Determination of 16 Per and Polyfluoroalkyl Substances PFAS in Processed Food using Liquid Chromatography-Tandem Mass Spectrometry LC-MS/MS

SCOPE OF APPLICATION

The method describes a procedure for measuring 16 PFAS in food using LC-MS/MS. The method has been single laboratory validated in the following food matrices:

• A method verification was performed with lettuce, milk, bread, and salmon to capture the matrices originally described in C-010.01. Samples were spiked in duplicate at 0.5 μg/kg and 2 μg/kg. All recoveries were between 70-130% with the exception of 11Cl-PF3OUdS in bread which had recoveries ranging from 26-34%. This analyte has known issues with certain matrices, which may reduce its confidence in certain food types.
• This method should be used by analysts experienced in the use of LC-MS/MS, including but not limited to operation of the instrumentation and software, data analysis and reporting results.
• Analysts should also be able to identify chromatographic and mass spectrometric interferences during sample analysis and take necessary actions following validated procedures for their correction to achieve reliable identification and quantitation.
• The method should be used only by personnel thoroughly trained in the handling and analysis of samples for the determination of trace contaminants in food and beverage products. PFAS chemicals are prevalent in all laboratory environments and special care must be taken to prevent false positives due to accidental and/or routine laboratory contamination.
• Only LC-MS grade solvents should be used unless otherwise noted in the procedure below. All solvents and complete method blanks should be analyzed on the LC-MS/MS instrument prior to sample analysis. If PFAS compounds are determined, complete method blank results should be subtracted from samples. Complete method blanks should be performed and analyzed daily, preferably in the same instrument sequence as the samples. Sources of potential contamination during sample preparation include; solvents, syringe filters, centrifuge tubes, SPE sorbents, septa, and others.
• A delay column should be used between the mobile phase mixer and sample injector to temporarily trap any system related interferences, which results in their elution at a later retention time than the analyte. This eliminates contamination from instrument tubing, mobile phase solvents, and solvent bottles.
• Due to the extreme low concentrations of detection required for this analysis, choice of MS/MS instrumentation is critical. Our analysis has been performed using Sciex 6500 and 6500 plus instrumentation platforms. We have not fully evaluated any Orbitrap MS systems and have not yet demonstrated adequate lower levels of quantitation (LLOQ) for these systems.

PRINCIPLE

REAGENTS

The use of trade names in this method constitutes neither endorsement nor recommendation by the U.S. Food and Drug Administration (FDA). Equivalent performance may be achievable using apparatus and materials other than those cited here.

• Formic acid, reagent grade >95% (Sigma Aldrich St. Louis, MO)
• LC/MS grade Optima water (Fisher Scientific, Hampton, NH)
• LC/MS grade Optima acetonitrile (Fisher Scientific, Hampton, NH)
• LC/MS grade Optima methanol (Fisher Scientific, Hampton, NH)
• Acetic acid, ammonium salt, 98% for analysis (Acros Organic, Geel, Belgium)
• Original QuEChERS extraction salt ECMSSCFS-MP with 6000 mg MgSO4 and 1500 mg NaCl (UCT, Bristol, PA)
• QuEChERS dSPE ECMPSCB-MP with 900 mg MgSO4, 300 mg PSA, 150 mg graphitized carbon black (UCT, Bristol, PA) or ECMPSCB15-CT prefilled units
• Ammonium hydroxide, certified ACS Plus 14.8N (Fisher Scientific, Hampton, NH)

STANDARDS

• Isotopically labeled PFAS analytical standards (Wellington laboratories, Guelph, ON, Canada)
• Native PFAS analytical standards (Wellington laboratories, Guelph, ON, Canada)
• Both PFOA and PFOS were quantified using technical standards and reported as the sum of linear and branched isomers. All other analytes were reported as the concentration of the linear isomer (if applicable).

PREPARATION OF SAMPLES OR TEST PORTIONS

Prepare native PFAS stock solution at 1000 ng/mL, 100 ng/mL, 10 ng/mL and 1 ng/mL

1. Add 0.2 mL of each 50 μg/mL PFAS analytical standard (16 native compounds in Table 1) to 6.8 mL methanol. In the resulting solution, each compound has a concentration of 1000 ng/mL in methanol. Individual PFAS 50 μg/mL methanol standards were purchased from Wellington, but other sources are acceptable. This solution will be used for calibration curve preparation and single lab validation (SLV) spikes.
2. Add 1 mL of 1000 ng/mL stock solution to 9 mL of methanol to produce a 100 ng/mL stock solution. This solution will be used for calibration curve preparation and SLV spikes.
3. Add 1 mL of 100 ng/mL stock solution to 9 mL of methanol to produce a 10 ng/mL stock solution. This solution will be used for SLV and method detection limit (MDL) spikes.
4. Add 1 mL of 10 ng/mL stock solution to 9 mL of methanol to produce a 1 ng/mL stock solution. This solution will be used for calibration curve preparation.

