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Chromatographic and Spectroscopic Data of Cannabinoids from Cannabis sativa L

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  • Hazekamp Herbal Consulting

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Chromatographic and spectroscopic data was determined for 16 different major cannabinoids from Cannabis sativa plant material as well as 2 human metabolites of Δ‐tetrahydrocannabinol. Spectroscopic analysis included UV absorbance, infrared‐spectral analysis, (GC‐) mass spectrometry, and spectrophotometric analysis. Also, the fluorescent properties of the cannabinoids are presented. Most of this data is available from literature but scattered over a large amount of scientific papers. In this case, analyses were carried out under standardised conditions for each tested cannabinoid so spectroscopic data can be directly compared. Different methods for the analysis of cannabis preparations were used and are discussed for their usefulness in the identification and determination of separate cannabinoids. Data on the retention of the cannabinoids in HPLC, GC, and TLC are presented.
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Chromatographic and Spectroscopic Data of
Cannabinoids from Cannabis sativa L.
Arno Hazekamp, Anja Peltenburg, and Rob Verpoorte
Division of Pharmacognosy, Institute of Biology, Leiden University,
Leiden, The Netherlands
Christian Giroud
Laboratoire de Toxicologie et de Chimie Forensiques, Institute
Universitaire de Me
´
decine le
´
gale, Lausanne, Switzerland
Abstract: Chromatographic and spectroscopic data was determined for 16 different
major cannabinoids from Cannabis sativa plant material as well as 2 human meta-
bolites of D
9
-tetrahydrocannabinol. Spectroscopic analysis included UV absorbance,
infrared-spectral analysis, (GC-) mass spectrometry, and spectrophotometric analysis.
Also, the fluorescent properties of the cannabinoids are presented. Most of this data
is available from literature but scattered over a large amount of scientific papers.
In this case, analyses were carried out under standardised conditions for each tested
cannabinoid so spectroscopic data can be directly compared. Different methods for
the analysis of cannabis preparations were used and are discussed for their usefulness
in the identification and determination of separate cannabinoids. Data on the retention
of the cannabinoids in HPLC, GC, and TLC are presented.
Keywords: Cannabis sativa, Plant material, Cannabinoids, Metabolites, Chromato-
graphic data, Spectroscopic data
INTRODUCTION
In recent years a lot of research on the medical applications of Cannabis sativa L.
has been initiated, as several, mostly European countries, move towards a more
Address correspondence to Arno Hazekamp, Division of Pharmacognosy, Institute
of Biology, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands.
E-mail: ahazekamp@rocketmail.com
Journal of Liquid Chromatography & Related Technologies
w
, 28: 23612382, 2005
Copyright # Taylor & Francis, Inc.
ISSN 1082-6076 print/1520-572X online
DOI: 10.1080/10826070500187558
2361
liberal view on the use of Cannabis as a medicine.
[1]
Although more than 400
compounds have been identified in the Cannabis plant,
[2]
most studies have
focused on the effects of the cannabinoids, in particular (2 )-D
9
-(trans)-tetrahy-
drocannabinol (D
9
-THC). One reason is that the main pharmacological and
psychoactive effects of Cannabis have been attributed to D
9
-THC. For
instance, synthetic D
9
-THC (dronabinol, Marinol
TM
) has been shown to
possess anti-emetic properties useful in cancer therapy. However, in several
medical studies the effect of D
9
-THC or dronabinol alone could not match the
effects of a total Cannabis preparation,
[3]
indicating there might be other
active compounds present.
[4]
More than 60 cannabinoids
[2,5 7]
have been
identified in Cannabis, and occasionally new cannabinoids are being discov-
ered.
[8]
The chemical structures of the main cannabinoids from the Cannabis
plant are shown in Figure 1, and their physical properties are listed in Table 1.
Only a few of these cannabinoids have been studied in detail, although several
of these have been shown to possess some biological activity (reviewed by
[9]
).
To extend the knowledge of the therapeutic properties to cannabinoids
other than D
9
-THC, large amounts of pure compounds must be available.
Assessment of cannabinoids pharmacology is now almost restricted to the
few that are commercially available (e.g., D
8
-THC, D
9
-THC, CBD, and
CBN). Furthermore, pure cannabinoids must be available as reference
compounds for their unequivocal identification and determination. For that
purpose, chromatographic and spectroscopic methods and data are available
from scientific literature. Although these data have been published for most
known cannabinoids during isolation and identification experiments (see
[2]
for an overview), they are scattered over a huge amount of scientific papers.
Moreover, standardised data obtained under identical analytical conditions
have not been reported yet. As far as we know, the fluorescent properties of
the cannabinoids remain largely unknown.
[10]
This report lists the main chromatographic and spectroscopic data of 16
main cannabinoids and of two of their human metabolites obtained under
identical analytical conditions. Methods were kept as straightforward,
simple, and rapid as possible. The pros and cons of each method will also
be discussed. All analyses were carried out for each cannabinoid as far as
permitted by the amount available to us.
EXPERIMENTAL
Standards and Solvents
Reference compounds of (2 )-D
9
-tetrahydrocannabinol (D
9
-THC), cannabinol
(CBN), cannabidiol (CBD), cannabigerol (CBG), (2 )-D
9
-(trans)-tetra-
hydrocannabinolic acid A (THCA), cannabidiolic acid (CBDA), and cannabi-
gerolic acid (CBGA) were isolated from plant materials previously in our
laboratory.
[11]
A quantitative
1
H-NMR method was developed for their quanti-
A. Hazekamp et al.2362
fication.
