EPA method 521 by direct-inject LC-MS/MS Agustin Pierri, PhD NEMC 2017 Washington DC

Background

Polyfluoroalkyl Substances Polyfluoroalkyl Substances (PFASs) Aliphatic backbone—from C4 on up Typically per-fluorinated Acid functional group Unique environmental chemistry  Persistent Lipophilic Bioaccumulative Chemically stable: low reactivity, resistant to heat, oil, water, acid, almost anything!

Polyfluoroalkyl Substances (PFASs) Uses? Repellents Performance chemicals Consumer and industrial goods

Polyfluoroalkyl Substances (PFASs) Routes of exposure Direct contact with food products Environmental exposure EPA established a limit of 70 ppt in water to minimize lifetime exposure

Target compounds Carboxylic acids Sulfonic acids Sulfonamides 6:2 FTS 8:2 FTS EtFOSA MeFOSA FOSA EtFOSE MeFOSE PFBA PFBS PFDA PFDS PFHpA PFHpS PFHxA PFHxS PFNA PFOA PFOS PFPeA PFDoA PFTeDA PFTrDA PFUdA Carboxylic acids Sulfonic acids Sulfonamides Sulfonamido-ethanols

Sample prep

Sample prep approach SPE: QuEChERS: Only suitable for liquid samples Weak anion exchange offline SPE QuEChERS: Suitable for non-liquid samples

QuEChERS Screening: C18 d-SPE C18 d-SPE + hexanes 👎 PSA d-SPE + hexanes C18/GCB d-SPE C18/GCB d-SPE + hexanes PSA d-SPE 👎 👎 👎 👎 Semi isotope dilution methods 👎 👌👏

QuEChERS procedure Add 1.0 grams homogenized sample to 50 mL tube Add 10 mL H2O and 10 mL MeCN Add 4 g MgSO4 and 1 g NaCl Vortex 3 minutes, centrifuge 5 minutes Transfer 1 mL aliquot to tube with 150 mg MgSO4 and 50 mg PSA Semi isotope dilution methods Transfer 200 µL to conical vial for analysis

Secondary cleanup QuEChERS followed by SPE QuEChERS: 1.0 g  10 mL WAX SPE: 500 µL  500 µL

QuEChERS + SPE procedure Add 1.0 grams homogenized sample to 50 mL tube Add 4 g MgSO4 and 1 g NaCl Vortex 3 minutes, centrifuge 5 minutes Transfer 1 mL aliquot to tube with 150 mg MgSO4 and 50 mg PSA Transfer 500 µL, dilute to ~15 mL with H2O for SPE Add 10 mL H2O and 10 mL MeCN Condition Strata-XAW 200 mg/3 mL SPE cartridge with 0.3% NH4OH/MeCN Load diluted QuEChERS extract, wash with 5 mL H2O Elute with 4 mL 0.3% NH4OH/MeCN Evaporate to 500 µL, transfer to conical vial for analysis This extra cleanup affords a 20 uL injection, which gets me to a 10x lower MRL Transfer 200 µL to conical vial for analysis

Analysis: LC/MS

PFAS by LC/MS Unique analytical challenges Low levels Relative polarities Isomers Contamination

Sensitivity 1290 LC, 6460 QQQ Choosing the right mobile phase modifier UHPLC with the 1290 coupled with the 6460 gives us plenty of sensitivity for these.

Mobile phase modifiers for ESI Negative mode  Ammonium salts Ammonium acetate Ammonium formate Ammonium fluoride—the secret sauce (usually)

Mobile phase choice 👍 Separation/run time: Ammonium acetate Acetic acid Ammonium formate PFAS peak shape with different mobile phases Ammonium fluoride

Chromatographic conditions Column: Phenomenex Luna Omega PS C18 50 x 2.1 1.6 μm Injection volume: 2 μL or 20 μL Mobile phase: A: Water with 5 mM ammonium acetate B: 100% Acetonitrile Flow rate: 0.550 mL/min Time %B 0.0 40 0.5 3.0 90 3.1 100 4

Mid level chromatogram (0.5 ppb) Mid level CCV (0.5 ppb), equivalent to 5 ng/g in the sample

Low level (0.05 ppb) 👍 Low levels: Low level CCV (0.05 ppb in the standard), equivalent to 0.5 ng/g in the sample 👍 Low levels:

Solvent blank Solvent blank 👍 No instrument contamination:

Branched vs. linear PFBA PFHxS PFOS Solvent blank 👍 Isobars:

Dynamic range Calibrates up to 0.05 ppb – 1000 ppb PFPeA: 0.05 ppb to 5 ppb, but it is capable of going up to 1000 ppb. Calibrates up to 0.05 ppb – 1000 ppb

Method performance

Extracts Blank Butter Cheese Egg Milk Fish

QuEChERS recoveries, milk n=4, spiked at 2x the MRL: 100 ppt n=4, 1 ng/g

QuEChERS recoveries, egg n=4, spiked at 2x the MRL: 100 ppt n=4, 1 ng/g

QuEChERS recoveries, butter n=4, spiked at 2x the MRL: 100 ppt n=4, 1 ng/g

QuEChERS recoveries, tuna n=4, spiked at 2x the MRL: 100 ppt n=4, 1 ng/g

QuEChERS recoveries, cheese n=4, spiked at 2x the MRL: 100 ppt n=4, 1 ng/g

QuEChERS + SPE recoveries, egg n=4, spiked at 2x the MRL: 10 ppt n=4, 0.1 ng/g

Robust and versatile method for PFAS in food Challenges unique to PFAS Trace levels, MRL at 0.5 ng/g No instrument contamination Resolution Handle complicated matrix 0.05 ng/g

Agustin Pierri, PhD agustin.pierri@wecklabs.com Questions? Agustin Pierri, PhD agustin.pierri@wecklabs.com 626-336-2139 Weck Laboratories, Inc. Industry, CA 91745