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Quality Traits of “Cannabidiol Oils”: Cannabinoids Content, Terpene Fingerprint and Oxidation Stability of European Commercially Available Preparations

1 CRC-Ge.S.Di.Mont.—Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, CRC-Ge.S.Di.Mont.—Università degli Studi di Milano, Via Morino 8, Edolo, 25048 Brescia, Italy; [email protected] (R.P.); [email protected] (L.P.); [email protected] (A.G.)

2 Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

Giorgio Nenna

3 UPFARM-UTIFAR—The Professional Union of Pharmacists for Orphan Medicines; Piazza Duca d’Aosta, 14, 20124 Milan, Italy; [email protected]

Lorenzo Calvi

2 Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

Sara Panseri

4 Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy

Gigliola Borgonovo

5 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

Luca Giupponi

1 CRC-Ge.S.Di.Mont.—Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, CRC-Ge.S.Di.Mont.—Università degli Studi di Milano, Via Morino 8, Edolo, 25048 Brescia, Italy; [email protected] (R.P.); [email protected] (L.P.); [email protected] (A.G.)

2 Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

Giuseppe Cannazza

6 Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 103, 41121 Modena, Italy; [email protected]

Annamaria Giorgi

1 CRC-Ge.S.Di.Mont.—Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, CRC-Ge.S.Di.Mont.—Università degli Studi di Milano, Via Morino 8, Edolo, 25048 Brescia, Italy; [email protected] (R.P.); [email protected] (L.P.); [email protected] (A.G.)

2 Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

1 CRC-Ge.S.Di.Mont.—Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, CRC-Ge.S.Di.Mont.—Università degli Studi di Milano, Via Morino 8, Edolo, 25048 Brescia, Italy; [email protected] (R.P.); [email protected] (L.P.); [email protected] (A.G.)

2 Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

3 UPFARM-UTIFAR—The Professional Union of Pharmacists for Orphan Medicines; Piazza Duca d’Aosta, 14, 20124 Milan, Italy; [email protected]

4 Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy

5 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy; [email protected]

6 Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 103, 41121 Modena, Italy; [email protected]

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Abstract

Cannabidiol (CBD)-based oil preparations are becoming extremely popular, as CBD has been shown to have beneficial effects on human health. CBD-based oil preparations are not unambiguously regulated under the European legislation, as CBD is not considered as a controlled substance. This means that companies can produce and distribute CBD products derived from non-psychoactive hemp varieties, providing an easy access to this extremely advantageous cannabinoid. This leaves consumers with no legal quality guarantees. The objective of this project was to assess the quality of 14 CBD oils commercially available in European countries. An in-depth chemical profiling of cannabinoids, terpenes and oxidation products was conducted by means of GC-MS and HPLC-Q-Exactive-Orbitrap-MS in order to improve knowledge regarding the characteristics of CBD oils. Nine out of the 14 samples studied had concentrations that differed notably from the declared amount, while the remaining five preserved CBD within optimal limits. Our results highlighted a wide variability in cannabinoids profile that justifies the need for strict and standardized regulations. In addition, the terpenes fingerprint may serve as an indicator of the quality of hemp varieties, while the lipid oxidation products profile could contribute in evaluation of the stability of the oil used as milieu for CBD rich extracts.

1. Introduction

Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the most common cannabinoids in medical cannabis preparations [1]. The are both responsible for a variety of pharmacological actions that can have remarkable applications, but unlike THC, CBD does not possess any psychoactive effects [1]. Several studies suggest that CBD can be effective in treating epilepsy and other neuropsychiatric disorders, including anxiety and schizophrenia [2,3,4]. CBD may also be effective in treating post-traumatic stress disorder and may have anxiolytic, antipsychotic, antiemetic and anti-inflammatory properties [5,6,7]. This plethora of pharmacological activities has led to rapid changes in the cultural, social and political legal viewpoints regarding the utilization of cannabis-based preparations [8]. Although there is still a complicated legal milieu that calls for caution, it is undeniable that there is an enormous interest from consumers/patients in the utilization of CBD dietary supplements. This has created an exploding industry of CBD products in Europe and around the world. “CBD enriched oils”, obtained from extraction of different Cannabis sativa L. chemotypes with high content of CBD, are the most popular products used [9,10,11,12].

Since CBD, in contrast to THC, is not a controlled substance in the European Union [13] several companies produce and distribute CBD-based products obtained from inflorescences of industrial hemp varieties. However, due to the lack of specific regulations, no analytical controls are mandatory for CBD-based products, leaving consumers with no legal protection or guarantees about the composition and quality of the product they are acquiring. Currently, CBD-based products are not subject to any obligatory testing or basic regulatory framework to determine the indication area, daily dosage, route of administration, maximum recommended daily dose, packaging, shelf life and stability. Exceptions are galenical “CBD oil” prepared by pharmacists following medical prescriptions in several European Union countries such as Germany, Italy and Holland. The German Drug Codex (DAC), which is published by the Federal Union of German Associations of Pharmacists (ABDA) and functions as a supplementary book to the Pharmacopoeia, suggests a preparation of 5% CBD in medium chain triglycerides oil also indicating detailed analytical controls of galenic preparations [14].

