Testing PFAS by LC-MS/MS, From Carrots to Kibble
eBook
Published: August 24, 2026
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Food safety standards now name 30 per- and polyfluoroalkyl substances (PFAS) and set their limits at trace levels. Environmental agencies designed their PFAS methods for water and soil, so when food laboratories adapt those protocols for eggs and animal tissue, cholic acids elute right alongside perfluorooctanoic acid.
Designing chromatography around food creates an accurate template, so the instrument setup stays fixed while sample preparation absorbs matrix differences.
This eBook details an LC-MS workflow across 11 food categories, naming which extraction steps a matrix can bypass to cut processing time.
Download this eBook to explore:
- Which foods the method evaluated, from beef kidney to coffee
- Where offal and fish oil skip QuEChERS and hold their recovery
- How a delay column isolates background PFAS contamination from target peaks
LC-MS Solutions for
PFAS in Food
Accepted AOAC First Action Method to SMPR 2023.003
Table of Contents
Introduction............................................................................................................ 3
Instrument Setup.................................................................................................... 5
PFAS Target Limits and Matrix Categories Set by SMPR 2023.003 ........................... 8
Liquid Chromatography Optimization...................................................................... 9
Sample Preparation and Analysis........................................................................... 11
PFAS Confirmation Using QTOF LC/MS.................................................................. 12
Analysis Results..................................................................................................... 13
Results and Discussion........................................................................................... 15
Applications.......................................................................................................... 16
Baby Food....................................................................................................... 16
Egg................................................................................................................ 24
Fish................................................................................................................ 32
Protein Powder................................................................................................. 40
Pet Food......................................................................................................... 48
Produce.......................................................................................................... 56
Offal.............................................................................................................. 64
Milk............................................................................................................... 73
Fish Oil........................................................................................................... 81
Coffee............................................................................................................ 90
Seafood.......................................................................................................... 98
References.......................................................................................................... 106
Introduction
Per- and polyfluoroalkyl substances (PFAS) are
broad group of thousands of manufactured
chemicals characterized by fluorinated organic
chains. This includes perfluorooctanoic acid (PFOA)
and perfluorooctanesulfonic acid (PFOS), the two
most commonly found and historically most widely
used PFAS compounds. Widely known as “forever
chemicals”, PFAS are persistent pollutants taken up
by plants, they can bioaccumulate in animals, impact
our soils, waterways and farms, and have records of
adverse health effects.
PFAS have been used in manufacturing of consumer
goods like carpet, clothing and shoes, cookware, and
packaging for decades. Their effectiveness as both an
oil and water repellent, a lubricant, as well as a fire
retardant, made these substances popular for use in
furniture production, linings for take-out containers,
firefighting, medicine and countless other applications.
Many industries have moved away from using PFAS, but
due to that historic use, and since they are extremely
difficult to destroy, PFAS are prolific in our environment
as residue, leachate, or in legacy products.
With years of data on exposure, scientists have
determined adverse health effects stemming from
contact with some of the most prolific of these
chemicals, and further study is underway. The historical
and widespread use has resulted in finding PFAS in
the environment and in our food supply. Even after
phasing these out from new products, old materials
still end up in landfill, and in some industries, they’re
still used where we don’t have a suitable replacement.
Widespread contamination has led to a necessity of
testing, and many groups globally have been working
on solving this problem.
Many LC-MS/MS methods have been developed
for the targeted analysis of PFAS in environmental
samples. Most notably EPA Methods 537, 537.1,
533, 1633. These methods demonstrate testing in
the environmental space has been going on for nearly
two decades, as EPA 537 V1.1 was finalized in 2009
with years of work prior to putting it together. ASTM
International, a group working towards the creation
of voluntary consensus standards, also developed
methods: D7979, D8421, D7968, and D8535.(1-7) These
methods, similar to those developed by the EPA, are
focused on soils, biosolids, but none were designed for
testing food products. In the vacuum of food testing
methods, EPA 1633’s inclusion of tissue samples led
to the testing of farm animals using this method, but
ultimately, it was not designed for testing human food
products.
Methods developed by food protection agencies,
notably C-01.02 from the FDA and USDA’s CLG - PFAS
2.03(8-9) have been single laboratory validated for PFAS
in food. The FDA has recently published an update
C-010.03(10), a single laboratory validated method for
30 PFAS in 9 matrixes, expanding capabilities from the
C-01.02.
AOAC International, an industry-led standardization
body for food safety had called for methods to test
30 PFAS compounds from 11 food categories to
address the lack of standardized food centric methods.
The Standard Method Performance Requirements
document SMPR 2023.003 detailed the necessary
specifications. We present Shimadzu’s response, an
accepted First Action method to AOAC’s OMA: a
single LC-MS/MS method to address the needs of
routine testing labs.
03
Table 1: PFAS Target Analytes, Abbreviations, and CAS Numbers
Name Abbreviation CAS No.
Perfluorobutanoic acid PFBA 375-22-4
Perfluoropentanoic acid PFPeA 2706-90-3
Perfluorohexanoic acid PFHxA 307-24-4
Perfluoroheptanoic acid PFHpA 375-85-9
Perfluorooctanoic acid PFOA 335-67-1
Perfluorononanoic acid PFNA 375-95-1
Perfluorodecanoic acid PFDA 335-76-2
Perfluoroundecanoic acid PFUnA 2058-94-8
Perfluorododecanoic acid PFDoA 307-55-1
Perfluorotridecanoic acid PFTrDA 72629-94-8
Perfluorotetradecanoic acid PFTeDA 376-06-7
Perfluorobutanesulfonic acid PFBS 375-73-5
Perfluoropentansulfonic acid PFPeS 2706-91-4
Perfluorohexanesulfonic acid PFHxS 355-46-4
Perfluoroheptanesulfonic acid PFHpS 375-92-8
Perfluorooctanesulfonic acid PFOS 1763-23-1
Perfluorononanesulfonic acid PFNS 68259-12-1
Perfluorodecanesulfonic acid PFDS 335-77-3
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1
Perfluorododecanesulfonic acid PFDoS 79780-39-5
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8
Perfluorooctanesulfonamide PFOSA 754-91-6
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0
The list of PFAS in AOAC’s SMPR is covered largely by the same standards used in EPA method 1633, with the addition of PFUnDS, PFTrDS, and
10:2 FTS.
04
Instrument Setup
Two Shimadzu LCMS-8060NX triple quadrupole
LC/MS/MS instruments with heated electrospray
ionization (ESI) in negative mode, using Lab Solutions
Software, were optimized and coordinated in two
different locations. These were used for the collection
of all data to rapidly meet the requirements set in SMPR
2023.003 in a collaborative effort. Methods submitted
to AOAC undergo a review and approved methods
are published in the Official Methods of Analysis or
OMA. First they are published as First Action where
they have done a single lab validation. After a First
Action acceptance, an inter-laboratory study is needed
for submission for approval to a Final Action method.
Setting up our initial instrumentation parameters in two
locations and duplicating our settings sets a foundation
for future validation work.
Mass spectrometry source and ionization conditions
were first optimized by introducing the mixed PFAS
standard into the Shimadzu 8060NX MS detector. In
these experiments, the following were tested: collision
induced dissociation (CID) gas pressure, desolvation
line (DL) temperature, drying gas flow rate, heating
block temperature, heating gas flow rate, interface
temperature, interface voltage, and nebulizer gas flow
rate. Small changes in these parameters can affect
the sensitivity of the detector and the most optimal
conditions can differ compound-to-compound.
Determining the best setup for a given method provides
the most sensitive results from the detector.
The results were assessed to maximize the response of
the various PFAS, with particular focus given to the EU
regulated PFAS: PFOA, PFHxS, PFNA, and PFOS.
These optimization experiments were conducted on
both instruments at the two selected Shimadzu research
centers in Columbia, Maryland, USA and in Tokyo,
Japan. Thirty-eight different variables were tested
in each location on all 30 target PFAS analytes for a
total of 1984 computations. At the Maryland location,
the heating gas flow rate was also tested against the
response of PFOA, PFHxS, PFNA, and PFOS as these
targets required the most sensitive responses per the
SMPR. Confirmation of these optimized conditions
in two location allowed for duplicate systems to be
run concurrently and share the research workload.
LabSolutions CS software allows for real time sharing
of data across the globe to facilitate rapid decision
making and collaboration across teams.
LCMS-8060NX Triple Quadrupole Mass Spectrometer
Table 2: MRM Transitions, CE and Quantitation Isotopes
Analyte
Quantitation
Ion
Qualifier
Ion
Quantitation
Ion CE
Qualifier
Ion CE
Quantitation
labeled Isotope
PFBA 212.9 > 168.8 10 13C4-PFBA
PFPeA 262.9 > 218.8 8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 9 22 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 9 16 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 11 17 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 10 16 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 10 18 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13 17 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 19 26 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13 25 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 12 28 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 33 26 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 39 30 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 44 33 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 49 39 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 54 39 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 54 43 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 45 45 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 52 52 13C8-PFOS
PFDoS 699 > 80 699 > 99 54 48 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 54 53 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 31 25 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 25 25 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 27 28 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 7 27 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 12 29 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 31 31 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 36 36 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 26 46 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 27 45 13C7-PFUnA
06
Table 3: Isotopically Labeled Internal Standards
Analyte Quantitation Ion Qualifier Ion
Quantitation Ion
CE
Qualifier Ion CE
13C4-PFBA-ES 217 > 172 10
13C5-PFPeA-ES 268 > 222.9 8
13C5-PFHxA-ES 318 > 272.9 9
13C4-PFHpA-ES 367 > 321.9 9
13C8-PFOA-ES 421 > 375.9 9
13C9-PFNA-ES 472 > 426.9 10
13C6-PFDA-ES 519 > 473.8 11
13C7-PFUnA_ES 570 > 524.8 11
13C2-PFDoA_ES 615 > 569.8 11
13C2-PFTeDA_ESa 715 > 669.7 13
13C3-PFBS-ES 302 > 79.8 32
13C3-PFHxS-ES 402 > 79.8 42
13C8-PFOS_ES 507 > 79.8 55
13C8-PFOSA_ES 506 > 77.8 33
13C3-HFPO-DA-ES 287 > 168.8 7
13C2-4:2 FTS-ES 329 > 80.9 329 > 308.8 26 18
13C2-6:2 FTS-ESa 429 > 80.9 429 > 408.8 35 23
13C2-8:2 FTS-ESa 529 > 79.8 529 > 508.8 44 24
a not used in SLV, but optional. 13C2-isotopes tend to suffer more interference particularly for higher mass compounds
07
PFAS Target Limits and Matrix Categories Set by
SMPR 2023.003
SMPR
Category
Category 1
(ug/kg)
Category 2
(ug/kg)
Category 3
(ug/kg)
egg ≤0.3 ≤3 ≤3
seafood ≤0.3 ≤3 ≤3
meat & fish ≤0.1 ≤1 ≤1
offal ≤0.4 ≤4 ≤4
produce ≤0.01 ≤1 ≤0.1
coffee ≤0.3 ≤3 ≤3
milk ≤0.01 ≤1 ≤0.1
protein
powder
≤0.08 ≤1 ≤0.8
fish oil ≤0.5 ≤5 ≤5
baby food ≤0.01 ≤1 ≤0.1
pet food ≤0.5 ≤5 ≤5
Table 4: PFAS Target Limits Set by SMPR 2023.003
With AOAC being an international organization,
existing EU regulations were taken into
consideration. Four of the target PFAS are already
regulated in certain foods in Europe: PFOS,
PFOA, PFNA, and PFHxS. These are considered
in category 1 of table 4. These are some of the
most studied PFAS compounds with respect to
exposure and testing, so they require lower limits
of quantitation and stricter tolerances. They are
regulated in the first 4 matrices of the table: eggs,
seafood, meat, fish, and offal. Category 2 includes
only PFBA and PFPeA, these are PFAS with only one
specific MS/MS transition and have the highest
limits of all the targeted compounds. Category
3 contains the remaining 24 PFAS targets which
include perfluoroalkyl acids and their precursors.
For the study, we used representative examples
from each matrix category listed in the SMPR; the
products validated for the study per category are
shown in table 5.
SMPR Matrix Category Product validated in study
Produce Carrots
Coffee Organic brewed coffee
Milk (liquid) Whole Milk (3.7% fat)
Dairy powders and plant-based protein powder Organic plant-based protein powder
Eggs Whole eggs
Seafood Frozen shrimp
Fish meat and meat of terrestrial mammals Raw tuna filet
Edible offal of terrestrial animals Beef kidney
Fish oil Fish oil capsules
Foods for infants and young children Strained pumpkin and sweet potato
Pet food and animal feed Bagged dry dog food
Table 5: Matrix categories from the SMPR and corresponding products validated in this study
08
Liquid Chromatography
Optimization
In order to ensure the best possible outcomes, the chromatographic conditions, including flow rate, gradient, injection
volume were evaluated to find an optimal setup. The TQ 8060NX triple quadrupole LCMS was optimized across over
1900 parameters on two different units duplicated in two locations to ensure replication of the setup. The columns were
optimized, as were the gradient conditions and flow rates. The final conditions chosen and used in the study are shown
in Table 6. These conditions maximized separation of the PFAS analytes and their sensitivity while minimizing noise and
systemic contamination.
Instrument Parameter Value
Mobile Phase A 2mM Ammonium Acetate in 5 / 95 = MeCN / Water
Mobile Phase B MeCN
Analytical Column Shimpack Scepter C18-120, 3um, 2.1 x 100 mm
Delay Column Shimpack Scepter C18-120, 3um, 2.1 x 50 mm
Oven Temperature 40 °C
Table 6: Chromatographic instrument settings
09
Time, min Flow rate mL/min
Mobile Phase Composition
A % B %
0 0.3 80 20
10 0.3 0 100
10.01 - 12 0.6 0 100
12.01 - 15 0.3 80 20
An example chromatogram is shown from a sample of carrots which were used to demonstrate the extraction of PFAS
from produce. All 30 target PFAS and the respective isotopically labelled internal standards were separated in a nineminute
chromatographic run. A Shimpack Scepter C18-120 delay column was used to isolate possible background
contaminant PFAS compounds and avoid coelution with target analytes.
Chromatography Optimization and Interferences
Figure 2: Chromatogram of 0.1 ng/g PFAS in a carrot, representing extraction from produce, with separation of all peaks in nine
minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched
and linear isomers
Part of optimization for chromatography involved the identification and separation of
PFOA from potential cholic acid interferences, per the requirements set forth by AOAC
INTERNATIONAL. Three were noted in SMPR 2023.003, Taurodeoxycholic acid (TCDA),
Taurochenodeoxycholic acid (TCDCA) and Tauroursodeoxycholic acid (TUDCA).
Nexera HPLC, most used on
LCMS-8060NX
10
We found that for offal and fish oil, a QuEChERS
treatment was unnecessary, with better or equal
recovery and repeatability when compared to the
general method versus omitting this step. Similarly,
concentration post-SPE cleanup was unnecessary for
fish tissue, offal, fish oil, and animal feed or pet food.
The general workflow shown in chart 1 walks through
the general procedure of: homogenization, QuEChERS
where needed, followed by SPE for all samples, then
concentration (again only where needed) before
injection to an LC-MS/MS system. Were we could skip
a step, we highlighted to do so, so we could streamline
the procedure to save testing labs time and money.
Sample Preparation and Analysis
Consistent sample preparation is key to obtaining the
necessary repeatability to meet SMPR 2023.003. A
general method was therefore developed for sample
preparation involving cleanup with QuEChERS and SPE,
then preconcentrating the samples before running the
analysis on the Shimadzu LCMS-8060NX. Each matrix
type was run using the same instrument method.
Knowing the challenges faced in routine labs, the
goal in creating this method was to use the same
LCMS-8060NX method, keep the workflow of sample
preparation the same but simplify it wherever possible.
We chose to omit steps on matrices where we found
they were unnecessary, saving time, reagents, and
consumable costs. For some matrices some components
of the sample preparation did not improve results, so
we investigated omitting these steps. We evaluated
recovery and repeatability without the addition of
the QuEChERS reagents and process, and without
concentration after SPE extraction. In the case of milk,
dispersive SPE proved more efficient than cartridge SPE.
Chart 1: General workflow of PFAS sampling
PFAS Confirmation Using QTOF LC/MS
The presence of PFAS compounds that do not have a confirmation ion, such as PFBA and PFPeA, must be confirmed
if detected in a sample at or above the LOQ. Confirmation may be by using another column of a different stationary
phase, or by using a High Resolution Mass Spectrometer (HRMS), such as the Shimadzu LCMS-9050 Quadrupole Timeof-
Flight (QTOF) Liquid Chromatograph Mass Spectrometer. HRMS determines the atomic masses of organic molecules
with a high degree of accuracy.
Figure 4: Confirmation by LC-QTOF for analytes without reference ion, example in egg for PFBA showing both the chromatogram
as TIC and extracted ion chromatogram, followed by the mass spectrum compared to the theoretical reference.
Analysis using the LCMS-9050 begins by injecting the
extracted sample into the LC operating with the same
analytical conditions as used on the LC-MS/MS. Once
the analytes elute from the column, they are ionized. The
ions travel along the spectrometer’s length, separating
by their relative charge and mass and a ratio of chargeto-
mass is recorded to five decimal places. This accurate
mass from the unconfirmed target analyte in the
sample can be compared to theoretical values for the
mass of the target analyte or to the retention time and
accurate mass of a calibration standard injected under
the same conditions. Figure 4 shows and extracted ion
chromatogram for the exact mass of PFBA (212.9792
m/z) in an egg matrix.
LCMS-9050 Quadrupole Time-of-Flight
Liquid Chromatograph Mass Spectrometer
12
Analysis Results
We used the SMPR as guide for the required LOQ and tested in 3 concentration points: one below the estimated LOQ,
and two between 2 – 100 times the estimated LOQ.
Figure 5 shows the recoveries and RSD results for the fish matrix samples, it is used as an example as fish is one of the
matrices regulated in the EU. The RSD limit for fish for the 4 EU-regulated compounds was 20%, the scale on the right
for RSD does not exceed 20% demonstrating results were well within the required limit. The recovery was required
between 80-120%, and on the left scale for recovery rate, this was met for 5, 1 and 0.1 ug/kg level spiked samples.
