Introduction: Understanding PFAS Exposure During Early Life
Per- and polyfluoroalkyl substances (PFAS), commonly known as forever chemicals, are a large group of synthetic chemicals that have attracted increasing attention because of their environmental persistence, widespread occurrence and potential implications for human health.
Used in a variety of industrial applications and consumer products, PFAS can enter the environment and contribute to human exposure through food, drinking water, household dust and other environmental sources.
One important area of environmental health research is the transfer of PFAS from mothers to infants during pregnancy and breastfeeding. Human milk provides essential nutrition and numerous health benefits for infants, but it can also represent a route of early-life exposure to environmental contaminants, including PFAS. Understanding the concentrations of these chemicals in breast milk is therefore important for maternal exposure assessment, infant exposure research and population-level biomonitoring [1,2].
However, accurately measuring PFAS in human milk presents considerable analytical challenges. Traditional methods can require relatively large sample volumes and labour-intensive preparation procedures, limiting their suitability for large-scale studies.
A new study published in Analytical Chemistry addresses these limitations by introducing a scalable, semi-automated method for measuring PFAS in human milk. Patel and colleagues developed a high-throughput analytical workflow capable of quantifying 23 different legacy and replacement PFAS compounds using just 300 µL of human milk [3].
By combining automated sample preparation with sensitive mass spectrometry, the method offers an important opportunity to expand research into PFAS exposure during early life.
Why Is Measuring PFAS in Breast Milk Important?
PFAS are a diverse group of synthetic chemicals characterised by exceptionally strong carbon-fluorine bonds. These bonds contribute to their environmental persistence, meaning that many PFAS compounds resist degradation and can remain present in the environment for extended periods.
Human exposure can occur through several routes, including contaminated drinking water, food, household dust and consumer products. Some PFAS can also accumulate in the human body, making biomonitoring an important tool for understanding exposure.
Human milk is a particularly relevant biological sample for investigating early-life exposure. Measuring PFAS concentrations in breast milk can help researchers understand maternal body burdens, chemical transfer during lactation and the potential contribution of breastfeeding to infant exposure [1,2].
Previous research has identified PFAS in human breast milk and highlighted the importance of developing analytical methods specifically suited to this complex biological matrix [1].
Importantly, detecting PFAS in breast milk does not mean that breastfeeding is unsafe. Breastfeeding provides well-established nutritional and health benefits, and research into environmental contaminants should be viewed as part of a broader effort to understand and reduce avoidable chemical exposure.
To investigate these questions effectively, researchers need analytical methods that can detect multiple PFAS compounds at very low concentrations, even when only small quantities of human milk are available.
The Challenge of PFAS Analysis in Human Milk
Analysing PFAS in human milk is technically challenging because milk is a complex biological matrix containing proteins, lipids and other substances that can interfere with chemical detection.
Traditional PFAS analysis methods have often required relatively large sample volumes alongside extensive manual sample preparation. These requirements can create significant limitations for population-level biomonitoring studies [1].
There are several important challenges:
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Limited sample availability: Human milk samples are valuable biological resources, and collecting large volumes can be difficult.
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Matrix interference: Milk contains fats, proteins and other components that can affect analytical performance.
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Low chemical concentrations: PFAS may be present at picogram-per-millilitre concentrations, requiring highly sensitive analytical techniques.
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Labour-intensive preparation: Manual processing can increase analysis time and introduce variability.
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Limited sample throughput: Conventional workflows can make it difficult to analyse the large sample numbers needed for epidemiological research.
Previous studies have explored different approaches to PFAS analysis in breast milk, including solid-phase extraction, liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry [1,4,5].
These studies demonstrate the importance of developing more efficient analytical approaches that combine high sensitivity, reliable quantification and scalable sample processing.
A New High-Throughput Method for Measuring 23 PFAS Compounds
In their study, Patel and colleagues developed and validated a semi-automated analytical workflow for quantifying 23 legacy and replacement PFAS compounds in human milk [3].
One of the most significant features of the method is its ability to analyse samples using just 300 µL of human milk. This represents a substantial reduction compared with conventional approaches requiring approximately 1–5 mL.
The workflow was designed to improve laboratory efficiency without compromising the sensitivity required for low-level PFAS detection.
- Automated sample preparation: The researchers used an Opentrons Flex automated liquid-handling system to process human milk and quality-control samples. Automation helps standardise sample preparation, reduces the need for repetitive manual handling and makes it easier to process larger numbers of samples consistently. The system uses a 96-well processing format, allowing multiple samples to be prepared in parallel.
- Enhanced matrix removal: A major challenge in PFAS analysis is removing interfering substances from the biological sample before instrumental analysis. To address this, the researchers incorporated enhanced matrix removal (EMR) mixed-mode clean-up into the workflow. This approach helps remove unwanted matrix components that could otherwise interfere with the detection and quantification of PFAS. Effective matrix clean-up is particularly important when working with human milk because its complex composition can affect analytical performance.
- Liquid chromatography-high-resolution mass spectrometry: Following sample preparation, the researchers analysed the samples using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Liquid chromatography separates individual chemical compounds within a sample, while high-resolution mass spectrometry enables their detection and measurement based on their mass-to-charge characteristics. The combination provides a powerful analytical approach for identifying and quantifying multiple PFAS compounds at very low concentrations. Previous research has also demonstrated the value of LC-HRMS for measuring PFAS and other environmental contaminants in human milk [4].
Together, these technologies create a workflow designed to deliver sensitive, reliable and high-throughput PFAS analysis.
How Sensitive and Accurate Is the New PFAS Analysis Method?
An important consideration when developing a new analytical method is whether it can reliably detect and quantify chemicals at environmentally relevant concentrations.
