https://dx.doi.org/10.21577/0103-5053.

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Review J. Braz. Chem. Soc. 2024, 35, 10, e-20240129, 1-25

©2024 Sociedade Brasileira de Química

Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

Julieth G. Herrera, a
Larissa A. Rolim, b
Ricardo S. Honorato c
and Maria Fernanda Pimentel *,d
a
Departamento de Química Fundamental, Universidade Federal de Pernambuco (UFPE),
Av. Jornalista Aníbal Fernandes, Cidade Universitária, 50740-560 Recife-PE, Brazil
b
Central de Análise de Fármacos, Medicamentos e Alimentos,
Universidade Federal do Vale do São Francisco (UNIVASF),
Av. José de Sá Maniçoba, s/n, 56304-917 Petrolina-PE, Brazil
Sector Técnico Científico, Polícia Federal (PF), Cais do Apolo 321, 50030-907 Recife-PE, Brazil
c

d
Departamento de Engenharia Química, Universidade Federal de Pernambuco, (UFPE),
Av. dos Economistas, Cidade Universitária, s/n, 50740-590 Recife-PE, Brazil

Cannabis plant has been known for its medicinal use for centuries. Recent research into its
pharmacology has revealed medicinal properties, promoting the use of cannabis for therapeutic
purposes and consequently the growth in the production of medicinal cannabis-based products.
The potency of cannabinoids in products for therapeutic purposes is essential to assess their effects
on health. Medicinal cannabis products, based on extracts from the cannabis plant, however, lack
standardization in both preparation and quality control. In this review, chromatographic methods
and alternative strategies are described for qualitative and quantitative analysis of cannabinoids in
cannabis-based products. The direct application of portable spectroscopic techniques on cannabis
farms for monitoring plant growth is also reported. Finally, a special section has been dedicated to
the use of chemometric tools that have been employed in the field of cannabis including methods
utilizing chemometric tools such as exploratory analysis, multivariate classification, and calibration.

Keywords: Cannabis sativa L., quality control, methods, hemp, chemometrics

1. Introduction recognized in cannabis, and 144 distinct cannabinoids

have been isolated from the plant. In plant tissues,
Cannabis, also known as marijuana and hemp, has three cannabinoids are synthesized in the form of carboxylic
different varieties: Cannabis sativa, Cannabis indica, and acids. The most common acidic cannabinoids found are
Cannabis ruderalis. It is a dioecious annual plant belonging Δ9-tetrahydrocannabinoic acid A (THCA-A), cannabidiolic
to the Cannabaceae family. As dioecious species exhibit acid (CBDA) and cannabigerolic acid (CBGA). The last
inflorescences exclusively male or female on separate one, CBGA is the direct precursor of THCA, CBDA and
plants, the differentiation between male and female plants cannabichromenic acid (CBCA). CBGA derivatives are
occurs in the early stages of their vegetative development. formed from different enzymes that cyclize the terpene
Exclusively in female plants, the development of trichomes fraction. The carboxyl group exhibits low stability and,
on the bracts of the flowers is observed. These trichomes when subjected to heat or light, can readily lost as CO2.
consist of a unique set of terpenophenolic compounds This process leads to the formation of the corresponding
found in cannabis, called cannabinoids, also known as neutral cannabinoids: Δ9-tetrahydrocannabinol (THC),
phytocannabinoids.1-3 cannabidiol (CBD), and cannabigerol (CBG). In other
C. sativa is one of the plants capable of producing words, neutral cannabinoids arise from photochemical,
cannabinoids with more than a hundred compounds oxidation, or isomerization reactions that may take place
described. Currently, over 550 compounds have been within the plant, or as a result of external conditions after
harvest as, for instance, during heating, drying of harvested
*e-mail: [email protected] plant material, storage or when cannabis is smoked.
Editor handled this article: Brenno A. D. Neto Cannabinol (CBN) is a common degradation product of

This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

THC, found in higher quantities in cannabis samples that 1.1. Legal aspects
have been stored for a prolonged period.1,4-6 The mechanism
of cannabinoids is shown in Figure 1. Following alcohol, cannabis stands as the second most
frequently utilized psychotropic substance in the United
States. 10 In Europe, its consumption surpasses other
substances by approximately fivefold, making it the most
commonly used illicit drug.11 C. sativa can grow under
different climatic conditions and has excellent genetic
adaptability. These characteristics allow the creation of new
varieties of genotypes enriched for a specific cannabinoid.
As a result, strain varieties have been produced in abundance
with limited taxonomic classification, popularly referred
to as either medicinal type or marijuana, or industrial type
or hemp.9 To overcome this situation, the United Nations
Office on Drugs and Crime12 has proposed the use of an
index based on the amount of the three main cannabinoids
to classify cannabis plants. The index is calculated with the
relative proportions, that is, the peak area, of THC, CBN
and CBD obtained by chromatography, in the expression:
{[THC] + [CBN]/[CBD]}. If the index value is greater than 1,
THC is predominant and the plant is classified as “drug type”
or chemotype I (Δ9-THC/CBD >> 1.0). When the content of
THC and CBD is balanced, a Δ9-THC/CBD ratio close to 1.0
is for “intermediate type” or chemotype II plants. On the other
hand, if the index value is less than 1, CBD is predominant
and the plant is classified as “fiber type” or chemotype III (Δ9-
THC / CBD << 1.0). Other chemotypes are also classified as
Figure 1. The most common cannabinoids and their biosynthetic pathway type IV, when cannabigerol (CBG) predominates, and type V
from CBGA to THCA and CBDA, conversion of THC and CBD through
light and/or heat, and the oxidative degradation process converting THC when there is an undetectable amount of phytocannabinoids.
into CBN. This approach enables differentiation of the plants based on
the proportions of specific cannabinoids and helps classify
Interest in the chemistry of cannabis metabolites cannabis varieties.13-15
increased following the discovery of phytocannabinoids, Cannabis with a predominant THC profile is extensively
a product of chemical studies carried out in the 1940s and cultivated globally. Approximately 147 million people,
1960s. Since the early 1940s, researchers7 have identified constituting around 2.5% of the world population, consume
and characterized at least 90 cannabinoids from cannabis. it.16 Cannabis (fiber type) known as hemp is used for food,
A significant discovery occurred in 1964 when Raphael textile, and medicinal purposes. The plant is employed for
Mechoulam8 successfully isolated and characterized THC, industrial purposes in 50 countries across Europe, Asia,
the primary psychoactive component of cannabis, for the North, and South America. The industrial use of cannabis
first time. Due to the intoxicating effects resulting from focuses on the production of more than 2,500 products
the psychotropic activity of THC, cannabis has become used in the fields of agriculture, energy, the paper industry,
the most widespread drug of abuse, so much so that it is textiles, recycling, personal care products, construction
considered illegal in many countries due to its potential materials, and medical supplements. Regulations by
for abuse. On the other hand, cannabis has been known different countries vary the maximum limit of THC in
for centuries around the world for its medicinal properties. plants allowed. Producing countries require that varieties
Research into the biological activities of the plants, contain less than 1% THC. In the European Union, the
chemistry and pharmacology has confirmed medicinal maximum legal limit for cultivation is 0.2% THC, with
properties. For this reason, the study of the plant and its some exceptions such as the Czech Republic and Austria,
terpenophenolic compounds has sparked increasing interest with < 0.3%, and Switzerland with 1.0%.17,18
in the development of medicines and cannabis preparations In the United States, before 1950, hemp was freely
for various medical applications.1,2,4,6,9 cultivated, mainly to produce fiber for industrial

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

applications. In 1970, the Federal Controlled Substances stimulus and reduction in violence linked to gangs and
Act19 made the medicinal and recreational use of cannabis, drug trafficking. On the other hand, there are opponents
as well as the cultivation of hemp, illegal. This situation, who highlight issues such as chemical dependency, risks to
however, changed over time. By February 2022, Medical mental health, passive exposure and possible worsening of
Cannabis Acts had been enacted in 37 states and the mental illnesses. The impact of legalization continues to be
District of Columbia, each with notable variations in the subject of debate, with research underway to explore the
specific provisions.20 As of November 2021, Recreational implications in different jurisdictional areas, although the
Marijuana Acts had been passed in 18 states and the scientific evidence of the therapeutic potential of cannabis
District of Columbia.20 Additionally, in December 2018, is already well established.33
the U.S. Congress passed the 2018 Farm Bill,21 legalizing
industrial hemp as an agricultural product. Since then, in the 1.2. Medicinal cannabis
United States, hemp has been defined by law21 as C. sativa
that contains no more than 0.3% (m/m) of total THC, For centuries, cannabis has been recognized for its
calculated as: (Δ-9-THC) + 0.877 × (THCA) based on dry medicinal applications. In recent years, there has been a
weight. Additionally, and in accordance with Department of notable surge in research exploring the medical applications
Agriculture rules,22 flower samples from hemp cultivation of cannabis, with a number of countries adopting a
must be tested 30 days before the expected harvest date. more flexible stance towards its use as a medicine.
Testing is carried out in registered facilities using traditional Cannabinoids are the main active constituents of the plant
chromatography methods.23-25 and are associated with a broad range of pharmacological
In recent years, Cannabis has garnered increased activities. Medical cannabis is the term used to describe
attention in medical research as a therapeutic option, with the therapeutic use of cannabis or cannabinoids, to treat
numerous countries worldwide legalizing its medicinal illnesses or alleviate medical symptoms. Medicinal
use. Despite the therapeutic promise of cannabinoids, cannabis extracts are obtained from the botanical raw
legislative measures tied to the 1961 United Nations material of the cannabis plant and contain cannabinoids at
Single Convention on Narcotic Drugs26 and the 1971 different levels of purification and refinement.32,34,35
Convention on Psychotropic Substances27 have significantly The most common cannabinoids, which have
impeded research on the pharmacological and therapeutic paradoxical effects on the central nervous system, are
applications of Cannabis. Nevertheless, shifts in national THC and CBD. THC is psychoactive and euphoric,
policies concerning medicinal cannabis have taken place while CBD is a depressant and has anticonvulsant
in various countries. Between 2012 and 2021, 41 countries and anxiolytic properties. Clinical studies involving
(23 in Europe) legalized the use of C. sativa and/or cannabis, cannabinoids, and synthetic analogues have
cannabis-based products for medical purposes.18 In 2016, been reported to be efficient in treating conditions such
Australia authorized the medicinal use of cannabis, with as chronic neuropathic pain, appetite loss in cancer or
ongoing investigations into the potential legalization for AIDS (acquired immunodeficiency syndrome) patients,
recreational use.28 There is confusion regarding the legality and multiple sclerosis. For this reason, medicinal cannabis
and availability of these products, both within and among has been used for various therapeutic purposes, seeking to
legal regulatory bodies. Prescription and over-the-counter offer relief and treatment for specific medical conditions.
medicines marketed and approved by regulatory agencies, Cannabis containing elevated THC levels is employed for
such as the European Medicines Agency (EMA) and the managing conditions such as Tourette syndrome, glaucoma,
Food and Drug Administration (FDA), are standardized and nausea. A mixture of THC and CBD is utilized to
products and formulated in dosages with proven quality alleviate pain and muscle spasms. CBD mitigates pain,
and safety.29-31 Products from plant-based formulations or inflammation, and the psychoactive side effects of THC;
artisanal extracts, however, have not been subjected to the as well, CBD is used to treat various forms of epilepsy.2,36,37
quality controls generally associated with legal approval Cannabinoids can be administered in a variety of ways,
for the marketing of medicines.5,18,32 such as orally, sublingually, topically, or by being smoked,
Despite having been legalized in some countries, inhaled, added to food, or prepared as a tea. They can be
there are still concerns about how cannabis consumption used in the form of natural herbs, extracted directly from
may affect society. In favor of legalization, there are the plant, obtained through the isomerization of cannabidiol
those who highlight the extinction/minimization of the or produced synthetically.35 These compounds can activate
illegal market, improvement in quality control of inputs or modulate the endocannabinoid system (EC), which is
and medicines, increase in tax collection, commercial extensively integrated into various organ systems of the

