Accepted Manuscript

Title: Pharmaceutical and biomedical analysis of cannabis

extracts: a critical review

Authors: Cinzia Citti, Daniela Braghiroli, Maria Angela

Vandelli, Giuseppe Cannazza

PII: S0731-7085(17)31189-5
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2017.06.003
Reference: PBA 11308

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 10-5-2017

Revised date: 1-6-2017
Accepted date: 2-6-2017

Please cite this article as: Cinzia Citti, Daniela Braghiroli, Maria Angela
Vandelli, Giuseppe Cannazza, Pharmaceutical and biomedical analysis of
cannabis extracts: a critical review, Journal of Pharmaceutical and Biomedical
Analysishttp://dx.doi.org/10.1016/j.jpba.2017.06.003

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 Pharmaceutical and biomedical analysis of cannabis extracts: a critical review

Cinzia Cittia,b, Daniela Braghirolic, Maria Angela Vandellic, Giuseppe Cannazzab,c,1

a
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via per Monteroni,
73100 Lecce, Italy.
b
CNR NANOTEC, Campus Ecoteckne dell’Università del Salento, Via per Monteroni, 73100 Lecce, Italy.
c
Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 103, 41125 Modena,
Italy.

Highlights

 Sample preparation strategies for the extraction of cannabinoids are described for plant and
biological matrices
 Techniques for cannabinoids analysis are described with advantages and drawbacks
 Chromatographic methods are compared in terms of selectivity and sensitivity
 Detection methods are presented based on the specific aim of the cannabinoids analysis

Abstract
Cannabis products have recently regained much attention due to the high pharmacological potential
of their cannabinoid content. In this review, the most widely used sample preparation strategies for
the extraction of cannabinoids are described for the specific application to either plant materials or
biological matrices. Several analytical techniques are described pointing out their respective
advantages and drawbacks. In particular, chromatographic methods, such as TLC, GC and HPLC,
are discussed and compared in terms of selectivity and sensitivity. Various detection methods are also
presented based on the specific aim of the cannabinoids analysis. Lastly, critical considerations are
mentioned with the aim to deliver useful suggestions for the selection of the optimal and most suitable
method of analysis of cannabinoids in either biomedical or cannabis derived samples.
Keywords: cannabis, cannabinoids, liquid chromatography, gas chromatography.
Abbreviations: AcOH, acetic acid; ACN, acetonitrile; APCI, atmospheric pressure chemical
ionization; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CBCA, cannabichromenic acid; CBC,
cannabichromene; CBDA, cannabidiolic acid; CBD, cannabidiol; CBDVA, cannabidivarinic acid;
CBDV, cannabidivarin; CBE, cannabielsoin; CBGA, cannabigerolic acid; CBGAM, CBGA
monomethyl ether; CBG, cannabigerol; CBGM, CBG monomethyl ether; CBGV, cannabigerovarin;
CBLA, cannabicyclolic acid; CBL, cannabicyclol; CBLV, cannabicyclolvarin; CBNA, cannabinolic
acid; CBN, cannabinol; CBT, cannabitriol; CBV, cannabivarin; CFL-A, cannaflavin A; CFL-B,
cannaflavin B; CHCl3, chloroform; Chex: cyclohexane; CL, chemiluminescence; CPE, cloud point
extraction; DAD, diode array detector; DCM, dichloromethane; EI, electron impact; ESI, electrospray
ionization; EtOAc, ethyl acetate; EtOH, ethanol; Et2O, diethyl ether; FA, formic acid; FID, flame
ionization detector; FLD, fluorescence detector; FT-IR, Fourier Transform infrared spectroscopy;

1
Corresponding author: Giuseppe Cannazza. Tel: +39 059 2055013. Fax +39 059 2055750. E-mail address:
[email protected]
FUSE, focused ultrasound extraction; GC, gas chromatography; hex, hexane; HFIP,
hexafluoroisopropanol; HILIC, hydrophilic interaction LC; HPLC, high performance LC; HPTLC,
high performance TLC; HS-SPME, headspace solid phase microextraction; iPrOH, isopropanol; IT,
ion trap; LC, liquid chromatography; LLE, liquid-liquid extraction; LOD, limit of detection; LOQ,
limit of quantification; MeOH, methanol; MEPS, microdialysis-extraction packed sorbent; MS, mass
spectrometry; MSTFA, N-methyl-(trimethylsilyl) trifluoroacetamide; NCI, negative chemical
ionization; NH4OH, ammonium hydroxide; NMR, nuclear magnetic resonance; 1H NMR, proton
NMR; OF, oral fluids; o.n., overnight; PFPA, pentafluoropropionic anhydride; PFPOH,
pentafluoropropanol; PLE, pressurized liquid extraction; QqQ, triple quadrupole; Q-ToF,
quadrupole-time of flight; RP, reverse phase; r.t., room temperature; SFE, supercritical fluid
extraction; SLE, solid-liquid extraction; SPE, solid phase extraction; TFAA, trifluoroacetic
anhydride; THCA, tetrehydrocannabinolic acid; THC (or Δ9-THC), tetrahydrocannabinol; THC-
COOH, 11-nor-9-carboxy-THC; THC-COOH-gluc, THC-COOH-glucuronide; THC-gluc, THC-
glucuronide; THC-OH, 11-hydroxy-THC; THCVA, tetrahydrocannabidivarinic acid; THCV,
tetrahydrocannabidivarin; TLC, thin layer chromatography; TMCS, trimethylchlorosilane; UPLC,
ultra-performance LC; UV, ultraviolet.
1. Introduction
Cannabis sativa L. can be considered as the most controversial plant in our society [1]. It is the most
widespread drug of abuse due to its intoxicating effects resulting from the psychotropic activity of
the best known component (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC or simply THC) [2-6]. At the
same time, cannabis has been known for centuries all over the world for its undeniable medicinal
properties. Nowadays its applications in the clinical world span from multiple sclerosis to epilepsy,
neuropathic pain, arthritis, nausea and vomiting due to chemotherapy, appetite stimulation in
HIV/AIDS, depression, anxiety disorders, sleep disorders, psychosis, glaucoma, and Tourette
syndrome [7-11]. The interest in the chemistry and pharmacology of this annual dioecious plant
belonging to the family of Cannabaceae is continuously increasing after the discovery of a unique
group of terpenophenolic compounds named phytocannabinoids. At least 90 cannabinoids have been
isolated from cannabis and characterized since the early 1940s [2]. An important breakthrough was
made in 1964 when Raphael Mechoulam isolated and characterized for the first time the main
psychoactive component of cannabis, THC [12,13]. Whether cannabis is intended either as a source
of fibers and/or seed production or for therapeutic purposes depends on the ratio between THC and
cannabidiol (CBD), which is known to possess several pharmacological properties but not the
psychotropic one of THC. In particular, analgesic, antioxidant and antiepileptic activities have been
attributed to this compound, which seems also to reduce THC side effects [14-16]. Although CBD
and THC have such relevance when talking about cannabis, these molecules are not biosynthesised
in the plant, which instead produces cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid
(THCA). A chemical reaction triggered by heat leads to the decarboxylation of these compounds to
get the corresponding decarboxylated (or neutral) species CBD and THC. The latter are the bioactive
components, whereas still very little is known about the activity of the two acid forms.[17-20] CBDA
and THCA are the major components of cannabis inflorescence. Other minor cannabinoids are
cannabichromenic acid (CBCA), cannabigerolic acid (CBGA), the “stem cell” of the other
cannabinoid acids, and cannabinolic acid (CBNA). All these compounds upon decarboxylation lead
to the neutral derivatives, respectively cannabichromene (CBC), with anti-inflammatory,
antibacterial and antifungal activity, cannabigerol (CBG) with analgesic, antibacterial and antifungal
activity, and cannabinol (CBN), which derives from the oxidation of THC as a result of prolonged
storage and has potent sedative properties [21]. A schematic representation of the biosynthetic route
of THCA and CBDA, their conversion respectively into THC and CBD and the oxidation of THC to
CBN is reported in Figure 1.
Beyond cannabinoids, a substantial part of the about 500 compounds present in this complex matrix
is represented by other types of molecules, such as terpenes, flavonoids, stilbenoids, amino acids,
fatty acids, alkaloids, hydrocarbons, carbohydrates, and phenols [22]. Terpenes represent the volatile
component of the plant and were proved to have a synergic action with cannabinoids [21,23].
As a consequence of the increase in the development of medicinal cannabis preparations, there is an
increasing demand of the development of qualitative and quantitative methods for the analysis of the
bioactive components of cannabis. As a general rule, the analytical method employed for the
determination of cannabinoids needs to match the application required. Analysis of plant material is
generally performed for the determination of the type of cannabis (fiber or drug type), for the quality
control of the material used for medicinal purposes, or for biosynthetic studies within
biotechnological research [24]. Conversely, analysis of biological matrices, such as urine, blood, hair,
etc., are mainly necessary to provide evidence of drug abuse or for pharmacokinetics studies [24].
Different aims claim different techniques to be used for both sample preparation and analysis. The
literature on the determination of THC and its six main metabolites in human body materials until
2002 has been fully covered in the review by Raharjo and Verpoorte [24], whereas Battista et al.
evaluated the analytical approaches for the determination of THC and the endocannabinoids
anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol in several human matrices
[25]. This review focuses on the analytical methods employed to analyse both plant materials and
biological matrices with respect to both cannabinoid content and other bioactive substances contained
in cannabis. The review mainly relates the progress that has been done in the past fifteen years (2002-
2016) in the sample preparation and analytical techniques employed.2

2. Experimental techniques
2.1 Extraction methodologies and sample preparation
1.1.1 Plant material
Figure 2 shows the molecular structure of the most common cannabinoids and cannabis flavones. In
plant material, which commonly corresponds to the plant inflorescence, the most widely employed
method of extraction is the solid-liquid extraction (SLE), which involves the use of an appropriate
solvent with great affinity for cannabinoids. In order to obtain a selective extraction of either
cannabinoid acids or neutral cannabinoids, it is necessary to undertake a different extraction
procedure. Acid and neutral cannabinoids can be extracted using common organic solvents or a
mixture of more solvents. The most common solvent is ethanol (EtOH) since it has a high extracting
efficiency due to its high affinity for cannabinoid molecular structure [2,26,27]. Indeed, a method of
extraction with EtOH 96% (v/v) has been recently proposed on a draft of Cannabis Flos monograph
of the German Pharmacopoeia [28].

