Potential Obstacles in Chromatographic Analyses Distinguishing Marijuana from Hemp
LCGC International's April series for National Cannabis Awareness Month concludes with a discussion with Walter B. Wilson from the National Institute of Standard and Technology’s (NIST’s) Chemical Sciences Division regarding recent research his team conducted investigating chromatographic interferences that can potentially inflate the levels of Δ9-THC in Cannabis sativa plant samples, and possible solutions to avoid this problem.
Researchers at the National Institute of Standards and Technology’s (NIST's) Chemical Sciences Division have been working on improving analytical methods to distinguish between hemp and marijuana, especially after the 2018 Agriculture Improvement Act legally defined hemp as Cannabis sativa with ≤0.3% total Δ9-THC (including its precursor Δ9-THCA). This responsibility now largely falls to forensic and cannabis testing labs. NIST previously validated a reliable method using liquid chromatography with photodiode array detection (LC–PDA) to measure Δ9-THC, Δ9-THCA, and related cannabinoids. A recent study extends that method to analyze 16 commercial hemp and 20 seized cannabis samples, reflecting real-world testing scenarios. It revealed that compounds like cannabinolic acid (CBNA) and synthetic Δ8-THC by-products can interfere with Δ9-THC readings, potentially causing hemp to be misidentified as marijuana. Analysis of over 7,400 samples from 2023 showed these interferences are common. The study demonstrates how adjusting chromatographic methods, using selective detection, and applying peak deconvolution can resolve such interferences and improve accuracy.
Your recently published paper (1) stems from the National Institute of Standards and Technology (NIST) evaluation of existing and developing new analytical methods for the differentiation of hemp and marijuana since the passage of the Agriculture Improvement Act of 2018. What are the basic differences between the two?
This new legislation defined hemp as the Cannabis sativa plant containing 0.3 % or less of decarboxylated-Δ9-tetrahydrocannabinol (Δ9-THC) and removed hemp from the United States Drug Enforcement Agency controlled substance list. The United States Department of Agriculture later clarified that this threshold must be representative of the total Δ9-THC, which includes the Δ9-THC acidic precursor Δ9-tetrahydrocannabinolic acid (Δ9-THCA). Δ9-THCA is converted to Δ9-THC via decarboxylation after heating. As a result, the burden of making the distinction between hemp and marijuana fell to forensic and Cannabis testing laboratories.
What is Δ9-THC and why is it important?
Δ9-THC is the primary psychoactive compound present in Cannabis plants responsible for its intoxicating effects through altering a user’s perception, mood, and behavior.
You state in your paper that the NIST Chemical Sciences Division (CSD) has previously demonstrated accurate and precise analytical measurements for Δ9-THC, Δ9-THCA, and nine other related cannabinoids in well-characterized samples from interlaboratory studies at NIST by liquid chromatography with photodiode array detection (LC–PDA) following a methanolic extraction. The new research you are reporting on expands this method for the first time to include 16 commercial hemp samples and 20 seized Cannabis samples, simulating the types of samples typically analyzed by forensic and Cannabis testing laboratories. What motivated this expansion of the method?
After the passage of the new legislation, NIST developed an integrated measurement services program (2) to support forensic and Cannabis testing laboratories by developing Reference Materials (RMs) and implementing an interlaboratory studies program (Cannabis Laboratory Quality Assurance Program, CannaQAP). To provide these services, NIST needed to implement or develop analytical methodologies to accurately measure Δ9-THC, Δ9-THCA, total Δ9-THC, and other related cannabinoids in Cannabis samples. The initial LC-PDA method implemented at NIST was demonstrated to provide accurate measurements for hemp plant and oil samples (3). Later, this method was used for assigning cannabinoid mass fractions on the first NIST hemp plant RM 8210 (4,5) and test samples in CannaQAP (6,7). Despite the relatively short analysis time of 10 min, this method was not transferable to forensic laboratories because it required approximately 70 min of sample preparation time. For this reason, NIST was awarded a research grant from the National Institute of Justice (NIJ) to optimize the sample preparation method for routine analysis of Cannabis plant samples in forensic laboratories, as highlighted in a recent publication reducing the sample preparation time to 15 min (8). To thoroughly investigate this method's capabilities, NIST needed to analyze commercial and seized Cannabis plant samples to determine any issues that may arise before implementation into forensic laboratories.
