A Critical Review on Cannabinoids, Legislation, and Analytical Methods
Cannabis sativa L., one of the most fascinating and controversial plants in human history, is gaining a prominent place in the global scientific and regulatory landscape. With a history dating back more than 4000 years of medicinal use in China, where the legendary Emperor Shen Nung documented its therapeutic applications around 2700 BC 1 , cannabis is now emerging from the shadows of prohibition to become the focus of innovative scientific research and a complex international regulatory puzzle.
Cannabis has been used for medicinal purposes for over 4000 years, with documented use in ancient China 1 .
Today, cannabis is at the center of scientific research with over 500 identified compounds, about 100 of which are unique cannabinoids 1 .
In recent years, we have witnessed a quiet revolution in how cannabis is perceived, studied, and regulated. While laboratories worldwide examine the more than 500 compounds already identified in this unique plant, governments face the challenge of creating legal frameworks that balance the plant's therapeutic potential with the risks associated with its recreational use.
At the heart of scientific interest in cannabis are cannabinoids, unique terpenophenolic compounds of this plant . These molecules, which act primarily through our body's endocannabinoid system - a complex signaling network discovered only in the late 20th century - are responsible for the plant's therapeutic and psychoactive effects 1 .
The main psychoactive component of cannabis, responsible for the characteristic "high" sensation. THC acts as a partial agonist of CB1 receptors in the brain 5 , triggering a cascade of effects that include not only psychoactivity but also pain relief, nausea reduction, and appetite stimulation 1 5 .
A non-psychoactive cannabinoid with a remarkably diverse pharmacological profile. CBD has demonstrated anticonvulsant, anxiolytic, anti-inflammatory, and neuroprotective properties 5 . Unlike THC, CBD has little affinity for classical cannabinoid receptors, acting through other mechanisms including modulation of serotonin receptors and inhibition of endocannabinoid reuptake 1 5 .
In addition to cannabinoids, cannabis produces an impressive variety of terpenes - aromatic compounds that give the plant its distinctive odoriferous character 2 . These compounds, also found in other aromatic plants such as pines and citrus, are not merely ornamental; they actively contribute to the therapeutic effects of cannabis through what has been called the "entourage effect" 2 .
This phenomenon describes the synergistic action between cannabinoids and terpenes, where the plant's non-psychoactive compounds, including terpenes and flavonoids, modulate and complement the effects of the main cannabinoids.
It's crucial to understand that these cannabinoids exist in the plant mainly in their acidic forms (THCA and CBDA), which convert to neutral forms (THC and CBD) through a decarboxylation process, typically induced by heat . This transformation is essential for activating THC's psychoactive properties, explaining why raw cannabis does not produce the same effects as when heated.
The global legal landscape of cannabis is undergoing a profound and accelerated transformation. What was once a marginal issue has become a complex regulatory puzzle with significant economic and public health implications.
| Country | Medical Use Status | Adult Use Status | Relevant Notes |
|---|---|---|---|
| Germany | Legalized & Expanding | Legalized (since 2024) | Adults can cultivate up to 3 plants; imports jumped from 32 tons (2023) to >70 tons (2024) 4 |
| Portugal | Legalized (since 2018) | Decriminalized | Largest medical exporter in Europe (32 tons in 2024), but local access limited by high prices 4 |
| Spain | Under Regulation | Social Clubs Tolerated | Royal Decree project submitted to EC in Jan 2025; >600 social clubs operating in de facto tolerance 4 |
| Netherlands | Legalized | Experimentation Ongoing | "Closed Coffee Shop Chain Experiment" in 75 municipalities; 7 licensed growers 4 |
| United Kingdom | Medical Prescription (NHS & private) | Illegal | Rapidly growing medical market; annual sales >200 million GBP 4 |
| Poland | Legal, but Restricted | Illegal | Medical consumption increased 224% in 2023; private telemedicine banned in Nov 2024 4 |
| USA | Varies by State | Varies by State | Federal rescheduling process stopped in April 2025 4 |
The European medicinal cannabis market is estimated to grow from USD 2.59 billion in 2024 to USD 12.65 billion by 2033, representing a compound annual growth rate (CAGR) of 18.33% 4 .
Projected CAGR for European Medicinal Cannabis Market (2024-2033)
Gas chromatography is one of the most established methods for the analysis of cannabinoids and terpenes . This technique involves vaporizing the sample and separating its components in a capillary column under high temperatures.
GC is typically coupled with flame ionization detectors (FID) for routine quantification, or mass spectrometers (MS) for unambiguous compound identification .
Liquid chromatography has emerged as a powerful alternative to GC, particularly for cannabinoid analysis. The main advantage of HPLC lies in its ability to quantify both acidic and neutral forms of cannabinoids without the need for derivation, since separation occurs at lower temperatures that do not cause decarboxylation .