2
The literature references discussed and listed herein were found in scopus and web of science databases by searching
for keywords related to: cannabis analysis, cannabinoids analysis, cannabinoids extraction, cannabis and gas
chromatography, cannabis and liquid chromatography, LC-MS of cannabinoids, etc. Moreover, the research was limited
to the time interval 2002-2016.
Methanol (MeOH), and ethyl acetate (EtOAc) are also widely employed alone or in combination with
other solvents (for example MeOH:CHCl3 9:1 (v/v)) for both chemotype distinction [29,30] and
quality control purposes [31,32].
Another solvent which has high lipophilicity that is employed for the extraction of cannabinoids for
quality control purposes is hexane [32], or hexane:iPrOH 9:1 (v/v) for chemotaxonomic analysis [33].
Hexane has also been used by Mariotti et al. in order to classify the plant material on the basis of its
THC content, which can be considered a marker of the plant age [6]. Peschel and Politi explored
different extraction solvents and combination for chemotype distinction [34]. They proposed a
complex procedure involving an extraction with ethyl acetate (EtOAc) and ethanol (EtOH) 40% (v/v)
and a parallel defatting with heptane and exhaustive extractions with methanol (MeOH) 70% (v/v).
Each extract underwent a fractionation between water and an organic solvent in a liquid-liquid system
in several steps, first with dichloromethane (DCM), and secondly with EtOAc. EtOAc extracts were
fractionated into a hexane, and an aqueous (8% MeOH (v/v)) fraction [34]. The authors also included
in the extracts profiling the characteristic cannabis derived prenylated flavones cannaflavin A (CFL-
A) and B (CFL-B).
When only cannabinoid acids are the target of analysis, it is necessary to perform the extraction at
room temperature, which ensures no conversion of the actual cannabinoids composition of the plant
material. Conversely, in order to make cannabis extract for medicinal use it is important to ensure the
presence of active principles represented by neutral cannabinoids. To this end, it is necessary to
perform the extraction at high temperature or pass through a preliminary decarboxylation step, which
can be carried out in the presence (in water at 100 °C for 2 hours) or absence of a solvent [26,35].
The decarboxylation is a critical step because it does not provide the conversion of cannabinoid acids
into equivalent amounts of neutral cannabinoids when it is conducted in an open reactor. The
temperature is a key parameter that dramatically affects the conversion process. In order to get the
total consumption of cannabinoid acids, it is necessary to heat the sample at a temperature that causes
the evaporation and/or decomposition of the neutral cannabinoids [24]. A closed reactor, high
temperatures and short time would certainly prevent these side events [24]. A very common hot
extraction that can be employed for different purposes like taxonomical species identification,
forensic classification and source tracing, consists of the use of a soxhlet apparatus, which however
is solvent and time consuming (more than two extraction cycles are generally required) [29,32].
An interesting alternative method for quality control analysis has been proposed by Ameur et al.,
which consists of a cloud point extraction (CPE) of THC from cannabis resin [36]. This method
involves the use of a non-ionic surfactant (Dowfax 20B102) mixed with cannabis resin, a salt
(Na2SO4) and deionized water. The mixture is shaken and heated at an appropriate temperature (40-
90 °C). A separation of two phases, aqueous and surfactant-rich phase, is reached upon heating (45
°C), addition of the salt and centrifugation. In this way, a 60% extraction yield was achieved for THC
within one hour (it did not increase over time). The authors suggest that CPE can be a good alternative
to other traditional processes and offers many interesting advantages, such as the possibility of
extracting and pre-concentrating analytes in a simple single-step procedure, without the use of
expensive and potentially toxic organic solvents. Moreover, it does not require the evaporation of the
solvent and does not cause any analyte loss, and the extract is compatible with the mobile phase used
in reverse phase HPLC.
Supercritical fluid extraction (SFE) is a very efficient way to extract cannabinoids and terpenes from
cannabis inflorescence. Supercritical CO2 is the solvent used to extract the terpene component, while
cannabinoids are extracted by means of a co-solvent, usually ethanol (10-20% in CO2) [5,37]. The
parameters involved in this process are temperature and pressure, which require a fine tune in order
to obtain a high extraction efficiency of all compounds. SFE is a useful technique for preserving the
stability of thermos-labile and light-sensitive compounds and is scalable up to industrial size [5].
Moreover, Omar et al. ensure an extraction yield of cannabinoids up to 90% with EtOH as co-solvent
(less than 40% with only CO2) [37]. SFE is generally employed to separate the aromatic part of
cannabis (terpenes), which is cannabinoid free, from the pharmacologically active fraction, which is
cannabinoid rich. The latter can be used for cannabis varieties distinction which are to be correlated
to the therapeutic effects.
When dealing with medicinal cannabis, common toxic organic solvents are to be avoided. Cannabis
tea is indeed a popular preparation to consume medicinal cannabis [38]. However, it is reasonable
that the amount of cannabinoids is quite low due their scarce solubility even in hot water [35,39].
Oily preparations are becoming also very popular but there is still the need for a standardized
extraction protocol [39,40]. Only recently, a research work has been published by our research group
regarding the extraction procedure and the analysis of cannabinoids from medicinal cannabis
inflorescence [41]. The extraction procedure proposed involves the heat of the cannabis inflorescence
at 110 °C in olive oil and at 78 °C in ethyl alcohol. All the vapours produced are cooled down with a
condenser and refluxed into the stirring mix. In this way, it is possible to preserve the terpene
component. The results indicated that the procedure is quite efficient due to the reflux and the
cannabinoids extraction yield is close to 100% in ethyl alcohol in less than one hour and 70% in olive
oil in about two hours [41].

2.1.1 Biological matrices

A recent review regarding the analytical approaches used for the determination of phytocannabinoids
and endocannabinoids in human matrices has been published by Battista et al. [25]. The most
commonly analysed human samples for the detection of cannabinoids are blood (whole blood, serum
and plasma), urine and hair. The aim of the analysis in the case of human matrices is generally to
prove the precedent or present consumption of illicit drugs. It is important to take into account the
time elapsed since the last use, because not all cannabinoids can be detected after a certain time. For
example, after 10-12 hours from cannabis consumption THC can no longer be detected [24]. The
compounds generally analysed in blood and urine are 11-hydroxy-Δ9-THC (THC-OH) and 11-nor-9-
carboxy-Δ9-THC (THC-COOH), which are the major metabolites of THC. THC-COOH undergoes
glucuronidation and can be found in urine as the most abundant metabolite (in both conjugated, THC-
COOH-gluc, and non-conjugated form). CBD and CBN are generally not suitable analytes for
cannabis consumption proof in human matrices [25]. The only example of the detection of these two
phytocannabinoids was reported by Milman et al. in 2012 suggesting that CBD and CBN positive
samples indicate unambiguously recent cannabis consumption [42]. The main THC metabolites are
reported in Figure 3.
The extraction step of cannabinoids in biological matrices is very critical since it needs to be highly
reproducible, highly efficient (in terms of analyte recovery), highly selective and to eliminate as much
interference as possible deriving from the matrix.
Detection of phytocannabinoids and their metabolites in human matrices always requires a step of
deproteination. It can be achieved by treatment with either formic acid in methanol or ice-cold
acetonitrile, or enzymatically [42-53].
The deproteinized samples can then be extracted following two different procedures: liquid-liquid
extraction (LLE) or solid phase extraction (SPE). LLE is usually time-consuming and makes the
quantification of cannabinoids very difficult in samples where their level is generally very low, while
SPE seems to be a more common technique for biological matrices [3,25]. With SPE the target
compounds are absorbed onto a stationary solid phase material, allowing a pre-concentration of the
analytes before analysis [51]. Among the advantages of SPE there are high reproducibility, easy
automation of the technique and less solvent waste compared to LLE [25,54,55]. The solvents used
to elute cannabinoids from SPE are similar to those generally employed in LLE: MeOH, EtOH,
hexane and EtOAc, in some case with the addition of acetic acid depending on the nature of the SPE
stationary phase. More recently, a μ-SPE clean-up procedure has been proposed by Sergi et al., which
involves minimal volumes of organic solvents and only 100 μL of plasma [47]. The difference with
the classical SPE consists of a packed sorbent in a pipette tip that does not require vacuum neither for
loading or elution. This technique dramatically reduces the time needed for the extraction.
A particular biological matrix that requires a long procedure due to its complexity is hair. The sample
preparation involves a wash procedure to eliminate any possible external contamination and a
digestion step to liberate the analytes from the matrix [53,56]. Finally, a pre-concentration step
generally with SPE has to be included in most cases or, alternatively, a headspace solid phase
microextraction (HS-SPME) can be applied in order to extract the analytes of interest [57,58]. The
latter consists of dipping a fiber material directly into the digested solution to let the analytes absorb
onto it [58].

2.2 Analytical techniques

3.1.1 Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) presents some advantages compared to other more sophisticated
technologies. In particular, it is largely employed for a preliminary semi-quantitative analysis of the
cannabinoid content of plant extracts [26]. The aforementioned draft of the Cannabis Flos monograph
reports a TLC based method for the qualitative determination of the main cannabinoids in the plant
inflorescence (see paragraph 2.1.1) [28]. Hazekamp et al. developed and validated a simple rapid
high performance TLC (HPTLC) method for the quantification of THC, which was proved to be
accurate and reproducible. Moreover, it allowed for the qualitative analysis of other main neutral
cannabinoids found in cannabis [26]. The identification of cannabinoids is generally based on the
comparison of the retention factor (RF) value with that of authentic standards, whereas the visual
evaluation is obtained by dipping the TLC plate into aqueous fast blue B solution (FBB), which is a
selective stain for cannabinoids [26]. In addition, this method can be applied to both polar and non-
polar C18 silica gel plates, which provide opposite elution orders. Nonetheless, TLC has some
limitations in terms of specificity and sensitivity, which result fairly low compared to other analytical
platforms and thus the results must be taken with caution.

4.1.1 Gas Chromatography (GC)

Gas chromatography (GC) is one of the most commonly used approaches for the analysis of
cannabinoids in both plant materials and biological matrices [33,59,60]. However, this analytical
platform does not allow for the direct analysis of the extracted sample because it involves the heating
of the sample at high temperature (about 280 °C) prior to the chromatographic separation in order to
transform the liquid sample into its gaseous phase. The heating of the sample leads unavoidably to
the decarboxylation of the cannabinoids acids to get the corresponding neutral cannabinoids.
Therefore, the result is the sum of acid and neutral form. In order to avoid this phenomenon a
preliminary step of derivatization of the cannabinoid acids is necessary and the distinction between
acid and neutral form is possible. However, it is important to take into account that a 100% yield for
the derivatization of cannabinoid acids by GC is difficult to obtain [61]. Moreover, it has been
demonstrated that the thermal conversion of the cannabinoid acids into their neutral derivatives in the
GC injection port is only partial yielding an underestimation of the total amount of cannabinoids [62].
In fact, the authors suggest the exact total cannabinoids value should be measured by determining
acid and neutral form separately [62]. Nevertheless, GC is the method officially employed by the
authorities for the determination of cannabinoids.
GC is generally interfaced to a flame ionization detector (FID) or to a mass spectrometry (MS)
detector. The advantage of FID consists of a more accurate quantitative response with respect to MS.
In fact, whilst the former bases the quantification of cannabinoids on the use of authentic standards,
the latter needs the use of the corresponding deuterated standards, which are expensive and not
commercially available for all minor cannabinoids. Anyway, MS allows for a higher specificity
compared to FID when similar species co-elute. The sensitivity of GC-FID is also remarkably lower
than that of GC-MS. Specifically, the sensitivity of GC-FID is only slightly below 1 μg/mL [2,27,63],
whereas it is possible to reach values in the order of or even below 1 ng/mL with GC-MS [33,64,65].
For hair samples, along with LC-ESI-MS, GC-MS with electron impact ionization (GC-EI-MS) is
the most employed technique for the analysis of THC and its metabolite THC-COOH. However, the
level of the latter are lower than that of its parent drug because of the weak incorporation of the acidic
metabolite into the hair matrix. Since it is important to determine this metabolite in cases when
discrimination of the external contamination from cannabis consumption is required, the
aforementioned technique is not suitable. Given that the cut-off concentration for THC-COOH is 0.2
pg/mg, it is possible to overcome the limitation of GC-MS by using a negative chemical ionization
(GC-NCI-MS) with triple quadrupole (QqQ) MS/MS that increases the limit of quantification (LOQ)
to 0.05 pg/mg [66,67].
Some authors have suggested that one-dimension GC does not offer enough resolution in the
separation of cannabinoids, which actually present very similar chemical structures [5]. Hence,
hyphenated techniques, such as two-dimension GC (GC×GC), are much more efficient in the
evaluation of the chemical profile of cannabis samples [5,68]. One example was published by Lowe
et al. in 2007, where a two-dimensional (2D) gas chromatography/electron impact-mass spectrometry
(GC/EI-MS) method was developed and validated for simultaneous quantification of THC, THC-OH
and THC-COOH in human plasma [69]. The method employs 2D capillary GC (in series) and
cryofocusing for enhanced resolution and sensitivity [69]. Limits of quantification (LOQ) reached
with this technique were 0.125, 0.250 and 0.125 ng/mL for THC, THC-OH, and THC-COOH,
respectively [69].

