Effective determination of the principal non-psychoactive cannabinoids in fiber-type Cannabis sativa L. by UPLC-PDA following a comprehensive design and optimization of extraction methodology

Abstract

Cannabidiol (CBD) and cannabidiolic acid (CBDA) represent the most abundant non-psychoactive cannabinoids in fiber-type Cannabis sativa L. (hemp) and both have demonstrated high therapeutic potential. Hence, efficient extraction coupled with reliable determination of these compounds is crucial for informed utilization of hemp and is increasingly needed in the present state of harmonization efforts. In this context, a systematic approach for extraction optimization was followed, which initially involved comparison of three widely available extraction techniques, i.e. ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and dynamic maceration (DM). These were applied on samples of different hemp varieties (n = 3) using ethanol as a safe and efficient solvent. UAE showed the most promising results and was further optimized by means of response surface methodology (RSM), based on a circumscribed central composite design. The conditions maximizing CBD, CBDA, and total CBD content as well as extraction yield were determined with high desirability (0.97) and were experimentally confirmed. The optimized UAE method was also compared with a previously reported extraction procedure, demonstrating superior performance. For the quantitation of CBD and CBDA in hemp extracts, a reversed-phase UPLC-PDA method was developed and validated. Chromatographic separation was achieved in less than 10 min, while satisfactory results for linearity (R² > 0.996), precision (RSD < 2.0%), and accuracy (recovery rates of 93.1-101.0%) were obtained for both analytes. Limits of detection were determined as 0.07 and 0.04 μg mL^-1 for CBD and CBDA, respectively, indicating sufficient sensitivity. The good performance of the method was verified by the evaluation of additional parameters (e.g. matrix effect, extraction recovery), which was largely enabled by the use of isolated standards. The whole analytical workflow, involving both optimized UAE extraction and UPLC-PDA determination, entails simplified manipulation and may offer a reliable and cost-effective approach for routine quality control of hemp regarding the principal cannabinoids.

Keywords: Cannabidiol; Cannabidiolic acid; Cannabis sativa L.; Multivariate optimization; Ultra-performance liquid chromatography; Ultrasound-assisted extraction.