Ya-Chih Cheng

The legalization of hemp cultivation in the United States has raised the need for reliable methods to distinguish between legal hemp and illegal marijuana. Genetic analysis has emerged as a powerful tool, surpassing traditional chemical methods in specific aspects, such as analyzing trace amounts, aged samples, and different parts of the sample. Genetic differences in cannabinoid synthase genes offer promise for precise crop type determination, particularly focusing on genes like tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and cannabichromenic acid synthase (CBCAS). However, previous research faced several challenges in developing discriminatory genetic markers, including limited sample sizes, high similarity between the synthase genes, and the presence of pseudo synthase genes. A comprehensive study using Next‐Generation Sequencing (NGS) introduced a differentiation flowchart based on THCAS, CBDAS, and THCAS pseudogenes. To bridge the gap between NGS and the practical requirements of crime laboratories, two rapid genotyping assays were developed: a CE‐based SNaPshot™ assay and a TaqMan™ real‐time PCR assay. While the SNaPshot™ assay effectively differentiated various hemp and marijuana types, differentiation was limited with marijuana samples containing THC% close to the 0.3% legal threshold (0.3%–1%). The TaqMan™ qPCR SNP genotyping assay provided quicker results, making it an efficient choice for crime laboratories. However, this method had the same limitations as the SNaPshot™ assay with addtional challenges in differentiating edible hemp seed samples, and it did not provide additional CBD information. The study also highlighted the influence of two variants of one THCAS pseudogene on chemotype determination, emphasizing the necessity for precise genetic analysis for accurate categorization of cannabis varieties.

Gas chromatography–mass spectrometry (GC‐MS) is widely used for the identification of cannabinoids in seized plant material. Conditions used for instrumental analysis should maximize decarboxylation, while minimizing the in situ production of Δ9‐THC inside the GC inlet. In this study, decarboxylation of the acidic Δ9‐THC precursor and in situ degradation of cannabidiol (CBD) were investigated using seven commercial GC liners with different deactivation chemistries and geometries. While the inlet temperature was previously optimized at 250°C in a previously validated assay, we systematically examined the temperature‐dependent decarboxylation of tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) and cyclization of CBD between 230°C and 310°C using different liners using favorable and unfavorable conditions. Significant differences in decarboxylation rate and CBD cyclization were observed between different liner types. While no temperature‐dependent differences in decarboxylation rate were observed within liner type, liner‐dependent differences were observed (α = 0.05), particularly between those with different geometry. In contrast, temperature and liner‐dependent differences were observed for in situ formation of Δ9‐THC (α = 0.05). This was influenced by liner geometry and to a smaller extent by surface deactivation. Effects were exacerbated with liner usage. While significant differences were observed using new and used GC liners, differences between liners of the same type but different lot numbers were not observed. Inter‐instrument differences using the same liner were also evaluated and had minimal effect. Liner‐ and temperature‐dependent effects were also confirmed using more than 20 cannabis plant extracts. Careful selection of liner, inlet conditions, and regular preventive maintenance can mitigate the risks associated with in situ formation Δ9‐THC from CBD.

The Controlled Substances Act (CSA) classifies marijuana (Cannabis sativa) as a Schedule I illicit drug. However, the recent Agriculture Improvement Act of 2018 (U.S. Farm Bill) removed hemp from the definition of marijuana in the CSA, making it a legal crop. As a result, many hemp products are now available, including strains of hemp buds high in other cannabinoids such as cannabidiol (CBD) or cannabigerol (CBG). The genetic inheritance of chemical phenotype (chemotype) has been widely studied, with the tetrahydrocannabinolic acid (THCA) synthase gene at the forefront. Previous studies have speculated that there are two forms of the THCA gene, one that produces an active enzyme (present in marijuana) and one that cannot produce a functional enzyme (present in hemp). A DNA analysis method is desirable for determining crop type in sample types inconducive to chemical analysis, such as immature crops, trace residues, small leaf fragments, seeds, and root material. This study optimized and evaluated a previously reported single nucleotide polymorphism (SNP) assay for determining C. sativa crop type. Furthermore, the presence or absence of 15 cannabinoids, including THC and THCA, was reported in cannabis reference materials and 15 legal hemp flower samples. The SNP assay correctly identified crop type in most samples. However, several marijuana samples were classified as hemp, and several hemp seeds were classified as marijuana. Two strains of legal CBG hemp flowers were also classified as marijuana, indicating that factors other than the genetic variation of the THCA synthase gene should be considered when determining crop type.

Cannabis sativa (marijuana and hemp) is one of the most controversial crops worldwide. In the USA, the state-specific legalization of marijuana and recently legalized hemp pose a problem for law enforcement. This study seeks to utilize chloroplast hSTRs, INDEL, and SNPs markers to develop genotyping methods to aid in the differentiation of legal hemp from illicit marijuana and also for tracking the flow of trafficked marijuana. Three polymorphic regions: trnK-matK-trnK, ycf3, and accD-psal, of the C. sativa chloroplast genome were evaluated in order to distinguish crop type and biogeographic origin. A total of nine polymorphic sites were genotyped from five distinct populations (hemp from the USA and Canada, marijuana from Chile and USA-Mexico, and medical marijuana from Chile) with a custom fragment and SNaPshot^TM assay. The study also combined genotype results from the same sample set using 21 additional polymorphic markers from previous studies. The effectiveness of these multi-locus assays to distinguish sample groups was assessed using haplotype analysis, phylogenetic analysis, pairwise comparisons, and principal component analysis. Results indicated a clear separation of Canadian hemp using only the nine polymorphic sites developed in this study. The additional 21 markers were able to separate US hemp from both marijuana groups to a significant level (p < 0.05) when assessing average Fixation Indices (F_ST). This study demonstrated the applicability of these organelle markers for the determination of crop type and biogeographic origin of C. sativa. However, a more extensive database is needed to evaluate the true discriminatory power of these markers.