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

brain and body. The EC system plays diverse roles in the When it comes to the quality of medicinal cannabis
homeostatic regulation of humans and certain animals.38 products, however, other parameters need to be considered
Various medicinal cannabis products and magisterial in addition to cannabinoid content. Other classes of
preparations with varying ratios of Δ9-THC and CBD analytes found in the plant require analysis: terpenes, other
are available on the market. Common formulations secondary metabolites, heavy metals, residual solvents,
include filter bags for cannabis decoction, single-dose microorganisms, pesticides, herbicides, fungicides,
inhalation formulations, and cannabis extracts, primarily rodenticides, and other chemical adulterants that can be
suspended in olive oil or ethanol.39 Cannabis oil is the harmful to health and the environment. The relevance of
preferred form of preparation, given its ease of adjusting their analysis is related to the possible harm that these
individual administration doses throughout the treatment compounds may inflict if they are ingested directly from
period and the increased bioavailability of its active the plant or if they are co-extracted with cannabis’s primary
constituents. 37 In addition to the medicinal cannabis components and later detected in processed cannabis
products mentioned above, several cannabis-based products.44,45
medicines have been developed. Among them, Marinol® Given these problems, consumers may inadvertently
(Solvay Pharmaceuticals, Belgium), an oral preparation of purchase products with undesirable properties. In fact, the
Δ9-THC, Nabilone® (Valeant Pharmaceuticals International, Food and Drug Administration (FDA) has been issuing
USA), a synthetic analogue of Δ9-THC, and Sativex® (GW warning letters since 201546 regarding unwarranted and
Pharmaceuticals, UK) are the most known, as well as an illicit medical claims concerning CBD formulations
oral mucosal spray that contains a balanced proportion of produced or distributed by US companies. Cases have been
Δ9-THC and CBD.2 reported47 in which some products contained significantly
Cannabinoid concentrations may vary depending on the lower cannabinoid content than that indicated on the label,
product. For example, CBD oil products marketed in Japan while others contained significantly higher amounts of THC
have CBD concentrations between 29 and 109 mg g-1, with than labeled, placing patients at risk of adverse effects.
THC detected in trace concentrations of 0.002 mg g-1.40 Research on the quantification of cannabinoids in Brazilian
E-liquids for electronic cigarettes sold in Switzerland medicinal extracts, carried out by Carvalho et al.,36 in 2020,
have THC concentrations of less than 0.2%, while CBD showed a wide variability in the composition of medicinal
varies between 0.182 and 3.346%.41 In extracts from extracts used to treat the same disease. In preparing the
two strains of Cannabis sativa, Bedrocan and Bedrolite, extracts, different vehicles can be used to dissolve the
CBD concentrations range from 0.38 to 39.2% and cannabis resin: olive oil, sunflower oil, coconut oil, soybean
THC concentrations from 18.93 to 86.99%.42 Cannabis oil and even oil mixtures. Differences in phytocannabinoid
oils for medicinal purposes distributed in Brazil have content have also been reported; in addition to CBD, the
THC concentrations of around 10 mg mL-1 and CBD extracts were found to have other cannabinoids such as
concentrations of 3 mg mL-1.36 Techniques used for these THC, CBN, THCA and CBDA. The presence of CBN
analyses include liquid chromatography-tandem mass indicates the degradation of THC; the presence of acid
spectrometry (LC-MS/MS), ultra-performance liquid cannabinoids (THCA and CBDA) indicates that the
chromatography with diode array detection (UPLC-DAD), decarboxylation of the raw material was inadequate.
Fourier-transform infrared spectroscopy (FTIR), and The lack of established guidelines for use, restrictions
high-performance liquid chromatography with diode array on varieties and chemical residues, as well as the absence
detection (HPLC-DAD). To detect low concentrations, of proven methods for assessing cannabis products, can
the LC-MS/MS technique is more suitable due to its high expose users to harmful substances or residue levels higher
sensitivity, while HPLC-DAD and UPLC-DAD are more than would be permitted if there were clear regulatory
affordable and widely used. guidelines and regulations.47
The raw material used in the production of medicinal The objective of this literature review is to record the
extracts are the flowers of pistillate specimens rich in versatility and evolution of analytical methods used in the
THCA and CBDA, which are heated to obtain the active quantitative and qualitative analysis of phytocannabinoids
ingredients, THC and CBD. These 4 cannabinoids, together in cannabis-based products (especially products with
with CBN, are widely used as criteria for the following: medicinal purposes), and cannabis extracts. The use
to characterize Cannabis plant products; provide useful of chemometric tools in data processing and analysis
information about the plant such as age, potency (amount will also be discussed, as well as the development of
of cannabinoids) and possible geographic origin; and to cannabinoid prediction models and their application in
control the quality of medicinal products.36,43 quality control.

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2. Methods for the Review of the ions of the separated analytes, thus overcoming the
low specificity of conventional detectors.49,50 When coupled
To find relevant publications for the construction of this to gas chromatography, mass spectrometry also provides
review, databases such as Scopus, Google Academic and detailed structural information about mass spectrometry
Web of Science were consulted over a period of 6 months. analytes, in addition to GC-MS systems having high
A combination of keywords such as “medical cannabis”, sensitivity.51
“cannabinoids”, “analysis”, “control”, “quantification” and One of the primary challenges in analytical assessments
“methods” were searched. In total, 113 articles published for quality control, however, is the proficiency of
between 1970 and 2023 in English, Portuguese or Spanish laboratories to do the analysis. Standardized tests are
were selected. The focus of this review was on the analytical necessary that meet analytical criteria that would be
part, the methods used to analyze cannabinoids in the approved by the competent control authorities. Laboratories
cannabis plant and its extracts, and cannabis-based products also face challenges related to the cost and accessibility
(especially with medicinal purposes). Therefore, articles of some cannabinoid standards. Another difficulty is
were disregarded which had a different perspective such related to the complexity of the matrices of medicinal
as clinical studies, classification, and taxonomy, as well as cannabis products. For example, an oily matrix can lead
uses and cultivation of cannabis, analysis of cannabinoids to technical damage to analytical instruments and diminish
in biological matrices. Altogether, 98 articles were read the operational lifespan of chromatographic columns.7,18,52,53
to prepare this work, including 16 articles that were cited For the development of analytical methods applied
only in the introduction part, such as book chapters and to cannabis-based products, it is necessary to extract
review articles. cannabinoids from plant material, prepare the obtained
extract, and then perform detection followed by
3. Analysis of Cannabinoids quantification of the concentration of each cannabinoid
found in the plant.54 The focus of this review is on qualitative
Due to the growth in production of medicinal and quantitative analytical methods for cannabinoids in
cannabis formulations, there is an increasing need for the plant extracts and products used for medicinal applications.
development of analytical methods. Accurate qualitative
and quantitative analyses of phytocannabinoids are 3.1. Chromatographic techniques
indispensable for associating medicinal effects and potential
negative health impacts associated with the potency of Traditionally, the determination of cannabinoid content
specific phytocannabinoids and other compounds, such has been conducted using HPLC and GC coupled to MS;
as terpenoids. Chromatographic methods, such as liquid alternatively, flame ionization detection (FID) has been
chromatography (LC) and gas chromatography (GC), used for GC or UV-DAD for HPLC, as detectors. The GC
are the most used mainly due to their ability to separate method has been widely used in the quantitative analysis
analytes. THC was isolated for the first time in 1964 by of cannabinoids, but it is not capable of distinguishing
Raphael Mechoulam,8 using alumina chromatography between cannabinoids and their carboxylic counterparts
for analysis. With the advancement of technology and without prior derivatization, because as the method involves
analytical techniques, cannabinoid analysis methods have subjecting the sample to heat, causing the acidic forms of
evolved significantly. Currently, advanced techniques such cannabinoids to undergo decarboxylation and transform
as high-performance liquid chromatography (HPLC) and into their neutral counterparts. On the other hand, the
ultra-performance liquid chromatography (UPLC) are used, HPLC method makes it possible to determine the original
coupled with ultraviolet diode array detection (UV-DAD), composition of cannabinoids in the plant through direct
which enable the accurate identification of cannabinoids by analysis. Unlike GC, no decomposition of cannabinoids
their chromophore groups. At the same time, these detectors occurs during HPLC analysis.1,17,34,55 The most common
reveal the presence of interfering compounds in various analytical technique for determining cannabinoids in plants
sample matrices.48 Furthermore, the introduction of liquid in most laboratories worldwide is high-performance liquid
chromatography coupled with mass spectrometry (LC‑MS) chromatography coupled with either diode array detection
has revolutionized the analysis of polar and unstable or mass spectrometry. These are preferred for analyzing
compounds, increasing sensitivity and selectivity in the the phytocannabinoid profile due to their robustness,
detection of analytes in complex matrices. This coupling reproducibility, sensitivity and speed.18,56 In addition to
has made it possible to identify and quantify compounds in cannabinoid analysis, liquid chromatography has been used
complex mixtures by measuring the mass-to-charge ratio in terpene analysis. Caruso et al.57 reported for the first

