Expanding Human Hair Biomonitoring for PFAS: Broadening Analyte Coverage to Emerging Compounds

Per- and polyfluoroalkyl substances (PFAS) are widely used synthetic chemicals that accumulate in humans through food, water, air, and consumer products, binding to proteins and concentrating in organs and breast milk. Traditional human biomonitoring (HBM) relies on blood, serum, and urine, but hair offers a promising, non-invasive alternative with advantages for large-scale studies. Despite this, only a handful of studies have assessed PFAS in hair, focusing mainly on legacy compounds such as perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs), with limited data on emerging contaminants. Concentrations in hair are typically lower than in serum, and uncertainties remain about external contamination and incorporation mechanisms.

A recent study expanded PFAS biomonitoring in hair to 26 analytes—including fluorotelomer sulfonic and carboxylic acids, FTUCAs, diPAPs, diSAmPAP, FOSA, N-EtFOSAA, and the ionic liquid NTf₂—to characterize exposure patterns in non-occupationally exposed individuals and explore links with physical and lifestyle factors. LCGC International spoke to Miriam Haußecker, lead author of the paper (1) that resulted from this study.

What chromatographic techniques were most critical for separating and quantifying the broad range of PFAS analytes, including emerging compounds like NTf₂, in human hair samples?

In our study, we applied ultrahigh-performance liquid chromatography (UHPLC) with a C18 reversed phase (RP) column coupled to a triple quadrupole mass spectrometer (QqQ) for the analysis of 26 target PFAS in 45 human hair samples. The target analytes included the well-known perfluoroalkyl acids (PFAAs) as well as several precursors and the emerging compound NTf2. The most critical point is the separation of the analytes from matrix components which was achieved by a highly efficient column with 1.7 µm particles and an optimized gradient elution. For specific detection the use of unique mass transitions in MS/MS detection is important.

What specific challenges did you encounter with matrix effects in hair compared to more conventional biomonitoring matrices like blood or serum, and how were these addressed chromatographically?

Hair is a complex matrix because it consists of various components (proteins, lipids, pigments) and can be affected by external contaminants that vary greatly between individuals. We applied several measures to minimize matrix effects on separation and quantification:

Pre-washing the hair by water and acetone.

Methanol extraction of the analytes and SPE clean-up and pre-concentration on weak ion exchange material.

Use of isotope labelled standards (so far available), measurements of three replicates plus a spiked sample (fortified with all target analytes).

This procedure enabled us to correct for reduced time (RT) shifts resulting from the high matrix load as well as matrix effects on quantification as far as possible. Furthermore, we incorporated an 8 min flushing step with 100 % organic phase in the gradient elution program to remove the matrix from the column after each injection.

Could you explain how chromatography enabled the detection and distinction of branched versus linear isomers of PFOS, PFOA, and N-EtFOSAA, and why this is important for source tracking?

Branched and linear isomers of PFOS, PFOA, and N-EtFOSAA are predominantly distinguished by chromatographic separation. Therefore, chromatographic separation is the key. However, due to the lack of standards and due to limited separation efficiency, we couldn´t identify the specific branched isomers and were only able to perform a semi-quantitative estimation of the sum of the branched isomers based on the response factor of the linear reference standards. It must be noted that different isomers also have different response factors, and, in some cases, specific mass transitions must be considered (2).

It is important to differentiate between linear and branched isomers because they exhibit different physicochemical properties and subsequently, their fate and effects on human health are different (3). The isomer signature of technical produced PFAS is known; PFOS produced via electrochemical fluorination (ECF) consists roughly of 70% linear and 30% branched isomers. Thus, the detection of different isomer distributions in human hair samples can be a hint to exposure sources. For example, increased levels of branched PFOS isomers in human matrices may indicate exposure to branched precursors instead of branched PFOS being the original substance (4). However, many different factors must be considered when drawing a conclusion here, such as the different partitioning behavior of PFOS precursors compared to PFOS within the human body and the different half-lives of the isomers (5). There is still some uncertainty about the fate of PFAS isomers within the human body and it would be a very interesting topic for follow-up research.

How did you ensure sufficient sensitivity and selectivity in your chromatographic method to detect low PFAS concentrations given the small hair/serum transfer ratios?