5.1.1 High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography coupled to mass spectrometry (HPLC-MS) has been
recently become the method of choice for the qualitative and quantitative determination of
cannabinoids in both plant materials and biological fluids [46,70-79]. In contrast to GC, LC based
techniques do not encounter decomposition of the sample as they work at room temperature allowing
the direct analysis of cannabinoid acids in the extracted sample [61].
The most commonly used columns are based on reverse phase (RP) C18 stationary phases, although
hydrophilic interaction liquid chromatography (HILIC) stationary phases have also been employed
[80]. In our laboratory, we have tested different stationary phases like amino, cyano, and different
C18 columns (unpublished results). Our results confirmed what reported in the literature on fused core
C18 columns (e.g. Poroshell C18), which have a high resolution power [27,41,81-83]. This aspect is
remarkably important in the case of analytes extracted from cannabis due to the presence of numerous
co-eluting cannabinoids. In particular, it is difficult to obtain a baseline resolution for Δ9-THC and
Δ8-THC, for CBDA and CBGA, and for CBD and CBG [1,27]. Several methods have been recently
developed with UPLC columns, which have a sub-2 μm diameter of the particles [27,67,84-95]. The
great advantage in the use of such columns lies in fast analyses and high separation efficiency.
Ultimately, the stationary phases that have proved to provide an optimal separation of the main
cannabinoids in both plant materials and biological fluids are RP C18.
It is noteworthy to point out that phytocannabinoids are optically pure in the plant. Anyway, very few
works report the chiral separation of cannabinoids [96]. Hence, it is of utmost important for the
scientific community and for the ultimate users of cannabis products to investigate the stereostabilty
of cannabinoids not only in the solvents used for the extraction but also in the biological fluids after
in vivo administration since they are known to undergo a series of metabolic transformations. There
are many research works regarding the rapid inversion of configuration of compounds favoured by
both solvents [97-104] and biological fluids [105][106]. Indeed, it is known that enantiomers
generally possess different pharmacological activities [101,106]. In the particular case of
cannabinoids, it has been demonstrated that (+)-CBD, which is the non-natural cannabinoid, has an
affinity with CB1 receptors similar to that of THC. On the other hand, the natural enantiomer, ()-
CBD, has shown no preferential affinity for either CB1 or CB2 receptors [107]. An example of chiral
column with the ability of offering an optimal separation of the enantiomers of the main cannabinoids
is based on the amylose tris(3,5-dimethylphenylcarbamate) stationary phase [96,108].
A considerable improvement in the separation power can be achieved using 2D chromatography [29].
The technique involves the combination of two dimensions of different separation mechanisms in
series. The whole eluate (comprehensive 2D chromatography) or selected fractions (“heart-cut” 2D-
chromatography) from the first dimension are collected and injected into the second dimension, where
they are further separated by an orthogonal separation mechanism [109]. This analytical trick is
particularly useful when chromatographic resolution of numerous compounds is desired, especially
for cannabinoids, many of which are isomers difficult to separate by only one separation mechanism
[29].
The separation of the main cannabinoids by HPLC is not a trivial task, especially with an isocratic
elution. In fact, most papers report their separation by gradient elution
[1,5,27,31,34,44,47,50,51,60,69-71,73,74,77,78,110-112], and only very few works describe
isocratic elution methods maintaining a good resolution for the main cannabinoids [41,63,75,76]. It
is interesting to note that the relative elution time of the acidic cannabinoids can be influenced by the
pH of the eluent, while the order of elution for the neutral cannabinoids remains the same on RP C18
columns [61].
With HPLC different types of detectors can be employed, such as ultraviolet (UV), fluorescence
(FLD) and mass spectrometry (MS). UV detection is the most used for the analysis of cannabinoids
in plant materials, where the amount of the main cannabinoids is relatively high
[1,27,29,34,36,60,63]. Only few scientific works report the use of this detector for the analysis of
biological samples as they require more sensitive detectors like MS due to the low level of the analytes
of interest [3,113]. UV detection is based on the absorption of the chromophore of the substituted
phenolic ring, as this is a common structural element among the tested cannabinoids. The alkyl
sidechain does not influence the UV absorbance, as there is no difference between THCA (C5-
sidechain) and THCVA (C3-sidechain). The cyclization of the non-phenolic part of the cannabinoids
also has no influence on the absorbance, except when it implies the formation of another aromatic
ring (CBN and CBNA) or a conjugated double bond (CBC and CBCA). The draft of the Cannabis
Flos monograph of the German Pharmacopoeia describes a LC-UV method for the purity test of the
main cannabinoids, CBDA, Δ9-THCA, CBD, Δ9-THC, Δ8-THC and CBN [28]. Although UV
spectrophotometer is the most widely employed detector, it presents some drawback related to the
scarce sensitivity and specificity. This is why it is scarcely used for the qualitative and quantitative
determination of cannabinoids in biological fluids. However, a recent study has reported a SPE
method for the pre-concentration of the sample to the UV level of sensitivity [3]. The low specificity
could be overcome by the use of a photodiode array detector (PDA) since the cannabinoid acids
present an absorption spectrum different from that of neutral cannabinoids. Specifically, the
wavelength used for neutral cannabinoids is about 220 nm, while cannabinoid acids also show
absorption peaks at about 270 and 310 nm [41,61]. Anyway, by setting the UV response at 228 nm it
is possible to detect both acid and neutral cannabinoids. The UV detector, however, does not allow
to discriminate neutral cannabinoids like CBG and CBD, which result very difficult to separate. In
this case, MS provides a higher level of detection since it distinguishes the various cannabinoids
depending on the m/z of their molecular ion. Given that CBG and CBD have different m/z, they could
be identified by this method. This is not true for isomers of cannabinoids like Δ8-THC and Δ9-THC,
which cannot resolved even by MS detection. In fact, the performance of MS depends on the type of
mass analyzer used in the analysis. In the case of isomers, a high-resolution fragmentation spectrum
could help in the identification on the account of the fragments generated [5]. In this respect, time-of
flight analyzers (ToF) are often used in order to obtain the structural information of the target
compounds. Coupled to a quadrupole mass filter, Q-ToF mass spectrometers provide accurate mass
identification (<5 ppm accepted accuracy threshold for confirmation of elemental composition) for
both the precursor and the product ions. This allows differentiating between two different compounds
with the same nominal mass but with different elemental composition [5]. One of our ongoing studies
has highlighted the presence of five cannabinoids with the same m/z 315.2294 (Δppm<5), identical
to that of THC and CBD (unpublished results) in Bediol® oil and EtOH extracts. Among these
cannabinoids, some co-elute, thus making their analysis difficult. The only way to be distinguished
is by their high-resolution MS/MS spectrum. Unfortunately, the studies on the high-resolution
MS/MS fragmentation of both major and minor cannabinoids are quite poor in the literature [5].
Nowadays, the most widely used analyzers for quantitative determination of cannabinoids and their
respective metabolites in biological fluids are QqQ instruments, which have excellent sensitivity and
selectivity as they work on multiple reaction monitoring (MRM) transitions. However, since they
provide only nominal mass measurements and not a structural identification of non-target
compounds, they require the use of deuterated analytical standards (-d3, -d6 and -d9). The use of
deuterated standard is essential in order to obtain accurate data, as the major drawback of MS is the
matrix effect [114]. Only unreliable values are obtained without these analytical standards, especially
for cannabinoid acids, which are generally ionized at about 300 °C in the electrospray ionization
(ESI) interface and thus are decomposed to the neutral forms.
MS has proved to be very useful also in the evaluation of the chemical stability of the analytical
standards used for the quantitative analysis. In fact, one of our recent works pointed out that the
analytical standard purchased in methanol solution (1 mg/mL) of CBD undergo a sort of
decomposition with the formation of a new peak of m/z 347.0222 in positive ionization mode [41].
The peak was not observed in the standard solution of CBD stored in ethanol [41].
Another MS detector used for the analysis of cannabinoids is the ion trap, which has the advantage
of adding other levels of fragmentation (MS3, MS4,…, MSn) of the fragments generated from the
fragmentation of the parent molecular ion, thus providing important additional information on the
chemical structure of unknown compounds [31].
Very few studies have reported the use of HPLC coupled to FLD since fluorescence spectra of
cannabinoids are strongly affected by the pH of the mobile phase [61]. In fact, cannabinoid acids lose
completely their fluorescence properties in acidic conditions, CBC does not show any fluorescence
signal in a basic environment, and CBN has no fluorescence at all [61].

6.1.1 Other techniques

An alternative method to the conventional HPLC and GC analysis for the determination of
cannabinoids is nuclear magnetic resonance (NMR) spectroscopy [34,111,115,116]. In fact,
quantitative NMR has been considered as a highly accurate and reproducible technique with relatively
short analysis time. In contrast to LC and GC, the major advantage of such technique is the lack of
sensitivity toward impurities present in the plant material such as chlorophyll and lipids [35,111,115].
However, this technique is not commonly employed due to the high instrumental costs and to the
necessity of highly specialized personnel.
Very few applications to qualitative and quantitative analysis of cannabinoids have been developed
and reported with Fourier Transform Infrared (FT-IR) spectroscopy. To the best of our knowledge,
the scientific research in this field has been limited to the paper published by Dorado et al. in 2001,
which describes the analysis of the changes in C/N-modified lignocellulosic substrates from
Cannabis sativa L. during microbial transformation of hemp [117].
Another technique employed in the determination of cannabinoids is immunoassay (IA). This
technique generally provides scarce selectivity due to the difficulty in finding antibodies that are
specific for each cannabinoid. It is rather common to have an antibody that recognizes a class of
compounds with similar chemical structure. Therefore, an IA is suitable for a preliminary assessment
of drug abuse, but a positive IA should always be confirmed with other more sensitive and specific
techniques such as either GC-MS or LC-MS [118]. On the other hand, several IA based methods have
been recently developed because it offers a rapid screening of synthetic cannabinoids in biological
fluids [118-120].

7.1.1 New frontiers in the analysis of cannabis extracts

Cannabis sativa L. is an important medicinal plant of great pharmacological interest. Indeed, it is
currently prescribed in form of either oil, tea or tincture for a series of pathologies [7-10].
Nonetheless, the scientific community is still very far from a thorough understanding of its
comprehensive chemical composition. So far, about 90 cannabinoids and 500 compounds belonging
to different chemical classes have been identified [22]. In order to extend the knowledge on this
powerful plant, metabolomics has been used as a new analytical tool for the identification of unknown
compounds in both plant materials and biological matrices [121]. In the past few years dramatic
developments in high-throughput metabolomics have been achieved, especially due to the aid of
bioinformatics technologies [121].
Metabolomics studies can be carried out by using several analytical platforms, such as high-resolution
1
H NMR, GC-MS, and LC-MS. In particular, 1H NMR has been widely employed for classification
of cannabis cultivars and for structure elucidation exploiting J-resolved, 1H-1H COSY, and 1H-13C
HMBC spectroscopy [34,115,122-125]. GC-MS is also employed for the discrimination of cannabis
cultivars [2,6,126]. However, a superior level of accuracy and precision for metabolite identification
with very low ppm error (<5) is undoubtedly provided by HPLC-MS/MS with detectors like Q-ToF
or Orbitrap [127-129]. The great advantage of these detectors is the capability of generating a
molecular formula from the molecular ion isotopic pattern. The acquisition of the high-resolution
fragmentation spectra and the match with the corresponding authentic standard allows for an
unambiguous identification of the compounds under investigation. In spite of this outstanding
progress, the chemical composition of cannabis medicinal extracts is dramatically variable due to
different temperatures, time and solvents used in the extraction process as reflected in the HPLC-MS
chromatogram reported in Figure 4 (Citti et al., unpublished results) [130]. In this regard,
metabolomics can be considered as a very powerful tool in the scientists’ hands for the evaluation of
the most significant metabolite changes that affect the pharmacological activity of the extracts. This
approach could make a significant breakthrough in the comprehensive chemical characterization of
cannabis medicinal extracts and could eventually pave the way towards a standardized extraction
procedure.

3. Conclusions
In summary, the methods of choice for the determination of cannabinoids in both plant materials and
biological matrices are chromatography-based techniques. Between gas and liquid chromatography,
the latter should be preferred as it allows for the determination of the actual cannabinoid composition
(acid and neutral species) without the necessity of a derivatization step. If GC is employed without a
preliminary derivatization reaction, it unavoidably transforms the cannabinoid acids into their
corresponding neutral forms, thus providing a total value of the two species. Furthermore, recent
works have suggested that the decarboxylation of the cannabinoid acids is only partial and the results
is an underestimation of the actual value. Much attention is to be paid on both the purity of the
analytical standards and their storage conditions, as they are easily degradable by light and heat. Their
authenticity needs to be assessed each time in order to obtain accurate and reproducible results among
different analytical laboratories. Moreover, when a UV spectrophotometer is used as detector,
particular attention is to be paid on having a reasonable resolution of cannabinoids that could co-
elute. When a co-elution occurs, a mass spectrometer is more suitable as it provides both molecular
ions and fragmentation spectra of the cannabinoids under investigation. On the other hand, with a
mass spectrometer the use of appropriate deuterated standards is mandatory in order to compensate
for the matrix effect especially with an ESI source. Often, these standards are either not commercially
available or very expensive. If not all these requisites are satisfied, the analysis is not to be considered
as accurate and reliable and would only generate discordant results among different analytical
laboratories.

Acknowledgements

We are indebted to the pharmacist Dr. Alfredo Tundo (Farmacia Tundo Alfredo, Alliste (LE), Italy)
for his engaging enthusiasm, his continued help and valuable advice.

