PFAS Testing Methods and Guidance for Sample Filtration

Recommended filtration products for PFAS testing

Table 1. Millex^® syringe filter devices and cut disc membrane filters appropriate for PFAS testing-related sample preparation and mobile phase filtration. Recommendations are based on testing water samples using modified EPA537.1 and methanol samples with modified EPA 1633. *Hydrophilic polypropylene can be used for sample/mobile phase filtration of aqueous solvents. Hydrophobic polypropylene is recommended for sample/mobile phase filtration of organic solvents.

Filter Format	Membrane	Pore Size (µm)	Diameter (mm)	Product
Syringe Filter ·       For sample filtration	Millipore PES Express®	0.22	33	SLGP033N
		0.45	33	SLHP033N
	Nylon	0.20	33	SLGN033
	Nylon with glass fiber prefilter	0.20	25	SLGNM25
Cut Disc Membrane Filter* For sample, mobile phase, and buffer filtration	Hydrophilic and hydrophobic polypropylene	0.20, 0.45	13, 25, 47, 90	Polypropylene membrane filters

What Are Poly- and Perfluorylalkyl Substances (PFAS)?

PFAS are poly- and perfluorylalkyl substances known as “forever chemicals” and comprise a group of over 4,000 varieties of long- and short-chain perfluorinated compounds.¹ PFAS are used in a variety of industries for their excellent oil-, water-, temperature-, chemical-, and fire-resistant properties and noted for their use in polymerization reactions of fluoropolymers such as Teflon^® by companies such as 3M and Dupont. Products that contain PFAS and related compounds are ubiquitous in industrial and consumer products, including product packaging, cosmetics, non-stick cookware, stain repellents, polishes, paints, coatings, and firefighting foams.

The excellent properties and broad use of PFAS have led to the persistent accumulation of these man-made chemicals in environmental and biological matrices, recently linked to liver damage, cancer, weakened immune system, and high cholesterol in humans.^1-3

Sample Filtration in PFAS Testing

Increasing concern about their threat to human health has sparked a cascade of regulatory action in the US and Europe. The primary focus is on water matrices starting with drinking water and then focusing on higher particulate water matrices and beyond. The primary analytical method for testing PFAS in water is solid phase extraction followed by liquid chromatography tandem mass spectrometry.

In all analytical methods, sample preparation should be carefully considered. However, in PFAS workflows, additional factors could complicate PFAS analyses downstream. These include potential PFAS contamination from filters or other consumables that contact the samples and adsorption of PFAS compounds onto consumables, leading to loss of recovery. Thus, we tested PES, nylon, and nylon-HPF syringe filter devices within PFAS detection workflows EPA 537.1 and EPA Draft 1633 to determine levels of contamination resulting from membrane extractables. We also tested polypropylene cut disc membrane filters (0.2 µm and 0.45 µm hydrophilic and hydrophobic polypropylene for EPA 537.1 and 0.2 µm hydrophilic polypropylene for EPA Draft 1633). We found that none of the filters had any detectable levels of PFAS contamination above the reporting limits (RL). Adsorption of internal standards leading to some loss of recovery occurred primarily for nylon and hydrophobic polypropylene membrane materials, which varied according to PFAS type, chain length, and filtrate material (methanol vs. water). Filtration in methanol demonstrated better recoveries of the same standards for nylon. This supports the suggestion that rinsing with methanol can reduce binding of PFAS compounds to filter materials. Hydrophilic polypropylene performed similar in both methanol and water.

Therefore, when filtration of higher particulate samples is needed in a PFAS workflow, PES, nylon, and nylon-HPF Millex^® syringe filters, as well as polypropylene cut disc membrane filters, provide a suitable option.

Concerns about PFAS contamination from sample collection and preparation materials

In recent studies, there has been concern about adding PFAS contamination to samples from a variety of sources including collection bottles, solvents, storage vials, tubing components, and any other plastic that contacts the sample. This includes membrane filters and syringe filter housing used for clearing particulates from sample matrices. Some filters may show trace amounts of contamination that may interfere with LC-MS/MS detection of PFAS and resulting data, especially among increasing sensitivity requirements.⁶ Another concern specifically for consumables is the adsorption of PFAS compounds, such as onto filtration media or SPE sorbent. For filter devices, this is dependent on many attributes - most importantly the filter type, the solvent being filtered, and the type of PFAS molecule.^4-5 For example, in some cases, both contamination and sorption onto filtration media can be reduced by washing with methanol.^6-7

Testing Millex^® syringe filters using Modified EPA 537.1 & Modified Draft EPA 1633

Testing Millex^® Syringe Filters with Water Samples using Modified EPA 537.1

Materials and Methods

To alleviate this concern, three lots of polyethersulfone (PES), three lots of nylon, and two lots of nylon-HPF (nylon membrane with a glass fiber prefilter for high-particulate filtration) Millex^® syringe filter devices were tested for PFAS extractables according to EPA Method 537.1. Some PFAS compounds not required by the method, including next-generation PFAS compounds and fluorotelomer sulfonates, were also included. The compounds tested are listed in Table 1. EPA 537.1 does not require sample filtration, but filtration was used to provide a clean sample to test extractable contamination levels from the syringe filter. A comparison of those compounds tested to the compounds required by ASTM D7979-19, SW-846 Method 8327, ISO 21675, and EPA Draft Method 1633 are also included in Table 1.

