Forensic Cannabis Analysis: Differentiating Hemp from Marijuana

Forensic drug analysis is instrumental in criminal investigations involving drug-related offenses. It helps law enforcement determine whether a substance adheres to legal standards, guiding decisions related to criminal charges and sentences.

Image Credit: 24K-Production/Shutterstock.com

Image Credit: 24K-Production/Shutterstock.com

Cannabis is the most widely produced and used illicit drug globally.1 A challenging aspect of analyzing cannabis evidence is the differentiation between marijuana and hemp, which are the major varieties of Cannabis sativa (the primary species in the Cannabis genus).2,3 While both marijuana and hemp contain the psychoactive compound delta-9-tetrahydrocannabinol (THC), the quantity of this molecule differs between them.

In 2018, the US federal guidelines outlined that Cannabis sativa containing 0.3 % THC or less is considered a legal agricultural product (classified as hemp), while samples with more than 0.3 % THC are the scheduled drug type (classified as marijuana).2

This threshold has imposed significant challenges on forensic laboratories, such as an increased workload due to the need to analyze the THC content of all incoming Cannabis sativa samples. Defining the error cutoff for the 0.3 % designation is also problematic for samples with threshold quantities of THC.2

Traditionally, marijuana and hemp are differentiated based on THC content using chromatography-based approaches, such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GS-MS), and GS-flame ionization detection (GS-FID).2,4

While these techniques offer distinct advantages, the recent regulatory changes have created a need for more high-throughput and sensitive methodologies that can accurately identify and quantify THC in Cannabis sativa.1

Recent investigations have found that combining ambient ionization techniques, such as direct analysis in real-time—high-resolution mass MS (DART-HRMS), with advanced statistical analysis aids in the rapid analysis and differentiation of marijuana and hemp plant material.2

These innovative techniques will be discussed at this year’s Pittcon as a part of the ‘Cannabis and Psychedelics track. In addition to talks from experts in cannabis analysis, Pittcon will host representatives from CPI International and LECO Corporation, who will showcase novel analytical instrumentation and systems for forensic analysis.

Pittcon 2024: Differentiating marijuana and hemp with DART-HRMS and advanced chemometrics

DART-HRMS quickly examines samples for the presence of cannabinoids, such as THC, while distinguishing them from samples that do not. This is via the detection of high-resolution masses of deprotonated or protonated precursor molecules.5

At Pittcon 2024, Dr. Rabi Musah, Professor of Chemistry at the State University of New York at Albany, will present a talk titled “Deep into the Weeds – A Combined DART-HRMS and Chemometric Approach for Differentiating Hemp and Marijuana Varieties of Cannabis sativa.”

In this presentation, Dr. Musah will provide insight into a recent study that analyzed marijuana samples (obtained from Drug Enforcement Administration-registered suppliers) and hemp plant materials (purchased from licensed vendors) using DART-HRMS with no sample pretreatment. Exploratory statistical analyses, including principal component analysis (PCA) and random forest, were also used to differentiate marijuana and hemp with high accuracy.2

PCA revealed chemical fingerprint signatures that distinguish between hemp and marijuana with a high level of certainty: a peak at nominal mass-to-charge ratio (m/z) 315 is indicative of THC presence. The identities of other m/z values have also been confirmed via analysis under ambient conditions, including those of known cannabinoids or fragments of major cannabinoids and terpenes.

This combined DART-HMRS and chemometric approach could enhance the analysis and differentiation of Cannabis sativa materials before the launch of confirmatory chromatographical tests. This will save both time and resources.

However, DART-HRMS cannot differentiate THC from other cannabinoid isomers like cannabinol (CBD), which occurs in high quantities in hemp under soft ionization conditions without sample pretreatment (such as derivatization).5

In one study that examined purified THC and CBD reference standards using DART-HMRS, a peak at nominal m/z 629 was observed, suggesting the presence of protonated dimers [2M + H+].

Further investigations using high-level density functional theory (DFT) calculations revealed that the THC-THC protonated homodimer complex is more stable than other dimer combinations (CBD-CBD and CBD-THC) by approximately 1-3 kcal/mole. However, with increasing temperatures, populations of the THC-THC and CBD-THC dimers decline, becoming insignificant at 200 K and above, while that of the CBD-CBD dimer rises.

Results suggested that, at a specific temperature, the protonated dimer peak at m/z 639 corresponds to CBD-CBD when CBD levels were high, as seen in hemp. In contrast, in marijuana samples with high THC content and minimal CBD, no m/z 629 peak is apparent in the DART-HRMS spectra.

Subsequent DART-HRMS analysis, categorized by the THC thresholds for hemp (≤0.3 %) and marijuana (>0.3 %), confirmed this distinction. Thus, m/z 629 could be utilized to rapidly differentiate marijuana and hemp using DART-HMRS analysis.

This research will be discussed in further detail by Dr. Musah in an additional talk at Pittcon, titled “Towards Development of a Rapid Approach for Differentiating Marijuana from Hemp—Insights from High-level Density Functional Theory Calculations.”

Cannabis analysis at Pittcon

Recent regulatory changes have imposed severe challenges on forensic laboratories, which are now tasked with the differentiation of Cannabis sativa plant materials based on their THC content.3 The expectation to conduct an entire quantitative analysis on all seized cannabis samples is irrational and unattainable, given current analytical methodologies.

The need for accurate, high-throughput THC quantification has led to advancements in analytical technology and systems, including the combined DART-HMRS and chemometric approach. This will be discussed further at Pittcon 2024, where attendees can expect to gain a comprehensive perspective on the future of cannabis analysis from experts in the field.

To find out more or to register for this year’s event, visit the Pittcon website.

References and further reading

  1. Slosse, A., et al. (2022). Analytical Strategies for Herbal Cannabis Samples in Forensic Applications: A Comprehensive Review. WIREs Forensic Science. doi.org/10/1002/wfs2.1479.
  2. Chambers, MI., et al. (2023). Combined Ambient Ionization Mass Spectrometric and Chemometric Approach for the Differentiation of Hemp and Marijuana Varieties of Cannabis Sativa. Journal of Cannabis Research. doi.org/10.1186/s42238-023-00173-0.
  3. Ya-Chih, C., et al. (2023). Differentiation of Hemp From Marijuana Using a Qualitative Decision-Point Assay. Forensic Chemistry. doi.org/10.1016/j.forc.2023.100541.
  4. Chambers, MI., et al. (2023). DART-MS Facilitated Quantification of Cannabinoids in Complex Edible Matrices─Focus on Chocolates and Gelatin-Based Fruit Candies. ACS Omega. doi.org/10.1021/acsomega.2c08172.
  5. Chambers, MI., et al. (2022). DART-HMRS as a Triage Approach for the Rapid Analysis of Cannabinoid-Infused Edible Matrices, Personal-Care Products and Cannabis Sativa Hemp Plant Material. Forensic Chemistry. doi.org/10.1016/j.forc.2021.100382.

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.

Having grown beyond its roots in analytical chemistry and spectroscopy, Pittcon has evolved into an event that now also serves a diverse constituency encompassing life sciences, pharmaceutical discovery and QA, food safety, environmental, bioterrorism and cannabis/psychedelics. 


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Last updated: Jan 29, 2024 at 11:45 AM

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