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

time, a two-dimensional liquid chromatography method for multiple reaction monitoring (MRM) allowed verification
the simultaneous separation of terpenes and cannabinoids of the legality and screening of cannabinoids in CBD
in cannabis plant material. The proposed method makes oils.52 When analyzing galenic preparations of cannabis
it possible to identify 21 terpenes and 10 cannabinoids oil, Cas et al.37 reported that cannabinoid content was
in cannabis samples, resolving between 40 and 54 peaks significantly linked to cannabis chemotypes and extraction
in just 65 min. This is due to the distinctive advantage of protocols. The authors evaluated samples with and without
two-dimensional liquid chromatography, which lies in its a previous decarboxylation step. With the help of selected
superior ability to separate multiple compounds in complex ion monitoring (SIM) by a mass detector, it was possible to
samples. evaluate which oils were richer in the neutral active form
of cannabinoids (CBD, THC) through a decarboxylation
3.1.1. Gas chromatography (GC) step. Ciolino et al.61 identified cannabinoids in cannabis-
GC is a chromatographic technique employed based consumer products in the United States. Commercial
for the separation and analysis of compounds from products analyzed included foods, sweets, beverages,
various matrices that can undergo vaporization without topicals, vapes/e-liquids, oral supplements; hemp seed
decomposition. GC is one of the most frequently used oils of different origins and generic dronabinol capsules.
chromatographic techniques for the quantitative analysis Most of these products presented CBD as the predominant
of phytocannabinoids in plant material and biological cannabinoid.
matrices due to its robustness and reproducibility. When
coupled with mass spectrometry, it adds high sensitivity 3.1.2. Liquid chromatography (HPLC/UPLC)
and the ability to provide detailed structural information HPLC is recognized as a modern, powerful, and
about the analytes.18,51,58 versatile chromatographic separation technique. It is
The most commonly used solvents for extracting the most employed analytical separation method for
cannabinoids, prior to chromatographic analysis by qualitatively and quantitatively assessing compounds
GC, are methanol,37,43,59,60 and ethanol.61-65 Hexane14 and in natural product extracts, fractions, or end products.
dichloromethane66 have also been used. Regarding the UPLC, an advanced liquid chromatography technique,
columns, non-polar or low-polarity stationary phases, requires a short analysis time and uses a minimal quantity
such as dimethylpolysiloxane, have been employed59 as of solvent(s) as the mobile phase. UPLC instruments are
well as mixtures with phenyl, dimethylpolysiloxane 95%/ characterized by smaller particles, less than 2 microns,
phenyl 5%.60,66,67 Regarding the mobile phase, the carrier compared to larger particles, between 2.5 and 10 microns,
gas mostly used is helium; in fact, of the studies mentioned in conventional HPLC systems. Due to the smaller particle
in this article, only one of them used hydrogen.64 The size, UPLC operates at higher pressures (above 6,000 psi)
temperature ramps used mostly range up to 300 °C with and offers greater separation efficiency of analytes from
analysis times from 1267 to 39.9 min.61 Due to the need for samples due to the shorter diffusion path between them
high temperatures in the chromatographic column, acidic and the stationary phase. Several types of detection
cannabinoids undergo decarboxylation during analysis, systems can be used in liquid chromatography; for
therefore, in GC, the total amount of cannabinoids in a cannabinoid analysis, however, the DAD have emerged
sample is the sum of the acidic and neutral components.9,58 as the standard for cannabinoid potency testing, as well
The literature gives examples of cannabinoid analysis as the use of liquid chromatography coupled to mass
by GC for various purposes, for example, Broséus et al.14 spectrometry.24,68 In fact, a bibliometric analysis carried
developed a method for distinguishing between drug-type out on publications that contained key words such as
and fiber-type cannabis based on the relative proportions analysis and cannabinoids or cannabis, showed a strong
of the main compounds found in the leaves of cannabis connection to liquid chromatography and quality control
seedlings. With the proposed method, it would not be when the word “cannabinoids” was chosen as the key word
necessary to wait for the plant to flower to determine the on the map, as illustrated in Figure 2. This indicates that
cannabis chemotype. The chemical profile of cannabis liquid chromatography is one of the most recurring topics
was studied using high-resolution mass spectrometry and in publications relating to cannabinoid analysis. There are
two-dimensional analysis, making it possible to identify the also other links to cannabis, hemp, THC, synthetic THC
chemical structures of cannabinoids and other compounds (dronabinol), and CBD and its extracts.
of interest, such as pesticides and degradation products. In the published literature1,24,43,44,54,57,59,61-63,66,70-73 on
With the GC-MS system, it is possible to use libraries for cannabinoid analysis employing chromatographic methods,
structural identification.59,60,64 Likewise, GC-MS/MS with the majority of studies reported have analyzed extract of the

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

that dynamic maceration with ethanol at room temperature

for 45 min was the best technique among those tested. On
the other hand, in a different study, Tzimas et al.85 also
evaluated cannabinoid extraction techniques: extraction
assisted by ultrasound, microwaves and dynamic
maceration in fiber-type cannabis inflorescences. The
researchers reported more promising results for ultrasound-
assisted extraction, given that acoustic cavitation favors
the penetration of the solvent into the plant tissue and
the diffusion of solutes within the extraction medium.
Similarly, Baranauskaite et al.63 pointed out ultrasound-
assisted extraction was the best technique for extracting
cannabinoids compared to maceration and heat extraction
by reflux. Ultrasound-assisted extraction was considered
Figure 2. Map based on bibliographical data with co-occurrence analysis the ideal technique as it required less time, energy and cost.
of keywords, highlighting the connections with the word cannabinoids.
Figure obtained from Vosviewer (Centre for Science and Technology Correia et al.73 also highlight the use of ultrasound in the
Studies Leiden University).69 extraction process. The authors proposed and optimized a
methodology for ultrasound-assisted solid-liquid extraction.
plant material. Some studies have analyzed cannabinoids The extraction process was optimized using experimental
from the leaves 14,74 and most of them have analyzed design to evaluate different solvents, volumes, steps and
the flowers.5,13,60,64,65,75-86 Other products have also been sonication time. The mixture of ethanol and acetonitrile
analyzed, such as hemp nuts,87 CBD oil,40,52 hemp seed (50:50) provided the highest extraction yield, with 1 step
oil,61,62,88 oil preparations, which include diluting the plant of 1 min of sonication and a volume of 10 mL of solvent.
extract in oil, or extracting the cannabinoids directly into Sample preparation varies depending on the matrix.
oil36,37,39,67,71,89 and generic capsules.62,70,90 Among cannabis products for medicinal purposes, the oily
Prior to a chromatographic analysis, it is necessary to matrix is the most common. Preparing cannabis oil for
extract the cannabinoids from their respective matrices. analysis involves accurately measuring a specific amount
The solvent most commonly used for this purpose is of oil, typically between 50 and 100 mg, and dissolving
methanol, followed by ethanol and lastly mixtures of it in suitable solvents such as methanol, isopropanol,
methanol with various solvents, for example methanol/ dichloromethane, acetonitrile, or solvent mixtures such as
chloroform;1,17,34,54,76,89 methanol/acetonitrile;71,81 methanol/ methanol (9:1 v/v). After adding the solvent, the mixture
hexane36,44 and methanol/acetone.60 The use of hexane as is vortexed and, in some cases, subjected to ultrasound to
an extraction solvent,72 as well as the mixture of ethanol ensure complete homogenization. The samples are then
and acetonitrile in a 50:50 ratio,73 have also been reported. centrifuged and filtered using Teflon or nylon filters with
Methods for extracting non-psychoactive cannabinoids a porosity of 0.22 to 0.45 µm. Some protocols include
such as CBD, CBDA, CBG, CBGA from fiber-type plant cooling to -20 or -41 °C to facilitate phase separation.
material have been evaluated. Brighenti et al.77 tested This step helps freeze the oil layer, allowing the liquid
4 techniques to optimize the extraction of cannabinoids solvent layer to be pipetted into a new container. Finally,
in pharmaceuticals and hemp. The techniques tested samples can be diluted and mixed with internal standards
were ultrasound-assisted extraction, microwave-assisted before being transferred to HPLC vials and stored at 4 °C
extraction, supercritical fluid extraction and dynamic until analysis.36,53,75,80,91
maceration. Ultrasound and microwave assisted extraction Published literature shows that for cannabinoid analysis
are techniques employing sound waves or microwaves with chromatography, the stationary phase most used has
to accelerate the extraction process and enhance the been a C18 reversed phase column. Some authors used
yield of metabolites. Supercritical fluid extraction offers the following C18 column types with complementary
the advantage of eliminating flammability or toxicity selectivity: C18-AR;24,64,75 EC-C18, C18 stationary phase
concerns since the solvent is completely removed. Dynamic with superficially porous particles; 78,88 or the SB-C18
maceration involves extracting plant material using a and XB-C18, C18 stationary phase with fully porous
solvent and multiple agitations. The extraction time and particles.13,39,65,82 The vast majority of mobile phases were
temperature were optimized for each technique and were used with gradients of water acidified with formic acid
compared under their optimal conditions. Studies report77 (0.1% v/v) and acetonitrile or ammonium formate and