Prepare isotopically labeled PFAS surrogate stock solution (SS) at 1000 ng/mL

1. Add 0.2 mL of each 50 μg/mL analytical standard (8 isotopically labeled PFAS in Table 1) to 8.4 mL methanol. Individually labeled PFAS 50 μg/mL methanol standards were purchased from Wellington but other sources are acceptable. This stock solution was used for both sample analysis and calibration curve preparation.

Prepare isotopically labeled internal standard solution (IS) at 200 ng/mL

1. Add 0.04 mL of N-ethyl-d5-perfluoro-1-octanesulfonamidoacetic acid (d5-N-EtFOSAA) 50 μg/mL analytical standard to 9.96 mL methanol. The individual d5-N-EtFOSAA standard was purchased from Wellington but other sources are acceptable.

Prepare mobile phase A (5 mM ammonium acetate in water) and 5mM 1-methyl piperidine

1. Weigh out 0.38 ± 0.01 g of ammonium acetate.
2. Add to mobile phase bottle with 1000 mL of LC/MS Optima water.
3. Add 0.5 mL of 1-methyl piperidine.
4. Invert several times to mix.

Prepare mobile phase B (100% methanol)

1. Add ~ 1000 mL of LC/MS Optima methanol to a mobile phase bottle.

Continuing Calibration Verification (CCV) standard

1. A duplicate solution was prepared of the 1 ng/mL calibration standard and used as the CCV standard (Table 2).

Solution for solid phase extraction (SPE) clean-up

1. Add 6 mL of a 14.8 N ammonium hydroxide solution to 1000 mL volumetric flask and fill to volume with methanol to make up a 0.3 % w/w solution.

Calibration Standards

1. Calibration standards are prepared at concentrations of 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, 10, and 25ng/mL according to Table 2 below.

Preparation of Samples or Test Portions

1. The samples used for method development were previously homogenized by FDA’s Kansas City lab. The sample size for analysis was 5 grams.
2. The samples used for method verification (lettuce, milk, bread, and salmon) were homogenized using an IKA tube mill with a disposable 100 mL polypropylene grinding chamber. Samples were ground at 5000 rpm for approximately 2 minutes.

APPARATUS/INSTRUMENTATION

1. Digital pulse mixer/vortexer (Glas-Col, Terre Haute, IN) capable of 1500 rpm with pulse 70
2. Sorvall legend XTR centrifuge (Thermo Fisher Scientific, Waltham, MA)
3. Nitrogen evaporation system (Turbovap LV, Biotage, Uppsala, Sweden)
4. Nexera X2 (Shimadzu, Kyoto, Japan) with binary pump, degasser, autosampler, and thermostatted column compartment
5. A Sciex 6500 plus QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer with an electrospray ESI ion source (Sciex, Toronto, ON Canada)
6. Analyst® Software version 1.7.1
7. ScieX OS Version 2.0.0.45330
8. Falcon 50 mL polypropylene (PP) conical centrifuge tubes (Thermo Fisher Scientific, Waltham, MA)
9. Falcon 15 mL polypropylene (PP) conical centrifuge tubes (Thermo Fisher Scientific, Waltham, MA)
10. 300 μL PP autosampler vials (SUN Sri, Rockwood, TN)
11. PP autosampler vial caps (SUN Sri, Rockwood, TN)
12. 0.2 μm Acrodisc nylon syringe filters (Pall Corporation, Port Washington, NY)
13. 5 mL PP/PE luer lock syringes (Sigma Aldrich, St. Louis, MO)
14. Nano filter vials 0.2 μm nylon without cap (Thomson Instrument Company, Oceanside, CA)
15. PP vial caps (Sun Sri, Rockwood, TN)
16. Analytical column – 150 mm x 2.1 mm, 3.5 μm XBridge C18 (Waters Corp, Milford, MA)
17. Guard column – 2.1 mm x 5 mm, 1.7 μm Vanguard™ Acquity BEH C18 (Waters Corp, Milford, MA)
18. Delay column – 2.1 mm x 50 mm, 5 μm Atlantis T3 (Waters Corp, Milford, MA)
19. SPE cartridge – Strata™-XL-AW 100 μm Polymeric Weak Anion 200 mg / 3 mL, Tubes (Phenomenex, Torrance, CA)