Rabi A. Musah*, Megan I. Chambers, Samira Beyramysoltan, and Benedetta Garosi | University at Albany – State University of New York Abstract: Hemp (fiber-type) and marijuana (drug-type) are the two major varieties of Cannabis sativa. Although both varieties contain Δ9-tetrahydrocannabinol, or THC, (i.e., the primary psychoactive component of this species), they differ in the amount of this active ingredient present. Currently, federal laws designate that C. sativa materials that contain greater than 0.3% THC are classified as marijuana (i.e., a Schedule I controlled substance) while C. sativa that has less than or equal to 0.3% THC is hemp (i.e., a legal agricultural commodity). In the context of analyzing Cannabis materials in a forensic laboratory, distinguishing between these two varieties can be difficult. Furthermore, the increased workload associated with differentiating between hemp and marijuana, which derives from the requirement to quantify the THC content in all C. sativa samples submitted as evidence, has imposed additional challenges. Therefore, one of the primary goals of this National Institute of Justice–funded project was to develop a method to rapidly distinguish between hemp and marijuana plant materials. To accomplish this, advanced chemometric techniques were applied to data obtained by direct analysis in real time–high-resolution mass spectrometry (DART-HRMS). A diverse set of C. sativa plant materials was acquired, including commercial hemp flower from multiple vendors, marijuana samples from two Drug Enforcement Agency–registered suppliers (i.e., the National Institute on Drug Abuse and the National Institute of Standards and Technology), and recreational marijuana flower. All plant materials were analyzed by DART-HRMS in positive-ion mode under soft ionization conditions (i.e., orifice 1 voltage of 20 V). No sample pretreatment steps were required prior to interrogating the samples by DART-HRMS, which was accomplished by simply introducing the closed end of a glass melting point capillary tube into the sample and presenting the coated surface of the tube to the open-air gap between the ion source and mass spectrometer inlet (i.e., DART gas stream). After obtaining the mass spectral chemical profiles for all samples, chemometric techniques (i.e., principal component analysis and random forest) were applied to optimally differentiate between the hemp and marijuana samples with high accuracy. A set of external validation samples (i.e., samples that were not used to develop the model) were used to assess the model’s prediction ability to classify “unknowns.” The assessment resulted in 100% accuracy for determining the varieties of the C. sativa unknowns. During the development of the model, a set of m/z values was identified as diagnostic for differentiating between the two varieties. During investigations to determine the identities of all variables found to be crucial for this distinction, it was found that several markers had masses that were consistent with cannabinoid and terpene molecules known to be present in Cannabis. Furthermore, fragments of these major molecules, which are formed during the ambient ionization analysis by DART-HRMS, also appear to aid in the differentiation of these varieties. In summary, the developed mass spectrometric and chemometric approach for differentiating between hemp and marijuana would assist crime laboratories in the rapid identification of unknown C. sativa material prior to the launch of time-consuming and resource-intensive confirmatory methods.