As one of the oldest cultivated plants Cannabis sativa has been grown for millennia under different climatic conditions making it a particularly versatile plant [1] with many facets ranging from intoxicant to medicine. On the one hand, it remains the most commonly used drug with approx. 244 million consumers worldwide in 2023 equalling 4.6% of the global population between 15 and 64 years of age [2]. On the other hand, 64 countries have developed provisions in their national legislation or at least corresponding guidelines for the medicinal use of cannabis by 2021 [3]. In Germany, an amendment of the law in March 2017 allowed the prescription of medicinal cannabis flowers which are not approved under medicinal law [4]. Since then, a trend towards diversification of flowers with increasingly high Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) levels can be observed on the German medicinal cannabis market. While in 2017 there were only 14 products with a maximum of 22 wt‐% Δ⁹‐THC [5], currently more than 900 products containing up to 35 wt‐% Δ⁹‐THC are available [6]. Application patterns of patients reflect high‐dose inhalation of cannabis flowers with high Δ⁹‐THC contents [7], creating the impression that trends from the recreational sector overflow the medicinal cannabis market and blur the boundaries between both. Depending on national jurisdiction, the introduction of cannabis as medicine may lead to issues with other legislations, for example, drug driving regulations under traffic law [8, 9, 10]. In Germany, medicinal cannabis patients are allowed to take part in road traffic as long as they do not show signs of impairment [10]. Specific forensic questions to differentiate recreational and medicinal use may arise, which are to be addressed by an expert witness in court. From a forensic point of view, there is hence an increasing interest in finding markers that enable the differentiability of cannabis flower material [11, 12].

For this reason, it is worth developing a deeper knowledge of the numerous systems that can be found in the literature to classify the great diversity of cannabis. According to its main cannabinoid cannabis can broadly, though legally helpful, be categorised into chemotypes [13, 14]. In the context of illegal/recreational cultivation, however, unscientific descriptions are used with terms that have adopted different meanings in the vernacular than their original scientific definition was [1, 15]. Especially Δ⁹‐THC‐dominant chemotypes are often further subdivided using the terms ‘sativa’, ‘indica’ or ‘hybrid’, which are originally derived from botanical taxonomy [16]. ‘Sativa’ plants are said to have a high Δ⁹‐THC content with a sweetish aroma causing a euphoric, stimulating ‘head‐high’. ‘Indica’ plants with moderate Δ⁹‐THC levels and a sour‐biting aroma were reported to be better tolerated and to cause a ‘body‐high’ with sedative properties [1, 17]. Since the 1970s, an excessive number of roughly estimated > 700 [13] to > 8000 [18] cannabis cultivars (‘strains’) have been bred [1, 17, 19]. Since these strains mainly originate from illegality, information on genetic relationships between them is usually incomplete [20] and very few are sufficiently described to be considered real ‘cultivars’ in the botanical sense [21]. Despite the questionable scientific evidence, medicinal cannabis is still marketed today with strain names, classified as ‘sativa’, ‘indica’ or ‘hybrid’. The recording of a comprehensive chemical fingerprint has been recently proposed as an objective way, to classify medicinal cannabis flowers into ‘chemovars’ [13]. For the characterisation of chemovars, terpenes are currently used as a second substance class alongside cannabinoids [13, 19, 22, 23, 24, 25, 26].

Terpenes are a particularly diverse, sometimes even referred to as the largest [27], group of plant chemicals with up to 30,000 characterised substances [28]. The volatile hydrocarbons follow a modular construction principle of isoprene subunits; for example, representatives with two isoprene subunits are called monoterpenes (C₁₀H₁₆, M = 136 g/mol), while terpenes with three isoprene units are referred to as sesquiterpenes (C₁₅H₂₄, M = 204 g/mol). If they also contain oxygen, for example, in the form of alcohols, ethers, esters or epoxides, they are called terpenoids (Figure 1) [28, 29]. At least 120 terpenes have been identified in the cannabis plant so far, among them 61 monoterpenes and 51 sesquiterpenes [30], making them the second largest group of constituents after phytocannabinoids. As terpenes occur ubiquitously in the plant kingdom, their investigation in cannabis has received substantially less research interest in the past than phytocannabinoids which are specific to them [31]. However, apart from the recent increase in medicinal interest in them, for example due to possible entourage effects between phytocannabinoids and terpenes [32], terpenes have also occasionally been studied in cannabis material in the context of other backgrounds. From a forensic perspective, comprehensive analytical profiles including terpenes, were recorded as an approach to trace the geographical origin of illegally confiscated material [33, 34, 35, 36, 37, 38, 39]. As volatile terpenes contribute to the odour of cannabis, they have also been the focus of studies aimed at uncovering its characteristic smell [40, 41, 42]. In addition to genetics, terpene profiles have been investigated as an approach to an objective chemotaxonomic classification [43, 44, 45].

While analysis of cannabinoids is already well implemented in forensics by either gas chromatography mass spectrometry (GC–MS) [46] or liquid chromatography tandem mass spectrometry (LC–MS/MS) [47, 48], the additional detection of minor cannabinoids besides Δ⁹‐THC could not sufficiently answer all complex questions to differentiate between medicinal cannabis and illegal/recreational cannabis [12, 49]. The detection of terpenes as a second dimension could therefore be a helpful approach to objectively and analytically scrutinise statements regarding seized/consumed cannabis material. Due to the recent increase in research interest in terpenes, the number of scientific publications on terpene analysis in cannabis flowers, mainly using GC, has increased significantly in the past few years although the quality of the analytical approaches differs greatly. Isomerism of terpenes makes analysis challenging, including difficulties in identifying co‐elutions and unclear defined compositions of reference substances. Early studies were often performed using identification via mass spectrometry (MS) libraries without reference standards; percentage values were estimated using relative responses delivered after flame ionisation detection (FID) [50]. When quantifying using FID, often only a few standards were calibrated, and calibration functions were used as surrogates for other analytes, since similar structures exhibited similar response factors [13, 51]. Comparing different studies, discrepancies regarding major constituents and concentration ranges of individual terpenes have been noticed [52]. Since some studies also report terpenes that are missing in other studies, misidentification of isomers may be assumed [19]. From an analytical point of view, there are differences with regard to the detection method [53] as well as sample application techniques [52], including solvent extraction with subsequent liquid injection [54] or headspace‐based techniques such as static headspace (SHS) and solid phase microextraction (SPME) [55]. The full evaporation technique (FET) is a special form of SHS and has recently been proposed in literature for terpene analysis in cannabis flowers [56, 57]. Unlike conventional SHS, FET is not based on establishing an equilibrium in the vial, but rather on complete evaporation of volatile components in the sample. Usually, very small sample quantities are thermostatted at high temperatures for a short period of time. FET was developed specifically for applications where no analyte‐free matrix is available [58]. This study places a strong focus on a structured and careful development of a HS‐FET‐GC/MS methodology, its validation and exemplary application for the detection of terpenes in cannabis flowers according to forensic guidelines.