The Detection of Flavonoids in Hemp Flower by LC-MS/MS

Hemp is a chemically complex matrix that contains a wide array of bioactive compounds whose synergistic interactions give hemp and cannabis their therapeutic effects. Flavonoids are a class of compounds endogenous in hemp and cannabis that are antioxidant rich and can affect the flavor profile. Routine testing in cannabis labs oftentimes does not include flavonoid testing but as the market for both cannabis analysis and products grows, industry leaders may choose to implement new tests that not only aid the advancement of the science of cannabis but also to differentiate their services or products from their competitors. In this work, a method to detect 19 flavonoids in CBD and CBG dominant hemp flower was developed using LC-MS/MS. Extraction of flavonoids was performed by weighing out 0.5 g of ground hemp flower. 80/20 methanol/water (10 mL) was added to the hemp flower and vortexed. The samples were sonicated for 15 min, centrifuged at 4200 rpm for 5 min, and diluted 50-fold. The analytical method was developed using LC-MS/MS with electrospray ionization in positive and negative ion modes. Mobile phase A consisted of 0.1 % formic acid in water and mobile phase B consisted of 0.1% formic acid in methanol. The method uses gradient conditions on a Raptor Biphenyl 100 x 2.1, 2.7 µm column equipped with a guard cartridge. The chromatographic method was able to resolve all isobars with an overall analysis time of 7 minutes. Ten flavonoids were detected in the CBG hemp flower sample and 12 flavonoids were detected in the CBD hemp flower samples. Single point recovery experiments were also performed at 100 ppb using two isotopically labeled internal standards. The results of the single point recovery ranged from 80–104%. The developed method was able to resolve isobars within a cycle time of 7 minutes, and acceptable recoveries were achieved for both hemp flower samples, indicating this is an effective procedure for the extraction of flavonoids from hemp flower.