The researchers evaluated the analytical performance of their workflow using method detection limits, limits of quantification and accuracy measurements.
The results demonstrated encouraging analytical sensitivity.
- Detection limits: Method detection limits ranged from 0.53 to 9.19 pg/mL across the 23 PFAS compounds investigated. Importantly, 22 of the 23 compounds achieved limits of quantification (LOQs) of 5 pg/mL or lower [3]. This demonstrates the method’s ability to measure very low concentrations of a broad range of PFAS compounds in human milk.
- Analytical accuracy: The method also demonstrated good accuracy during validation. Average accuracy was reported as 93 ± 10% at the 5 pg/mL concentration level, increasing to 90–98% at higher spiking concentrations [3]. These results support the method’s suitability for quantitative PFAS analysis and demonstrate its potential for application in larger biomonitoring studies.
- Analysing 72 Samples in a 20-Hour Batch: One of the most important advantages of the new approach is its potential to increase laboratory sample throughput. Using the semi-automated 96-well workflow, the researchers demonstrated the ability to process 72 samples within a 20-hour analytical batch [3]. This is particularly relevant for population-level environmental exposure research, where hundreds or thousands of biological samples may need to be analysed. By reducing sample preparation time and increasing processing capacity, the workflow could help researchers investigate PFAS exposure across larger and more diverse populations. The reduced sample volume is another significant benefit. Requiring only 300 µL of human milk makes it easier to work with limited sample collections and potentially allows more extensive chemical analysis from the same biological sample.
Together, these improvements could make large-scale PFAS biomonitoring more practical.
What Did the Researchers Find When Analysing Human Milk Samples?
To demonstrate the practical application of their analytical method, the researchers analysed paired human milk samples collected from 36 mothers at two different postpartum time points: one month and six months after birth [3].
This allowed the team to investigate PFAS concentrations over time and explore how chemical exposure patterns might change during lactation.
The analysis identified four PFAS compounds that were detected above their respective limits of quantification in at least 50% of mothers at one or both sampling time points.
An interesting finding was that individual PFAS concentrations showed compound-specific changes between one and six months postpartum.
However, despite these changes in individual compounds, the summed concentration of the detected PFAS remained relatively unchanged between the two sampling periods.
The researchers also observed differences in the quantification of short-chain PFAS compounds, with detection frequencies shifting noticeably between the sampling time points [3].
These findings highlight the complexity of PFAS exposure during lactation and demonstrate why monitoring individual chemical compounds is important.
What Does This Research Mean for Maternal and Infant Health?
Understanding PFAS concentrations in human milk is an important part of investigating environmental chemical exposure during early life.
Infants may encounter environmental contaminants through several pathways, and researchers need reliable analytical tools to understand the contribution of different sources.
The development of a sensitive, high-throughput method for PFAS analysis in human milk provides an opportunity to generate more detailed exposure data.
In particular, the method could support future research into:
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Maternal PFAS exposure and body burden.
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PFAS transfer from mothers to infants during lactation.
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Changes in PFAS concentrations throughout breastfeeding.
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Differences in exposure between populations and geographical regions.
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The occurrence of legacy and replacement PFAS compounds in biological samples.
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Relationships between environmental exposure and potential health outcomes.
However, it is important to distinguish between measuring exposure and demonstrating health effects.
This study primarily focuses on analytical method development and validation. Although it provides valuable information about PFAS concentrations in a group of human milk samples, it was not designed to establish whether these concentrations cause adverse health outcomes in infants.
Larger epidemiological studies will be needed to investigate these questions further.
The Future of PFAS Biomonitoring: Why Scalable Analytical Methods Matter
As awareness of PFAS contamination continues to grow, the need for reliable environmental and human biomonitoring methods is becoming increasingly important.
Traditional analytical workflows can be expensive, time-consuming and difficult to scale. These limitations can restrict the number of samples available for research and make it harder to understand exposure patterns across entire populations.
The method developed by Patel and colleagues offers a potential solution by combining several important analytical improvements:
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Reduced human milk sample requirements.
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Automated and standardised sample preparation.
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Effective removal of interfering biological matrix components.
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Sensitive detection using LC-HRMS.
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Simultaneous quantification of multiple PFAS compounds.
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Increased laboratory sample throughput.
The ability to quantify both legacy and replacement PFAS is particularly relevant. As the use of certain historically common PFAS has been restricted or phased out in various applications, alternative fluorinated chemicals have become increasingly important subjects of environmental monitoring.
Being able to monitor a broader range of PFAS compounds will help researchers develop a more comprehensive understanding of human exposure.
Ultimately, scalable analytical methods could support larger biomonitoring programmes, improve the quality of environmental exposure data and help inform future public health research.
Conclusion: A Significant Step Forward in PFAS Analysis in Human Milk
The study by Patel and colleagues represents an important development in the analysis of PFAS in human milk.
By combining automated liquid handling, enhanced matrix removal and liquid chromatography-high-resolution mass spectrometry, the researchers have developed a semi-automated method capable of quantifying 23 PFAS compounds using only 300 µL of human milk.
With detection limits as low as 0.53 pg/mL and the ability to process 72 samples in a 20-hour batch, the workflow addresses several important limitations associated with conventional PFAS analysis.
Its application to paired samples from 36 mothers also demonstrates how this approach can be used to investigate changes in PFAS concentrations during lactation.
Although further research is needed to understand the broader implications of maternal and infant PFAS exposure, improved analytical methods are an essential first step.
By making PFAS analysis more sensitive, efficient and scalable, this research could help advance environmental biomonitoring and improve our understanding of chemical exposure during one of the most important stages of human development.
The bigger picture: Better measurement leads to better exposure data, and better exposure data provides a stronger foundation for understanding and addressing environmental chemical contamination.