[12]
(2 )-D
8
-tetrahydrocannabinol (D
8
-THC) was obtained from
Sigma. The main human metabolites of D
9
-THC, i.e., 11-hydroxy-THC (11-
OH-THC) and 11-carboxy-THC (THC-COOH) were purchased from
Cambridge isotope laboratories (CIL, Innerberg, Switzerland) and from
Figure 1. Structures of the cannabinoids.
Chromatographic and Spectroscopic Data of Cannabinoids 2363
Lipomed (Arlesheim, Switzerland), respectively. All these cannabinoids were
available as certified and calibrated reference standards. The other cannabi-
noids used for this study were obtained by preparative HPLC on extracts of
Cannabis sativa plant materials and identified by comparing their chromato-
graphic and spectroscopic data with literature,
[13 15]
and by a search in
Table 1. Physicochemical properties of the cannabinoids
# Cannabinoid Full name (description) MW (calc.)
Molecular for-
mula
CHO
Neutral cannabinoids
1 d9-THC trans-(2 )-delta-9-tetrahy-
drocannabinol
314.472 21 30 2
2 d8-THC trans-(2 )-delta-8-tetrahy-
drocannabinol
314.472 21 30 2
3 THV trans-(2 )-delta-9-tetra-
hydrocannabivarin
(C3-isomer of THC)
286.418 19 26 2
4 CBD cannabidiol 314.472 21 30 2
5 CBN cannabinol 310.440 21 26 2
6 CBG cannabigerol 316.488 21 32 2
7 CBC cannabichromene 314.472 21 30 2
8 CBL cannabicyclol 314.472 21 30 2
Acidic cannabinoids
9 THCA trans-(2 )-delta-9-tetra-
hydrocannabinolic acid A
358.482 22 30 4
10 THCA-C4 trans-(2 )-delta-9-tetra-
hydrocannabinolic acid-
C4 (C4-isomer of THCA)
344.455 21 28 4
11 THVA trans-(2 )-delta-9-tetra-
hydrocannabivarinic acid
(C3-isomer of THCA)
330.428 20 26 4
12 CBDA cannabidiolic acid 358.482 22 30 4
13 CBNA cannabinolic acid 354.450 22 26 4
14 CBGA cannabigerolic acid 360.498 22 32 4
15 CBCA cannabichromenic acid 358.482 22 30 4
16 CBLA cannabicyclolic acid 358.482 22 30 4
Human metabolites
17 11-OH-THC 11-hydroxy-tetrahydrocan-
nabinol (metabolite of
THC)
330.471 21 30 3
18 THC-COOH 11-carboxy-tetrahydrocan-
nabinol (metabolite of
THC)
344.455 21 28 4
A. Hazekamp et al.2364
UV
[16]
and mass spectra databases.
[17,18]
All organic solvents (analytical
or HPLC reagent grade) were purchased from J.T. Baker (Deventer,
The Netherlands) or from Fluka Chemie (Buchs, Switzerland).
Thin Layer Chromatography (TLC)
Samples in ethanol were spotted on 10 20 cm silica plates. Two different
TLC systems were used. For the non-polar system, reversed phase (C
18
)
silicagel plates F254 No. 105559 (Merck, Darmstadt, Germany) were used
with methanol/5% acetic acid 19 : 1 (v/v) as the eluent. For the polar
system, normal phase silicagel plates F254 No. 105554 (Merck, Darmstadt,
Germany) were used with chloroform/methanol 19 : 1 (v/v) as the eluent.
Plates were developed in saturated normal chambers (saturation time 15
minutes). Absorption of chromatographic spots was evaluated under UV
254 nm. General visualisation of compounds was done by spraying with
modified anisaldehyde-sulphuric acid spray reagent.
[19]
For selective visual-
isation of cannabinoids, the TLC plate was sprayed with 0.5% fast blue B
salt (o-dianisidine-bis-(diazotized)-zinc double salt) (Sigma) in water,
followed by 0.1 M NaOH.
[20]
Gas Chromatography-Mass Spectrometry (GC-MS)
To obtain GC retention times, molecular weights, and fragmentation spectra
of cannabinoids, GC-MS analyses were performed on a Varian 3800 gas
chromatograph, coupled to a Varian Saturn 2000 GC/MS apparatus. The
system was controlled with Varian Saturn GC/MS workstation version 5.2
software. The GC was fitted with two different types of columns; a
Durabond fused silica capillary column (30 m 0.25 mm inner diameter)
coated with DB-1 at a film thickness of 0.1 mm, and a similar column,
coated with HP-50þ at a film thickness of 0.15 mm (J&W Scientific Inc.,
Rancho Cordova, CA). The oven temperature was programmed from 1008C
to 2808C at a rate of 108C/min. The oven was then kept at 2808C until the
end of a 30 min run time. The injector and detector port temperatures were
maintained at 2808 C and 2908C, respectively. Helium was used as the
carrier gas at a pressure of 65 kPa. The injection split ratio was 1/50.
Elution time of D
9
-THC was used as internal reference to determine the
relative retention times of all cannabinoids.
High-Performance Liquid Chromatography (HPLC)
with Diode-Array and Fluorescence Detection
The HPLC profiles were acquired on an Agilent 1100 series HPLC, consisting
of a G1322A solvent degasser, a G1311A quaternary solvent pump, and a
Chromatographic and Spectroscopic Data of Cannabinoids 2365
G1313A autosampler. The column was kept at constant temperature by using a
G1316A column oven. The analytical column was a Waters XTerra MS C
18
(2.1 150 mm, 3.5 mm) fitted with a XTerra MS C
18
(2.1 10 mm,
3.5 mm) guard column. Light absorption and emission were detected by a
G1315B UV-diode array detector (DAD) and a G1321A fluorescence
detector (FLD). The system was controlled through a Vectra VL 420 DT
computer equipped with Agilent A09.01 software. UV-spectra were
measured on-line by DAD in the range of 195 400 nm with a slit of 2 nm.