In Italy medical cannabis represents a multifaceted reality [9,10,11,12]. At present Dutch Bedrocan varieties (Bedrocan, Bediol, Bedica and Bedrolite as representative) [15] and the new strain FM2 produced by Military Pharmaceutical Chemical Works of Florence, Italy (authorized in November 2015 by a Ministerial Decree) can be prescribed to treat a wide range of pathological conditions [16]. Indeed. Italian galenic pharmacies are authorized to prepare precise cannabis doses for vaping, herbal teas, resins, micronized capsules and oils. The oil preparation has received considerable attention since it is easy to adjust the individual administration dose required throughout the treatment period, and due to the enhanced bioavailability of its active compounds [9,10,11,12]. Among abovementioned strains, Bedrolite with CBD and THC contents of 9% and

Cannabis sativa L. has been cultivated throughout the world for industrial and medical purposes. The European Union permits the cultivation of plants for hemp products based on the THC content being less than 0.2%. EU Regulation 1307/2013 [17] states that hemp farmers are required to use seeds of cannabis varieties included in the European Union catalogue. In general, specialized extraction procedures, among which the most common is supercritical CO2 extraction, are used to draw out an extract rich in CBD from the cannabis to obtain CBD oil formulations [18,19]. This product also contains other biological active compounds such as omega-3 fatty acids, vitamins, terpenes, flavonoids and other phytocannabinoids like cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN) and cannabidivarian (CBCV) [10,11,12].

Among non-cannabinoids compounds, special attention must be paid to terpenes that represent the largest group (more than 100 different molecules) of cannabis phytochemicals [20,21,22,23]. Monoterpenes, diterpenes, triterpenes and sesquiterpenes are important components present in the cannabis resin responsible for its unique aromatic properties. Due to their ability to easily cross cell membranes and the blood-brain barrier, they can also influence the medicinal quality of different cannabis chemotypes [24]. Several therapeutic approaches based on the combined use of cannabinoids and terpenes have been developed recently. Particularly, treatment of sleeping disorders and social anxiety by adding caryophyllene, linalool and myrcene to CBD/THC extracts gave encouraging results [25]. In addition, differences between the pharmaceutical properties of diverse Cannabis sativa L. varieties have been attributed to strict interactions, defined as ‘entourage effects’, between cannabinoids and terpenes as a result of synergic action [25]. Recently Pagano et al. [26] investigated the different effect of a pure CBD preparation versus a standardized Cannabis sativa extract with the same concentration of cannabidiol (CBD) in the remission of mucosal inflammation in a mouse models of colitis. The author reveled that under the same experimental conditions, pure CBD just partially ameliorated colitis, while Cannabis sativa L. extract almost entirely reduced the injuries. These findings sustain the rationale of the ‘entourage effect’ achievable by combining CBD with other minor Cannabis constituents.

The quality of Cannabis macerated oils has already been investigated in previous research demonstrating the importance of selecting correct preparation methods and conditions as well as studying the evolution of major and minor compounds (cannabinoid and terpenes) during storage in order to define the ideal shelf-life and management guidelines (storage temperature) [12]. Oxidation products derived from fatty acid degradation during the storage period of macerated oils are critical for overall formulation stability [27,28]. Galenic preparations are usually prepared by using pharmacopeia grade olive oil (FU) to minimise the formation of large quantities of aldehydes and ketones that can also influence the digestibility of the macerated oil [9,12,29].

Since the production of CBD-based oils as dietary supplements has increased rapidly, and since they are frequently used for therapeutic purposes, the main scope of this study was to assess the overall quality of 14 CBD oil preparations produced in different European countries and purchased on the Internet and highlight possible criticisms. Moreover, a Bedrolite macerated oil prepared as a galenic product was used as a reference therapeutic formulation. In order to define and increase knowledge about the characteristic of CBD oils, an in-depth chemical profiling of cannabinoids, terpenes and oxidation compounds by means of GC-MS and HPLC-Q-Exactive-Orbitrap-MS analytical platforms was presented herein.

2. Results and Discussion

2.1. Cannabinoids Content

Current ambiguous all-purpose regulations allow huge variations in the quality and safety of the CBD-based preparations available on the market and clear labelling regarding the exact concentration of CBD is not yet mandatory. Our results demonstrate that CBD concentrations were not always in accordance with producer information ( Table 1 ). As a matter of fact, nine out of 14 tested samples presented concentrations that differed notably from the declared amount, while the remaining five preserved CBD levels within optimal limits (the variation was less than 10%). Our analysis also revealed that two preparations (particularly oils 8 and 10) exhibited higher levels of CBD than those specified by producers, while in another two (samples Oil_3 and Oil_14) the CBD content was far inferior to the stated values. In one sample, the theoretical CBD concentration was not indicated on the label and therefore values obtained could not be compared to the producer’s statement. Taken together, the results highlighted the extreme variability of the commercialised CBD oil preparations, justifying the need for stricter regulations/controls. Precise information regarding the composition of each lot that is available on the market is crucial for consumers who have to be able to properly adapt the recommended dose to the available/purchased preparation [9]. These results are in agreement with those obtained from a preliminary study toward the labeling accuracy of cannabidiol extracts preparations from products available on the US market. In the tested products, 26% contained less CBD than labeled, which could negate any potential clinical response [30]. The over labeling of CBD products in the study was similar in magnitude to levels that triggered warning letters to 14 businesses in 2015–2016 from the US Food and Drug Administration suggesting that there is a continued need for federal and state regulatory agencies to take steps to ensure label accuracy of these consumer products.

