High-Throughput LC–MS/MS Quantification of Eighteen Cannabinoids in Hemp Flowers with Baseline Separation of Structural Isomers

Following the passage of the Agriculture Improvement Act of 2018, demand for accurate cannabinoid quantification in hemp flowers has increased to ensure regulatory compliance. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a triple-quadrupole mass spectrometer provides high sensitivity and selectivity and is well suited for this purpose; however, a review of the literature indicates that many published LC–MS/MS methods target only a limited number of cannabinoids, and reliable differentiation of structural isomers remains challenging. In this study, an LC–MS/MS method was developed for the simultaneous quantification of eighteen cannabinoids in hemp flowers. Baseline chromatographic separation of structural isomers enabled reliable differentiation of compounds with highly similar fragmentation patterns and allowed the use of the most sensitive multiple reaction monitoring (MRM) transitions for quantification. Both positive and negative ionization modes were employed to achieve optimal sensitivity using dynamic polarity switching within a single analytical run. Following validation in accordance with ISO/IEC 17025, the method was applied to a proficiency test hemp sample and six commercial hemp samples, demonstrating excellent time efficiency (11 min for 18 cannabinoids) and an exceptionally wide calibration range (8–5000 ng/mL, corresponding to 0.032–20% (w/w) for all cannabinoids).