Can bile acids found in plasma, eggs, liver and other biological samples interfere with PFAS analysis? Yes! But there are ways to avoid these interferences and produce reliable data.

Bile acids and their salts are either synthesized in the liver or formed from bacterial activity. They are the major constituents of bile and help digest fats and oils. Some of them can also interfere with PFOS analysis, especially the ones highlighted in the image above, the taurine-conjugated bile acids.

The Problem

1. Three bile acids, Taurochendesoxycholic Acid (TCDCA), Taurodeoxycholic Acid (TDCA) and Taurorsodeoxycholic Acid (TUDCA) all have a molecular mass of 499.2, which is <1 amu from PFOS and contribute to the PFOS precursor ion in unit-resolution LC-MS/MS.
2. Both PFOS and these bile acids are sulfonic acids. The PFOS product ion of 80 m/z is the same as their product ion, as it is a loss of the sulfonate group in both cases.
3. Given their similar chemistry, they will come through most extract cleanup used for PFAS including weak anion exchange, and most likely, carbon cleanup as well.
4. In the case of a coelution, because of their much larger abundance than PFOS, they will result in a false-positive in the 499->80 transition used for quantifying PFOS even though their nominal mass is marginally different, especially due to large contributions from the natural 13C portion (M+1 ion for the bile acids). A small part of a large peak can still be large.

All this sounds worrying, but best-practice PFAS methods that use appropriate techniques with confirming transitions and good chromatography can easily resolve these interferences.

Solution

• The only guaranteed solution in unit resolution LC-MS/MS is to separate these interferences chromatographically. As shown in the image below, the default PFAS method run at SGS AXYS separates the bile acids from PFOS by more than 2 minutes, which is a very long time in the high-efficiency separation world of UPLC. This is the 100% error-free option, no chance of interference whatsoever. The SGS AXYS method does not start acquiring PFOS transitions till well after the bile acids elute.

• Cleanup: There is anecdotal evidence that cleanup using graphitic carbon could potentially remove bile acid interferences. Nonetheless, this was not found to be true in our laboratory, so if you go this route, you will definitely need to confirm using bile acid standards that you’ve actually removed the interferences. Chromatographic separation is the safest solution.
• Use high resolution mass spectrometry. However, these instruments are not common commercially and there is likely a drop in sensitivity from using a high resolution mass-spectrometer for low-level PFOS quantitation. If chromatographic separation is achieved, high resolution is not necessary.

Diagnostics

• Always use multiple transitions to confirm detects. Bile acids do not have a response in the 499->99 transition which is used for PFOS. Therefore, a PFOS detect that has a response ratio much higher than the calibration standard for 499->80/499->99 clearly has an interference. However, this is only a diagnostic, and a check back on historical data. If the peak did not meet ratio and was reported as such, the data is not usable.

Questions to ask your data provider

1. Have you checked for bile acid interferences in your PFAS method?
2. Did you do this by running bile acid standards? Can you provide the data showing separation?
3. If you use a cleanup technique to remove bile acids from the extract, can you show using spiked standard data that the interferences were actually removed?
4. Do you acquire multiple transitions and check response ratios?
5. With historical data, were multiple transitions acquired and reported? It is possible that they were acquired and shown in the chromatograms, and evaluated qualitatively. This is still okay as a chromatogram will provide adequate information to assess interferences in the absence of a quantitative ratio check.

If the answer to any of these questions is no, then there would be significant data quality issues to raise.

Please note, these concerns are not new. Benskin et al. 2007 published on this issue [1]; and SGS AXYS had a SETAC presentation in 2010 [2] talking about bile acids and interferences.

References