Figure 5: Recoveries and RSD results for the fish matrix samples
The following page organizes the 30 PFAS included in the AOAC SMPR into groupings based on existing regulations in
Europe for category 1, and differing requirements for PFBA and PFPeA than the remaining PFAS targets. EU-regulated
matrices for PFAS include eggs, seafood, fish meat and meat of terrestrial animals, and offal. These food groups have
stricter limits for PFOS, PFOA, PFNA and PFHxS ,and make up the first quadrant of the table. Category 2 includes only
PFBA and PFPeA, these are PFAS with only one specific MS/MS transition and have the highest limits of all the targeted
compounds. Category 3 contains the remaining 24 PFAS targets which include perfluoroalkyl acids and their precursors.
Limits for each target in each category are followed by the limits of quantitation achieved. Each limit in category 1 was
met, in some cases over an order of magnitude lower than the requested value, even as they were some of the lowest
concentrations due to existing EU regulations. All limits in category 2 and 3 were met at the SMPR level or we could
quantitate lower.
13
PFAS Egg Seafood Fish Offal Produce Coffee Milk
Protein
powder
Fish oil
Baby
food
Pet
food
Limit (ppb) in SMPR
per matrix for all
Category A
≤0.3 ≤0.3 ≤0.1 ≤0.4 ≤0.01 ≤0.3 ≤0.01 ≤0.08 ≤0.5 ≤0.01 ≤0.5
PFOA 0.006 0.006 0.1 0.2 0.006 0.006 0.01 0.055 0.25 0.01 0.5
PFOS 0.055 0.055 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.01 0.5
PFNA 0.006 0.006 0.1 0.2 0.006 0.006 0.01 0.006 0.25 0.01 0.5
PFHxS 0.006 0.006 0.1 0.2 0.006 0.006 0.01 0.006 0.25 0.01 0.5
Limit (ppb) in SMPR
per matrix for all
Category B
≤3 ≤3 ≤1 ≤4 ≤1 ≤3 ≤1 ≤1 ≤5 ≤1 ≤5
PFBA 0.055 0.055 0.1 0.2 0.055 0.055 0.01 0.055 0.25 0.1 0.5
PFPeA 0.055 0.006 0.1 0.2 0.055 0.006 0.01 0.055 0.25 0.1 0.5
Limit (ppb) in SMPR
per matrix for all
Category C
≤3 ≤3 ≤1 ≤4 ≤0.1 ≤3 ≤0.1 ≤0.8 ≤5 ≤0.1 ≤5
PFHxA 0.055 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.1 0.5
PFHpA 0.006 0.055 0.1 0.2 0.006 0.006 0.01 0.055 0.25 0.01 0.5
PFDA 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.006 0.25 0.01 0.5
PFUnA 0.006 0.006 0.1 0.2 0.055 0.006 0.01 0.006 0.25 0.01 0.5
PFDoA 0.006 0.055 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.01 0.5
PFTrDA 0.006 0.055 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.1 0.5
PFTeDA 0.055 0.055 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.1 0.5
PFBS 0.006 0.055 0.1 0.2 0.055 0.006 0.01 0.055 0.25 0.01 0.5
PFPeS 0.006 0.055 0.1 0.2 0.006 0.006 0.01 0.055 0.25 0.01 0.5
PFHpS 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.006 0.25 0.01 0.5
PFNS 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.01 0.5
PFDS 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.01 0.5
PFUnDS 0.055 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.1 0.5
PFDoS 0.055 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.25 0.01 0.5
PFTrDS 0.055 0.055 0.1 0.2 0.055 0.55 0.01 0.55 0.25 0.1 0.5
PFOSA 0.55 0.055 0.1 0.2 0.006 0.55 0.01 0.55 0.25 0.1 0.5
9Cl-PF3ONS 0.006 0.006 0.1 0.2 0.006 0.006 0.01 0.006 0.25 0.01 0.5
11Cl-PF3OUdS 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.006 0.25 0.01 0.5
HFPO-DA 0.006 0.055 0.1 0.2 0.006 0.055 0.01 0.55 0.25 0.01 0.5
DONA 0.006 0.006 0.1 0.2 0.006 0.006 0.01 0.006 0.25 0.01 0.5
4:2 FTS 0.55 0.055 0.1 0.2 0.055 0.055 0.01 0.055 0.25 0.01 0.5
6:2 FTS 0.006 0.55 0.1 0.2 0.055 0.006 0.01 0.055 0.25 0.01 0.5
8:2 FTS 0.006 0.006 0.1 0.2 0.006 0.55 0.01 0.006 0.25 0.01 0.5
10:2 FTS 0.006 0.006 0.1 0.2 0.006 0.055 0.01 0.055 0.5 0.01 0.5
Table 7: Obtained LOQs and SMPR target limits for all matrices Values shown in ug/kg
14
Results and Discussion
The same procedure from the test samples was used
to process the calibration standards. A linear model
provided the best fit and best recoveries of analytes.
Residuals of each point in the curve were ±25% of the
expected value. Branched and linear isomers of PFHxS
and PFOS were integrated together. Blank matrixes
were analyzed in triplicate, depending on the matrix.
Four different concentrations from below the LOQ
required by the SMPR, and ranging up to 500 times the
estimated LOQ, were also analyzed in triplicate to assess
recovery and repeatability throughout the calibration
and across matrices. Recovery and repeatability at the
LOQ in egg matrix for each analyte in category A are
given in table 8 to highlight existing EU regulations.
Remaining data is appended.
Table 8: Recovery and repeatability for select PFAS at each concentration level in eggs.
Analyte Spike conc. (ppb) Average conc. (ppb)
Standard
Deviation
%RSD
Average
Recovery%
PFOA
Blank 0
0.006 0.005 5.5 6.19 88.9
0.055 0.054 2.41 2.46 97.9
0.55 0.557 1.11 1.1 101.3
5.5 6.338 2.33 2.02 115.2
PFNA
Blank 0
0.006 0.005 1.66 1.67 99.1
0.055 0.056 2.05 2.01 102
0.55 0.592 0.59 0.54 107.6
5.5 6.122 8.8 7.91 111.3
PFOS
Blank 0.002
0.006 0.006 5.17 5.28 98.1
0.055 0.051 2.3 2.49 92.4
0.55 0.52 0.29 0.31 94.6
5.5 5.563 1.78 1.76 101.2
PFHxS
Blank -0.001
0.006 0.006 21.39 19.16 111.7
0.055 0.051 1.16 1.24 93.7
0.55 0.533 1.27 1.32 96.9
5.5 5.779 1.15 1.09 105.1
The LOQs for all matrices and compounds were determined according to the SMPR, with retention time, recovery,
repeatability, S/N > 3 for the qualifier ion and an ion ratio of ±30% all taken into consideration. Figure 6 includes
examples of the LOQ spike for PFHxS, PFNA, PFOA, and PFOS and their corresponding internal standards. The LOQ for
each analyte was determined based on the specifications listed in the SMPR. The appended application notes detail
the process and provide data for each matrix category.
Figure 6: LOQ peaks for PFHxS, and PFOA and their
corresponding internal standards
15
User Benefits
Application
News
◆ Validated method for 30 PFAS in baby food meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Foods for Infants and Young
Children (Baby Food) by Liquid Chromatography Triple
Quadrupole Mass Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in pumpkin
and sweet potato puree baby food using the QuEChERS
technique followed by analysis using the Shimadzu Nexera
Liquid Chromatograph coupled to a Shimadzu LCMS-
8060NX triple quadrupole mass spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at four concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
1 Shimadzu Scientific Instruments, Inc., 2 Shimadzu Corporation
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1 Nozomi Maeshima2, Manami Kobayashi2
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.1
Perfluoropentanoic acid PFPeA 2706-90-3 0.1
Perfluorohexanoic acid PFHxA 307-24-4 0.1
Perfluoroheptanoic acid PFHpA 375-85-9 0.01
Perfluorooctanoic acid PFOA 335-67-1 0.01
Perfluorononanoic acid PFNA 375-95-1 0.01
Perfluorodecanoic acid PFDA 335-76-2 0.01
Perfluoroundecanoic acid PFUnA 2058-94-8 0.01
Perfluorododecanoic acid PFDoA 307-55-1 0.01
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
■ Sample Preparation and Analysis Conditions
Samples were prepared by spiking blended baby food
samples in triplicate at four different concentrations with 30
native PFAS (Table 1) and 16 isotopically labeled internal
standards. Calibration curves for use in the quantitative
analysis were prepared using baby food test portions spiked
with concentrations of 0.005, 0.01, 0.05, 0.10, 0.50 and 1.0
ng/g. Quantitation was carried out on additional baby food
samples spiked in triplicate at 0.01, 0.10, 0.50 and 1.0 ng/g.
Since standards were extracted in a baby food matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
Multiple jars of baby food were purchased, combined, and
blended. Ten-gram portions were weighed, spiked with
target analytes and internal standards, and 10 mL of
acetonitrile was added. The samples were shaken for 1
minute and a QuEChERS packet was added. The sample was
shaken for 1 minute, allowed to cool to room temperature
and then centrifuged for 5 minutes at 4000 rpm. An aliquot
of the acetonitrile layer was transferred to a tube and diluted
5 times with PFAS-free reagent water. The sample was then
passed through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with
basic methanol. For greater sensitivity, the extract was
concentrated to less than 1 mL, then adjusted to a final 1
mL in a methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.1
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.1
Perfluorobutanesulfonic acid PFBS 375-73-5 0.01
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.01
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.01
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.01
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.01
Perfluorononanesulfonic acid PFNS 68259-12-1 0.01
Perfluorodecanesulfonic acid PFDS 335-77-3 0.01
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.1
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.01
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.1
Perfluorooctanesulfonamide PFOSA 754-91-6 0.1
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.01
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.01
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.01
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.01
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.01
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.01
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.01
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.01
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.1 ng/g PFAS in a baby food matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
0
100000
200000
300000
400000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Conc. (ng/g) %Accuracy
0.005 101
0.01 96.6
0.05 104
0.1 102
0.5 97.1
1 99.0
Blank matrixes and at least three different concentrations
ranging from the SMPR required LOQ to 100 times the
estimated LOQ were analyzed in triplicate. Recovery and
repeatability for each analyte at each concentration are given
in Table 3. The LOQ for each analyte was estimated by
spiking at concentrations at, or below, the required LOQs
listed in SMPR. The spiked samples were analyzed in triplicate
and the mean and repeatability standard deviation were
calculated. Then, the standard deviation was divided by the
mean and multiplied by 100% to calculate the repeatability
percent relative standard deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.005 98.3
0.01 102
0.05 106
0.1 102
0.5 94.8
1 96.2
Conc. (ng/g) %Accuracy
0.005 94.4
0.01 110
0.05 106
0.1 99.2
0.5 93.5
1 96.8
Conc. (ng/g) %Accuracy
0.005 97.5
0.01 103
0.05 110
0.1 98.5
0.5 94.6
1 96.1
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery (%)
PFBA
Blank 0.000
0.1 0.095 3.90 4.10 95.1
0.5 0.506 0.38 0.37 101.2
1 1.006 0.46 0.46 100.6
PFPeA
Blank 0.008
0.01 0.011 6.52 5.76 113.3
0.1 0.101 0.91 0.90 100.9
0.5 0.481 0.53 0.55 96.1
1 0.976 0.40 0.41 97.6
PFHxA
Blank ND
0.01 0.010 2.82 2.89 97.5
0.1 0.099 1.85 1.86 99.4
0.5 0.475 1.26 1.32 95.0
1 0.977 1.37 1.41 97.7
PFHpA
Blank ND
0.01 0.010 3.20 3.33 96.2
0.1 0.104 2.60 2.49 104.1
0.5 0.488 1.90 1.95 97.6
1 0.990 0.35 0.35 99.0
PFOA
Blank ND
0.01 0.010 3.04 3.07 99.3
0.1 0.102 1.95 1.92 101.7
0.5 0.495 0.95 0.96 99.0
1 0.994 0.38 0.38 99.4
PFNA
Blank 0.000
0.01 0.011 5.44 5.13 106.2
0.1 0.100 2.35 2.36 99.6
0.5 0.480 2.24 2.33 96.1
1 0.975 1.83 1.88 97.5
PFDA
Blank ND
0.01 0.010 5.19 5.04 103.0
0.1 0.102 1.17 1.15 102.0
0.5 0.490 2.71 2.76 98.0
1 1.000 0.52 0.52 100.0
PFUnA
Blank ND
0.01 0.010 8.96 8.91 100.6
0.1 0.104 0.97 0.94 103.6
0.5 0.495 1.99 2.01 98.9
1 0.995 3.20 3.22 99.5
PFDoA
Blank ND
0.01 0.012 10.48 9.10 115.2
0.1 0.103 6.81 6.63 102.8
0.5 0.467 1.61 1.72 93.4
1 0.957 3.90 4.08 95.7
PFTrDA
Blank ND
0.1 0.103 1.04 1.01 103.0
0.5 0.504 1.34 1.33 100.8
1 1.024 3.89 3.80 102.4
PFTeDA
Blank ND
0.1 0.117 5.89 5.03 117.3
0.5 0.557 3.35 3.01 111.3
1 1.139 3.42 3.00 113.9
PFBS
Blank ND
0.01 0.010 15.82 15.43 102.5
0.1 0.107 3.90 3.64 107.2
0.5 0.503 2.70 2.68 100.7
1 1.023 3.93 3.84 102.3
PFPeS
Blank 0.000
0.01 0.011 5.20 4.62 112.6
0.1 0.104 2.75 2.63 104.4
0.5 0.483 3.68 3.80 96.7
1 0.969 1.64 1.70 96.9
PFHxS
Blank 0.001
0.01 0.011 7.99 7.45 107.4
0.1 0.101 2.16 2.15 100.5
0.5 0.457 2.40 2.62 91.5
1 0.941 2.06 2.19 94.1
PFHpS
Blank ND
0.01 0.009 2.86 3.10 92.2
0.1 0.098 2.71 2.75 98.6
0.5 0.461 4.58 4.97 92.1
1 0.949 3.25 3.42 94.9
PFOS
Blank ND
0.01 0.009 5.72 6.56 87.2
0.1 0.099 3.74 3.78 99.1
0.5 0.462 0.06 0.06 92.3
1 0.943 1.66 1.76 94.3
PFNS
Blank ND
0.01 0.010 4.62 4.59 100.8
0.1 0.103 2.44 2.37 103.0
0.5 0.495 1.33 1.34 99.1
1 0.991 2.71 2.74 99.1
PFDS
Blank ND
0.01 0.008 1.62 2.04 79.3
0.1 0.106 6.18 5.83 105.9
0.5 0.508 1.40 1.38 101.6
1 1.020 3.31 3.24 102.0
PFUnDS
Blank ND
0.1 0.098 3.71 3.76 98.5
0.5 0.498 0.55 0.55 99.6
1 1.005 1.80 1.79 100.5
PFDoS
Blank ND
0.01 0.008 10.92 13.51 80.8
0.1 0.097 2.81 2.91 96.7
0.5 0.407 0.85 1.05 81.4
1 0.808 1.21 1.50 80.8
PFTrDS
Blank ND
0.1 0.100 2.46 2.45 100.4
0.5 0.504 5.22 5.17 100.8
1 1.028 4.62 4.50 102.8
PFOSA
Blank ND
0.01 0.010 10.68 10.71 99.7
0.1 0.101 4.27 4.24 100.9
0.5 0.478 1.86 1.94 95.6
1 0.961 0.25 0.26 96.1
9Cl-PF3ONS
Blank 0.000
0.01 0.011 7.75 7.30 106.2
0.1 0.104 3.58 3.44 104.0
0.5 0.485 2.06 2.13 96.9
1 0.987 2.03 2.06 98.7
11Cl-PF3OUdS
Blank ND
0.01 0.010 0.69 0.66 104.4
0.1 0.099 3.76 3.79 99.2
0.5 0.478 2.25 2.35 95.7
1 0.941 4.44 4.72 94.1
HFPO-DA
Blank ND
0.01 0.011 18.97 17.88 106.1
0.1 0.108 1.46 1.35 107.7
0.5 0.503 0.93 0.92 100.6
1 1.009 1.21 1.19 100.9
DONA
Blank ND
0.01 0.011 3.65 3.49 104.5
0.1 0.103 2.15 2.09 103.0
0.5 0.486 1.91 1.96 97.3
1 0.995 0.40 0.41 99.5
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
PFOA PFHxS PFNA PFOS
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a baby food matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4:2 FTS
Blank 0.000
0.01 0.012 11.06 9.41 117.5
0.1 0.105 3.82 3.64 104.9
0.5 0.492 2.05 2.09 98.2
1 0.999 0.47 0.47 99.9
6:2 FTS
Blank ND
0.01 0.011 7.86 7.17 109.6
0.1 0.104 4.90 4.72 103.9
0.5 0.492 1.23 1.25 98.5
1 0.981 2.00 2.04 98.1
8:2 FTS
Blank 0.000
0.01 0.011 1.06 0.94 112.5
0.1 0.101 4.33 4.29 100.9
0.5 0.451 1.90 2.11 90.2
1 0.895 3.04 3.40 89.5
10:2 FTS
Blank ND
0.01 0.012 10.15 8.88 114.3
0.1 0.104 3.84 3.69 104.0
0.5 0.470 2.59 2.76 94.0
1 0.911 2.92 3.20 91.1
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-163 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Egg meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Egg by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in eggs using
the QuEChERS technique followed by analysis using the
Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at four concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.055
Perfluoropentanoic acid PFPeA 2706-90-3 0.055
Perfluorohexanoic acid PFHxA 307-24-4 0.055
Perfluoroheptanoic acid PFHpA 375-85-9 0.0055
Perfluorooctanoic acid PFOA 335-67-1 0.0055
Perfluorononanoic acid PFNA 375-95-1 0.0055
Perfluorodecanoic acid PFDA 335-76-2 0.0055
Perfluoroundecanoic acid PFUnA 2058-94-8 0.0055
Perfluorododecanoic acid PFDoA 307-55-1 0.0055
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Dominika Gruszecka1, Toshiya Matsubara1
■ Sample Preparation and Analysis Conditions
Large brown eggs were purchased locally. Samples were
prepared cracking and removing the whole egg from the
shell and grinding with dry ice for 30 seconds at 4000 rpm.
The ground material was placed in a beaker. Test portions
were spiked in triplicate at four different concentrations with
30 native PFAS (Table 1) and 16 isotopically labeled internal
standards. Calibration curves for use in the quantitative
analysis were prepared using 10- gram test portions spiked
with concentrations of 0.001, 0.01, 0.1, 1.0 and 10.0 ng/g.
Quantitation was carried out on additional whole egg
samples spiked in triplicate at 0.0055, 0.055, 0.55 and 5.5
ng/g. Since standards were extracted in an egg matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 10 mL of acetonitrile was added.