We also thank Linnea (Locarno, Switzerland) and Crystal Hemp (Lugano, Switzerland) for financial
support.
 4. References
[1] B. De Backer, B. Debrus, P. Lebrun, L. Theunis, N. Dubois, L. Decock, A. Verstraete, P.
Hubert, C. Charlier, Innovative development and validation of an HPLC/DAD method for the
qualitative and quantitative determination of major cannabinoids in cannabis plant material, J.
Chromatogr. B Anal. Technol. Biomed. Life Sci. 877 (2009) 4115–4124.
doi:10.1016/j.jchromb.2009.11.004.
[2] J.T. Fischedick, A. Hazekamp, T. Erkelens, Y.H. Choi, R. Verpoorte, Metabolic fingerprinting
of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug
standardization purposes, Phytochemistry. 71 (2010) 2058–2073.
doi:10.1016/j.phytochem.2010.10.001.
[3] L. Mercolini, A. Musenga, I. Comin, C. Baccini, M. Conti, M.A. Raggi, Determination of
plasma and urine levels of Δ9-tetrahydrocannabinol and its main metabolite by liquid
chromatography after solid-phase extraction, J. Pharm. Biomed. Anal. 47 (2008) 156–163.
doi:10.1016/j.jpba.2007.12.023.
[4] M. Taschwer, M.G. Schmid, Determination of the relative percentage distribution of THCA
and Δ(9)-THC in herbal cannabis seized in Austria - Impact of different storage temperatures
on stability., Forensic Sci. Int. 254 (2015) 167–71. doi:10.1016/j.forsciint.2015.07.019.
[5] O. Aizpurua-Olaizola, J. Omar, P. Navarro, M. Olivares, N. Etxebarria, A. Usobiaga,
Identification and quantification of cannabinoids in Cannabis sativa L. plants by high
performance liquid chromatography-mass spectrometry, Anal. Bioanal. Chem. 406 (2014)
7549–7560. doi:10.1007/s00216-014-8177-x.
[6] K. de C. Mariotti, M.C.A. Marcelo, R.S. Ortiz, B.T. Borille, M. dos Reis, M.S. Fett, M.F.
Ferr??o, R.P. Limberger, Seized cannabis seeds cultivated in greenhouse: A chemical study by
gas chromatography-mass spectrometry and chemometric analysis, Sci. Justice. 56 (2016) 35–
41. doi:10.1016/j.scijus.2015.09.002.
[7] B.S. Koppel, J.C.M. Brust, T. Fife, J. Bronstein, S. Youssof, G. Gronseth, D. Gloss, Systematic
review: Efficacy and safety of medical marijuana in selected neurologic disorders: Report of
the Guideline Development Subcommittee of the American Academy of Neurology,
Neurology. 82 (2014) 1556–1563. doi:10.1212/WNL.0000000000000363.
[8] L.M. Borgelt, K.L. Franson, A.M. Nussbaum, G.S. Wang, The pharmacologic and clinical
effects of medical cannabis, Pharmacotherapy. 33 (2013) 195–209. doi:10.1002/phar.1187.
[9] D.R. Blake, P. Robson, M. Ho, R.W. Jubb, C.S. McCabe, Preliminary assessment of the
efficacy, tolerability and safety of a cannabis-based medicine (Sativex) in the treatment of pain
caused by rheumatoid arthritis, Rheumatology. 45 (2006) 50–52.
doi:10.1093/rheumatology/kei183.
[10] P.F. Whiting, R.F. Wolff, S. Deshpande, M. Di Nisio, S. Duffy, A. V Hernandez, J.C.
Keurentjes, S. Lang, K. Misso, S. Ryder, S. Schmidlkofer, M. Westwood, J. Kleijnen,
Cannabinoids for medical use a systematic review and meta-analysis, Jama. 313 (2015) 2456.
doi:10.1001/jama.2015.6358.
[11] E.P. Baron, Comprehensive Review of Medicinal Marijuana, Cannabinoids, and Therapeutic
Implications in Medicine and Headache: What a Long Strange Trip It’s Been, Headache.
(2000) 885–916.
[12] Y. Gaoni, R. Mechoulam, Isolation, Structure, and Partial Synthesis of an Active Constituent
of Hashish, J. Am. Chem. Soc. 86 (1964) 1646–1647. doi:10.1021/ja01062a046.
[13] E.B. Russo, History of cannabis and its preparations in saga, science, and sobriquet, Chem.
Biodivers. 4 (2007) 1614–1648. doi:10.1002/cbdv.200790144.
[14] E.B. Russo, Cannabinoids in the management of difficult to treat pain, Ther. Clin. Risk Manag.
4 (2008) 245–259.
[15] E.B. Russo, Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid
entourage effects, Br. J. Pharmacol. 163 (2011) 1344–1364. doi:10.1111/bph.2011.163.
[16] F. Grotenhermen, K. Müller-Vahl, Das therapeutische Potenzial von Cannabis und
Cannabinoiden, Dtsch. Arztebl. Int. 109 (2012) 495–501. doi:10.3238/arztebl.2012.0495.
[17] I.N. T. Yamauchi, Y. Shoyama, H. Aramaki, T. Azuma, Tetrahydrocannabinolic acid, a
genuine substance of tetrahydrocannabinol, Chem Pharm Bull. 15 (1967) 1075–1076.
[18] R. RK, Structure-Activity Relationships of the Cannabinoids, Pharmacol Rev. 38 (1986) 75–
149.
[19] S.H. Burstein, The Cannabinoid Acids : Nonpsychoactive Derivatives with Therapeutic
Potential, Pharmacol. Ther. 82 (1999) 87–96.
[20] M.B. and V.D.M. Alessia Ligresti, Aniello Schiano Moriello, Katarzyna Starowicz, Isabel
Matias, Simona Pisanti, Luciano De Petrocellis, Chiara Laezza, Giuseppe Portella, Antitumor
Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast
Carcinoma, J. Pharmacol. Exp. Ther. 318 (2006) 1375–1387. doi:10.1124/jpet.106.105247.
[21] J.M. McPartland, E.B. Russo, Cannabis and Cannabis Extracts: Greater Than the Sum of Their
Parts?, Cannabis Ther. HIV/AIDS. 1 (2001) 103–132. doi:10.1300/J175v01n03_08.
[22] R. Brenneisen, Chemistry and Analysis of Phytocannabinoids and Other Cannabis
Constituents, Forensic Sci. Med. Marijuana Cannabinoids. (2007) 17–49. doi:10.1007/978-1-
59259-947-9_2.
[23] E.B. Russo, J.M. McPartland, Cannabis is more than simply delta(9)-tetrahydrocannabinol.,
Psychopharmacology (Berl). 165 (2003) 431-432-434. doi:10.1007/s00213-002-1348-z.
[24] T.J. Raharjo, R. Verpoorte, Methods for the analysis of cannabinoids in biological materials:
a review., Phytochem Anal. 15 (2004) 79–94. doi:10.1002/pca.753.
[25] N. Battista, M. Sergi, C. Montesano, S. Napoletano, D. Compagnone, M. Maccarrone,
Analytical approaches for the determination of phytocannabinoids and endocannabinoids in
human matrices, Drug Test. Anal. 6 (2014) 7–16. doi:10.1002/dta.1574.
[26] J.T. Fischedick, R. Glas, A. Hazekamp, R. Verpoorte, A qualitative and quantitative HPTLC
densitometry method for the analysis of cannabinoids in Cannabis sativa L., Phytochem. Anal.
20 (2009) 421–426. doi:10.1002/pca.1143.
[27] M.W. Giese, M.A. Lewis, L. Giese, K.M. Smith, Development and validation of a reliable and
robust method for the analysis of cannabinoids and terpenes in cannabis, J. AOAC Int. 98
(2015) 1503–1522. doi:10.5740/jaoacint.15-116.
[28] F. Institute, for D. and M. (BfArM) Devices, German Cannabis Monograph draft_EN, (2016)
1–9.
[29] J. Pandohee, B.J. Holland, B. Li, T. Tsuzuki, P.G. Stevenson, N.W. Barnett, J.R. Pearson,
O.A.H. Jones, X.A. Conlan, Screening of cannabinoids in industrial-grade hemp using two-
dimensional liquid chromatography coupled with acidic potassium permanganate
chemiluminescence detection, J. Sep. Sci. 38 (2015) 2024–2032. doi:10.1002/jssc.201500088.
[30] K.W. Hillig, P.G. Mahlberg, A chemotaxonomic analysis of cannabinoid variation in Cannabis
(Cannabaceae), Am. J. Bot. 91 (2004) 966–975. doi:10.3732/ajb.91.6.966.
[31] A.A.M. Stolker, J. Van Schoonhoven, A.J. De Vries, I. Bobeldijk-Pastorova, W.H.J. Vaes, R.
Van Den Berg, Determination of cannabinoids in cannabis products using liquid
chromatography-ion trap mass spectrometry, J. Chromatogr. A. 1058 (2004) 143–151.
doi:10.1016/j.chroma.2004.08.089.
[32] D. Wianowska, A.L. Dawidowicz, M. Kowalczyk, Transformations of Tetrahydrocannabinol,
tetrahydrocannabinolic acid and cannabinol during their extraction from Cannabis sativa L., J.
Anal. Chem. 70 (2015) 920–925. doi:10.1134/S1061934815080183.
[33] M. Pellegrini, E. Marchei, R. Pacifici, S. Pichini, A rapid and simple procedure for the
determination of cannabinoids in hemp food products by gas chromatography-mass
spectrometry, J. Pharm. Biomed. Anal. 36 (2005) 939–946. doi:10.1016/j.jpba.2004.07.035.
[34] W. Peschel, M. Politi, 1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction,
extract profiling and specification, Talanta. 140 (2015) 150–165.
doi:10.1016/j.talanta.2015.02.040.
[35] M. Politi, W. Peschel, N. Wilson, M. Zloh, J.M. Prieto, M. Heinrich, Direct NMR analysis of
cannabis water extracts and tinctures and semi-quantitative data on D 9 -THC and D 9 -THC-
acid, Phytochemistry. 69 (2008) 562–570. doi:10.1016/j.phytochem.2007.07.018.
[36] S. Ameur, B. Haddou, Z. Derriche, J.P. Canselier, C. Gourdon, Cloud point extraction of Δ9-
tetrahydrocannabinol from cannabis resin, Anal. Bioanal. Chem. 405 (2013) 3117–3123.
doi:10.1007/s00216-013-6743-2.
[37] J. Omar, M. Olivares, M. Alzaga, N. Etxebarria, Optimisation and characterisation of
marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction
and retention time locking GC-MS, J. Sep. Sci. 36 (2013) 1397–1404.
doi:10.1002/jssc.201201103.
[38] A. Hazekamp, K. Bastola, H. Rashidi, J. Bender, R. Verpoorte, Cannabis tea revisited: A
systematic evaluation of the cannabinoid composition of cannabis tea, J. Ethnopharmacol. 113
(2007) 85–90. doi:10.1016/j.jep.2007.05.019.
[39] R. Pacifici, E. Marchei, F. Salvatore, L. Guandalini, F.P. Busardò, S. Pichini, Evaluation of
cannabinoids concentration and stability in standardized preparations of cannabis tea and
cannabis oil by ultra-high performance liquid chromatography tandem mass spectrometry,
Clin. Chem. Lab. Med. 0 (2017) 1–9. doi:10.1515/cclm-2016-1060.
[40] L.L. Romano, A. Hazekamp, Cannabis Oil: chemical evaluation of an upcoming cannabis-
based medicine, Cannabinoids. 1 (2013) 1–11. doi:10.1002/dta.407.
[41] C. Citti, G. Ciccarella, D. Braghiroli, C. Parenti, M.A.M.A. Vandelli, G. Cannazza, Medicinal
Cannabis: principal cannabinoids concentration and their stability evaluated by a high
performance liquid chromatography coupled to diode array and quadrupole time of flight mass
spectrometry method, J. Pharm. Biomed. Anal. 128 (2016) 201–209.
doi:10.1016/j.jpba.2016.05.033.
[42] G. Milman, D.M. Schwope, D.A. Gorelick, M.A. Huestis, Cannabinoids and metabolites in
expectorated oral fluid following controlled smoked cannabis, Clin. Chim. Acta. 413 (2012)
765–770. doi:10.1016/j.cca.2012.01.011.
[43] A. Molnar, S. Fu, J. Lewis, D.J. Allsop, J. Copeland, The detection of THC, CBD and CBN in
the oral fluid of Sativex® patients using two on-site screening tests and LC-MS/MS, Forensic
 Sci. Int. 238 (2014) 113–119. doi:10.1016/j.forsciint.2014.03.004.
[44] N.A. Desrosiers, K.B. Scheidweiler, M.A. Huestis, Quantification of six cannabinoids and
metabolites in oral fluid by liquid chromatography-tandem mass spectrometry, Drug Test.
Anal. 7 (2015) 684–694. doi:10.1002/dta.1753.
[45] G. Milman, A.J. Barnes, R.H. Lowe, M.A. Huestis, Simultaneous quantification of
cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass
spectrometry, J. Chromatogr. A. 1217 (2010) 1513–1521. doi:10.1016/j.chroma.2009.12.053.
[46] M. Concheiro, D. Lee, E. Lendoiro, M.A. Huestis, Simultaneous Quantification of Δ9-
Tetrahydrocannabinol, 11-nor-9-carboxy-Tetrahydrocannabinol, Cannabidiol and Cannabinol
in Oral Fluid by MicroFlow-Liquid Chromatography- High Resolution Mass Spectrometry, J.
Chromatogr. A. 454 (2007) 42–54. doi:10.1097/OPX.0b013e3182540562.The.
[47] M. Sergi, C. Montesano, S. Odoardi, L. Mainero Rocca, G. Fabrizi, D. Compagnone, R. Curini,
Micro extraction by packed sorbent coupled to liquid chromatography tandem mass
spectrometry for the rapid and sensitive determination of cannabinoids in oral fluids, J.
