Rapid Characterization of Per- and Polyfluoroalkyl Substances (PFAS) by IMS-MS.

[NNadir]

The paper I'll discuss in this post is from the "ASAP" section (as of 3/08/20) of the journal Analytical Chemistry:

Rapid Characterization of Per- and Polyfluoroalkyl Substances (PFAS) by Ion Mobility Spectrometry–Mass Spectrometry (IMS-MS) (Erin Baker et al, Anal Chem ASAP 3/08/20)

I had the privilege of seeing Dr. Baker speak on two occasions, once in Boston and once in New Jersey. At that time she was at the PNNL (Pacific Northwest National Laboratory) in Richland, Washington; she has now joined the faculty at North Carolina State.

She is an outstanding rising young scientist from whom the world can expect great things.

Dr. Baker's work when I saw her speak concerned the "Exposome" which is an attempt to measure the effect of non-genetic chemicals on human health.

The Human Exposome Project

This is about the physiological impact of pollutants.

In a purely analytical sense, determinations of the concentration of pollutants in various matrices, blood, plasma, tissue and environmental matrices such as air, water, soil and even household dust is very challenging because the concentrations are low enough as to be inaccessible to spectroscopic dependent detection, such as straight up HPLC or UV/VIS or IR detection, but high enough to have profound health effects. A more sensitive method of detection, in which one can typically see concentrations on the order of a picogram per milliliter is mass spec. Unfortunately, mass spec is poor at differentiating compounds that are not identical but have the same molecular weight: We call these "isobaric species."

The last lecture I attended where Dr. Baker was speaking was on the topic of perfluoro compounds such as PFOA, PFOS, and similar compounds. These used to be considered "emerging pollutants." Arguably they have fully emerged into a full blown problem. While the structure and molecular weight of the most common forms of these are known, it is not generally possible by traditional mass spec means to determine the precise (or better put "most likely&quot structure of a particular degradatant. Let's see if I can explain this clearly. Here is the structure of PFOA, perfluorooctanoic acid:



It is possible to completely mineralize this molecule by exposure of it to gamma radiation under the right conditions over a period of time, but in the environment, where radiation is not generally available, it is mostly partially defluorinated, usually where one or two of the fluorine atoms in the molecule is replaced by a hydrogen (or a hydroxide). The question is which fluorine? If one replaces the fluorine on carbon 2, the one immediately adjacent to the carboxylic group, this will be a different molecule than one in which the fluorine replaced is on carbon 4, which will be different than both if the carbon replaced is on carbon 6. Then there are cases where two fluorines are replace, or three, and so on.

These are all different molecules and all can have very different physiological effects.

Yet any species in which only one fluorine is replaced will have the same molecular weight as any other species.

Enter Doctor Baker and her work with ion mobility spectroscopy. In this approach, one takes advantage of slightly different cross sections in an electric field, the molecules of the same molecular weight will move at different rates depending on how the charges are distributed over the molecules surface area.

From the introduction to the paper:

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants characterized by extensive fluorination along extended aliphatic chains, rendering them chemically inert and thermally stable. Their physiochemical properties are also uniquely suited for many household and industrial applications, such as the production of stain-resistant carpeting, nonstick cookware, and aqueous film-forming foams (AFFF).(1,2) The production of long chain PFAS, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), began in the U.S. during the 1950s and continued into the early 2000s. However, toward the end of this time period, manufacturers began to phase out long chain chemistries because of evidence that PFAS exposure is related to various adverse health outcomes, including ulcerative colitis,(3) thyroid disease,(4) cancer,(5) elevated cholesterol,(6) and decreased immune function,(7) and corresponding pressure from regulatory agencies.(8,9) In the U.S., enforceable drinking water standards for PFAS are lacking at the federal level, but the U.S. EPA published a lifetime health advisory level for the sum concentration of PFOA and PFOS at 70 parts-per-trillion (or ng L–1) in May 2016.(10) While long chain PFAS production continues in emerging ADONA (see Supporting Information Figure S1).(11−13) Although toxicological data of emerging PFAS are scarce, the occurrence and persistence of PFECAs in the environment is already being documented.(12−17)

While the chemical structure and isomeric diversity of legacy PFAS are well-characterized, understanding the evolving structure of emerging fluorinated compounds is a challenge.(14) Legacy PFAS isomers result largely from impure chemical syntheses, where the linear form is the desired target (e.g. PFOS, PFOA), but side reactions also produce branched isomers.(18) Emerging PFAS possess both linear and branched structures, typically characterized by flexible ether linkages within the CF2 branches (e.g. GenX, see Supporting Information). To date, several analytical strategies have been employed to characterize the isomeric PFAS.(19−21) These techniques predominantly consist of gas or liquid chromatography (GC or LC) coupled with mass spectrometry (MS). In isomer characterization studies, spatial annotation of branched isomers is particularly challenging as described by previous studies.(22,23) Highly selective chromatographic methods are often employed in these studies and many take over 1 h of analysis time per sample. Analyte derivatization is also regularly utilized, further lengthening the required analysis time.(21,24) Similar fragmentation profiles (MS/MS) for the branched PFAS isomers also complicate the analyses and make the isomer distinction even more difficult (see Supporting Information Figure S2). Additionally, these analyses require reference standards, which are not always available, for replacement PFAS chemistries and manufacturing byproducts.

