Accepted Manuscript

Title: Quantitative determination of CBD and THC and their

acid precursors in confiscated cannabis samples by
HPLC-DAD

Authors: Marianne Hädener (Conceptualization), Stefan

König, Wolfgang Weinmann (Conceptualization)

PII: S0379-0738(19)30131-8
DOI: https://doi.org/10.1016/j.forsciint.2019.03.046
Reference: FSI 9741

To appear in: FSI

Received date: 15 January 2019

Revised date: 15 March 2019
Accepted date: 28 March 2019

Please cite this article as: Hädener M, König S, Weinmann W, Quantitative

determination of CBD and THC and their acid precursors in confiscated
cannabis samples by HPLC-DAD, Forensic Science International (2019),
https://doi.org/10.1016/j.forsciint.2019.03.046

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Quantitative determination of CBD and THC and their acid precursors in
confiscated cannabis samples by HPLC-DAD

Authors:
Marianne Hädener, Stefan König, Wolfgang Weinmann

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Affiliation and Address:
Institute of Forensic Medicine Department of Forensic Toxicology and Chemistry University of
Bern Bühlstrasse 20 3012 Bern Switzerland

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Corresponding author:

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Wolfgang Weinmann Institute of Forensic Medicine Department of Forensic Toxicology and
Chemistry University of Bern Bühlstrasse 20 3012 Bern Switzerland
E-mail address: wolfgang.weinmann@irm.unibe.ch Telephone number: +41 (0)31 631 56 68

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Highlights
 New HPLC-DAD method for forensic analysis as well as quality control of cannabis.
 Quantitative determination of THC, THCA, CBD, CBDA, and CBN.
 Baseline resolution of cannabinoid analytes on a core-shell C8 stationary phase.
 Confirmation of method performance through extensive validation.
 Successful analysis of herbal cannabis and hashish samples from forensic cases.

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ORCID:
Marianne Hädener 0000-0002-3665-3703
Stefan König 0000-0002-7851-1973
Wolfgang Weinmann 0000-0001-8659-1304

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Abstract
Analysis of cannabis has gained new importance worldwide, mainly for quality control within the

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legalized recreational and medical cannabis industry, but also for forensic differentiation between
drug-type cannabis and legal products such as fiber hemp and CBD-rich/THC-poor cannabis. We
herein present an HPLC-DAD method for quantitative analysis of major neutral and acidic

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cannabinoids in herbal cannabis and hashish: Δ9-tetrahydrocannabinol (THC), Δ9-
tetrahydrocannabinolic acid A (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and
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cannabinol (CBN). Plant material was dried, homogenized and extracted with a mixture of
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methanol/hexane. Chromatographic separation of the analytes was achieved on a core-shell C8
column using gradient elution with water/acetonitrile containing 0.1% formic acid. The analytical
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run time was 13 min and analytes were detected at 210 nm. The method is selective, sensitive,
accurate, and precise, as confirmed through validation according to ICH and AOAC guidelines.
Linearity in herbal cannabis ranged from 0.04 – 4.00% for the neutral cannabinoids, and from 0.40
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– 20% for the acids. Linear ranges in hashish samples were 0.13 – 13.33% and 1.33 – 66.66%,
respectively. The presented method was successfully applied to characterize 110 cannabis samples
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seized by the Swiss police, demonstrating its applicability for routine cannabis potency testing in
the forensic setting.
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Keywords
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Cannabis, Cannabinoids, THC, CBD, potency testing, HPLC-DAD analysis

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1. Introduction
Since ancient times, cannabis has been used as a medicinal and recreational drug, as well as for
industrial purposes (fiber, food and oil) across the world [1, 2]. Nowadays, cannabis is the most
widely cultivated, trafficked and abused recreational drug worldwide [3]. In recent years, however,
there has been a resurgence of interest in the therapeutic potential of this versatile plant [4]. At least
70 of the over 400 identified cannabis constituents are phytocannabinoids, a class of

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terpenophenolic compounds which are largely responsible for the plant’s pharmacological
properties [5]. Δ9-Tetrahydrocannabinol (THC) exhibits the greatest psychoactivity and produces
feelings of euphoria and relaxation, but has also been shown to possess analgesic, anti-
inflammatory, appetite stimulant, and antiemetic properties [6]. However, chronic THC
consumption has been linked to severe side effects, including cognitive deficits, anxiety, paranoia,
chronic psychosis and dependence [7-10]. Another major cannabinoid with great therapeutic
potential is cannabidiol (CBD), a non-psychoactive isomer of THC, which was found to have anti-

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inflammatory, anticonvulsive, anxiolytic, analgesic, neuroprotective, anticancer and antioxidant
effects. Furthermore, studies suggest that CBD has a modulating role on the psychoactivity of THC,
thus mitigating the adverse psychological effects of THC [11, 12].