Chromatogr. A. 1301 (2013) 139–146. doi:10.1016/j.chroma.2013.05.072.
[48] N. Samyn, M. Laloup, G. De Boeck, Bioanalytical procedures for determination of drugs of
abuse in oral fluid, Anal. Bioanal. Chem. 388 (2007) 1437–1453. doi:10.1007/s00216-007-
1245-8.
[49] C. Lacroix, E. Saussereau, Fast liquid chromatography/tandem mass spectrometry
determination of cannabinoids in micro volume blood samples after dabsyl derivatization, J.
Chromatogr. B Anal. Technol. Biomed. Life Sci. 905 (2012) 85–95.
doi:10.1016/j.jchromb.2012.08.006.
[50] D.M. Schwope, K.B. Scheidweiler, M.A. Huestis, Direct quantification of cannabinoids and
cannabinoid glucuronides in whole blood by liquid chromatography-tandem mass
spectrometry, Anal. Bioanal. Chem. 401 (2011) 1273–1283. doi:10.1007/s00216-011-5197-7.
[51] S. König, B. Aebi, S. Lanz, M. Gasser, W. Weinmann, On-line SPE LC-MS/MS for the
quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human
peripheral blood by liquid chromatography tandem mass spectrometry., Anal. Bioanal. Chem.
400 (2011) 9–16. doi:10.1007/s00216-011-4708-x.
[52] A. Gronewold, G. Skopp, A preliminary investigation on the distribution of cannabinoids in
man, Forensic Sci. Int. 210 (2011) e7–e11. doi:10.1016/j.forsciint.2011.04.010.
[53] T. Baciu, F. Borrull, C. Aguilar, M. Calull, Recent trends in analytical methods and separation
techniques for drugs of abuse in hair, Anal. Chim. Acta. 856 (2015) 1–26.
doi:10.1016/j.aca.2014.06.051.
[54] E. Jagerdeo, J.E. Schaff, M.A. Montgomery, M.A. LeBeau, A semi-automated solid-phase
extraction liquid chromatography/tandem mass spectrometry method for the analysis of
tetrahydrocannabinol and metabolites in whole blood, Rapid Commun. Mass Spectrom. 23
(2009) 2697–2705.
[55] A.A. Elian, J. Hackett, Solid-phase extraction and analysis of THC and carboxy-THC from
whole blood using a novel fluorinated solid-phase extraction sorbent and fast liquid
chromatography-tandem mass spectrometry., J. Anal. Toxicol. 33 (2009) 461–8.
doi:10.1093/jat/33.8.461.
[56] L. Mercolini, R. Mandrioli, M. Protti, M. Conti, G. Serpelloni, M.A. Raggi, Monitoring of
chronic Cannabis abuse: An LC-MS/MS method for hair analysis, J. Pharm. Biomed. Anal. 76
 (2013) 119–125. doi:10.1016/j.jpba.2012.12.015.
[57] E.S. Emídio, V. de Menezes Prata, H.S. Dórea, Validation of an analytical method for analysis
of cannabinoids in hair by headspace solid-phase microextraction and gas chromatography-ion
trap tandem mass spectrometry, Anal. Chim. Acta. 670 (2010) 63–71.
doi:10.1016/j.aca.2010.04.023.
[58] T. Nadulski, F. Pragst, Simple and sensitive determination of ??9-tetrahydrocannabinol,
cannabidiol and cannabinol in hair by combined silylation, headspace solid phase
microextraction and gas chromatography-mass spectrometry, J. Chromatogr. B Anal. Technol.
Biomed. Life Sci. 846 (2007) 78–85. doi:10.1016/j.jchromb.2006.08.015.
[59] M. Tayyab, D. Shahwar, GCMS analysis of Cannabis sativa L. from four different areas of
Pakistan, Egypt. J. Forensic Sci. 5 (2015) 114–125. doi:10.1016/j.ejfs.2014.07.008.
[60] N. Uchiyama, R. Kikura-Hanajiri, J. Ogata, Y. Goda, Chemical analysis of synthetic
cannabinoids as designer drugs in herbal products, Forensic Sci. Int. 198 (2010) 31–38.
doi:10.1016/j.forsciint.2010.01.004.
[61] A. Hazekamp, A. Peltenburg, R. Verpoorte, C. Giroud, Chromatographic and Spectroscopic
Data of Cannabinoids from Cannabis sativa L., J. Liq. Chromatogr. Relat. Technol. 28 (2005)
2361–2382. doi:10.1080/10826070500187558.
[62] F.E. Dussy, C. Hamberg, M. Luginbühl, T. Schwerzmann, T.A. Briellmann, Isolation of Δ9-
THCA-A from hemp and analytical aspects concerning the determination of Δ9-THC in
cannabis products, Forensic Sci. Int. 149 (2005) 3–10. doi:10.1016/j.forsciint.2004.05.015.
[63] V. Gambaro, L. Dell’Acqua, F. Farè, R. Froldi, E. Saligari, G. Tassoni, Determination of
primary active constituents in Cannabis preparations by high-resolution gas
chromatography/flame ionization detection and high-performance liquid chromatography/UV
detection, Anal. Chim. Acta. 468 (2002) 245–254. doi:10.1016/S0003-2670(02)00660-8.
[64] P.F. Nadulski T, Sporkert F, Schnelle M, Stadelmann AM, Roser P, Schefter T, Simultaneous
and sensitive analysis of THC, 11-OH-THC, THC-COOH, CBD, and CBN by GC-MS in
plasma after oral application of small doses of THC and cannabis extract, J. Anal.Toxicol. 29
(2005) 782–789.
[65] U.F. Coles R, Clements TT, Nelson GJ, McMillin GA, Simultaneous analysis of the Delta9-
THC metabolites 11-nor-9-carboxy-Delta9-THC and 11-hydroxy-Delta9-THC in meconium
by GC-MS, J. Anal.Toxicol. 29 (2005) 522–527.
[66] C. Moore, F. Guzaldo, T. Donahue, Determination of 11-nor-D9-tetrahydrocannabinol-9-
carboxylic acid in hair using gas chromatography/ tandem mass spectrometry in negative ion
chemical ionization mode, Rapid Commun. Mass Spectrom. 21 (2007) 1339–1342.
doi:10.1002/rcm.
[67] A. Thomas, C. Widmer, G. Hopfgartner, C. Staub, Fast gas chromatography and negative-ion
chemical ionization tandem mass spectrometry for forensic analysis of cannabinoids in whole
blood, J. Pharm. Biomed. Anal. 45 (2007) 495–503. doi:10.1016/j.jpba.2007.08.019.
[68] R. Andrews, S. Paterson, A validated method for the analysis of cannabinoids in post-mortem
blood using liquid-liquid extraction and two-dimensional gas chromatography-mass
spectrometry, Forensic Sci. Int. 222 (2012) 111–117. doi:10.1016/j.forsciint.2012.05.007.
[69] R.H. Lowe, E.L. Karschner, E.W. Schwilke, A.J. Barnes, M.A. Huestis, Simultaneous
quantification of Δ9-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, and 11-nor-
Δ9-tetrahydrocannabinol-9-carboxylic acid in human plasma using two-dimensional gas
 chromatography, cryofocusing, and electron impact-mass spectromet, J. Chromatogr. A. 1163
(2007) 318–327. doi:10.1016/j.chroma.2007.06.069.
[70] E. Marchei, D. Escuder, C.R. Pallas, O. Garcia-Algar, A. Gómez, B. Friguls, M. Pellegrini, S.
Pichini, Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast
milk by liquid chromatography tandem mass spectrometry, J. Pharm. Biomed. Anal. 55 (2011)
309–316. doi:10.1016/j.jpba.2011.01.028.
[71] J. Jung, J. Kempf, H. Mahler, W. Weinmann, Detection of D9-tetrahydrocannabinolic acid A
in human urine and blood serum by LC-MS/MS, J. Mass Spectrom. 42 (2007) 354–360.
doi:10.1002/jms.
[72] B. Maralikova, W. Weinmann, Simultaneous determination of Δ9-tetrahydrocannabinol, 11-
hydroxy-Δ9-tetrahydrocannabinol and 11 -nor-9-carboxy-Δ9-tetrahydrocannabinol in human
plasma by high-performance liquid chromatography/tandem mass spectrometry, J. Mass
Spectrom. 39 (2004) 526–531. doi:10.1002/jms.616.
[73] S.B. Grauwiler, A. Scholer, J. Drewe, Development of a LC/MS/MS method for the analysis
of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa
extracts, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 850 (2007) 515–522.
doi:10.1016/j.jchromb.2006.12.045.
[74] M. Fabritius, C. Staub, P. Mangin, C. Giroud, Distribution of free and conjugated cannabinoids
in human bile samples, Forensic Sci. Int. 223 (2012) 114–118.
doi:10.1016/j.forsciint.2012.08.013.
[75] H. Teixeira, A. Verstraete, P. Proença, F. Corte-Real, P. Monsanto, D.N. Vieira, Validated
method for the simultaneous determination of Δ9-THC and Δ9-THC-COOH in oral fluid, urine
and whole blood using solid-phase extraction and liquid chromatography-mass spectrometry
with electrospray ionization, Forensic Sci. Int. 170 (2007) 148–155.
doi:10.1016/j.forsciint.2007.03.026.
[76] M. del Mar Ramirez Fernandez, G. De Boeck, M. Wood, M. Lopez-Rivadulla, N. Samyn,
Simultaneous analysis of THC and its metabolites in blood using liquid chromatography-
tandem mass spectrometry, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 875 (2008)
465–470. doi:10.1016/j.jchromb.2008.09.032.
[77] N. Roth, B. Moosmann, V. Auwärter, Development and validation of an LC-MS/MS method
for quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), THC, CBN and CBD in
hair, J. Mass Spectrom. 48 (2013) 227–233. doi:10.1002/jms.3152.
[78] W. Swift, A. Wong, K.M. Li, J.C. Arnold, I.S. McGregor, Analysis of Cannabis Seizures in
NSW, Australia: Cannabis Potency and Cannabinoid Profile, PLoS One. 8 (2013) 1–9.
doi:10.1371/journal.pone.0070052.
[79] N.B. Tiscione, R. Miller, X. Shan, J. Sprague, D.T. Yeatman, An Efficient, Robust Method for
the Determination of Cannabinoids in Whole Blood by LC–MS-MS, J. Anal.Toxicol. 40
(2016) 639–648.
[80] C.H. Hung, J. Zukowski, D.S. Jensen, A.J. Miles, C. Sulak, A.E. Dadson, M.R. Linford,
Separation of cannabinoids on three different mixed-mode columns containing
carbon/nanodiamond/amine-polymer superficially porous particles, J. Sep. Sci. 38 (2015)
2968–2974. doi:10.1002/jssc.201500156.
[81] C. Citti, U.M. Battisti, G. Ciccarella, V. Maiorano, G. Gigli, S. Abbate, G. Mazzeo, E.
Castiglioni, G. Longhi, G. Cannazza, Analytical and preparative enantioseparation and main
 chiroptical properties of Iridium(III) bis(4,6-difluorophenylpyridinato)picolinato, J.
Chromatogr. A. 1467 (2016) 335–346. doi:10.1016/j.chroma.2016.05.059.
[82] J.N. Fairchild, K. Horvath, J.R. Gooding, S.R. Campagna, G. Guiochon, Two-dimensional
liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble
metabolites, J. Chromatogr. A. 1217 (2010) 8161–8166. doi:10.1016/j.chroma.2010.10.068.
[83] V. Brighenti, S.F. Groothuis, F.P. Prencipe, R. Amir, S. Benvenuti, F. Pellati, Metabolite
fingerprinting of Punica granatum L. (pomegranate) polyphenols by means of high-
performance liquid chromatography with diode array and electrospray ionization-mass
spectrometry detection, J. Chromatogr. A. 1480 (2017) 20–31.
doi:10.1016/j.chroma.2016.12.017.
[84] S. Hegstad, S. Hermansson, I. Betn??r, O. Spigset, B.M.H. Falch, Screening and quantitative
determination of drugs of abuse in diluted urine by UPLC-MS/MS, J. Chromatogr. B Anal.
Technol. Biomed. Life Sci. 947–948 (2014) 83–95. doi:10.1016/j.jchromb.2013.12.014.
[85] H.B. Klinke, I.B. Müller, S. Steffenrud, R. Dahl-Sørensen, Two cases of lysergamide
intoxication by ingestion of seeds from Hawaiian Baby Woodrose, Forensic Sci. Int. 197
(2010) 1–5. doi:10.1016/j.forsciint.2009.11.017.
[86] S.A. Legrand, S. Houwing, M. Hagenzieker, A.G. Verstraete, Prevalence of alcohol and other
psychoactive substances in injured drivers: Comparison between Belgium and the Netherlands,
Forensic Sci. Int. 220 (2012) 224–231. doi:10.1016/j.forsciint.2012.03.006.
[87] S. Ozturk, Y.E. Ozturk, O. Yeter, B. Alpertunga, Application of a validated LC-MS/MS
method for JWH-073 and its metabolites in blood and urine in real forensic cases, Forensic
Sci. Int. 257 (2015) 165–171. doi:10.1016/j.forsciint.2015.08.013.
[88] S. Schneider, R. Bebing, C. Dauberschmidt, Detection of pesticides in seized illegal cannabis
plants, Anal. Methods. 6 (2014) 515–520. doi:10.1039/C3AY40930A.
[89] K.G. Shanks, T. Dahn, A.R. Terrell, Detection of JWH-018 and JWH-073 by UPLC-MS-MS
in postmortem whole blood casework, J. Anal. Toxicol. 36 (2012) 145–152.
doi:10.1093/jat/bks013.
[90] M. Styrczewska, A. Kulma, K. Ratajczak, R. Amarowicz, J. Szopa, Cannabinoid-like anti-
inflammatory compounds from flax fiber., Cell. Mol. Biol. Lett. 17 (2012) 479–99.
