Decoding Cannabis: Differentiating Hemp and Marijuana

In this interview conducted at Pittcon 2024 in San Diego, Rabi Musah delves into her innovative use of Direct Analysis in Real-Time – High-Resolution Mass Spectrometry (DART-HRMS) and chemometric analysis to differentiate between hemp and marijuana varieties of Cannabis sativa, exploring the forensic and legal implications of these findings.

Could you start by introducing yourself and sharing a bit about your background and what led you to focus on the analysis of cannabis evidence in forensic science?

Several years ago, we developed a keen interest in psychoactive plants within the forensics field, with a specific focus on the identification of these plants by crime scene analysts during criminal investigations. There are over 400 plants that can induce a high, and in any particular country, only a very small subset, usually less than 20, are either scheduled or outlawed.

As a result, people import these plants with impunity, attempting to circumvent the laws governing drugs that are scheduled, such as marijuana. Due to the lack of laws outlawing these drugs, people overdose, experience psychosis, and suffer negative health impacts, making it very difficult for the judicial system to deal with these issues.

Part of the problem stems from the need for a definitive method to identify these plant-based substances. When people grind up roots, leaves, or other plant parts, it becomes very difficult to identify them because they have lost all their morphological features.

We began by developing this approach where we take advantage of the fact that the genomic differences between plants are, of course, species-specific, and that translates into species-specific changes in metabolome or small-molecule profiles.

If you apply statistical analysis and processing to the data that you get from the profiles, you can readily differentiate species. The challenge with that approach is its time-consuming nature, particularly when it comes to building databases of chemical signatures specific to plants. We discovered a method that employs a fast mass spectrometric-based analytical technique to accomplish this, and it has significantly aided our efforts.

This technique is known as Direct Analysis in Real Time, High-Resolution Mass Spec, or DART-MS for short. It turns out that you can get these chemical signatures very quickly. Shortly after, we realized that we could use this knowledge to answer the questions associated with cannabis. Since then, we have been applying what we have learned about identifying psychoactive plants, specifically cannabis.

In your Pittcon talk abstract, you discussed the challenges of differentiating hemp and marijuana in forensic laboratories. What are the key factors that differentiate hemp and marijuana?

This is a challenging problem because hemp and marijuana are simply varieties of the same plant species, cannabis sativa. While genetic testing has revealed clear genetic differences between the two, it is important to remember that they belong to the same species. Therefore, it is not feasible for crime labs to use genetic testing to differentiate between the two.

One way to distinguish between hemp and marijuana is by measuring the presence of the psychoactive component delta-9-tetrahydrocannabinol, commonly known as THC. Both hemp and marijuana contain THC, but the amount of marijuana is significantly higher. In contrast, the THC content in hemp is considerably lower.

This is the main distinction between hemp and marijuana. While there are other differences, this is the key distinction that the government focuses on. The Schedule 1 status of marijuana is directly related to its higher THC content.

What are the current challenges and limitations that make this differentiation between these two varieties of Cannabis in forensic labs so difficult?

Cannabis has been illegal in the United States for a long time, regardless of whether it is the low-THC form, like hemp, or the high-THC form, such as marijuana. However, hemp has traditionally been recognized as a valuable agricultural product worldwide due to its versatility in producing a variety of goods. American farmers sought access to this lucrative market.

In 2018, the US Congress passed the Farm Bill, establishing hemp as an agricultural product. This legislation necessitated that crime labs analyze every cannabis sample they received. The government stipulated that plant material containing 0.3 % THC by mass or less would be classified as hemp, while anything higher would be considered marijuana.

Accurately quantifying the amount of THC present in samples became essential for proper classification. Crime labs with extensive THC quantification experience excel at this task. However, the methods used are time-consuming and become less effective when handling large volumes of samples. This creates significant challenges, such as testing backlogs, lengthy experiment times, and substantial resource demands, including staffing and consumables.

There is an urgent need for rapid testing methods to enable crime labs to efficiently triage samples, reserving resources for those identified as marijuana. Addressing these challenges is a critical focus for us.

Could you tell us about the DART-HRMS approach you developed for differentiating between hemp and marijuana, as well as some of the most significant technical hurdles that you faced?

DART-HRMS, which stands for Direct Analysis in Real Time, High-Resolution Mass Spectrometry, is a mass spectrometry method that utilizes ambient ionization. Unlike other mass spectrometry methods that operate under high vacuum, this method operates at atmospheric temperature and pressure in open air. This not only simplifies sample analysis but also allows the analysis of samples in their native form.

One of the challenges that crime labs currently face with many conventional approaches is the extensive sample pretreatment needed to transform the samples into a form suitable for analysis. With DART-MS, you can simply analyze the sample in its native form. Additionally, you can obtain a spectrum within three to five seconds.

If you want to apply statistical analysis to the generated data or create a database of chemical signatures, you need hundreds or thousands of these signatures to build a robust database. DART-MS facilitates this by enabling the rapid generation of signatures.

Another advantage of this technique is its ability to provide information on a range of small molecules or metabolites present in the plant material. This includes the identification of highly polar compounds as well as the detection of highly non-polar compounds. This high-information content approach is beneficial for statistical analysis, especially when trying to identify which particular subset of molecules is important for distinguishing one type of sample from another. This is precisely the goal of differentiating hemp from marijuana.

The DART-HRMS and statistical analysis approach we developed revolutionizes current practices in forensic laboratories for Cannabis sativa analysis by addressing several key challenges. This method dramatically accelerates the analysis process in crime labs, leading to significant cost and time savings. By enabling quicker analyses, resources can be more efficiently allocated to samples that actually contain marijuana, streamlining the entire workflow.