The samples were vortexed for 1 minute and a QuEChERS
packet was added. The sample was shaken for 1 minute and
then centrifuged for 5 minutes at 4000 rpm. An aliquot of
the acetonitrile layer was transferred to a tube and diluted 5
times with PFAS-free reagent water. The sample was then
passed through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with
basic methanol. For greater sensitivity, the extract was
concentrated to dryness and dissolved in 0.4 mL of a
methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.0055
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.055
Perfluorobutanesulfonic acid PFBS 375-73-5 0.0055
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.0055
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.0055
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.055
Perfluorononanesulfonic acid PFNS 68259-12-1 0.0055
Perfluorodecanesulfonic acid PFDS 335-77-3 0.0055
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.055
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.055
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.055
Perfluorooctanesulfonamide PFOSA 754-91-6 0.55
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.0055
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.0055
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.55
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.0055
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.0055
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.0055
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.55 ng/g PFAS in an egg matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0min
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and four different concentrations ranging
from below the SMPR required LOQ to approximately 20
times the estimated LOQ were analyzed in triplicate.
Recovery and repeatability for each analyte at each
concentration are given in Table 3. The LOQ for each analyte
was estimated by spiking at concentrations at, or below, the
required LOQs listed in SMPR-2023_003. The spiked samples
were analyzed in triplicate and the mean and repeatability
standard deviation were calculated. Then, the standard
deviation was divided by the mean and multiplied by 100%
to calculate the repeatability percent relative standard
deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.001 101
0.01 91.2
0.1 97.3
1 102
10 108
Conc. (ng/g) %Accuracy
0.001 101
0.01 92.4
0.1 96.6
1 99.8
10 110
Conc. (ng/g) %Accuracy
0.001 99.7
0.01 103
0.1 106
1 108
10 84.0
Conc. (ng/g) %Accuracy
0.001 102
0.01 84.4
0.1 98.0
1 100
10 116
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank 0.002
0.055 0.051 1.38 1.48 93.3
0.55 0.528 1.30 1.36 95.9
5.5 5.664 0.26 0.26 103.0
PFPeA
Blank 0.004
0.0055 0.006 10.48 9.67 108.4
0.055 0.050 4.96 5.42 91.5
0.55 0.486 0.50 0.57 88.3
5.5 5.364 0.87 0.90 97.5
PFHxA
Blank 0.004
0.0055 0.005 4.52 4.82 93.8
0.055 0.055 0.62 0.62 100.3
0.55 0.550 1.37 1.36 100.0
5.5 6.107 1.10 0.99 111.1
PFHpA
Blank 0.000
0.0055 0.005 4.39 4.62 94.9
0.055 0.053 1.04 1.09 95.6
0.55 0.542 0.49 0.50 98.5
5.5 6.271 0.92 0.80 114.0
PFOA
Blank 0.000
0.0055 0.005 5.50 6.19 88.9
0.055 0.054 2.41 2.46 97.9
0.55 0.557 1.11 1.10 101.3
5.5 6.338 2.33 2.02 115.2
PFNA
Blank 0.000
0.0055 0.005 1.66 1.67 99.1
0.055 0.056 2.05 2.01 102.0
0.55 0.592 0.59 0.54 107.6
5.5 6.122 8.80 7.91 111.3
PFDA
Blank ND
0.0055 0.005 3.66 3.91 93.7
0.055 0.055 0.98 0.99 99.7
0.55 0.569 2.10 2.03 103.5
5.5 6.085 7.05 6.37 110.7
PFUnA
Blank 0.000
0.0055 0.005 3.20 3.35 95.5
0.055 0.057 2.15 2.10 102.8
0.55 0.587 0.85 0.80 106.8
5.5 6.281 8.37 7.33 114.2
PFDoA
Blank ND
0.0055 0.004 12.06 16.81 71.7
0.055 0.047 6.41 7.53 85.1
0.55 0.504 1.88 2.05 91.6
5.5 5.892 1.60 1.49 107.1
PFTrDA
Blank 0.000
0.0055 0.005 9.21 10.21 90.2
0.055 0.047 3.12 3.63 85.8
0.55 0.515 0.81 0.86 93.6
5.5 6.927 3.26 2.59 125.9
PFTeDA
Blank 0.002
0.0055 0.006 24.12 21.48 112.3
0.055 0.040 0.61 0.83 73.5
0.55 0.508 0.91 0.98 92.4
5.5 7.220 3.10 2.36 131.3
PFBS
Blank ND
0.0055 0.004 7.72 10.25 75.4
0.055 0.051 0.61 0.66 93.1
0.55 0.534 2.17 2.23 97.1
5.5 5.763 1.50 1.43 104.8
PFPeS
Blank 0.001
0.0055 0.005 17.67 18.11 97.6
0.055 0.051 4.60 4.99 92.2
0.55 0.516 0.67 0.71 93.9
5.5 5.494 1.49 1.49 99.9
PFHxS
Blank ND
0.0055 0.006 21.39 19.16 111.7
0.055 0.051 1.16 1.24 93.7
0.55 0.533 1.27 1.32 96.9
5.5 5.779 1.15 1.09 105.1
PFHpS
Blank 0.001
0.0055 0.005 2.93 2.98 98.3
0.055 0.057 3.84 3.73 103.0
0.55 0.515 1.29 1.38 93.6
5.5 5.058 3.91 4.25 92.0
PFOS
Blank 0.002
0.0055 0.006 5.17 5.28 98.1
0.055 0.051 2.30 2.49 92.4
0.55 0.520 0.29 0.31 94.6
5.5 5.563 1.78 1.76 101.2
PFNS
Blank 0.000
0.0055 0.005 10.26 11.84 86.7
0.055 0.048 1.71 1.94 88.1
0.55 0.523 2.52 2.65 95.1
5.5 6.736 3.30 2.69 122.5
PFDS
Blank ND
0.0055 0.005 2.25 2.47 91.3
0.055 0.050 2.42 2.67 90.5
0.55 0.503 1.82 1.99 91.5
5.5 5.541 3.26 3.23 100.8
PFUnDS
Blank 0.001
0.0055 0.005 4.47 5.03 88.9
0.055 0.049 3.86 4.36 88.7
0.55 0.536 2.55 2.62 97.4
5.5 6.074 2.62 2.37 110.5
PFDoS
Blank ND
0.0055 0.004 7.73 11.46 67.5
0.055 0.048 1.70 1.94 87.7
0.55 0.548 1.64 1.65 99.7
5.5 5.444 3.69 3.72 99.0
PFTrDS
Blank ND
0.055 0.045 1.86 2.25 82.5
0.55 0.514 1.37 1.46 93.4
5.5 6.457 3.92 3.34 117.4
PFOSA
Blank ND
0.0055 0.005 10.12 12.42 81.5
0.055 0.054 2.96 3.00 98.7
0.55 0.539 1.73 1.77 98.1
5.5 5.676 2.21 2.14 103.2
9Cl-PF3ONS
Blank 0.000
0.0055 0.006 6.82 6.82 100.0
0.055 0.048 1.26 1.45 86.6
0.55 0.507 1.59 1.73 92.2
5.5 7.191 2.75 2.10 130.7
11Cl-PF3OUdS
Blank 0.002
0.0055 0.006 4.92 4.23 116.2
0.055 0.045 0.96 1.18 81.4
0.55 0.494 2.01 2.23 89.9
5.5 7.332 3.60 2.70 133.3
HFPO-DA
Blank 0.000
0.0055 0.005 10.01 10.88 92.0
0.055 0.052 0.50 0.53 94.7
0.55 0.533 1.25 1.29 96.9
5.5 5.695 1.62 1.56 103.5
DONA
Blank 0.000
0.0055 0.005 3.26 3.24 100.6
0.055 0.057 0.53 0.51 104.2
0.55 0.583 0.31 0.29 106.1
5.5 6.148 9.36 8.38 111.8
4:2 FTS
Blank 0.017
0.0055 0.005 3.67 4.43 82.7
0.055 0.052 3.28 3.44 95.5
0.55 0.548 2.03 2.04 99.6
5.5 5.733 2.18 2.09 104.2
ND = average results less than zero
6:2 FTS
Blank 0.000
0.0055 0.005 14.57 15.09 96.6
0.055 0.053 2.80 2.88 97.2
0.55 0.538 1.99 2.03 97.9
5.5 5.480 1.60 1.61 99.7
8:2 FTS
Blank 0.000
0.0055 0.004 2.76 3.47 79.5
0.055 0.056 3.32 3.29 101.2
0.55 0.613 2.81 2.52 111.6
5.5 4.938 3.12 3.47 89.8
10:2 FTS
Blank 0.000
0.0055 0.005 10.90 13.79 79.1
0.055 0.052 1.12 1.17 95.5
0.55 0.596 2.71 2.50 108.3
5.5 4.553 4.08 4.93 82.8
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in an egg matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
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LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-165 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Fish meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Fish by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in fish using
the QuEChERS technique followed by analysis using the
Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at three concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.1
Perfluoropentanoic acid PFPeA 2706-90-3 0.1
Perfluorohexanoic acid PFHxA 307-24-4 0.1
Perfluoroheptanoic acid PFHpA 375-85-9 0.1
Perfluorooctanoic acid PFOA 335-67-1 0.1
Perfluorononanoic acid PFNA 375-95-1 0.1
Perfluorodecanoic acid PFDA 335-76-2 0.1
Perfluoroundecanoic acid PFUnA 2058-94-8 0.1
Perfluorododecanoic acid PFDoA 307-55-1 0.1
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc., 2 Shimadzu Corporation
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1, Nozomi Maeshima2, Kota Ishioka2,
Manami Kobayashi2
■ Sample Preparation and Analysis Conditions
Samples were prepared by dicing the edible portion of tuna
filets and grinding with dry ice. The ground material was
placed in a freezer overnight. Test portions were spiked in
triplicate at three different concentrations with 30 native
PFAS (Table 1) and 16 isotopically labeled internal standards.
Calibration curves for use in the quantitative analysis were
prepared using 10- gram test portions spiked with
concentrations of 0.05, 0.1, 0.5, 1.0 and 5.0 ng/g.
Quantitation was carried out on additional tuna samples
spiked in triplicate at 0.1, 1.0, and 5.0 ng/g. Since standards
were extracted in a tuna matrix and carried through the
same procedure, the final concentration of each PFAS in the
sample can be calculated directly from the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 10 mL of acetonitrile was added.
The samples were shaken by hand for 1 minute and a
QuEChERS packet was added. The sample was shaken for 1
minute and then centrifuged for 5 minutes at 4000 rpm. An
aliquot of the acetonitrile layer was transferred to a tube and
diluted 5 times with PFAS-free reagent water. The sample
was then passed through a weak anion exchange (WAX)
Solid Phase Extraction (SPE) cartridge and the PFAS were
eluted with basic methanol. A 0.5 mL aliquot was transferred
to a LC vial and acidified with formic acid.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.1
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.1
Perfluorobutanesulfonic acid PFBS 375-73-5 0.1
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.1
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.1
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.1
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.1
Perfluorononanesulfonic acid PFNS 68259-12-1 0.1
Perfluorodecanesulfonic acid PFDS 335-77-3 0.1
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.1
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.1
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.1
Perfluorooctanesulfonamide PFOSA 754-91-6 0.1
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.1
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.1
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.1
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.1
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.1
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.1
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.1
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.1
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 1.0 ng/g PFAS in a fish matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and three different concentrations ranging
from the SMPR required LOQ to 50 times the estimated LOQ
were analyzed in triplicate. Recovery and repeatability for
each analyte at each concentration are given in Table 3. The
LOQ for each analyte was estimated by spiking at
concentrations at, or below, the required LOQs listed in
SMPR-2023_003. The spiked samples were analyzed in
triplicate and the mean and repeatability standard deviation
were calculated. Then, the standard deviation was divided by
the mean and multiplied by 100% to calculate the
repeatability percent relative standard deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.05 100
0.1 98.6
0.5 105
1 97.0
5 98.8
Conc. (ng/g) %Accuracy
0.05 94.6
0.1 110
0.5 105
1 97.7
5 92.9
Conc. (ng/g) %Accuracy
0.05 102
0.1 95.6
0.5 106
1 95.9
5 100
Conc. (ng/g) %Accuracy
0.05 99.9
0.1 99.2
0.5 107
1 96.2
5 97.7
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank 0.004
0.1 0.103 2.51 2.43 103.4
1 0.970 0.21 0.21 97.0
5 4.830 0.79 0.82 96.6
PFPeA
Blank ND
0.1 0.096 2.11 2.20 96.2
1 0.976 0.46 0.47 97.6
5 4.920 0.61 0.62 98.4
PFHxA
Blank 0.002
0.1 0.099 1.64 1.66 98.8
1 0.957 0.55 0.58 95.7
5 4.883 1.72 1.77 97.6
PFHpA
Blank ND
0.1 0.100 0.44 0.44 100.1
1 0.980 1.72 1.76 98.0
5 4.998 1.42 1.42 100.0
PFOA
Blank ND
0.1 0.100 1.50 1.51 99.6
1 0.960 1.43 1.49 96.0
5 4.852 0.81 0.84 97.0
PFNA
Blank ND
0.1 0.101 2.74 2.72 100.9
1 0.984 1.11 1.12 98.4
5 5.039 2.25 2.24 100.8
PFDA
Blank 0.000
0.1 0.098 3.36 3.42 98.2
1 0.936 1.39 1.48 93.6
5 4.800 0.57 0.59 96.0
PFUnA
Blank 0.002
0.1 0.100 1.65 1.65 99.8
1 0.932 1.44 1.55 93.2
5 4.896 1.63 1.66 97.9
PFDoA
Blank ND
0.1 0.093 10.48 11.29 92.9
1 0.939 2.17 2.31 93.9
5 4.833 1.42 1.47 96.6
PFTrDA
Blank 0.004
0.1 0.107 8.56 8.05 106.4
1 0.996 3.93 3.95 99.6
5 4.889 4.60 4.71 97.8
PFTeDA
Blank 0.009
0.1 0.114 8.25 7.23 114.1
1 1.029 12.47 12.12 102.9
5 4.860 5.60 5.76 97.2
PFBS
Blank ND
0.1 0.100 2.77 2.78 99.7
1 0.945 1.76 1.86 94.5
5 4.836 1.26 1.30 96.7
PFPeS
Blank ND
0.1 0.092 9.10 9.85 92.4
1 0.990 2.20 2.22 99.0
5 4.857 1.96 2.01 97.2
PFHxS
Blank 0.002
0.1 0.105 6.51 6.21 104.9
1 0.968 2.02 2.09 96.8
5 4.838 3.11 3.21 96.7
PFHpS
Blank 0.001
0.1 0.099 3.85 3.88 99.1
1 0.925 2.29 2.47 92.5
5 4.613 0.35 0.38 92.2
PFOS
Blank 0.003
0.1 0.102 1.89 1.85 102.2
1 0.968 0.60 0.62 96.8
5 4.834 0.90 0.93 96.7
PFNS
Blank ND
0.1 0.101 6.29 6.26 100.4
1 0.929 2.48 2.68 92.9
5 4.840 1.61 1.66 96.8
PFDS
Blank ND
0.1 0.097 4.10 4.24 96.7
1 0.982 2.63 2.68 98.2
5 4.877 3.65 3.74 97.6
PFUnDS
Blank 0.000
0.1 0.106 13.25 12.58 105.3
1 1.054 7.89 7.49 105.4
5 4.759 6.56 6.90 95.2
PFDoS
Blank 0.005
0.1 0.114 16.66 14.59 114.1
1 1.084 20.30 18.72 108.4
5 4.694 2.70 2.88 93.9
PFTrDS
Blank 0.008
0.1 0.119 14.78 12.41 119.1
1 1.113 24.60 22.11 111.3
5 4.911 7.02 7.15 98.2
PFOSA
Blank 0.000
0.1 0.096 2.16 2.25 96.0
1 0.945 0.97 1.03 94.5
5 4.898 0.67 0.68 98.0
9Cl-PF3ONS
Blank ND
0.1 0.097 1.14 1.17 97.2
1 0.965 0.44 0.45 96.5
5 4.901 1.82 1.86 98.0
11Cl-PF3OUdS
Blank ND
0.1 0.097 6.63 6.82 97.3
1 0.943 3.67 3.89 94.3
5 4.796 3.06 3.20 95.9
HFPO-DA
Blank 0.004
0.1 0.102 6.03 5.91 101.9
1 0.964 1.36 1.41 96.4
5 4.778 0.21 0.22 95.5
DONA
Blank 0.001
0.1 0.100 1.42 1.43 99.6
1 0.956 0.90 0.94 95.6
5 4.974 1.10 1.11 99.5
4:2 FTS
Blank 0.000
0.1 0.099 3.73 3.77 98.9
1 0.955 1.62 1.69 95.5
5 4.718 1.86 1.97 94.4
6:2 FTS
Blank 0.001
0.1 0.098 2.91 2.98 97.7
1 0.944 1.03 1.09 94.4
5 4.664 1.00 1.07 93.3
8:2 FTS
Blank 0.010
0.1 0.107 4.31 4.02 107.2
1 1.096 9.63 8.79 109.6
5 5.041 19.12 18.97 100.8
10:2 FTS
Blank 0.008
0.1 0.110 5.70 5.21 109.4
1 1.029 7.31 7.10 102.9
5 4.028 8.96 11.12 80.6
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a fish matrix and compared
to criteria set by AOAC SMPR 2023.003. Chromatography
conditions and the mass spectrometer were optimized to
achieve excellent separation of all analytes, baseline
resolution between linear and branched isomers, and a twominute
separation between PFOS and potentially interfering
cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
SHIMADZU Scientific Instruments Inc.
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LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-167 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Protein Powder meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Protein Powder by
Liquid Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in protein
powder using the QuEChERS technique followed by
analysis using the Shimadzu Nexera Liquid
Chromatograph coupled to a Shimadzu LCMS-8060NX
triple quadrupole mass spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at four concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.055
Perfluoropentanoic acid PFPeA 2706-90-3 0.055
Perfluorohexanoic acid PFHxA 307-24-4 0.055
Perfluoroheptanoic acid PFHpA 375-85-9 0.055
Perfluorooctanoic acid PFOA 335-67-1 0.055
Perfluorononanoic acid PFNA 375-95-1 0.0055
Perfluorodecanoic acid PFDA 335-76-2 0.0055
Perfluoroundecanoic acid PFUnA 2058-94-8 0.0055
Perfluorododecanoic acid PFDoA 307-55-1 0.055
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Dominika Gruszecka1, Toshiya Matsubara1
■ Sample Preparation and Analysis Conditions
Organic, plant-based protein powder was purchased locally.
