Clinical research analysis of perfluoroalkyl substances (PFAS) in human serum by LC-MS/MS using Captiva EMR-Lipid cleanup

Efficient sample preparation before LC-MS/MS analysis of perfluoroalkyl substances (PFAS) is an important consideration for research laboratories performing clinical analysis. Phospholipids (PPLs) have been identified as a major cause of matrix effects in the LC-MS/MS analysis of serum samples.

In this SelectScience webinar, now available on demand, Joseph Homan, Senior Scientist III at NMS labs, describes serum sample preparation and LC-MS/MS analysis of PFAS using protein precipitation followed by PPL removal using the Agilent Captiva EMR-Lipid cartridges.

Read on for highlights from the Q&A discussion and register now to watch the webinar on demand

Can you use LC-MS-grade ACN with molecular sieves to remove water rather than anhydrous ACN?

JH: The reason that I used anhydrous acetonitrile in the method had nothing to do with the fact that it was anhydrous. It had everything to do with the fact that the bottle that the anhydrous acetonitrile is packaged in has a crimp top with a silicone rubber seal on the top. The reason that I used the anhydrous is that the seal is silicone.

Most acetonitrile is packaged in glass bottles with a screw-top cap, and that cap has a Teflon seal in it. And that Teflon seal is often enough to contaminate that solvent with PFAS. So, the solvent doesn't have to be anhydrous. What it must be is low enough in PFAS so that when you concentrate it, you don't have positive blank samples.

Can Captiva EMR-Lipid be used for matrices other than serum?

JH: I would imagine, especially if you're doing an offline protein precipitation step before loading onto the Captiva EMR-Lipid cartridges, anything that would yield you a clear supernatant that you could then load onto the Captiva EMR-Lipid cartridges should work. Regarding water testing, you probably would still want to use a traditional SPE cartridge because you're typically concentrating a very large volume of water, and that would be difficult to dry down if you're just passing through a cartridge and you're not concentrating the analyte on the cartridge.

Can the method be used for the analysis of PFAS in matrices like whole blood or dried blood spots, or even teeth?

JH: You could certainly do whole blood. You'll end up with a supernatant that's pink in color, depending on how fresh the blood is. And then you're going to concentrate that after you pass it through the Captiva EMR-Lipid cartridge. You certainly can extract out lipids with the Captiva EMR-Lipid cartridges in whole blood and you will not extract out the PFAS.

The question will be how clean is that extract after you reconstitute it, and whether you want to inject that onto an LC-MS. Typically the extraction from dried blood spots tends to be relatively clean because it's very low volume. But since you can extract PFAS from dried blood spots using an organic solvent, you could certainly then put that organic solvent through a Captiva EMR-Lipid cartridge to concentrate the PFAS.

For samples of teeth, you would have to get the sample into the organic solvent to put it through the Captiva cartridge. If you could do that, then I would think that you could also use Captiva EMR-Lipid for that purpose. Captiva EMR-Lipid is used to remove the matrix and not the PFAS. So once you get your sample into an organic solvent you can pass it through the Captiva EMR-Lipid cartridge, and it should work for that matrix.

LC vials are made of glass. Can these bind to the analyte of interest after extraction and cleanup?

JH: There could be some non-specific binding. I used polypropylene vials for PFAS because I had observed a difference between glass and plastic for certain PFAS compounds, with some PFAS compounds more prevalent to bind to glass than others. If you're in a glass vial and your reconstitution solution is aqueous enough, you may start to see loss due to the vial.

How long was the delay column? And did this have the same stationary phase as the analytical column?

JH: It did have the same stationary phase just a larger particle size, and that's to reduce some of the back pressure on the system. I'm not worried about the peak shapes for the PFAS that are being delayed so I used a larger particle size, 2.7 micron instead of the 1.9 micron. It was also a 50 mm column, and you may be able to get away with an even shorter column. A lot of people use a shorter delay column than they do an analytical column for back pressure reasons as well.

Does it have to be the same stationary phase? I don't think it has to be the same stationary phase, but you don't want to have a situation where you have separation on your delay column that you don't have on your analytical column. So, most of the time, people use the same stationary phase for their delay column as they use for their analytical column.

To learn more about Captiva EMR-Lipid cleanup, watch the full webinar here>>

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