Analysis of PFAS in Water according to EPA 1633

Abstract

Per- and polyfluoroalkyl substances (PFAS), known for their environmental persistence and potential health impacts, are a significant concern in water quality management. This study demonstrates the utilization of the EPA Method 1633 to analyze and quantify 40 PFAS chemicals in water samples. The technique utilizes liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) after a solid-phase extraction (SPE) using Supelclean™ ENVI-WAX™ SPE cartridges. The procedure allows for highly sensitive detection and quantitation of PFAS analytes in various matrices. Performance evaluation demonstrated that recoveries for all 40 PFAS compounds and 24 isotopically labeled standards (EIS) were within the typical EPA acceptable range of 70-130% with low corresponding standard deviations, indicating high precision and robustness. The method's suitability for comprehensive PFAS analysis in environmental samples underscores its value for regulatory compliance and environmental monitoring.

Section Overview:

• Introduction
• Experimental
• Results
• Conclusion
• Related Products

INTRODUCTION

Per- and polyfluoroalkyl substances (PFAS), also referred to as “forever chemicals,” are a broad category of more than 4700 synthetic fluorinated aliphatic compounds that have been applied in consumer goods since the 1950s. Known for their resistance to lipids and water, PFAS are highly stable due to the strong carbon-fluorine bond. They are commonly used as surface-active agents in products like stain repellents and firefighting foams, persist in the environment due to their slow degradation rate, however.

With their widespread use in various consumer and commercial applications, PFAS have drawn significant attention regarding the contamination of water, soil, and even the bloodstreams of humans and animals. This emphasizes their persistence and impact on environmental and human health. The United States Environmental Protection Agency (US EPA) and the European Union (EU) played key roles in developing stringent regulatory guidelines for PFAS testing, which are essential for protecting ecosystems and human populations.^1,2,3

The US EPA adopted a comprehensive strategy, as demonstrated by the development of established analytical methods such as EPA Methods 533 and 537.1 for drinking water, to thoroughly evaluate PFAS levels in various environmental samples. At the same time, the EU has implemented strict regulations under the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) framework.⁴ These regulations use advanced analytical tools such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) to monitor PFAS in different chemicals substances, showing a dedication to improving analytical capabilities and safeguarding the environment and human health.

The EPA Method 1633¹ is a laboratory-validated approach that employs LC-MS/MS to analyze aqueous, solid, biosolid, and tissue samples for 40 PFAS across nine compound classes. This "performance-based" method allows for condition adjustments to improve performance if all requirements are met, providing a stable platform for precise calibration and quantification of PFAS analytes using isotopically labeled standards. With the inclusion of analytes from EPA drinking water Methods 533 and 537.1, EPA Method 1633 tackles emerging PFAS classes, filling important voids in testing consistency, scope, sensitivity, and applicability across different sample types, helping to address the complex challenges posed by PFAS contaminations and ensuring public health and environmental protection.

The objective of this application note is to demonstrate the determination of 40 PFAS analytes from PFAS-spiked water samples by solid-phase extraction (SPE) using Supelclean™ ENVI-WAX™ SPE cartridges on a PTFE-free Visiprep™ SPE vacuum manifold followed by LC/MS-MS analysis employing Fused-Core^® Ascentis^® Express PFAS columns (analytical and delay) and compare the obtained method performance characteristics to the criteria stated in EPA 1633.

EXPERIMENTAL

Solutions and Standards Preparation

The sample collection and preparation followed the EPA Method 1633 procedure.

Native (40) and isotopically (31) labeled PFAS standards were used as methanolic 50 µg/mL stock solutions. The labeled compounds are either used as extracted internal standards (EIS, 24 compounds) or non-extracted internal standards (NIS, 7 compounds). Following the recommendations of the EPA1633 method, these stock solutions were used to prepare seven calibration solutions containing the native PFAS compounds in various concentrations (CS1 - CS7; compound specific concentrations ranging from 0.2 - 5 ng/mL for CS1 to 62.5 - 1560 ng/mL for CS7 as indicated in Table 4 of the EPA1633 method) to cover the working range of the MS instrument.