Test portions were weighed directly from the store packaging
and spiked in triplicate at four different concentrations with
30 native PFAS (Table 1) and 16 isotopically labeled internal
standards. Calibration curves for use in the quantitative
analysis were prepared using 10- gram protein powder test
portions spiked with concentrations of 0.001, 0.01, 0.1, 1.0
and 10.0 ng/g. Quantitation was carried out on additional
protein powder samples spiked in triplicate at 0.0055, 0.055,
0.55 and 5.5 ng/g. Since standards were extracted in a
protein powder matrix and carried through the same
procedure, the final concentration of each PFAS in the
sample can be calculated directly from the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, 10 mL of water and 10 mL of
acetonitrile was added. The samples were shaken for 1
minute and a QuEChERS packet was added. The sample was
shaken again for 1 minute and then centrifuged for 5
minutes at 4000 rpm. An aliquot of the acetonitrile layer was
transferred to a tube and diluted 13 times with PFAS-free
reagent water. The sample was then passed through a weak
anion exchange (WAX) Solid Phase Extraction (SPE) cartridge
and the PFAS were eluted with basic methanol. For greater
sensitivity, the extract was concentrated to dryness and
dissolved in 0.4 mL of a methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.055
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.055
Perfluorobutanesulfonic acid PFBS 375-73-5 0.055
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.055
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.0055
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.055
Perfluorononanesulfonic acid PFNS 68259-12-1 0.055
Perfluorodecanesulfonic acid PFDS 335-77-3 0.055
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.055
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.055
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.55
Perfluorooctanesulfonamide PFOSA 754-91-6 0.55
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.0055
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.55
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.055
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.055
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.0055
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.055
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.055 ng/g PFAS in a protein powder matrix with separation of all peaks in nine minutes.
The chromatogram shows other co-eluting peaks between 2.7 and 3.6 minutes, but they did not interfere.
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000
275000
300000
325000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and at least three different concentrations
ranging from below the SMPR required LOQ to
approximately 70 times the estimated LOQ were analyzed in
triplicate. Recovery and repeatability for each analyte at each
concentration are given in Table 3. The LOQ for each analyte
was estimated by spiking at concentrations at, or below, the
required LOQs listed in SMPR-2023_003. The spiked samples
were analyzed in triplicate and the mean and repeatability
standard deviation were calculated. Then, the standard
deviation was divided by the mean and multiplied by 100%
to calculate the repeatability percent relative standard
deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.001 101
0.01 90.2
0.1 104
1 101
10 103
Conc. (ng/g) %Accuracy
0.001 99.7
0.01 102
0.1 109
1 104
10 85.7
Conc. (ng/g) %Accuracy
0.001 101
0.01 88.6
0.1 102
1 106
10 102
Conc. (ng/g) %Accuracy
0.01 99.1
0.1 109
1 108
10 84.0
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank ND
0.055 0.071 6.83 5.31 128.7
0.55 0.599 2.41 2.22 108.9
5.5 6.077 0.70 0.64 110.5
PFPeA
Blank ND
0.055 0.061 3.70 3.36 110.2
0.55 0.598 0.29 0.27 108.6
5.5 6.275 2.88 2.53 114.1
PFHxA
Blank ND
0.055 0.049 2.50 2.81 89.1
0.55 0.546 2.08 2.10 99.2
5.5 5.758 1.32 1.26 104.7
PFHpA
Blank 0.005
0.055 0.057 1.46 1.41 103.3
0.55 0.541 4.25 4.32 98.4
5.5 5.339 2.40 2.47 97.1
PFOA
Blank 0.000
0.055 0.060 4.83 4.40 109.7
0.55 0.614 2.34 2.10 111.6
5.5 5.962 2.80 2.58 108.4
PFNA
Blank ND
0.0055 0.006 10.70 10.53 101.7
0.055 0.060 0.67 0.61 109.2
0.55 0.604 2.45 2.23 109.8
5.5 6.158 2.84 2.54 111.9
PFDA
Blank ND
0.0055 0.005 4.86 5.09 95.6
0.055 0.058 1.52 1.45 104.8
0.55 0.589 3.72 3.48 107.1
5.5 5.996 3.88 3.55 109.0
PFUnA
Blank ND
0.0055 0.007 21.51 18.99 113.3
0.055 0.059 6.09 5.73 106.3
0.55 0.595 2.56 2.36 108.2
5.5 5.655 4.91 4.78 102.8
PFDoA
Blank ND
0.055 0.060 10.10 9.34 108.2
0.55 0.597 6.61 6.09 108.6
5.5 5.748 7.59 7.26 104.5
PFTrDA
Blank ND
0.0055 0.004 12.72 17.77 71.6
0.055 0.052 2.20 2.30 95.5
0.55 0.561 2.15 2.10 102.0
5.5 5.583 3.40 3.35 101.5
PFTeDA
Blank 0.000
0.0055 0.005 4.51 4.94 91.2
0.055 0.049 5.97 6.69 89.3
0.55 0.568 1.33 1.29 103.3
5.5 5.432 4.41 4.46 98.8
PFBS
Blank ND
0.055 0.062 11.70 10.33 113.2
0.55 0.620 7.16 6.35 112.7
5.5 5.952 6.67 6.16 108.2
PFPeS
Blank 0.001
0.055 0.063 3.61 3.14 114.8
0.55 0.602 6.16 5.62 109.5
5.5 6.168 0.86 0.77 112.2
PFHxS
Blank ND
0.0055 0.006 6.85 6.02 113.8
0.055 0.059 3.86 3.60 107.1
0.55 0.587 1.17 1.10 106.7
5.5 5.486 2.16 2.17 99.7
PFHpS
Blank 0.001
0.0055 0.006 15.90 14.87 106.9
0.055 0.058 2.05 1.94 105.6
0.55 0.563 3.63 3.55 102.3
5.5 6.008 7.58 6.94 109.2
PFOS
Blank ND
0.055 0.057 17.30 16.79 103.0
0.55 0.603 4.60 4.20 109.6
5.5 5.325 2.41 2.49 96.8
PFNS
Blank ND
0.055 0.054 4.15 4.24 98.0
0.55 0.610 8.84 7.97 110.9
5.5 6.174 5.02 4.48 112.3
PFDS
Blank 0.003
0.055 0.056 18.37 18.08 101.6
0.55 0.532 3.72 3.85 96.6
5.5 5.808 6.93 6.56 105.6
PFUnDS
Blank 0.001
0.0055 0.005 10.13 12.93 78.4
0.055 0.056 3.07 3.03 101.4
0.55 0.561 3.32 3.25 101.9
5.5 4.688 3.45 4.04 85.3
PFDoS
Blank 0.003
0.0055 0.006 7.39 6.50 113.7
0.055 0.058 4.98 4.75 104.8
0.55 0.523 3.60 3.79 95.0
5.5 4.703 1.74 2.04 85.5
PFTrDS
Blank ND
0.55 0.695 2.08 1.65 126.4
5.5 6.531 6.31 5.32 118.8
PFOSA
Blank ND
0.055 0.053 7.62 7.91 96.3
0.55 0.614 3.36 3.01 111.7
5.5 5.397 2.50 2.55 98.1
9Cl-PF3ONS
Blank 0.000
0.0055 0.006 3.21 2.76 116.4
0.055 0.063 2.40 2.10 114.3
0.55 0.607 3.84 3.48 110.3
5.5 5.687 1.52 1.47 103.4
11Cl-PF3OUdS
Blank ND
0.0055 0.004 9.37 12.62 74.2
0.055 0.060 7.15 6.56 109.0
0.55 0.557 0.90 0.89 101.3
5.5 5.777 3.99 3.80 105.0
HFPO-DA
Blank 0.030
0.55 0.663 8.31 6.89 120.5
5.5 6.199 8.09 7.18 112.7
DONA
Blank ND
0.0055 0.005 5.20 5.91 88.0
0.055 0.060 3.83 3.54 108.2
0.55 0.546 4.27 4.30 99.3
5.5 5.671 1.33 1.29 103.1
4:2 FTS
Blank 0.012
0.055 0.054 5.74 5.85 98.0
0.55 0.589 2.02 1.89 107.1
5.5 5.963 5.17 4.77 108.4
6:2 FTS
Blank 0.004
0.055 0.071 15.56 12.10 128.6
0.55 0.582 4.10 3.87 105.9
5.5 5.729 4.83 4.64 104.1
8:2 FTS
Blank 0.000
0.0055 0.005 7.43 7.54 98.6
0.055 0.055 7.09 7.10 99.8
0.55 0.553 5.34 5.32 100.5
5.5 5.004 4.06 4.46 91.0
10:2 FTS
Blank ND
0.055 0.055 10.56 10.61 99.5
0.55 0.673 8.99 7.35 122.3
5.5 5.601 11.11 10.91 101.8
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a protein powder matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-172 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Pet Food meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, Low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Pet Food by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in pet food
using the QuEChERS technique followed by analysis using
the Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at three concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.5
Perfluoropentanoic acid PFPeA 2706-90-3 0.5
Perfluorohexanoic acid PFHxA 307-24-4 0.5
Perfluoroheptanoic acid PFHpA 375-85-9 0.5
Perfluorooctanoic acid PFOA 335-67-1 0.5
Perfluorononanoic acid PFNA 375-95-1 0.5
Perfluorodecanoic acid PFDA 335-76-2 0.5
Perfluoroundecanoic acid PFUnA 2058-94-8 0.5
Perfluorododecanoic acid PFDoA 307-55-1 0.5
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc., 2 Shimadzu Corporation
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1, Nozomi Maeshima2, Yui Higashi2,
Manami Kobayashi2
■ Sample Preparation and Analysis Conditions
Dog food was purchased locally. Sub samples were removed
from the bag and crushed with dry ice. The ground material
placed into containers and stored in a freezer overnight to
allow the dry ice to sublimate away completely. Test portions
were spiked in triplicate at three different concentrations
with 30 native PFAS (Table 1) and 16 isotopically labeled
internal standards. Calibration curves for use in the
quantitative analysis were prepared using 2- gram test
portions spiked with concentrations of 0.25, 0.5, 2.5, 5, 12.5,
and 25 ng/g. Quantitation was carried out on additional dog
food samples spiked in triplicate at 0.5, 5.0, and 25 ng/g.
Since standards were extracted in a pet food matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
2-gram portions were weighed, spiked with target analytes
and internal standards, 10 mL of water and 10 mL of
acetonitrile was added. The samples were shaken by hand
for 1 minute and a QuEChERS packet was added. The
sample was shaken for 1 minute and then centrifuged for 5
minutes at 4000 rpm. An aliquot of the acetonitrile layer was
transferred to a tube and diluted 5 times with PFAS-free
reagent water. The sample was then passed through a weak
anion exchange (WAX) Solid Phase Extraction (SPE) cartridge
and the PFAS were eluted with basic methanol. A 0.5 mL
aliquot of the eluted solution was transferred to a vial and
acidified with formic acid.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.5
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.5
Perfluorobutanesulfonic acid PFBS 375-73-5 0.5
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.5
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.5
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.5
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.5
Perfluorononanesulfonic acid PFNS 68259-12-1 0.5
Perfluorodecanesulfonic acid PFDS 335-77-3 0.5
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.5
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.5
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.5
Perfluorooctanesulfonamide PFOSA 754-91-6 0.5
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.5
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.5
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.5
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.5
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.5
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.5
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.5
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.5
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 5 ng/g PFAS in a pet food matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
0
250000
500000
750000
1000000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and three different concentrations ranging
from the SMPR required LOQ to 50 times the estimated LOQ
were analyzed in triplicate. Recovery and repeatability for
each analyte at each concentration are given in Table 3. The
LOQ for each analyte was estimated by spiking at
concentrations at, or below, the required LOQs listed in
SMPR-2023_003. The spiked samples were analyzed in
triplicate and the mean and repeatability standard deviation
were calculated. Then, the standard deviation was divided by
the mean and multiplied by 100% to calculate the
repeatability percent relative standard deviation (RSD).
The LOQs for all compounds were determined using an
Excel worksheet that compared each of the requirements
of the SMPR including retention time, recovery,
repeatability, S/N > 3 for the qualifier ion and an ion ratio
of ±30%. PFBA, PFPeA, and PFOSA LOQ were set at the
minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.25 97.5
0.5 105
2.5 99.1
5 100
12.5 99.8
25.0 98.2
Conc. (ng/g) %Accuracy
0.25 102
0.5 95.5
2.5 106
5 100
12.5 104
25.0 92.6
Conc. (ng/g) %Accuracy
0.25 97.2
0.5 106
2.5 97.6
5 104
12.5 97.0
25.0 98.3
Conc. (ng/g) %Accuracy
0.25 98.9
0.5 103
2.5 96.2
5 101
12.5 104
25.0 97.3
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank ND
0.5 0.541 13.70 12.66 108.2
5 4.956 1.69 1.71 99.1
25 22.753 2.35 2.58 91.0
PFPeA
Blank ND
0.5 0.514 0.65 0.63 102.8
5 5.071 0.59 0.58 101.4
25 23.462 0.29 0.31 93.9
PFHxA
Blank ND
0.5 0.502 2.66 2.65 100.3
5 5.088 0.25 0.25 101.8
25 24.247 1.07 1.10 97.0
PFHpA
Blank ND
0.5 0.559 3.74 3.35 111.8
5 5.073 0.85 0.84 101.4
25 23.973 0.36 0.38 95.9
PFOA
Blank ND
0.5 0.525 2.82 2.68 105.0
5 5.147 0.81 0.79 102.9
25 24.564 0.42 0.42 98.3
PFNA
Blank ND
0.5 0.513 0.76 0.74 102.8
5 5.101 0.95 0.93 102.0
25 24.739 0.64 0.65 99.0
PFDA
Blank ND
0.5 0.525 1.19 1.14 105.1
5 5.197 1.80 1.74 103.9
25 24.522 0.76 0.78 98.1
PFUnA
Blank ND
0.5 0.541 1.25 1.16 108.3
5 5.141 2.02 1.97 102.8
25 23.863 0.76 0.79 95.4
PFDoA
Blank ND
0.5 0.482 4.82 5.00 96.4
5 4.919 3.08 3.13 98.4
25 23.291 1.75 1.88 93.2
PFTrDA
Blank ND
0.5 0.490 6.38 6.51 97.9
5 4.921 5.49 5.58 98.4
25 24.337 0.87 0.90 97.4
PFTeDA
Blank ND
0.5 0.508 1.01 0.99 101.7
5 4.858 2.87 2.96 97.2
25 24.158 1.93 2.00 96.6
PFBS
Blank ND
0.5 0.533 11.52 10.80 106.7
5 5.091 1.32 1.30 101.8
25 22.671 0.89 0.98 90.7
PFPeS
Blank ND
0.5 0.428 13.61 15.89 85.6
5 4.429 0.35 0.39 88.6
25 19.906 5.80 7.28 79.6
PFHxS
Blank ND
0.5 0.539 2.23 2.07 107.7
5 4.900 2.96 3.02 98.0
25 22.645 2.36 2.60 90.6
PFHpS
Blank ND
0.5 0.494 2.61 2.64 98.7
5 5.026 0.82 0.81 100.5
25 24.918 1.45 1.46 99.7
ND = average results less than zero
PFOS
Blank ND
0.5 0.475 6.50 6.84 95.0
5 4.930 3.29 3.34 98.6
25 22.823 1.41 1.55 91.3
PFNS
Blank ND
0.5 0.512 7.18 7.01 102.3
5 4.926 2.71 2.75 98.5
25 22.611 0.29 0.32 90.4
PFDS
Blank ND
0.5 0.512 9.79 9.57 102.3
5 4.921 1.68 1.71 98.4
25 23.794 2.55 2.68 95.2
PFUnDS
Blank ND
0.5 0.531 3.02 2.84 106.1
5 5.145 1.75 1.70 102.9
25 24.117 1.78 1.84 96.5
PFDoS
Blank ND
0.5 0.502 5.59 5.57 100.5
5 5.103 2.54 2.49 102.1
25 23.501 3.05 3.25 94.0
PFTrDS
Blank ND
0.5 0.511 5.99 5.87 102.1
5 4.752 5.22 5.49 95.0
25 23.836 1.86 1.95 95.3
PFOSA
Blank ND
0.5 0.523 0.95 0.91 104.7
5 5.139 0.62 0.61 102.8
25 24.134 0.55 0.57 96.5
9Cl-PF3ONS
Blank ND
0.5 0.494 2.51 2.54 98.9
5 5.095 1.51 1.49 101.9
25 22.288 0.81 0.91 89.1
11Cl-PF3OUdS
Blank ND
0.5 0.501 1.86 1.86 100.1
5 4.919 3.34 3.39 98.4
25 23.572 1.67 1.78 94.3
HFPO-DA
Blank ND
0.5 0.360 1.91 2.66 72.0
5 5.044 1.04 1.03 100.9
25 24.698 2.86 2.89 98.8
DONA
Blank ND
0.5 0.493 6.51 6.59 98.7
5 4.744 2.14 2.25 94.9
25 21.780 7.33 8.41 87.1
4:2 FTS
Blank ND
0.5 0.582 4.99 4.29 116.4
5 5.084 1.85 1.82 101.6
25 23.506 0.26 0.28 94.0
6:2 FTS
Blank ND
0.5 0.545 3.78 3.47 109.0
5 5.080 2.71 2.66 101.6
25 24.619 6.15 6.24 98.5
8:2 FTS
Blank ND
0.5 0.543 0.85 0.79 108.6
5 5.626 3.15 2.80 112.5
25 23.754 3.35 3.52 95.0
10:2 FTS
Blank ND
0.5 0.502 10.12 10.08 100.4
5 5.372 0.85 0.79 107.4
25 22.176 3.46 3.90 88.7
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a pet food matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-170 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in produce meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, Low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Produce by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in carrots
using the QuEChERS technique followed by analysis using
the Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at four concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.055
Perfluoropentanoic acid PFPeA 2706-90-3 0.055
Perfluorohexanoic acid PFHxA 307-24-4 0.0055
Perfluoroheptanoic acid PFHpA 375-85-9 0.0055
Perfluorooctanoic acid PFOA 335-67-1 0.0055
Perfluorononanoic acid PFNA 375-95-1 0.0055
Perfluorodecanoic acid PFDA 335-76-2 0.0055
Perfluoroundecanoic acid PFUnA 2058-94-8 0.055
Perfluorododecanoic acid PFDoA 307-55-1 0.0055
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc., 2 Shimadzu Corporation
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1, Nozomi Maeshima2, Kota Ishioka2,
Manami Kobayashi2
■ Sample Preparation and Analysis Conditions
Samples were prepared by dicing the tuber portion of carrots
and grinding with dry ice. The ground material was placed in
a freezer overnight. Test portions were spiked in triplicate at
four different concentrations with 30 native PFAS (Table 1)
and 16 isotopically labeled internal standards. Calibration
curves for use in the quantitative analysis were prepared
using 10- gram carrot test portions spiked with
concentrations of 0.001, 0.01, 0.1, 1.0 and 10.0 ng/g.
