**4. Conclusions**

One of the most relevant problems in analytical determinations for quality control, especially when there are legal problems related with quantitation, such as for cannabis, relates to the proficiency of laboratories. Therefore, detailed and validated procedures that are freely available are essential for the full understanding of any analytical step and its careful application. This is also true for "daily" methods that can be easily applied for quality control, carried out using traditional RP-HPLC and UV-Vis detectors, with less e fficient performance than diode-array detectors but with lower costs, rendering them a ffordable even for small laboratories.

The validated method described herein allows the quantitative determination of the 10 most relevant cannabinoids using a single wavelength (220 nm) in 8 min. A full separation is obtained, even in the elution sequence of a difficult resolution, of the group of peaks related to CBGA, CBG, CBD, and THCV (from 3.5 to 4.5 min).

The method is applied to cannabis inflorescences and involves extraction in methanol/ chloroform, drying of the extract, taking it up in acetonitrile and injection into an HPLC. The method has sensitivity and accuracy to discriminate samples with amounts of Δ-9- and Δ-8-THC (total THC content) that are below the limit of 0.2% from those that are subjected to legal restrictions in many EU countries, with a total THC content above 0.6%, which cannot be classified as hemp. Due to its simplicity and rapidity, it can be used to check raw material or crops during the harvesting period.

A detailed standard operating procedure (SOP), as a supplementary information file, is also available, so that any operator with basic knowledge of HPLC can easily apply it and make all the elution and calibration control checks using commercially available mixtures of standards, which are more affordable and sustainable than single cannabinoid standards in terms of costs and solvents used for calibration.

**Supplementary Materials:** The following are available online. File S1: Standard operating procedure (SOP) of the method presented in this article, Table S1: Calibration curves relating to the standard solution of 10 cannabinoids determined by RP-HPLC-UV method, Figure S1: Calibration curves relating to the standard solution of 10 cannabinoids determined by RP-HPLC-UV method, File S2: Preliminary tests carried out for development of the analytical procedure by RP-HPLC-UV.

**Author Contributions:** Conceptualization, T.G.T., M.M. and M.T.; Methodology, M.M.; Software, M.M. and S.S.; Validation, M.M.; Formal analysis, M.M.; Investigation, T.G.T. and M.M.; Resources, T.G.T. and M.M.; Data curation, M.M., T.G.T. and M.T.; Writing—original draft preparation, M.M., M.T. and T.G.T.; writing—review and editing, T.G.T. and S.S.; Visualization, T.G.T.; Supervision, T.G.T.; project administration, T.G.T.; funding acquisition, T.G.T.

**Funding:** This research received no external funding; this trial received financial support from Enecta Srl.

**Acknowledgments:** The authors gratefully acknowledge Enecta Srl for providing samples. The experimentation was conducted in the context of a PhD project entitled *"Harmonized procedures of analysis of medical, herbal, food and industrial cannabis: development and validation of cannabinoids' quality control methods, of extraction and preparation of derivatives from the plant raw material, according to the product destination"* and funded by ENECTA Srl.

**Conflicts of Interest:** The authors declare no conflict of interest.