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The ratio of the different cannabinoids is largely determined by the genetics of the plant (strain
type), but also depends on environmental and harvesting conditions (e.g. cultivation and storage

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conditions, state of maturity at harvest) [13]. THC and CBD are enzymatically biosynthesized as
pharmacologically inactive carboxylic acids, Δ9-tetrahydrocannabinolic acid A (THCA) and
cannabidiolic acid (CBDA), respectively (Fig. 1). The active, neutral cannabinoids are formed via

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decarboxylation occurring naturally as the plant ages and when it is exposed to light or heat (e.g.
smoking, baking, vaporizing) [14, 15]. Therefore, the pharmacological activity or potency of
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cannabis is best represented by the total cannabinoid content, i.e. the sum of the free cannabinoid
and its respective acidic precursor (corrected for weight of the carboxylic acid groups and
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expressed as weight percentage of dry plant material). As THC is thermolabile and photolabile, it
undergoes oxidative degradation to cannabinol (CBN) upon prolonged storage. Hence, the ratio of
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CBN to THC can serve as indicator for the age of the stored cannabis sample [16-18].
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Over the last few decades, cannabis cultivation has been biased towards plants with highest THC
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content and negligible CBD levels that give greatest psychoactive effects. However, due the
growing interest in the therapeutic effects of CBD, recent efforts have been directed to breeding
CBD-rich strains. Nowadays, plants with up to 25% total CBD and less than 1% total THC are
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bred [19-21].
The legal status of cannabis varies significantly from country to country. The European Union (EU)
allows cultivation of non-psychoactive fiber-type hemp varieties with total THC levels below 0.2%
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[22]. Although being the main cannabinoid, CBD is generally found at low levels in fiber hemp (<
4%). True CBD-rich strains, which have recently been bred, usually express 0.3 – 0.7% total THC
[21, 23] and are thus illegal in the EU. In Switzerland, however, only plant material with a total
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THC content of 1% or higher is controlled by the narcotics legislation [24]. Therefore, CBD-
rich/THC-poor cannabis products can be legally sold as tobacco substitutes, cosmetics, foodstuffs
or without an intended use via the Internet and in supermarkets, and their sale has massively
increased over the past year.
Reliable distinction between legal CBD- and illegal THC-cannabis is not possible by appearance
or smell, but requires determination of the total THC content by validated laboratory methods.
However, potency testing of cannabis is not only performed for forensic and legal purposes, but

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has also become an important part of quality control in the cannabis industry of countries or states
that have legalized cannabis for medical and/or recreational use. Accurate testing and labeling is
crucial to ensure proper dosing and choice of an appropriate product. While medical patients may
seek cannabis with a high CBD:THC ratio, recreational users rather prefer the opposite. In October
2018, Canada become the second country in the world after Uruguay to fully legalize cannabis [25,
26]. As of December 2018, ten US states have legalized cannabis for recreational use and 33 states
as well as Australia have allowed the use of cannabis for medical purposes [27-29]. With the

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growing demand for cannabis potency testing, laboratories have to cope with an increasing number
of samples and numerous analytical laboratories have recently emerged to keep up with demand.
Various analytical methods have been reported for the analysis of cannabinoids in cannabis plant

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material, as recently reviewed by Citti et al. [30]. The most commonly used techniques are gas
chromatography (GC) combined with flame ionization detection (FID) or mass spectrometry (MS),

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and high performance liquid chromatography (HPLC) with UV or MS detection. GC is generally
considered faster and simpler than HPLC, but only provides the total cannabinoid content, as acidic
cannabinoids are converted to their neutral forms in the heated injection port. Thermal

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decarboxylation during GC analysis may be partial, leading to an underestimation of the actual
value [31]. Separate determination of acidic and free cannabinoids by GC requires a time-
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consuming derivatization step prior to analysis. However, this procedure can lead to ambiguous
results as well, since derivatization may also be incomplete. HPLC has the advantage of allowing
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direct analysis of acidic and neutral cannabinoids and thus determination of the original
composition of the plant. Alternative methods reported for the quantitative analysis of cannabinoids
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in cannabis products are ultra-high performance supercritical fluid chromatography (UHPSFC)

with UV/MS detection [32], 1H NMR spectroscopy [33], and ion mobility spectrometry (IMS)
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coupled to MS [34].
In this work, we developed and validated a straightforward and sensitive HPLC-DAD method for
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quantitative analysis of THC, THCA, CBD, CBDA, and CBN to determine the psychoactive and
therapeutic potency of herbal cannabis and hashish. The method offers a short analytical run time
and produces accurate and repeatable results, as confirmed through validation, and thus is a
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valuable addition to existing procedures for cannabis testing. The presented method was
successfully applied to the analysis of forensic cannabis samples seized by the Swiss police,
including several samples which were claimed to be legal CBD-rich/THC-poor cannabis flowers.
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2. Material and Methods

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2.1 Chemicals

THCA, THC, CBDA, CBD, and CBN reference standards were purchased from Lipomed
(Arlesheim, Switzerland). Methanol (absolute, HPLC grade) was obtained from Biosolve (Dieuze,
France), hexane (EMSURE®) from Merck (Darmstadt, Germany) and acetonitrile (HPLC gradient
grade, 99.9%) from Acros Organics (Geel, Belgium). Formic acid solution (puriss p.a., 50% in

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water) was acquired from Sigma-Aldrich (Buchs, Switzerland). Ultrapure water was generated in-
house with a Direct-Q water purification system from Millipore (Zug, Switzerland).