Forensic laboratories have historically used gas chromatography mass spectrometry (GC–MS) for this type of analysis. Your team used liquid chromatography (LC) coupled with ultraviolet (UV) detection. What benefits did LC–UV offer?
Based on statistics published from over 200 participants in the Cannabis Laboratory Quality Assurance Program (CannaQAP) (7), LC–UV is the primary analytical technique used in Cannabis testing laboratories. It uses either a single UV wavelength detector or multiple wavelength detectors called photodiode arrays. The primary benefit of LC–UV versus GC–MS is that it can accurately measure Δ9-THCA in Cannabis samples independently from Δ9-THC. The same can be accomplished by combining LC with MS or MS/MS, as Andrea Yarberry at NIST demonstrated in recent publications (1,7), but it comes with a significantly higher instrumental cost. GC-MS requires a derivatization step to be used before analysis to prevent the decarboxylation of Δ9-THCA to Δ9-THC in the GC inlet. This procedure would require additional sample preparation steps and consumables that are unwanted by forensic laboratories.
What were your main findings? Was there anything surprising or interesting?
NIST discovered that chromatographic interferences existed for Δ9-THC for the previously published LC–PDA method from synthetic Δ8-THC by-products in commercial hemp samples and cannabinolic acid (CBNA) in seized samples. Three of the commercial hemp plant samples analyzed in this study had elevated levels of Δ8-THC on the product labels; however, natural levels of Δ8-THC are at extremely low levels in Cannabis plant samples. The presence of high levels of Δ8-THC is only going to exist if the Cannabis plant is adulterated through the addition of synthetic Δ8-THC, which also includes the possibility of many other by-products with similar chromatographic behavior to Δ9-THC. In the case of CBNA, these samples were stored for at least four years in the dark at room temperature, permitting the slow degradation of Δ9-THCA to produce CBNA and then eventually decarboxylate to cannabinol (CBN).
The measurements performed here were limited to a small sample size. To better understand the prevalence of these chromatographic interferences, NIST obtained a more extensive data set from collaborator Stephen Goldman, the Chief Scientific Officer at Kaycha Labs. Data was provided from their Denver, Colorado location, which is accredited to analyze hemp/marijuana plant samples and its LC-UV method separates 21 cannabinoids, including the interfering cannabinoids. Δ8-THC and CBNA were detected in approximately 0.5 % and 80 %, respectively, of the almost 7500 Cannabis plant samples analyzed in 2023. These results supported the previous statements for Δ8-THC being present at low levels. However, they contradicted the belief that CBNA was only present due to the poor storage conditions because these samples were predominantly fresh with minimal degradation of Δ9-THCA. Additionally, ≈ 94 % of these samples had a total Δ9-THC mass fraction above the 0.3 % threshold, suggesting that Cannabis and forensic laboratories should anticipate most marijuana samples to have some CBNA present.
What were the main analytical challenges you encountered and how did you overcome them?
As a result of the challenges highlighted above, NIST had to implement a detailed procedure for evaluating the purity of chromatographic peaks of Δ9-THC and explore potential modifications to help resolve these chromatographic interferences. Δ9-THC was only quantitatively measured if it had matching retention times and absorbance spectra across the entirety of the chromatographic peak to a reference standard. NIST highlighted the benefits of modifying the chromatographic mobile phase by using methanol instead of acetonitrile or a combination of both, using selective detection methods (PDA, MS, and MS/MS), and peak deconvolution to resolve chromatographic interferences of Δ9-THC.
Were there any advantages to performing your research in a government facility as opposed to one from academia or the private sector?
As a non-regulatory agency, NIST has been uniquely positioned to be impartial and build working relationships with all the different sectors of the Cannabis industry that academic institutions or other organizations might not be able to. The research highlighted in the recent publication here included key collaboration in forensics using obtained seized Cannabis plant samples previously adjudicated from a local police department. The collaboration agreement NIST was able to form with Kaycha Labs provided access to extensively larger data sets, helping NIST better understand the Cannabis industry as it continues to evolve and help assist NIST in developing future RMs.
