA

N-EtFOSAA
HFPO-DA

NaDONA

NaDONA
L-PFHxS

L-PFHxS

L-PFOS
L-PFOS

PFTeDA
L-PFBS

L-PFBS

PFTrDA
PFHpA

PFUdA
PFUdA

PFDoA

PFDoA
PFHxA

PFHxA

PFOA

PFOA

PFNA

PFDA

PFDA

Gemini C18 2.42 2.55 2.97 3.88 4.45 5.55 5.47 7.25 8.89 9.08 9.10 9.82 10.24 10.43 11.85 12.96 13.15 14.40

Luna Omega Polar C18 1.83 1.97 2.25 2.92 3.43 4.31 4.51 5.75 7.14 7.35 7.35 7.98 8.44 8.45 9.69 10.84 10.88 12.01

Luna omega Ps C18 1.87 1.99 2.34 3.12 3.66 4.69 4.87 6.29 7.74 7.90 8.02 8.71 8.91 9.10 10.38 11.46 11.71 12.91

Kinetex C18 1.42 1.47 1.69 2.30 2.79 4.12 4.31 6.40 8.28 8.45 8.54 9.28 9.62 9.90 11.36 12.51 12.72 14.00

Kinetex Polar C18 1.32 1.39 1.58 1.99 2.38 3.13 3.29 4.45 5.90 6.09 6.16 6.80 7.21 7.29 8.53 9.62 9.65 10.76

Elution Order Shifts from “EPA 533 Similar”

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

L-PFOS

PFTeDA
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Kinetex XB-C18 1.37 1.42 1.63 2.23 2.69 3.68 3.76 5.27 6.80 6.87 7.26 7.94 7.96 8.20 9.52 10.57 10.82 12.08

Elution Order Shifts from “EPA 533 Similar”

 10. Column Chemistry Considerations (continued)

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA
New Analytical Frontiers

PFNA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
Kinetex® PAH 0.76 0.76 0.83 0.95 1.05 1.33 1.34 2.19 3.44 3.49 3.78 4.30 4.31 4.78 6.10 6.90 7.47 8.82

Elution Order Shifts from “EPA 533 Similar”

The methanol:acetonitrile mobile phase also resulted in more The 533 Acetonitrile Altered method also showed increased
overlapping pairs and changes in elution order with the C8 col- overlapping peaks in all phenyl and F5 stationary phases (Fig-
umns (Figure 11). The elution order was consistent between the ure 12), although the shorter run times may have contributed
two C8 columns using this method, but there were many shifts significantly to these increases. All things considered, the meth-
in elution order compared to the methanol-only eluent. Finally, anol:acetonitrile data demonstrate that mobile phase polarity (in
the methanol:acetonitrile method and the methanol-only meth- conjunction with stationary phase chemistry) has a great deal of
od showed similar but not identical elution orders in the Kinetex influence over the sorption behavior of the different classes of
Biphenyl and Phenyl-Hexyl Columns. The elution order with Ki- PFAS compounds and could be a powerful tool with which to
netex F5 was less comparable with Kinetex Biphenyl and Phe- influence chromatographic behavior.
nyl-Hexyl columns with the acetonitrile altered eluent than pre-
viously seen with the methanol-only eluent. However, with the
acetonitrile altered eluent, Kinetex F5 was more similar in elution
56
order to the C18 columns than to the phenyl stationary phases.

Figure 9.
[Link]/PFAS

C8 Summary

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

L-PFOS

PFTeDA
L-PFBS

PFTrDA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Luna® C8 2.05 2.15 2.34 3.22 3.55 4.51 5.07 6.22 7.48 7.53 8.04 8.28 9.05 9.09 10.25 11.33 11.36 12.42

Kinetex C8 2.14 2.22 2.41 3.30 4.10 5.19 5.34 7.01 8.32 8.35 8.47 9.11 9.50 9.53 11.06 12.17 12.14 13.17

Elution Order Shifts from “EPA 533 Similar”

Figure 10.
Phenyl Stationary Phase Summary

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFUdA
PFHpA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Gemini® C18 2.42 2.55 2.97 3.88 4.45 5.55 5.47 7.25 8.89 9.08 9.10 9.82 10.24 10.43 11.85 12.96 13.15 14.40

Benchmark: C 18 “EPA 533 Acetonitrile Altered”