Fluorescence (FL) spectra were recorded on-line by the FLD in the range of
280650 nm with a step of 5 nm after excitation at 222 nm. Retention times
were expressed as relative to D
9
-THC.
DAD and FLD data of cannabinoids were recorded under acidic con-
ditions, with a mobile phase consisting of a mixture of methanol-water
containing 25 mM of formic acid (pH + 3). The proportion of methanol
was linearly increased from 65 to 100% over 25 minutes, and then kept
constant for 3 minutes. Analysis under basic conditions was obtained with a
mobile phase consisting of a mixture of acetonitrile-phosphate buffer
(10 mM, pH 7.5). The acetonitrile concentration was increased from 40 to
100% in 25 minutes, and then kept constant for 3 minutes. After each run
the column was re-equilibrated under initial conditions for 10 minutes. The
flow rate was 0.3 mL/min and the total run time was 38 minutes. All determi-
nations were carried out at 308C.
Spectrophotometric Analysis (Extinction Coefficients)
Cannabinoids that were available as calibrated certified standards were diluted
to a concentration of 0.01 mg/ mL in ethanol to determine molar extinction
coefficients in the range of 200 to 400 nm. A blank measurement was
obtained with ethanol. UV-spectra were recorded using a Varian Cary 1 Bio
UV-Visible spectrophotometer controlled by Cary 1/3E system software,
version 3.02. A sample cell of 10 mm was used for all measurements.
Infrared Spectroscopy (IR)
Infrared spectra of cannabinoids that were available in sufficient amounts were
measured using a Perkin Elmer paragon 1000PC FT-IR instrument, which was
controlled by Perkin Elmer spectrum IR V2.00 software. Concentrated
ethanolic solutions of the cannabinoids (25 mL) were mixed with finely
ground KBr (Merck, IR-grade), and ethanol was evaporated under vacuum
for 10 minutes. After proper calibration of the apparatus, IR-spectra were
measured as an average of 4 scans in the wave number range of 500 to
4000 cm
21
. After acquisition, the spectra were smoothened by using the
software.
A. Hazekamp et al.2366
RESULTS AND DISCUSSION
Spectroscopic and chromatographic data is shown for 18 different cannabi-
noids that were available to us. However, not all cannabinoids were
available in large enough quantities to obtain exploitable data in all
analyses that were carried out. Therefore, the presented data is not complete
for all cannabinoids.
TLC
By using two TLC-systems (polar and apolar system) in combination with fast
blue B spray reagent, it was possible to distinguish and detect all tested
compounds. The Rf-values of the cannabinoids in both TLC-systems and
their spot colour after spraying with fast blue B are shown in Table 2. The
use of fast blue B as a selective detection reagent for cannabinoids
[20]
results in differently coloured spots for some compounds. Unfortunately,
these colours also depend on the concentration of the substance and on the
presence of interfering compounds, the results must be, therefore, considered
with caution. Nevertheless, we found that fast blue B was more sensitive for
detection of cannabinoid spots than UV-detection under 254 nm. For example,
the detection limit for D
9
-THC was 0.5 mg/mL (2 mL spotted) with UV-
detection under 254 nm, and around 0.002 mg/mL with fast blue B detection.
The main advantages of TLC are its ability to detect all spotted
compounds, while analysing several samples simultaneously under identical
conditions within a short timeframe. Lack in selectivity can sometimes be
Table 2. Relative retention (Rf) values of the cannabinoids in a polar (silica-gel) and
non-polar (C
18
) TLC-system. The colours of chromatographic spots after spraying with
the cannabinoid-selective spray reagent fast blue B (FBB) are indicated
Color FBB
Nonpolar TLC system
(RP-18) Polar TLC system (silica)
Cannabinoid Rf-value Cannabinoid Rf-value
Red CBDA 0.68 D
9
-THC 0.65
Brown CBGA 0.67 D
8
-THC 0.65
Orange-brown CBG 0.59 CBD 0.64
red-brown CBD 0.58 CBN 0.62
Purple CBN 0.48 CBG 0.61
Red D
9
-THC 0.44 CBC 0.58
Red D
8
-THC 0.43 THCA 0.39
Red THCA 0.40 CBDA 0.37
Purple CBC 0.37 CBGA 0.31
Purple CBCA 0.35 CBCA 0.25
Chromatographic and Spectroscopic Data of Cannabinoids 2367
overcome by the use of selective detection reagents. However, in the case of
cannabinoids it seems impossible to obtain a good separation with positive
identification of all cannabinoids when complex mixtures (e.g., plant
extracts) are analysed. Several TLC systems are therefore needed for
tentative identification. For instance, CBDA and CBGA or CBD and CBG,
which were not separated in the non polar system, could be distinguished
when using silica as stationary phase. On the other hand, D
8
-THC and
D
9
-THC were found to co-elute on both systems (see Table 2). In conclusion,
TLC is very useful to rapidly screen many samples for the presence of canna-
binoids in crude plant extracts, or in eluting fractions collected after prepara-
tive chromatography. However, reproducibility of TLC depends on several
parameters, e.g., relative humidity. Compared to other separation methods,
TLC performances are also very low. Consequently, unequivocal identifi-
cation of cannabinoids spots requires further methods.
GC/MS
Two different capillary column phases were used for GC analysis (HP-50þ
and DB-1). The HP-50þ column was a medium-polar column, resulting in
relatively longer retention times compared to the nonpolar DB-1 column.
Simultaneous injection on both columns enables the distinction of all tested
cannabinoids. Retention times (relative to D
9
-THC) of the analysed cannabi-
noids are shown in Table 3. All cannabinoids eluted well after other major
cannabis components like terpenoids.