Table 1

Cannabinoid content (expressed as % w/w and in μg/g) in investigated CBD oils (average ± S.D., n = 2).

(% w/w) Cannabinoids Content (μg/g)
Samples Code Deviation from Declared CBD Percentage Declared CBD 1 Revealed CBD 2 CBD THC CBN CBG CBDA THCA CBGA
Average ±SD Average ±SD Average ±SD Average ±SD Average ±SD Average ±SD Average ±SD
Oil_1 3 9.00 0.9 0.89 8143 170.2 232 4.9 14 0.3 / 884 18.8 123 2.6 7 0.1
Oil_2 8.49 4 3.66 36,567 257.3 1908 13.5 208 1.5 716 5.1 42 0.3 1 0.0 12 0.1
Oil_3 21.21 1 0.79 3247 241.7 148 10.5 40 2.8 16 1.1 5282 373.5 191 13.5 693 49.0
Oil_4 15.29 5 4.24 42,352 2395.8 0.01 0.0 3 0.2 / 6 0.3 196 11.1 19 1.1
Oil_5 10.53 4 4.42 43,509 3076.6 533 37.7 69 4.9 / 802 56.7 17 1.2 27 1.9
Oil_6 4.44 3 2.87 28,536 1008.9 3546 125.4 481 17.0 / 152 5.4 29 1.0 8 0.3
Oil_7 8.27 4 4.33 42,601 1807.4 526 22.3 65 2.8 / 804 34.1 12 0.5 26 1.1
Oil_8 35.41 3 4.06 39,962 3108.3 695 54.1 62 4.8 / 753 58.6 47 3.7 22 1.7
Oil_9 7.63 3 3.23 32,212 683.3 1607 34.1 345 7.3 23 0.5 88 1.9 25 0.5 6 0.1
Oil_10 23.89 4 4.96 48,879 1036.9 557 11.8 79 1.7 / 774 16.4 58 1.2 23 0.5
Oil_11 / / 0.24 1875 68.9 36 1.3 7 0.3 / 634 23.3 32 1.2 18 0.7
Oil_12 19.28 2 1.61 12,758 180.4 494 7.0 188 2.7 6 0.1 3862 54.6 107 1.5 97 1.4
Oil_13 36.20 4 2.55 24,444 2419.8 568 56.2 1105 109.4 624 61.8 1229 121.7 27 2.7 22 2.2
Oil_14 38.14 5 3.09 23,186 655.8 524 14.8 67 1.9 460 13.0 8828 249.7 358 10.1 216 6.1
Oil_15 24.33 3 2.27 22,692 320.9 / / 5687 80.4 9 0.1 / 4 0.1

1 CBD declared on labels, 2 CBDtot (sum of CBD +0.877 × CBDA); 3 Bedrolite oil extract prepared as galenic product—detailed description of the method and its suitability was given previously by Calvi et al., 2018 [12].

Although CBD is a principal constituent of the examined cannabis oil extracts, the original plant is only capable of producing its acid form, cannabidiolic acid (CBDA). Decarboxylation of CBDA catalysed by thermal exposure during extraction conditions leads to the conversion of CBDA to the CBD as the corresponding decarboxylated (neutral) counterpart. Therefore, the determination of CBDA is important in order to evaluate the CBDA decarboxylation rate and effectiveness of the reaction during the extraction process [31]. Interestingly, looking through the web sites of the CBD oil producers enrolled in this survey, it can be found that some of them published an analytical report in which only the total CBD content as the sum of CBD + (0.877 CBDA) is reported. This is quite problematic as the biological effects of the neutral and acidic forms are remarkably different [5]. Generally, expressing the CBD content as a sum of the acidic and neutral forms is conditioned by the analytical method applied. Concretely, it occurs when gas chromatography (GC), one of the most commonly used analytical platforms for cannabinoid analysis, is used [31]. It involves the heating of the sample at high temperature in the injector prior to the chromatographic separation that leads inevitably to the decarboxylation of the cannabinoid acids. Therefore, the analytical result is the sum of the acid and neutral forms. The GC method is still officially employed by the authorities for the determination of cannabinoids, but obviously is unsuitable. A few research groups continue to suggest that an accurate cannabinoid profile should be evaluated by determining the acid and neutral forms separately [12,31]. Results obtain in this study confirms this necessity. Employing the LC-HRMS technology, we were able to distinguish the acidic form from neutral CBD, and to examine the wide concentration range. As can be seen in Table 1 , in the majority of the samples the CBDA concentration was found to be negligible compared to the amount of CBD (for example, samples Oil_4 and Oil_15). On the contrary, there were a few samples with a significant amount of CBDA. A striking example is Oil_3, in which the CBDA content exceeded CBD, and only the sum of both forms justified the CBD percentage declared by the producer. Furthermore, it is evident that the label concentration of CBD in Oil_12 is reached only when the sum of both forms is considered, bearing in mind the significant amount of CBDA.