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFUdA
PFHpA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Kinetex F5 1.27 1.32 1.45 1.93 2.29 3.12 3.26 4.55 5.92 6.05 6.54 6.84 7.03 7.22 8.44 9.24 9.63 10.76

Elution Order Shifts from C18 Acetonitrile Altered

11Cl-PF3OUdS
9-Cl-PF3ONS
N-MeFOSAA

N-EtFOSAA

11Cl-PF3OUdS
HFPO-DA

9-Cl-PF3ONS
NaDONA

L-PFHxS

N-MeFOSAA
L-PFOS

PFTeDA
L-PFBS

PFTrDA
N-EtFOSAA
PFHpA

PFUdA

PFDoA
PFHxA

PFOA

PFNA

PFDA
HFPO-DA

NaDONA
L-PFHxS

PFTeDA
L-PFOS
L-PFBS

PFTrDA
PFUdA
PFHpA

PFDoA
PFHxA

PFOA

PFNA

PFDA

Phenyl-
Kinetex 1.05 1.14 1.18 1.39 1.60 1.99 2.25 2.96 4.09 4.33 4.47 4.82 5.16 5.46 6.17 7.09 7.45 7.95
Hexyl
Kinetex Biphenyl 0.85 0.90 0.90 0.99 1.07 1.20 1.32 1.55 2.11 2.32 2.37 2.68 2.81 3.23 3.59 4.31 4.83 5.05

Elution Order Shifts from C18 Acetonitrile Altered

 10. Column Chemistry Considerations (continued)
New Analytical Frontiers

Conclusions References
The HPLC methodology in EPA methods 537, 537.1 and 533 are 1. National Institute of Environmental Health Sciences. NIEHS Perfluorinate
all based upon a C18 stationary phase and a methanol-water Chemicals (PFCs) fact sheet, 2012. [Link]
mobile phase. In this study we have shown that the use of alter- pease/documents/perflourinated_chemicals_508.pdf (accessed Dec 1,
2019).
native stationary phases of varying surface chemistry and elu-
ents of varying polarity can significantly alter the sorption-elution 2. United States Environmental Protection Agency. Per- and Polyfluoroalkyl
Substances (PFAS). [Link] (accessed Dec 1, 2019).
characteristics of different classes of PFAS compounds. This
orthogonal approach to PFAS HPLC chromatography should 3. Agency for Toxic Substances and Disease Registry (ATSDR). An Overview
of Perfluoroalkyl and Polyfluoroalkyl Substances and Interim Guidance for
serve as a fruitful avenue for future method development. As Clinicians Responding to Patient Exposure Concerns, 2019. [Link]
analyte lists increase in size and complexity, a variety of HPLC [Link]/pfas/docs/ATSDR_PFAS_ClinicalGuidance_12202019.pdf
column chemistries and eluent compositions will be needed to (accessed Apr 6, 2020).
accommodate the wide range of PFAS related compounds that 4. PFAS List of PFAS Substances (Version 2). CompTox Chemicals Dash-
might be encountered such as polar acids, non-polar acids, es- board | PFASMASTER Chemicals ([Link]) Accessed April 7, June 9,
2001.
ters, amides, sulfonamides, and telomere length, all of which
can be complicated with branched vs. linear isomers.

The work presented here is merely illustrative and should be

considered a starting point for column chemistry and mo-
57 bile phase considerations for PFAS HPLC methodology. Even
though the demonstration sample contained a nice mix of PFAS
compounds with varied functional groups, there are certainly
[Link]/PFAS

many more compounds in the 9000-strong (and growing) PFAS

inventory that will challenge LC-MS methodology. National and
state PFAS analyte panels are constantly being updated and
expanded. There is increasing emphasis on identifying and
quantifying PFAS related isomers, unique functional groups and
degradation products across a wide range of sample matrices.
With regulated detection and quantitation limits being driven
lower and lower, sensitivity is a significant issue. The choice of
HPLC column chemistry will play a significant role in successful-
ly meeting all these future challenges.
 Product Guide
Table 1.
Phenomenex PFAS Products Referenced or Applicable in Official Methods

Regulatory Method Product Part Number

Strata® SDB-L 500 mg/6 mL 8B-S014-HCH

USEPA 537.1: Determination of Selected Per-and Polyfluorinated Alkyl Substances in Drinking Water by
Gemini® 3 μm C18, 50 x 3 mm or 00B-4439-B0
Solid Phase Extraction and Liquid Chromatography/ Tandem Mass Spectrometry (LC/MS/MS) (5)
Luna® Omega 1.6 μm PS C18 100 x 2.1 mm 00D-4752-AN
Product Guide