Because no derivatization was used in our case, the mass-spectra obtained
by GC-MS (Figure 2) are similar for the acids and their corresponding neutral
Table 3. Relative retention time (RRT) of
cannabinoids in GC using a non-polar (DB-1)
and medium-polar (HP-50) column
GC column type
DB-1 HP-50
Cannabinoid RRT RRT
THV 0.885 0.902
CBL 0.922 0.907
CBD 0.942 0.935
THC-C4 0.942 0.948
CBC 0.956 0.924
D
8
-THC 0.988 0.981
D
9
-THC 1 1
CBG 1.026 1.012
CBN 1.033 1.046
A. Hazekamp et al.2368
Figure 2. Mass-spectra in the range of M/Z 50-335 obtained by LC-MS.
(continued)
Chromatographic and Spectroscopic Data of Cannabinoids 2369
Figure 2. Continued.
A. Hazekamp et al.2370
cannabinoids (e.g., THCA and D
9
-THC). Although CBD is structurally quite
distinct from CBC and CBL, these three cannabinoids nonetheless show
similar MS spectra (compare spectra of Figure 2) with identical base peak
(m/z ¼ 231) and molecular ion (m/z ¼ 314). Also their retention times in
GC were quite similar (Table 3), but their separation is good enough to dis-
tinguish them. Cannabidiol differs from CBC and CBL with one significant
fragment at m/z ¼ 246. A retro-Diels-Alder reaction accounts for the
formation of the minor ion at m/z ¼ 246. Subsequent loss of a methyl
fragment results in a contribution to the ion at m/z ¼ 231.
[15]
As can be
seen in Figure 2, the base peak of all tested cannabinoids (except D
8
-THC)
doesn’t correspond to the molecular ion, but to a fragment, indicating that
these cannabinoids are easily fragmented by GC-MS.
In the absence of derivatization, the high temperature that is applied in
GC causes the decarboxylation of acidic cannabinoids to their corresponding
neutral form.
[21]
Since the cannabis plant mainly contains the (carboxylic-)
acidic forms of cannabinoids,
[22]
GC analysis is not the method of choice
to establish the metabolic profile of a cannabinoid sample. To avoid
decarboxylation, the acids must be derivatized, e.g., by silylation or
formation of the alkylboronates.
[19]
However, a 100% derivatization yield
is difficult to obtain. Moreover, we believe that thermo-degradation
(oxidation, isomerization) of cannabinoids in the injector port and column
may also occur. In the case of D
9
-THC, a significant amount of D
8
-THC
and CBN was detected in the GC-chromatogram, whereas other analyses
(HPLC, NMR, TLC) did not show these compounds, which are known degra-
dation products of D
9
-THC (data not shown). Despite these problems associ-
ated with GC, it remains a very useful method for the analysis of
cannabinoids.
[21]
HPLC with UV/FLD Detection
With gradient-elution, most cannabinoids were base-line separated as sharp
peaks with excellent peak purity level, yielding fully exploitable UV and
fluorescence spectra. The retention times of cannabinoids (relative to
D
9
-THC) are shown in Table 4. It is interesting to note that the relative
elution time of the acidic cannabinoids can be influenced by changing the
pH of the eluent, while the order of elution for the neutral cannabinoids
remains the same.
[23]
Notwithstanding these pH differences, the elution
order of THCCOOH and THC was not modified. In this way, overlap
between chromatographic peaks of acid and neutral cannabinoids can be
decreased by changing the elution pH.
Although the UV-spectra of the analysed cannabinoids (Figure 3a) were
left unchanged when the pH was changed from 3.0 to 7.5, the FL-spectra differ
drastically (Figure 3b). Acidic cannabinoids completely lose their fluor-
escence under acidic conditions, while CBC has no fluorescence under
Chromatographic and Spectroscopic Data of Cannabinoids 2371
basic conditions, and CBN has no fluorescent properties at all. The fluorescent
properties of the other analysed cannabinoids are not influenced by pH. The
UV absorption and FL yield in Figure 3a and b cannot be compared
because no standardised concentrations of the cannabinoids were used.
Standardised UV-spectra were obtained using a spectrophotometer (see
below and Figure 4).
In some cases, partially unresolved peaks could not be identified because
their UV and fluorescence spectra were identical. This can be seen with Table 4
and in Figure 3a and b with CBD and CBG or D
8
-THC and D
9
-THC, which are
characterised by very close retention times and identical UV and fluorescence
spectra.
The chromophore of the cannabinoids corresponds to its substituted
phenolic ring, as this is a common structural element among the tested canna-
binoids. The UV spectrum of D
9
-THC is identical to that of olivetol, which
shows the same phenolic ring structure and is the precursor of D
9
-THC
and the other cannabinoids. The alkyl-side chain does not influence the
UV-absorbance, as there is no difference between THCA (C
5
-side chain)
and THVA (C
3
-side chain). The cyclization of the non-phenolic part of the
cannabinoids also has no influence on the absorbance, except when another
aromatic ring (CBN, CBNA) or a conjugated double bond (CBC, CBCA) is
introduced.