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

acetonitrile buffers. Some studies have reported the use by extraction of cannabinoids from the matrix and
of acetonitrile acidified with formic acid5,44,64,81-83,85,87,89 chromatographic analysis, the purpose or motivations
or acetonitrile acidified with acetic acid.62,86,91 Liquid for these studies have been different. For example,
chromatography has emerged as the gold standard for for quality control, Carvalho et al.36 quantified THC,
cannabinoid analysis using DAD as detector, as well CBD, CBN, THCA, and CBDA in artisanal medicinal
as coupled to MS. Several types of mass spectrometry preparations produced in Brazil by patients’ families. The
analyzers are used in cannabinoid analysis, each with its authors found that only 10 of the 68 extracts analyzed
specific advantages. The triple quadrupole (tQ) provides had a predominant CBD profile and, even so, the levels
high sensitivity and selectivity at a low cost and is were much lower than those determined in American
commonly used for cannabinoid quantification in routine extracts. Furthermore, high levels of THCA, CBDA,
methods due to its robustness.5,83,89 Time of flight (ToF) and CBN were found in the national samples analyzed.
analyzers provide high resolution and rapid analysis Similarly, de Backer et al.1 analyzed cannabinoids to
and are used for the identification and characterization evaluate the psychoactive potency and quality control
of cannabinoids due to their ability to provide accurate of medicinal samples. Li et al.90 developed a method to
mass data.24,92,93 The orbitrap provides better resolution evaluate the quality and stability of medical cannabis
and accuracy of mass/charge ratio data, making it ideal products produced in New York and to confirm the potency
for identifying new cannabinoids and analyzing complex described on the labels. Takashina et al.40 tested the
samples.13 quality of CBD oil products sold in Japan, by evaluating
Additionally, it is possible to observe that recently the CBD profile and identifying and quantifying THC
some works78,82,83 have reported using sequential mass residues. Some of the samples that revealed traces of
spectrometry (MS/MS) systems due to the possibilities THC exhibited concentrations of 0.007 and 0.002 mg g-1.
of obtaining more detailed structural information. This These values are in line with the limits established by the
ability to fragment selected ions, enables the acquisition of European Union,95 which has set the maximum THC limit
detailed information about the structure of the cannabinoids in CBD oils at 0.0075 mg g-1. Citti et al.88 carried out a
and better analytical sensitivity and selectivity. Sequential kinetic study of the decarboxylation of CBDA present
mass analysis improves detection sensitivity and selectivity, in hemp seed oil. The reported results suggest that the
reducing interference and increasing quantification best storage condition is 5 °C, in order to preserve the
accuracy. stability of the oil for longer periods. Analysis of THC
In summary, mass spectrometry is an essential technique and CBD and other less abundant cannabinoids has been
for analyzing cannabinoids in medicinal samples, offering used to distinguish strains of the cannabis plant;13,79 for
high sensitivity, specificity, and the ability to provide assessment of therapeutic potency;44 for evaluation of new,
detailed information about molecular structure. The use faster and more efficient methods to extract cannabinoids
of different types of analyzers and the combination with and terpenes from plant material;71 for classification of
HPLC systems further improve the accuracy and quality medicinal cannabis samples based on their cannabinoid
of the data obtained. Table 1 lists works on cannabinoid and terpene profiles;65 to study cannabinoids present in
analysis using liquid and gas chromatography as the main different parts of the cannabis plant;59 to identify hemp
technique. retailers based on the chemical content of cannabinoids
The methods developed for qualitative and quantitative in hemp samples;74 to determine the best solvent for
analysis of cannabinoids by liquid chromatography extraction;75,78,81 as well as to determine the best extraction
have also been validated in terms of linearity, limit of technique.63,77,85 The time of chromatographic analysis
detection (LOD), limit of quantification (LOQ), accuracy, has also been evaluated, with methods lasting 30 min,1,36
precision, recovery and selectivity.60,73,75 Validation of the 10 min74,76 and 8 min per sample.5,70
methods was carried out in accordance with International
Organization for Standardization (ISO) 1702594 and 3.1.3. Disadvantages of chromatographic techniques
SFSTP (French Society of Pharmaceutical Sciences and Despite being accurate and standardized for certification
Techniques);1 the AOAC (Association of Official Analytical purposes, as mentioned above, chromatographic methods
Chemists) and ASTM (American Society for Testing and (LC and GC) have disadvantages such as sample
Materials),71 as well as the ICH (International Council for destruction, high sample preparation costs and run times,
Harmonization).53,54,85,91 involving the extraction of active ingredients with organic
Although, in general, the methodologies for solvents, which limit their application in the location where
cannabinoid analysis have been the same, that is, a quick and non-destructive process is preferred. Also

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

Table 1. Chromatographic methods for analyzing cannabinoids in cannabis extracts and medicinal products

Stationary phase (SP) and

Method Matrix Sample preparation Analyte Chemometrics tool Reference
mobile phase (MP)
SP: 2 columns,
dimethylpolysiloxane and
GC×GC-FID cannabis resin methanol extraction chemical profile PCA, HCA 59
polyethylene glycol
MP: helium
three ethanol extraction
techniques: maceration, SP: capillary column
GC-FID hemp plant material ultrasound-assisted Rxi-5 MS CBD, CBG N/A 63
extraction and reflux MP: helium
heat extraction
SP: capillary column
DB-5MS UI 5%
cannabis oil
GC-FID, GC-MS not reported diphenyl/95% THC, THCA, CBD N/A 67
preparations
dimethylpolysiloxane
MP: helium
SP: capillary column LDA and SVM
leaves from different ultrasound-assisted THV, CBD, THC,
GC-MS HP-5 ms (support vector 14
cannabis chemotypes hexane extraction CBN
MP: helium machines)
ultrasound-assisted SP: capillary column THC, CBD, CBN,
GC-MS plant material ANOVA and HCA 43
methanol extraction J&W HP5 MP: helium CBG
SP: capillary column
CBD, CBDA, THC,
GC-MS cannabis oil methanol extraction Rxi-5ms PCA, HCA, PLS-DA 37
THCA, CBN
MP: helium
SP: fused silica capillary
GC-FID hemp flowers extraction in hexane column (SPB-5) THC, CBD N/A 72
MP: helium
SP: fused silica capillary
GC-MS/MS CBD oil QuEChERS column VF-5 MS CBD, THC, CBN N/A 52
MP: helium
SP: 2 columns,
nonpolar Rxi-5MS
(5% diphenyl-95%
dimethylpolysiloxane
GC×GC-LR TOF Cannabis flowers extraction in water/
phase) and midpolar
MS; GC×GC-HR (Indica, sativa and methanol/acetone THC, CBD, CBN PCA, HCA, DOE 60
Rxi-17Sil MS (equivalent
TOF MS hybrid types) mixture (5:4:1)
to a 50% diphenyl-50%
dimethylpolysiloxane
phase)
MP: helium
GC-MS
SP: Restek Rxi-35Sil MS
THCA-rich plant
extraction in variations MP: helium
materials, commercial D9-THC, D8-THC,
of ethanol (95% HPLC-DAD
GC-MS cannabis consumer THCA, CBD, CBDA,
aqueous or pure) or SP: C18-AR. MP: N/A 61,62
HPLC-DAD products, hemp seed CBN, CBG, CBGA,
acetonitrile (83-91% acetonitrile 66:34:
oil, and generic CBDV, THCV, CBC
aqueous or pure) 0.5% acetic acid (no pH
dronabinol capsules
adjustment, nominal pH
2.9)
GC-MS
SP: DB-5 capillary
column (5% phenyl, 95%
dimethylpolysiloxane) CBDA, CBGA, CBG,
inflorescences of MP: helium CBD, THCV, CBN,
GC-MS
different chemovars of ethanol extraction HPLC-DAD D8-THC, D9‑HC, PLS-DA 65
HPLC-DAD
medicinal cannabis SP: XB-C18 CBL, CBC, THCA,
MP: formic acid and CBCA
20mM ammonium
formate buffer (pH 2.9)
and acetonitrile

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

Table 1. Chromatographic methods for analyzing cannabinoids in cannabis extracts and medicinal products (cont.)

Stationary phase (SP) and

Method Matrix Sample preparation Analyte Chemometrics tool Reference
mobile phase (MP)
GC-FID
SP: column Elite 5MS
THCA, CBDA, THC,
MP: hydrogen
GC-FID, CBG, CBD, CBDVA,
cannabis flowers ethanol extraction HPLC-DAD N/A 64
HPLC-DAD CBDV, CBGA,
SP: C18
THCV, THCVA, CBC
MP: 0.1% of formic acid
in water and 0.1%
GC-FID
SP: DB-5 MS capillary
column MP: helium
cannabis plant (aerial
HPLC-DAD
GC-FID, parts) GC: dichloromethane THCA, THC, CBDA,
SP: CORTECS Shield N/A 66
HPLC-DAD (agricultural hemp extraction CBD, CBN
RP18
and smoking product)
MP: 0.1% formic acid
in water (v/v) and
acetonitrile
dried pistillate SP: aluminum sheets pre-
CBDA, THCA,
inflorescences coated with silica gel 60
ultrasound-assisted CBGA, CBD, CBG,
TLC of cannabis, F245. MP: hexane-ethyl DOE 84
methanol extraction CBN, D8-THC, D9-
THC‑dominant acetate-methanol ternary
THC
chemotype system (70:20:10 v/v/v)
SP: EC-C18 THCA, THC, CBDA,
commercial hemp
HPLC-UV dilution in 2-propanol MP: 0.1% of formic acid CBD, CBN, CBG, N/A 88
seed oil
in water and acetonitrile CBDV
SP: Poroshell 120 EC-
C18
cannabis (plant MP: 0.1% (v/v) formic THC, CBD, CBN,
HPLC-UV extraction in olive oil ANOVA 71
material) acid aqueous phase and THCA, CBDA
0.05% (v/v) formic acid
in methanol
SP: C18. MP: water/
acetonitrile in a ratio of
THCA, D9-THC, D8-
9:31 (v/v), with 0.1%
THC, THCV, CBDA.
HPLC-UV hemp plant material ethanol extraction formic acid (v/v) and N/A 70
CBD, CBDV, CBG,
10 mM ammonium
CBN, CBC
formate (without
pH adjustment)
SP: C18
MP: methanol and water THC, THCA, CBD,
drug and fiber methanol: chloroform
HPLC-DAD containing 50 mM CBDA, CBG, CBGA, DOE 1
cannabis (9:1) extraction
ammonium formate (pH CBN, D8-THC
5.19)
SP: C18 D9-THC, D8-THC,
dried flowers and MP: acetonitrile and 10 THCA, CBD, CBDA,
HPLC-DAD methanol extraction DOE 75
cannabis oil mM ammonium formate CBN, CBG, CBC,
(pH 3.6) THCV
SP: C18
D9-THC, D8-THC,
methanol: chloroform MP: 25 mM ammonium
HPLC-DAD cannabis flowers THCA, CBD, CBDA, N/A 76
(9:1) extraction acetate solution (pH 5.75)
CBGA, CBG, CBN
and methanol
SP: C18 THC, CBD, THCA,
cannabis flowers of extraction in 80% MP: 10 mM ammonium CBDA, CBN,
HPLC-DAD PCA, MLR 79
different strains methanol formate (pH 3.6) and CBG, CBGA, CBC,
acetonitrile CDBVA
SP: 120 SB-C18
MP: ACN/water mixture
cannabis oil
dilution in THF and containing 5 mM K 2
HPLC-DAD preparations/cannabis CBD, THC AQbD, DOE 39
methanol HPO 4 adjusted to pH
olive oil extracts
3.45 (range 3.11-3.50) at
75/25 v/v ratio

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

Table 1. Chromatographic methods for analyzing cannabinoids in cannabis extracts and medicinal products (cont.)