An overview of the modified method EPA 537.1 is described in Figure 1, with the LC-MS/MS conditions in Table 4. Briefly, a 250 mL PFAS-free DI water sample was spiked with surrogates. The internal standard spike of 0.08 ppb was used for QC blanks. To determine if sample filtration media contributes to PFAS contamination, the entire sample was passed through the filter and into a styrene divinylbenzene (SDVB) SPE cartridge. The sample bottles and tubes were rinsed with basic methanol, passed through the filter and into the cartridge. The entire sample was then subjected to SPE and concentrated to 1 mL in 96:4% (v/v) methanol:water prior to LC-MS/MS analysis using a C18 column. Analysis was performed using internal standards. C-13 labeled standards were used in this study. The filters tested included: three lots of Millex®-GP syringe filters (non-sterile, 33 mm filter with Millipore Express® PLUS PES membrane) in both 0.22 µm (Cat. No. SLGP033N and 0.45 µm (Cat. No. SLHP033N) pore sizes, three lots of Millex^® nylon syringe filters (non-sterile, 33 mm filter with nylon membrane) in 0.20 µm pore size (Cat. No. SLGN033N), and two lots of Millex^® nylon-HPF syringe filters (non-sterile, 25 mm filter with nylon membrane and glass fiber prefilter) in 0.20 µm pore size (Cat. No. SLGNM25). 

Figure 1. Overview of the modified EPA 537.1 method.

• 250 mL water sample + surrogates – QC blank internal standard spike = 0.08 ppb
  
  
• Filtration with 0.22 µm or 0.45 µm Millex^® syringe filter
  
  
• SDVB SPE cartridge extraction (Methanol)
  
  
• Concentrate samples to 1 mL in 96:4% v/v Methanol:Water
  
  
• LC-MS/MS, C18 column
  
• Analysis by internal standards

Table 4. LC and MS/MS conditions used in this study.

Column:	C18, 100 x 2.1mm ID, 2.7 µm superficially porous particles
Mobile phase:	[A] DI Water, 0.1% (v/v) acetic acid; [B] Methanol (MeOH), 0.1% (v/v) acetic acid
Gradient	Time (min)	A %	B %		Flow (mL/min)
	0-0.0	65%	35%		0.4
	0-7.0	0%	100%		0.4
	7.0-10.0	0%	100%		0.7
	10.0-11.0	0%	100%		0.7
	11.0-15.0	65%	35%		0.4
Column temp:	50.0 °C
Injection volume	3-5 µL autosampler injection
Sample:	SPE eluate concentrated to 1 mL methanol:water, 96:4% (v/v)
MS/MS conditions	Parameter			Value
	Gas Temp (°C)			250
	Gas Flow (l/min)			10
	Nebulizer (psi)			50
	Sheath Gas Heater			275
	Sheath Gas Flow (L/min)			10
	Capillary (V)			4500
	V Charging			600
	Ionization Mode			Neg ESI
	Collision Cell Gas (PSI)			40
	Collision Cell Gas			UHP N2

Results

There were no detectable PFAS contaminants in any of the Millex^® devices tested using modified EPA 537.1 above the reporting limits (RL) or the minimum detection limits (MDL), even given the very low thresholds of 0.0020-0.0080 ppb and 0.0010-0.0020 ppb, respectively, depending on the compound (Table 5). The results were the same for the three different lots of the 0.22 and 0.45 µm PES membranes, three different lots of 0.20 µm nylon membranes, and two different lots of 0.20 µm nylon-HPF membranes. These results suggest that PES, nylon and nylon-HPF Millex^® filtration devices are reliable and appropriate to utilize in the filtration of water samples in preparation for the analysis of these PFAS compounds.

View Table 5 

Table 5. Detection of PFAS contaminants after filtration with Millex® PES and nylon devices using modified EPA 537.1.

Recovery

In this study, sample tubes and sample bottles were rinsed with basic methanol. The recoveries of the C-13-labeled standards were within the acceptable QC range for the method. However, recovery varied from filter material to filter material (Figure 2) and from compound to compound. For example, nylon-based filter devices demonstrated lower recoveries than those with PES. In the case of nylon membranes, non-specific adsorption of internally spiked standards and sample can be reduced using a methanol rinse.⁶ In fact, lower relative adsorption of most compounds was observed when the filtration was performed in methanol (EPA Draft 1633 results). Similar results were found using PES membranes alone (without housing and filtered using a 25 mm Swinnex^® filter holder device).

Figure 2. The average percent recovery of C-13 labeled standards for PFBS, PFBA, PFOA, PFOS and PFNA after filtration with nylon (blue, mean ± standard deviation (STDEV), n=9 replicates over 3 lots), nylon-HPF (green mean±STDEV, n=6 replicates over 2 lots) and PES (yellow, mean±STDEV, n=9 replicates over 3 lots) Millex^® syringe filter devices. The acceptable QC range for recovery of internal standards is demonstrated by the purple bars to the left for each compound.