Quantitation was carried out on additional carrot samples
spiked in triplicate at 0.0055, 0.055, 0.55 and 5.5 ng/g.
Since standards were extracted in a carrot matrix and carried
through the same procedure, the final concentration of each
PFAS in the sample can be calculated directly from the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 10 mL of acetonitrile was added.
The samples were vortexed for 1 minute and a QuEChERS
packet was added. The sample was shaken for 1 minute and
then centrifuged for 5 minutes at 4000 rpm. An aliquot of
the acetonitrile layer was transferred to a tube and diluted 5
times with PFAS-free reagent water. The sample was then
passed through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with
basic methanol. For greater sensitivity, the extract was
concentrated to dryness and dissolved in 0.4 mL of a
methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.0055
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.0055
Perfluorobutanesulfonic acid PFBS 375-73-5 0.055
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.0055
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.0055
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.0055
Perfluorononanesulfonic acid PFNS 68259-12-1 0.0055
Perfluorodecanesulfonic acid PFDS 335-77-3 0.0055
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.0055
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.0055
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.055
Perfluorooctanesulfonamide PFOSA 754-91-6 0.0055
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.0055
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.0055
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.055
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.055
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.0055
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.0055
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.055 ng/g PFAS in a carrot matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
0
100000
200000
300000
400000
500000
600000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Conc. (ng/g) %Accuracy
0.001 99.8
0.01 102
0.1 102
1 103
10 93.5
Conc. (ng/g) %Accuracy
0.001 100
0.01 98.3
0.1 91.7
1 96.0
10 114
Conc. (ng/g) %Accuracy
0.001 99.1
0.01 109
0.1 104
1 112
10 76.3
Conc. (ng/g) %Accuracy
0.001 99.4
0.01 107
0.1 91.8
1 92.1
10 110
Blank matrixes and at least three different concentrations
ranging from below the SMPR required LOQ to
approximately 500 times the estimated LOQ were analyzed
in triplicate. Recovery and repeatability for each analyte at
each concentration are given in Table 3. The LOQ for each
analyte was estimated by spiking at concentrations at, or
below, the required LOQs listed in SMPR-2023_003. The
spiked samples were analyzed in triplicate and the mean and
repeatability standard deviation were calculated. Then, the
standard deviation was divided by the mean and multiplied
by 100% to calculate the repeatability percent relative
standard deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank 0.000
0.055 0.047 1.74 2.03 85.5
0.55 0.504 0.40 0.44 91.7
5.5 5.485 0.32 0.32 99.7
PFPeA
Blank ND
0.0055 0.005 9.98 11.62 85.9
0.055 0.051 0.40 0.44 92.4
0.55 0.519 1.91 2.02 94.3
5.5 5.895 0.52 0.48 107.2
PFHxA
Blank 0.001
0.0055 0.005 9.51 11.41 83.4
0.055 0.057 3.03 2.94 103.0
0.55 0.533 1.36 1.40 96.9
5.5 6.089 0.92 0.83 110.7
PFHpA
Blank 0.000
0.0055 0.005 2.80 3.20 87.5
0.055 0.054 1.46 1.50 97.1
0.55 0.558 0.87 0.86 101.4
5.5 6.131 1.21 1.09 111.5
PFOA
Blank 0.001
0.0055 0.006 3.55 3.30 107.3
0.055 0.055 1.16 1.16 99.8
0.55 0.560 0.65 0.64 101.7
5.5 5.954 0.45 0.42 108.2
PFNA
Blank 0.001
0.0055 0.006 1.45 1.38 104.8
0.055 0.057 0.67 0.64 103.6
0.55 0.592 2.73 2.54 107.7
5.5 5.168 0.60 0.64 94.0
PFDA
Blank 0.000
0.0055 0.005 14.07 15.03 93.6
0.055 0.058 1.85 1.77 104.7
0.55 0.606 0.85 0.78 110.1
5.5 6.447 1.36 1.16 117.2
PFUnA
Blank 0.000
0.0055 0.004 4.53 5.65 80.3
0.055 0.052 1.79 1.89 94.8
0.55 0.548 2.22 2.23 99.6
5.5 6.023 0.49 0.45 109.5
PFDoA
Blank 0.000
0.0055 0.005 6.79 7.44 91.2
0.055 0.044 4.99 6.22 80.2
0.55 0.483 16.54 18.85 87.8
5.5 4.796 1.74 2.00 87.2
PFTrDA
Blank 0.000
0.0055 0.005 7.05 8.34 84.6
0.055 0.041 1.95 2.58 75.4
0.55 0.477 11.69 13.46 86.9
5.5 4.357 2.07 2.61 79.2
PFTeDA
Blank 0.001
0.0055 0.005 5.88 6.00 97.9
0.055 0.043 1.99 2.51 79.3
0.55 0.484 10.52 11.96 88.0
5.5 4.333 1.80 2.29 78.8
PFBS
Blank 0.003
0.055 0.050 2.00 2.21 90.6
0.55 0.520 0.56 0.59 94.5
5.5 5.669 0.06 0.06 103.1
PFPeS
Blank 0.000
0.0055 0.005 1.13 1.16 97.5
0.055 0.054 2.88 2.95 97.7
0.55 0.504 1.66 1.82 91.6
5.5 5.637 1.20 1.17 102.5
PFHxS
Blank 0.000
0.0055 0.005 6.82 7.21 94.5
0.055 0.050 1.82 1.98 91.8
0.55 0.514 1.02 1.09 93.5
5.5 5.182 10.95 11.62 94.2
PFHpS
Blank 0.002
0.0055 0.007 10.64 8.67 122.8
0.055 0.060 3.50 3.22 108.8
0.55 0.545 6.17 6.22 99.1
5.5 6.013 0.38 0.35 109.3
PFOS
Blank ND
0.0055 0.006 3.72 3.77 98.9
0.055 0.050 2.49 2.75 90.4
0.55 0.511 1.08 1.16 93.0
5.5 5.627 0.15 0.15 102.3
PFNS
Blank 0.000
0.0055 0.005 18.78 20.65 90.9
0.055 0.048 1.61 1.86 86.5
0.55 0.488 12.40 13.98 88.7
5.5 4.823 2.00 2.28 87.7
PFDS
Blank 0.000
0.0055 0.004 5.30 6.84 77.5
0.055 0.046 3.56 4.24 84.1
0.55 0.501 10.91 11.96 91.2
5.5 5.495 3.06 3.06 99.9
PFUnDS
Blank 0.001
0.0055 0.005 0.86 0.91 95.0
0.055 0.055 2.30 2.31 99.7
0.55 0.594 1.35 1.25 107.9
5.5 6.127 0.92 0.82 111.4
PFDoS
Blank 0.000
0.0055 0.006 18.94 16.15 117.3
0.055 0.064 1.96 1.68 117.0
0.55 0.638 13.06 11.26 116.0
5.5 6.836 1.31 1.05 124.3
PFTrDS
Blank 0.000
0.0055 0.005 1.42 1.45 97.9
0.055 0.051 0.89 0.95 93.2
0.55 0.529 7.67 7.97 96.2
5.5 5.722 4.76 4.58 104.0
PFOSA
Blank ND
0.0055 0.005 4.88 5.54 88.1
0.055 0.053 1.66 1.71 96.6
0.55 0.528 1.40 1.46 96.0
5.5 5.828 2.20 2.07 106.0
9Cl-PF3ONS
Blank 0.000
0.0055 0.006 3.20 2.93 109.1
0.055 0.059 1.42 1.33 106.2
0.55 0.604 2.48 2.26 109.8
5.5 6.603 0.15 0.13 120.0
11Cl-PF3OUdS
Blank 0.000
0.0055 0.005 0.95 0.97 97.4
0.055 0.050 1.57 1.73 91.0
0.55 0.530 11.47 11.91 96.4
5.5 6.030 2.40 2.19 109.6
HFPO-DA
Blank 0.000
0.0055 0.005 4.01 4.32 92.9
0.055 0.052 1.37 1.43 95.4
0.55 0.538 0.79 0.81 97.8
5.5 5.755 0.75 0.72 104.6
DONA
Blank 0.000
0.0055 0.006 5.41 5.26 102.8
0.055 0.056 0.30 0.30 101.5
0.55 0.590 0.87 0.81 107.3
5.5 5.373 0.51 0.53 97.7
4:2 FTS
Blank ND
0.0055 0.006 11.31 11.28 100.2
0.055 0.049 0.96 1.08 88.8
0.55 0.544 0.21 0.21 98.9
5.5 5.793 0.25 0.24 105.3
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a carrot matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
6:2 FTS
Blank 0.002
0.055 0.051 0.85 0.92 92.4
0.55 0.493 0.67 0.74 89.7
5.5 5.107 1.99 2.14 92.8
8:2 FTS
Blank 0.000
0.0055 0.006 3.26 2.78 117.2
0.055 0.057 3.83 3.68 104.0
0.55 0.649 7.42 6.29 118.1
5.5 6.487 2.75 2.33 117.9
10:2 FTS
Blank 0.000
0.0055 0.005 3.15 3.39 93.0
0.055 0.050 0.86 0.95 90.4
0.55 0.512 11.03 11.85 93.1
5.5 4.647 2.25 2.66 84.5
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LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-171 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Offal meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Offal by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in beef kidney
using Acetonitrile and Solid Phase Extraction (SPE)
followed by analysis using the Shimadzu Nexera Liquid
Chromatograph coupled to a Shimadzu LCMS-8060NX
triple quadrupole mass spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at five concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.2
Perfluoropentanoic acid PFPeA 2706-90-3 0.2
Perfluorohexanoic acid PFHxA 307-24-4 0.2
Perfluoroheptanoic acid PFHpA 375-85-9 0.2
Perfluorooctanoic acid PFOA 335-67-1 0.2
Perfluorononanoic acid PFNA 375-95-1 0.2
Perfluorodecanoic acid PFDA 335-76-2 0.2
Perfluoroundecanoic acid PFUnA 2058-94-8 0.2
Perfluorododecanoic acid PFDoA 307-55-1 0.2
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Dominika Gruszecka1, Eishi Imoto1, Toshiya Matsubara1
■ Sample Preparation and Analysis Conditions
Samples were prepared by cutting locally purchased beef
kidney into slices, freezing and grinding with dry ice. The
ground material was placed in a freezer overnight. Test
portions were spiked in triplicate at five different
concentrations with 30 native PFAS (Table 1) and 16
isotopically labeled internal standards. Calibration curves for
use in the quantitative analysis were prepared using 10-
gram kidney test portions spiked with concentrations of 0.1,
0.5, 1.5, 5.0 and 15 ng/g. Quantitation was carried out on
additional kidney samples spiked in triplicate at 0.2, 0.4, 1, 4,
and 10 ng/g. Since standards were extracted in a kidney
matrix and carried through the same procedure, the final
concentration of each PFAS in the sample can be calculated
directly from the curve.
10-gram test portions were weighed, spiked with target
analytes and internal standards, and 10 mL of acetonitrile
was added. The samples were vortexed for 1 minute and
then centrifuged for 5 minutes at 4000 rpm. An aliquot of
the acetonitrile layer was transferred to a tube and diluted 5
times with PFAS-free reagent water. The sample was then
passed through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with a
basic methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.2
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.2
Perfluorobutanesulfonic acid PFBS 375-73-5 0.2
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.2
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.2
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.2
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.2
Perfluorononanesulfonic acid PFNS 68259-12-1 0.2
Perfluorodecanesulfonic acid PFDS 335-77-3 0.2
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.2
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.2
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.2
Perfluorooctanesulfonamide PFOSA 754-91-6 0.2
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.2
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.2
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.2
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.2
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.2
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.2
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.2
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.2
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.4 ng/g PFAS in a beef kidney matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
0
50000
100000
150000
200000
250000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and five different concentrations ranging from
below the SMPR required LOQ to 25 times the estimated
LOQ were analyzed in triplicate. Recovery and repeatability
for each analyte at each concentration are given in Table 3.
The LOQ for each analyte was estimated by spiking at
concentrations at, or below, the required LOQs listed in
SMPR-2023_003. The spiked samples were analyzed in
triplicate and the mean and repeatability standard deviation
were calculated. Then, the standard deviation was divided by
the mean and multiplied by 100% to calculate the
repeatability percent relative standard deviation (RSD).
The LOQs for all compounds were determined using an
Excel worksheet that compared each of the requirements
of the SMPR including retention time, recovery,
repeatability, S/N > 3 for the qualifier ion and an ion ratio
of ±30%. PFBA, PFPeA, and PFOSA LOQ were set at the
minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.1 101
0.5 93.0
1.5 99.9
5 95.5
15 110
Conc. (ng/g) %Accuracy
0.1 99.3
0.5 104
1.5 99.0
5 93.6
15 104
Conc. (ng/g) %Accuracy
0.1 99.8
0.5 102
1.5 95.1
5 104
15 98.3
Conc. (ng/g) %Accuracy
0.1 99.5
0.5 104
1.5 97.3
5 97.2
15 102
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank 0.002
0.2 0.212 3.23 3.04 106.3
0.4 0.442 1.78 1.61 110.5
1.0 1.131 5.12 4.52 113.1
4.0 4.102 2.55 2.49 102.5
10 10.121 3.84 3.80 101.2
PFPeA
Blank 0.010
0.2 0.213 3.58 3.35 106.7
0.4 0.434 1.53 1.41 108.5
1.0 1.068 4.36 4.08 106.8
4.0 3.908 2.66 2.72 97.7
10 9.583 2.29 2.38 95.9
PFHxA
Blank 0.003
0.2 0.216 1.08 1.00 107.9
0.4 0.454 4.87 4.29 113.5
1.0 1.195 6.03 5.04 119.5
4.0 4.441 3.84 3.46 111.0
10 10.797 4.18 3.87 108.0
PFHpA
Blank 0.007
0.2 0.208 4.47 4.30 103.8
0.4 0.412 0.46 0.45 102.9
1.0 1.014 5.75 5.67 101.4
4.0 3.690 1.90 2.06 92.3
10 9.120 3.12 3.42 91.2
PFOA
Blank 0.012
0.2 0.221 3.89 3.52 110.7
0.4 0.453 2.65 2.34 113.3
1.0 1.174 6.21 5.29 117.4
4.0 4.331 2.69 2.48 108.3
10 10.503 3.84 3.65 105.0
PFNA
Blank 0.007
0.2 0.205 3.75 3.67 102.1
0.4 0.435 2.77 2.55 108.9
1.0 1.139 7.18 6.30 113.9
4.0 4.204 4.53 4.31 105.1
10 10.421 2.91 2.79 104.2
PFDA
Blank 0.020
0.2 0.211 3.42 3.25 105.3
0.4 0.433 0.55 0.51 108.1
1.0 1.053 8.41 7.98 105.3
4.0 3.936 2.52 2.56 98.4
10 9.547 3.61 3.78 95.5
PFUnA
Blank 0.011
0.2 0.213 1.89 1.77 106.4
0.4 0.433 4.47 4.12 108.3
1.0 1.094 4.58 4.18 109.4
4.0 4.070 2.89 2.84 101.7
10 9.848 4.64 4.71 98.5
PFDoA
Blank 0.016
0.2 0.226 3.26 2.88 113.2
0.4 0.462 3.03 2.62 115.5
1.0 1.124 2.03 1.80 112.4
4.0 4.365 2.26 2.07 109.1
10 10.375 3.51 3.38 103.7
PFTrDA
Blank 0.001
0.2 0.214 2.86 2.66 107.3
0.4 0.433 3.99 3.69 108.2
1.0 0.973 7.74 7.95 97.3
4.0 3.927 1.39 1.41 98.2
10 9.297 2.55 2.74 92.9
PFTeDA
Blank 0.008
0.2 0.205 2.66 2.60 102.5
0.4 0.427 0.99 0.92 106.8
1.0 0.990 4.18 4.22 99.0
4.0 3.880 0.98 1.02 97.0
10 9.041 0.56 0.62 90.4
PFBS
Blank 0.011
0.2 0.194 11.88 12.27 96.8
0.4 0.415 9.99 9.64 103.6
1.0 1.035 4.94 4.78 103.5
4.0 3.824 0.17 0.18 95.6
10 9.594 7.39 7.71 95.9
PFPeS
Blank 0.025
0.2 0.225 6.70 5.95 112.8
0.4 0.455 2.64 2.31 113.9
1.0 1.042 3.84 3.69 104.2
4.0 4.009 0.78 0.78 100.2
10 9.704 3.97 4.09 97.0
PFHxS
Blank 0.026
0.2 0.203 6.37 6.29 101.3
0.4 0.383 1.65 1.72 95.7
1.0 0.962 5.37 5.58 96.2
4.0 3.953 4.07 4.12 98.8
10 9.357 3.12 3.34 93.6
PFHpS
Blank 0.025
0.2 0.216 6.23 5.77 107.9
0.4 0.456 5.34 4.68 114.0
1.0 1.110 7.64 6.88 111.0
4.0 4.110 2.21 2.15 102.8
10 9.886 4.35 4.40 98.9
PFOS
Blank 0.027
0.2 0.212 1.90 1.80 105.9
0.4 0.429 1.97 1.84 107.1
1.0 1.092 4.66 4.27 109.2
4.0 4.071 5.24 5.15 101.8
10 10.156 7.04 6.94 101.6
PFNS
Blank 0.015
0.2 0.223 2.50 2.24 111.4
0.4 0.437 4.84 4.43 109.4
1.0 1.142 7.23 6.33 114.2
4.0 4.119 1.61 1.56 103.0
10 9.991 5.09 5.09 99.9
PFDS
Blank ND
0.2 0.214 6.66 6.23 106.8
0.4 0.444 7.66 6.91 110.9
1.0 1.122 11.22 10.00 112.2
4.0 4.189 0.80 0.77 104.7
10 9.983 4.23 4.24 99.8
PFUnDS
Blank 0.030
0.2 0.239 9.94 8.31 119.6
0.4 0.438 3.92 3.59 109.5
1.0 1.005 5.98 5.95 100.5
4.0 4.038 3.74 3.71 101.0
10 9.503 5.35 5.63 95.1
PFDoS
Blank 0.016
0.2 0.232 4.32 3.73 115.9
0.4 0.459 2.71 2.36 114.7
1.0 1.081 0.83 0.77 108.1
4.0 4.281 3.52 3.29 107.0
10 9.839 0.96 0.98 98.4
PFTrDS
Blank 0.009
0.2 0.222 5.77 5.20 111.0
0.4 0.475 12.42 10.46 118.7
1.0 1.088 2.72 2.50 108.8
4.0 4.264 4.76 4.47 106.6
10 9.680 2.69 2.78 96.8
PFOSA
Blank ND
0.2 0.232 24.78 21.34 116.1
0.4 0.419 13.60 12.98 104.7
1.0 1.110 6.80 6.12 111.0
4.0 4.157 5.28 5.09 103.9
10 10.422 6.27 6.02 104.2
9Cl-PF3ONS
Blank 0.016
0.2 0.202 2.37 2.34 101.1
0.4 0.428 5.16 4.83 106.9
1.0 1.143 4.80 4.20 114.3
4.0 4.194 2.87 2.74 104.9
10 10.461 6.84 6.54 104.6
11Cl-PF3OUdS
Blank 0.008
0.2 0.210 2.53 2.41 104.9
0.4 0.458 8.34 7.29 114.5
1.0 1.123 7.60 6.77 112.3
4.0 4.352 2.84 2.61 108.8
10 10.038 3.95 3.94 100.4
ND = average results less than zero
HFPO-DA
Blank 0.016
0.2 0.207 3.86 3.72 103.6
0.4 0.418 4.55 4.36 104.5
1.0 1.084 6.85 6.32 108.4
4.0 3.938 4.03 4.09 98.4
10 9.601 3.20 3.34 96.0
DONA
Blank 0.011
0.2 0.211 1.14 1.08 105.2
0.4 0.428 0.55 0.51 107.0
1.0 1.042 6.69 6.43 104.2
4.0 3.827 1.91 2.00 95.7
10 9.590 2.75 2.86 95.9
4:2 FTS
Blank 0.016
0.2 0.213 5.91 5.54 106.7
0.4 0.422 6.26 5.92 105.6
1.0 1.056 6.91 6.54 105.6
4.0 3.881 1.99 2.05 97.0
10 9.579 3.63 3.79 95.8
6:2 FTS
Blank 0.028
0.2 0.218 15.62 14.33 109.1
0.4 0.431 2.45 2.27 107.7
1.0 1.149 5.08 4.42 114.9
4.0 4.304 3.14 2.92 107.6
10 10.209 6.68 6.55 102.1
8:2 FTS
Blank 0.022
0.2 0.164 2.83 3.46 81.8
0.4 0.340 4.56 5.36 85.0
1.0 0.952 1.18 1.24 95.2
4.0 3.974 9.63 9.70 99.3
10 9.327 3.07 3.29 93.3
10:2 FTS
Blank 0.016
0.2 0.217 9.19 8.47 108.5
0.4 0.489 14.16 11.58 122.4
1.0 1.251 8.26 6.61 125.1
4.0 4.956 4.67 3.77 123.9
10 11.777 6.18 5.25 117.8
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in an offal matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
SHIMADZU Scientific Instruments Inc.