doi:10.2478/s11658-012-0023-6.
[91] L.V. Tose, N.A. Santos, R.R.T. Rodrigues, M. Murgu, A.F. Gomes, G.A. Vasconcelos, P.C.T.
Souza, B.G. Vaz, W. Romäo, Isomeric separation of cannabinoids by UPLC combined with
ionic mobility mass spectrometry (TWIM-MS) -part I, Int. J. Mass Spectrom. (2016).
doi:10.1016/j.ijms.2016.10.018.
[92] T. Van der Linden, P. Silverans, A.G. Verstraete, Comparison between self-report of cannabis
use and toxicological detection of THC/THCCOOH in blood and THC in oral fluid in drivers
in a roadside survey, Drug Test. Anal. 6 (2014) 137–142. doi:10.1002/dta.1517.
[93] S.M. Wille, V. Di Fazio, M.M. Ramírez-Fernandez, N. Kummer, N. Samyn, Driving under the
influence of cannabis: Pitfalls, validation, and quality control of a UPLC-MS/MS method for
the quantification of tetrahydrocannabinol in oral fluid collected with statsure, quantisal, or
certus collector, Ther. Drug Monit. 35 (2013) 101–111.
[94] S.M. Wille, V. Di Fazio, S.W. Toennes, J.H. van Wel, J.G. Ramaekers, N. Samyn, Evaluation
of Δ9-tetrahydrocannabinol detection using DrugWipe5S® screening and oral fluid
quantification after QuantisalTM collection for roadside drug detection via a controlled study
 with chronic cannabis users, Drug Test. Anal. 7 (2015) 178–186. doi:10.1002/dta.1660.
[95] C. Jamey, E. Szwarc, A. Tracqui, B. Ludes, Determination of cannabinoids in whole blood by
UPLC-MS-MS., J. Anal. Toxicol. 32 (2008) 349–354. doi:10.1093/jat/32.5.349.
[96] S. Levin, S. Abulafi, J. Zahalka, R. Mechoulam, Resolution of Chiral Cannabinoids on
Amylose Tris(3,5-Dimethylphenylcarbamate) Chiral Stationary-Phase - Effects of Structural
Features and Mobile-Phase Additives, J. Chromatogr. A. 654 (1993) 53–64. doi:Doi
10.1016/0021-9673(93)83064-Y.
[97] G. Cannazza, U. Battisti, M.M. Carrozzo, L. Brasili, D. Braghiroli, C. Parenti, Evaluation of
Stereo and Chemical Stability of Chiral Compounds, Chirality. 23 (2011) 851–859.
doi:10.1002/chir.
[98] G. Cannazza, M.M. Carrozzo, U. Battisti, D. Braghiroli, C. Parenti, A. Troisi, L. Troisi,
Determination of Kinetic Parameters of Enantiomerization of Benzothiadiazines by
DCXplorer, Chirality. 22 (2010) 789–797. doi:10.1002/chir.
[99] U.M. Battisti, M.M. Carrozzo, G. Cannazza, G. Puia, L. Troisi, D. Braghiroli, C. Parenti, K.
Jozwiak, Molecular modeling studies, synthesis, configurational stability and biological
activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine
1,1-dioxide., Bioorg. Med. Chem. 19 (2011) 7111–9. doi:10.1016/j.bmc.2011.09.063.
[100] G. Cannazza, M.M. Carrozzo, U. Battisti, D. Braghiroli, C. Parenti, On-line racemization by
high-performance liquid chromatography, J. Chromatogr. A. 1216 (2009) 5655–5659.
doi:10.1016/j.chroma.2009.05.057.
[101] M.M. Carrozzo, U.M. Battisti, G. Cannazza, G. Puia, F. Ravazzini, A. Falchicchio, S. Perrone,
C. Citti, K. Jozwiak, D. Braghiroli, C. Parenti, L. Troisi, Design, stereoselective synthesis,
configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-
1H-benzo[e]pyrrolo[2,1- c][1,2,4]thiadiazine 5,5-dioxide, Bioorganic Med. Chem. 22 (2014).
doi:10.1016/j.bmc.2014.07.017.
[102] M.M. Carrozzo, G. Cannazza, U. Battisti, D. Braghiroli, L. Troisi, C. Parenti, Epimerization
and hydrolysis of 3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-
dioxide, J. Chromatogr. A. 1217 (2010) 8136–8145. doi:10.1016/j.chroma.2010.10.044.
[103] G. Cannazza, M.M. Carrozzo, D. Braghiroli, C. Parenti, Enantiomerization and hydrolysis of
(±)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide by stopped-flow
multidimensional high-performance liquid chromatography, J. Chromatogr. A. 1212 (2008)
41–47. doi:10.1016/j.chroma.2008.09.087.
[104] G. Cannazza, M.M. Carrozzo, D. Braghiroli, C. Parenti, Enantiomerization of chiral 2,3,3a,4-
tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide by stopped-flow
multidimensional HPLC, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 875 (2008) 192–
199. doi:10.1016/j.jchromb.2008.05.009.
[105] G. Cannazza, U.M. Battisti, M.M. Carrozzo, A.S. Cazzato, D. Braghiroli, C. Parenti, L. Troisi,
Development of an in vitro liquid chromatography-mass spectrometry method to evaluate
stereo and chemical stability of new drug candidates employing immobilized artificial
membrane column, J. Chromatogr. A. 1363 (2014) 216–225.
doi:10.1016/j.chroma.2014.05.073.
[106] U.M. Battisti, C. Citti, G. Rastelli, L. Pinzi, G. Puja, F. Ravazzini, G. Ciccarella, D. Braghiroli,
G. Cannazza, An unexpected reversal in the pharmacological stereoselectivity of
benzothiadiazine AMPA positive allosteric modulators, Medchemcomm. 7 (2016).
 doi:10.1039/c6md00440g.
[107] L.O. Hanus, S. Tchilibon, D.E. Ponde, A. Breuer, E. Fride, R. Mechoulam, Enantiomeric
cannabidiol derivatives: synthesis and binding to cannabinoid receptors., Org. Biomol. Chem.
3 (2005) 1116–23. doi:10.1039/b416943c.
[108] S. Abu-Lafi, M. Sterin, S. Levin, Role of hydroxyl groups in chiral recognition of cannabinoids
by carbamated amylose, J. Chromatogr. A. 679 (1994) 47–58. doi:10.1016/0021-
9673(94)80310-2.
[109] U.M. Battisti, C. Citti, M. Larini, G. Ciccarella, N. Stasiak, L. Troisi, D. Braghiroli, C. Parenti,
M. Zoli, G. Cannazza, “Heart-cut” bidimensional achiral-chiral liquid chromatography applied
to the evaluation of stereoselective metabolism, in vivo biological activity and brain response
to chiral drug candidates targeting the central nervous system, J. Chromatogr. A. 1443 (2016)
152–161. doi:10.1016/j.chroma.2016.03.027.
[110] S.S. Simões, A.C. Ajenjo, M.J. Dias, Qualitative and quantitative analysis of THC, 11-
hydroxy-THC and 11-nor-9-carboxy-THC in whole blood by ultra-performance liquid
chromatography/tandem mass spectrometry., Rapid Commun. Mass Spectrom. 25 (2011)
2603–10. doi:10.1002/rcm.5165.
[111] N. Happyana, S. Agnolet, R. Muntendam, A. Van Dam, B. Schneider, O. Kayser, Analysis of
cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and
cryogenic NMR, Phytochemistry. 87 (2013) 51–59. doi:10.1016/j.phytochem.2012.11.001.
[112] W. Weinmann, S. Vogt, R. Goerke, C. Müller, A. Bromberger, Simultaneous determination of
THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS, Forensic Sci. Int.
113 (2000) 381–387. doi:10.1016/S0379-0738(00)00210-3.
[113] A. Zgair, J.C.M. Wong, A. Sabri, P.M. Fischer, D.A. Barrett, C.S. Constantinescu, P.
Gershkovich, Development of a simple and sensitive HPLC–UV method for the simultaneous
determination of cannabidiol and 9 -tetrahydrocannabinol in rat plasma, J. Pharm. Biomed.
Anal. J. Pharm. Biomed. 114 (2015) 145–151. doi:10.1016/j.jpba.2015.05.019.
[114] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards in
quantitative bioanalysis using liquid chromatography/mass spectrometry: Necessity or not?,
Rapid Commun. Mass Spectrom. 19 (2005) 401–407. doi:10.1002/rcm.1790.
[115] A. Hazekamp, Y.H. Choi, R. Verpoorte, Quantitative analysis of cannabinoids from Cannabis
sativa using 1H-NMR., Chem. Pharm. Bull. (Tokyo). 52 (2004) 718–721.
doi:10.1248/cpb.52.718.
[116] Y.H. Choi, A. Hazekamp, A.M.G. Peltenburg-Looman, M. Frédérich, C. Erkelens, A.W.M.
Lefeber, R. Verpoorte, NMR assignments of the major cannabinoids and cannabiflavonoids
isolated from flowers of Cannabis sativa, Phytochem. Anal. 15 (2004) 345–354.
doi:10.1002/pca.787.
[117] J. Dorado, G. Almendros, J.A. Field, R. Sierra-Alvarez, Infrared spectroscopy analysis of
hemp (Cannabis sativa) after selective delignification by Bjerkandera sp. at different nitrogen
levels, Enzyme Microb. Technol. 28 (2001) 550–559. doi:10.1016/S0141-0229(00)00363-X.
[118] M. Sundström, A. Pelander, I. Ojanperä, Comparison between drug screening by immunoassay
and ultra-high performance liquid chromatography/high-resolution time-of-flight mass
spectrometry in post-mortem urine, Drug Test. Anal. 7 (2015) 420–427. doi:10.1002/dta.1683.
[119] G. Tassoni, M. Cippitelli, G. Ottaviani, R. Froldi, M. Cingolani, Detection of Cannabinoids by
ELISA and GC–MS Methods in a Hair Sample Previously Used to Detect Other Drugs of
 Abuse, J. Anal. Toxicol. 40 (2016) 408–413.
[120] K. Tsujikawa, F. Saiki, T. Yamamuro, Y.T. Iwata, R. Abe, H. Ohashi, R. Kaigome, K.
Yamane, K. Kuwayama, T. Kanamori, Development of a novel immunoassay for herbal
cannabis using a new fluorescent antibody probe, “Ultra Quenchbody,” Forensic Sci. Int. 266
(2016) 541–548.
[121] T.F. Jorge, J.A. Rodrigues, C. Caldana, R. Schmidt, J.T. van Dongen, J. Thomas-Oates, C.
António, Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress,
Mass Spectrom. Rev. (2015). doi:10.1002/mas.21449.
[122] R.V. Isvett Josefina Flores-Sanchez, Young Hae Choi, Metabolite Analysis of Cannabis sativa
L. by NMR Spectroscopy, Methods Mol. Biol. 815 (2011) 363–375.
[123] R. Muntendam, N. Happyana, T. Erkelens, Time Dependent Metabolomics and
Transcriptional Analysis of Cannabinoid Biosynthesis in C annabis sativa var . Bedrobinol and
Bediol Grown under Standardized Condition and with Genetic Homogeneity, Online Int. J.
Med. Plants Res. 1 (2012) 31–40.
[124] Y.H. Choi, H.K. Kim, A. Hazekamp, C. Erkelens, A.W.M. Lefeber, R. Verpoorte,
Metabolomic Differentiation of Cannabis sativa Cultivars Using 1 H NMR Spectroscopy and
Principal Component Analysis, J. Nat. Prod. 67 (2004) 953–957.
[125] C.S. Elaine Holmes, Huiru Tang, Yulan Wang, E. Holmes, H. Tang, Y. Wang, C. Seger, The
assessment of plant metabolite profiles by NMR-based methodologies, Planta Med. 72 (2006)
771–785. doi:10.1055/s-2006-946682.
[126] A. Hazekamp, J.T. Fischedick, Cannabis - from cultivar to chemovar, Drug Test. Anal. (2012).
doi:10.1002/dta.407.
[127] E. Grata, D. Guillarme, G. Glauser, J. Boccard, P.A. Carrupt, J.L. Veuthey, S. Rudaz, J.L.
Wolfender, Metabolite profiling of plant extracts by ultra-high-pressure liquid
chromatography at elevated temperature coupled to time-of-flight mass spectrometry, J.
Chromatogr. A. 1216 (2009) 5660–5668. doi:10.1016/j.chroma.2009.05.069.
[128] J.-L. Wolfender, G. Marti, A. Thomas, S. Bertrand, Current approaches and challenges for the
metabolite profiling of complex natural extracts, J. Chromatogr. A. 1382 (2015) 136–164.
doi:10.1016/j.chroma.2014.10.091.
[129] G. Marti, S. Schnee, Y. Andrey, C. Simoes-pires, P. Carrupt, J. Wolfender, K. Gindro, Study
of Leaf Metabolome Modifications Induced by UV-C Radiations in Representative Vitis,
Cissus and Cannabis Species by LC-MS Based Metabolomics and Antioxidant Assays,
Molecules. 19 (2014) 14004–14021. doi:10.3390/molecules190914004.
[130] C. Citti, U.M. Battisti, D. Braghiroli, G. Ciccarella, M. Schmid, M.A. Vandelli, G. Cannazza,
A metabolomic approach applied to a liquid chromatography coupled to high-resolution
tandem mass spectrometry method (HPLC-ESI-HRMS/MS): towards the comprehensive
evaluation of the chemical composition of cannabis medicinal extracts, submitted to
Phytochem. Anal. (2017).
Figure 1. Schematic representation of the biosynthetic route of THCA and CBDA from CBGA, formation of THC and CBD by light
and /or heat and oxidation of THC to CBN.
Figure 2. Molecular structure of the most common acid and neutral cannabinoids and flavonoids (cannaflavin A and cannaflavin B).