2.2 Cannabis material

Cannabis samples (flowers, mixed material consisting of leaves and stems without flowers,
hashish) were obtained from forensic cases. The material had been seized by the police from
recreational users, street-level dealers and larger indoor or outdoor cultivation sites and had been

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submitted to our laboratory for routine potency testing.

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2.3 Sample preparation

Sample preparation was carried out according to a previously published procedure [35]. Briefly,

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plant material was dried at 40 °C in an oven and then homogeneously ground (Retsch Grindomix
GM 200, Haan, Germany). The resulting powder was extracted with a mixture of methanol/hexane
9:1 (v/v) by ultrasonication for 20 min (flowers, mixed material: 500 mg / 10 mL; hashish: 300 mg

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/ 20 mL). The extracts were diluted 20-fold with extraction solvent for subsequent HPLC-DAD
analysis.

2.4 HPLC-DAD analysis

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The analysis was carried out using an UltiMate 3000 HPLC system (Dionex, Olten, Switzerland)
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consisting of a HPG-3400RS binary pump, a WPS-3000TRS autosampler, a TCC-3000RS column

compartment and a DAD-3000RS detector. The autosampler and column compartment were
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thermostatted at 8 and 25°C, respectively. Chromeleon software version 6.8 (Thermo Scientific,
Reinach, Switzerland) was used for data acquisition and analysis. Chromatographic separation was
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achieved using a Kinetex 2.6 µm C8 column (100 x 2.1 mm) protected by a KrudKatcher Ultra in-
line filter (Phenomenex, Aschaffenburg, Germany) and gradient elution with 0.1% formic acid in
water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. The flow rate was
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0.6 mL/min and the gradient conditions were as follows: 0-2 min, hold at 50% B; 2-9 min, increase
to 65% B; 9-10 min, hold at 65% B; 10-10.1 min, decrease to 50% B; 10.1-13 min, hold at 50% B.
The injection volume was 5 µL. Full spectra were recorded from 200 to 800 nm. For quantification,
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the detection wavelength was set at 210 nm.

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2.5 Method validation

Method validation was performed according to the ICH and AOAC guidelines [36, 37] by
evaluating the following parameters: selectivity, linearity, limit of detection (LOD), lower limit of
quantification (LLOQ), accuracy, intra- and inter-assay precision, and carry-over. Microsoft Excel
2010 and GraphPad Prism 6.05 software for Windows were used for statistical analysis of the
validation data.

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Analyte peak identification was achieved by comparing the retention times with those of the
reference standards. The retention time was required to be within ± 0.1 min of that observed for
the highest calibration standard in the same run.
Selectivity was assessed by determining peak purity as well as the resolution of the analyte peaks
from neighboring peaks in 14 cannabis extracts with different cannabinoid contents. Peak
resolution ≥ 1.5 was considered acceptable. Peak purity was estimated based on the peak purity
match factor (PPM) and the percent relative standard deviation (%RSD) of the peak purity index

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(PPI) calculated by the Chromeleon software. The PPM describes the correlation of the spectrum
at the peak maximum and the spectra at the peak flanks. A PPM of 1000 describes identical spectra.
The PPI represents the central wavelength of a spectrum, i.e. the wavelength where the areas of the

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spectrum to the left and right are equal. For a pure peak, the PPI values should be the same for all
spectra acquired across the peak. The RSD of the PPI values over a peak provides a measure of
peak purity. [38] Peaks were considered pure if PPM ≥ 950 and %RSD PPI ≤ 1%.

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Furthermore, selectivity was evaluated by analyzing five different non-cannabis plants (nettle
(Urtica dioica, which belongs to the same order as Cannabis sativa L. [39]), dandelion

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(Taraxacum), spinach (Spinacia oleracea), coriander (Coriandrum sativum L.), and yuka (Yucca
gigantea)) which had been processed as described for cannabis flowers and mixed material.
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Extracts of non-cannabis plants should be analyte-free. Eventual peaks present at the retention
times of analytes could not meet LOD criteria.
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Calibration standards prepared in methanol were analyzed ten times on eight different days to
determine linearity. Concentration levels for THC, CBD, and CBN were 1, 5, 10, 20, 50, and 100
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µg/mL. For the acids, separate calibration standards with concentrations of 10, 50, 100, 200, 300,
400, and 500 µg/mL were used. Calibration curves were obtained by fitting the analyte
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concentration (x) versus the peak area by unweighted linear least-squares regression and linear
least-squares regression using a 1/x and 1/x2 weighting factor, respectively. The calibration models
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were evaluated based on the percentage relative error (%RE) which compares the concentration
calculated from the regression equation with the nominal value [40]. Linearity was acceptable if
the correlation coefficient (r) exceeded 0.99 and if back-calculated calibrator concentrations were
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within ± 15% of the target value.