 

PFAS methods for clean aqueous samples, such as EPA 537.1 or EPA 533 for drinking water, do not require filtration of samples. However, as more particulate-laden samples (such as wastewater, soil, and biosolids) require testing, filtration of aqueous samples becomes an increasingly important consideration to ensure data quality and protect instrument lifetime. When filtration becomes part of the analytical workflow, it is important to ensure that the filtration device does not introduce PFAS contaminants that would compromise the integrity of the data generated. 

Testing Millex^® Filtration Devices with Methanol using Modified EPA Draft 1633

Materials and Methods

To investigate PFAS extractables in methanol solvent, PFAS contamination of Millex^® syringe filter devices was tested according to a modified version of EPA Draft 1633 in collaboration with SGS North America (Orlando, FL location).

EPA Draft 1633 requires the detection of a large number of PFAS compounds (40 compounds, compared to EPA 537.1) in high-particulate matrices - either aqueous, solids, or tissues - and thus requires filtration. However, different sample processing and extraction methods are required by the method depending on the percent solids of the samples. Despite this, every sample matrix requires a filtration step after the addition of non-extracted internal standards (NIS) and/or carbon cleanup with a 0.20 µm nylon membrane, which is performed in methanol solvent.

An overview of the method is described in Figure 3, with the LC-MS/MS conditions in Table 6. Briefly, a 5 mL methanol sample was spiked with C-13 labeled extracted (EIS) and non-extracted (NIS) internal standards ranging from 1.25 - 10 ppb, depending on the compound, according to EPA Draft 1633. To determine if sample filtration media contributes to PFAS contamination, the entire sample was passed through the filter. The filtrate was collected and analyzed with LC-MS/MS using a C18 column. Analysis was performed using internal standards. The filters tested included: two lots of Millex^® -GP syringe filters (non-sterile, 33 mm filter with PES membrane) in 0.22 µm pore size (Cat. No. SLGP033N), two lots of Millex^® nylon syringe filters (non-sterile, 33 mm filter with nylon membrane) in 0.20 µm pore size (Cat. No. SLGN033N), two lots of Millex^® nylon-HPF syringe filters (non-sterile, 25 mm filter with nylon membrane and glass fiber prefilter) in 0.20 µm pore size (Cat. No. SLGNM25), and for comparison, two lots of a nylon syringe filter device in 0.20 µm pore size from the manufacturer listed in EPA 1633 (Pall).

Figure 3. Overview of the method used for modified EPA Draft 1633.

• 5 mL methanol sample + EIS and NIS mixes
  
  
• Filtration with Millex^® syringe filters (SLGP033N, SLGN033N, SLGNM25) or PPHG cut discs in SWINNEX
  
  
• LC-MS/MS, C18 column
• Analysis by internal standards

Table 6. LC and MS/MS conditions used in this study.

Column	C18, 2.7 µm, 100 x 2.1 mm ID, superficially porous column
Mobile phase	[A] Acetonitrile [B] 2 mM ammonium acetate in 95:5 water:acetonitrile
Gradient	Time (min)	A %	B %		Flow (mL/min)
	0.20	10.0%	90.0%		0.350
	4.00	30.0%	70.0%		0.350
	7.00	55.0%	45.0%		0.350
	9.00	75.0%	25.0%		0.350
	10.00	95.0%	5.0%		0.400
	10.30	95.0%	5.0%		0.400
	10.40	2.0%	98.0%		0.400
	11.80	2.0%	98.0%		0.400
	13.00	2.0%	98.0%		0.350
Column temp	50.0 °C
Injection volume	6-10 µL autosampler injection
MS/MS conditions	Parameter			Value
	Gas Temp (°C)			250
	Gas Flow (l/min)			10
	Nebulizer (psi)			50
	Sheath Gas Heater			300
	Sheath Gas Flow (L/min)			10
	Capillary (V)			3500
	V Charging			500
	Ionization Mode			Neg ESI
	Collision Cell Gas (PSI)			40
	Collision Cell Gas			UHP N2

Results

There were no detectable PFAS contaminants in any of the Millex^® syringe filters or other devices tested for modified EPA Draft 1633 above the reporting limit (RL) for the 40 compounds in any of the replicates and lots tested in methanol. However, in the category of perfluoroalkyl carboxylic acids (PFCAs), the first replicate device in Lot 1 of Millex^® nylon-HPF syringe filter devices demonstrated hits that were below the RL but above the minimum detection limit (MDL) of the instrument (Table 7). Because of this, one additional replicate device was tested and PFCAs were not detected above RL or MDL in that device. None of the other devices or lots tested for this material demonstrated hits for any PFCAs or other compounds. The Millex^® nylon syringe filter device lots performed the same as the nylon syringe filters from Pall. Regarding membrane material selection, researchers should always investigate whether levels of chemical extractables that may come after exposure to organic solvents are at appropriate levels.

View Table 7 

Table 7. Detection of PFAS contaminants after filtration with Millex® PES and nylon devices and nylon from a different manufacturer using LC-MS/MS according to modified EPA Draft 1633.