Stationary phase (SP) and

Method Matrix Sample preparation Analyte Chemometrics tool Reference
mobile phase (MP)
SP: Poroshell® 120
capsules, tablets,
MP: 0.1% (v/v) formic THCA, CBDA,
sublingual oral
acid in 25 mM aqueous CBGA, CBDV, THC,
HPLC-DAD solutions, tinctures extraction in methanol N/A 90
ammonium formate; and CBD, CBN, CBC,
and vaporizer
0.1% (v/v) formic acid in CBG, THCV
cartridges
acetonitrile
SP: C18
extraction in methanol:
cannabis and hashish MP: 0.1% formic acid in THC, THCA, CBD,
HPLC-DAD hexane (9:1) assisted by N/A 44
plant material water and 0.1% formic CBDA, CBN
ultrasound
acid in acetonitrile
extraction in methanol: MP: ammonium formate
medicinal extracts THC, CBD, CBN,
HPLC-DAD hexane (9:1) assisted by buffer as solvent, 50 mM, N/A 36
in oils THCA, CBDA
ultrasound (pH 5.19) and methanol
SP: C18
CBD, CBDA, THC,
HPLC-DAD hemp oil supplements acetonitrile extraction MP: 0.5% acetic acid in N/A 91
CBN
water and acetonitrile
SP: C18
CBDVA, CBGA,
MP: 0.05% acetic acid in
CBDA, THCA,
dried pistillate flowers water adjusted to
ultrasound-assisted CBDV, THCV, CBD,
HPLC-DAD of predominant thc pH 4.40 ± 0.05 with DOE, (CCD) 86
methanol extraction CBG, CBN,
chemotype ammonium hydroxide
D8 -THC, D9 -THC,
(30% solution); and
CBC
acetonitrile
SP: C18
MP: phase A (Milli-Q
water buffered with
20 mM ammonium
extraction in D9 -THC, D8 -THC,
dried cannabis formate and 0.1% formic
acetonitrile: methanol THCA-A, CBN,
HPLC-DAD inflorescences and acid), mobile phase B N/A 53
(4:1 v/v) assisted by CBD, CDBA, CBC,
cannabis oil (acetonitrile) and mobile
ultrasound CBDV, CBG, CBGA
phase C (methanol
buffered with 10 mM
ammonium formate and
0.05% formic acid)
2 extraction methods
and 2 solvents
ultrasound-assisted SP: C18
cannabis (plant CBD, CBD, THC,
HPLC-DAD extraction and turbo- MP: 0.1% formic acid and N/A 54
material) THCA, CBDA
extraction. methanol: methanol solution
chloroform (9:1) and
ethanol
SP: C18
MP: isocratic separation
ultrasound-assisted D9-THC, CBD,
cannabis herbal composed of 75%
HPLC-DAD acetonitrile: ethanol D8-THC, CBN, D9- DOE 73
samples acetonitrile and 25%
(50:50, v:v) extraction THCA, CBDA
ultrapure water with 0.1%
formic acid (pH ca. 2.8)
4 extraction techniques
evaluated: dynamic SP: C18
HPLC-DAD
female inflorescence maceration, extraction MP: water acidified with CBDA, CBD, CBGA,
HPLC-ESI-MS and N/A 77
of fiber type C. sativa assisted by ultrasound, 0.1% formic acid and CBG
MS2
microwaving and acetonitrile
supercritical fluids
CBDVA, CBDV,
CBDA, CBGA, CBG,
SP: column Raptor
CBD, THCV, ACBD,
1 placebo, 8 samples ARC-18
CBCV, THCVA,
HPLC-DAD, of plant material, ultrasound-assisted MP: 0.5 mM ammonium
CBN, CBNA, D9- N/A 24
ESI/TOFMS cigarette samples, methanol extraction formate plus 0.02% (v/v)
THC, D8-THC, CBL,
hemp flowers formic acid (pH 3.0) and
CBC, D9-THCA,
acetonitrile
D8-THCA, CBCA,
CBLA, CBT

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

Table 1. Chromatographic methods for analyzing cannabinoids in cannabis extracts and medicinal products (cont.)

Stationary phase (SP) and

Method Matrix Sample preparation Analyte Chemometrics tool Reference
mobile phase (MP)
SP: C18
ultrasound-assisted MP: water and Design of Experiment,
HPLC-MS powdered hemp nuts THC, CBD, CBN 87
isopropanol extraction acetonitrile containing HCA and PCA
0.1% formic acid
SP: column Acquity®
extracts of UPLC HSS T3
Bedrocan®, Bediol®, extraction in olive oil MP: ACN: water (75: 25 THC, CBD, THCA,
HPLC-MS N/A 80
Bedrolite® and mixed dilution in isopropanol + 0.05% formic acid) and CBDA, CBN
preparations isopropanol:ACN (80: 20
+ 0.05%)
SP: EC-C18
medicinal cannabis extraction in ethanol CBDA, CBGA,
MP: water acidified with
HPLC-MS/MS extracts, Bediol and olive oil THCA, CBD, THC, PCA 78
0.1% formic acid and
inflorescences dilution in 2-propanol CBG, CBN
acetonitrile
SP: C18-XB
THC, CBD, CBC,
MP: water containing
ultrasound-assisted CBG, CBN, CBDV,
HPLC-MS/MS hemp flowers 5 mM formic acid and PCA, PLS-DA 82
ethanol extraction THCA, CBGA,
acetonitrile with 5 mM
CBDA
formic acid
CBDV, THCV, THC,
CBD, CBC, CBG,
SP: C18-amide
CBN, CBL, CBDVA,
cannabis and hemp extraction in methanol: MP: 100:0.1 water:
HPLC-MS/MS THCVA, THCA, N/A 83
flowers water (80:20) formic acid and 100:0.1
CBDA, CBCA,
acetonitrile: formic acid
CBGA, CBNA,
CBLA
SP: Inertsil ODS-HL
dilution with column CBD, D8-THC, CBN,
LC-MS/MS CBD oils products isopropanol and water/ MP: 0.1% formic acid in CBG, CBDA, CBGA, N/A 40
methanol water and 0.1% formic CBD, D9-THC
acid in acetonitrile
CBDVA, CBDV,
SP: C18 CBDA, CBGA,
MP: water with 0.1% CBG, CBD, THCV,
dried cannabis
LC-QQQ-MS extraction in methanol formic acid and THCVA, CBN, N/A 5
inflorescences
acetonitrile with 0.1% CBNA, THC, Δ8-
formic acid THC, CBL, CBC,
THCA-A, CBCA
3 ethanol extraction
SP: C18-PFP
techniques: dynamic
fiber-type cannabis MP: water and
maceration, ultrasound-
UPLC-DAD inflorescences of acetonitrile, both CBD, CBDA DOE 85
assisted extraction and
different chemotypes containing 0.1% (v/v)
microwave-assisted
formic acid
extraction
SP: C18
ultrasound-assisted
MP: water containing THC, CBG, CBD,
extraction in
UPLC-UV-MS cannabis flowers 0.05% formic acid and THCA, CBGA, N/A 81
acetonitrile: methanol
acetonitrile with 0.05% CBDA
(80:20)
formic acid
SP: column ACQUITY
CBL, CBD, D8-THC,
UPC2 BEH 2-EP (2-ethyl
cannabis extracts, THCV, D9-THC,
acetonitrile: methanol pyridine)
UHPSFC/PDA-MS flower, leaf and CBC, CBN, CBG, PCA and PLS-DA 74
(80:20) extraction MP: CO2 as solvent and
hashish samples THCA-A, CBDA,
isopropanol: acetonitrile
CBGA
(80:20) with 1% water
SP: SB-C18
MP: 0.1% (v/v) of
extraction in 96% phytocannabinoid
UPLC-HRMS cannabis flowers aqueous solution PCA, PLS-DA 13
ethanol profile
of formic acid and
acetonitrile

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Table 1. Chromatographic methods for analyzing cannabinoids in cannabis extracts and medicinal products (cont.)

Stationary phase (SP) and

Method Matrix Sample preparation Analyte Chemometrics tool Reference
mobile phase (MP)
SP: C18
THC, THCA, CBD,
methanol: chloroform MP: acetonitrile and
UPLC-MS/MS cannabis oil and tea CBDA, CBN, CBG, N/A 89
(9:1) extraction acidified water (0.1%
CBC
formic acid)
ANOVA: analysis of variance; AQbD: analytical quality by design; CBL: cannabicyclol; CBLA: cannabicyclolic acid; CBD: cannabidiol; CBDA: cannabidiolic
acid; CBDV: cannabidivarin; CBDVA: cannabidivarinic acid; CBG: cannabigerol; CBGA: cannabigerolic acid; CBNA: cannabinolic acid; CBC: cannabichromene;
CBCA: cannabichromenic acid; CCD: central composite design; DA: discriminant analysis; DAD: diode array detector; DOE: design of experiments; ESI: electrospray
ionization; FID: flame ionization detector; HCA: hierarchical cluster analysis; HPLC: high-performance liquid chromatography; LC-QQQ-MS: liquid chromatography
triple quadrupole mass spectroscopy; LDA: linear discriminant analysis; MLR: multiple linear regression; MS: mass spectrometry; PCA: principal component analysis;
PFP: pentafluorophenyl; PLS: partial least squares; SVM: support vector machine; THC: delta-9-tetrahydrocannabinol; THCA-A: delta-9-tetrahydrocannabinolic
acid A; THCVA: tetrahydrocannabivarinic acid; THCV: tetrahydrocannabivarin; THV: tetrahydrocannabivarinic acid; UHPSFC: ultra-high-performance supercritical
fluid chromatography; UPLC: ultra-performance liquid chromatography; N/A: not applicable.

required are complex and expensive instruments, well- 3.2.1. Vibrational spectroscopy
trained technicians, use of hazardous chemicals, and long Analytical tools such as infrared (IR) and Raman
chemical analysis times, which represent major challenges spectroscopy techniques have become instrumental in
for implementing regulatory testing, making them harder monitoring attributes of quality within the pharmaceutical
to use for quality control within design and manufacturing industry. These techniques excel in rapidly screening
facilities.1,23,34,96 substantial sample volumes, offering a versatile, non-
These constraints have prompted the quest for quicker, destructive approach for qualitative and quantitative
more user-friendly alternatives to HPLC and GC. At the profiling, as well as identifying growth stages of cannabis
same time, the development of the hemp and medicinal plants and extracts.14 In recent years, there has been a
cannabis industry has presented a demand for fast, robust growing use of non-destructive vibrational spectroscopy
and cheap methods for large-scale testing, thus encouraging methods, such as infrared spectroscopy or Raman, for the
alternative analytical techniques to cover this need.17,97 analysis of marijuana.98
Mid-infrared spectroscopy (MIR, 4000-400 cm -1)
3.2. Alternative methods is a method used to analyze the molecular structure of
unknown compounds. By obtaining these patterns, it is
The number of publications on cannabinoid analyses possible to obtain information about the molecular structure
has grown considerably since 2004 (Figure 3a). With the by comparing spectra to identify specific fingerprints,
advancement of technology, the growing popularity of the which is useful in drug quality control. Furthermore, MIR
use of alternative methods is evident when comparing these can analyze complex mixtures such as cells and food,
with studies that employ classical methods for analysis, identifying small changes in the samples.99
as illustrated in Figure 3b. The techniques that make up Vibrational spectroscopy techniques combined with
these alternative methods will be discussed below, as well chemometric tools have become the favored technology
as their applications. for detecting and quantifying materials in the agricultural

Figure 3. Number of articles published per year on analysis of cannabinoids in cannabis extracts and cannabis-based products (a). Distinction of the types
of analysis method used (b).

