NCSU METRIC - 20211103 - OE240 PFAS Method Validation

OE240 PFAS Method Validation
  • Instrument: Orbitrap Exploris 240
  • SpikeIn: No
  • Keywords: PFAS, Orbitrap, validation
  • Lab head: Jeffrey Enders Submitter: Jeffrey Enders
The detection and quantitation of per- and polyfluoroalkyl substances (PFAS) is becoming an increasingly important aspect of environmental remediation processes. While, historically, two disparate mass spectrometry technologies, triple quadrupole and high-resolving power platforms, have risen to the forefront for either quantitation or detection, the use of a single platform to perform both functions would be highly advantageous to forwarding these efforts. Additionally, the highly cooperative nature of these environmental missions would be greatly aided by the use of vendor-neutral software for data analysis and data sharing/collaboration. Here we present an absolute quantitative LC-MS method performed on a high-resolving power orbitrap instrument utilizing Skyline software for data analysis. This method was validated for 45 PFAS compounds (incorporating 23 internal standards) across 7 different chemical classes using recommended guidelines found in EPA Method 537.1. It achieves limits on par with previous methods, but importantly does so without the need for upfront pre-concentration/extraction.
Experiment Description
LC-MS Method A reversed-phase LC setup consisting of a Thermo Scientific Vanquish LC system (Germering, Germany) was used. Aqueous (solvent A: water with 5% ACN and 0.1% formic acid) and organic (solvent B: ACN with 5% water and 0.1% formic acid) solvents were run at 500 μL/min using the following gradient: 0 min: 1% B, 2 min 1% B, 13 min: 70% B, 13.01 min: 99%B, 17 min: 99% B, 17.01 min: 1% B, 20 min 1% B. A Phenomenex Kinetex 2.6 μm particle, F5 100 Å, 100 × 2.1 mm analytical column (part number 00D-4723-AN) was used for separation. A Thermo Scientific 1.9 µm particle Hypersil GOLD column, 50 × 3.0mm (25002-053030) was placed in the flow path after the binary pumps but before the autosampler to delay any potential PFAS present in the LC solvents and thus prevent their integration into the sample peak area. The autosampler was operated at 10 °C, while the column compartment was set to 45 °C. A 100 μL sample loop was used to accommodate a 100 μL injection volume. The system was also upgraded to be “PFAS ready” by Thermo Fisher Scientific. For mass analysis an Orbitrap Exploris 240 (Thermo Fisher Scientific, Bremen, Germany) was used. A negative mode full scan event at 60,000 resolving power at m/z = 200FWHM was “triggered” for MS2 by detection of an exact mass as dictated by a list of analytes which was entered into a Targeted Mass Filter. The MS2 scan was run at 15,000 resolving power at m/z = 200FWHM with dynamic exclusion turned off. A second identical but positive mode event was run from minutes 9.5-11.5 in order to capture the zwitterions (N-AP-FHxSA, N-CMAmP-6:2FOSA and N-TAmP-FHxSA). Optimized settings for the various analytes in this panel were often mutually exclusive (i.e., the most ideal settings for some analytes were the least ideal for other analytes). These differences often manifested according to analyte mass/chain length/hydrophobicity. As such, dynamic source settings were utilized. Negative spray voltage was (-) 1000 V from 0 min to 11.6 min and (-) 1500 V from 11.6 min to 20 min. From 0 min to 11.6 min sheath gas flow/Aux gas flow/Sweep gas flow were set to 50/12/0.5 and from 11.6 min to 20 min, these same settings were at 30/10/1. The ion transfer tube temperature was 240 °C and the vaporizer temperature was 300 °C. Additionally two MS1 mass ranges were used. For the lower mass analytes a range of 50-600 Da was optimal, however, these settings rendered many high mass analytes undetectable. For this reason a second MS1 event that incorporated a mass range of 150-1500 Da was used. Although this mass range also captured the lower mass analytes their sensitivity was drastically reduced under these conditions. Skyline Data Processing Skyline Daily, version was used for data processing. Thermo data (.RAW) were imported into Skyline without any need for manipulation or conversion. PFAS target lists were generated by using the “Edit → insert → transition list” function. Analyte precursor masses were entered (based on molecular formulas) as well as product ion masses that were observed upon infusion of pure standards (after collision energy optimization) on the instrument. Method Validation The method was validated in neat solvents (i.e., water and methanol), roughly in accordance with guidance detailed in EPA Method 537.1. Minimum reporting limit (MRL) and detection limit (DL) were determined as is detailed in this same method. Briefly, 5-10 replicates of the calibration curve were prepared and run. By treating one of the replicates as the calibration curve and the others as unknowns, a large amount of replicate calibration curve data is produced. The half range for the prediction interval of results (HRPIR) was calculated according to: 〖HR〗_PIR=s × t_(df,1- .01/2 ) × (1+ 1/N) Where, s is the standard deviation, t_(df,1- .01/2 ) is the two-tailed 99% confidence interval student t value for the appropriate degrees of freedom and N is the number of replicates used. Then, to determine the MRL, a calibration curve point’s replicate data was required to meet the following criteria: The upper PIR limit must be ≤ 150% recovery (Mean+ 〖HR〗_PIR)/(Actual Concentration) ×100% ≤150% The lower PIR limit must be ≥ 50% recovery (Mean- 〖HR〗_PIR)/(Actual Concentration) ×100% ≥50% The DL was determined by subjecting the MRL concentration’s data to the following calculation: DL=s × t_(df,1- .01/2 ) The initial demonstration of precision (IDP) is the relative standard deviation of the replicate data at a mid-level concentration (in this case 500 ng/L), while the initial demonstration of accuracy (IDA) was the accuracy of the replicate data compared to the prepared concentration at a mid-level concentration (in this case 500 ng/L).
