Detecting Anabolic-Androgenic Steroids in E-cigarettes Seized from Scottish Prisons using GC–MS

The detection of anabolic-androgenic steroids (AAS) in seized e-cigarette cartridges has revealed an unexpected development in the illicit drug landscape within Scottish prisons. In a recent study published in Forensic Science International, ¹ researchers at the Leverhulme Research Centre for Forensic Science (LRCFS), Dundee, Scotland reported the identification of two AAS compounds, mestanolone and oxandrolone, in five seized e-cigarette cartridges The findings highlight not only an unusual potential route of administration, but also the importance of broad, non-targeted drug screening in identifying emerging patterns that might otherwise go unnoticed.

In this interview, with LCGC International, Lorna Nisbet, Principal Investigator for Forensic Toxicology at LRCFS, discusses the research behind the discovery and the analytical challenges involved in identifying the compounds. She explains why gas chromatography–mass spectrometry (GC–MS) was selected, how researchers confirmed the presence of the steroids, and why analysing the cartridges without opening them provided a safer and practical approach. She also reflects on what the findings reveal about the changing nature of illicit drug markets and the importance of being prepared to detect substances that researchers may not initially expect.

With expertise spanning forensic toxicology, new psychoactive substances and seized-drug analysis, Nisbet also discusses how the research will inform future testing and the wider development of forensic drug-analysis capabilities in Scotland

What was the rationale behind your paper Detection of Anabolic-Androgenic Steroids in e-cigarettes Seized from Prisons: A Case Study¹?

Leverhulme Research Centre for Forensic Science (LRCFS) has been involved in the testing of non-judicial samples seized from the Scottish Prison estate since 2019 and so have been monitoring the changing drug patterns within this quite specific ecosystem for some time. When I joined LRCFS in 2022, I brought a PhD student with me from Anglia Ruskin University whose research focused on developing analytical methods for detecting anabolic-androgenic steroids in forensic toxicology. Detection of these compounds is still limited to a relatively small number of specialist laboratories, and he has been a strong advocate for increasing detection capabilities. As a result, the possibility of encountering anabolic steroids was something that was regularly discussed within our research group.

We had identified some anabolic androgenic steroids previously as part of the prison study, however quite unexpectedly, we began identifying anabolic-androgenic steroids in seized e-cigarettes. This was a particularly unusual route of administration and not something that had previously been reported. We felt it was important to publish these findings, not because we had all the answers, but because the discovery raised a number of important questions about how these substances are potentially being used and distributed within prisons.

Why was gas chromatography–mass spectrometry (GC–MS) chosen as the analytical technique for this study?

GC–MS was chosen because it is a well-established and widely used technique for routine seized drug analysis. In this study, we were not dealing with the trace concentrations that are typically encountered in forensic toxicology; we were analysing seized drug material, where the concentrations are generally much higher. This meant that we did not necessarily need the more sophisticated analytical platforms that might be required for highly sensitive toxicological applications. Another important advantage of GC–MS was its ability to support non-targeted screening. When looking at samples from the illicit drug market, we do not always know in advance what substances we are going to find, and the drug landscape can change rapidly. Using a technique that allows us to screen more broadly means that we are better placed to recognise unexpected or emerging compounds rather than simply looking for a predefined target list. There is also a practical consideration. GC–MS is a robust and relatively accessible technology that is available in many forensic laboratories internationally. While more advanced techniques undoubtedly have their advantages, for seized drug analysis you do not always need all the “bells and whistles” to answer the forensic question effectively. This is particularly relevant when considering laboratories in lower-resource settings or developing countries, where access to highly advanced instrumentation may be limited. Using established “fit-for-purpose” analytical approaches can therefore make findings more transferable and the methodology more realistic for laboratories operating with more limited resources.

What were the main challenges developing this method from an analytical perspective and how did you overcome them?

The method itself was relatively straightforward because we were working with seized drug material rather than biological samples. This meant that we did not require a derivatization step, and we were primarily looking for the parent compounds rather than metabolites, as would typically be required when developing a toxicology method. This simplified both the sample preparation and the analytical process considerably.

