2.2.7. Linearity

In order to evaluate the linearity of the method, eight di fferent points of standard mixture solutions were analyzed in triplicate by HPLC-UV.

The following equations are related to the calibration curves in a concentration range between 0.01–100 μg/mL: CBDA, y = 18955x − 1612.6 (*r*2 = 0.9999); CBGA, y = 19796x − 3475.7 (*r*2 = 0.9999); CBG, y = 18094x − 9195.3 (*r*2 = 0.9995); CBD, y = 13703x − 6009.5 (*r*2 = 0.9995); THCV, y = 18534x − 15213 (*r*2 = 0.9989); CBN, y = 34148x − 7943.1 (*r*2 = 0.9999); Δ9 − THC, y = 19893x − 31896 (*r*2 = 0.9981); Δ8-THC, y = 17526x − 18267 (*r*2 = 0.9987); CBC, y = 18590x − 4777.1 (*r*2 = 0.9999); THCA, y = 18239x − 8969.3 (*r*2 = 0.9998) (Table 1).

With the aid of the equation obtained from the calibration curve, the quantity of each cannabinoid was calculated.

To express the data relative to the content of the individual cannabinoid as a percentage (%, p/p) referred to the dried material, it is necessary to refer to the weight of the sample considering the dilution factor. The linearity in the concentration range analyzed was good for cannabinoid standards, being *r*2 > 0.998, as reported before.

#### *2.3. Cannabinoids in Hemp Samples*

The method developed in this study was applied to quali-quantitative analysis of main cannabinoids in two samples of hemp inflorescences. The samples analyzed, belonging to the same variety of *Cannabis sativa* L., did not show a significant di fference in the concentration of the target compounds. As shown in Table 2, CBDA is the only cannabinoid for which a di fferent concentration was determined. The other cannabinoids had a similar or the same concentration (e.g., CBGA, CBG, CBN, Δ-9-THC, and Δ-8-THC) in both samples. THCV was not found in the hemp inflorescence samples analyzed, as shown in Figure 2 and Table 2. Δ-9-THC and Δ-8-THC were found at a low concentration, below the legal limit. Under the current legislation regarding *Cannabis sativa* L. cultivation [6,29], in fact, the total content of THC must not be higher than 0.2% and in any case within 0.6%. Indeed, only the hemp varieties reported in the *Common catalogue of varieties of agricultural plant species* can be

cultivated without authorization [6,7]. These kinds of results confirmed that the analyzed samples were correctly classified as hemp, since the quantity of Δ8-THC and Δ9-THC was found to be lower than the limits established by the legislation. According to what is indicated in literature [30], in the hemp variety considered (Futura 75), the most present compound was CBDA, followed by CBD; all the other compounds were in very low amounts ranging from 0.01 to 0.06%. CBGA is the compound from which all other cannabinoids are biosynthesized [5], which is probably why it was found at a low concentration in both samples examined.

The number of cannabinoids in hemp samples is reported in Table 2.


**Table 2.** Number of cannabinoids in hemp samples.

#### **3. Materials and Methods**

#### *3.1. Chemicals, Standards and Apparatus*

All chemicals used were of analytical grade. Methanol p.a CAS 67-56-1, chloroform p.a CAS 67-66-3, acetonitrile CAS 75-05-8, water CAS 7732-18-5, and orthophosphoric acid CAS 7664-38-2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitrogen, pure gas for analysis CAS 7727-37-9 was purchased from SIAD Spa (Bergamo, Italy). Standard mixture of phytocannabinoids 0.1% in acetonitrile: Cannabidiolic acid (0.01%) CAS 1244-58-2, cannabigerolic acid (0.01%) CAS 25555-57-1, cannabigerol (0.01%) CAS 25654-31-3, cannabidiol (0.01%) CAS 13956-29-1, tetrahydrocannabivarin (0.01%) CAS 31262-37-0, cannabinol (0.01%) CAS 521-35-7, tetrahydrocannabinolic acid (0.01%) CAS 23978-85-0, Δ-9-tetrahydrocannabinol (0.01%) CAS 1972-08-3, Δ-8-tetrahydrocannabinol (0.01%) CAS 5957-75-5, cannabichromene (0.01%) CAS Number 20675-51-8, were purchased from Cayman Chemical Company, (Ann Arbor, MI, USA). Cannabidiol 1.0 mg/mL in methanol CAS 13956-29-1: LGC Standards S.r.l., (Milan, Italy).

Analytical mill, IKA A11 Basic (IKA® Werke GMBH & Co. KG, Germany). Analytical balance with precision of 0.1 mg, mod. E42, (Gibertini, Italy). Vortex vibrating shaker, mod. ST5, (Janke & Kunkel, Germania). Centrifuge mod. ALC, PK 120 (Thermo Electron Corporation, Massachusetts, USA). Termoblock heating block, mod. A120, (Falc, Italy). Natural ventilation stove. Sieve with 1 mm meshes. Tilting shaker. Ultrasound bath Branson 2150, (Danbury-CT, USA). Volumetric flasks of 1, 2, 10 and 25 mL. SOVIREL-type tubes with screw cap. Glass syringes with luer lock attachment, 0.45 μm nylon membrane filters. Microsyringes from 1 to 1000 μL. HPLC Cannabis Analyzer for Potency Prominence-i LC-2030C equipped with a reverse phase C18 column, Nex-Leaf CBX Potency 150 × 4.6 mm, 2.7 μm with a guard column Nex-Leaf CBX 5 × 4.6 mm, 2.7, UV detector and acquisition software LabSolutions version 5.84 (Shimazu, Kyoto, Japan).
