Analyzing PFAS and Androgens through HPLC and Radiochromatography

🎤 Andrea Andress Huacachino

Dive into the world of environmental toxicology with Andrea Andress Huacachino, a PhD student at the University of Pennsylvania. In this fascinating interview, Andrea discusses her groundbreaking research on PFAS (per- and polyfluoroalkyl substances) and their impact on androgen regulation. Learn about:

• The novel finding of PFOA's effect on pre-receptor androgen regulation
• Innovative use of radio chromatography in hormone analysis
• Challenges in developing HPLC methods for androgen separation
• The broader implications of androgen dysregulation on human health

Discover how environmental pollutants affect hormones in all humans and why this research matters for everyone. Perfect for science enthusiasts, students, and anyone interested in the intersection of chemistry and human health.

Video Transcription

Environmental pollutants and hormonal regulation

Interviewer: I know you’ve studied the impact of environmental pollutants such as PFAS on hormonal regulation. Could you give us an overview of that work?

Andrea: Our work falls under environmental toxicology, which looks at contaminants in the environment and how they affect human health. We’re particularly interested in human health, and our lab studies PFAS in the context of hormonal regulation.

Hormones are chemical messengers in the body. They interact with receptors on cells and trigger biological responses, such as the production of specific proteins. Because of this signaling role, even small disruptions can have significant effects.

In addition to hormones and receptors, there are also enzymes known as pre-receptor regulators. These enzymes control hormone levels before the hormones even reach their receptors. Depending on the physiological needs of the body, hormones can be activated or inactivated at this stage.

Our lab focuses on these pre-receptor regulators. It has long been speculated that PFOA (perfluorooctanoic acid), a member of the PFAS family, can interfere with hormonal balance, particularly with androgens. Androgens include hormones such as testosterone, DHT, and DHEA. Although they’re often associated with male physiology, they are present in all sexes and play important roles across the population.

There has been epidemiological evidence suggesting a link between PFOA exposure and androgen dysregulation, but it was unclear where this disruption occurred—whether at the androgen receptor itself or earlier in the pathway.

What we discovered is that the effect occurs at the pre-receptor level. PFOA inhibits an enzyme called aldo-keto reductase 1C2 (AKR1C2). This enzyme normally inactivates a potent androgen before it can bind to the androgen receptor. When PFOA inhibits this enzyme, androgen levels remain elevated, leading to altered biological signaling.

Why HPLC and radiochromatography were used

Interviewer: You used HPLC to analyze androgens. Why was this the right analytical approach?

Andrea: We use a range of analytical techniques in our lab, including different types of chromatography and mass spectrometry. In this particular case, chromatography was essential because there was no existing assay for the specific enzymatic reaction we wanted to study. We had to develop a method from scratch.

Many androgens can be detected using UV/Vis spectroscopy, but the androgen we were most interested in—DHT—doesn’t have a chromophore. That meant we couldn’t detect it optically. To solve this, we used radioactive labeling and combined HPLC with radioactive detection, a technique known as radiochromatography.

Radiochromatography allows you to separate compounds chromatographically and then detect them based on radioactivity. While this approach has been used historically, it’s less common today, so there was a learning curve—but also a lot of excitement in revisiting the technique.

One challenge was obtaining standards. The parent androgen was commercially available in radioactive form, but the downstream metabolites were not. We had to synthesize them ourselves. Fortunately, previous work in our lab had established an enzymatic synthesis pathway with nearly quantitative yield, which we adapted for our needs. We confirmed purity using HPLC before proceeding.

Separating the compounds chromatographically was one of the most difficult aspects. Androgens are structurally very similar—sometimes differing only in stereochemistry. We tested multiple columns, solvents, and flow rates before finally achieving reliable separation using a C8 column.

Radiochromatography also introduces additional complexity at the detection stage. The radioactive detector relies on liquid scintillation counting, which requires a second solvent stream. Both chromatographic flow and scintillation flow must be optimized. Too slow and peaks broaden; too fast and you waste expensive scintillation fluid. After extensive optimization, we developed a robust method that worked well across all parameters.

Sample preparation workflow

Interviewer: Could you describe your sample preparation workflow?

Andrea: Our samples come from cultured cells. After treating the cells with radioactive androgens and allowing metabolism to occur, we collect media samples. The androgens are extracted using diethyl ether. We separate the organic layer, dry it down, and repeat the extraction multiple times.

Once fully dried, the extracts are reconstituted in 60% acetonitrile, which is compatible with our chromatographic method. Drying is performed using nitrogen blowdown, which we’ve found to be the most efficient approach.

Typically, we extract about 2 mL per cycle, and because we repeat the extraction two to three times, we evaporate a total of 4–6 mL down to complete dryness. Diethyl ether is highly volatile, so evaporation is relatively fast.

Using radioactivity significantly reduces sample manipulation. In contrast, mass spectrometric detection of androgens requires derivatization because they ionize poorly, which adds time and complexity. Starting with a radioactive androgen simplifies the workflow considerably.

Challenges in HPLC method development

Interviewer: What were the main challenges in developing the HPLC method?

Andrea: The biggest challenge is that androgens are extremely similar structurally. Many differ only by small functional group changes or stereochemistry. In our case, one reaction converted a ketone to an alcohol. You might expect predictable changes in polarity, but chromatography doesn’t always behave as expected.

Some alcohols eluted before ketones, which was counterintuitive. You can theorize how changing flow rate or solvent composition should affect separation, but in practice, it often behaves differently. That trial-and-error process is one of the biggest lessons I’ve learned—and also one of the reasons I enjoy chromatography so much.

Interestingly, compounds that differed only by stereochemistry were not the hardest to separate. The most challenging separations involved compounds that appeared more distinct on paper. It reinforced how empirical chromatographic method development really is.

Broader relevance of the research

Interviewer: What would you like people to take away from your work?

Andrea: One important message is that androgens affect everyone. Even though they’re often framed in a male or fitness context, they play essential roles across all sexes. Environmental factors that disrupt androgen regulation can therefore affect the entire population.

Environmental exposure to chemicals like PFAS may contribute to diseases such as polycystic ovary syndrome (PCOS), which involves elevated androgen levels, or conditions like prostate cancer, which are linked to androgen signaling. Understanding how these chemicals interfere with hormonal pathways is crucial for public health.

Conferences and PFAS research context

Interviewer: Have you worked with PFAS in the context of EPA methods?

Andrea: Not directly. PFAS research is extremely broad. My work is more focused on the biological effects rather than analytical detection in environmental samples.

Interviewer: Which conferences do you usually attend?

Andrea: I typically attend meetings of the Society of Toxicology and the American Chemical Society, particularly within the Chemical Toxicology Division.

Although our lab has methods to detect PFAS such as PFOA and PFOS, my personal research focuses on androgen metabolism. Still, it’s encouraging to see PFAS research gaining attention across many disciplines.

This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.

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