In efforts to quantify phytocannabinoids in hemp, liquid chromatography diode array detector (LC-DAD) methods are favored, but their selectivity is dependent on baseline separation of all phytocannabinoids along with unknown compounds in the extract. Therefore, the development of a LC-DAD method with a different selectivity has become highly desirable. Most current LC-DAD methods use a water/acetonitrile eluting system, whereas this study aimed to use a water/methanol eluting system. A systematic investigation of various chromatographic parameters on LC separation of eighteen phytocannabinoids, the maximum that has been quantified in hemp at the time of this study, plus two internal standards, led to a four-step isocratic mobile phase that was capable of baseline separation for all twenty compounds with a significantly different eluting order from published methods. The changes in the mobile phase caused baseline drift, yet difficulty in quantification was avoided through detection at wavelengths longer than 230 nm. This study’s method was validated according to the ISO 17025 guidelines, calibrated between 0.04 and 50 µg/mL, and used to analyze phytocannabinoids in nine strains of hemp flowers that were extracted using methanol between 0.04 and 50% (w/w). Extraction recovery was tracked by spiking the samples with one of two potential internal standards utilized in the study. Method selectivity was further assessed by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS), indication minimum interferences. Additionally, five untargeted/unknown phytocannabinoids were identified by ESI/TOFMS, including two structural isomers of Δ9 -tetrahydrocannabinol (Δ9 -THC), two structural isomers of Δ 9 -tetrahydrocannabinolic acid (Δ9 -THCA), and one structural isomer of Δ9 -THC acetate.
(Author abstract provided.)