Strata-X-AW 500 mg/6 mL 8B-S038-HCH

USEPA Method 533: Determination of Per- and Polyfluoroalkyl Substances in Drinking Water by Isotope
Gemini 3 μm C18 50 x 2 mm or 00B-4439-B0
Dilution, Anion Exchange Solid Phase Extraction and LC-MS/MS. (1)
Luna Omega 1.6 μm PS C18 100 x 2.1 mm 00D-4752-AN

US Food and Drug Administration: Determination of 16 Perfluoroalkyl and Polyfluoroalkyl

Strata-XL-AW 200 mg/3 mL 8B-S051-FBJ
Substances(PFAS) in Food using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). (2)

US Department of Agriculture: Screening, Determiation and Confirmation of PFAS by UPLC-MS-MS (3) Luna C8(2) 3 μm 50 x 2 mm 00B-4248-B0

Strata PFAS (WAX/GCB)

200 mg/50 mg/6 mL, 30/box CSO-9207
US Department of Defense: Quality Systems Manual (QSM) for Environmental Laboratories (4) 500 mg/50 mg/6 mL, 30/box CSO-9208

58 Gemini 3 μm C18 50 x 2 mm 00B-4439-B0

References
[Link]/PFAS

1. Method 537.1: Determination of Selected Per- and Polyfluorinated Alkyl

Substances in Drinking Water by Solid Phase Extraction and Liquid Chro-
matography/Tandem Mass Spectrometry (LC/MS/MS) | Science Inventory
| US EPA
2. 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 | Methods Ap-
proved to Analyze Drinking Water Samples to Ensure Compliance with
Regulations | US EPA
3. Determination of 16 Perfluoroalkyl and Polyfluoroalkyl Substances in Food
using Liquid Chromatography-Tandem Mass Spectrometry ([Link])
4. Screening, Determination and Confirmation of PFAS by UPLC-MS-MS
([Link])
5. [Link]

Table 2.
Recommended HPLC Products for Routine PFAS Analysis

Description and Function Product Part Number

Kinetex® 5 µm EVO C18 100 x 2.1 mm 00D-4633-AN

Analytical Column (UHPLC) Luna Omega C18 1.6 μm 50 x 2.1 00B-4752-AN

Gemini 3 μm C18 50 x 3 mm 00B-4439-Y0

Analytical Column Gemini 3 μm C18 50 x 3 mm 00B-4439-Y0

Analytical Column (> 100 µL injection) Gemini 3 μm C18 100 x 3 mm 00D-4439-Y0

Analytical Column (improved. lmwt acids) Luna Omega 3 μm PS C18 50 x 3 mm 00B-4758-Y0

00B-4633-AN
Delay Column Kinetex 5 µm EVO C18, 50 x 2.1 mm
00A-4252-YO

Luna Omega PS C18

SecurityGuard 4 x 3.0/10 pack for ID: 3.2-8.0 mm AJ0-7606
4 x 2.0/10 pack for ID: 2.0-3.0 mm AJ0-7605
 Product Guide (continued)
Table 3.
Recommended SPE Products

Description and Function Product Part Number

SPE Cartridge for EPA 537.1 Strata® SDB-L 500 mg/6 mL, 30/box 8B-S014-HCH

Strata-X-AW 33um Polymeric Weak Anion,

SPE Cartridge for EPA 533 8B-S038-HCH
500 mg/6 mL, 30/box

SPE Cartridge (Rev. Phase, High Perf.) Strata-XL 500 mg/6 mL, 30/box 8B-S043-HCH
Product Guide

Strata PFAS (WAX/GCB)

SPE Stacked Cartridge (DOD QSM 5.3) CS0-9207
200 mg/50 mg/6 mL, 30/box

Strata PFAS (WAX/GCB)

SPE Stacked Cartridge (DOD QSM 5.3) CS0-9208
500 mg/50 mg/6 mL, 30/box

SPE Cartridge (WAX for DOD QSM 5.3) Strata-XL-AW 500 mg/6 mL, 30/box 8B-S051-HCH

GCB** Cartridge (GCB for DOD QSM 5.3) Strata GCB 250 mg/6 mL, 30/box 8B-S528-FCH

SPE Cartridge (WAX* for FDA Method) Strata-XL-AW 100 µm 200 mg/3 mL, 50/box 8B-S051-FBJ