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

sector.96 Specifically, near-infrared spectroscopy (NIR), a fast, to make accurate prediction of cannabinoids in cannabis
economical, versatile, robust, and sustainable tool, has been plants as raw material. Geskovski et al.42 demonstrated the
widely used in various fields for qualitative and quantitative potential of mid-infrared spectroscopy (MIR) as a process
analysis of key parameters such as proteins, fats, moisture, analytical technology (PAT) in the quantification of THC
ash, starch or sugar, raw materials related to the quality and CBD in extracts and flowers from different origins,
of agricultural products. In contrast to chromatographic considered critical quality parameters in plant production
methods, NIR offers numerous benefits, including and cannabis extracts. NIR has also been used to distinguish
streamlined sample preparation, swift data acquisition, legal and illegal industrial hemp. Su et al.25 developed
non-destructiveness, cost-effective instrumentation, and models for discriminant analysis that achieved an overall
reduced consumable usage. These advantages position accurate classification of 94%, with independent validation,
NIR as a viable alternative for the prompt analysis of hemp demonstrating an 84% correct classification for legal and
oil samples, enabling users to make swift and informed illegal industrial hemp.
adjustments during product quality control assessments.17,100 In addition to plant material, NIR has also been used
NIR employs the absorption of electromagnetic to quantify cannabinoids in hemp oil,58,99 commercial
radiation within the range of 780 to 2500 nm to create a products,41 and pharmaceuticals.103 Chen et al.100 presented
distinctive spectral fingerprint for each sample. NIR spectra an application of NIR spectroscopy combined with
associated with multivariate chemometrics techniques chemometrics to quantify concentrations of CBD and total
are used to build models to predict composition or for CBD (CBD and CBDA) in hemp oil samples examined
classification purposes.97 NIR has the capability to measure through a glass container. Grafinger et al.41 used Fourier
multiple chemical parameters simultaneously. When transform infrared (FTIR) to analyze CBD-rich e-liquids
coupled with multivariate analysis techniques, it has been sold in Switzerland. All samples complied with legal
applied for both qualitative and quantitative analyses of a requirements, with a THC content of less than 1%. On
wide range of plant materials. This feature makes NIR one the other hand, they observed a deviation in the quantified
of the most appealing non-destructive analytical tools in CBD content in relation to the labeled CBD content in half
the fields of agriculture and food industries.23 In reflectance of the analyzed samples. The manufacturer’s information
mode, NIR spectroscopy enables rapid scanning of cannabis on the label and packaging varied drastically in relation
to generate NIR spectra. These spectra can be employed for to the determined CBD content, ranging from 45.9 to
predicting cannabinoid content or distinguishing between 117.9%. Grekopoulos103 developed and validated a NIR
cultivars with distinct chemotype profiles.55 spectroscopic method for the release of pharmaceutical-
Studies that used NIR spectroscopy to analyze grade CBD-based liquid formulations. The method enabled
cannabinoids in industrial hemp samples have shown the direct measurement and rapid quantification of active
ability of the NIR technique to quantify cannabinoids pharmaceutical ingredients. In this study, NIR spectroscopy
quickly, sensitively, accurately, reliably and non- emerged as a pivotal choice for the quality control of
destructively.15,17,96,101,102 Additionally, NIR enables the pharmaceutical products derived from cannabis, attributed
simultaneous prediction of numerous constituents using to its versatility and efficacy.
the same spectral data.25 One of the advantages of the NIR technique is that
Jarén et al.17 identified the main bands of the NIR measurements in the field can be carried out using
spectra of hemp samples. The spectrum region examined portable devices.96,101,104 Risoluti et al.105,106 proposed
exhibited distinct absorption bands at approximately a method based on a miniaturized spectrophotometer,
1210, 1450, 1736, 1762, 1820, 1940, 2060, and 2090 nm, the MicroNIR On-Site, for monitoring cannabinoids in
which are indicative of proteins, lipids, water, and various hemp seed oil106 and monitoring the residual content of
compounds found in hemp, originating from OH, NH, cannabinoids in hemp flour.105 Deidda et al.104 evaluated
CH, and other bonds. Notably, the band at 1736 nm, 2 portable spectrophotometers (NIR-S-G1 and MicroNIR)
corresponding to aromatic hydrocarbons of terpenes, is for the quantitative analysis of THC in whole cannabis
linked to CBD content, as cannabinoids are classified as inflorescences and cannabis resins. Tran et al.55 performed
terpene-phenolic compounds. statistical analyses of inflorescences from 734 cannabis
Sample variability has also been studied with NIR and plants, using NIR and LC-MS data to create predictive
MIR. Gloerfelt et al.97 showed the effectiveness of NIR in models for 14 cannabinoids. Using different multivariate
quantifying cannabinoids in plant material from different analysis techniques, they compared two NIR instruments:
origins from at least 22 countries. The results highlight the Bruker MPA II (bench) and the MicroNIR (handheld).
the feasibility of NIR in conjunction with chemometrics The benchtop instrument showed better results; The

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

MicroNIR, however, yielded accurate predictions for the in plant material, such as chlorophyll and lipids. Aside
primary cannabinoid precursors, such as CBDA and THCA, from providing shorter analysis times when compared to
and was precise in the identification of other cannabinoids, traditional chromatographic methods, direct NMR analysis
including CBD, THC, CBNA, CBN, and CBCA. Similarly, circumvents potential alterations in sample composition
Duchateau et al.101 created 2 classification methods in and loss of analytes during sample preparation. Moreover,
accordance with European and Swiss laws107,108 using benchtop NMR enable simultaneous determination of multiple
NIR and a portable device on aerial parts of cannabis. These analytes. Despite these potential benefits, the infrequent
models were accurate by 91 and 95% for the set of tests utilization of NMR methods in cannabinoid determination
obtained with bench and portable devices, respectively. is attributed to elevated instrumental costs and the need for
Raman spectroscopy has also been used as a non- highly specialized personnel.9,88,109,110
invasive and non-destructive means of cannabinoid analysis Studies in the literature that have used NMR for
with the aim of differentiating cannabis plants according to cannabinoid analysis include various purposes, such as
type and gender before flowering. Ramos‑Guerrero et al.98 quality control of cannabis cultivars. Choi et al.111 carried
discriminated between different varieties of marijuana. By out metabolomic analysis of cannabis cultivars using NMR
comparing the Raman spectra of standard cannabinoids with and multivariate analysis. The authors analyzed flowers
five types of marijuana, they identified common spectral and leaves of the plants taking 12 min for each procedure.
bands useful for plant characterization. The OPLS‑DA The results showed that the cannabinoid content in the
(orthogonal partial least squares discriminant analysis) leaves was lower than in the flowers; the main factors
model developed provided a classification accuracy of that contributed to this differentiation were carbohydrates
100%, making it possible to distinguish different varieties and amino acids. NMR combined with chemometrics
of Sativa marijuana based on their Raman spectra. On the was also used for rapid screening and authentication of
other hand, Goff et al.3 explored the feasibility of Raman cannabis samples based on the chemical profile of plant
spectroscopy to differentiate between hermaphrodite, extracts,112 discrimination of marijuana seized according
male and female cannabis plants. They collected Raman to the capture period, using CBN as a marker,113 screening
spectra from these plants, discovering that the differences in for cannabinoids in CBD oils111 and studying the quality
biochemical profiles enabled this differentiation. Carotenoid of hemp seed oils during storage under different conditions
levels are notably higher in female plants, whereas male (2-8 and 30 °C) for 30 days.110
plants exhibit lower concentrations. Hermaphrodites have The performance of cannabinoid analysis by NMR has
lower carotenoid concentrations compared to male and been verified with the reference method, chromatography.
female plants. Using chemometrics, the authors achieved Brighenti et al.56 compared the efficiency of HPLC-
99.6% accurate differentiation between the chemotypes DAD and NMR analytical techniques for studying the
of cannabis. The portability and sensitivity of Raman main cannabinoids in different samples of cannabis
spectroscopy makes it possible to use it directly on cannabis inflorescences. In general, agreement was reported between
farms to monitor and control plant growth. Higgins et al.16 the two methods regarding quantitative cannabinoid data,
have also been successful in differentiating young male indicating their reliability in determining active compounds
and female hemp plants. The analysis identified, with 90 in cannabis extracts. This study underscores the potential
and 94% accuracy, male and female plants, respectively, of NMR as a promising tool for accurately determining
before flowering. The non-invasive and fast technique was cannabinoids in both plant material and derived products.
based on variations in the levels of lutein, an important
carotenoid, revealed by Raman spectra. This differentiation 3.2.3. Mass spectrometry
can be made non-destructively and accurately in a matter Mass spectrometry also stands out as an emerging
of seconds using a portable spectrometer, paving the way technique for the direct analysis of complex mixtures. It
to monitor and control hemp production directly on farms. facilitates the identification of compounds in extracts or
medicinal preparations by differentiating analytes by the
3.2.2. NMR spectroscopy mass/charge ratio (m/z). Modern spectrometers operate by
Nuclear magnetic resonance (NMR) spectroscopy ionizing the molecules of interest and separating the ions
presents a viable alternative to traditional chromatographic based on this ratio (m/z), enabling the detection, counting,
analysis for assessing cannabinoids. This technique is and characterization of atoms and molecules with different
fast, reliable and does not require special processing or compositions and sizes.114,115
sample preparation. Unlike LC and GC, this method’s Borille et al.116 combined the electrospray ionization (ESI)
primary benefit lies in its insensitivity to impurities found technique with Fourier transform ion cyclotron resonance