Could you discuss the role of advanced statistical processing in your research and its significance in improving the accuracy of differentiating hemp and marijuana?

One way statistical analysis has manifested itself in forensic science is with DNA technology. The chances of you and me having the same DNA profile are about one in 6.5 million, indicating a high degree of success if you have the DNA profile. It would be amazing if we could get these kinds of numbers for other types of evidence.

Now, this will be very challenging, but it would be highly beneficial to apply a certain level of certainty to our identifications. When we encounter chemical signatures that could indicate either hemp or marijuana, it would be excellent to express our confidence levels. For instance, being able to state that we are 100 % certain a sample is hemp or 92.3 % sure a sample is marijuana would significantly enhance the reliability and credibility of our analyses.

The application of statistical analysis to metabolome profiles allows us to identify materials with a high degree of certainty and objectivity. This is crucial when the stakes are high, as it can potentially lead to jail sentences or aid in exonerating innocent people.

In your study, you identified several m/z values corresponding to known cannabinoids or fragments of major cannabinoids and terpenes. How does this knowledge contribute to the broader understanding of Cannabis sativa?

While it is true that hemp has generally lower levels of delta-9-THC than marijuana, it is also important to understand the other ways in which the two plants differ. The more you understand these differences, the better able you are to perform confirmatory testing to distinguish between the two. It also paves the way for the discovery of cost-effective methods for distinguishing between them.

In our case, for example, we observed differences in the terpenes present in both plants. It would be amazing if we could correlate the differences in terpene profiles to the distinctions in the amounts of THC present in the two plants since this can lead to more cost-effective ways to distinguish between them.

Based on your research findings, what are the next steps in addressing the challenges that crime labs face due to the legal distinction between hemp and marijuana?

The first step in advancing this work is for researchers to publish their results for proper evaluation. Once this happens, criminologists can begin using this technology to solve crimes, and it can become an accepted part of evidence in trials. This will help courts and the judicial system become more comfortable with the technology.

Forensic scientists typically resist change, and this is not a criticism because there is a good reason for it. They rely on tried and tested methods to ensure successful prosecutions, adhering to court-accepted gold standards. If a case uses DART-MS data to prove the identity of materials and the court accepts this evidence, it will likely encourage more crime labs to embrace this technology. This can ultimately aid in exonerating innocent people and prosecuting criminals more effectively.

Pittcon Thought Leader: Rabi Ann MusahPlay

As we mark the 75^th anniversary of Pittcon, could you share your first memory of attending this conference and how it impacted your view of the scientific community?

I first attended Pittcon in 2017. Aside from the renowned presenters conducting cutting-edge research, one distinguishing feature of Pittcon is the presence of vendors, which I find truly remarkable. While vendors attend most conferences, those at Pittcon bring operational equipment and offer on-site sample analysis.

Since 2017, I have attended every year, and each time, I learn about new, cutting-edge technology from different companies. We also make purchases that further our research, making it a fantastic conference from that perspective.

Additionally, the conference provides an invaluable opportunity for the individuals I mentor, whether they are post-docs or graduate students, to interact with the authors of the textbooks we use for teaching.

These interactions allow them to learn from practitioners about how their research intersects with existing studies and how to adapt it to meet the needs of professionals, such as forensic scientists.

In an academic lab, we may develop approaches that work brilliantly at the bench but are impractical for practitioners unless modified according to their instructions. This feedback is crucial, enabling us to translate our research into something usable by professionals in the field.

What are you most looking forward to at Pittcon 2024 in San Diego?

I am very interested in seeing what the vendors have to offer, especially as we are moving our research into new areas, such as the detection of cannabis intoxication. Many individuals here are also working in this field, which presents amazing opportunities to learn from their experiences and insights.

We have encountered some new findings in our lab that we find puzzling, and we suspect others may have addressed similar issues. You mentioned earlier that some of the molecules, or m/z values, were detected using mass spectrometry to differentiate between hemp and marijuana. It appears other researchers are also involved in this area, and I am eager to see what they have discovered.

Additionally, I have a post-doc and a graduate student from my lab with me, and I am excited to expose them to the resources available here. They are also giving talks, which is incredible. It is a significant achievement to present at Pittcon as a student or post-doc, highlighting the importance and recognition of their work.

About Rabi Musah

Areas of focus in her laboratory include forensic chemistry (of psychoactive plants; wildlife forensics to address illegal trafficking of endangered species; development of novel approaches to address concerns in forensic entomology; and environmental forensics); the environmental impact of terrestrial plant-derived and anthropogenic volatile chemical emissions; and the development of disease diagnosis tools through analysis of non-traditional biological matrices. Her scientific research has been featured on the PBS science documentary series NOVA and has been highlighted in Scientific American, The New Scientist (UK), Forensic Magazine, Chemical and Engineering News, Mental Floss, Science, Ms. Magazine, numerous Times Union Newspaper stories, and the Mirror Newspaper (UK), among other publications. Professor Musah teaches both undergraduate and graduate courses in organic chemistry, biochemistry, medicinal chemistry and forensics chemistry, as well as sub-disciplines of these areas. She also conducts research on development and implementation of sustainable strategies that can be readily expanded, replicated and applied to increase engagement and retention of students in science, technology, engineering and math (STEM) majors in higher education.

About Pittcon

Pittcon is the world’s largest annual premier conference and exposition on laboratory science. Pittcon attracts more than 16,000 attendees from industry, academia and government from over 90 countries worldwide.

Their mission is to sponsor and sustain educational and charitable activities for the advancement and benefit of scientific endeavor.

Pittcon’s target audience is not just “analytical chemists,” but all laboratory scientists — anyone who identifies, quantifies, analyzes or tests the chemical or biological properties of compounds or molecules, or who manages these laboratory scientists.

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