These results suggest that PES and nylon filtration devices are reliable and appropriate to use in the filtration with EPA Draft 1633 for analysis of PFAS compounds. Nylon-HPF Millex^® filtration devices should be considered when samples require pre-filtration, and researchers should always be aware of the required reporting limits for the method.

Chemical extractables (apart from PFAS compounds listed in Table 7) were not tested in this study. To get the best possible data quality, researchers should always investigate the levels of chemical extractables that may come after certain membrane materials are exposed to organic solvents to make sure that they are at acceptable levels.

Recovery

For filtration of C-13 labeled standards in water (see section for EPA 537.1), the average percent recovery of various standards for nylon-based syringe filter devices versus PES syringe filter devices was lower, indicating that there may be some binding of PFAS compounds onto the filter material or housing. This is a property of the nylon membrane material and has been shown with proteins as well. However, for EPA Method 1633 testing in methanol solvent, the percent recoveries for filtrates from nylon-based syringe filter devices versus PES-based syringe filter devices were at similar levels (Figure 4). This supports what has been reported in literature for methanol helping to dissociate PFAS molecules from filtration media.

Figure 4. The average percent recovery (mean ± standard deviation (STDEV), n=6 replicates over 2 lots) of C-13 labeled standards for PFBS, PFBA, PFOA, PFOS and PFNA after filtration with Millipore^® nylon (blue, mean±STDEV, n=9 replicates over 3 lots), nylon from another manufacturer (pink), nylon-HPF Millex^® (green) and PES Millex^® (yellow) syringe filter devices. The acceptable QC range for recovery of internal standards is demonstrated by the purple bars to the left for each compound.

Testing Millipore^® polypropylene membrane filters using EPA 537.1 & Modified Draft EPA 1633

The Relevance of Disc Membrane Filters in PFAS Workflows

Syringe filter devices are the most recommended and preferred format for filtering samples for LC-MS/MS analysis of PFAS due to ease of use and the range of small volumes that can be processed (10-100 mL). Further, syringe filter devices are compatible with larger throughput devices such as Samplicity^® G2 Filtration System, a vacuum driven device that can prepare multiple samples at once for LC-MS/MS.

There are instances, however, when syringe filters may not be the best option for filtration; for example, when there are no commercially available syringe filters suitable for a specific application. In these instances, an alternative must be considered. A syringe filter-like device, such as a Swinnex^® holder, is a viable alternative. This pressure-driven device holds any cut disc membrane filter of a specific size (13 mm or 25 mm diameter) and is operated in the same way as a traditional syringe filter, thus converting any membrane material into a syringe filter format.

Another instance where syringe filters might not be a suitable option would be for filtration of larger sample volumes (>100 mL). In these cases, a cut disc membrane filter, available in many different sizes and materials, with the appropriate filter holder, is a better choice. Cut disc membrane filters are commonly used in environmental methods for air matrices, to capture the desired compounds in the particulate phase, and then are extracted using a variety of different methods prior to chromatographic, gravimetric, or microscopic analyses. For LC-MS/MS-based workflows, cut disc membrane filters are also commonly used for the preparation of mobile phase mixtures and buffers. Filtration of the mobile phase ensures that particulates are not introduced into sensitive equipment, which is crucial for maintaining data quality for complex analyses. To filter mobile phases, filters are used in vacuum-based glassware filtration setups with flasks or vacuum-based plastic filtration setups using Millicup™-FLEX vacuum filtration devices with bottles to filter larger volumes of mobile phase prior to use in chromatography.

Testing Polypropylene Membranes for up to 40 PFAS Extractables

ASTM D7979 (for PFAS detection in water matrices excluding drinking water) and ASTM D7968 (for PFAS detection in environmental solids) both suggest the use of polypropylene for sample preparation and filtration. Polypropylene is a durable material compatible with a broad range of solvents and temperatures and demonstrates low extractables, making it appropriate specifically for PFAS-related sample and mobile phase preparation. A challenge with polypropylene is that it is naturally hydrophobic, which makes it challenging to filter aqueous samples. Most commercially available polypropylene disc filters are hydrophobic, such as Millipore^® polypropylene membrane filters Cat. No. PPTG04700 and Cat. No. PPTH04700. Though appropriate for solvents such as methanol, it can be challenging to filter aqueous samples. In a few cases, polypropylene can be found in a hydrophilic format (Millipore^® hydrophilic polypropylene membrane filters Cat. No. PPHG04700 and Cat. No. PPHH04700). These filters are suited to handle aqueous samples. Thus, realizing the potential for polypropylene material to be used within the context of a variety of PFAS workflows, including mobile phase filtration, we determined the level of PFAS extractables that these filter discs release.

Testing Millipore^® Polypropylene Membrane Filters with Water Samples using Modified EPA 537.1

Materials and Methods

Swinnex^® filter holder assembly

Hydrophilic and hydrophobic Millipore^® polypropylene (PP) membrane filters of 0.2 µm and 0.45 µm pore sizes were tested for PFAS extractable content. Swinnex^® devices (25 mm diameter) were used to convert the various disc membrane filters into a Luer-lock based syringe filter device, according to Figure 5. Once assembled, the Swinnex^® device can be connected to a Luer-lock syringe barrel with the material to be filtered. Filtration was then performed as with other syringe filter devices. For every disc replicate, a new and clean Swinnex^® device was used.