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

mass spectrometry (FT-ICR-MS) to analyze the chemical and extracts. The application of each of these methods is
profile of cannabis. The results obtained forecast the summarized below.
growth duration of the plants with an approximate Comeau et al.119 created a sensor utilizing organic
prediction error of one week (root mean square error of thin-film transistors (OTFT), comprising two organic
calibration‑RMSEC 0.34 week and root mean square semiconductor materials, CuPc and F16-CuPc, coated
error of prediction-RMSEP 1.01 week). The ESI FT-ICR with an alkaline thin film based on Fast blue BB (4-amino-
MS approach was efficient in detecting a wide range of 2,5-diethoxybenzanilide zinc diazotized double salt, or
cannabinoids, providing detailed and necessary information FBBB). This sensor is designed for precisely detecting and
about the characteristics and chemical profile of cannabis, characterizing the main cannabinoid components found in
without the need for prior information about the samples. The the cannabis plant. The profile and chemical compositions
ESI technique offers soft ionization, capturing ions directly (chemotyping) of cannabis extracts was also studied by an
from the sample solution into the gaseous environment ultraviolet microplate reader method.120
of the mass spectrometers. ESI was also used to classify Lu et al.23 used hyperspectral imaging technology to
cannabis samples according to their origin and composition, measure the levels of cannabinoids in industrial hemp floral
employing flow injection analysis (FIA-ESI-MS/MS) and and leaf materials. Using the wavelength range of 400 to
chemometric tools.6 1000 nm, they identified samples rich/poor in CBD and
Contreras et al.117 introduced an innovative method, legal/illegal in THC with 99 and 97% accuracy, respectively,
thermal desorption ion mobility spectrometry (TD-IMS), to using linear discriminant analysis. Full-spectrum PLS
analyze cannabinoids directly from cannabis plant extracts. models identified both CBD and THC satisfactorily in
This method offers fast spectral fingerprints. The TD-IMS floral tissues. Predictions for CBG and its acidic forms,
technique was combined with chemometric tools to identify however, were unsatisfactory; these were improved with
cannabis chemotypes by their chemical characteristics, parsimonious PLS models, employing a wavelength selection
using data pre-processing. With this approach, cannabis procedure to minimize collinearity of variables. This study
samples were successfully discriminated, grouped pointed out the potential of hyperspectral imaging for rapid
according to chemotypes, psychoactivity and cannabinoid quantification of cannabinoids in industrial hemp. On the
profiles. This technique allows for quick analysis (less other hand, Nicolas et al.121 used hyperspectral imaging to
than 2 min) and can be used on-site, making it especially classify cannabis chemotypes in several cultivars during
attractive for cannabis growers. the cultivation cycle of the plants. Due to the non-invasive
Dong et al.118 developed a method involving direct and non-destructive nature of the technique, the authors
thermal desorption analysis coupled with real-time mass were able to perform in situ measurements and classify
spectrometry (TD-DART-MS) for the classification of diverse cannabis plants into their corresponding chemotype based
cannabis hemp cultivars through multivariate analysis. The on the quantification of the cannabinoids present in them.
method proved to be simple and fast, without the need for This methodology was developed with the aim of ensuring
chromatography and solvent extraction. The PLS-DA model quality control throughout the cultivation process. This non-
obtained performed well in the classification rate (> 99%). destructive technique represents a promising alternative for
The findings of this study indicate that the TD‑DART-MS monitoring chemotype in cannabis crops.
method, when combined with chemometrics, was able to Birenboim et al.122 introduced a new method that
classify cannabis cultivars. Chambers et al.93 also reported combined fluorescence spectroscopy with parallel factor
promising results using direct real-time analysis coupled analysis modeling (PARAFAC) to identify and quantify
to high-resolution mass spectrometry (DART-HRMS) and key cannabinoids in fresh cannabis inflorescences. The
chemometrics to differentiate between hemp and marijuana five‑component PARAFAC model was successful in
plant materials. The model achieved an internal accuracy of predicting four acidic cannabinoids and one neutral
98% and 100% accuracy for external validation samples, cannabinoid. The identity of the cannabinoids was
showing its ability to differentiate accurately C. sativa plant confirmed by comparison with pure standards and by
materials. concentrations measured by HPLC. The study showed that
there is ample information in the fluorescent spectral region
3.2.4. Other analytical methods to construct prediction models for cannabinoids in cannabis
Analytical methods such as sensors based on organic extracts, demonstrating the viability of this approach as a
semiconductors, hyperspectral imaging, and fluorescence simple, economical and rapid alternative for cannabinoid
spectroscopy, combined with chemometric tools, have been analysis. Table 2 compiles the studies that have employed
used for the analysis of cannabinoids in cannabis plants alternative methods for cannabinoid analysis.

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Table 2. Alternative methods for analyzing cannabinoids in cannabis extracts and medicinal products

Method Matrix Sample preparation Reference method Analyte Chemometrics tools Reference
CBD, CBDA, THC,
CBD-rich e-liquids N/A UPLC-DAD N/A 41
THCA, CBN
cannabis flowers and
N/A HPLC-DAD THC, CBD PLS 42
FTIR extracts
THC, THCA, CBD,
drug-type and fiber-
N/A HPLC-MS/MS CBDA, CBG, PLS 102
type cannabis flowers
CBGA, CBN
hemp samples (plant THCA, THC, CBDA,
N/A UHPLC-MS/MS PLS 96
material) CBD
CBDV, Δ9-THCV,
cannabis leaves and CBD, CBC, Δ8-THC,
N/A GC-FID PCA, PLS 15
inflorescences Δ9- THC, CBG
and CBN
CBD-based liquid
formulations in
N/A HPLC-UV-Vis CBD PLS 103
different doses and
flavors
hemp seed oil and
N/A GC-MS THC, THCA, CBD PCA, PLS-DA, PLS 105, 106
hemp flour
aerial parts of
N/A GC-FID THC PLS-DA, SIMCA 101
cannabis
female inflorescences
NIR N/A UHPLC-UV THC PLS 104
of plants and resins
hemp oil N/A HPLC CBD, CBDA SOSVEN, PLS 100
Kompolti variety
N/A HPLC-DAD THC, CBD PCA, PLS 17
hemp
CBDA, THCA,
CBD, THC, CBC,
particle size
CBGA, CBG,
plant material reduction (ground HPLC-MS PCA, PLS, PLS-DA 55
CBDVA, CBDV
plant material)
THCV, THCVA,
CBN, CBNA CBCA
Δ9-THCA, Δ9-
THC, CBDA, CBD,
plant material from stacked ensemble,
CBGA, CBG,
different origins N/A HPLC-UV gradient boosting 97
THCVA, THCV,
(different countries) machine
CBDVA, CBDV,
CBN, CBC
UV-Vis dry industrial hemp physical and PCA, artificial neural
ethanol extraction N/A 123
NIR (plant material) chemical properties networks
whole and ground THC, CBD, CBN,
1
H NMR NIR N/A GC-FID PLS 25
hemp seeds CBG, CBC
plant material, four extraction in
THC, CBD, CBN,
different cannabis methanol/chloroform GC-FID N/A 34
THCA, CBDA
cultivars (9:1, v/v)
extraction in
leaves and flowers of methanol, water THC, CBD, CBN,
N/A PCA 111
1
H NMR 12 cannabis cultivars and chloroform THCA, CBDA
(25:25:50, v/v)
chemical profile of
cannabis plant buds extraction in CDCl3 N/A LDA, SVM 112
extracts
D8-THC, D9-THC, PCA, PLS-DA,
marijuana samples methanol extraction N/A 113
THCA, CBN, CBV OPLS-DA

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

Table 2. Alternative methods for analyzing cannabinoids in cannabis extracts and medicinal products (cont.)

Method Matrix Sample preparation Reference method Analyte Chemometrics tools Reference
cannabis CBDA, CBGA,
inflorescences of dilution in CDCl3 HPLC-DAD CBG, CBD, CBN, N/A 56
different chemotypes THC, THCA
1
H NMR CBD, THC, CBN,
hemp seed CBD oil dilution in CDCl3 N/A N/A 109
CBG, THCV
quality of oils due to
hemp seed oil dilution in CDCl3 N/A PCA, OPLS-DA 110
differences in spectra
hemp plant (plant THCA, CBDA,
material, leaves and N/A HPLC-UV CBGA, THC, CBD, PLS 23
Hyperspectral flowers) CBG
imaging
images were captured
cannabis plant HPLC-DAD THC, CBD PLS-DA, HPLS-DA 121
in situ
cannabis (plant
THCA, THC, CBDA,
material) of 4 sativa N/A N/A OPLS-DA 98
CBD, CBN
genetic varieties
Raman
cannabis plants N/A N/A biochemical profile PLS-DA 3
fresh hemp plant and
N/A HPLC-PDA biochemical profile PLS-DA 16
collected leaves
CBD, CBDV, THCV,
cannabis plant
CBC, D8-THC, D9-
of different ultrasound-assisted
TD-IMS N/A THC, CBG, CBN, PCA-LDA 117
chemotypes (female n-hexane extraction
THCA, CBDA,
inflorescence)
CBGA
flowers of hemp THC, THCA, THCV,
TD-DART-MS N/A HPLC-MS PCA, PLS-DA 118
chemotypes CBD, CBDA, CBDV
Ultraviolet cannabis extracts extraction and chemical profile of
N/A PLS-DA, SVM 120
microplate reader from various species dilution in CDCl3 extracts
extraction and
Thin-film organic
cannabis plant dilution in HPLC-DAD CBD, THC N/A 119
transistors
acetonitrile
ultrasound-assisted chemical profile of
ESI(±)-FT-ICR MS plant material N/A GA-PLS 116
acetonitrile extraction extracts
cannabis samples D8-THC, D9-THC,
(aerial parts and extraction in THCA, CBDA,
FIA-ESI-MS HPLC-DAD HCA, PCA, PLS-DA 124
pressed plant methanol CBD, CBN, CBGA,
material) CBG
cannabis THCA, CBDA,
Fluorescence
inflorescences of 16 dilution in ethanol HPLC-DAD CBGA, THC, CBD, PARAFAC 122
spectroscopy
different chemistries CBG, CBCA
hemp and marijuana
DART-HRMS N/A N/A biochemical profile random forest, PCA 93
plant material
The sample preparation listed in the table corresponds to the main method and not the reference method. CBD: cannabidiol; CBDA: cannabidiolic acid;
CBDV: cannabidivarin; CBDVA: cannabidivarinic acid; CBG: cannabigerol; CBGA: cannabigerolic acid; CBNA: cannabinolic acid; CBC: cannabichromene;
CBCA: cannabichromenic acid; DA: discriminant analysis; DAD: diode array detector; DART-HRMS: direct analysis in real-time- high resolution mass
spectrometry; ESI: electrospray ionization; FID: flame ionization detector; FT-ICR-MS: Fourier transform ion cyclotron resonance mass spectrometry;
FTIR: Fourier transform infrared; GA: genetic algorithm; GC: gas chromatography; HCA: hierarchical cluster analysis; HPLC: high-performance liquid
chromatography; IR: infrared; LDA: linear discriminant analysis; MS: mass spectrometry; NIR: near-infrared; NMR: nuclear magnetic resonance;
OPLS: orthogonal partial least squares; PARAFAC: parallel factor analysis; PCA: principal component analysis; PLS: partial least squares; SIMCA: soft
independent modeling of class analogy; SOSVEN: self-optimizing support vector elastic net; SVM: support vector machine; TD-DART-MS: thermal
desorption direct analysis in real time mass spectrometry; TD-IMS: thermal desorption ion mobility spectrometry; THC: delta-9-tetrahydrocannabinol;
THCA-A: delta-9-tetrahydrocannabinolic acid A; THCVA: tetrahydrocannabivarinic acid; THCV: tetrahydrocannabivarin; UPLC: ultra-performance liquid
chromatography; N/A: not applicable.