www.ssi.shimadzu.com
Shimadzu Corporation
www.shimadzu.com/an/
Founded in 1875, Shimadzu Corporation, a leader in the
development of advanced technologies, has a distinguished
history of innovation built on the foundation of contributing to
society through science and technology. Established in 1975,
Shimadzu Scientific Instruments (SSI), the American subsidiary of
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For information about Shimadzu Scientific Instruments and to
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www.ssi.shimadzu.com
LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
For Research Use Only. Not for use in diagnostic procedures.
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to change without notice.
LCMS-8045RX LCMS-2050
02-SSI-LCMS-169 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Milk meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Milk by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in milk using
the QuEChERS technique followed by analysis using the
Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at three concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.01
Perfluoropentanoic acid PFPeA 2706-90-3 0.01
Perfluorohexanoic acid PFHxA 307-24-4 0.01
Perfluoroheptanoic acid PFHpA 375-85-9 0.01
Perfluorooctanoic acid PFOA 335-67-1 0.01
Perfluorononanoic acid PFNA 375-95-1 0.01
Perfluorodecanoic acid PFDA 335-76-2 0.01
Perfluoroundecanoic acid PFUnA 2058-94-8 0.01
Perfluorododecanoic acid PFDoA 307-55-1 0.01
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc., 2 Shimadzu Corporation
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1, Nozomi Maeshima2, Yui Higashi2,
Manami Kobayashi2
■ Sample Preparation and Analysis Conditions
Whole milk (3.7 % fat) was purchased and sampled directly
from the carton. Test portions were spiked in triplicate at
three different concentrations with 30 native PFAS (Table 1)
and 16 isotopically labeled internal standards. Calibration
curves for use in the quantitative analysis were prepared
using 10- gram test portions spiked with 0.005, 0.01, 0.05,
0.10, 0.50, and 1.0 ng/g of each target analyte. Quantitation
was carried out on additional whole milk samples spiked in
triplicate at 0.01, 0.10 and 1.0 ng/g. Since standards were
extracted in milk matrix and carried through the same
procedure, the final concentration of each PFAS in the
sample can be calculated directly from the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 150 μL of formic acid and 10 mL
of acetonitrile was added. The samples were shaken by hand
for 10 seconds and a QuEChERS packet was added. The
sample was shaken again for 5 minutes and then centrifuged
for 5 minutes at 4000 rpm. An aliquot of the acetonitrile
layer was transferred to a tube to which additional
QuEChERS reagent was added. The sample was shaken for
10 seconds by hand, for 5 minutes on a shaker, and then
centrifuged for 5 minutes at 4000 rpm. An aliquot was
removed to another tube and concentrated to less than 1 ml
under nitrogen, reconstituted to 1 ml in a methanol-water
mixture, transferred to a 1.5 mL tube and centrifuged for 10
minutes at 15,000 rpm. The supernatant was transferred to
an LC vial for analysis.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.01
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.01
Perfluorobutanesulfonic acid PFBS 375-73-5 0.01
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.01
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.01
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.01
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.01
Perfluorononanesulfonic acid PFNS 68259-12-1 0.01
Perfluorodecanesulfonic acid PFDS 335-77-3 0.01
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.01
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.01
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.01
Perfluorooctanesulfonamide PFOSA 754-91-6 0.01
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.01
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.01
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.01
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.01
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.01
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.01
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.01
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.01
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.1 ng/g PFAS in a milk matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
0
250000
500000
750000
1000000
1250000
1500000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and three different concentrations ranging
from the SMPR required LOQ to 100 times the estimated
LOQ were analyzed in triplicate. Recovery and repeatability
for each analyte at each concentration are given in Table 3.
The LOQ for each analyte was estimated by spiking at
concentrations at, or below, the required LOQs listed in
SMPR-2023_003. The spiked samples were analyzed in
triplicate and the mean and repeatability standard deviation
were calculated. Then, the standard deviation was divided by
the mean and multiplied by 100% to calculate the
repeatability percent relative standard deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.005 97.1
0.01 104
0.05 109
0.10 99.9
0.50 96.5
1.0 93.6
Conc. (ng/g) %Accuracy
0.005 93.8
0.01 111
0.05 111
0.10 98.8
0.50 94.4
1.0 91.5
Conc. (ng/g) %Accuracy
0.005 96.6
0.01 105
0.05 109
0.10 97.8
0.50 95.2
1.0 95.8
Conc. (ng/g) %Accuracy
0.005 98.5
0.01 101
0.05 114
0.10 96.7
0.50 98.3
1.0 91.7
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank 0.000
0.01 0.012 8.20 7.02 116.8
0.1 0.103 2.10 2.02 103.5
1 0.973 0.57 0.58 97.3
PFPeA
Blank 0.000
0.01 0.011 2.63 2.45 107.1
0.1 0.099 0.66 0.66 99.3
1 0.947 0.38 0.40 94.7
PFHxA
Blank ND
0.01 0.010 1.74 1.67 104.4
0.1 0.096 0.93 0.97 96.1
1 0.947 0.10 0.11 94.7
PFHpA
Blank 0.001
0.01 0.011 7.89 6.79 116.2
0.1 0.099 0.45 0.45 99.4
1 0.968 1.50 1.55 96.8
PFOA
Blank ND
0.01 0.010 11.84 11.35 104.3
0.1 0.098 2.89 2.93 98.5
1 0.977 1.00 1.02 97.7
PFNA
Blank 0.000
0.01 0.011 0.96 0.86 112.2
0.1 0.100 0.92 0.93 99.5
1 0.976 0.61 0.62 97.6
PFDA
Blank 0.001
0.01 0.011 2.91 2.59 112.7
0.1 0.104 0.42 0.40 104.2
1 0.994 2.71 2.73 99.4
PFUnA
Blank 0.000
0.01 0.011 1.20 1.08 111.5
0.1 0.101 0.84 0.83 100.9
1 0.980 0.72 0.74 98.0
PFDoA
Blank 0.000
0.01 0.012 13.65 11.65 117.1
0.1 0.101 1.54 1.52 101.3
1 0.951 1.47 1.55 95.1
PFTrDA
Blank 0.000
0.01 0.011 2.40 2.24 106.9
0.1 0.101 1.60 1.59 100.9
1 0.994 0.68 0.68 99.4
PFTeDA
Blank 0.000
0.01 0.011 4.14 3.80 108.8
0.1 0.098 0.67 0.68 98.2
1 0.974 3.40 3.49 97.4
PFBS
Blank ND
0.01 0.012 8.49 7.19 118.1
0.1 0.102 1.65 1.62 101.6
1 0.958 0.25 0.26 95.8
PFPeS
Blank ND
0.01 0.010 11.26 11.41 98.7
0.1 0.102 1.05 1.03 101.9
1 0.953 1.62 1.70 95.3
PFHxS
Blank 0.000
0.01 0.010 10.50 10.76 97.6
0.1 0.099 3.88 3.89 99.6
1 0.956 1.36 1.42 95.6
PFHpS
Blank ND
0.01 0.010 7.26 6.85 106.0
0.1 0.105 2.27 2.17 104.4
1 0.983 2.10 2.14 98.3
ND = average results less than zero
PFOS
Blank ND
0.01 0.010 6.44 6.54 98.4
0.1 0.099 2.57 2.59 99.2
1 0.961 1.32 1.37 96.1
PFNS
Blank ND
0.01 0.010 10.24 10.20 100.4
0.1 0.102 2.86 2.81 101.8
1 0.992 2.58 2.60 99.2
PFDS
Blank ND
0.01 0.009 3.74 3.95 94.8
0.1 0.103 6.13 5.95 103.0
1 1.015 1.22 1.20 101.5
PFUnDS
Blank ND
0.01 0.011 2.19 2.01 109.3
0.1 0.105 4.78 4.56 105.0
1 0.988 3.49 3.54 98.8
PFDoS
Blank ND
0.01 0.010 8.88 9.15 97.0
0.1 0.101 2.24 2.22 100.6
1 0.963 2.27 2.36 96.3
PFTrDS
Blank ND
0.01 0.010 5.12 4.83 106.0
0.1 0.099 3.30 3.32 99.4
1 0.990 2.08 2.10 99.0
PFOSA
Blank ND
0.01 0.011 3.59 3.36 106.8
0.1 0.099 1.83 1.85 99.0
1 0.950 0.55 0.58 95.0
9Cl-PF3ONS
Blank 0.000
0.01 0.010 4.42 4.31 102.5
0.1 0.103 0.96 0.94 102.5
1 0.971 3.15 3.25 97.1
11Cl-PF3OUdS
Blank ND
0.01 0.009 5.32 5.61 94.8
0.1 0.101 1.10 1.09 100.8
1 0.992 1.22 1.23 99.2
HFPO-DA
Blank ND
0.01 0.010 7.16 7.19 99.6
0.1 0.099 2.17 2.18 99.3
1 0.932 0.80 0.86 93.2
DONA
Blank 0.000
0.01 0.010 0.80 0.77 103.9
0.1 0.099 0.42 0.42 98.9
1 0.961 2.12 2.21 96.1
4:2 FTS
Blank ND
0.01 0.011 4.97 4.63 107.5
0.1 0.102 2.75 2.69 102.1
1 0.989 1.28 1.29 98.9
6:2 FTS
Blank 0.001
0.01 0.012 11.91 10.39 114.6
0.1 0.106 5.06 4.80 105.5
1 0.984 1.40 1.43 98.4
8:2 FTS
Blank 0.001
0.01 0.012 8.01 6.86 116.7
0.1 0.097 1.53 1.57 97.1
1 0.888 0.23 0.26 88.8
10:2 FTS
Blank 0.000
0.01 0.011 10.94 10.09 108.4
0.1 0.095 3.27 3.43 95.4
1 0.862 1.22 1.42 86.2
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a high fat milk matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-168 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in fish oil meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Fish Oil by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in fish oil
followed by analysis using the Shimadzu Nexera Liquid
Chromatograph coupled to a Shimadzu LCMS-8060NX
triple quadrupole mass spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at five concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.25
Perfluoropentanoic acid PFPeA 2706-90-3 0.25
Perfluorohexanoic acid PFHxA 307-24-4 0.25
Perfluoroheptanoic acid PFHpA 375-85-9 0.25
Perfluorooctanoic acid PFOA 335-67-1 0.25
Perfluorononanoic acid PFNA 375-95-1 0.25
Perfluorodecanoic acid PFDA 335-76-2 0.25
Perfluoroundecanoic acid PFUnA 2058-94-8 0.25
Perfluorododecanoic acid PFDoA 307-55-1 0.25
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Dominika Gruszecka1, Eishi Imoto1, Toshiya Matsubara1
■ Sample Preparation and Analysis Conditions
Fish oil soft gels were purchased locally. Samples were
prepared by cutting the tip of the capsule with scissors and
squeezing the oil out into a 50 ml tube. Test portions were
spiked in triplicate at five different concentrations with 30
native PFAS (Table 1) and 16 isotopically labeled internal
standards. Calibration curves for use in the quantitative
analysis were prepared using 10- gram fish oil test portions
spiked with concentrations of 0.1, 0.5, 1.5, 5.0 and 15.0
ng/g. Quantitation was carried out on additional fish oil
samples spiked in triplicate at 0.25, 0.5, 1.0, 5.0, and 10
ng/g. Since standards were extracted in a fish oil matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 10 mL of acetonitrile was added.
The samples were vortexed for 1 minute and then
centrifuged for 5 minutes at 4000 rpm. An aliquot of the
acetonitrile layer was transferred to a tube and diluted 5
times with PFAS-free reagent water. The sample was then
passed through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with
basic methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.25
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.25
Perfluorobutanesulfonic acid PFBS 375-73-5 0.25
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.25
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.25
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.25
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.25
Perfluorononanesulfonic acid PFNS 68259-12-1 0.25
Perfluorodecanesulfonic acid PFDS 335-77-3 0.25
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.25
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.25
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.25
Perfluorooctanesulfonamide PFOSA 754-91-6 0.25
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.25
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.25
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.25
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.25
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.25
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.25
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.25
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.5
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.5 ng/g PFAS in a fish oil matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
0
50000
100000
150000
200000
250000
300000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Conc. (ng/g) %Accuracy
0.1 99.5
0.5 104
1.5 95.8
5 98.6
15 102
Blank matrixes and five different concentrations ranging from
below the SMPR required LOQ to 20 times the estimated
LOQ were analyzed in triplicate. Recovery and repeatability
for each analyte at each concentration are given in Table 3.