Figure 5. Assembling Swinnex^® device with a polypropylene cut disc membrane filter.

Modified EPA 537.1

 In this part of the study, four types of 25 mm cut disc membrane filters were tested:

• 0.2 µm hydrophobic PP (Cat. No. PPTG02500)
• 0.45 µm hydrophobic PP (Cat. No. PPTH02500)
• 0.2 µm hydrophilic PP (Cat. No. PPHG02500)
• 0.45 µm hydrophilic PP (Cat. No. PPHH02500)

Once each cut disc membrane filter was placed securely into a Swinnex^® device, 250 mL water samples spiked with surrogates were passed through each filter and into a styrene divinylbenzene (SDVB) SPE cartridge using EPA 537.1 for drinking water matrices as a guideline. The entire sample was subjected to SPE and concentration, according to procedures outlined in Figure 1. LC-MS/MS with a C18 column according to conditions in Table 4 was performed and analysis was carried out using internal standards to determine if there were levels of extractables present in the PP cut disc membrane filters. One lot (n=3 filters per lot) was tested per membrane type.

It is important to note that it is difficult to flow pure water through hydrophobic PP cut disc membrane filters; thus, for improved water flow for these samples, cut disc membrane filters were pre-wetted in methanol prior to filtering 250 mL water. There was no need to pre-wet hydrophilic PP cut disc membrane filters.

Results

As was found for Millex^® syringe filter devices, there were no detectable PFAS contaminants in any of the polypropylene cut disc membrane filters according to modified EPA 537.1 above the RL, ranging from 0.0020-0.0080 ppb, or the MDL, ranging from 0.0010-0.0020 ppb (Table 8). This indicates that these membranes do not have PFAS extractables at these limits and could be used for PFAS applications where filtration is needed for sample preparation.

However, for only the hydrophobic polypropylene membranes, there were four compounds (perfluoro-n-dodecanoic acid (PFDoDA), perfluoro-n-tridecanoic acid (PFTrDA), perfluoro-n-tetradecanoic acid (PFTeDA), and N-ethyl perfluorooctanesulfonamidoacetic acid (N-EtFOSAA)) where there were no detectable PFAS compounds, but the associated standards of ¹³C₂-PFDoA, ¹³C₂-PFTeDA and D₅-NEtFOSAA, demonstrated recoveries outside of the control limits of 40-140%, 30-130%, and 40-140%, respectively. These compounds demonstrated average recoveries of approximately ~15-25% (for PFDoDA, PFTrDA, and PFTeDA) and 30-33% (for N-EtFOSAA). This indicates that non-specific adsorption of these compounds onto hydrophobic polypropylene could be significant in water. Considering the long chain lengths and bulky functional groups of these compounds, there is a potential for hydrophobic and steric interactions with filtration media and other consumables. Interestingly, hydrophilic polypropylene recoveries remained within control limit ranges for all compounds, indicating that hydrophilization of the membrane material reduced any non-specific interactions with PFAS standards.

View Table 8 

Table 8. Detection of PFAS contaminants in water after filtration with hydrophobic 0.2 µm and 0.45 µm polypropylene disc filters and hydrophilic 0.2 µm and 0.45 µm polypropylene disc filters in Swinnex® devices using modified EPA 537.1.

Testing Millipore^® Polypropylene Membrane Filters with Methanol using Modified EPA Draft 1633

Materials and Methods

Swinnex^® filter holder assembly

Only the hydrophilic 0.2 µm polypropylene cut disc membrane filter (Cat. No. PPHG02500) was used in this study, assembled in a Swinnex^® filter holder as described for the modified EPA 537.1 study.

Modified Draft 1633

Three lots of the hydrophilic 0.2 µm polypropylene cut disc membrane filters were tested, using n=3 filters per lot. This was because, unlike EPA 537.1 which had to be modified to include filtration for this study, EPA Draft 1633 requires filtration with a hydrophilic nylon syringe filter device (0.2 µm pore size). Thus, 0.2 µm hydrophilic polypropylene was chosen as part of this study (for 0.2 µm nylon Millex^® syringe filter results, see “Testing Millex^® Syringe Filters with Methanol using Modified EPA Draft 1633” section.)

As described previously, EPA Draft 1633 outlines slightly different strategies of extraction and cleanup for each high-particulate matrix tested. Thus, this study focused on just the filtration step required for all matrices after the addition of non-extracted internal standards (NIS) and/or carbon cleanup performed in a methanol solvent. This was done to focus specifically on whether the cut disc membrane filters tested contained any contamination of the 40 PFAS compounds outlined in the method. This modified method is depicted in Figure 3.

Once each cut disc membrane filter was placed securely into a Swinnex^® device, 5 mL methanol samples spiked with C-13 labeled extracted (EIS) and non-extracted (NIS) internal standards according to the Method were passed through each filter. The filtrate was collected for LC-MS/MS using a C18 column using conditions described in Table 6 and analyzed by internal standards. Since the filtration was performed in methanol, none of the cut disc membrane filters required pre-wetting.