Nevertheless, all producers underline that their manufacturing methods yield the so called full spectrum extract, which means that hemp extracts contain different phyto-cannabinoids, including THC, CBN, CBG, THCA, CBGA and others, depending from cannabis strain and extraction method. In order to achieve full-spectrum in a hemp extract, the profile of bioactive compounds that a plant flower contains must be transferred into the extract itself without compromising any aspect of the profile.

In comparison with previous works available on cannabis oil [9,10,11,31] we employed a HRMS method that provided more complete information regarding the cannabinoids profile and amount in the oil composition. Actually, besides CBD as a principal cannabinoid, we were able to detect and to quantify the six most significant cannabinoids, including the essential ones (THC, THCA and CBDA) along with quantification of CBN, CBG and CBGA. The obtained results clearly show that 12 out of 14 samples contained THC which is attention-grabbing because of its potential intoxicating activity. The THC content showed the considerable variability in the analysed samples, but was mainly at the levels describable as low (0.2%) [17]. Only one among all THC-positive sample (Oil_6) contained a considerable amount of THC (0.35%), which is matter of concern because the manufacturer declared the product to be THC-free. This result highlights the importance of also specifying the amount of THC or any another intoxicating cannabinoid present in commercialised CBD oils.

CBN was quantifiable in the vast majority of samples (except Oil_14). Its detection is of great importance as it is not considered to be a natural cannabinoid but rather an artefact formed by THC oxidation during plant aging, by use of an inadequate extraction procedure or inappropriate storage conditions [32]. Therefore, its determination may assist in the evaluation of the quality of CBD oils with regards to the raw plant material used, extraction method applied and storage. For example, in the sample Oil_13 the quantity of the CBN was more than twice the amount of THC. Considering CBN as a degradation product of THC, it would be better to think through the sum of THC+CBN as a relevant parameter for the evaluation of initial THC concentration in the oil extract. In addition, CBN, though much less psychoactive than THC, express sedative effects [33,34] which is why its content should be indicated on the label along with THC. It is well known that THC derives from the decarboxylation of tetrahydrocannabinolic acid (THCA) [20], and this is the reason why the amount of THCA was quantified in this study. Our results did not reveal any significant presence of either THCA ( Table 1 ) or cannabigerolic acid (CBGA) which is the precursor of the all other cannabinoid acids. CBGA gives by decarboxylation cannabigerol (CBG) that either was completely absent or present in minor quantities. The quantification of CBGA and CBG did not turn out to be imperative, but their presence could serve as a confirmation that the oil sample contains a natural, full spectrum cannabis extract. Furthermore, employing the retrospective analysis, several other minor “untargeted” compounds were detected by means of the Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA) ® analyser. Among others (data not shown) it is important to highlight the persistent occurrence of CBDV in all analysed samples. Figure 1 shows the fragmentation pattern of CBDV and CBD. Bearing in mind that this compound has expressed significant physiological activity [33] and that accompanies the CBD as its analogue, it should be included in any quality evaluation of full spectrum CBD oil preparations. Besides, we noticed that when the hemp seed oil was used as matrix, the signal of CBDV augments notably, which means that maybe one portion of CBDV derives from hemp oil, not from flower extract [34].

Retrospective data analysis reveals the occurrence of CBDV: full MS-dd-MS 2 chromatogram and relative fragmentation pattern of parent ion (287.20048) obtained in dd-MS 2 acquisition mode. For the comparison, the CBD signal and fragmentation pattern is also presented.

Bedrolite oil extract (Oil_1) obtained by a recently published procedure [12], is a defined galenic formulation that has been used for distinct therapeutic purposes. It was included in this study as a “reference material” from a well-defined starting material (cannabis plant variety) and made using a standardized/authorized preparation procedure. There are at least two reasons to use the cannabinoid profile of Bedrolite oil extract as a reference point in the evaluation of CBD-rich hemp oils. Firstly, it can be considered as a full-spectrum extract that preserves the natural ratios of cannabinoids, any impurities that can compromise the experiments should be absent. Secondly, many consumers tend to replace galenic oil preparations (such Bedrolite oil extract) with CBD-rich hemp oil extract, due to the fact that a medical prescription is required for the former. Our study revealed that Bedrolite oil extract contains 0.8% of CBD. This is in agreement with theoretical percentage (0.9%) that should be found in the Bedrolite oil extract: the inflorescence contains 9% of CBD and the dilution ratio during the extraction is 1:10. However, as regards the cannabinoids profile ( Table 1 ), it is evident that the quantities of CBDA, THC, THCA and CBGA are inferior compared with CBD-rich hemp oil extracts. These data are of great importance as they highlight the reduced concentration of all cannabinoids in Bedrolite oil extract compared to CBD hemp oil extract.