The LOQ for each analyte was estimated by spiking at
concentrations at, or below, the required LOQs listed in
SMPR-2023_003. The spiked samples were analyzed in
triplicate and the mean and repeatability standard deviation
were calculated. Then, the standard deviation was divided by
the mean and multiplied by 100% to calculate the
repeatability percent relative standard deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.1 99.4
0.5 105
1.5 92.8
5 99.8
15 103
Conc. (ng/g) %Accuracy
0.1 101
0.5 99.0
1.5 94.3
5 99.1
15 107
Conc. (ng/g) %Accuracy
0.1 100
0.5 102
1.5 92.5
5 104
15 102
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank ND
0.25 0.254 6.17 6.07 101.7
0.5 0.434 7.21 8.31 86.8
1.0 1.017 3.90 3.83 101.7
5.0 5.338 7.41 6.94 106.8
10 10.414 3.17 3.04 104.1
PFPeA
Blank ND
0.25 0.249 2.87 2.88 99.7
0.5 0.447 5.37 6.00 89.5
1.0 1.025 2.20 2.15 102.5
5.0 5.247 8.12 7.74 104.9
10 10.238 2.98 2.91 102.4
PFHxA
Blank 0.008
0.25 0.264 4.76 4.50 105.6
0.5 0.483 4.25 4.40 96.6
1.0 1.103 1.17 1.06 110.3
5.0 5.537 6.70 6.05 110.7
10 10.836 5.22 4.81 108.4
PFHpA
Blank ND
0.25 0.266 3.07 2.87 106.7
0.5 0.482 4.35 4.51 96.4
1.0 1.089 2.63 2.42 108.9
5.0 5.556 8.68 7.81 111.1
10 10.898 4.01 3.68 109.0
PFOA
Blank 0.009
0.25 0.270 3.15 2.92 108.0
0.5 0.470 2.78 2.96 94.0
1.0 1.022 1.14 1.11 102.2
5.0 5.292 5.17 4.88 105.8
10 10.508 1.59 1.51 105.0
PFNA
Blank ND
0.25 0.261 1.91 1.83 104.3
0.5 0.473 2.11 2.23 94.7
1.0 1.054 2.91 2.76 105.4
5.0 5.453 3.67 3.36 109.0
10 10.879 1.39 1.27 108.8
PFDA
Blank 0.006
0.25 0.265 6.96 6.57 105.9
0.5 0.491 3.98 4.05 98.2
1.0 1.054 0.81 0.77 105.4
5.0 5.568 3.46 3.11 111.4
10 10.804 1.37 1.26 108.1
PFUnA
Blank 0.010
0.25 0.259 6.56 6.34 103.4
0.5 0.529 4.65 4.39 105.9
1.0 1.040 3.08 2.96 104.0
5.0 5.453 4.99 4.58 109.0
10 11.149 1.21 1.08 111.5
PFDoA
Blank 0.011
0.25 0.252 6.11 6.06 100.8
0.5 0.552 2.93 2.65 110.5
1.0 1.076 7.91 7.35 107.6
5.0 5.845 4.88 4.17 116.9
10 11.709 1.20 1.02 117.1
PFTrDA
Blank 0.019
0.25 0.235 18.57 19.79 93.8
0.5 0.544 9.18 8.43 108.9
1.0 1.064 1.30 1.22 106.4
5.0 5.459 4.67 4.27 109.2
10 10.772 1.80 1.67 107.8
PFTeDA
Blank 0.028
0.25 0.202 19.65 24.26 81.0
0.5 0.472 2.76 2.93 94.3
1.0 0.994 7.50 7.54 99.4
5.0 5.695 7.96 6.99 113.9
10 11.144 2.97 2.67 111.4
PFBS
Blank ND
0.25 0.240 3.96 4.13 96.0
0.5 0.467 6.35 6.79 93.5
1.0 0.920 0.46 0.50 92.0
5.0 4.573 4.12 4.51 91.4
10 9.796 2.10 2.15 98.0
PFPeS
Blank ND
0.25 0.276 7.43 6.72 110.5
0.5 0.471 3.21 3.41 94.1
1.0 1.079 4.42 4.10 107.9
5.0 5.282 2.68 2.53 105.6
10 11.338 4.10 3.62 113.4
PFHxS
Blank 0.013
0.25 0.262 4.08 3.89 104.9
0.5 0.482 1.97 2.04 96.4
1.0 0.941 1.99 2.12 94.1
5.0 4.740 4.58 4.83 94.8
10 9.739 0.49 0.51 97.4
PFHpS
Blank ND
0.25 0.266 3.21 3.02 106.3
0.5 0.464 5.18 5.59 92.8
1.0 1.066 2.45 2.30 106.6
5.0 5.037 0.35 0.35 100.7
10 11.747 3.06 2.61 117.5
PFOS
Blank 0.008
0.25 0.242 5.29 5.46 96.8
0.5 0.464 3.63 3.90 92.9
1.0 0.922 3.99 4.33 92.2
5.0 4.614 4.50 4.88 92.3
10 9.836 2.56 2.60 98.3
PFNS
Blank ND
0.25 0.256 5.54 5.41 102.4
0.5 0.488 4.03 4.14 97.5
1.0 1.011 3.78 3.74 101.1
5.0 4.918 0.85 0.86 98.4
10 11.269 1.63 1.44 112.7
PFDS
Blank 0.004
0.25 0.261 2.69 2.58 104.2
0.5 0.480 7.88 8.21 96.0
1.0 0.986 3.78 3.84 98.6
5.0 4.884 2.71 2.77 97.7
10 11.074 2.04 1.84 110.8
PFUnDS
Blank 0.038
0.25 0.229 11.65 12.74 91.4
0.5 0.471 3.69 3.92 94.2
1.0 0.936 5.08 5.43 93.6
5.0 4.868 5.07 5.21 97.3
10 9.633 2.03 2.10 96.3
PFDoS
Blank 0.014
0.25 0.240 17.49 18.23 95.9
0.5 0.463 3.94 4.26 92.5
1.0 1.008 8.40 8.34 100.8
5.0 5.083 3.24 3.19 101.7
10 10.273 3.84 3.74 102.7
PFTrDS
Blank 0.013
0.25 0.217 10.40 11.99 86.8
0.5 0.449 2.72 3.03 89.9
1.0 1.017 8.78 8.64 101.7
5.0 5.471 5.86 5.36 109.4
10 11.963 3.60 3.01 119.6
PFOSA
Blank ND
0.25 0.254 5.16 5.08 101.7
0.5 0.497 7.54 7.59 99.4
1.0 0.998 6.46 6.48 99.8
5.0 5.054 3.82 3.78 101.1
10 10.768 3.10 2.88 107.7
9Cl-PF3ONS
Blank 0.007
0.25 0.261 4.09 3.91 104.5
0.5 0.500 1.76 1.76 100.1
1.0 0.976 3.38 3.46 97.6
5.0 4.989 5.16 5.17 99.8
10 10.804 1.33 1.23 108.0
11Cl-PF3OUdS
Blank 0.004
0.25 0.276 0.75 0.68 110.5
0.5 0.496 3.36 3.39 99.3
1.0 1.036 9.19 8.87 103.6
5.0 5.483 4.11 3.75 109.7
10 12.079 4.51 3.73 120.8
HFPO-DA
Blank 0.010
0.25 0.251 4.55 4.53 100.4
0.5 0.469 7.34 7.81 93.9
1.0 0.876 3.07 3.50 87.6
5.0 4.538 4.21 4.63 90.8
10 9.461 3.98 4.21 94.6
DONA
Blank ND
0.25 0.244 3.92 4.01 97.7
0.5 0.466 4.58 4.92 93.1
1.0 1.026 1.17 1.14 102.6
5.0 5.390 9.33 8.65 107.8
10 10.210 3.70 3.62 102.1
4:2 FTS
Blank ND
0.25 0.250 10.10 10.10 99.9
0.5 0.480 2.93 3.05 96.0
1.0 1.107 7.81 7.05 110.7
5.0 5.443 9.61 8.83 108.9
10 10.788 7.37 6.83 107.9
6:2 FTS
Blank ND
0.25 0.267 12.51 11.70 106.9
0.5 0.511 13.05 12.78 102.1
1.0 1.079 9.92 9.19 107.9
5.0 5.342 8.95 8.38 106.8
10 10.826 9.54 8.81 108.3
8:2 FTS
Blank 0.009
0.25 0.225 9.23 10.25 90.1
0.5 0.483 0.38 0.39 96.6
1.0 1.016 0.85 0.84 101.6
5.0 5.945 10.53 8.86 118.9
10 10.140 3.49 3.44 101.4
10:2 FTS
Blank 0.003
0.5 0.478 10.34 10.82 95.6
1.0 1.060 8.61 8.13 106.0
5.0 5.939 9.65 8.13 118.8
10 11.502 2.70 2.35 115.0
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a fish oil matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
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LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-166 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in Coffee meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, with SPE cleanup
◆ Simple and rapid extraction
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Coffee by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in brewed
coffee followed by analysis using the Shimadzu Nexera
Liquid Chromatograph coupled to a Shimadzu LCMS-
8060NX triple quadrupole mass spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at four concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.055
Perfluoropentanoic acid PFPeA 2706-90-3 0.0055
Perfluorohexanoic acid PFHxA 307-24-4 0.055
Perfluoroheptanoic acid PFHpA 375-85-9 0.0055
Perfluorooctanoic acid PFOA 335-67-1 0.0055
Perfluorononanoic acid PFNA 375-95-1 0.0055
Perfluorodecanoic acid PFDA 335-76-2 0.055
Perfluoroundecanoic acid PFUnA 2058-94-8 0.0055
Perfluorododecanoic acid PFDoA 307-55-1 0.055
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Dominika Gruszecka1, Toshiya Matsubara1
■ Sample Preparation and Analysis Conditions
Organic brewed coffee was purchased locally. Samples were
prepared by shaking the bottle and removing aliquots directly
from the original container. Test portions were spiked in
triplicate at four different concentrations with 30 native PFAS
(Table 1) and 16 isotopically labeled internal standards.
Calibration curves for use in the quantitative analysis were
prepared using 10- gram test portions spiked with
concentrations of 0.001, 0.01, 0.1, 1.0 and 10.0 ng/g.
Quantitation was carried out on additional samples spiked in
triplicate at 0.0055, 0.055, 0.55 and 5.5 ng/g. Since
standards were extracted in a brewed coffee matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
10-gram portions were weighed, spiked with target analytes
and internal standards, and 10 mL of acetonitrile was added.
The sample was shaken for 1 minute and then centrifuged
for 5 minutes at 4000 rpm. An aliquot of the acetonitrile
layer was transferred to a tube and diluted 5 times with
PFAS-free reagent water. The sample was then passed
through a weak anion exchange (WAX) Solid Phase
Extraction (SPE) cartridge and the PFAS were eluted with
basic methanol. For greater sensitivity, the extract was
concentrated to dryness and dissolved in 0.4 mL of a
methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.055
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.055
Perfluorobutanesulfonic acid PFBS 375-73-5 0.0055
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.0055
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.055
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.055
Perfluorononanesulfonic acid PFNS 68259-12-1 0.055
Perfluorodecanesulfonic acid PFDS 335-77-3 0.055
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.055
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.055
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.55
Perfluorooctanesulfonamide PFOSA 754-91-6 0.55
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.055
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.055
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.055
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.0055
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.55
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.055
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C3-PFBS
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C2-PFDoA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.55 ng/g PFAS in a coffee matrix with separation of all peaks in nine minutes
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
0
250000
500000
750000
1000000
1250000
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Blank matrixes and at four different concentrations ranging
from below the SMPR required LOQ to approximately 20
times the estimated LOQ were analyzed in triplicate.
Recovery and repeatability for each analyte at each
concentration are given in Table 3. The LOQ for each analyte
was estimated by spiking at concentrations at, or below, the
required LOQs listed in SMPR-2023_003. The spiked samples
were analyzed in triplicate and the mean and repeatability
standard deviation were calculated. Then, the standard
deviation was divided by the mean and multiplied by 100%
to calculate the repeatability percent relative standard
deviation (RSD).
The LOQs for all compounds were determined using an
Excel worksheet that compared each of the requirements
of the SMPR including retention time, recovery,
repeatability, S/N > 3 for the qualifier ion and an ion ratio
of ±30%. PFBA, PFPeA, and PFOSA LOQ were set at the
minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Conc. (ng/g) %Accuracy
0.001 101
0.01 89.5
0.1 98.6
1 98.8
10 112
Conc. (ng/g) %Accuracy
0.001 102
0.01 84.8
0.1 95.8
1 102
10 116
Conc. (ng/g) %Accuracy
0.001 100
0.01 100
0.1 97.1
1 96.0
10 107
Conc. (ng/g) %Accuracy
0.001 99.4
0.01 107
0.1 93.0
1 93.9
10 107
Table 3: Recovery and repeatability for each analyte at each spike concentration
Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery%
PFBA
Blank ND
0.055 0.049 4.19 4.75 88.1
0.55 0.526 2.00 2.09 95.8
5.5 5.553 0.31 0.30 101.0
PFPeA
Blank 0.001
0.0055 0.005 8.23 8.41 97.9
0.055 0.051 3.42 3.67 93.1
0.55 0.504 1.34 1.46 91.7
5.5 5.274 1.07 1.12 95.9
PFHxA
Blank 0.001
0.0055 0.005 17.58 18.38 95.7
0.055 0.054 1.08 1.11 97.2
0.55 0.534 3.22 3.32 97.2
5.5 5.744 1.98 1.89 104.4
PFHpA
Blank 0.001
0.0055 0.005 16.77 17.85 94.0
0.055 0.053 1.46 1.53 96.0
0.55 0.530 2.95 3.07 96.3
5.5 5.549 2.10 2.08 100.9
PFOA
Blank ND
0.0055 0.005 2.36 2.62 89.9
0.055 0.053 0.55 0.58 95.7
0.55 0.555 0.67 0.66 100.9
5.5 5.798 2.46 2.33 105.4
PFNA
Blank 0.000
0.0055 0.005 2.91 3.48 83.4
0.055 0.052 4.07 4.26 95.4
0.55 0.523 4.73 4.97 95.2
5.5 5.581 3.29 3.24 101.5
PFDA
Blank 0.000
0.0055 0.004 14.51 17.41 83.4
0.055 0.051 3.33 3.56 93.6
0.55 0.521 2.63 2.78 94.7
5.5 5.445 2.10 2.12 99.0
PFUnA
Blank 0.001
0.0055 0.005 10.28 11.12 92.4
0.055 0.052 3.02 3.22 93.8
0.55 0.520 0.78 0.82 94.6
5.5 5.420 4.17 4.23 98.5
PFDoA
Blank 0.001
0.055 0.056 11.37 11.21 101.4
0.55 0.504 1.40 1.53 91.7
5.5 5.587 3.54 3.48 101.6
PFTrDA
Blank 0.001
0.0055 0.005 13.20 14.63 90.2
0.055 0.043 3.55 4.53 78.2
0.55 0.450 1.87 2.29 81.9
5.5 5.272 3.37 3.51 95.9
PFTeDA
Blank ND
0.055 0.037 2.40 3.59 66.9
0.55 0.440 2.75 3.43 80.1
5.5 5.363 4.01 4.12 97.5
PFBS
Blank 0.000
0.0055 0.005 10.02 10.89 92.0
0.055 0.051 1.92 2.07 92.7
0.55 0.515 1.65 1.76 93.5
5.5 5.359 0.78 0.80 97.4
PFPeS
Blank 0.000
0.0055 0.006 9.29 9.22 100.7
0.055 0.060 2.99 2.75 108.9
0.55 0.547 1.80 1.81 99.5
5.5 5.546 2.99 2.96 100.9
PFHxS
Blank ND
0.0055 0.006 12.64 12.35 102.3
0.055 0.054 1.14 1.16 97.8
0.55 0.538 1.75 1.79 97.8
5.5 5.752 1.80 1.72 104.6
PFHpS
Blank 0.001
0.055 0.052 6.88 7.25 94.9
0.55 0.484 1.62 1.84 88.1
5.5 4.930 1.72 1.91 89.6
PFOS
Blank 0.000
0.0055 0.005 14.22 15.79 90.1
0.055 0.048 1.11 1.26 88.1
0.55 0.493 2.30 2.57 89.7
5.5 5.395 7.45 7.59 98.1
PFNS
Blank 0.001
0.0055 0.005 18.55 20.76 89.3
0.055 0.056 1.30 1.28 101.6
0.55 0.573 4.06 3.89 104.2
5.5 5.419 4.92 4.99 98.5
PFDS
Blank 0.007
0.055 0.051 8.80 9.47 93.0
0.55 0.497 2.55 2.83 90.3
5.5 5.096 4.15 4.48 92.7
PFUnDS
Blank 0.005
0.055 0.047 10.89 12.89 84.5
0.55 0.401 4.99 6.85 72.9
5.5 5.537 9.92 9.85 100.7
PFDoS
Blank 0.003
0.055 0.047 13.08 15.26 85.7
0.55 0.478 5.59 6.44 86.8
5.5 4.933 1.55 1.73 89.7
PFTrDS
Blank 0.001
0.055 0.038 6.92 10.10 68.5
0.55 0.437 3.70 4.65 79.5
5.5 5.021 3.90 4.27 91.3
PFOSA
Blank ND
0.055 0.046 12.00 14.31 83.9
0.55 0.605 7.45 6.77 110.0
5.5 4.924 3.98 4.45 89.5
9Cl-PF3ONS
Blank 0.000
0.0055 0.006 13.50 13.00 103.8
0.055 0.054 0.32 0.33 98.0
0.55 0.538 1.85 1.89 97.8
5.5 5.888 7.98 7.45 107.0
11Cl-PF3OUdS
Blank 0.003
0.0055 0.007 13.02 11.27 115.5
0.055 0.047 9.48 11.20 84.6
0.55 0.451 1.20 1.46 82.0
5.5 4.916 3.70 4.14 89.4
HFPO-DA
Blank ND
0.055 0.046 2.80 3.33 84.3
0.55 0.550 3.03 3.03 99.9
5.5 5.770 2.77 2.64 104.9
DONA
Blank 0.000
0.0055 0.006 3.27 3.18 102.8
0.055 0.053 3.57 3.69 96.7
0.55 0.547 2.87 2.89 99.4
5.5 5.721 1.55 1.49 104.0
4:2 FTS
Blank 0.005
0.055 0.051 6.44 6.97 92.5
0.55 0.557 1.34 1.33 101.2
5.5 5.534 1.07 1.06 100.6
6:2 FTS
Blank 0.001
0.0055 0.005 6.71 7.33 91.5
0.055 0.056 16.30 16.11 101.2
0.55 0.559 10.64 10.46 101.7
5.5 5.406 4.45 4.53 98.3
8:2 FTS
Blank 0.000
0.0055 0.006 19.62 20.35 96.4
0.055 0.054 8.94 9.00 99.3
0.55 0.609 2.99 2.69 110.9
5.5 7.143 6.46 4.97 129.9
10:2 FTS
Blank 0.001
0.055 0.043 10.17 12.82 79.4
0.55 0.443 3.81 4.73 80.4
5.5 5.042 6.27 6.84 91.7
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a coffee matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
4.5 5.0
0
25000
50000
19:412.90>168.80(-)
19:412.90>368.90(-)
4.5 5.0
0
1000000
20:421.00>375.90(-)
5.0 5.5
0
2500
5000
7500
21:398.80>98.90(-)
21:398.80>79.80(-)
5.0 5.5
0
100000
200000
22:402.00>79.80(-)
5.0 5.5
0
25000
50000 23:462.90>218.60(-)
23:462.90>418.50(-)
5.0 5.5
0
500000
24:472.00>426.90(-)
PFOA PFHxS PFNA PFOS
6.0 6.5
0
5000
10000
31:498.80>98.90(-)
31:498.80>79.80(-)
6.0 6.5
0
250000
30:507.00>79.80(-)
Copyright © 2024 Shimadzu Corporation and/or its affiliates. All rights reserved.
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LCMS-8050RX LCMS-8060RX LCMS-2020 Q-TOF LCMS-9030/9050
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LCMS-8045RX LCMS-2050
02-SSI-LCMS-164 First Edition: September 2024
User Benefits
Application
News
◆ Validated method for 30 PFAS in seafood (shrimp) meeting all criteria of AOAC SMPR 2023.003
◆ High precision, excellent recovery, low Limit of Quantification (LOQ)
◆ Simple and rapid extraction using QuEChERS
LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer
Nexera series High Performance Liquid Chromatograph
Determination of 30 PFAS in Seafood by Liquid
Chromatography Triple Quadrupole Mass
Spectrometry (LC-MS/MS)
■ Introduction
Per-and polyfluorinated substances (PFAS) are a diverse
group of man-made chemicals used in numerous products
since the 1950s. PFAS can enter the food supply by
contact in environmentally contaminated areas, during
food processing, or exposure to packaging. Because PFAS
have been linked to serious health effects, accurate
methodology is needed. In this application news, we
describe a single laboratory validation study with a rapid
extraction of low concentrations of 30 PFAS in shrimp
using the QuEChERS technique followed by analysis using
the Shimadzu Nexera Liquid Chromatograph coupled to a
Shimadzu LCMS-8060NX triple quadrupole mass
spectrometer (Figure 1).
We optimized the chromatography and instrument
operating parameters to achieve excellent peak shape,
separation, and sensitivity. Sensitivity was improved for 4
PFAS, PFOA, PFHxS, PFNA, and PFOS.
