**1. Introduction**

*Cannabis* is classified into the family of Cannabaceae and initially encompassed three main species: *Cannabis sativa*, *Cannabis indica*, and *Cannabis ruderalis* [1]. Nowadays, Cannabis has only one species due to continuous crossbreeding of the three species to generate hybrids. In fact, all plants are categorized as belonging to *Cannabis sativa* and classified into chemotypes based on the concentration of the main cannabinoids. Depending on the THCA/CBDA ratio, some chemotypes have been distinguished. In particular, chemotype I or "drug-plants" have a TCHA/CBDA ratio >1.0, plants that exhibit an intermediate ratio are classified as chemotype II, chemotype III or "fiber-plants" have a THCA/CBDA ratio <1.0, plants that contain cannabigerolic acid (CBGA) as the main cannabinoid are classified as chemotype IV, and plants that contain almost no cannabinoids are classified as chemotype V [2–5].

Recently, in Italy the interest in *Cannabis sativa* L. has increased mainly due to the latest legislation (Legge n. 242 del 2 dicembre 2016) [6]. As a consequence, there is a request to develop cost-e ffective and easy-to-use quantitative and qualitative methods for analysis of cannabinoids.

The Italian regulatory framework has classified two types of *Cannabis sativa* L. depending on the content of Δ9-THC. In particular, fiber-type plants of *Cannabis sativa* L., also called "hemp", are

characterized by a low content of Δ9-THC (<0.2% *w*/*w*). If the content of Δ9-THC is >0.6% *<sup>w</sup>*/*<sup>w</sup>*, it is considered as drug-type, also called "therapeutic" or "marijuana".

Industrial hemp is used in several sectors, such as in the pharmaceutical, cosmetic, food, and textile industries, as well as in energy production and building. In general, fiber-type plants are less used in the pharmaceutical field, where drug-type plants are more often employed [5]. However, there is also an increased interest in hemp varieties containing non-psychoactive compounds. In fact, the European Union has approved 69 varieties of *Cannabis sativa* L. for commercial use [7].

Hemp has a complex chemical composition that includes terpenoids, sugars, alkaloids, stilbenoids, quinones, and the characteristic compounds of this plant, namely cannabinoids. *Cannabis sativa* L. has several chemotypes, each of which is characterized by a different qualitative and quantitative chemical profile [5]. The cannabinoids, terpenes, and phenolic compounds in hemp are formed through secondary metabolism [3,8]. The term "cannabinoid" indicates terpenophenols derived from *Cannabis*. More than 90 cannabinoids are known, and some are derived from breakdown reactions [8]. Gaoni and Mechoulam [9] were the first to define cannabinoids "as a group of C21 compounds typical of and present in *Cannabis sativa*, their carboxylic acids, analogs, and transformation products". Currently, cannabinoids have been classified according to their chemical structure, mainly seven types of cannabigerol (CBG); five types of cannabichromene (CBC); seven types of cannabidiol (CBD); the main psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) in nine different forms including its acid precursor (Δ9-tetrahydrocannabinolic acid, Δ9-THCA); Δ8-tetrahydrocannabinol (Δ8-THC), which is a more stable isomer of Δ9-THC but 20% less active; three types of cannabicyclol (CBL); five different forms of cannabielsoin (CBE); seven types of Cannabinol (CBN), which is the oxidation artifact of Δ9-THC; cannabitriol (CBT); cannabivarin (CBDV); and tetrahydrocannabivarin (THCV) [10,11]. THC, CBD, CBG, CBN, and CBC are not biosynthesized in *Cannabis sativa*, and the plant produces the carboxylic acid forms of these cannabinoids (THCA, CBDA, CBGA, CBNA, and CBCA). Cannabinoid acids undergo a chemical decarboxylation reaction triggered by different factors, mainly temperature. This decarboxylation reaction leads to the formation of the respective neutral cannabinoids (THC, CBD, CBG, CBN, and CBC) [12,13].

There are several methods to quantify cannabinoids [14–21], some of which require expensive mass spectrometry detectors [22–25]. Furthermore, there is a grea<sup>t</sup> deal of uncertainty around the use of gas chromatography (GC) for the titration of cannabinoids due to the high temperature of the injector and detector that can lead to the decarboxylation of cannabinoid acids if not derivatized correctly [26]. Moreover, recent studies have reported that cannabinoid acid decarboxylation is only partial, and as result the actual value is underestimated. An HPLC system allows for determination of the actual cannabinoid composition, both neutral and acid forms, without the necessity of the derivatization step [13].

It is necessary, in addition to honed methods, to develop new procedures with a view to discriminate different *Cannabis* varieties in order to identify and titrate cannabinoids in a simple way. These methods should ideally be fast, easy, robust, and cost-efficient as they can be used not only by research laboratories but also by small companies with a view on quality control.

This study focuses on the development, validation, and step-by-step explanation of a rapid and simple HPLC-UV method for identification and quantification of the main cannabinoids in hemp inflorescences that can be easily reproduced and applied. The method described is focused on the quantification of CBD but can also be applied to check the levels of THC.

#### **2. Results and Discussion**