The reasons for all the abovementioned variations between examined samples are numerous and multiple. The final composition of CBD-rich hemp oil extracts depends on the chemotype and quality of the industrial hemp used, but it is also conditioned by the extraction method applied. Unfortunately, not all producers indicate the extraction method used. Only four declared the use of supercritical CO2 fluid extraction, which is shown to be the method of choice in that the low temperature and inert atmosphere results in higher CBD yields [18,19]. However, the main drawback of this technology is its high cost, and it is reasonable to assume that solvent extraction is also used for the inexpensive industrial processing. However, it is questionable if this is a correct choice for a product for human consumption because residual solvents (typically hexane, ethanol, isopropyl alcohol, toluene, benzene, xylene and acetone) may contaminate the final product [32]. Without having complete information on the methods of CBD oils preparation, we investigated the occurrence of the most frequently used extraction solvents as solvent residues. Our analysis revealed the sporadic incidence of acetone ( Table 2 , ketones section) that is more probably present as a lipid oxidation product rather than as a true residual solvent. Nevertheless, the presence of some volatile compounds that might be considered as problematic impurities from solvents residues was detected ( Table 2 , miscellaneous section). Namely, the samples Oil_4 and Oil_6 showed the presence of 1,3-dimethylbenzene while in the sample Oil_3, 1,2,4-trimethylbenzene was detected. Those aromatic compounds were not present in galenic preparation (Oil_1).

Table 2

Volatile compounds profile extracted by using HS-SPME and GC/MS from CBD oils samples.