(*WAX = Weak Anion Exchange)

(**GCB = Graphitized Carbon Black)
59

Table 4.
[Link]/PFAS

Recommended QuEChERs Products

Description and Function Product Part Number

QuEChERs Extraction (Soil/Sediment) roQ QuEChERs Extraction Kit KS0-8911

QuEChERs dSPE (Soil/Sediment) roQ QuEChERs dSPE Kit, 15 mL KS0-9516

QuEChERs Extraction (Dairy/Eggs/Fish) roQ QuEChERs Extraction Kit KS0-8910

QuEChERs dSPE (Dairy/Eggs/Fish) roQ QuEChERs dSPE Kit KS0-9511

Table 5.
Recommended Accessories

Description and Function Product Part Number

SPE Sample Reservoir 75 mL Sample Reservoir H0-7005

Large Volume SPE Adaptor Cap for 12,20, 60 mL SPE Tubes AH0-7379

Autosampler Vials Polypropylene, 300 µm + PE Starburst Cap AR0-9995-12-C

Polypropylene Vials Vial 9 mm Screw Thd PP 2 mL, 1000 Pk AR0-89C7-13

Vial Caps Cap 9 mm Solid Top Black Unlined 8B-S528-FCH

PEEK Capillary Tubing Capillary Tubing Kit, Various Sizes AT0-1964

PEEK Tubing Cutter Cutter for PEEK Capillary Tubing AT0-1110

 roQ™ An Easier QuEChERS Solution
Ordering Information

roQ™ Extraction Kits

Extraction kits contain fifty easy-pour salt packets and fifty 50 mL stand-alone centrifuge tubes
Ordering Information
Description Unit Part No.
AOAC 2007.01 Method Extraction Kits
6.0 g MgSO4, 1.5 g NaOAc 50/pk KS0-8911*
EN 15662 Method Extraction Kits
4.0 g MgSO4, 1.0 g NaCl, 1.0 g SCTD, 0.5 g SCDS 50/pk KS0-8909*
Original Non-Buffered Method Extraction Kits
4.0 g MgSO4, 1.0 g NaCl 50/pk KS0-8910
6.0 g MgSO4, 1.5 g NaCl 50/pk KS0-8912
*AOAC and EN Extraction Kits also available in traditional non-collared 50 mL centrifuge tubes, Part No.: KS0-8911-NC and KS0-8909-NC
60
roQ dSPE Kits
dSPE kits contain pre-weighed sorbents/salts inside 2 mL or 15 mL centrifuge tubes
[Link]/PFAS

Ordering Information
Description Unit Part No.
2 mL dSPE Kits
150 mg MgSO4, 25 mg PSA, 25 mg C18E 100/pk KS0-9504
150 mg MgSO4, 25 mg PSA, 2.5 mg GCB 100/pk KS0-9505
150 mg MgSO4, 25 mg PSA, 7.5 mg GCB 100/pk KS0-9506
150 mg MgSO4, 25 mg PSA 100/pk KS0-9503
150 mg MgSO4, 50 mg PSA, 50 mg C18E, 50 mg GCB 100/pk KS0-9514
150 mg MgSO4, 50 mg PSA, 50 mg C18E 100/pk KS0-9512
150 mg MgSO4, 50 mg PSA, 50 mg GCB 100/pk KS0-9513
150 mg MgSO4, 50 mg PSA 100/pk KS0-9511
15 mL dSPE Kits
900 mg MgSO4, 150 mg PSA, 150 mg C18E 100/pk KS0-9508
900 mg MgSO4, 150 mg PSA, 15 mg GCB 100/pk KS0-9509
900 mg MgSO4, 150 mg PSA, 45 mg GCB 100/pk KS0-9510
900 mg MgSO4, 150 mg PSA 100/pk KS0-9507
1200 mg MgSO4, 400 mg PSA, 400 mg C18E, 400 mg GCB 100/pk KS0-9518
1200 mg MgSO4, 400 mg PSA, 400 mg C18E 100/pk KS0-9516
1200 mg MgSO4, 400 mg PSA, 400 mg GCB 100/pk KS0-9517
1200 mg MgSO4, 400 mg PSA 100/pk KS0-9515

roQ Extraction Salt Packets

Salt packets only. Centrifuge tubes not included.
Ordering Information
Description Unit Part No.
AOAC 2007.01 Method Extraction Packets
6.0 g MgSO4, 1.5 g NaOAc 50/pk AH0-9043
EN 15662 Method Extraction Packets
4.0 g MgSO4, 1.0 g NaCl, 1.0 g SCTD, 0.5 g SCDS 50/pk AH0-9041
Original Non-Buffered Method Extraction Packets
4.0 g MgSO4, 1.0 g NaCl 50/pk AH0-9042
6.0 g MgSO4, 1.5 g NaCl 50/pk AH0-9044