Sample Preparation

PFAS Analysis Method Performance Assessment

In accordance with the EPA 1633 method, the method performance was investigated for water samples. 500 mL of water (tap fresh water from a Milli-Q^® system) was collected in HDPE bottles with liner-less polypropylene caps. The water sample was fortified at three different levels (2x CS1, 12.5x CS1, 40x CS1) with 40 native PFAS and spiked with 24 isotopically (¹³C or D) labeled standards (extracted internal standards – EIS) according to EPA 1633 (Table 3). For the extraction by SPE, Supelclean™ ENVI-WAX™ SPE tubes (500 mg/6 mL, 54057-U) were equipped with large volume SPE reservoirs (25 mL, 54258-U) and placed on a PTFE-free Visiprep™ vacuum manifold (57030-U). The tubes were conditioned with 15 mL of 1.0% NH₄OH in MeOH and equilibrated with 5 mL of aqueous 0.3 M formic acid. After the water sample (500 mL) was loaded and passed through the cartridge, 2x 5 mL of water and 5 mL of 0.1M formic acid/methanol (1:1 (v/v)) were added as a washing step. The cartridge was subsequently dried for 1 min before 5 mL 1.0% NH₄OH in MeOH was used to elute the analytes. For further clean-up 25 µL concentrated acetic acid and approximately 10 mg loose/bulk Supelclean™ ENVI-Carb™ (57210-U) were added to the eluate and mixed for less than 5 minutes using a vortex shaker, followed by centrifugation for 10 minutes at 4000 g. Subsequently, the supernatants were filtered using Millex^® Nylon 0.2 µm syringe filters (SLGNX13) into collection tubes containing additional 7 isotopically labeled (¹³C or ¹⁸O) labeled standards (non-extracted internal standards – NIS) prior to LC-MS/MS analysis. The sample preparation represents an enrichment of 1:100 from the original water sample to the final extract.

Instrumental Analysis

LC-MS/MS analysis was performed using an Agilent 1290 Infinity II instrument coupled to an Agilent 6495C triple quadrupole mass spectrometer (Table 1). Chromatographic separation was achieved using an Ascentis^® Express PFAS 90 Å (5 cm x 2.1 mm, 2.7 µm, 53557-U) analytical column. In addition, an Ascentis^® Express PFAS Delay column 90 Å (5 cm x 3.0 mm, 2.7 µm, 53572-U) was installed after the mixing valve and before the autosampler to offset PFAS contamination potentially originating from the LC system (e.g. pump, tubing, fittings, filters). Polypropylene snap cap vials were used instead of standard glass vials to avoid possible PFAS adsorption to the glass surface.

For the quantification of the native PFAS based on stable-isotope dilution, the MRM transitions shown in Table 2 were used. The EIS compounds were allocated in accordance with what is stated in EPA1633.

RESULTS AND DISCUSSION

A chromatogram of the calibration solution 5 (CS5) containing the 40 native compounds is shown in Figure 1; as evident from Table 3, linear calibration curves with R² ≥ 0.99 were obtained for all PFAS analytes.

The targeted native PFAS analytes were quantified based on stable-isotope dilution using extracted internal standards (EIS) which were added to the sample before the SPE extraction. The recovery of the EIS surrogates was determined using the non-extracted internal standards (NIS) which were spiked to the concentrated extract after the clean-up step; IS allocation was in accordance with the EPA 1633 method. Tables 3 and 4 display the recoveries and RSDs from the experimental study where 40 native PFAS compounds (Table 2) were spiked to the water samples at three different concentration levels and 24 EIS surrogates (Table 4) were added prior to extraction. All recoveries were between 84.0% and 110.7% and RSDs ranged from 0.2% to 18.1%. All were well within the acceptance limits for recovery from aqueous matrices as listed in Tables 5 and 6 of EPA Method 1633. 

CONCLUSIONS

This application note presents the workflow for EPA method 1633 to analyze 40 PFAS compounds in water samples by applying SPE extraction using a Supelclean™ ENVI-WAX™ cartridge, followed by additional dispersive clean-up with bulk Supelclean™ ENVI-Carb™ adsorbents and quantification by LC-MS/MS employing Ascentis^® Express PFAS analytical and delay columns. The method demonstrated robust performance with excellent recoveries for all 40 PFAS compounds and 24 extracted internal standards (EIS) well within acceptance limits for aqueous matrices listed in the method at the three recommended fortification levels. The calculated RSDs were below 20%, further indicating a satisfactory precision. This proves the suitability of PTFE-free Visiprep™ SPE Vacuum Manifold with Supelclean™ ENVI-WAX™ SPE cartridges for the extraction of PFAS compounds from water samples and of Supelclean™ ENVI-Carb™ for clean-up prior to subsequent LC-MS/MS analysis according to EPA 1633.

See more on PFAS testing at SigmaAldrich.com/PFAS