The LODs were estimated by visual examination of the chromatograms of methanolic solutions
containing decreasing concentrations of analytes. The LOD was determined as the concentration
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with a signal-to-noise ratio (S/N) of at least 3. The LLOQ was defined as the lowest standard on
the calibration curve.
Accuracy was evaluated by spiking aliquots of pre-analyzed cannabis extracts with analytes at
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concentrations of approximately 3, 5 and 10 times the initial concentrations found in the extract (n
= 6 per concentration level). Accuracy was reported as the percentage of the mean concentration
of all replicates from the corresponding nominal value and was considered acceptable if within the
range of 80 - 120%. Furthermore, accuracy was assessed by analyzing four samples obtained from
last year’s annual national proficiency test for determination of the total THC content of cannabis.
Precision measurements were performed using nine authentic cannabis samples having different
cannabinoid profiles. Intra-assay precision was determined from six replicates per sample in a

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single run. For the calculation of the inter-assay precision, six replicates per sample were prepared
and analyzed on three different days (n total = 18). Replicates were prepared by diluting aliquots
of a single cannabis extract since the available amounts of powdered cannabis samples did not
allow for 18 individual extractions. Precision was expressed as percent relative standard deviation
(%RSD) and was expected to be ≤ 20 %RSD.
Carry-over was tested in six independent runs by injection of pure extraction solvent directly after
the highest calibrators. The analyte response in the solvent sample was required to be ≤ 20% of the

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LLOQ response to prove absence of carry-over.

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2.6 Analysis of forensic cannabis samples

The presented method was applied to the analysis of 110 cannabis samples from forensic cases,

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including 45 samples which were claimed to be legal, commercially available CBD-rich cannabis
flowers and were labelled as such when seized by the police. Every sample was extracted and
analyzed in duplicate. To verify the performance of the newly developed method, 151 of the

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prepared extracts were also measured by the HPLC-DAD method which until now has been used
in our laboratory for routine analysis of seized cannabis material [35]. Since CBDA is not
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determined by this existing method and CBD and CBN are determined only semiquantitatively,
the method comparison was limited to the THC and THCA levels.
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3. Results and Discussion
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3.1 Method development and optimization

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Drying and homogenization followed by ultrasonic extraction using an organic solvent has proven
to be an efficient, reproducible and simple sample preparation method for cannabis potency testing
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[39, 41, 42] and is the procedure recommended by both the United Nations Office on Drugs and
Crime (UNODC) [43] and the American Herbal Pharmacopoeia (AHP) [44]. Numerous organic
solvents have been used for cannabinoid extraction from powdered cannabis material, ranging from
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polar solvents such as methanol, ethanol, and ethyl acetate, to less polar solvents such as
chloroform, benzene, toluene, petroleum ether, and hexane as well as solvent combinations such
as methanol/chloroform 9:1 (v/v), methanol/hexane 9:1 (v/v) or hexane/isopropanol 9:1 (v/v) [30,
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45]. Compared to alcohol-based solvents, non-polar solvents such as hexane and petroleum ether
give a relatively clean extract due reduced co-extraction of chlorophyll and other plant constituents,
but they only extract the neutral cannabinoids quantitatively [43, 46]. Alcohol-based solvents were
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found to be superior to chloroform and petroleum ether for most of the cannabinoids [46]. We
chose to adopt the extraction procedure routinely used in our laboratory for forensic analysis of
cannabis [35] as this has produced satisfactory proficiency testing results for the total THC content
over several years.
Cannabis extracts are mixtures of complex composition and thus their analysis requires highly
selective methods. Mass spectrometric detection would offer greater selectivity and sensitivity than
UV and FID detection. However, MS instrumentation is expensive and requires more maintenance
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and expertise to operate. The UV spectra of the five cannabinoids of interest show several distinct
absorption maxima (Supplementary Fig. S1). The spectra of the acids differ from those of neutral
cannabinoids, with additional maxima at about 270 and 310 nm. Since absorption decreases with
increasing wavelength, cannabinoids are typically detected at 210 – 230 nm. We selected 210 nm
as detection wavelength as this provides highest sensitivity for THC and CBD.
Due to the inherent lack of selectivity of UV detection, especially at short wavelengths, adequate
chromatographic separation of the cannabinoid analytes and matrix components is crucial for

