Liguo Song* | Western Illinois University Abstract: There are two primary species of Cannabis: marijuana is the cultivated plant used as a psychotropic drug because of high concentrations of ∆9-tetrahydrocannabinol (Δ9-THC) whereas hemp is characterized by a low concentration of Δ9-THC and high concentration of cannabidiol (CBD), which is antipsychoactive. The Federal Controlled Substance Act of 1970 defined Cannabis as a Schedule I substance, which made both marijuana and hemp illegal. The 2018 Farm Bill excluded hemp from the statutory definition of cannabis with a total concentration of Δ9-THC and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) not more than 0.3% (w/w) on a dry basis, which made hemp compliance testing mandatory. To meet this demand, a liquid chromatography diode array detector (LC-DAD) method has been developed and validated. So far, published LC-DAD methods have focused on the quantification of 17 or fewer cannabinoids. In addition, resolution of critical pairs of cannabinoids (e.g., cannabigerol [CBG]/CBD, Δ9-/Δ8-THC) often did not meet the required minimum resolution of 1.5 for appropriate method validation, which was especially true when LC separation was carried out at high concentration of cannabinoids or involved a large number of cannabinoids. In our study, a thorough separation optimization led to baseline separation of both critical pairs. Precision and accuracy were assessed using quality control samples at three concentration levels (0.02, 0.5, and 12.5 µg/mL) in triplicates with inter-day and intra-day precision of less than 15% relative standard deviation (RSD) and accuracy of less than ±15% relative error, therefore meeting the requirements by the ISO 17025 standards. Unlike most published methods that had to analyze the same sample at more than one concentration because of a narrow linear calibration range, samples were analyzed with this method at one concentration (50 µg/mL in methanol) because of a wide linear calibration range (i.e., 0.02–25 µg/mL or 0.04%–50% [w/w]). In the literature, published recovery experiments were limited by the unavailability of cannabinoid-free matrix and the high cost of cannabinoid standards. This problem was solved by spiking abnormal CBD, a cannabinoid not naturally present in Cannabis products and commercially available with a reasonable price, into the samples. The researcher’s assessment in triplicate showed that the recovery ranged from 93.6% to 106.1% while the RSD values ranged from 1.1% to 7.0%. The method had overall good specificity with only a few minor interferences from compounds in the samples, which was verified by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS). Additionally, ESI/TOFMS has discovered seven unknown cannabinoids, including one structural isomer of CBG, one structural isomer of cannabinolic acid, four structural isomers of Δ9-THC, and one structural isomer of Δ9-THC acetate. The method was applied in the analysis of cannabinoids in 23 samples of plant materials of Cannabis. Eighteen cannabinoids were found at concentrations higher than the limit of quantification and ranged from 0.04 to 26.97% (w/w) in the samples. The analysis also uncovered that one of the two samples of Δ8-THC fortified hemp flowers contained 5.16% (w/w) Δ9-THC, an alarm to the current Δ8-THC craze by the public.