Modern HPLC systems generally use C18 stationary phase columns and mobile phases that combine methanol or acetonitrile with water acidified with formic acid .
| Parameter | Gas Chromatography (GC) | Liquid Chromatography (HPLC) |
|---|---|---|
| Sample Preparation | May require derivation for acids | Generally straightforward |
| Analysis Temperature | High (causes decarboxylation) | Ambient or moderate |
| Acidic Cannabinoid Forms | Not directly quantified | Directly quantified |
| Neutral Cannabinoid Forms | Directly quantified | Directly quantified |
| Terpene Analysis | Excellent | Limited |
| Sensitivity | High | Very high (with MS/MS) |
| Instrumental Cost | Moderate | High (for LC-MS/MS) |
One of the most significant analytical challenges in the field of cannabis science is the simultaneous analysis of terpenes and cannabinoids, compounds with drastically different physicochemical properties. An innovative study published in 2020 sought to overcome this challenge through the development of a unified gas chromatography method for the quantification of both groups of compounds in plant samples and their extracts 2 .
The research team faced two main obstacles: the large differences in polarity and volatility between terpenes and cannabinoids, and the need for exhaustive decarboxylation of the acidic forms of cannabinoids without degradation of volatile terpenes 2 .
Researchers tested several solvents before selecting acetone as the ideal extraction solvent, offering an effective compromise between the recovery of polar terpenes and lipophilic cannabinoids. Any solvent evaporation step after extraction was intentionally avoided to prevent significant losses of volatile terpenes 2 .
A ratio of 1:17 (300 mg sample to 5 mL solvent) was established as the ideal balance between extraction efficiency and adequate terpene concentrations for quantification without the need for concentration steps 2 .
To solve the critical problem of overlap between cannabichromene (CBC) and cannabidiol (CBD), researchers selected a column with a 50% phenyl 50% dimethylpolysiloxane stationary phase, significantly more polar than the conventional 5% phenyl columns typically used in cannabinoid analysis 2 .
The temperature program was carefully optimized to accommodate the wide range of volatilities, from highly volatile monoterpenes to heavy, low-volatility cannabinoids.
The developed method demonstrated robust performance for both analyte groups. The detection limits ranged between 120-260 ng/mL for terpenes and 660-860 ng/mL for cannabinoids 2 . Parallel validation with established HPLC methods confirmed that the results for cannabinoids were comparable, validating the method's accuracy for routine applications.
| Compound | Detection Limit (ng/mL) | Linear Range (μg/mL) | Notes |
|---|---|---|---|
| β-myrcene | 120 | 1-100 | Predominant terpene in indica varieties |
| α-pinene | 150 | 1-100 | Contributes to "spicy" aroma |
| limonene | 180 | 1-100 | Common in citrus varieties |
| THC | 750 | 10-1500 | Main psychoactive |
| CBD | 660 | 10-1500 | Non-psychoactive, therapeutic |
| CBC | 820 | 10-1500 | Minor cannabinoid |
Modern cannabis analysis requires a variety of specialized reagents, solvents and reference materials to ensure accurate and reproducible results.
| Item | Function | Specific Application | Considerations |
|---|---|---|---|
| Certified Cannabinoid Standards | Calibration and quantification | GC, HPLC, LC-MS/MS | Must include acidic and neutral forms; critical for accuracy |
| HPLC Grade Solvents | Extraction and mobile phase | Sample preparation, chromatography | Low UV-abs for UV detection; free of interferences |
| Acetonitrile and Methanol | Mobile phases | HPLC, LC-MS | High purity grade to avoid contamination |
| Acetone | Extraction solvent | Simultaneous extraction of terpenes and cannabinoids | Selected as ideal compromise 2 |
| Derivatizing Agents (e.g., BSTFA) | Protection of functional groups | GC for acidic forms | Stabilizes acidic cannabinoids against decarboxylation |
| Chromatographic Columns | Compound separation | GC, HPLC | 50% phenyl phase for improved separation 2 |
| Acidified Mobile Phases | Improve separation and peak shape | HPLC | 0.1% formic acid common |
| Quality Control Materials | Method validation | Quality assurance | Matrix reference materials |
| 2,4-dimethyl-9H-pyrido[2,3-b]indole | Bench Chemicals | Bench Chemicals | |
| 4,5,5-trifluoropent-4-enoic Acid | Bench Chemicals | Bench Chemicals | |
| 2,4-bis(2-phenylpropan-2-yl)phenol | Bench Chemicals | Bench Chemicals | |
| 2-Hexynyl-NECA | Bench Chemicals | Bench Chemicals | |
| 4-hydroxy-N-methylproline | Bench Chemicals | Bench Chemicals |
Cannabis sativa L. has traveled a remarkable path from its ancient roots to becoming a focus of modern scientific and regulatory innovation. As research continues to unravel the mysteries of this complex plant, we are presented with new therapeutic applications and analytical challenges.
Will continue to evolve, with multidimensional chromatography and high-resolution mass spectrometry offering increasingly detailed views of the plant's chemical complexity.
Will deepen into the synergy between cannabinoids and terpenes, potentially leading to specific formulations for particular medical conditions.
Will face the challenge of harmonizing rigorous quality control with equitable access for patients.
As cannabis continues to transition from controlled substance to agricultural commodity and pharmaceutical, collaboration between scientists, regulators, healthcare professionals, and patients will be crucial to realizing the full potential of this remarkable plant. The path ahead will require not only scientific rigor but also open and informed social discussion about cannabis' place in our societies.