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accurate quantification. During method development, we evaluated HPLC columns with different
reversed-phase (RP) stationary phases (C18, C8, C5, C4, Biphenyl, pentafluorophenyl, RP-select
B) and different dimensions (length 50 – 125 mm, ID 2.0- 4.6 mm, particle size 2.6 – 5.0 µm)

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which were obtained from different manufacturers. Columns were tested under different mobile
phase and gradient conditions (mobile phase solvents: methanol, acetonitrile, water; additives: 25

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mM triethylammonium phosphate, 25 mM ammonium acetate, 10 mM ammonium formate, 0.1%
formic acid) in order to achieve optimum separation in a short run time.
The best results were obtained by using a Kinetex C8 column and gradient elution with

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water/acetonitrile containing 0.1% formic acid. This core-shell stationary phase led to a
considerable improvement of chromatographic performance in terms of both resolution and
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sensitivity compared to conventional columns packed with fully porous particles. With core-shell
particles, sources of band broadening (mainly the A and B terms of the van Deemter equation) are
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reduced compared to fully porous particles. The unusually low value of the A term (Eddy diffusion)
can be explained by the reduction of trans-column velocity biases due to the marked roughness of
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the particle surface. The decrease of the B term (longitudinal diffusion) can be attributed to the fact
that about 20% of the column volume is occupied by a solid, nonporous spherical core, through
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which analytes cannot diffuse. Columns packed with core-shell particles can hence provide
efficiency similar to columns packed with sub-2 μm fully porous particles, with the advantage of
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maintaining low backpressure [47-49]. To improve peak shape and shorten the analysis time, we
operated the column at a flow rate of 0.6 mL/min which generated a backpressure of approximately
460 bar and thus required a mid-pressure range HPLC instrument having an operating pressure
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limit > 400 bar.

Controlling mobile phase pH when analyzing ionizable compounds is important. THCA and
CBDA have pKa values of approximately 3.3 and 3.4, respectively (calculated using Advanced
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Chemistry Development ACD/labs Software V11.02), and thus an acidic pH is required to bring
them to their protonated, noncharged forms which will have improved retention on non-polar RP
stationary phases. When going from neutral to acidic conditions, the relative retention times of the
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acids were modified, while those of the neutral cannabinoids barely changed. Bringing the mobile
phase to pH 2.7 with 0.1% formic acid resulted in improved resolution of THCA and better peak
shapes.
The optimized method provided baseline separation of all analytes within a favorably short run
time of 13 min (Fig. 2). Since the employed mobile phase solvents are MS compatible, the method
could be easily transferred from UV to MS detection, if improved sensitivity and selectivity are

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required, e.g. for the detection and identification of minor cannabinoids or other cannabis
constituents.
3.2 Validation data
Before implementation, the method was thoroughly validated using authentic cannabis samples
with different cannabinoid contents. Analyte retention times were highly reproducible and stable
over a large number of injections. For all analytes, the %RSD of the retention time in the calibration
standards of nine runs was less than 0.7%.

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THC, THCA, CBD, and CBDA peaks proved to be baseline separated and pure (Table 1).
Resolutions ranged from 1.9 to 5.3 in the 14 sample extracts tested, and PPM and %RSD PPI values
were greater than 986 and less than 0.9%, respectively (≤ 1.1% for THC). For CBN, peak resolution

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and purity criteria were not fulfilled in all samples, but selectivity was still deemed acceptable. The
analytes could not be detected in any of the extracts of non-cannabis plants (Supplementary Fig.

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S2a).
Initial experiments were performed with ten sets of calibration curves fit by unweighted linear
least-squares regression and linear least-squares regression applying a 1/x and 1/x2 weighting