Bulk roQ QuEChERS Sorbents

Ordering Information
Phase 10 g 100 g
C18-E — 04G-4348
GCB (Graphitized Carbon Black) 04D-4615 04G-4615
PSA — 04G-4610

For Additional Food Resources Visit:

[Link]/food
 Strata -X Polymeric SPE ®

Strata-X Strata-XL
Ordering Information Ordering Information
Ordering Information

Format Sorbent Mass Part Number Unit Format Sorbent Mass Part Number Unit
Tube Tube
30 mg 8B-S100-TAK** 1 mL (100/box) 30 mg 8B-S043-TAK 1 mL (100/box)
30 mg 8B-S100-TBJ 3 mL (50/box) 60 mg 8B-S043-UBJ 3 mL (50/box)
60 mg 8B-S100-UBJ** 3 mL (50/box) 100 mg 8B-S043-EBJ 3 mL (50/box)
100 mg 8B-S100-EBJ 3 mL (50/box) 200 mg 8B-S043-FBJ 3 mL (50/box)
100 mg 8B-S100-ECH 6 mL (30/box) 200 mg 8B-S043-FCH 6 mL (30/box)
200 mg 8B-S100-FBJ 3 mL (50/box) 500 mg 8B-S043-HCH 6 mL (30/box)
200 mg 8B-S100-FCH 6 mL (30/box) Giga Tube
500 mg 8B-S100-HBJ 3 mL (50/box) 2g 8B-S043-KDG 12 mL (20/box)
500 mg 8B-S100-HCH 6 mL (30/box) 2g 8B-S043-KEG 20 mL (20/box)
Giga™ Tube 5g 8B-S043-LEG 20 mL (20/box)
500 mg 8B-S100-HDG 12 mL (20/box) 5g 8B-S043-LFF 60 mL (16/box)
1g 8B-S100-JDG 12 mL (20/box) 10 g 8B-S043-MFF 60 mL (16/box)
1g 8B-S100-JEG 20 mL (20/box) 30 mg 8E-S043-TGB 2 Plates/Box
2g 8B-S100-KEG 20 mL (20/box)
5g 8B-S100-LFF 60 mL (16/box) * To control flow rate with Strata-XL, use a stopcock (AH0-6048)
when processing samples with a vacuum manifold.
Teflon® Tube
200 mg 8B-S100-FBJ-T 3 mL (50/box)
61 200 mg 8B-S100-FDG-T 12 mL (20/box)
[Link]/PFAS

On-line Extraction Cartridge

Description Part Number Unit/Box
Strata-X on-line extraction 00M-S033-B0-CB ea
cartridge, 20 x 2.0 mm
Cartridge holder, 20 mm CH0-5845 ea

**Tab-less tubes available. Contact Phenomenex for details.

Strata Solid Phase Extraction (SPE)

PFAS
(WAX/GCB)
Consists of a stacked single cartridge solution filled with polymeric
WAX (200 mg) and GCB sorbents (50 mg) that functions to meet the
DOD guidelines for PFAS testing. It is ideal for complex biota matri-
ces and reduces the need for multiple extraction tubes.