What are the next steps in this research?
Even with the benefits of LC-based methods discussed in previous questions, many forensic laboratories are still relying on GC-MS to differentiate between hemp and marijuana, referring to them as “semi-quantitative” or “threshold methods” at 1 % instead of 0.3 %. For this reason, Jerome Mulloor at NIST has performed extensive studies on understanding the decarboxylation issues in the GC inlet and has developed a GC-MS method to accurately measure total Δ9-THC without derivatization that has been submitted for peer review. Additionally, NIST CSD has expanded its research by adopting the previously developed analytical methods for Cannabis plant samples for Cannabis-derived finished products like vapes, oils, and gummies. This required NIST to develop new sample preparation procedures to combine with these methods through additional grant funding from NIJ. NIST has started disseminating these results at conferences and is working on several publications to be released in the future.
References
1. Wilson, W. B.; Yarberry, A. J.; Goldman S. Chromatographic Interferences Potentially Inflating the Levels of Δ9-THC in Cannabis Sativa Plant Samples and Possible Solutions. J. Chromatogr. A 2025, 1748, 465871. DOI: 10.1016/j.chroma.2025.465871
2. NIST Tools for Cannabis Laboratory Quality Assurance. NIST website.https://www.nist.gov/programs-projects/nist-tools-cannabis-laboratory-quality-assurance (accessed 2025-04-07)
3. Wilson, W. B.; Abdur-Rahman, M. Determination of 11 Cannabinoids in Hemp Plant and Oils by Liquid Chromatography and Photodiode Array Detection. Chromatographia 2022, 85, 115. DOI: 10.1007/s10337-021-04114-y
4. “NIST’s New Hemp Reference Material Will Help Ensure Accurate Cannabis Measurements.” NIST website.https://www.nist.gov/news-events/news/2024/07/nists-new-hemp-reference-material-will-help-ensure-accurate-cannabis (accessed 2025-04-07)
5. Bryan Sallee, C. E.; Wilson, W. B.; Barber, C. A.; Johnson, M. E.; Klingsick, J. R.; Mulloor, J.; Toman, B.; Wood, E. S. C.; Wood, L. J.; Yarberry, A. J. Characterization of Reference Material 8210: Hemp Plant. NIST Special Publication 260 2024, NIST SP 260-248. DOI: 10.6028/NIST.SP.260-248
6. Abdur-Rahman, M.; Phillips, M. M.; Wilson, W. B. Cannabis Quality Assurance Program: Exercise 1 Final Report. NIST Interagency Report 2021, NISTIR 8385. DOI: 10.6028/NIST.IR.8385
7. Yarberry, A. J.; Phillips, M. M.; Wilson, W. B. Cannabis Quality Assurance Program: Exercise 2 Cannabinoid Final Report. NIST Interagency Report 2024, NISTIR 8519. DOI: 10.6028/NIST.IR.8519
8. Wilson, W. B.; Urbas, A. A.; Jensen, H.; Sander, L. C. High-throughput LC-PDA method for determination of Δ9-THC and related cannabinoids in Cannabis sativa. Forensic Chemistry. 2024, 41, 100610. DOI: 10.1016/j.forc.2024.100610
Walter Wilson is a research chemist who coordinates the Cannabis research program in the Chemical Sciences Division at NIST with a focus on developing analytical methods, Cannabis reference materials, and the administration of a Quality Assurance Program. Walter is involved with the development of chromatographic methods for separating natural and synthetic cannabinoids in complex Cannabis matrixes such as dried plants, extracts, concentrates, and edibles. Wilson also has responsibilities in other aspects of natural product research at NIST involving tobacco and dietary supplements. He has currently published 42 peer-reviewed journal articles, 7 NIST special publications, and 2 book chapters. He was previously recognized in the 50th-anniversary issue of Chromatographia as a Rising Star in Chromatography.
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