Oil Samples 1 2 3 4 5 6 7 8
Matrix FU Oil Hemp Seed Oil Olive Oil MCT Oil Olive Oil Hemp Seed Oil Olive Oil Hemp Seed Oil
RI a R.T b Compound Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Alcohols
831 20.63 1-Hexanol 2.08 0.14 5.15 0.71 n.d. n.d. 2.55 0.16 8.10 0.12 2.58 0.03 11.52 0.37
868 21.43 3-Hexen-1-ol 0.66 0.07 0.67 0.11 n.d. 0.55 0.02 1.47 0.08 1.46 0.04 1.76 0.05 n.d.
849 22.02 2-Hexen-1-ol n.d. n.d. n.d. n.d. 0.77 0.11 n.d. 1.10 n.d.
969 23.07 1-Octen-3-ol n.d. 3.90 0.47 n.d. n.d. 0.73 0.15 1.73 0.03 1.09 0.51 n.d.
960 23.17 1-Heptanol n.d. 0.83 0.13 n.d. n.d. 0.50 0.01 2.49 0.10 n.d. n.d.
1059 25.48 1-Octanol n.d. n.d. 1.29 0.11 n.d. 0.59 0.03 5.23 0.63 n.d. n.d.
1068 27.98 3,3,6-Trimethyl-1,5-heptadien-4-ol 2.39 0.46 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1036 31.59 α-Toluenol 0.42 0.03 2.74 0.59 n.d. n.d. 2.36 0.16 3.96 0.47 3.22 0.23 n.d.
1136 32.06 Benzeneethanol 0.51 0.04 1.39 0.32 n.d. n.d. 2.73 0.16 5.05 0.58 3.43 0.22 0.76 0.01
Total 6.06 14.68 1.29 0.55 11.70 28.01 13.18 12.28
Aldehydes
508 2.31 Propanal n.d. n.d. 0.77 0.03 n.d. 0.96 0.04 1.10 0.08 0.97 0.05 n.d.
574 3.23 2-Methyl-2-propenal n.d. n.d. 0.61 0.03 n.d. n.d. n.d. n.d. n.d.
643 3.74 2-Methyl-butanal 1.73 0.02 0.56 0.02 n.d. n.d. n.d. n.d. n.d. n.d.
643 3.83 3-Methyl-butanal 0.90 0.16 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
785 10.12 Hexanal 0.70 0.02 5.57 0.14 10.15 0.29 n.d. 2.97 0.17 6.89 0.01 2.60 0.15 3.42 0.03
905 15.05 Heptanal 0.76 0.03 n.d. 1.03 0.03 n.d. n.d. n.d. 0.67 0.10 0.62 0.03
814 16.24 2-Hexenal n.d. n.d. n.d. n.d. 1.19 0.12 n.d. 1.41 0.03 n.d.
1005 18.74 Octanal n.d. n.d. 2.47 0.02 n.d. 2.03 0.23 17.40 0.16 1.87 0.03 n.d.
913 19.72 2-Heptenal n.d. 3.10 0.05 2.61 0.22 n.d. 1.86 0.09 6.44 0.41 1.59 0.03 1.22 0.05
1104 21.67 Nonanal 0.73 0.16 n.d. 2.63 0.30 n.d. n.d. 4.92 0.03 1.07 n.d.
1013 22.53 2-Octenal n.d. n.d. 0.68 0.05 n.d. n.d. 2.07 0.08 n.d. 0.61 0.02
921 24.01 2,4-Heptadienal n.d. 1.27 0.05 0.74 0.14 n.d. n.d. 7.97 0.34 n.d. n.d.
982 24.77 Benzaldehyde n.d. n.d. n.d. n.d. 1.86 0.01 25.84 0.45 n.d. n.d.
1174 28.04 3,7-Dimethyl-2,6-octadienal n.d. n.d. n.d. n.d. n.d. 44.49 4.40 n.d. n.d.
Total 4.82 10.50 21.68 n.d. 10.87 117.14 10.19 5.88
Esters
487 2.63 Acetic acid-methyl ester 0.78 0.02 0.72 0.04 n.d. n.d. n.d. n.d. n.d. n.d.
586 3.33 Acetic acid-ethyl ester n.d. 8.70 0.12 302.74 12.32 n.d. n.d. n.d. n.d. n.d.
686 5.35 Acetic acid-propyl ester n.d. n.d. 1.45 0.07 n.d. n.d. n.d. n.d. n.d.
721 6.77 Acetic acid-2-methyl-propyl ester n.d. n.d. 5.39 0.06 n.d. n.d. n.d. n.d. n.d.
785 9.76 Acetic acid-buthyl ester n.d. n.d. 1.17 0.11 n.d. n.d. n.d. n.d. n.d.
820 12.27 1-Butanol-3-methyl acetate n.d. n.d. 7.46 0.17 n.d. n.d. 16.98 0.21 n.d. n.d.
992 19.62 3-Hexen-1-ol-acetate n.d. n.d. n.d. n.d. n.d. n.d. 0.64 0.02 n.d.
1183 22.24 Butanoic acid-hexyl ester n.d. n.d. n.d. 0.60 0.02 n.d. n.d. n.d. n.d.
Total 0.78 9.42 318.21 0.60 n.d. 16.98 0.64 n.d.
Ketones
455 2.50 2-Propanone 1.76 0.13 3.50 0.47 8.56 0.57 n.d. 2.46 0.51 5.03 0.65 1.78 0.14 0.96 0.01
1161 13.19 1-(1,3-dimethyl-3-cyclohexen-1-yl)-Ethanone n.d. 1.89 0.18 n.d. n.d. n.d. 2.07 0.15 n.d. 0.90 0.06
853 14.89 2-Heptanone n.d. 3.32 0.09 0.71 0.02 n.d. 2.69 0.16 n.d. 2.74 0.06 1.00 0.06
952 18.57 2-Octanone n.d. 4.63 0.64 n.d. n.d. n.d. n.d. n.d. n.d.
960 19.99 6-Octen-2-one n.d. 0.86 0.11 n.d. n.d. n.d. n.d. n.d. n.d.
987 20.17 6-Methyl-5 hepten-2 one 1.25 0.04 8.97 1.20 10.55 0.42 n.d. 2.28 0.11 17.04 0.14 1.63 0.00 2.26 0.13