Ordering Information
Format Sorbent Mass Part Number Unit
Tube
200 mg / 50 mg CS0-9207 6 mL (30/box)
 Gemini pH Flexible LC Columns ®

Ordering Information
3 μm Microbore‚ Minibore and MidBore™ Columns (mm) SecurityGuard™ Cartridges (mm)
Phases 50 x 1.0 20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0* /10pk
C18 00B-4439-A0 00M-4439-B0 00A-4439-B0 00B-4439-B0 00D-4439-B0 00F-4439-B0 00B-4439-Y0 00D-4439-Y0 00F-4439-Y0 AJ0-7596
Ordering Information

for ID: 2.0-3.0 mm

3 μm Analytical Columns (mm) SecurityGuard™ Cartridges (mm)
Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0* /10pk
C18 00A-4439-E0 00B-4439-E0 00D-4439-E0 00F-4439-E0 00G-4439-E0 AJ0-7597
for ID: 3.2-8.0 mm

62 Kinetex Core-Shell LC Columns ®

Ordering Information
[Link]/PFAS

2.6 μm Micro LC Columns (mm)

Phases 30 x 0.3 50 x 0.3 100 x 0.3 150 x 0.3 50 x 0.5 150 x 0.5
EVO C18 –– 00B-4725-AC –– 00F-4725-AC 00B-4725-AF —

2.6 μm MercuryMS™ LC-MS Cartridges (mm) MercuryMS Cartridge Holders

Phases 20 x 2.0 20 x 4.0 Part No. Description Unit
Biphenyl 00M-4622-B0-CE 00M-4622-D0-CE CH0-7188 Direct-Connect Cartridge Holder, 20 mm ea
CH0-5845 Standard Cartridge Holder, 20 mm ea

SecurityGuard
2.6 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 75 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 00A-4725-AN 00B-4725-AN –– 00D-4725-AN 00F-4725-AN AJ0-9298
for 2.1 mm ID

SecurityGuard
2.6 μm MidBore™ Columns (mm) ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 75 x 3.0 100 x 3.0 150 x 3.0 3/pk 2010 R&D 100
Award Recipient
EVO C18 00A-4725-Y0 00B-4725-Y0 –– 00D-4725-Y0 00F-4725-Y0 AJ0-9297
for 3.0 mm ID
Ordering Information
SecurityGuard™
5 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 00A-4633-AN 00B-4633-AN 00D-4633-AN 00F-4633-AN AJ0-9298
for 2.1 mm ID

SecurityGuard
5 μm MidBore™ Columns (mm) ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 100 x 3.0 150 x 3.0 3/pk
EVO C18 00A-4633-Y0 00B-4633-Y0 00D-4633-Y0 00F-4633-Y0 AJ0-9297
for 3.0 mm ID

SecurityGuard
5 μm Analytical Columns (mm) ULTRA Cartridges‡
Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk
EVO C18 00B-4633-E0 00D-4633-E0 00F-4633-E0 00G-4633-E0 AJ0-9296
for 4.6 mm ID

‡
SecurityGuard ULTRA Cartridges require holder‚
Part No.: AJ0-9000
***SemiPrep SecurityGuard Cartridges require holder‚
Part No.: AJ0-9281
*PREP SecurityGuard Cartridges require holder‚
Part No.: AJ0-8223
 Luna One of The World’s Leading LC Columns
®
Ordering Information

Luna C18
Ordering Information
5 μm MidBore and Analytical Columns (mm) SecurityGuard™ Cartridges (mm)
Phases 30 x 3.0 50 x 3.0 150 x 3.0 250 x 3.0 30 x 4.6 50 x 4.6 75 x 4.6 4 x 2.0* 4 x 3.0*
/10pk /10pk
C18(2) 00A-4252-Y0 00B-4252-Y0 00F-4252-Y0 00G-4252-Y0 00A-4252-E0 00B-4252-E0 00C-4252-E0 AJ0-4286 AJ0-4287
for ID: 2.0-3.0 mm 3.2-8.0 mm

5 μm Analytical and Semi-Prep Columns (mm) SecurityGuard™ Cartridges (mm)

Phases 100 x 4.6 150 x 4.6 250 x 4.6 250 x 10 4 x 3.0* 10 x 10‡
/10pk /3pk
C18(2) 00D-4252-E0 00F-4252-E0 00G-4252-E0 00G-4252-N0 AJ0-4287 AJ0-7221
for ID: 3.2-8.0 mm 9-16 mm
*SecurityGuard™ Analytical Cartridges require holder‚ Part No.: KJ0-4282
63 ‡
SemiPrep SecurityGuard™ Cartridges require holder‚ Part No.: AJ0-9281

Luna Omega PS C18 and Luna C18

[Link]/PFAS

Ordering Information
1.6 μm Microbore Columns (mm)
Phases 50 x 1.0 100 x 1.0 150 x 1.0
PS C18 — 00D-4752-A0 —
C18 00B-4742-A0 00D-4742-A0 00F-4742-A0