Sample Description
Standards All experiments were performed on an Orbitrap Exploris 240 (Thermo Scientific, Bremen, Germany) incorporating a Thermo Scientific Vanquish LC system (Germering, Germany). Two unlabeled PFAS mixtures were acquired from Cambridge Isotope Laboratories (Tewksbury, MA). Mix 1 (2 µg/mL each) contained 1H,1H,2H,2H-Perfluorohexane sulfonate, Na salt (4:2 FTS), 1H,1H,2H,2H-Perfluorooctane sulfonate, Na salt (6:2 FTS), 1H,1H,2H,2H-Perfluorodecane sulfonate, Na salt (8:2 FTS), 1H,1H,2H,2H-Perfluorododecane sulfonate, Na salt (10:2 FTS), N-Ethylperfluoro- octanesulfonamidoacetic acid (mixed isomers) (NEtFOSAA), N-Methylperfluoro- octanesulfonamidoacetic acid (mixed isomers) (NMeFOSAA), Perfluorooctanesulfonamide (PFOSA), N-Methylperfluorooctanesulfonamide (MeFOSA), Perfluorobutane sulfonate, K salt (PFBS), Perfluorohexane sulfonate (mixed isomers), K Salt (PFHxS), Perfluorooctane sulfonate (mixed isomers) (PFOS), Perfluorobutyric acid (PFBA), Perfluoropentanoic acid, Na salt (PFPeA), Perfluorohexanoic acid, Na salt (PFHxA), Perfluoroheptanoic acid (PFHpA), Perfluorooctanoic acid (PFOA), Perfluorononanoic acid (PFNA), Perfluorodecanoic acid, Na salt (PFDA), Perfluoroundecanoic acid, Na salt (PFUdA), Perfluorododecanoic acid, Na salt (PFDoA), Perfluorotetradecanoic acid (PFTeDA), and Tetrafluoro-2-(heptafluoropropoxy)propanoic acid (HFPO-DA [Gen-X]). Mix 2 (1 µg/mL each) contained Perfluorobutane sulfonamide (FBSA), Perfluorohexane sulfonamide (FHxSA), Perfluorohexadecanoic acid (PFHxDA), Perfluorooctadecanoic acid (PFODA), Perfluoropentane sulfonate - Na salt (PFPeS), Perfluoroheptane sulfonate - Na Salt (PFHpS), Perfluorononane sulfonate (linear) - Na Salt (PFNS), Perfluorodecane sulfonate - Na Salt (PFDS), Perfluorotridecanoic acid (PFTrDA), 7:3 Fluorotelomer carboxylic acid, FHpPA, 3-Perfluoroheptyl propanoic acid(7:3) (7:3 FT(C)A), N-(3-dimethylaminopropan- 1-yl)perfluoro-1-hexane-sulfonamide (N-AP-FHxSA), N-(carboxymethyl)-N,N- dimethyl-N-[3-(1H,1H,2H,2H-perfluoro-1- octanesulfonamido)propan- 1-yl]ammonium (N-CMAmP-6:2FOSA [6:2 FTAB]), N-[3-(perfluoro-1- hexanesulfonamido)propan-1-yl]-N,N,N-trimethylammonium (N-TAmP-FHxSA), Sodium dodecafluoro-3H-4,8-dioxanonanoate (NaDONA), Ethanesulfonic acid, 2-[1-[difluoro[(1,2,2-trifluoroethenyl)oxy]methyl]- 1,2,2,2-tetrafluoroethoxy]-1,1,2,2-tetrafluoro, Perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid (Nafion by product 1, also known as PS Acid), Ethanesulfonic acid, 2-[1-[difluoro(1,2,2,2-tetrafluoroethoxy)methyl]- 1,2,2,2-tetrafluoroethoxy]-1,1,2,2- tetrafluoro-polymer with 1,1,2,2-tetrafluoroethene, 7H-Perfluoro-4-methyl-3,6- dioxaoctanesulfonic acid (Nafion by product 2), 11-chloroeicosafluoro-3-oxaundecane-1-sulfonic acid (F53B Minor [11Cl-PF3OUdS]), 9-chlorohexadecafluoro-3-oxanone-1-sulfonic acid (F53B Major [9Cl-PF3ONS]) and Perfluoro-2-methoxyacetic acid (PFMOAA). 