Figure 3. Molecular structure of the most common Δ9-THC metabolites.

Figure 4. 3D Total Ion Chromatogram (TIC) of a Cannabis sativa L. oil extract in positive ionization (ESI+) mode. The retention time
(min) is represented on the x axis, the peak intensity (ion counts) on the y axis and the m/z values on the z axis (Citti et al., unpublished
results) [130].
 Table 1. Analytical methods for the analysis of cannabis plant material

Analytical technique Matrix Identified analytes Extraction methodology Sensitivity Reference

Δ -THCA, CBDA, CBGA, Δ9-
9
LOQ 0.125 (THCA,
Seized cannabis and THC, CBD, CBG, CBN CBGA, CBDA, THC,
HPLC-DAD MeOH/CHCl3 9:1 (v/v) [1]
fiber-type plants (quantitative), Δ8-THC CBN), 0.188 (CBD),
(qualitative) 0.375 μg/mL (CBG)
Medicinal cannabis
Terpenes, Δ9-THC, Δ8-THC,
GC-FID buds (Bedrocan ®, 0.4-0.5 (terpenes), 0.6
CBD, CBG, CBC, THCV, EtOH [2]
GC-MS Bedropuur® and mg/g (cannabinoids)
CBDV, CBGM
Bediol®)
Medicinal cannabis Δ9-THC, CBN (quantitative) Decarboxylation (H2O, 100
HPTLC cultivars (Bedrocan® CBD, Δ8-THC, THCV, CBG, °C, 2 h) and extraction with LOD 10 ng, LOQ 50 ng [26]
and Bediol®) CBC (qualitative) EtOH
CBDVA, CBDV, THCV,
CBGA, THCVA (tentative ID),
HPLC-DAD
Flowers CBDA, CBG, CBD, CBN, Δ9- EtOH LOQ 5-8 μg/mL [27]
GC-FID
THC, Δ8-THC, CBC, THCA,
terpenes
CBDV, CBCV, CBV, CBLV,
CBGV, CBN, CBC, CBD,
2D-LC-UV-CL EtOAc 78 °C, 1.5 h with
Industrial-grade hemp CBL, CBG, CBE, CBT, CBNA, - [29]
HPLC-ESI-ToF Soxhlet
CBCA, CBDA, CBLA, CBGA
(tentative ID)
0.1 (CBD), 0.04 (CBDA),
THC, THCA, CBD, CBDA,
LC-APCI-IT Plant MeOH/CHCl3 9:1 (v/v) 0.03 (CBN), 0.28 (THC), [31]
CBN
9.9 g/kg (THCA)
Soxhlet or PLE with MeOH
GC-FID Seized plants THC, THCA, CBN - [32]
or hex
 Inflorescence (from CBG, CBN, CBD, CBC, THC, SLE with hex and
GC-MS - [6]
seized seeds) terpenes ultrasonication
Hemp products
Hex/iPrOH 9:1 (v/v) and
(pastilles, seeds, 1 (THC, CBN), 2 ng/mL
GC-MS THC, CBD, CBN derivatization with MSTFA [33]
scented grass, beer, CBD
and TMCS
liqueur, oil)
1 THC-type, CBD-type, THC, CBD, CBN, THCA, Fractionated extraction with LOQ 0.5 mg/g (of
H NMR
CBG-type, fiber CFL-A, CFL-B, phenols, EtOAc, EtOH, heptane, extract), 10 mg/mL for [34]
HPLC-DAD
(CBD)-type plants flavonoids MeOH peak separation
Bedrocan®, illicit
1 material, CBD-rich THC, THCA (semi- Hot and cold H2O extracts
H NMR - [35]
and non-cannabinoid quantitative) and tinctures (EtOH/H2O)
type plants
Cloud point extraction with
HPLC-DAD Resin THC Dowfax 20B102, deionized LOD 0.04 μg/mL [36]
H2O and Na2SO4
CBD, THCA, THCV, CBN,
THC, CBG (quantitative)
Cannabicoumaric acid, CBCA, LOD 0.2 (CBD, THCA)
HPLC-QTOF
Plants 10-EtO-9-OH-Δ6a-THC, 4- SFE (CO2/10%EtOH) 0.05 (THCV, CBN, THC), [5]
HPLC-QqQ
AcetoxyCBC, CBGA, 0.02 ng/mL (CBG)
CBGAM, THCA-C4 (tentative
ID)
FUSE with iPrOH:Chex 1:1
(v/v) or SFE CO2 (for
GC-MS Plants Terpenes, THC, CBN, CBD terpenes) then LOQ 1 μg/mL [37]
CO2/20%EtOH (for
cannabinoids)
Medicinal cannabis THCA, CBDA, THC, CBD,
UHPLC-QqQ Tea and oil extracts LOD 0.3, LOQ 1 μg/mL [39]
inflorescence CBC, CBG, CBN
 Medicinal cannabis
HPLC-DAD THCA, CBDA, THC, CBD, LOD 0.05, LOQ 0.1
inflorescence Oil and ethyl alcohol extracts [41]
HPLC-Q-ToF CBN μg/mL
(Bediol®)
Hexane for 10 days then
GC-MS Plant THC, CBD, CBN - [59]
sonication
UPLC-DAD-ESI-MS MeOH and ultrasonication
Herbal products Synthetic cannabinoids LOQ 10 μg/mL [60]
GC-EI-MS 10 min
Dried fresh
HPLC-DAD hemp plant, dried EtOAc and sonication 15
THCA, THC - [62]
GC-FID hemp flowers and min
hashish
HRGC-FID: 0.034 (THC),
0.041 (CBD), 0.026
HRGC-FID mg/mL (CBN)
Hashish THC, CBD, CBN MeOH [63]
HPLC-UV HPLC-UV: 0.044 (THC),
0.014 (CBD), 0.018
mg/mL (CBN)
Laser-microdissected MeOH, sonication 10 min LC-MS: LOQ 3 (CBD,
HPLC-QqQ THCA, CBDA, THC, CBD,
trichomes of medicinal and incubation o.n. at r.t. (for CBG, CBN), 30 ng/mL [111]
Cryogenic 1H NMR CBG, CBN, CBC
Cannabis (Bediol®) LC-MS), CDCl3 (for NMR) (CBC)
1 NMR: LOQ 0.2 (THCA,
H NMR Medicinal cannabis THCA, CBDA, THC, CBD, MeOH/CHCl3 9:1 (v/v) and
CBDA, CBD), 0.1 mg/mL [115]
GC-MS inflorescence CBN ultrasonication 2 min 4 °C
(THC, CBN)
Fractionated extraction with
MeOH/CHCl3 1:1 (v/v),
1 Medicinal cannabis THCA, CBDA, THC, CBD,
H NMR 90%MeOH, hex, then - [116]
inflorescence CBN, CBG, CFL-A, CFL-B
stepwise gradient of EtOH in
acetone
 Medicinal cannabis
Δ9-THCA, CBDA, CBNA,
HPLC-UV inflorescence MeOH and ultrasonication
1 CBGA, Δ9-THC, Δ8-THC, - [123]
H NMR (Bediol®, 10 min
CBD, CBG, CBN, CBC
Bedrobinol®)
1 Medicinal cannabis
H NMR THCA, THC, CBD 50%MeOH:CHCl3 1:1 (v/v) - [124]
inflorescence

Table 2. Analytical methods for the analysis of cannabis in biological samples

Analytical technique Matrix Identified analytes Extraction methodology Sensitivity Reference