Results

As was found for Millex^® syringe filter devices, there were no detectable PFAS contaminants in any of the hydrophilic polypropylene cut disc membrane filters tested according to modified EPA Draft 1633 above the RL, ranging from 0.2-5 ppb, or the MDL, ranging from 0.05-1 ppb (Table 9). This indicates that these membranes do not have PFAS extractables at these limits and could be used for PFAS applications where filtration is needed for sample preparation. There were no challenges observed with recovery of any of the internal standards for EPA Draft 1633 in methanol, which is unsurprising. Our studies, and previous studies⁶ with nylon membranes indicated that exposure to methanol solvent reduced non-specific adsorption of PFAS compounds and internal standards.

View Table 9 

Table 9. Detection of PFAS contaminants in methanol samples after filtration with three different lots of hydrophilic 0.2 µm polypropylene disc filters in Swinnex^® devices in methanol solvent using modified EPA Draft 1633.

Recovery

Hydrophobic versus Hydrophilic Polypropylene Membranes. In water samples (modified EPA 537.1 study), the hydrophilic polypropylene membranes tested demonstrated recovery of internal standards within the QC range, while the hydrophobic membranes did not (Figure 6). The recoveries for ¹³C₂-PFDoDA, ¹³C₂-PFTeDA and D₅-EtFOSAA consistently demonstrated recovery outside of the QC range for hydrophobic polypropylene, making it difficult to quantify the true PFAS content for these compounds. Given that PFDoDA, PFTeDA and PFTrDA are long-chain PFCA molecules, observed loss may be due to steric hindrance and hydrophobic interactions with the membrane material or Swinnex^® housing material.

Figure 6. The average percent recovery of C-13 labeled standards for hydrophilic versus hydrophobic polypropylene cut disc membrane filters in water, for the perfluoroalkyl carboxylic acid class of PFAS only. Values are mean ± standard deviation, n=3 replicates. The QC range for the standards varies by compound, from left to right: 35-135% ( ¹³C₄-PFBA), 50-150% (¹³C₅-PFPeA through ¹³C₆-PFDA), 40-140% (¹³C₂-PFDoDA), 30-130% (¹³C₂-PFTeDA).

 

Water versus Methanol Samples. Using syringe filter devices, a trend was observed of increased recovery for nylon devices when filtering methanol versus water, which has also been observed in published studies.⁶ However, according to the results observed here, not all membrane materials may respond in the same way to filtering in water versus methanol. For example, hydrophilic polypropylene demonstrated similar recovery within the acceptable QC range of all internal standards for both methanol and water, and in some cases (for example, shorter chain PFCA and PFSA compounds) even higher recovery in water than methanol, which was not observed with either PES or nylon materials (see Figure 7).

Figure 7. The average percent recovery of all C-13 labeled standards for hydrophilic polypropylene cut disc membrane filters in water vs. methanol. For water, values are mean ± standard deviation, n=3 replicates of one lot tested. For methanol, values are mean ± standard deviation, n=3 replicates across three lots, for a total of nine discs.

 

Overall, hydrophilic polypropylene and hydrophobic polypropylene cut disc membrane materials do not demonstrate levels of PFAS contamination. There are nuances in binding and retention of PFAS compounds leading to different recoveries in the filtrate, as observed with polypropylene, nylon and polyethersulfone membrane materials (Figure 8). Recovery in methanol samples filtered through 0.2 µm hydrophilic polypropylene is similar to that using 0.20 µm nylon and 0.22 µm polyethersulfone. Together, this indicates that polypropylene cut disc membrane filters can be paired with Swinnex^® devices to offer an alternative sample filtration method to syringe filter formats. For PFAS methods where the mobile phase needs to be filtered, polypropylene membrane filters may be used with the appropriate filter holder. Recovery of certain PFAS compounds should be carefully considered as part of this workflow.

Figure 8. The average percent recovery of select C-13 labeled standards in methanol according to EPA Draft 1633 for 0.20 µm nylon syringe filters (blue), 0.22 µm PES syringe filters (yellow), and 0.2 µm hydrophilic polypropylene cut disc membrane filters (green). All values are mean ± standard deviation across three lots, for a total of nine discs.

Many PFAS test methods require filtration

In response, agencies in the US and Europe have taken regulatory action. The Stockholm Convention proposed regulations for two of the most common PFAS compounds — perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) — with certain exemptions, effective in 2020. The US Environmental Protection Agency (EPA) published an Action Plan in 2019 followed by recommendations for testing water matrices for PFAS compounds under the Safe Drinking Water Act in early 2020, with a drinking water advisory concentration of 70 parts per trillion (ppt). In October 2021, the EPA published its PFAS Strategic Roadmap, which outlines their extensive approach to addressing PFAS from 2021 through 2024. Most recently, the EPA released drinking water advisories for four PFAS compounds (PFOA, PFOS, Hexafluoropropylene Oxide (HFPO) dimer acid and its ammonium salt, and PFBS and its potassium salt). The European Union (EU) drinking water directive, which includes a limit of 0.5 µg/L for all PFAS, took effect in January 2021. Additionally, the European Chemicals Agency (ECHA) submitted a restriction proposal in January 2022 for PFAS in firefighting foams, with several other proposals expected through 2023. Additional PFAS substances are on the list for evaluation under REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals). In response to rapidly evolving regulatory proposals and actions, academic and industrial testing labs have developed analytical methods for testing and monitoring PFAS in a variety of matrices, such as those listed in Table 2. These regulations are important to understanding the extent of human exposure and environmental contamination to inform future remediation efforts.

