User Benefits
Application
News
The optimized procedure for pretreatment and LC-MS/MS analytical conditions enable accurate quantification of thirty major
PFASs targeted by AOAC SMPR from 0.1 µg/kg.
The method allows the initiation of PFASs analysis in food.
Analysis of Per- and Polyfluoroalkyl Substances
(PFAS) using Triple Quadrupole Mass Spectrometer
Part 1 -Fish Fillet-
Nozomi Maeshima
High Performance Liquid chromatograph Mass Spectrometer LCMS-8060NX
Introduction
Per- and Polyfluoroalkyl Substances (PFASs) are a collective
name for more than four thousand organofluorine compounds.
Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid
(PFOA) are representative compounds of PFAS. They are used in
a wide range of applications, such as fire retardants, food
packaging materials, and non-stick coatings, due to their water-
repellent, oil-repellent, heat-resistant, and chemical-resistant
properties. Due to their structural stability, PFAS widely remains
in the environment. There are concerns about health risks
caused by human ingestion of fish that have ingested seawater,
river water, or feed contaminated with PFAS. Therefore,
quantitative assessment of PFAS levels in fish should be
important.
This application news introduces a quantitative analysis of PFAS
in tuna fillets with LC-MS/MS. Thirty principal PFASs targeted by
AOAC INTERNATIONAL, a North American organization that
standardizes food testing methods and verifies analytical
methods, were analyzed using newly created method, which
was investigated from the pretreatment procedure then
validated by recovery test. By reducing losses during the
pretreatment and optimizing the analytical conditions, good
recovery rates were obtained for all compounds.
Sample and pretreatment
As standards and internal standards (ISTDs), L-PFUdS, L-PFTrDS,
L-PFDoS, and 10:2 FTS and two standard mixtures of
PFAC30PAR and MPFAC-HIF-ES were purchased from
Wellington Laboratories. Tuna fillet was purchased at a local
grocery store, and frozen and crushed with dry ice using a freeze
grinder FST-4000 (AiSTI SCIENCE). The samples were left in a
freezer overnight, and the dry ice was removed before the
pretreatment.
Extraction was performed using the QuEChERS method as a
reference. The procedure is shown in Fig. 2. 10 g of frozen
crushed sample was weighed, added ISTDs and 10 mL of
acetonitrile, and shaken vigorously for 1 min. One packet of
QuEChERS extraction salt (Restek, P/N: 25849) was added and
immediately shaken vigorously by hand for 1 min then
centrifuged at 4,000 rpm for 5 min at room temperature.
Acetonitrile layer was collected then diluted 5-fold with water
and used as extract.
EVOLUTE PFAS (Biotage, P/N: 614-0015-CP, 150 mg/6 mL) and a
positive pressure manifold PRESSURE+ (Biotage) were used for
solid-phase extraction. The procedure is shown in Fig. 3. After
conditioning with 5 mL of 28% ammonium hydroxide/
methanol (1:100, v/v) and 5 mL of formic acid/methanol (1:1000,
v/v), 40 mL of extraction solution (equivalent to 8 g of fish
sample) was loaded. Following washing with 5 mL of water and
5 mL of formic acid/methanol/water (1:400:600, v/v/v), the
analytes were eluted with 5 mL of 28% ammonium
hydroxide/methanol/water (1:90:10, v/v/v). The eluate was
taken 500 µL and mixed with 2 µL of formic acid, then analyzed
by LC-MS/MS.
Table 1 Analytical conditions of LC-MS/MS
[HPLC conditions] Nexera X3
Column : Shim-pack Scepter
TM
C18-120
(100 mm x 2.1 mm I.D., 3 µm)
*1
Delay column : Shim-pack Scepter C18-120
(50 mm x 2.1 mm I.D., 3 µm)
*2
Mobile phase A : 2 mmol/L Ammonium Acetate in Acetonitrile/Water
(5:95, v/v)
Mobile phase B : Acetonitrile
Flow rate : 0.3 mL/min (0.6 mL/min only between 10.01-12 min)
Gradient program : B conc. 20% (0 min) → 100% (10-12 min) → 20%
(12.01-15 min)
The flow was loaded onto the mass spectrometer
between 1 to 9.6 min using a flow switching valve.
Column temp. : 40˚C
Injection volume : 5 µL
[MS conditions] LCMS-8060NX
Ionization : ESI, Negative mode
Nebulizing gas : 2 L/min
Heating gas : 10 L/min
Drying gas : 10 L/min
DL temp. : 200˚C
Interface temp. : 250˚C
Heat block temp. : 400˚C
Probe position : +2 mm
*1 227-31014-05
*2 227-31014-03
Analytical conditions
Analysis was performed using a triple quadrupole mass
spectrometer LCMS-8060NX equipped with an ultra-high
performance liquid chromatograph Nexera
TM
X3 UHPLC (Fig. 1).
The analytical conditions are shown in Table 1. To prevent
interference caused by PFAS contamination from analytical
equipment, solvents and glassware, a delay column was
installed between mixer and autosampler using SUS piping (300
mm x 0.3 mm I.D., P/N: 228-69955-41). The delay column
increased the elution time of PFAS derived from the LC system,
allowing it to be separated from the PFAS in the sample. PP vials
(Shimadzu GLC, P/N: GLC-IVS-100) confirmed to have no
detectable PFAS were observed.
Fig. 1 Nexera
TM
X3 and LCMS-8060NX
