Rapid Extraction of Low Concentrations of PFAS in Food
App Note / Case Study
Last Updated: August 2, 2024
(+ more)
Published: August 1, 2024
Credit: iStock
Per-and polyfluorinated substances (PFAS) have been linked to serious health effects and can enter the food supply by contact in contaminated areas, during food processing or from packaging.
Therefore, testing for PFAS in food is crucial to ensure the safety of these products.
This app note highlights a solution for the rapid extraction of low concentrations of 30 PFAS in shrimp.
Download this app note to discover a method that:
- Achieves excellent peak shape, separation and sensitivity
- Offer high precision, excellent recovery and a low limit of quantification
- Provides simple and rapid extraction
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 LCMS8060NX 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 µg/kg 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/ kg) %Accuracy
0.001 101
0.01 92.8
0.1 94.8
1 99.6
10 112
Conc. (ng/ kg) %Accuracy
0.001 102
0.01 82.4
0.1 97.0
1 102
10 116
Conc. (ng/ kg) %Accuracy
0.001 99.9
0.01 100
0.1 101
1 108
10 90.3
Conc. (ng/ kg) %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 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.
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
© Shimadzu Scientific Instruments, 2024
First Edition: May2024
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