Table 2. Selected analytical methods for the detection of PFAS compounds in various matrices.

Method	Update/Revision	Matrix/Matrices	Sample Preparation	Analytical Method
EPA 537.1	Jun. 2020	Drinking water	SPE	LC-MS/MS
EPA 533	Dec. 2019	Drinking water	SPE	LC-MS/MS
SW-846 Method 8327*	Jul. 2021	Non-potable groundwater, surface water, wastewater	SPE, filtration	LC-MS/MS
ASTM D7968-17a	Sep. 2017	Environmental solids	Solvent extraction, filtration	LC-MS/MS
ASTM D7979-19	Sep. 2021	Water matrix (no drinking water)	Solvent extraction, filtration	LC-MS/MS
ISO 21675	Oct. 2019	Drinking, natural and wastewater	SPE, filtration as needed	LC-MS/MS
FDA C-010.02	Dec. 2021	Foods	QuEChERS, SPE, filtration	LC-MS/MS
OTM-45	Jan. 2021	Air Emissions (stationary sources)	Sampling train: filtration, impingers	LC-MS/MS
EPA Draft 1633	Feb. 2022	Aqueous, soil, biosolids, sediment, tissue	SPE, filtration	LC-MS/MS
EPA Draft 1621*	Apr. 2022	Aqueous matrices	TSS, GAC column cleanup	CIC
*Screening method Abbreviations: SPE = solid phase extraction; TSS = total suspended solids determination; GAC = granular activated carbon; CIC = combustion ion chromatography

Comparison of PFAS compounds tested in EPA 537.1, EPA Draft 1633, ASTM D7979-19, SW-846 Method 8327, & ISO 21675

Currently, most analytical methods for water matrices involve solid phase extraction (SPE) sample preparation after the addition of internal standards and fortification of samples with surrogate standards, prior to liquid chromatography and tandem mass spectrometry (LS/MS/MS) for the detection of selected per- and polyfluorinated alkyl substances (usually 20-30 compounds) (Table 2).

Drinking water is considered a “clean” matrix and often does not require filtration ahead of sample preparation, such as in methods EPA 537.1 or EPA 533. However, methods such as SW-846 Method 8327, ASTM D7968-17a, ASTM D7979-20, ISO 21675 and EPA Draft 1633 involve matrices such as wastewater that could have a higher degree of particulates in them. One of the more recent methods, EPA Draft 1633 is undergoing multi-lab validation for the analysis of 40 PFAS compounds in a variety of high-particulate matrices, including tissue samples. Particulates in solution must be removed prior to LC/MS/MS as they can be detrimental to sample analysis, column longevity, and overall instrument function. These methods either require or suggest the need for filtration using membranes in a syringe filter format. The PFAS compounds required for each of these methods vary and are compared in Table 3.

Table 3. A comparison of the PFAS compounds required to be tested using EPA 537.1 for drinking water compared with those required by filtration-relevant methods, EPA Draft 1633, ASTM D7979-19, SW-847 Method 8327, and ISO 21675.