Table 4. Relative retention time (RRT) of cannabinoids in HPLC using a
reversed phase column (C
18
) and eluent with a basic (7.5) or acidic (3) pH
Acidic HPLC system Basic HPLC system
Cannabinoid RRT Cannabinoid RRT
11-OH-THC 0.70 THC-COOH 0.26
THC-COOH 0.76 CBDA 0.34
CBD 0.76 THVA 0.36
THV 0.77 CBGA 0.40
CBG 0.78 THCA-C4 0.42
CBDA 0.82 CBNA 0.50
CBGA 0.92 THCA-A 0.51
CBN 0.93 CBLA 0.53
D
9
-THC 1.00 CBCA 0.61
D
8
-THC 1.03 CBD 0.83
THVA 1.04 CBG 0.83
CBC 1.12 CBN 0.95
THCA-C4 1.13 D
9
-THC 1.00
CBNA 1.21 D
8
-THC 1.01
THCA-A 1.25 CBC 1.08
CBLA 1.32 11-OH-THC 1.31
CBCA 1.34
A. Hazekamp et al.2372
Figure 3. (a) UV-spectra in the range of 190400 nm obtained in two HPLC-systems
with acidic and basic pH. (b) Fluorescence spectra in the range of 280 650 nm
obtained in two HPLC-systems with acidic and basic pH.
(continued)
Chromatographic and Spectroscopic Data of Cannabinoids 2373
Figure 3. Continued.
A. Hazekamp et al.2374
Figure 3. Continued.
Chromatographic and Spectroscopic Data of Cannabinoids 2375
In the case of HPLC peak overlap, the use of MS-detection coupled to
HPLC (LC-MS) and furthermore, LC-MS-MS can provide better clues about
cannabinoid structure and identity. In the acid system (pH ¼ 3), formic acid
was used to make the eluent compatible with mass spectrometry. In contrast
to HPLC-DAD or Fl, which are carried out at room temperature, LC-MS
with ionspray ionisation at a relatively high temperature (e.g., 5008 C) may
result in partial thermal decomposition of acid cannabinoids. An example of
a LC-MS separation of a large array of THC metabolites in body fluids at a con-
centration of 50 ng/mL is shown in Figure 6. For separation, we used a Waters
XTerra C
8
microbore column. In contrast to GC-MS operating in the EI mode,
the mass spectra are very simple with one prominent [MH]
þ
or [M-H]
2
pseudo-
molecular ion and very little fragmentation. For better sensitivity, the data were
recorded in the Selected Ion Monitoring (SIM) mode. Except THC
([MH]
þ
¼ 315.2), all cannabinoids were measured in the negative ionisation
mode. The monohydroxylated (8
b
-OH- and 11-OH-THC) and dihydroxylated
8
b
-11-diOH THC metabolites were well resolved from the acid inactive metab-
olite (THCCOOH) and its conjugated derivative (THCCOOH-glucuronide).
Spectrophotometric Analysis (Extinction Coefficients)
Very few UV-absorption spectra of calibrated cannabinoids are given in the
scientific literature.
[16]
They are generally characterised by a few parameters
Figure 3. Continued.
A. Hazekamp et al.2376
(maxima and minima, shoulders of the UV spectra). The extinction coeffi-
cients are very seldom presented. Because most cannabinoids differ in their
UV with several absorption peaks, many wavelengths can be selected for
quantification. Figure 5 shows that absorption generally decreases with
increasing wavelength. A better sensitivity can be obtained in the low
200210 nm range, while selecting a higher wavelength will increase the
selectivity by diminishing the risk of measuring interfering compounds. In
order to get a rough estimate on the concentration of cannabinoids from any
selected wavelength, a spectrum measured at 0.01 mg/ mL between 200 and
400 nm is presented for 7 major cannabinoids (Figure 5). The extinction coef-
ficients (1) at 3 different maxima are also indicated.
Infrared Spectroscopy (IR)
Infrared spectroscopy has been a common tool for the identification and
structure elucidation of cannabinoids and derivatives in isolation and
synthesis experiments. As with UV-spectra, usually IR-spectra are reported
by presenting a few maximum absorbance peaks only. Obviously, reported
IR-spectra have been measured with a large variety of IR-spectrometers. In
Figure 4. Chromatogram of a separation and identification of cannabinoid metab-
olites from human blood in a single chromatographic run, by using LC-MS. All canna-
binoids can be identified because of the high selectivity of the mass-detector. The top
chromatogram shows the total ion current (TIC).
Chromatographic and Spectroscopic Data of Cannabinoids 2377
Figure 5. Extinction coefficients in the range of 200 400 nm at a concentration of
0.01 mg/mL in ethanol. Absorption values at maxima or shoulders are indicated.
A. Hazekamp et al.2378
this report (Figure 6) we present the full IR-spectra of 8 common natural can-
nabinoids measured on a single modern FT-IR-spectrometer.
CONCLUSION
A growing interest in Cannabis as a source of medicinal compounds has
emerged during the last few years. Several crude preparations or synthetic
Figure 6. IR-spectra in the range of 500 4000 cm
21
obtained by Fourier-transform
(FT)-IR spectrometry.
(continued)
Chromatographic and Spectroscopic Data of Cannabinoids 2379
drugs derived from Cannabis are under development, or in the clinical
pipeline for distribution on the market. For carrying out all these investi-
gations, pharmacologically pure cannabinoids must be available in large
quantities. Reference compounds for analytical research must also be
present. Chromatographic and spectroscopic data are, therefore, a prerequi-
site for their determination and identification.
The analytical data presented here makes it possible to positively identify
the major cannabinoids found in the Cannabis plant. Presenting all analytical
parameters measured under standardised conditions should facilitate the
Figure 6. Continued.
A. Hazekamp et al.2380
identification of cannabinoids isolated from, or present in, Cannabis prep-
arations. Unequivocal identification of cannabinoids cannot totally rely on
only one of the tested methods because confusion of some common cannabi-
noids always remains possible.
Finally, we believe that the use of LC-MS, and especially LC-MS-MS,
should make it possible to identify all tested cannabinoids in one single
analysis, even in the low ng/mL concentration range.
