The long coexistence between mankind and Cannabis sativa L. led to its early domestication, with the plant soon showing a plethora of possible uses, making it an emblematic example of polyvalent culture [1]. Over the centuries, this plant has gone from robust and durable textile fibers to recreational drugs used by artists and writers in the 19th century [2], until the beginning of a long period defined by the prohibition of its production, which continued until recent years. In fact, the contemporary re-evaluation of this crop has been stimulated by a number of studies that highlighted its agricultural features, together with its beneficial properties due to its undervalued richness in phytochemicals, in addition to fiber. In this context, many countries have authorized the cultivation and processing of hemp varieties (generally referred to as industrial hemp) with very low content of psychotropic compounds [3].

In particular, the food chain has mainly considered hemp seed intake, as well as that of the oil extracted therefrom and baked products derived from flour. Several studies crowned the hemp seed as a rich source of nutrients with health-promoting properties. The chemical composition and the nutraceutical nature of these fruits have been recently reviewed, pointing out that its functional benefits are still far from being fully understood [4]. In fact, besides essential polyunsaturated fatty acids with a ratio nearly equal to 3:1 (ω-6: ω-3), which characterize also the oil together with vitamins, minerals, amino acids and phytosterols, the presence of antioxidant (poly)phenols is worth mentioning, not only for their preservation of oil stability and quality but also for their numerous health benefits for consumers [5,6,7].

As the commercial interest (from farmers to producers) has increased, at the same time, the safety of products intended for human consumption has begun to be regulated at the legislative level. Italian legislation, with the circular of the Ministry of Health on 23 May 2009, has allowed the production and marketing of hemp seed products for use in the human nutrition sector, thus allowing the use of C. sativa, or parts thereof, in foodstuffs such as bread flour, sweets, oils and supplements [8]. Then, other decree laws followed, all focused on the Cannabis sativa plant and its cultivation. The first Italian regulation which fixed the maximum permitted levels of total THC in hemp-derived food dates back to 4 November 2019 and refers to hemp seeds (2.0 mg/kg), the oil (5.0 mg/kg) and flour therefrom (2.0 mg/kg) and also hemp-based supplements (2.0 mg/kg) [9].

Indeed, the growing expansion of marketed hemp foodstuffs, beyond seeds and derived products, requires the development and application of multiple quantitative analytical methods to determine the cannabinoid content in a wide range of matrices. Gas chromatography (GC) coupled with mass spectrometry (MS) or a flame ionization detector (FID), although widely used, suffers limitations in its ability to identify and quantify acid cannabinoids (e.g., cannabigerolic acid—CBGA, cannabidiolic acid—CBDA, and tetrahydrocannabinolic acid—THCA), because, during the analyses, they could decarboxylate in their neutral forms, due to high temperatures [10]. In this sense, liquid chromatography (LC) appears to be a more suitable tool for analyzing the native composition of the hemp plant. For the quantification of both acid and neutral cannabinoids, methods based on HPLC-MS, HPLC with a Diode-Array Detector (DAD) and HPLC-DAD-MS techniques have been reported, as well as the application of supercritical fluids in chromatographic separation (SFC-DAD-MS), developed for plant materials and plant extracts/oils, including those derived from illicit, medicinal and/or industrial varieties [11]. In particular, LC separation combined with tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) could be considered as the method of choice in the quali-quantitative determination of phytocannabinoids present in very low amounts, mainly due to the high signal-to-noise ratio and selectivity. For accurate mass estimation, time of flight (TOF) or linear ion trap-Orbitrap (LTQ-Orbitrap) hybrid mass analyzers have been successfully used [12].

In light of the above, herein, we applied ultra-high-performance liquid chromatography (UHPLC) techniques with high resolution tandem mass spectrometry (HR-MS/MS) detection to the analysis of three espresso coffees from mixtures of ground coffee enriched with shredded inflorescences from three different dioecious genotypes of C. sativa L. (cvs. Antal, Kompolti and Tiborszallasi), commonly cultivated for industrial purposes. The thermodynamics measurement of the coffee extraction process by “moka” (the most common Italian coffee maker) led to the distinction of two distinct phases [13]. In the first one, called “regular extraction”, liquid–solid extraction occurs, which is characterized by variable temperature and water flow rate over time. In this phase, the extraction is guided by the increase of the air vapor pressure above the water level in the lower tank of the device. In the second phase, known as the “strombolian phase”, intense evaporation takes place, resulting in the extraction of soluble compounds which are generally detrimental to the quality of the final product.

The experimental work described herein was aimed at the chemical characterization of the main phytocannabinoids extracted during the “moka” process and their quantification in the coffee drink obtained, also compared to the natural phenolic constituents of coffee (chlorogenic acids) and caffeine, concluding with discussion of the possible benefits deriving from this enrichment. In addition, the targeted analysis of neutral phytocannabinoids allowed us to verify possible decarboxylation of the acid cannabinoids during the “extraction” process due to high temperature and pressure. This study opens up a new concrete possibility to raise industrial hemp inflorescences, converting the by-product production chain into a high value chain.