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factor, respectively. The %RE values were plotted versus nominal concentration and the sum of
absolute %RE values was calculated. Linear least-squares regression with a 1/x2 weighting factor
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was found to be the most appropriate calibration model as it gave rise to the narrowest distribution
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around the concentration axis and the lowest %RE sum across the entire concentration range
(Supplementary Fig. S3). The method demonstrated excellent linearity from 1 to 100 μg/mL for
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the neutral cannabinoids and from 10 to 500 µg/mL for the acidic cannabinoids, with correlation
coefficients r ≥ 0.9981 (1/x2 weighting) for all calibration curves (Supplementary Table S1). The
corresponding calibration ranges in weight percentages are summarized in Table 2. Calibration
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ranges for neutral and acidic cannabinoids were different since the biogenic acid precursors are
present in herbal cannabis and hashish at much higher concentrations than the neutral cannabinoids.
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If necessary, the calibration ranges can be expanded by using different sample weights and dilution
factors than indicated in section 2.3. For all analytes, back-calculated calibrator concentrations
were within ± 8.6% of the target value. The detection and quantitation limits for each cannabinoid
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are listed in Table 2. The LODs were determined based on visual examination of the
chromatograms (S/N ≥ 3) while the LLOQs were equivalent to the lowest calibration standard (S/N
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≥ 10). Chromatograms of the analytes at the LOD and LLOQ, respectively, are shown in
Supplementary Fig. S2 and Fig. 2.
Method accuracy and precision were acceptable for all cannabinoids. The results of these
measurements are summarized in Tables 3 and 4. Accuracy ranged from 95.8 – 111.7% recovery
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of the spiked amounts. It should be noted, however, that these values do not take into account the
extraction efficiency since cannabinoid standards were spiked into cannabis extracts. Method
accuracy was therefore further demonstrated by analyzing four proficiency testing samples.
Measured total THC contents were within 89.2 – 94.4% of the interlaboratory mean values (n = 12
laboratories). Precision was only calculated if the analyte concentration in the authentic sample
was ≥ LLOQ. Inter-assay precision was better than 16.4 %RSD for all analytes and samples,
whereas intra-assay precision was > 20 %RSD in a few cases (max. 22.2 %RSD).
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Carry-over was found to be insignificant as analyte signals in solvent samples injected after the
highest calibration standards were ≤ 20% of the LLOQ response (≤ 3.9% for THC, ≤ 1.0% for
THCA, ≤ 2.5% for CBD, ≤ 1.4% for CBDA, and ≤ 2.5% for CBN).

3.3 Determination of cannabinoid contents in seized cannabis samples and method comparison

The results obtained from the analysis of the 110 seized cannabis samples are shown in

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Supplementary Tables S2 – S4. Presented data are the mean of duplicate measurements.
Chromatograms of authentic samples are shown in Fig. 2.
Only one of the 45 flower samples which were in packagings of commercially available CBD-rich

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products when seized had a total THC content above 1% (total THC: 1.30%, total CBD: 11.32)
and was thus illegal according to Swiss law. The total THC content of the other samples ranged

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from 0.11 - 0.69% (median 0.45%) and the total CBD content from 3.22 - 18.17% (median
12.24%), indicating that in the vast majority of the cases the packagings had not been misused to
hide illegal THC-rich cannabis. In general, the measured total THC and total CBD contents were

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lower than declared on the product packaging or in the product description found on the Internet,
or were at least at the lower end of the specified range (Supplementary Table S2). The measured
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total CBD content was up to 4 times lower than the specified value. Most online shops assert that
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their cannabis products have been lab-tested for potency - probably in one of the private cannabis
testing laboratories that have emerged in Switzerland. However, since most of the packagings were
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already open when seized by the police, it cannot be ruled out that the material has been tampered
with, i.e. replaced or mixed with a different CBD-rich product. Therefore, it would be wrong to
assume that the CBD contents are over-reported on purpose or due to inaccurate analytical results.
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To avoid making any false accusations, we refrain from listing the suppliers and product names
found on the seized packagings. It should also be kept in mind that herbal cannabis products are an
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extremely inhomogeneous material. Subsamples of a batch of cannabis may exhibit different

potencies [50]. The values reported on the packagings possibly correspond to the average potency
of the batch or to the highest measured value. So the observed differences between measured and
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reported potencies could stem from heterogeneity in the cannabis material.

Among the 110 seized samples there were another 17 flower samples and 11 mixed samples (leaves
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and stems without flowers) which also showed total THC contents below the Swiss legal limit of
1% (Supplementary Table S3). The median total CBD content of the mixed material was markedly
lower than that of the flower samples (1.22 vs. 9.72%). It is commonly known that the absolute
cannabinoid levels vary in different parts of the plant and are higher in flowers and younger leaves
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than in older leaves and the stems [51]. Five of the flower samples had a total CBD content ≤ 3.59%
and a total THC content ≤ 0.07%. While these samples rather stem from fiber hemp varieties, the
other flower samples (median total CBD content 11.04%; range 7.14 – 14.45%) can be considered
to be true CBD-rich cannabis [23, 52].
Thirty-seven of the analyzed cannabis samples had a total THC content above the Swiss 1% legal
limit (Supplementary Table S4). Whereas one flower samples could be ascribed to a mixed strain
with a more even balance of both total THC and total CBD (THC:CBD ratio of 0.6), the rest of the
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flowers, hashish and mixed samples were clearly THC predominant with very low total CBD
contents. Analyzed flower samples showed total THC levels up to 17.19% (median 9.20%) and
total CBD levels ≤ 0.40% (the majority of the samples had undetectable CBDA and/or CBD levels).
Hashish samples were even more potent with a total THC content between 18.01 and 35.07%.
These results reflect trends reported in other countries towards the use of high potency cannabis
with very low CBD levels [19].
CBN levels of the 110 seized samples were undetectable or very low (≤ 0.49%), suggesting that