4. Chemometric Tools for Cannabinoid on the use of multivariate statistical analysis for the
Analysis qualitative and quantitative analysis of cannabinoids. This
was particularly evident in sub-section 3.2. “Alternative
Throughout section 3, several methods were reported Methods”, because of the technological advancement of

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

instrumental methods. This is due to the ability of modern analogy) and its variations, such as DD-SIMCA;131 linear
analytical techniques to provide high-dimensional data, discriminant analysis LDA, and PLS-DA. The first is
where many variables for each sample can be measured at the most popular one-class classification method, which
a high speed and by frequency. These alternative methods focuses on similarities between samples within the same
have increasingly required the use of tools that assist in category, as opposed to variations between categories.
the processing, evaluation and interpretation of data.125,126 LDA and PLS-DA are known as discriminant classification
It is in this context that chemometrics is important. methods, utilized to establish delineations between the
Chemometrics is often defined as an area of chemistry assessed classes.127
that employs mathematical and statistical methods for Among the applications of supervised methods for
the treatment of multivariate data obtained from chemical cannabinoid analysis, the most commonly used is PLS-DA
systems to extract the maximum amount of information with several purposes, including identification of hemp
from these systems, enhancing the range of understanding retailers based on the chemical content of cannabinoids in
about what the data means. Some of the notable multivariate hemp samples, 92% of samples were correctly classified;85
methods that are planning, supervised and unsupervised classification of medical cannabis samples based on
pattern recognition, and multivariate calibration, which is their cannabinoid and terpene profiles with a correct
one of the most used techniques.127 classification rate of 96 and 100% for the cross-validation
Unsupervised pattern recognition methods are used and prediction datasets respectively;65 classification of
for exploratory data analysis, to evaluate correlations various hemp cultivars with classification rate (> 99%);118
and similarities/differences between samples. Thus, no and classification of illegally seized samples.6
pre‑existing knowledge about the identity or groupings Cannabis sample classification models using the SIMCA
of these samples is considered. The main unsupervised algorithm have also been reported. Duchateau et al.101
techniques are: hierarchical cluster analysis (HCA) and formulated PLS-DA and SIMCA models for the
principal component analysis (PCA). PCA is one of the most classification of cannabis samples based on European and
popular techniques employed, generally in the first contact Swiss legislative standards,107,108 employing both benchtop
with the data. It recognizes relationships between variables and portable infrared devices. The SIMCA models
and between samples, identifies patterns and detects and demonstrated accuracy of 91 and 93% for the benchtop
interprets anomalous samples present in the data.128 and portable devices, respectively. The PLS-DA models
Applications of unsupervised methods for cannabinoid achieved accuracy of 91% and 95% for the benchtop and
analysis include discrimination, according to cannabinoid portable devices, respectively.
content of hemp seed powder products,87 plant materials,43,55 The main objective of multivariate calibration
hemp seed oil,106 hemp flours,105 cannabis cultivars,111 techniques is to establish a mathematical relationship
and even samples of seized marijuana. Leite et al.113 between the instrumental data acquired from a sample, such
used PCA to differentiate among the samples according as a spectra data (X matrix) as a reference value represented
to their collection period using CBN as the marker: the by a vector y. The application of these techniques involves
concentration of CBN in the marijuana samples exhibited two phases. In the first, called calibration, the data from
a direct correlation with the seizure time. This study the X matrix, as well as the reference values contained
confirmed the use of CBN as a biological marker for in the y vector, are used to build the calibration model.
estimating the age of the plant or medicinal product. In the second stage, called prediction or validation, this
Chambers et al.93 used PCA to differentiate plant materials model is used to quantify the property of interest in
from hemp and marijuana. The application of PCA to hemp unknown samples.129 Among the most popular multivariate
and marijuana data revealed a clear grouping, enabling calibration methods, the following stand out: multiple linear
their distinction. regression (MLR), principal component regression (PCR),
In supervised pattern recognition techniques, additional partial least squares regression (PLS) and support vector
information about the identity and measurement of the machine regression (SVMR).132-134 PLS is the most used in
samples is required to categorize them. A training set the quantification of cannabinoids.
with samples from known classes is then used to develop PLS has been used to quantify cannabinoids using,
classification models, which are later used to identify for the most part, vibrational spectroscopic methods,
unknown samples.129,130 Some of the main supervised relating the spectra to the concentration of cannabinoids
pattern recognition techniques used in analytical chemistry determined by chromatography. In this way, PLS models
are: independent and flexible modeling by class analogy, have been developed to quantify cannabinoids in hemp
such as SIMCA (soft independent modeling of class samples of the kompolti variety,13 in liquid formulations

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Herrera et al. Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review

based on pharmaceutical grade CBD,103 in hemp seed and lastly, Design of Experiment (Figure 4b). The use of
oil,106 in cannabis extracts,42 in hemp oils,100 and in plant a variety of algorithms has been reported, including PCA,
material.23,55,96 HCA, PLS-DA. PCA-LDA, SIMCA, MLR, GA-PLS,
Geskovski et al. 42 demonstrated the capacity PLS, PARAFAC, SVM, and neural networks. The most
of mid-infrared spectroscopy (MIR) as a process frequently algorithms used, however, were PCA, followed
analytical technology (PAT) for quantifying the primary by PLS-DA, PLS and HCA (Figure 4c).
phytocannabinoids (THC and CBD). The concentrations of
CBD and THC in the samples ranged from 0.38 to 39.2% 5. Conclusions
and from 18.93 to 86.99%, respectively. Multivariate
models were built to predict THC and CBD content in The growing use of cannabis for therapeutic purposes has
samples of extracts and flowers originating from various encouraged the production of medicinal formulations that
sources. The PLS models accurately predicted THC and range from pharmaceutical products with certified quality
CBD concentrations in both cannabis extract samples and to artisanal products without any type of standardization
decarboxylated cannabis flowers. For cannabis extract and quality control. This has led to the development of
samples, the RY (the correlation coefficient of the Y analytical methods to enable quality control, whether in
matrix) values were 0.95 for THC and 0.99 for CBD, with pharmaceutical, artisanal or derived products, and with
corresponding RMSEC values of 4.67 and 1.21%, and crops in the growth phase.
RMSEP values of 3.79 and 1.44%, respectively. In the Since 2004, the number of publications on cannabinoid
models predicting THC and CBD from decarboxylated analysis has grown considerably. The standard analytical
cannabis flowers, the RY values were 0.99 for both method for quantitative and qualitative analysis of
compounds, with RMSEC values of 0.43% for THC and cannabinoids has been and continues to be chromatography,
0.21% for CBD, and RMSEP values of 2.32% for THC and specifically liquid chromatography with DAD detector as
1.33% for CBD, indicating robust predictive performance. well as coupled with MS. This is mainly because liquid
Similarly, Lu et al.23 built quantitative PLS-based models chromatography allows direct analysis to determine the
that achieved prediction accuracies of RPD (ratio of original composition of cannabinoids in the plant. Unlike
prediction to deviation) 2.5 and R2 0.84 for CBD and THC GC, there is no breakdown of cannabinoids during analysis
in hemp flowers. and the process does not require derivatization. LC-MS
In the studies that analyzed previously described analysis offers several important advantages: it allows
cannabinoids qualitatively and quantitatively, 63% used accurate identification of compounds, improves sensitivity
multivariate analysis tools, especially in studies employing for detection and quantitation at low concentrations,
alternative analytical methods. Of the publications where and provides high selectivity. These characteristics
alternative analytical techniques were employed, 85% make chromatography coupled with MS a powerful and
used chemometrics. And in the case of classic methods, efficient tool in the analysis of cannabis products. There
43% used chemometric tools (Figure 4a). In the group are limitations, however, such as the high cost of the
of studies that used chemometrics, unsupervised pattern instruments and the need for users to master analytical
recognition was the most common multivariate method, methodologies and the functioning of the equipment.
followed by supervised pattern recognition, calibration Therefore, these systems are not widely available or

Figure 4. Prevalence of analytical techniques for cannabinoid analysis using chemometrics (a). Comparison of the most used multivariate methods (b).
The most frequently used algorithms (c).

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Analysis of Cannabinoids in Medicinal Cannabis Products: A Comprehensive Review Herrera et al.

routinely used, being mainly intended for scientific research holds a Master and PhD in Pharmaceutical Sciences from
and technological development. UFPE. She works on research and innovation projects
Due to the limitations of chromatographic techniques as developing analytical methods in high-performance liquid
a whole, there is an increasing search for alternative methods chromatography, differential scanning calorimetry,
that are faster and simpler to use. With the technological thermogravimetry, absorption spectroscopy in infrared and
advancement of instrumental methods and the widespread ultraviolet regions, and mass spectrometry. She coordinates
use of chemometrics techniques, a variety of methodologies the Medicinal Cannabis Research Group and is doing a
have been developed for cannabinoid analysis. These post-doctoral internship in Chemistry at UFPE, focusing
methodologies include the use of vibrational spectroscopy on the safe therapeutic use of Cannabis sativa L.
such as NIR and Raman; NMR; and other emerging
techniques such as electrospray ionization coupled to Ricardo S. Honorato has a degree in
mass spectrometry, thermal desorption ion mobility Industrial Chemistry from the Federal
spectrometry, sensors based on organic semiconductors, University of Paraíba (1991), a PhD in
direct analysis in real time coupled to mass spectrometry, Chemistry from the Federal University of
hyperspectral imaging and fluorescence spectroscopy. Most Pernambuco (1999), post-doctorate from
of the proposed methods employ chemometric tools that are USP - Agricultural Nuclear Energy Center
essential for the processing, evaluation, and interpretation (2000) and the Universitat de València (2011). Federal
of the data generated. Chemometric tools are also used in Criminal Expert - Federal Police Department in Pernambuco
the construction of models that enable classifying and/or since 2003. Has experience in the areas of Forensic
predicting the concentration of cannabinoids. Chemistry and Analytical Chemistry.

Acknowledgments Maria Fernanda Pimentel has Engineering

degree in 1985, a master’s degree in
The authors would like to acknowledge Instituto Chemistry in 1992 and a PhD in Chemistry
Nacional de Tecnologias Analíticas Avançadas - INCTAA in 1996 from the Federal University of
(CNPq grants 573894/2008-6 and 465768/2014-8 and Pernambuco (UFPE). She is a full
FAPESP grants 2008/57808-1 and 2014/50951-4), CNPq professor at the Department of Chemical
(grants 408615/2023-0, 441493/2023-8 and 305127/2021- Engineering (UFPE), coordinates the Development and
7). The English text of this paper has been revised by Sidney Quality division of the Fuel Laboratory (LAC) and
Pratt, Canadian, MAT (The Johns Hopkins University), President-director of Pernambuco State Science and
RSAdip - TESL (Cambridge University). Technology Foundation (FACEPE). Her research experience
includes analytical chemistry/chemometrics with focus in
Julieth G. Herrera holds a degree in multivariate analysis and calibration, design of experiments,
chemistry (2018) from the Universidad del infrared spectroscopy, forensic chemistry, quality control
Atlántico (UA) in Colombia, and a of fuels and process analytical technology.
master’s degree in Analytical Chemistry
(2022) from the Federal University of References
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