Deproteination (ice-cold ACN), SPE:
2D-GC-EI-MS hex/acetone/EtOAc 60:30:20, v/v/v (THC,
(THC, THC-OH, THC-OH, CBD, CBN), hex/EtOAc/AcOH LOQ 0.25 (THC, THC-OH,
THC, THC-OH, THC-
CBD, CBN) OF 75:25:2.5, v/v/v (THC-COOH) and CBD), 1 μg/L (CBN), 5 ng/l [42]
COOH, CBD, CBN
2D-GC-NCI-MS derivatization: BSTFA (THC, THC-OH, (THC-COOH)
(THC-COOH) CBD, CBN) or HFIP and TFAA (THC-
COOH)
DDS buffer, 0.1 M Sørensen’s phosphate LOQ 1 (THC), 2 ng/mL
LC-ESI-QqQ OF THC, CBD, CBN [43]
buffer pH 6, hex/EtOAc 9:1 (v/v) 60 min (CBD, CBN)
THC, THC-OH, CBD, LOQ 0.2 μg/L (THC, THC-
Hydrolysis and SPE with
LC-APCI-Q-Trap OF CBG, THC-COOH, OH, CBD, CBG, THCV), 15 [44]
CH2Cl2:iPrOH:NH4OH 78:20:2 (v/v/v)
THCV ng/L (THC-COOH)
SPE with hex/acetone/EtOAc 60:30:20
2D-GC-EI-MS (v/v/v) for THC, THC-OH, CBD and CBN,
(THC, THC-OH, SPE with hex/EtOAc/AcOH 75:25:2.5 LOQ 0.5 (THC, THC-OH,
m THC, THC-OH, CBD,
CBD, CBN) OF (v/v/v) for THC-COOH, then derivatization CBD) and 1 ng/mL (CBN), [45]
CBN, THC-COOH
2D-GC-NCI-MS with BSTFA for THC, THC-OH, CBD and 7.5 pg/mL (THC-COOH)
(THC-COOH) CBN and with HFIP and TFAA for THC-
COOH
 LOQ 0.5 (THC, CBD, CBN)
THC, THC-COOH, Deproteination (ice-cold ACN) and SPE with
μ-flow-LC-Orbitrap OF m
and 0.015 ng/mL (THC- [46]
CBD, CBN hexane/EtOAc/AcOH 75:25:1 (v/v/v)
COOH)
LOQ 0.02 (THC-COOH),
Deproteination with 50 mM FA in MeOH
m THC, THC-OH, THC- 0.25 (THC), 0.30 (CBD,
LC-QqQ OF and elution on MEPS with 50 mM NH4OH [47]
COOH, CBD, CBN CBN) and 0.40 ng/mL
in MeOH
(THC-OH)
LOQ 0.25 (THC, THC-
Deproteination with ACN-d3, then on-line or
Blood, plasma THC, THC-OH, THC- COOH), 0.30 (THC-OH),
HPLC-ESI-μQqQ off-line derivatization with dabsyl chloride [49]
and serum COOH, CBD, CBN 0.40 (CBN) and 0.80 ng/mL
solution and 0.1 M NaOH
(CBD)
THC, CBN, THC- LOQ 1 (THC, THC-OH,
gluc, THC-COOH- Deproteination (MeOH, ACN) and SPE with THC-COOH, CBD, CBN),
LC-ESI-QqQ Whole blood [50]
gluc, THC-COOH, 1% AcOH in ACN (v/v) 0.5 (THC-gluc) and 5
THC-OH, CBD μg/mL (THC-COOH-gluc)
On-line SPE LC- Peripheral THC, THC-OH, THC- Deproteination with ACN and on-line SPE 0.50 (THC, THC-OH) and
[51]
QqQ blood COOH with ACN/H2O/FA 60:40:0.1 (v/v/v) 2.5 ng/mL (THC-COOH)
Whole blood,
urine, gall
bladder
fluid, LOQ 0.58 (THC), 0.47
cerebrospinal THC, THC-OH, THC- (THC-OH), 5.06 (THC-
Deproteination with ACN and addition of
LC-QqQ fluid, gastric COOH, THC-COOH- COOH), 41.1 (THC-COOH- [52]
0.25 M AcOH and EtOAc:hex 1:9 (v/v)
contents, gluc, CBD, CBN gluc), 0.60 (CBN), 0.44
cerebrum, ng/mL (CBD)
liver, lungs,
muscle and
kidneys
Plasma and Basic hydrolysis for urine, then SPE with LOQ 16 (THC) and 6.4
HPLC-UV THC, THC-COOH [3]
urine MeOH ng/mL (THC-COOH)
 LOD 6 (THC) and 2.5 ng/mL
(THC-COOH)
LOD 0.6 (THC), 1.1 (THC-
OH), 0.9 (THC-COOH,
Living and THC, CBD, CBN,
Protein precipitation with ACN and on-line CBD) and 2.5 ng/mL (CBN)
LC-QqQ post-mortem THC-OH, THC- [54]
SPE LOQ 1.8 (THC), 3.2 (THC-
whole blood COOH
OH), 2.8 (THC-COOH,
CBD) and 7.7 ng/mL (CBN)
Deproteination with ACN and SPE with
Fast LC-MS/MS Whole blood THC, THC-COOH LOQ 0.25 mg/mL [55]
EtOAc/hex 50:50 (v/v) and 2% acetic acid
Washing with MeOH, digestion with NaOH LOD 1 pg/mg
LC-ESI-QqQ Hair THC, THC-COOH [56]
2.5 M 60 °C and extraction with EtOAc LOQ 3 pg/mg
LOD 7 (CBD), 11 (CBN)
and 31 pg/mg (THC)
Washing with petroleum ether, H2O, DCM,
GC-IT Hair THC, CBD, CBN LOQ 0.012 (CBD), 0.030 [57]
digestion in 1 M NaOH and HS-SPME
(CBN) and 0.062 pg/mg
(THC)
LOD 0.012 (THC), 0.013
(CBD) and 0.016 ng/mg
Washing with H2O and acetone, digestion
(CBN)
GC-MS Hair THC, CBN, CBD with NaOH 1 M 80 °C, HS-SPME with iso- [58]
LOQ 0.037 (THC), 0.038
octane and derivatization with BSTFA
(CBD) and 0.048 ng/mg
(CBN)
LOQ 0.15-0.29 (THC, 11-
THC, THC-OH, THC-
GC-MS Plasma SPE and derivatization with BSTFA OH-THC, THC-COOH, [64]
COOH, CBD, CBN
CBD) and 1.1 ng/mL (CBN)
THC-OH, THC- LOD 5 ng/g
GC-MS Meconium SPE and hydrolysis [65]
COOH LOQ 10 ng/g
Washing with iPrOH, hydrolysis with 1 M LOD 0.02 pg/mg
GC-NCI-QqQ Hair THC-COOH [66]
NaOH (95 °C, 30 min), acidification, LOQ 0.05 pg/mg
 extraction with hex/EtOAc 9:1 (v/v) and
derivatization with PFPOH and PFPA
LOQ 0.5 (THC, THC-OH)
THC, THC-OH, THC- Hex/EtOAc 9:1 (v/v), then derivatization
Fast GC/NIC-QqQ Whole blood and 2.5 ng/mL (THC- [67]
COOH with TFAA and HFIP
COOH)
THC, CBD, CBN, 1 M phosphate buffer pH 4 and LLE with LOQ 0.25 (THC, CBN,
Post-mortem
2D-GC-EI-QqQ THC-OH, THC- hex/EtOAc 5:1 (v/v) and derivatization with THC-OH) and 0.5 ng/mL [68]
blood
COOH MSTFA (CBD, THC-COOH)
2D-GC: Enzymatic hydrolysis, deproteination with LOQ 0.125 (THC, THC-
THC, THC-OH, THC-
GC-FID and GC- Plasma ice-cold ACN, then SPE with EtOH and COOH) and 0.25 ng/mL [69]
COOH
cryotrap-MS derivatization with BSTFA (THC-OH)

THC, THC-OH, THC- Dilution with 100 mM ammonium acetate LOD 1 (THC-COOH) and
LC-QqQ Breast milk [70]
COOH pH 5.5 and SPE with MeOH 1.5 ng/mL (THC, THC-OH)
LOQ 5 ng/mL
LC-QqQ (THCA)
LOD 2.5 ng/mL
GC-MS (THC, Urine and THC, THCA, THC-
SPE with ACN LOQ 5 ng/mL in urine and [71]
THC-OH, THC- serum OH, THC-COOH
7.5 ng/mL in serum
COOH)
LOD 0.2 (THC, THC-OH)
and 1.6 ng/mL (THC-
THC, THC-OH, THC- COOH)
LC-QqQ Plasma SPE with MeOH and 0.1 M AcOH [72]
COOH LOQ 0.8 (THC, 11-OH-
THC) and4.3 ng/mL (THC-
COOH)
EDTA-plasma: anion exchange sorbent SPE LOD 0.1 (EDTA-plasma)
EDTA-
THC, THC-OH, THC- with ACN:ammonia 98:2 (v/v) and 0.5-1 ng/mL (urine)
LC-APCI-IT plasma and [73]
COOH, CBD, CBN Urine: enzymatic hydrolysis and LLE with LOQ 0.2 (EDTA-plasma)
urine
Et2O:EtOAc 50% (v/v) and 1-3 ng/mL (urine)
 THC, THC-OH, THC-
COOH, CBN, CBD, Dilution with 10 mM ammonium formate LOD 0.05-0.5 ng/mL
LC-ESI-QqQ Bile [74]
THCA, THC-COOH- buffer pH 6.5 and SPE with MeOH LOQ 0.3-0.8 ng/mL
gluc, THC-gluc
LOD 2 in OF (THC), 0.5 in
urine and blood (THC, THC-
COOH) and 20 ng/mL in
OF and urine: SPE with hex/EtOAc 80:20
OF, urine and THC, THC-OH, THC- blood and urine (THC-OH)
LC-ESI-MS (v/v) [75]
whole blood COOH LOQ 5 in OF (THC), 2 in
Blood: deproteination with ACN then SPE
urine and blood (THC, THC-
COOH) and 25 ng/mL in
blood and urine (THC-OH)
LOD and LOQ 0.5 (THC), 1
THC, THC-OH, THC- Deproteination with MeOH and extraction
LC-ESI-QqQ Whole blood (THC-OH) and 2 μg/L [76]
COOH with hex:EtOAc 90:10 (v/v)
(THC-COOH)
LOD and LLOQ 2.5 (THCA)
THCA, CBD, CBN, Washing with H2O, acetone and petroleum
LC-ESI-QqQ Hair and 20 pg/mg (THC, CBD, [77]
THC ether and extraction with MeOH
CBN)
LOD and LOQ 1 (THC,
THC, THC-OH, THC- Deproteination with MeOH and SPE with
LC-QqQ Whole blood THC-OH) and [79]
COOH hex/EtOAc/AcOH 88:10:2 (v/v/v)
5 ng/mL (THC-COOH)
THCA (and other Enzymatic hydrolysis and extraction with LOD 1 ng/mL
UPLC-ESI-QqQ Urine [84]
drugs of abuse) MeOH/H2O 60:40 (v/v) LOQ 2 ng/mL
Deproteination with MeOH and SPE with
UPLC-ESI-ToF Whole blood THC Not specified [85]
25%NH4OH/ACN/EtOAc 2:10:88 (v/v)
Synthetic
Whole blood LOD 0.08-0.13 ng/mL
UPLC-QqQ cannabinoids and their SPE with EtOAc [87]
and urine LOQ 0.11-0.17 ng/mL
metabolites
Post-mortem Synthetic LOD 0.01 ng/mL
UPLC-ESI-QqQ Na2CO3 buffer pH 10.2 and Et2O [89]
whole blood cannabinoids LOQ 0.05 ng/mL
 OF: LOD 0.25 ng/mL; LOQ
5 ng/mL (THC, CBD, CBN)
THC, CBD, CBN
UPLC-ESI-QqQ OF: DrugWipe5S® Serum: LOD 0.6 (THC), 0.3
(OF)
(OF) OF and serum Serum: SPE with methanol and (THC-OH) and 0.3 ng/mL [94]
THC, THC-OH, THC-
GC-MS (serum) derivatization with MSTFA (THC-COOH); LOQ 1
COOH (serum)
(THC), 0.3 (THC-OH) and 3
ng/mL (THC- COOH)
LOD 0.02 (THC), 0.05
(THC-OH) and 0.1 ng/mL
THC, THC-OH, THC- Deproteination with ACN and SPE with (THC-COOH)
UPLC-ESI-QqQ Whole blood [95]
COOH hex:EtOAc 80:20 (v/v) LOQ 0.05 (THC), 0.1 (THC-
OH) and 0.2 ng/mL (THC-
COOH)
LOD and LOQ 0.5 μg/L
THC, THC-OH, THC- Deproteination with MeOH and SPE with (THC-OH, THC-COOH)
UPLC-ESI-QqQ Whole blood [110]
COOH hex/EtOAc/AcOH 88:10:2 (v/v/v) LOD 0.2 and LOQ 0.5 μg/L
(THC)