960 21.96 3-Octen-2-one n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
962 22.51 Ketone 0.69 0.02 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
968 24.67 3,5-Octadien-2-one n.d. n.d. 4.94 0.41 n.d. n.d. n.d. n.d. n.d.
Total 3.70 23.17 24.77 n.d. 7.44 24.14 6.15 5.12
Terpenes
939 6.67 α-Pinene 14.25 0.55 21.47 0.38 n.d. 73.23 9.63 112.95 1.47 5883.13 22.05 119.09 0.23 52.84 1.11
932 7.03 α-Thujene 0.98 0.17 1.18 0.08 n.d. 0.79 0.08 2.86 0.19 44.54 1.08 3.24 0.10 n.d.
961 8.67 Camphene n.d. n.d. n.d. 0.94 0.27 1.57 0.09 65.97 3.91 1.60 0.01 0.95 0.03
989 10.75 β-Pinene 4.15 0.05 4.45 0.08 n.d. 23.36 4.71 35.65 2.06 625.28 4.35 37.08 0.48 17.91 0.28
985 11.66 Sabinene n.d. 1.26 0.07 n.d. n.d. 1.66 0.01 98.17 0.06 1.53 0.08 n.d.
879 11.74 2,4(10)-Thujadien n.d. 0.85 0.00 n.d. n.d. n.d. n.d. n.d. n.d.
1017 12.99 δ-3-Carene 1.73 0.03 0.61 0.09 n.d. 7.42 1.33 50.22 1.18 4.69 0.31 51.31 2.68 6.17 0.50
1015 13.81 α-Phellandrene 3.70 0.14 3.90 0.09 n.d. 2.91 0.65 6.78 0.01 4.97 0.04 8.06 0.18 1.85 0.09
991 14.24 β-Myrcene 108.13 0.09 34.63 1.00 9.14 0.48 419.53 33.32 389.37 20.60 2908.00 60.01 419.01 6.04 189.05 1.74
1026 14.44 α-Terpinene 3.60 0.38 10.01 0.56 n.d. 2.44 0.07 5.51 0.17 34.87 3.09 6.82 0.12 1.74 0.01
1038 15.31 Limonene 6.05 0.01 8.03 0.44 1.60 0.07 65.17 7.36 23.67 1.33 8841.42 171.34 20.17 0.26 23.97 0.34
1045 15.49 Eucalyptol 3.67 0.01 5.60 0.35 7.15 0.26 2.65 0.13 4.18 0.27 13.66 1.60 3.09 0.07 8.62 0.12
946 15.61 β-Phellandrene 7.16 0.14 4.80 0.52 n.d. 8.40 0.80 19.02 1.05 61.90 4.71 20.05 0.16 7.38 0.22
976 17.05 Cis-ocimene 0.48 0.02 2.17 0.17 0.48 0.00 16.55 1.07 21.17 0.88 7.13 0.24 19.24 0.23 5.50 0.11
1066 17.15 γ-Terpinene 5.38 0.01 5.84 0.58 0.59 0.06 2.11 0.09 4.80 0.23 499.58 9.26 4.61 0.08 2.48 0.04
1000 17.24 Terpene n.d. n.d. 2.48 0.24 n.d. 2.59 0.02 17.49 0.05 1.82 0.04 n.d.
1029 17.60 β-Ocimene 21.75 1.08 7.55 0.53 9.96 0.48 194.00 21.13 192.39 5.19 22.82 0.17 213.15 1.26 42.00 1.72
1034 18.01 p-Cymene 3.13 0.11 8.53 0.97 0.81 0.01 2.98 0.33 12.37 0.05 144.55 1.92 10.02 0.15 5.62 0.09
1094 18.43 α-Terpinolene 62.12 0.67 7.48 0.80 n.d. 111.31 14.58 265.95 16.97 33.73 0.11 297.20 3.11 73.16 3.13
1177 22.74 Para-cymenyl 13.97 0.88 11.28 0.16 n.d. 3.30 0.82 64.48 5.97 134.94 1.32 49.21 0.39 4.70 0.24
1136 23.01 Terpene n.d. n.d. n.d. 1.55 0.36 1.39 0.11 n.d. 1.43 0.05 n.d.
1083 23.35 4,8-Epoxy-p-menth-1-ene 1.30 0.08 2.08 0.23 n.d. 2.22 0.03 6.44 0.16 3.08 0.03 5.20 0.60 1.32 0.01
1164 23.48 Linalool oxide n.d. n.d. n.d. n.d. 1.11 0.03 17.95 0.94 1.51 0.01 n.d.
1221 23.68 α-Ylangene n.d. 1.09 0.23 n.d. n.d. 0.52 0.00 2.19 0.09 n.d. 1.18 0.07
1082 25.29 β-Linalool 5.52 0.81 2.22 0.32 10.55 1.27 0.82 0.01 5.19 0.01 1471.75 15.90 5.36 0.37 n.d.
1494 25.75 γ-Caryophyllene n.d. 5.87 1.68 2.02 0.24 1.03 0.06 1.81 0.05 30.72 1.10 1.32 0.04 4.40 0.17
1430 26.03 α-Bergamotene 2.42 0.21 11.89 3.95 1.75 0.08 2.21 0.19 2.31 0.02 40.96 0.21 1.01 0.08 9.44 0.07
1456 26.12 α-Guaiene 2.46 0.05 n.d. 7.19 0.57 n.d. n.d. 5.20 0.18 n.d. n.d.
1494 26.23 Trans-caryophyllene 17.34 1.81 159.92 49.08 90.68 7.15 40.67 3.44 48.77 1.93 425.63 7.84 32.06 4.48 110.41 1.33
Terpenes
1209 26.40 4-Terpineol 2.32 0.23 3.34 0.62 0.55 0.06 n.d. 2.80 0.24 15.34 1.66 1.71 0.18 n.d.
1440 26.59 Sesquiterpene n.d. 3.03 0.70 1.37 0.18 n.d. 1.64 0.07 17.73 0.11 n.d. 1.13 0.10
1386 27.21 Sesquiterpene n.d. 6.09 2.17 n.d. n.d. 0.58 0.05 12.62 0.59 n.d. 7.01 0.26
1131 27.47 Trans-pinocarveol n.d. 9.69 1.69 n.d. n.d. 3.04 0.07 10.99 0.49 2.30 0.08 5.12 0.08