Ion mobility spectrometry (IMS) is an emerging separation technique that distinguishes ions based on their size, shape, and charge state in the gas phase.(25) GC and LC differ from IMS in that they separate analytes based on differences in boiling point and polarity, while IMS separates ions based on differences in gas phase electrophoretic mobility. IMS separations occur post-ionization in a defined mobility separation region filled with an inert gas (also termed as the buffer gas). Ions traverse this region under the influence of an applied electric field, wherein their migration time through the gas (drift time) is directly correlated with ion size. The measured size is commonly described as the ion’s collision cross section (CCS, typically denoted in units of Å2)...

Some pictures from the text:

Here's a cartoon about the technology.



The caption:

Figure 1. LC-IMS-MS instrumentation and spectra utilized in this manuscript. (A) Instrumental schematic for the DTIMS-QTOF MS platform used in this work (Agilent 6560). (B) RPLC-IMS-MS workflow designed to separate and characterize PFAS isomers and subclasses. The RPLC, IMS, and MS stages are illustrated separately along with their corresponding mechanisms for structural discrimination. (C) IMS drift time profiles possible for isobaric and isomeric separations. Deprotonated ions for 6:2 FTS and Hydro-EVE are illustrated along with their resolution in a mixture. (D) Drift time aligned MS/MS fragments of the 6:2 FTS and Hydro-EVE isobars illustrating the different components for each molecule and their distinction with IMS at 5 V CID energy and mass selection of precursor ion 426.9 m/z (asterisk).

In this case, Dr. Baker is referring to an Agilent instrument; in much of her work she's been rather agnostic about particular manufacturers, and has worked with several of the major mass spec companies, including work on the Thermo Orbitrap instrument.

North Carolina is lucky to have her down there, since she's analyzing local samples and can tell them what's going on in their state:

Water samples were collected from the old wastewater outfall of a local chemical plant (Chemours, Fayetteville, NC) and a nearby lake (Marshwood Lake, Grays Creek, NC) to evaluate the robustness of the IMS CCS values for PFAS in complex matrices. Our sampling protocol was modeled after previous studies by Hopkins et al. using a modified variant of EPA method 537. This method is described in further detail in the Supporting Information (Table S2).(13) Briefly, 1 L of water was collected at the sampling site in 1 L polypropylene bottles, and five mL of 35% HNO3 was added as a biocide prior to storage. Fifty mL of the collected sample was then filtered through 0.45 μm glass microfiber filters, and PFAS were extracted using Oasis Wax Plus Short Cartridges (Waters Corporation, Milford, MA) as recommended by the manufacture protocol.(53) After extraction, samples were evaporated to near dryness using a Savant SPD131DDA SpeedVac Concentrator (ThermoFisher Scientific, Waltham, MA) and reconstituted to 0.5 mL in 18 MΩ water prior to LC-IMS-MS analysis...

Here's some of the stuff she found:



The caption:

Figure 2. Mass/CCS trendlines for the specific PFAS subclasses analyzed (e.g. FTSA, PFSA, etc.). Specific PFAS headgroups contribute significantly to the observed CCS for a given m/z in each PFAS subclass. The varying chain lengths of CF2 then produce the linear trendlines observed within each subclass. Specific compounds discussed in the main text (e.g. 6:2 FTS and Hydro-EVE) are noted in addition to the PFAS subclasses.

A note: You see that "Nalfion byproducts" stuff in there? "Nalfion" is a common proton transfer agent, a perfluoropolymer related to Teflon that is utilized widely in certain classes of hydrogen fuel cells, which are often discussed in terms of making so called "renewable energy" sustainable and effective and reliable, although it continues to fail at being any of these things. Nalfion, like Teflon, is a long term intractable persistent pollutant; without extreme conditions, the only really effective one being exposure to ionizing radiation, any of this stuff will be here forever.

Some commentary on calibrations using standards:

Relationships between Isomeric Branching, CCS, and Retention Time

As the trendlines for PFCA and PFSA subclasses shown in Figure 2 are based on primarily linear standards, another key focus of this work was to describe how isomeric branching affects CCS and causes deviations from the trendlines. To investigate this question, we obtained standards of specific branched isomers for PFOA and PFOS from Wellington Laboratories. Linear and branched isomers were characterized using both LC (retention time) and IMS (CCS) to examine the complementary separation of each method (Figure 3). For chemical structures of the singly methylated and dimethylated isomers, see Supporting Information Figure S5. In all cases, the dimethylated isomers (DMHxA and DMHxS) elute from the LC column first, followed by the singly methylated isomers (MHpA, MHpS) and finally each respective linear form (PFOA or PFOS, Supporting Information Figure S5). In the IMS analyses, the CCS values of the dimethylated isomers are also smaller than the singly methylated isomers (with the exception of 5,5 dm-HxS), analogous to the elution pattern noted with reverse-phase liquid chromatography (RPLC). This result makes qualitative sense as the dimethylated and methylated forms should be more compact than the linear forms of PFOA/PFOS and hence have smaller CCS values.