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THC had not suffered significant degradation during storage.
The THC and THCA concentrations of 151 sample extracts were compared to those obtained with
our routinely used HPLC-DAD method [35] which has produced satisfactory results in nation-wide

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proficiency tests over several years. Only values above the LLOQs were considered (n THC = 101,
n THCA = 68). The observed differences were compared to the maximum allowable difference

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between duplicate measurements as recommended by the Swiss Society of Legal Medicine
(Supplementary Tables S5 – S7). The THC and THCA results of the two methods were generally
in good agreement. Only ten extracts showed differences in THC concentrations lying outside the

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acceptable range. Six of these were hashish samples with a THC content > 8%. Observed
differences in THCA levels were larger than permitted in 13 cases, mainly for samples with THCA
contents > 6%. N
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4. Conclusion
We herein report a newly developed HPLC-DAD method that meets the increasing global demand
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for fast, cost-effective and reliable cannabis potency testing. HPLC offers advantages over
previously reported GC-based methods as neutral and acidic cannabinoids can be quantified
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separately, thus allowing direct determination of the original cannabinoid profile as well as more
accurate quantification of total cannabinoid contents. The presented method proved to be sensitive,
selective, accurate, and precise and was successfully used on herbal cannabis and hashish samples
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from forensic cases. Validation was limited to the five major cannabinoids of forensic and
therapeutic interest. However, the method might also be suitable for the analysis of additional
cannabinoids and other cannabis preparations such as oils, foods, or cannabis-based medicinal
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extracts. The method is broadly applicable and can be easily implemented in forensic chemistry
laboratories for differentiation between drug-type cannabis and fiber hemp as well as in quality
control laboratories in the legalized cannabis industry. However, when it comes to cannabis quality
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there are more parameters to consider than the cannabinoid content. Multiple analyses are often
required for quality control such as determination of pesticide/herbicide residues, residual solvents,
heavy metals, and terpenoids as well as microbial testing.

Authors contributions /Credit author statement:

11
Marianne Hädener
Conceptualization, data acquisition (sample work up, analysis), method adaption from previous
HPLC instrument, LC-MS/MS method validation, data generation. Writing of manuscript, original
draft including corrections from co-authors, revision of manuscript and all documents.

Stefan König
Methodology optimization, supervision of analytical instrumentation used, training for

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instrumentation, validation of results, correction of manuscript.

Wolfgang Weinmann

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Conceptualization, resources and funding during work and PhD thesis, of which this project was
one part, correction of draft and revisions.

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Author and co-authors disclose any potential conflict of interest.

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Conflict of Interest N
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The authors declare that they have no conflict of interest.
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Fig. 1. Structures of the cannabinoids analyzed by HPLC-DAD.

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Fig. 2. Representative chromatograms of a cannabinoid standards in pure solvent at the lower limits
of quantification and b-d extracts of cannabis samples (b THC-rich/CBD-poor flowers, c THC-
rich/CBD-poor hashish, d CBD-rich/THC-poor flowers). Peak assignment: 1, CBDA; 2, CBD; 3,
CBN; 4, THC; 5, THCA.

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Table 1. Resolution and peak purity of cannabinoid analytes in different cannabis extracts (flowers:
samples 1-12; hashish: samples 13, 14).
content concentration of
Analyte Sample resolution PPM %RSD PPI
(%) extract (μg/mL)
THC 1 0.10 2.1 2.6 986 0.6
2 0.06 1.4 2.4 998 0.2
4 0.13 2.9 2.4 998 0.4
5 0.16 4.4 3.6 999 0.2

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6 1.00 27.2 3.1 991 1.0
7 0.75 22.1 2.8 990 1.1
8 1.97 49.8 2.9 1000 0.1

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9 0.88 23.3 3.0 1000 0.0
10 1.50 38.0 3.0 999 0.2

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11 0.74 17.8 2.5 1000 0.1
12 2.54 65.2 2.7 1000 0.0
13 8.39 64.2 4.9 998 0.3
14 5.29 41.0 4.8 998 0.3

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THCA 3 0.88 23.1 1.9 999 0.3
5
6
0.46
7.78
11.8
207.0
3.0
3.3
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999
1000
0.3
0.2
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7 6.37 188.8 3.3 1000 0.1
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8 6.95 194.4 3.3 1000 0.2
9 6.17 162.2 2.4 1000 0.1
10 10.20 267.1 2.3 998 0.3
11 6.62 166.1 2.2 1000 0.1
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12 1.94 49.3 3.4 1000 0.1

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13 11.05 83.9 5.3 1000 0.1

14 27.02 209.8 5.2 999 0.3
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Table 1. continued
content concentration of
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Analyte Sample resolution PPM %RSD PPI

(%) extract (μg/mL)
CBD 1 1.24 24.9 2.4 1000 0.1
2 0.42 11.1 3.5 999 0.2
3 0.23 6.0 2.1 991 0.5
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4 2.44 53.2 2.4 1000 0.1