In this study, we spiked samples at three concentrations in
triplicate. For greater accuracy, standards were matrixmatched
and extracted and spikes were quantified using
the isotope dilution technique. Recovery and precision
were compared to the requirements of AOAC SMPR
2023.003. In addition, we determined the Limit of
Quantitation (LOQ) as the lowest concentration meeting
accuracy and precision, ion ratio, retention time, and
signal-to-noise ratio criteria of the qualifier ion. All
recovery, precision, and LOQ’s met the acceptance criteria
of the SMPR. The target analytes, their acronym, chemical
abstract number, and experimentally determined LOQ are
shown in Table 1.
1 Shimadzu Scientific Instruments, Inc.
William Lipps1, Toshiya Matsubara1, Dominika Gruszecka1
Figure 1: Nexera and LCMS -8060NX. The ion focus design
improves signal intensity with higher gas flows and higher effective
temperatures.
Analyte name Acronym CAS No. LOQ (ppb)
Perfluorobutanoic acid PFBA 375-22-4 0.055
Perfluoropentanoic acid PFPeA 2706-90-3 0.0055
Perfluorohexanoic acid PFHxA 307-24-4 0.0055
Perfluoroheptanoic acid PFHpA 375-85-9 0.055
Perfluorooctanoic acid PFOA 335-67-1 0.0055
Perfluorononanoic acid PFNA 375-95-1 0.0055
Perfluorodecanoic acid PFDA 335-76-2 0.0055
Perfluoroundecanoic acid PFUnA 2058-94-8 0.0055
Perfluorododecanoic acid PFDoA 307-55-1 0.055
Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ
■ Sample Preparation and Analysis Conditions
Samples were prepared by spiking prepared shrimp samples
in triplicate at three different concentrations with 30 native
PFAS (Table 1) and 16 isotopically labeled internal standards.
Calibration curves for use in the quantitative analysis were
prepared using shrimp test portions spiked with
concentrations of 0.001, 0.01, 0.10, 1.0, and 10.0 ng/g.
Quantitation was carried out on additional shrimp samples
spiked in triplicate at 0.0055, 0.055, 0.55, and 5.5 ng/g.
Since standards were extracted in a shrimp matrix and
carried through the same procedure, the final concentration
of each PFAS in the sample can be calculated directly from
the curve.
Frozen shrimp were thawed at room temperature and then
crushed in a grinder with dry ice for 30 seconds at 4000 rpm.
The ground sample was stored in a freezer overnight to
remove all the dry ice. Ten-gram portions were weighed, and
10 mL of acetonitrile was added. The samples were vortexed
for 1 minute and a QuEChERS packet was added. The
sample was shaken for 1 minute and then centrifuged for 5
minutes at 4000 rpm. An aliquot of the acetonitrile layer was
transferred to a tube and diluted 5 times with PFAS-free
reagent water. The sample was then passed through a weak
anion exchange (WAX) Solid Phase Extraction (SPE) cartridge
and the PFAS were eluted with basic methanol. For greater
sensitivity, 2 mL of the extract was concentrated in 400 μL of
a methanol-water mixture.
A volume suitable to obtain the required sensitivity of the
extract was injected onto a UHPLC system (Shimadzu
Nexera). Adequate separation of all compounds was
achieved in nine minutes. (Figure 2 chromatogram shows
the separation of all peaks).
For this study, Shimadzu evaluated 1984 different
instrument settings, and 6 different column and gradient
combinations, to achieve excellent peak shape and
resolution between peaks, as well as to maximize the
signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS.
Mass spectrometry was performed on a Shimadzu LCMS-
8060NX with heated electrospray ionization operated in
negative mode. Specific compound MRM transitions and
associated internal standards are listed in Table 2.
Chromatography was adjusted to provide sufficient
separation of PFOA from potential cholic acid interferences,
and to provide baseline resolution of branched and linear
isomers (Figure 3).
Perfluorotridecanoic acid PFTrDA 72629-94-8 0.055
Perfluorotetradecanoic acid PFTeDA 376-06-7 0.055
Perfluorobutanesulfonic acid PFBS 375-73-5 0.055
Perfluoropentansulfonic acid PFPeS 2706-91-4 0.055
Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055
Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.0055
Perfluorooctanesulfonic acid PFOS 1763-23-1 0.055
Perfluorononanesulfonic acid PFNS 68259-12-1 0.0055
Perfluorodecanesulfonic acid PFDS 335-77-3 0.0055
Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.0055
Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.0055
Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.055
Perfluorooctanesulfonamide PFOSA 754-91-6 0.055
9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.0055
Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.055
4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055
1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.055
1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.55
1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.0055
1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.0055
Analyte Quantitation Ion Qualifier Ion Internal Standard
PFBA 212.9 > 168.8 13C4-PFBA
PFPeA 262.9 > 218.8 13C5-PFPeA
PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA
PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA
PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA
PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA
PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA
PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA
PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA
PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA
PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA
PFBS 298.8 > 79.8 298.8 > 98.9 13C5-PFHxA
PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA
PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS
PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA
PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS
PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA
PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA
PFUnDS 649 > 99 649 > 80 13C8-PFOS
PFDoS 699 > 80 699 > 99 13C2-PFDoA
PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA
PFOSA 498 > 78 498 > 477.95 13C8-PFOSA
9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS
11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA
HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA
DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA
4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS
6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS
8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS
10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA
Figure 2: Chromatogram of 0.55 ng/g PFAS in shrimp matrix with separation of all peaks in nine minutes
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
0
250000
500000
750000
1000000
1250000
1500000
1750000
2000000
2250000
2500000
2750000
3000000
3250000
Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers
Table 2: MRM Transitions and Internal Standard Associations
■ Quantitative Analysis
Calibration standards were processed the same as samples.
A linear model not forced through zero isotopic dilution
calibration in matrix-matched standards provided the best fit
and best recoveries of analytes. Residuals of each point in the
curve were ±25% of the expected value. Calibration curves
for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6
respectively. Branched and linear isomers of PFHXS and PFOS
were integrated together.
Exact labeled analogs were used as isotope dilution
standards, except in a few cases where we found that 13C2
suffered matrix interferences. In these cases, another noninterfering
isotope was chosen. The analytes tested and the
associated calibration isotopes are shown in Table 3.
During the single laboratory validation, the shrimp matrix
contained an unknown interference coeluting with the
13C3-PFBS isotope so 13C5-PFHxA was used instead.
Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve
Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve
Conc. (ng/g) %Accuracy
0.001 101
0.01 92.8
0.1 94.8
1 99.6
10 112
Conc. (ng/g) %Accuracy
0.001 102
0.01 82.4
0.1 97.0
1 102
10 116
Conc. (ng/g) %Accuracy
0.001 99.9
0.01 100
0.1 101
1 108
10 90.3
Conc. (ng/g) %Accuracy
0.001 99.6
0.01 105
0.1 89.4
1 93.4
10 112
Blank matrixes and at least three different concentrations
ranging from below the SMPR required LOQ to
approximately 20 times the estimated LOQ were analyzed in
triplicate. Recovery and repeatability for each analyte at each
concentration are given in Table 3. The LOQ for each analyte
was estimated by spiking at concentrations at, or below, the
required LOQs listed in SMPR-2023_003. The spiked samples
were analyzed in triplicate and the mean and repeatability
standard deviation were calculated. Then, the standard
deviation was divided by the mean and multiplied by 100%
to calculate the repeatability percent relative standard
deviation (RSD).
The LOQs for all matrices and compounds were determined
using an Excel worksheet that compared each of the
requirements of the SMPR including retention time,
recovery, repeatability, S/N > 3 for the qualifier ion and an
ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set
at the minimum concentration, meeting recovery and
repeatability requirements and a S/N > 10. The lowest
concentration to meet all the requirements of the SMPR
was set as the LOQ. Figure 7 shows examples of the LOQ
spike for PFHxS, PFNA, PFOA, and PFOS and their
corresponding internal standards.
Analyte Spike conc. (ppb) Average conc. (ppb)
Standard
Deviation
%RSD Average Recovery (%)
PFBA
Blank 0.007
0.055 0.056 0.98 0.98 100.3
0.55 0.518 0.32 0.34 94.1
5.5 5.478 0.35 0.35 99.6
PFPeA
Blank ND
0.0055 0.005 3.52 3.81 92.6
0.055 0.054 2.17 2.23 97.6
0.55 0.517 0.67 0.71 94.1
5.5 5.751 0.26 0.25 104.6
PFHxA
Blank 0.001
0.0055 0.005 9.08 9.50 95.6
0.055 0.057 1.48 1.43 103.7
0.55 0.538 0.66 0.67 97.8
5.5 5.871 0.15 0.14 106.8
PFHpA
Blank 0.001
0.0055 0.005 3.90 4.06 96.1
0.055 0.051 0.75 0.82 92.0
0.55 0.518 0.26 0.28 94.2
5.5 6.161 1.25 1.12 112.0
PFOA
Blank ND
0.0055 0.006 6.16 5.89 104.6
0.055 0.053 0.67 0.69 95.8
0.55 0.543 1.59 1.61 98.8
5.5 6.303 0.67 0.58 114.6
PFNA
Blank 0.000
0.0055 0.006 7.32 6.77 108.1
0.055 0.056 0.68 0.67 102.1
0.55 0.583 0.55 0.52 106.0
5.5 5.340 2.53 2.61 97.1
PFDA
Blank 0.000
0.0055 0.006 4.45 4.38 101.6
0.055 0.056 0.72 0.71 101.9
0.55 0.572 0.86 0.83 104.0
5.5 5.827 2.00 1.89 105.9
PFUnA
Blank ND
0.0055 0.007 6.07 4.92 123.3
0.055 0.056 2.29 2.27 100.9
0.55 0.579 1.03 0.98 105.2
5.5 5.789 1.91 1.82 105.3
PFDoA
Blank 0.001
0.055 0.054 6.52 6.67 97.7
0.55 0.528 3.51 3.66 96.0
5.5 5.863 3.10 2.91 106.6
PFTrDA
Blank ND
0.055 0.063 2.31 2.01 115.0
0.55 0.586 3.89 3.65 106.6
5.5 6.215 2.56 2.26 113.0
PFTeDA
Blank 0.001
0.055 0.055 5.22 5.23 99.7
0.55 0.555 4.27 4.23 100.8
5.5 6.313 3.03 2.64 114.8
PFBS
Blank 0.001
0.055 0.051 5.55 5.93 93.6
0.55 0.527 2.83 2.95 95.8
5.5 5.940 2.31 2.14 108.0
PFPeS
Blank 0.002
0.0055 0.004 15.88 20.67 76.8
0.055 0.055 3.92 3.89 100.7
0.55 0.598 3.29 3.02 108.8
5.5 5.713 0.71 0.68 103.9
Table 3: Recovery and repeatability for each analyte at each spike concentration
PFHxS
Blank ND
0.0055 0.004 14.59 17.62 82.8
0.055 0.052 2.32 2.45 94.9
0.55 0.535 1.06 1.09 97.2
5.5 6.070 0.25 0.23 110.4
PFHpS
Blank 0.001
0.0055 0.006 20.96 18.27 114.8
0.055 0.057 9.51 9.14 104.0
0.55 0.590 8.60 8.02 107.2
5.5 6.223 4.30 3.80 113.1
PFOS
Blank 0.001
0.055 0.053 5.12 5.28 96.9
0.55 0.517 1.77 1.88 94.0
5.5 5.700 1.03 0.99 103.6
PFNS
Blank 0.001
0.0055 0.006 5.17 4.42 117.0
0.055 0.052 3.68 3.86 95.1
0.55 0.526 3.20 3.35 95.6
5.5 6.117 4.19 3.76 111.2
PFDS
Blank 0.000
0.0055 0.006 9.46 8.11 116.6
0.055 0.056 0.87 0.85 102.6
0.55 0.569 4.82 4.67 103.4
5.5 6.148 4.56 4.08 111.8
PFUnDS
Blank ND
0.0055 0.004 15.95 19.84 80.4
0.055 0.056 5.75 5.66 101.5
0.55 0.594 0.61 0.56 107.9
5.5 5.614 0.85 0.84 102.1
PFDoS
Blank 0.000
0.0055 0.007 14.15 11.90 118.9
0.055 0.056 5.90 5.80 101.7
0.55 0.607 4.68 4.24 110.3
5.5 5.949 2.90 2.68 108.2
PFTrDS
Blank 0.000
0.0055 0.005 8.10 7.94 102.0
0.055 0.057 4.16 4.03 103.2
0.55 0.581 5.25 4.97 105.7
5.5 6.228 3.96 3.50 113.2
PFOSA
Blank 0.001
0.0055 0.006 9.45 8.91 106.1
0.055 0.055 3.80 3.81 99.8
0.55 0.556 0.78 0.77 101.1
5.5 5.973 1.75 1.61 108.6
9Cl-PF3ONS
Blank 0.000
0.0055 0.004 7.22 8.57 84.3
0.055 0.054 1.65 1.67 98.6
0.55 0.570 0.78 0.75 103.6
5.5 5.896 2.62 2.44 107.2
11Cl-PF3OUdS
Blank 0.001
0.0055 0.006 5.66 5.06 112.0
0.055 0.054 1.81 1.85 97.8
0.55 0.546 3.03 3.06 99.2
5.5 6.022 3.61 3.30 109.5
HFPO-DA
Blank 0.002
0.0055 0.005 6.55 6.59 99.4
0.055 0.053 2.17 2.24 96.9
0.55 0.517 3.93 4.18 94.0
5.5 5.653 0.95 0.93 102.8
DONA
Blank 0.000
0.0055 0.005 2.04 2.07 98.7
0.055 0.060 3.96 3.59 110.1
0.55 0.619 2.37 2.10 112.6
5.5 5.714 1.95 1.87 103.9
4:2 FTS
Blank ND
0.055 0.052 1.92 2.05 93.6
0.55 0.535 0.35 0.36 97.2
5.5 5.663 0.80 0.78 103.0
6:2 FTS
Blank 0.018
0.055 0.048 4.12 4.66 88.4
0.55 0.495 1.60 1.78 90.0
5.5 5.290 1.26 1.31 96.2
8:2 FTS
Blank 0.000
0.0055 0.005 5.61 6.54 85.8
0.055 0.060 1.83 1.67 110.1
0.55 0.609 3.59 3.25 110.6
5.5 5.790 0.83 0.79 105.3
10:2 FTS
Blank 0.000
0.0055 0.006 13.17 12.02 109.5
0.055 0.065 2.75 2.34 117.8
0.55 0.641 2.69 2.31 116.6
5.5 6.102 4.75 4.28 110.9
ND = average results less than zero
Figure 7: LOQ Peaks with Internal Standards
4.25 4.50 4.75 5.00
0
5000
10000
15000
20000
25000
20:412.90>168.80(-)
20:412.90>368.90(-)
4.25 4.50 4.75 5.00
0
250000
500000
750000
19:421.00>375.90(-)
4.75 5.00 5.25 5.50
0
500
1000
1500
2000
22:398.80>98.90(-)
22:398.80>79.80(-)
4.75 5.00 5.25 5.50
0
25000
50000
75000
100000
125000
150000
21:402.00>79.80(-)
5.00 5.25 5.50 5.75
0
10000
20000
30000
40000
50000
60000
24:462.90>218.60(-)
24:462.90>418.50(-)
5.00 5.25 5.50 5.75
0
100000
200000
300000
400000
23:472.00>426.90(-)
5.75 6.00 6.25 6.50
0
2500
5000
7500
10000
30:498.80>98.90(-)
30:498.80>79.80(-)
5.75 6.00 6.25 6.50
0
25000
50000
75000
100000
125000
150000
31:507.00>79.80(-)
PFOA PFHxS PFNA PFOS
■ Conclusion
The Shimadzu LCMS-8060NX Triple Quadrupole Mass
Spectrometer coupled with a Shimadzu Nexera Liquid
Chromatograph was used in a single laboratory study to
measure 30 PFAS compounds in a shrimp matrix and
compared to criteria set by AOAC SMPR 2023.003.
Chromatography conditions and the mass spectrometer were
optimized to achieve excellent separation of all analytes,
baseline resolution between linear and branched isomers,
and a two-minute separation between PFOS and potentially
interfering cholic acids.
Precision and recovery and the experimentally determined
LOQ are well within the requirements of the SMPR.
■ Reference
1) AOAC SMPR 2023.003
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References
1. Shoemaker, J., Grimmet, P.E, and Boutin, P.K. Determination of Selected Perfluorinated Alkyl Acids in Drinking Water
by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) Method 537 Ver.
1.1. National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection
Agency; Cincinnati OH, 2009.
2. Shoemaker, J. and Dan Tettenhorst. Method 537.1: Determination of Selected Per- and Polyfluorinated Alkyl
Substances in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS). U.S. Environmental Protection Agency, Office of Research and Development, National Center for
Environmental Assessment, Washington, DC, 2018.
3. U.S. Environmental Protection Agency, Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid,
Biosolids, and Tissue Samples by LC-MS/MS, Method 1633, Office of Water, Office of Science and Technology
Engineering and Analysis Division 1200 Pennsylvania Avenue, NW Washington, DC 20460, January 2024
4. ASTM International, D7979, Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances in
Water, Sludge, Influent, Effluent and Wastewater by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/
MS), 2017, DOI: 10.1520/D7979-17
5. ASTM International, D8421, Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances (PFAS)
in Aqueous Matrices by Co-solvation followed by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS),
2021, 10.1520/D8421-21
6. ASTM International, D7968a, Standard Test Method for Determination of Polyfluorinated Compounds in Soil by
Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS), 2017, DOI: 10.1520/D7968-17A
7. ASTM International, D8535, Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances (PFAS)
in Soil/Biosolid Matrices by Solvent Extraction, Filtering, and Followed by Liquid Chromatography Tandem Mass
Spectrometry (LC/MS/MS), 2023, DOI: 10.1520/D8535-23
8. Genualdi, S. and deJager, L., Method C-010.02, Determination of 16 Per and Polyfluoroalkyl Substances (PFAS)
in Processed Food using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), U.S Food and Drug
Administration, 2021
9. United States Department of Agriculture, CLG - PFAS 2.03, Screening, Determination and Confirmation of PFAS by
UPLC-MS-MS, Food Safety and Inspection Service, Office of Public Health Science, Washington DC, 2021
10. Genualdi, S and deJager L., Method C-010.03, Determination of 30 Per and Polyfluoroalkyl Substances (PFAS)
in Food and Feed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), U.S. Food and Drug
Administration, 2024
11. https://www.aoac.org/resources/aoac-smpr-2023-003 [accessed 11 Mar 2024]
106
LC-MS Solutions for PFAS in Food
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