We optimized ultrahigh-pressure liquid chromatography (UHPLC) separation and tandem mass spectrometry (MS/MS) detection (dynamic multiple reaction monitoring [dMRM]) as far as possible. However, limitations occurred for specific analytes due to background concentrations in solvents and materials of lab equipment. To account for this, we included six procedural blanks per batch and several instrumental blanks per instrumental sequence. As a result, we couldn´t quantify ultra-short chain PFCAs in this work.

In your view, what role can advanced chromatographic techniques—such as two-dimensional liquid chromatography (2D-LC) or coupling with high resolution mass spectroscopy (HRMS)—play in expanding PFAS biomonitoring in hair beyond current capabilities?

In my opinion, PFAS biomonitoring in hair as well as in other matrices would definitively benefit from improved separation and detection which could be realized by 2D-LC and HRMS. Our study showed that PFAS human exposure goes beyond the typically applied target analyte lists which often include only PFAAs. The emerging compound NTf₂ was initially detected during pre-screening using a non-target approach which led to the inclusion of this substance into our target list. Ultimately, NTf₂ occurred in 44% of all hair samples which reveals a so far underestimated contribution to the PFAS body burden. It shows that non-target screening would be beneficial to gain a more complete picture of human PFAS exposure.

Two- or multidimensional LC is a powerful tool to considerably improve separation efficiency. However, it must be considered that method optimization and data evaluation may require much more effort and time.

What steps would you recommend toward harmonizing chromatographic protocols across laboratories to improve comparability of PFAS biomonitoring studies in hair?

I would recommend focusing strongly on harmonizing the sample preparation protocol. For a complex matrix such as hair, sample preparation and extraction include many different steps. Most studies on PFAS in human hair so far have used different techniques to homogenize, to pre-wash and to extract hair samples and furthermore used different clean-up processes. Therefore, a standardized analysis method would be very helpful to produce comparable results. This should include recommendations for hair sampling, and a harmonized target list of PFAS which goes beyond PFAAs. As our study showed, human exposure is caused by far more PFAS than PFAAs.

Do you foresee chromatographic segmental analysis of hair becoming a routine tool for reconstructing short- versus long-term PFAS exposure patterns?

In my opinion, using segmental hair analysis holds great potential for the time-resolved analysis of exposure because it is a method which was already established in using hair for drug analysis. However, to obtain a more complete picture of human exposure, PFAS blood-hair transfer factors and incorporation mechanisms should be established first. This is necessary to link internal exposure to hair concentrations (5). This knowledge combined with segmental analysis can be a relevant approach to assess human PFAS exposure by hair analysis.

Read more on this topic: Hot Topics in PFAS

References

1. Haußecker, M.; Zweigle, J.; Bugsel, B.; et al. Unveiling Novel and Legacy PFAS in Human Hair. Environ. Int. 2025, 202, 109714. DOI: 10.1016/j.envint.2025.109714
2. Londhe, K.; et al. The Need for Testing Isomer Profiles of Perfluoroalkyl Substances to Evaluate Treatment Processes. Environ. Sci. Technol. 2022, 56 (22), 15207–15219. DOI: DOI: 10.1021/acs.est.2c05518
3. Nilsson, S.; Thompson, J.; Mueller, J. F. et al. Apparent Half-Lives of Chlorinated-Perfluorooctane Sulfonate and Perfluorooctane Sulfonate Isomers in Aviation Firefighters. Environ. Sci. Technol. 2022, 56 (23), 17052–17060. DOI: DOI: 10.1021/acs.est.2c04637
4. Martin, J. W.; Asher, B. J.; Beesoon, S. et al. PFOS or PreFOS? Are Perfluorooctane Sulfonate Precursors (PreFOS) Important Determinants of Human and Environmental Perfluorooctane Sulfonate (PFOS) Exposure? J. Environ. Monit. 2010, 12 (11), 1979–2004. DOI: 10.1039/c0em00295j
5. Qiao, L.; et al. Analysis of Human Hair to Assess Exposure to Organophosphate Flame Retardants: Influence of Hair Segments and Gender Differences. Environ. Res. 2016, 148, 177–183. DOI: 10.1016/j.envres.2016.03.032