Development of a validated method for rapid quantification of up to sixteen cannabinoids using ultra-high-performance...

Due to the recent legalization of medical and recreational Cannabis in many countries in the world, there has been an increasing demand for accurate quantification of a growing number of cannabinoids. To meet this challenge, a method for rapid quantification of up to sixteen cannabinoids using ultra-high-performance liquid chromatography diode-array detector (UHPLC-DAD) has been developed, validated and used in the analysis of hemp concentrates. While published LC-UV methods were usually for twelve or less cannabinoids and might not achieve baseline separation of some critical pairs of cannabinoids, e.g., CBG/CBD (cannabigerol/cannabidiol) and Δ⁹-THC/Δ⁸-THC (tetrahydrocannabinol), in this study a systematic separation optimization led to a resolution of 1.7 for both pairs. The linear calibration range of all cannabinoids were between 0.02 to 25 µg/mL in methanol, leading to the quantification of 0.1 to 125% (w/w) individual cannabinoids in hemp concentrates after they were mixed with methanol at 20 µg/mL and analyzed after ultrasonication, centrifugation and filtration. The analytical results showed that none of the nine analyzed samples contained any of the seven acidic cannabinoids, but all the nine neutral cannabinoids were detected, with average content ranging from 0.10 to 93.23% (w/w) and RSD (relative standard deviation) values from 0.3 to 11.2% in triplicates. Particularly, two hemp concentrates, i.e., delta 8 hemp distillate and delta 8 hemp shatter contained 9.35 and 11.33% (w/w) Δ⁹-THC, respectively. While 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 plants and commercially available with a reasonable price, into the samples. The obtained average recovery ranged from 94.8 to 103.6% with RSD values from 1.5 to 9.0% in triplicates for the nine analyzed samples. Electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS) confirmed the good specificity of the UHPLC-DAD method, i.e., without any false positive identification of individual cannabinoids, and discovered six untargeted cannabinoids that were structural isomers of Δ⁹-THC in the nine hemp concentrate samples.