5 1.49 38.1 2.4 1000 0.1

CBDA 1 13.61 274.8 2.4 991 0.8

2 6.66 168.3 3.0 1000 0.1
3 13.65 361.0 3.5 989 0.8
4 9.08 233.9 2.9 999 0.3
5 13.77 351.0 2.1 987 0.9
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CBN 5 0.04 1.3 2.6 974 2.3
6 0.19 4.8 2.3 992 1.4
7 0.13 3.8 2.1 988 1.5
8 0.04 1.1 1.4 836 5.4
13 0.49 4.3 1.5 999 0.5
Resolution and peak purity were only assessed if the analyte concentration was ≥ LLOQ.
The indicated resolution is the mean of the resolution “analyte peak – previous peak” and the resolution “analyte peak
– next peak”.

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Table 2. Linear ranges, limits of detection (LOD), and lower limits of quantification (LLOQ) for
the determination of cannabinoids in herbal cannabis and hashish samples by HPLC-DAD.
Analyte Linear range LOD LLOQ
(µg/mL) (%)a (%)b (µg/mL) (%)a (%)b (µg/mL) (%)a (%)b
THC 1 - 100 0.04 – 4.00 0.13 - 13.33 0.3 0.012 0.04 1 0.04 0.13
THCA 10 - 500 0.40 - 20.00 1.33 - 66.66 0.5 0.020 0.07 10 0.40 1.33

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CBD 1 - 100 0.04 – 4.00 0.13 - 13.33 0.3 0.012 0.04 1 0.04 0.13
CBDA 10 - 500 0.40 – 20.00 1.33 - 66.66 0.5 0.020 0.07 10 0.40 1.33
CBN 1 - 100 0.04 – 4.00 0.13 - 13.33 0.3 0.012 0.04 1 0.04 0.13

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a
Herbal cannabis, given that the sample is prepared as described in section 2.3.
b
Hashish, given that the sample is prepared as described in section 2.3.

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Table 3. Accuracy data for cannabinoids spiked in cannabis extracts (flowers: samples 1, 3; mixed
material: samples 2, 4; hashish: sample 5).
Spiked Found Recovery
Sample Analyte
(μg/mL) (μg/mL, n = 6) (%)

1 THC 19.9 22.2 111.7

33.9 36.7 108.5
61.9 67.4 109.0

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2 THCA 105.4 109.6 104.0
175.4 185.3 105.7

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315.4 332.7 105.5

25.7

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3 CBD 25.3 98.2
44.2 43.4 98.2
81.2 77.8 95.8

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4 CBDA 66.1 67.6 102.2
111.1
201.1
112.8
201.9
101.6
100.4
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5 CBN 13.2 14.3 108.6
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22.7 24.7 108.8

41.7 46.3 111.0
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Table 4. Precision data for quantification of cannabinoids in cannabis extracts (flowers: samples
1, 3, 4, 5, 9; mixed material: 2, 6; hashish: 7, 8).
Intra-assay precision (%RSD, n = 6)
Content concentration of Inter-assay precision
Analyte Sample Day 1 Day 2 Day 3
(%) extract (μg/mL) (%RSD, n = 18)
THC 1 2.68 69.0 20.4 13.7 13.1 15.3
2 0.78 22.1 9.6 21.9 2.8 13.0

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3 1.06 28.8 2.5 8.9 13.6 9.5
4 0.23 5.8 16.3 9.4 8.8 11.4
0.0 2.8

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5 0.06 1.8 0.0 2.8
7 8.39 55.4 12.0 8.3 1.1 8.6
8 5.29 37.0 8.1 7.4 8.3 7.5

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9 2.82 68.3 8.0 8.5 0.7 6.4

THCA 1 16.50 412.2 19.7 13.1 12.4 14.8

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2 1.28 34.1 10.0 22.2 2.7 13.4
3 9.26 247.7 2.6 8.6 13.5 9.4
7 11.05 79.3 13.0 N 9.3 2.1 9.3
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8 27.02 204.9 7.6 6.8 7.8 7.0
9 0.88 27.9 8.6 10.5 2.1 7.5
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CBD 4 2.39 61.1 16.1 8.9 8.8 11.1

5 0.42 13.3 1.8 2.5 0.8 4.3
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6 0.09 2.3 11.7 3.6 7.3 8.1

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CBDA 4 10.96 286.4 15.9 8.7 8.7 10.9

5 6.66 191.9 1.8 2.4 0.6 3.1
6 0.75 21.1 12.2 3.6 7.6 8.2
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9 3.76 102.4 8.8 8.5 0.6 6.7

CBN 1 0.05 1.1 21.9 14.8 13.3 16.4

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7 0.49 3.5 12.8 9.0 2.0 9.2

9 2.54 63.6 8.1 8.6 0.3 6.5
Precision is only reported for analyte concentrations within the calibration range.
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