A graphic showing these effects:



The caption:

Figure 3. Correlation of RPLC retention time and CCS to specific constitutional isomers for PFOA (A) and PFOS (B). Dimethylated isomers elute earlier in RP-LC and tend to be more compact (smaller CCS) in comparison to singly methylated isomers or linear forms of PFOA and PFOS (C and D). Error bars for both variation in retention time and CCS are illustrated, though some error bars are within marker size.

Before the next graphic, a little text about what is happening:

When both LC and IMS separation are utilized cooperatively, it is possible to begin to characterize PFAS with multiple isomeric contributions, such as the manufacturing sample of PFOS we obtained from Synquest (see Figure 4). Previous characterizations of PFOS have illustrated as many as 16 isomers in a single sample.(22,56) Our analysis of PFOS produced 7 main isomeric contributors by LC-IMS-MS analyses which are labeled A, B, C, D, E, F, and G in Figure 4. The observed LC trace is very similar to previous studies which have characterized specific branched PFOS isomers in detail (Supporting Information Figure S6).(19,57) Interestingly the new data added from the IMS dimension shows that the PFOS isomers cover a wide range of CCS space for an individual chemical formula (6.7 Å2, a difference in CCS of 4.1%, Figure 4). Analysis of the LC-IMS data shows that the principal peak (component F) is the linear species, which previous publications cite as contributing ca. 70% of relative abundance.(18,22) Components A and B were annotated as dimethylated PFOS isomers, and components C, D, E, and G are singly methylated isomers.

The graphic:



The caption:

Figure 4. Separation of seven PFOS isomers by LC-IMS-MS. (I) While several publications have noted isomeric diversity for PFOS, this work notes 7 main contributors in our LC-IMS-MS analysis. The complementary separation mechanisms of analyte polarity from LC and molecular size (IMS) provide a more comprehensive snapshot of relative peak volume for each isomer (II). Converting observed drift times to CCS (inset table) further provides a metric for verification if previously measured standards have been analyzed. The retention time and CCS values from Figure 3B are used to make tentative annotations to the spectra. (*) While some components are easily annotated using both retention time and CCS for characterization, component A is very close to several DMHxS isomers and component C is close to both the 4th and 5th methylation site (4/5)-MHpS isomers.

The workflow for monitoring the environment:

Figure 5. (A) Experimental workflow for PFAS analysis of Marshwood Lake, spatially located near a chemical plant in North Carolina. After preconcentration with SPE and subsquent LC-IMS-MS analysis, measured CCS of detected PFAS correspond in good agreement to experimental standards (B). Retention time and accurate mass were also used for verification of annotated features (Supporting Information Table S4).

Some other commentary:

To evaluate the robustness of our LC-IMS-MS method in environmental samples, we analyzed samples collected from the wastewater outfall of a chemical plant (Chemours, Fayetteville, NC, Figure 5A) and nearby Marshwood Lake. This region of the Cape Fear River watershed has been the focus of prior investigations related to PFAS contamination and has been found to contain both legacy and emerging PFAS, such as GenX.(12−14) After preconcentration of 50 mL water using SPE, we detected several PFAS from the sulfonic acid subclass (PFSA) and several emerging PFESAs using LC-IMS-MS (Figure 5A and B). NVHOS and Nafion byproducts have recently been characterized by untargeted LC-MS/MS studies from McCord and Strynar,(14) and their presence is not surprising considering our sampling sites are located in the same geographical area. In addition to high mass measurement accuracy and retention time alignment afforded by our instrument setup (see ES1), observed CCS values for detected analytes in the environmental samples are in good agreement with CCS from standards (typically <0.5% difference) as shown in detail in the Supporting Information Table S4. Results such as these illustrate the potential benefits of adding the IMS separation to current workflows.

Mmmmmmmm...Nalfion...delicious...green...green...green...

Some concluding remarks from Dr. Baker and her co-workers:

As CCS values have been previously shown to be highly reproducible (often less than 1.0% different between laboratories),(48) CCS values in this manuscript can be used as reference standards for laboratories that do not have access to analytical standards for PFAS observed in our analyses, especially for manufacturing byproduct chemistries (e.g. NVHOS and Nafion Byproducts). Utilizing database CCS matching for unknown analytes provides additional molecular confidence in annotating unknown features in untargeted studies, especially when combined with other molecular descriptors such as mass defect analysis,(14) isotope ratio patterning, and fragmentation methods.(51) In addition, as IMS is a chemically independent separation, the methods described in this manuscript can be readily adapted for screening of other environmental pollutants, such as pesticides, PCBs, and PAHs.(31)

A wonderful paper from a wonderful scientist.

Have a nice day tomorrow.