ACKNOWLEDGMENTS
Mr Pascal Cardinal, chemist, University of Alberta, Canada, is thanked for his
fruitful discussion. The grower of certified cannabis plants, Bedrocan BV, The
Netherlands, is acknowledged for providing cannabis plant material.
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Received October 1, 2004
Accepted March 30, 2005
Manuscript 6511
A. Hazekamp et al.2382
... However, HPLC and LC-coupled chromatography techniques entail several limitations for cannabinoid identification. First, in HPLC with UV/FLD detection, acidic cannabinoids lose fluorescence abilities under acidic conditions (Hazekamp et al., 2005). ...
... HPLC coupled with UV (Zgair et al., 2015), diode-array detector (UV/DAD) (Ambach et al., 2014;De Backer et al., 2009;Hädener et al., 2019;Peschel & Politi, 2015), flame ionization detection (FID) (Gambaro et al., 2002), and electrospray ionization coupled with mass spectrometry detector (ESI-MS) (Berman et al., 2018) have also been utilized for identifying cannabinoids in hemp (Brighenti et al., 2017;Pellati et al., 2018). However, ion spray ionization is conducted at a high temperature (e.g., 500ºC), which can cause the partial thermal decomposition of acid cannabinoids (Hazekamp et al., 2005), which will distort the reconstruction of the original chemical composition conveyed by the plant. In addition, overpressured layer chromatographic (OPLC) methods were examined for cannabinoid identification, which combines the HPLC interface with the capacity of flash chromatography and multidimensionally of TLC (Hidvégi, 2013;Szabady et al., 2002). ...
... In addition, ∆ 9 -THC undergoes thermal degradation (i.e., oxidation, isomerization), which results in a significant detectable amount of ∆ 8 -THC and cannabinol (CBN), the ∆ 9 -THC prime degradation by-products. Despite these limitations, GC still remains a useful technique for the analysis of cannabis (Hazekamp et al., 2005). ...
Article
Half of the harvested food is lost due to rots caused by microorganisms. Plants emit various volatile organic compounds (VOCs) into their surrounding environment, and the VOC profiles of healthy crops are altered upon infection. In this study, a whole-cell bacterial biosensor was used for the early identification of potato tuber soft rot disease caused by the pectinolytic bacteria Pectobacterium in potato tubers. The detection is based on monitoring the luminescent responses of the bacteria panel to changes in the VOC profile following inoculation. First, gas chromatography-mass spectrometry (GC-MS) was used to specify the differences between the VOC patterns of the inoculated and non-inoculated potato tubers during early infection. Five VOCs were identified, 1-octanol, phenylethyl alcohol, 2-ethyl hexanol, nonanal, and 1-octen-3-ol. Then, the infection was detected by the bioreporter bacterial panel, firstly measured in a 96-well plate in solution, and then also tested in potato plugs and validated in whole tubers. Examination of the bacterial panel responses showed an extensive cytotoxic effect over the testing period, as seen by the elevated induction factor (IF) values in the bacterial strain TV1061 after exposure to both potato plugs and whole tubers. Moreover, quorum sensing influences were also observed by the elevated IF values in the bacterial strain K802NR. The developed whole-cell biosensor system based on bacterial detection will allow more efficient crop management during postharvest, storage, and transport of crops, to reduce food losses.
... However, HPLC and LC-coupled chromatography techniques entail several limitations for cannabinoid identification. First, in HPLC with UV/FLD detection, acidic cannabinoids lose fluorescence abilities under acidic conditions (Hazekamp et al., 2005). ...
... HPLC coupled with UV (Zgair et al., 2015), diode-array detector (UV/DAD) (Ambach et al., 2014;De Backer et al., 2009;Hädener et al., 2019;Peschel & Politi, 2015), flame ionization detection (FID) (Gambaro et al., 2002), and electrospray ionization coupled with mass spectrometry detector (ESI-MS) (Berman et al., 2018) have also been utilized for identifying cannabinoids in hemp (Brighenti et al., 2017;Pellati et al., 2018). However, ion spray ionization is conducted at a high temperature (e.g., 500ºC), which can cause the partial thermal decomposition of acid cannabinoids (Hazekamp et al., 2005), which will distort the reconstruction of the original chemical composition conveyed by the plant. In addition, overpressured layer chromatographic (OPLC) methods were examined for cannabinoid identification, which combines the HPLC interface with the capacity of flash chromatography and multidimensionally of TLC (Hidvégi, 2013;Szabady et al., 2002). ...
... In addition, ∆ 9 -THC undergoes thermal degradation (i.e., oxidation, isomerization), which results in a significant detectable amount of ∆ 8 -THC and cannabinol (CBN), the ∆ 9 -THC prime degradation by-products. Despite these limitations, GC still remains a useful technique for the analysis of cannabis (Hazekamp et al., 2005). ...
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Recent studies highlight the therapeutic virtues of cannabidiol (CBD). Furthermore, due to their molecular enriched profiles, cannabis inflorescences are biologically superior to a single cannabinoid for the treatment of various health conditions. Thus, there is flourishing demand for Cannabis sativa varieties containing high levels of CBD. Additionally, legal regulations around the world restrict the cultivation and consumption of tetrahydrocannabinol (THC)-rich cannabis plants for their psychotropic effects. Therefore, the use of cannabis varieties that are high in CBD is permitted as long as their THC content does not exceed a low threshold of 0.3%–0.5%, depending on the jurisdiction. These chemovars are legally termed ‘hemp’. This controlled cannabinoid requirement highlights the need to detect low levels of THC, already in the field. In this review, cannabis profiling and the existing methods used for the detection of cannabinoids are firstly evaluated. Then, selected valuable biosensor technologies are discussed, which suggest portable, rapid, sensitive, reproducible, and reliable methods for on-site identification of cannabinoids levels, mainly THC. Recent cutting-edge techniques of promising potential usage for both cannabis and hemp analysis are identified, as part of the future cultivation and agricultural improvement of this crop.
