*2.1. Method Development*

The aim of this work was to develop a new analytical method for determination of the main cannabinoids in hemp samples. In fact, the method described below can be used as a routine quality control procedure and can be applied by the pharmaceutical industry, small laboratories, or even small pharmacies.

A crucial aspect for accurate identification and quantification of analytes is optimization of separation conditions, and therefore various preliminary tests were carried out (e.g., mobile phase, detection wavelength). Di fferent mobile phases were tested, and trials were performed with di fferent compositions and gradient elution to optimize the separation of all 10 target compounds considered (File S2). The greatest di fficulty was that of separating CBD and THCV, which in many cases co-eluted. It was also di fficult to separate the isomers Δ9-THC and Δ8-THC. The best resolution of cannabinoids was obtained using a chromatographic column and, as an eluent mixture, water with 0.085% phosphoric acid and acetonitrile with 0.085% phosphoric acid.

The quantification of cannabinoids was made at 220 nm after testing di fferent wavelengths (File S2). This wavelength represents the best compromise for all the cannabinoids considered and was selected to detect and integrate all compounds of interest within the dedicated concentration range. As far as chromatographic analysis is concerned, before using the instrument, the system was conditioned for 20 min by fluxing the eluent mixture in the instrument under the same conditions as the method, and then a chromatographic run was performed by injecting 5 μL of acetonitrile to verify that the chromatographic system was adequately cleaned. Simultaneously with the analysis of the sample, standard solutions were injected at di fferent concentrations for the construction of calibration curves and to evaluate the separation and identification of each compound. The identification of cannabinoids was performed by comparing their retention times with those obtained by the injection of pure standards and by an enhancing procedure. Figure 1 shows a chromatogram of a standard mixture of cannabinoids and Figure 2 shows a chromatogram of a sample of hemp.

**Figure 1.** Chromatographic trace of a standard cannabinoid mixture analyzed by RP-HPLC-UV equipped with reverse phase C18 column.

Cannabinoids in di fferent varieties of *Cannabis sativa* L. can be present in very di fferent concentrations. In order to obtain good chromatographic separation and correct quantification, it may be necessary to dilute or concentrate the extract, performing two di fferent injections. For example, in the case of high levels of CBDA or CBD it will be necessary to dilute the extract. For THC, it is often found at low concentration in hemp inflorescences, so it may be necessary to concentrate the extract before injection. In our case, 2 mL of filtered extract was dried using a weak nitrogen flow, and the dry extract was recovered in 500 μL of acetonitrile.

**Figure 2.** Chromatographic trace of *Cannabis sativa* L. inflorescence extract analyzed by RP-HPLC-UV equipped with a reverse phase C18 column.
