What are the differences between EPA Method 533 vs 537.1?

Per- and polyfluoroalkyl substances (PFAS) are a group of harmful organic compounds that are very persistent in structure. What this means is PFAS compounds accumulate in the environment over time as they do not break down easily. This makes it a concern to regulate and test these compounds as they have been shown to have adverse effects. One of the most common ways that someone would come in contact with PFAS is through drinking water. There are two notable EPA regulated methods that laboratories can use to analyze PFAS compounds, EPA method 533 and 537.1. When evaluating how to handle these methods in your lab there are some key differences in how to approach PFAS testing. See our earlier blog extracting perfluorinated compounds from drinking water – why is it so challenging?

EPA method 533 is a compliment to method 537.1, including an additional 11 compounds and excluding 4 compounds from 537.1. When used together, twenty-nine compounds can be tested in drinking water. All of these can be visualized in the table below, showing the acronyms for each compound and what methods they are tested in. Specifically, method 533 focuses on PFAS compounds that have short carbon chains, which are those with carbon lengths of C4 to C12. The first major difference between the two methods is the type of solid-phase extraction media that is used. Method 533 uses polystyrene divinylbenzene with a positively charge diamnino ligand and isotope dilution whereas method 537.1 uses styrene-divinylbenzene (SDVB) media. So, when it comes to preparing for the extraction of these compounds it is important to ensure that you are using the right type of cartridge to get the best results. The other major difference that goes hand-in-hand with the media type, is how the extraction techniques differ. With method 533; methanol and 2% ammonium hydroxide are used for extraction elutions, evaporated to dryness with a nitrogen blowdown and water bath, and then reconstituted with 20% water in methanol. However, with method 537.1, just methanol is used for extraction elutions and after it has been concentrated to dryness it is reconstituted with a 96:4 methanol:water mixture instead.

In summary, while the overall extraction process is similar, the media type, elution solvents, and reconstitution process differ between the two methods. These are the key things that you need to keep in line so that the similar extractions do not get mixed up. The easiest part to keep together is the fact that despite the differences in the extraction methods, both are analyzed on LC-MS-MS. Hopefully, this helps you to get started on understanding the key differences between the two methods and how to extract them.

If you are looking to certify or currently running EPA method 533 and or 537.1 in your lab I have included links to Biotage solutions that can help to get you started and improve your laboratory’s workflow.

Evaporation/Concentration System

TurboVap® LV Automated Solvent Evaporation System

Method 537.1

ISOLUTE® 101 SPE column

Method 533

EVOLUTE® EXPRESS Wax in a 6-mL format (150 mg) or

EVOLUTE® EXPRESS WAX 500 mg bed mass