2,3,3,3-tetrafluoro-2-(1,1,2,2,2-pentafluoroethoxy)propanoate (PEPA), sodium nonafluoro-2,4,6-trioxaoctan-8-oate (PFO3OA), sodium undecafluoro-2,4,6,8-tetraoxadecan-10-oate (PFO4DA), and sodium tridecafluoro-2,4,6,8,10-pentaoxadodecan-12-oate (PFO5DoA) were acquired from Fluoryx Labs (Carson City, NV). A stable isotope labeled internal standard mixture (1 µg/mL) was also produced by Cambridge Isotope Laboratories and contained the following: 1H,1H,2H,2H-Perfluorohexane sulfonate, Na salt (13C2/D4) (13C2/D4-4:2 FTS), 1H,1H,2H,2H-Perfluorooctane sulfonate, Na salt (13C2/D4) (13C2/D4-6:2 FTS), 1H,1H,2H,2H-Perfluorodecane sulfonate, Na salt (13C2/D4) (13C2/D4-8:2 FTS), 1H,1H,2H,2H-Perfluorododecane sulfonate - Na salt (13C2/D4) (13C2/D4-10:2 FTS), N-Ethylperfluorooctanesulfonamidoacetic acid (D5) (D5-NEtFOSAA), N-Methylperfluorooctanesulfonamidoacetic acid (D3) (D3-NMeFOSAA), Perfluoro-1-[13C8]octanesulfonamide (13C8-PFOSA), N-Methylperfluorooctanesulfonamide (D3) (D3-MeFOSA), Perfluorobutane sulfonate, K salt (13C4) (13C4-PFBS), Perfluorohexane sulfonate, K salt (13C6) (13C6-PFHxS ), Perfluorooctane sulfonate, Na salt (13C8) (13C8-PFOS ), Perfluorobutyric acid, Na salt (13C3) (13C3-PFBA), Perfluoropentanoic acid, Na salt (13C5) (13C5-PFPeA), Perfluorohexanoic acid, Na salt (13C6) (13C6-PFHxA), Perfluoroheptanoic acid, Na salt (13C7) (13C7-PFHpA), Perfluorooctanoic acid (13C8) (13C8-PFOA), Perfluorononanoic acid (13C9) (13C9-PFNA), Perfluorodecanoic acid (13C9) (13C9-PFDA), Perfluoroundecanoic acid, Na salt (13C9) (13C9-PFUdA), Perfluorododecanoic acid, Na salt (13C12) (13C12-PFDoA), Perfluoro-n-[1,2-13C2]tetradecanoic acid (13C2-PFTeDA), Perfluoro-n-[1,2-13C2]hexadecanoic acid (13C2-PFHxDA), Tetrafluoro(heptafluoropropoxy)[13C3]propanoic acid (13C3-HFPO-DA [Gen-X]). Sample Preparation The internal standard was prepared at 10,000 ng/L by diluting the stock in 50:50 MeOH:Water with 0.1% formic acid using volumetric glassware. Calibrators were prepared by combining the appropriate volumes of mix 1 and mix 2 and diluting to mark with 50:50 MeOH:Water with 0.1% formic acid using volumetric glassware. Internal standard and calibrators were mixed 10:1 (e.g., 900 μL of calibrator mixed with 100 μL of IS) to produce a final sample composition of 50% MeOH. All solutions were stored in polypropylene (PP) conical tubes. Maintaining a minimum organic composition of 50% was necessary to insure solubility of the hydrophobic compounds to the walls of these PP tubes and LC vials.
Created on 12/22/21, 11:45 AM
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20211103 - Enders - OE240 PFAS Method 11:45:05140117165686140117