One of the more interesting challenges arose during the identification of mestanolone. Four of the samples were identified as containing mestanolone, but this compound has been reported as a potential breakdown product of oxymetholone, either through thermal degradation in the GC–MS injector or degradation in solution. This meant that we needed to be particularly careful about how we interpreted its detection. A simple identification of mestanolone could potentially raise the question of whether the original material had actually contained oxymetholone. We therefore had to consider the analytical behaviour of these structurally related steroids and ensure that the identification was sufficiently selective and reliable. In this case, because we detected mestanolone without evidence of oxymetholone, we considered it unlikely that oxymetholone was present in the samples analysed.

The researchers did not open the e-cigarette cartridges before extraction. Why might they have chosen this approach, and what are the potential advantages or disadvantages of analysing samples in this way?

There was actually quite a practical reason for this approach. Initially, we found it difficult to establish exactly how the e-cigarette cartridges were being tampered with and how the illicit substances were being introduced. From a health and safety perspective, we did not want to smash the cartridges open, particularly as this could introduce broken glass into the methanolic extraction and create additional handling risks.

We therefore found that rinsing the methanol through the cartridge provided a safer and more effective approach. It allowed us to recover the material of interest without having to physically dismantle the device.

Interestingly, after completing the work, we watched a BBC documentary about prison life in which someone with lived experience demonstrated exactly how these cartridges were being tampered with. It was a useful reminder that the chemistry can only answer part of the question.

How were the anabolic-androgenic steroids identified using GC–MS? Why are retention times, reference standards, and mass spectral library matching important for confirming compound identity?

Although we are a research laboratory operating within a university, we follow the same guidelines and standards as far as is feasibly possible when carrying out our analytical work. We want the findings we produce to be meaningful and acceptable to the wider forensic drug analysis community, rather than simply being research results generated in a university laboratory. For this reason, we followed established identification principles, including the guidance provided by the United Nations Office on Drugs and Crime (UNODC)andthe Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG). In this study, we therefore did not rely on a mass spectral library match alone. We compared the retention time of compounds detected in the seized samples with authentic reference standards and also compared their mass spectra with established libraries, including NIST14 and SWGDRUG. Using these different pieces of evidence together gives us greater confidence in the identification. This is particularly important for anabolic-androgenic steroids because they are structurally related and can therefore have similar analytical characteristics. The combination of retention time, reference standards and mass spectral data provides a much more robust identification than relying on any one characteristic in isolation. Ultimately, even though this was a research study, we wanted the analytical approach to be consistent with what would be expected within forensic drug analysis.

What anabolic-androgenic steroidswere identified?

We identified two anabolic-androgenic steroids across the five e-cigarette cartridges: mestanolone and oxandrolone. Mestanolone was detected in four of the samples, while oxandrolone was detected in one.

It was also interesting that the steroids were found alongside other drugs. All five samples contained nicotine, while four also contained another illicit substance, including SCRAs, Δ9-THC or cocaine.

The study reports that this may be the first detection of anabolic-androgenic steroids in e-cigarette cartridges. Are you planning to extend this research further?

At the moment, we do not have specific plans to undertake a dedicated follow-up study. Our focus is currently on establishing the National Drug Testing and Research Laboratory for Scotland, so that is understandably taking up a lot of our attention. What is encouraging from this work, however, is that we now know that we have the analytical capability to detect these compounds in seized material. We will therefore be including anabolic-androgenic steroids within our testing going forward, rather than treating them as something outside our usual screening scope. I think that is one of the important messages from this work: you cannot say you are not seeing something if you are not looking for it. This study started because we happened to be looking for AASs, and that allowed us to identify something that might otherwise have been missed.

Reference

1. Harries, R. L.; Norman, C.; Reid, R.; Nic Daéid, N.; Nisbet, L. A. Detection of Anabolic-Androgenic Steroids in e-Cigarettes Seized from Prisons: A Case Study. Forensic Sci. Int. 2024, 356, 111965. DOI: 10.1016/j.forsciint.2024.111965

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