1.6 μm Minibore Columns (mm) SecurityGuard™ ULTRA Cartridges‡

Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
PS C18 00A-4752-AN 00B-4752-AN 00D-4752-AN 00F-4752-AN AJ0-9508
C18 00A-4742-AN 00B-4742-AN 00D-4742-AN 00F-4742-AN AJ0-9502
for 2.1 mm ID

To find complete list of part numbers visit: FOr chat with a live technical expert at:
[Link] [Link]/chat
 Perfluoroalkyl Substances
(PFAS) Testing Guide
Australia Mexico
t: +61 (0)2-9428-6444 t: 01-800-844-5226
auinfo@[Link] tecnicomx@[Link]

Austria The Netherlands

t: +43 (0)1-319-1301 t: +31 (0)30-2418700
anfrage@[Link] nlinfo@[Link]

Belgium New Zealand

t: +32 (0)2 503 4015 (French) t: +64 (0)9-4780951
t: +32 (0)2 511 8666(Dutch) nzinfo@[Link]
beinfo@[Link]
Norway
Canada t: +47 810 02 005
t: +1 (800) 543-3681 nordicinfo@[Link]
info@[Link]
Poland
China t: +48 22 104 21 72
t: +86 400-606-8099 pl-info@[Link]
cninfo@[Link]
Portugal
Czech Republic t: +351 221 450 488
t: +420 272 017 077 ptinfo@[Link]
cz-info@[Link]
Singapore
Denmark t: +65 800-852-3944
t: +45 4824 8048 sginfo@[Link]
nordicinfo@[Link]
Slovakia
Finland t: +420 272 017 077
t: +358 (0)9 4789 0063 sk-info@[Link]
nordicinfo@[Link]
Spain
France t: +34 91-413-8613
t: +33 (0)1 30 09 21 10 espinfo@[Link]
franceinfo@[Link]
Sweden
Germany t: +46 (0)8 611 6950
t: +49 (0)6021-58830-0 nordicinfo@[Link]
anfrage@[Link]
Switzerland
Hong Kong t: +41 (0)61 692 20 20
t: +852 6012 8162 swissinfo@[Link]
hkinfo@[Link]
Taiwan
India t: +886 (0) 0801-49-1246
t: +91 (0)40-3012 2400 twinfo@[Link]
indiainfo@[Link]
Thailand
Indonesia t: +66 (0) 2 566 0287
t: +62 21 5010 9707 thaiinfo@[Link]
indoinfo@[Link]
United Kingdom
Ireland t: +44 (0)1625-501367
t: +353 (0)1 247 5405 ukinfo@[Link]
eireinfo@[Link]
USA
Italy t: +1 (310) 212-0555
t: +39 051 6327511 info@[Link]
italiainfo@[Link]
All other countries/regions
Japan Corporate Office USA
t: +81 (0) 120-149-262 t: +1 (310) 212-0555
jpinfo@[Link] info@[Link]

Luxembourg
t: +31 (0)30-2418700
nlinfo@[Link]

[Link]
Phenomenex products are available worldwide. For the distributor in your country/region,
contact Phenomenex USA, International Department at international@[Link]

Terms and Conditions

Subject to Phenomenex Standard Terms & Conditions, which may be viewed at
[Link]/TermsAndConditions.

Trademarks
Gemini, Strata, Luna, and Kinetex are registered trademarks and roQ, SecurityGuard, and
KrudKatcher are trademarks of Phenomenex. Agilent is a registered trademark and Ultivo is a
trademark of Agilent Technologies, Inc. SCIEX, QTRAP, and Analyst are registered trademarks and
Triple Quad, Turbo V, MultiQuant, ExionLC, and Scheduled MRM are trademarks of AB SCIEX Pte.
Ltd. Phenova is a trademark of Phenova, Inc.
Shimadzu is a registered trademark of Shimadzu Corporation. Millipore is a registered trademark
of Merck KGaA, Darmstadt, Germany. Thermo Scientific is a registered trademark and Orbitrap is a
trademark of Thermo Fisher Scientific.
Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145. Gemini and Kinetex EVO are
GU54960223_W

patented by Phenomenex. U.S. Patent Nos. 7,563,367 and 8,658,038 and foreign counterparts.
Disclaimer
FOR RESEARCH USE ONLY. Not suitable for clinical diagnostic procedures.
© 2023 Phenomenex, Inc. All rights reserved.