HomeLC-MS Analysis of PFAS Compounds in EPA Method 533 using Supelclean™ ENVI-WAX™ SPE

LC-MS Analysis of PFAS Compounds in EPA Method 533 using Supelclean™ ENVI-WAX™ SPE

Lara Rosenberger, Yannick Hövelmann, Olga Shimelis


Article from Analytix Reporter - Issue 15


Per- and polyfluoroalkyl substances (PFAS) have been in use since the 1940s. Consisting of over 4700 different compounds, PFAS substances are used in almost every facet of modern life. The utility of these compounds resulted in their rapid adoption in consumer goods manufacturing. PFAS compounds can now be found in food packaging, cookware, cosmetics, stain and water repellants, firefighting foams, and are commonly used in many manufacturing processes. While incredibly useful, these compounds also carry a risk to health that we have only recently started to understand clearly.

PFAS compounds are also commonly known as “forever chemicals” which means they do not break down in the environment like other chemicals. This persistence can result in the concentration of these compounds growing to levels that are unsafe for human exposure and that can cause negative health effects such as low infant birth weights, effects on the immune system, cancer, and thyroid hormone disruption.

PFAS detection plays therefore a crucial role in safeguarding public health and the environment. PFAS detection in water is essential for assessing water quality and to identifying potential health risks. To achieve accurate measurements and quantification of these contaminants in water samples, various PFAS analysis methods are employed.

Multiple regulatory methods, such as EPA 537 and 533, detail the extraction of PFAS analytes from drinking water using SPE cartridges followed by LC-MS/MS analysis. For EPA method 533, weak anion exchange (WAX) cartridges are specified and should contain 500 mg of the mixed-mode polymeric adsorbent. Supelclean™ ENVI-WAX™ SPE cartridges are direct equivalent to the specified SPE in EPA method 533. This application note demonstrates the extraction of 25 analytes from water using Supelclean™ ENVI-WAX™ SPE.


The procedure from EPA method 533 was followed for sample collection and sample preparation. Supelclean™ ENVI-WAX™ SPE 500 mg/6 mL cartridges (54057-U) were used with a Visiprep™ vacuum manifold (57030-U) for processing the samples. The large volume sampling kit (57275) was also used but the Teflon tubing was replaced with silicone tubing (1/8" diameter). The Teflon guides in the original manifold were replaced with stainless-steel solvent guides (57027). Analysis of the samples was done using an Agilent 6495C LC-MS/MS instrument. Ascentis® Express PFAS HPLC Column, 2.7 µm, 15 cm x 2.1 mm (53560-U) was used as an analytical column. In addition, an Ascentis® Express PFAS Delay Column, 2.7 µm, 5 cm x 3.0 mm (53572-U) was used (Table 1). The chromatogram of 25 compounds in a calibration standard is shown in Figure 1.

Table 1.LC-Conditions for analysis of 25 PFAS compounds
Table 2.MRM transition used for 25 PFAS compounds in EPA method 533
A chromatogram of a calibration standard having 25 PFAS compounds.

Figure 1.25 PFAS compounds at 50 ppb in 96:4 water:methanol (v/v) solvent.

UHPLC-MS grade water samples were tested for PFAS contamination and found to be free of 25 analytes as per the EPA method 533. The water was spiked at 10 or 40 ng/L with 25 analytes to demonstrate the performance of Supelclean™ ENVI-WAX™ SPE cartridges for this method. 250 mL of water samples were loaded onto 500 mg/6 mL SPE cartridges, and eluted using methanol with 2% (v/v) ammonium hydroxide; the resulting eluate was evaporated to dryness and reconstituted into 1.0 mL of 4% (v/v) methanol in water for LC-MS/MS detection.

Following the performance assessment of the method using Supelclean™ ENVI-WAX™ SPE, a tap water sample was analyzed using the same methodology for the presence of 25 PFAS compounds.

Results and Discussion

The background evaluation of the method using all SPE consumables and accessories resulted in excellent low background values (shown in Table 3). The result for screening all compounds in the UHPLC-MS solvent was at or below the lower limit of detection (LLOD) of the LC-MS/MS instrument.

Table 3.Results of background testing for the evaluation
1 LCMRL (Lowest Concentration Minimum Reporting Level) is 5.3 ng/L per EPA method 533
2 LCMRL is 3.8 ng/L per EPA method 533
*LLOD were 2-6 ppt for all compounds

Per EPA method 533 the recovery of the laboratory spiked blank water samples should fall in the range 70-130% with reproducibility of better than 20%. Figure 2 demonstrates the recoveries from laboratory spiked UHPLC-MS water blanks, where the recoveries for 25 compounds met the EPA method requirements. Figure 3 presents the %RSD for each of the 25 compounds indicating that the less than 20% RSD requirement was met.

A drinking water sample was also analyzed using EPA 533 method. No analytes were detected in the sample above 0.5 ng/L concentrations, and most were below LLOD.

Bar diagram showing % recoveries of the 25 PFAS analytes spiked into UHPLC-MS grade water samples.

Figure 2.Recoveries of 25 analytes spiked into UHPLC-MS grade water samples. Most analytes were spiked at 10 ng/L, perfluorosulfonic acids were spiked at 40 ng/L. 3 replicate measurements were performed.

Bar diagram showing %RSD for recoveries of the 25 PFAS analytes spiked into UHPLC-MS water samples.

Figure 3.%RSD for recoveries of the 25 analytes spiked into UHPLC-MS water samples. 3 replicate measurements were performed.


The workflow for EPA method 533 is presented in this article. All 25 compounds were recovered with acceptable accuracy and precision using Supelclean™ ENVI-WAX™ SPE cartridges, Visiprep™ vacuum manifold, Ascentis® Express PFAS columns and UHPLC-MS grade solvents. The background from all consumables and LC system was low and acceptable for detecting low levels of PFAS analytes.

Find more information on PFAS testing at

Reference Materials
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