... The UV-Vis PDA detectors are much cheaper, require less operator expertise, and are widely available. Since cannabinoids contain UV chromophores [27], they are amenable to PDA detection. Moreover, the UV spectra may assist with compound identity confirmation and the measurement of peak purity, which aids in quantification. ...
... To formalise the peak identification, and to demonstrate further gains in the inter-batch repeatability, the RRT were also pooled from the three analysts and were appended to Table 3. RRT should correct for inter-batch variabilities in retention times, provided that the variation in conditions proportionally affected all of the closely related analytes being studied [27]. As anticipated, the pooled RRT values for each cannabinoid had CV which ranged from 0.04 to 0.34%. ...
Article
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The quality control of medicinal cannabis should include quantification of as many cannabinoids as practicable in a routine analytical lab, to accurately reflect the quality of the product. However, the cost and availability of some cannabinoid standards is an impediment to their routine use. This work seeks to overcome this obstacle by analysing samples using relative retention times (RRT) and relative response factors (RRF), relative to CBD and CBDA reference standards which are readily available. A high-performance liquid chromatography-photodiode array method was developed to quantify ten cannabinoids (Δ⁹-THC, Δ⁸-THC, THCA-A, CBN, CBD, CDBA, CBC, CBDV, CBG, and CBGA) in dried cannabis inflorescence and cannabis oil. This method was validated according to ICH guidelines. The proposed method has detection limits ranging from 20 to 78 µg/g, which provided sufficient sensitivity for the panel of cannabinoids. Non-cannabinoid surrogate matrices were used for spike recovery studies to determine method accuracy – analyte recoveries for the inflorescence and oil ranged from 90.1% to 109.3% (inflorescence mean, 100.9%; oil mean, 99.6%). The RRT and RRF values determined independently by three analysts were comparable, indicating the method is robust. The validity of analysis using RRT and RRF was further confirmed by testing six inflorescence samples, as it was found that concentrations above the order of magnitude of the LoQ agreed satisfactorily (range, 95.0% to 111.9%; mean, 100.0%) with the concentrations obtained through the conventional approach of multipoint calibration using pure standards. The proposed method is therefore suitable for the rapid and simple determination of a panel of ten cannabinoids without having to repeatedly purchase every expensive pure standard. Accordingly, analysts in the medicinal cannabis field may explore the use of RRF and RRT for their methods and instruments.
... UV is cheaper and more straightforward than MS (Leghissa et al., 2018a). Neutral cannabinoids show a UV absorption peak around 220 nm and acidic cannabinoids peak in the range of 270-310 nm (Hazekamp et al., 2005;Citti et al., 2016). MS may provide higher specificity than UV, which makes it possible to analyze complex matrices of cannabis (Citti et al., 2018). ...
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“Hemp” refers to non-intoxicating, low delta-9 tetrahydrocannabinol (Δ9-THC) cultivars of Cannabis sativa L. “Marijuana” refers to cultivars with high levels of Δ9-THC, the primary psychoactive cannabinoid found in the plant and a federally controlled substance used for both recreational and therapeutic purposes. Although marijuana and hemp belong to the same genus and species, they differ in terms of chemical and genetic composition, production practices, product uses, and regulatory status. Hemp seed and hemp seed oil have been shown to have valuable nutritional capacity. Cannabidiol (CBD), a non-intoxicating phytocannabinoid with a wide therapeutic index and acceptable side effect profile, has demonstrated high medicinal potential in some conditions. Several countries and states have facilitated the use of THC-dominant medical cannabis for certain conditions, while other countries continue to ban all forms of cannabis regardless of cannabinoid profile or low psychoactive potential. Today, differentiating between hemp and marijuana in the laboratory is no longer a difficult process. Certain thin layer chromatography (TLC) methods can rapidly screen for cannabinoids, and several gas and liquid chromatography techniques have been developed for precise quantification of phytocannabinoids in plant extracts and biological samples. Geographic regulations and testing guidelines for cannabis continue to evolve. As they are improved and clarified, we can better employ the appropriate applications of this uniquely versatile plant from an informed scientific perspective.
... On a rudimentary level, the analytical method clearly has an impact on the detectability of cannabis ingredients and, therefore, the knowledge of their composition in the cannabis products [42,43]. In the cannabis plant, cannabinoids are mainly biosynthesized in their acidic forms, e.g., THC-acid (THCA). ...