Compound	Abbreviation	EPA 537.1a	EPA Draft 1633b	ASTM D7979-19b	SW-846 Method 8327b	ISO 21675b
Perfluoro-n-butanoic acid	PFBA		✓	✓	✓	✓
Perfluoro-n-pentanoic acid	PFPeA		✓	✓	✓	✓
Perfluoro-n-hexanoic acid	PFHxA	✓	✓	✓	✓	✓
Perfluoro-n-heptanoic acid	PFHpA	✓	✓	✓	✓	✓
Perfluoro-n-octanoic acid	PFOA	✓	✓	✓	✓	✓
Perfluoro-n-nonanoic acid	PFNA	✓	✓	✓	✓	✓
Perfluoro-n-decanoic acid	PFDA	✓	✓	✓	✓	✓
Perfluoro-n-undecanoic acid	PFUnDA	✓	✓	✓	✓	✓
Perfluoro-n-dodecanoic acid	PFDoDA	✓	✓	✓	✓	✓
Perfluoro-n-tridecanoic acid	PFTrDA	✓	✓	✓	✓	✓
Perfluoro-n-tetradecanoic acid	PFTeDA	✓	✓	✓	✓	✓
Perfluoro-n-hexadecanoic acid	PFHxDA					✓
Perfluoro-n-octadecanoic acid	PFOcDA / PFODA					✓
Perfluoro-n-butanesulfonic acid	PFBS	✓	✓	✓	✓	✓
Perfluoro-n-pentanesulfonic acid	PFPeS		✓		✓
Perfluoro-n-hexanesulfonic acid	PFHxS	✓	✓	✓	✓	✓
Perfluoro-n-heptanesulfonic acid	PFHpS		✓		✓	✓
Perfluoro-n-octanesulfonic acid	PFOS	✓	✓	✓	✓	✓
Perfluoro-n-nonanesulfonic acid	PFNS		✓		✓
Perfluoro-n-decanesulfonic acid	PFDS		✓		✓	✓
Perfluoro-n-dodecanesulfonic acid	PFDoS		✓
n-Decafluoro-4-(pentafluoroethyl) cyclohexanesulfonate	PFecHS			✓
4:2 Fluorotelomer sulfonic acid	4:2 FTS 4:2 FTSA		✓		✓
6:2 Fluorotelomer sulfonic acid	6:2 FTS 6:2 FTSA		✓		✓	✓
8:2 Fluorotelomer sulfonic acid	8:2 FTS 8:2 FTSA		✓		✓	✓
8:2 Fluorotelomer unsaturated carboxylic acid	8:2 FTUCa/ 8:2 FOUEA					✓
3-Perfluoropropyl propanoic acid	3:3 FTCA		✓
2H,2H,3H,3H-Perfluorooctanoic acid	5:3 FTCA		✓
8:2 Polyfluoroalkyl phosphate diester	8:2 diPAP					✓
Perfluorooctanesulfonamide	PFOSA/FOSA		✓		✓	✓
N-methyl-perfluorooctanesulfonamide	N-MeFOSA		✓			✓
N-ethyl-perfluorooctanesulfonamide	N-EtFOSA		✓			✓
N-methyl Perfluorooctanesulfonamidoacetic acid	N-MeFOSAA		✓		✓	✓
N-ethyl Perfluorooctanesulfonamidoacetic acid	N-EtFOSAA		✓		✓	✓
N-methyl perfluorooctanesulfonamidoethanol	N-MeFOSE		✓
N-ethyl perfluorooctanesulfonoamidoethanol	N-EtFOSE		✓
n-2-Perfluorohexyl ethanoic acid	FHEA / 6:2 FHEA / 6:2 FTCA			✓
n-2-Perfluorodecyl ethanoic acid	FDEA / 10:2 FDEA			✓
n-2H-Perfluoro-2-decenoic acid	FOUEA / 8:2 FTUCA			✓
n-3-Perfluoroheptyl propanoic acid / 3-Perfluoroheptyl propanoic acid	FHpPA /7:3 FTCA		✓	✓
n-2H-Perfluoro-2-octenoic acid	FHUEA / 6:2 FHUEA			✓
Hexafluoropropylene oxide dimer acid	GenX HFPO-DA		✓			✓
4,8-Dioxa-3H-perfluorononanoic acid	ADONA DONA		✓			✓
Perfluoro-3-methoxypropanoic acid	PFMPA		✓
Perfluoro-4methoxybutanoic acid	PFMBA		✓
Nonafluoro-3,6-dioxaheptanoic acid	NFDHA		✓
9-Chlorohexadecafluoro-3-oxanone-1-sulfonic acid	9Cl-PF3ONS F-53B Major		✓			✓
11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid	11Cl-PF3OUdS F-53B Minor		✓
Perfluoro(2-ethoxyethane)sulfonic acid	PFEESA		✓
aMethod does not require filtration bSample filtration is required and/or suggested by the Method

Alternative diameter PES Millex^® syringe filters and Millipore^® cut disc membranes are also available.

References

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2. Winchell LJ, Wells MJ, Ross JJ, Fonoll X, Norton JW, Kuplicki S, Khan M, Bell KY. 2021. Analyses of per- and polyfluoroalkyl substances (PFAS) through the urban water cycle: Toward achieving an integrated analytical workflow across aqueous, solid, and gaseous matrices in water and wastewater treatment. Science of The Total Environment. 774145257. https://doi.org/10.1016/j.scitotenv.2021.145257

3. Pérez F, Nadal M, Navarro-Ortega A, Fàbrega F, Domingo JL, Barceló D, Farré M. 2013. Accumulation of perfluoroalkyl substances in human tissues. Environment International. 59354-362. https://doi.org/10.1016/j.envint.2013.06.004

4. Lath S, Knight ER, Navarro DA, Kookana RS, McLaughlin MJ. 2019. Sorption of PFOA onto different laboratory materials: Filter membranes and centrifuge tubes. Chemosphere. 222671-678. https://doi.org/10.1016/j.chemosphere.2019.01.096

5. Labadie P, Chevreuil M. 2011. Biogeochemical dynamics of perfluorinated alkyl acids and sulfonates in the River Seine (Paris, France) under contrasting hydrological conditions. Environmental Pollution. 159(12):3634-3639. https://doi.org/10.1016/j.envpol.2011.07.028

6. So MK, Taniyasu S, Lam PKS, Zheng GJ, Giesy JP, Yamashita N. 2006. Alkaline Digestion and Solid Phase Extraction Method for Perfluorinated Compounds in Mussels and Oysters from South China and Japan. Arch Environ Contam Toxicol. 50(2):240-248. https://doi.org/10.1007/s00244-005-7058-x

7. Yamashita N, Kannan K, Taniyasu S, Horii Y, Petrick G, Gamo T. 2005. A global survey of perfluorinated acids in oceans. Marine Pollution Bulletin. 51(8-12):658-668. https://doi.org/10.1016/j.marpolbul.2005.04.026

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