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Cannabis sativa ( C. sativa ) is commonly chemically classified based on its Δ ⁹ -tetrahydrocannabinol (THC) and cannabidiol (CBD) content ratios. However, the plant contains nearly 150 additional cannabinoids, referred to as minor cannabinoids. Minor cannabinoids are gaining interest for improved plant and product characterization, e.g., for medical use, and bioanalytical questions in the medico-legal field. This study describes the development and validation of an analytical method for the elucidation of minor cannabinoid fingerprints, employing liquid chromatography coupled to high-resolution mass spectrometry. The method was used to characterize inflorescences from 18 different varieties of C. sativa , which were cultivated under the same standardized conditions. Complementing the targeted detection of 15 cannabinoids, untargeted metabolomics employing in silico assisted data analysis was used to detect additional plant ingredients with focus on cannabinoids. Principal component analysis (PCA) was used to evaluate differences between varieties. The overall purpose of this study was to examine the ability of targeted and non-targeted metabolomics using the mentioned techniques to distinguish cannabis varieties from each other by their minor cannabinoid fingerprint. Quantitative determination of targeted cannabinoids already gave valuable information on cannabinoid fingerprints as well as inter- and intra-variety variability of cannabinoid contents. The untargeted workflow led to the detection of 19 additional compounds. PCA of the targeted and untargeted datasets revealed further subgroups extending commonly applied phenotype classification systems of cannabis. This study presents an analytical method for the comprehensive characterization of C. sativa varieties. Graphical abstract
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Herbal products for smoking containing cannabidiol (CBD) are available as “low‐tetrahydrocannabinol cannabis products” in most EU countries. In Belgium, Δ9‐tetrahydrocannabinol (THC) content of these products must be less than 0.2 % w/w, which is also the limit for agricultural hemp. For agricultural hemp, the official and only valid method for European regulators is gas‐chromatography coupled to flame ionization detector (GC‐FID). There is no such method, for smoking products. Many of these herbal for smoking products are analyzed as part of their quality control and have certificate of analysis. During surveillance by official labs, discrepancies were seen between the official results and the certificate of analysis. In this study, a GC‐FID method based on the European method and a ultra‐high‐performance liquid chromatography coupled to diode array detection (UHPLC‐DAD) method were validated and applied for samples analysis in order to investigate these discrepancies. The GC‐FID method shows better results for the validation parameters, notably it has β‐ expectation tolerance limits within 10% with a β value of 95% while the validated UHPLC‐DAD method has β‐ expectation tolerance limits within 15% with a β value of 90%. Furthermore, the others parameters evaluated are generally better with the GC‐FID method. The statistic t‐test shows that the difference between both methods was significantly different for total‐THC, but not significantly different for the total‐CBD. The authors state that, as for agricultural hemp, the GC‐FID method is to be preferred for the analysis of THC and CBD in products for smoking.
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Full-text available
A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large‐scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (−)‐Δ‐(trans)‐tetrahydrocannabinol (Δ‐THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (−)‐Δ‐(trans)‐tetrahydrocannabinolic acid‐A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug‐type and a fiber‐type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material.
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Full-text available
An HPLC method with photodiode array detection (DAD) is described for the qualitative and quantitative determination of neutral and acidic cannabinoids in Cannabis sativa L. The complex chromatographic pattern can be used for the classification of Cannabis chemotypes, the monitoring of the psychotropic potency and the comparison of Cannabis products of different origin.
Chapter
The cannabinoids form a group of terpene-related compounds unique to the plant Cannabis sativa L., the source of the drug marijuana. To date, some 70 of these compounds have been identified (1), the major ones having the structures shown in Fig. 6.1. Of these, delta-9-tetrahydrocannabinol (delta-9-THC, I) is the major psychoactive constituent, cannabidiol (CBD, II) is its biosynthetic precursor, and cannabinol (CBN, III) is generally regarded as a chemical degradation product (2). Most of the major cannabinoids possess a pentyl chain, but the complexity of cannabinoid mixtures arises from the presence of lower homologues, of which the propyl series is the best known (3). Methyl (4) and butyl (5) cannabinoids have also been reported, but in low concentration. An interesting feature of the relative abundance of all of the cannabinoids is their dependence on geographical origin and age. Plants grown in temperate regions tend to have low THC contents, whereas those originating in tropical regions contain high concentrations of this cannabinoid. In addition, the relative concentration of the homologues displays a strong regional bias with, for example, propyl cannabinoids being particularly abundant in samples originating from the Indian subcontinent. Further complexity is imparted by the fact that most natural cannabinoids contain a carboxylic acid group either at C-2 (named acid A, IV) or at C-4 (acid B, V). This group is labile, is lost when the drug is smoked, and presents problems when analysis is performed by gas chromatography.
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Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.
Article
Various chromogenic chemical tests for Cannabis were evaluated as stains for the microscopical identification of the glandular trichomes of Cannabis sativa. Only vanillin in ethanolic sulphuric acid gave results comparable with the staining obtained with Fast Blue B. The specificity of these two stains was examined using 222 non-Cannabis species from 21 selected plant families. None of the species possessed glandular trichomes which stained with Fast Blue B. Two Mentha and three Callicarpa species had stained glandular trichomes with the vanillin reagent. It is concluded that Fast Blue B is a very specific stain for the cannabinoids in the glandular trichomes of C. sativa L.
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Stable cyclic methyl- and n-butylboronates were prepared from the cannabinolic acids and used as derivatives for the characterization of these compounds by combined gas chromatography mass spectrometry. Retention times of the methylboronates were comparable with those of the trimethylsilyl derivatives. The mass spectra of these derivatives were more characteristic of the cannabinoid skeleton than were the spectra of the trimethylsilyl derivatives where fragmentation was associated with the trimethylsilyl groups. Molecular weights were lower and abundant molecular ions were obtained. These derivatives also offered the possibility of distinguishing between isomeric cannabinolic acids where only isomers possessing adjacent phenolic and carboxylic functions form cyclic boronates.
Article
High-resolution capillary gas chromatography with flame ionization detection and mass spectrometry (GC and GC/MS) and high-performance liquid chromatography (HPLC) were used to establish complex chemical profiles (chemical signatures) of Cannabis samples of known origin. Over 100 compounds could be differentiated, including noncannabinoids (terpenes, alkanes) as well as minor and major cannabinoids and their acids. A characteristic peak pattern was found within a limited number of specimens of identical origin. Correlation studies on the basis of peak area ratios [A(x)/A(i.s.)] showed the feasibility of tracing Cannabis chemically to its country of origin. Several forensic science applications for the chromatographic and spectroscopic profiles of confiscated Cannabis samples are discussed, such as detection of additives (phencyclidine), differentiation of chemotypes, and monitoring of tetrahydrocannabinol (THC) potency.