1482 27.73 α-Humulene 6.28 0.44 45.95 15.88 21.61 1.49 10.13 1.54 9.62 0.69 117.14 3.27 5.94 0.88 32.25 1.15
1189 28.13 1,8-Menthadien-4-ol 6.23 1.02 21.18 4.16 n.d. 2.19 0.43 19.68 0.44 44.91 2.39 12.61 0.58 6.86 0.41
1209 28.32 α-Terpineol 2.79 0.43 3.31 0.85 1.13 0.02 n.d. 2.90 0.24 14.35 1.10 1.83 0.04 0.63 0.07
1189 28.40 Borneol 0.52 0.04 2.78 0.74 n.d. n.d. 0.64 0.04 1.59 2.25 n.d. n.d.
1490 28.65 δ-Guaiene 1.56 0.01 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1519 28.72 β-Selinene 0.85 0.01 11.64 4.28 1.65 0.02 0.96 0.19 1.46 0.13 41.02 1.77 0.85 0.17 8.15 0.36
1522 28.82 α-Selinene 1.04 0.01 7.51 2.69 1.06 0.14 n.d. 1.04 0.06 26.41 1.64 n.d. 4.51 0.20
1474 28.98 Sesquiterpene n.d. 1.88 0.58 n.d. n.d. n.d. 53.97 6.99 n.d. 0.92 0.02
1190 29.03 Carvone n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1507 29.86 Selina-3,7(11)-diene n.d. 6.79 2.41 n.d. 0.96 0.31 1.85 0.06 25.33 2.61 n.d. 3.78 0.21
1191 30.13 Myrtenol n.d. 1.49 0.26 n.d. n.d. 0.94 0.04 3.18 0.40 n.d. 0.80 0.03
1284 31.17 Cuminol 4.00 0.64 7.44 1.98 n.d. 0.54 0.07 7.87 0.61 30.26 3.84 7.02 0.08 1.46 0.10
1322 33.41 Humulene oxide n.d. 2.22 0.92 0.72 0.13 n.d. n.d. n.d. n.d. 1.36 0.14
1419 34.11 Sesquiterpene n.d. n.d. 1.54 0.03 n.d. n.d. n.d. n.d. n.d.
1392 34.86 Eugenol n.d. n.d. n.d. n.d. 0.82 0.00 1.12 0.05 1.17 0.02 n.d.
Total 314.90 457.03 174.05 1000.37 1339.56 21860.3 1367.63 644.70
Miscellaneous
906 11.03 3,3,6-Trimethyl-1,5-heptadiene n.d. n.d. n.d. n.d. n.d. 11.87 0.33 n.d. n.d.
907 12.72 1,3-Dimethyl-benzene n.d. 0.96 0.08 n.d. 2.17 0.38 n.d. 1.41 0.02 n.d. n.d.
1040 16.87 2-Pentyl-furan n.d. 1.82 0.20 n.d. n.d. 0.24 0.01 3.94 0.10 n.d. 1.99 0.07
1020 18.31 1,2,4,-Trimethyl-benzene n.d. n.d. 1.35 0.38 n.d. n.d. n.d. n.d. n.d.
894 19.54 2,5-Dimethyl-pyrazine 1.03 0.05 n.d. n.d. n.d. n.d. 1.23 0.04 n.d. n.d.
891 19.74 2,6-Dimethyl-pyrazine 0.82 0.03 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1176 20.72 1,4-Bis (1-methylethyl)-benzene n.d. n.d. n.d. n.d. n.d. 1.81 0.01 n.d. n.d.
985 21.89 2,6-Dimethyl-2,6-octadiene n.d. n.d. 3.25 0.14 n.d. n.d. n.d. n.d. n.d.
1081 22.10 Diethyl carbitol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1039 22.36 1,3,5-Trimethylenecycloheptane n.d. 1.07 0.26 n.d. 0.80 0.12 1.52 0.00 2.62 0.06 1.70 0.02 n.d.
986 28.45 5-Ethyldihydro-2(3H)-furanone n.d. 1.92 0.14 n.d. n.d. 3.89 0.41 27.39 2.75 6.63 0.41 0.96 0.15
1190 30.85 1-Methoxy-4(1-propenyl)-benzene n.d. n.d. n.d. 3.22 0.07 n.d. n.d. n.d. n.d.
Total 1.85 7.19 4.60 6.19 8.38 54.23 10.93 2.95
Oil Samples 9 10 11 12 13 14 15
Matrix Hemp Seed Oil Olive Oil Hemp Seed Oil Hemp Seed Oil Olive Oil Hemp Seed Oil Hemp Seed Oil
RI a R.T b Compound Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Average c
μg/g
SD
(±)
Alcohols
831 20.63 1-Hexanol 6.19 0.32 2.66 0.08 2.00 0.14 4.37 0.20 0.67 0.02 3.52 0.23 9.67 0.16
868 21.43 3-Hexen-1-ol n.d. 1.81 0.03 n.d. n.d. 0.65 0.01 0.86 0.06 n.d.
849 22.02 2-Hexen-1-ol n.d. 1.15 0.05 n.d. 0.62 0.07 0.63 0.00 n.d. n.d.
969 23.07 1-Octen-3-ol 1.08 0.03 0.79 0.01 1.26 0.13 1.23 0.07 n.d. 1.35 0.00 n.d.
960 23.17 1-Heptanol n.d. n.d. n.d. 0.72 0.04 n.d. n.d. n.d.
1059 25.48 1-Octanol n.d. 0.58 0.11 n.d. n.d. n.d. 2.30 0.25 n.d.
1068 27.98 3,3,6-Trimethyl-1,5-heptadien-4-ol n.d. n.d. n.d. n.d. n.d. 1.56 0.10 n.d.
1036 31.59 α-Toluenol n.d. 3.03 0.35 n.d. 1.39 0.15 n.d. 2.08 0.61 n.d.
1136 32.06 Benzeneethanol 0.62 0.03 3.07 0.62 n.d. 1.25 0.01 0.87 0.13 2.72 0.98 n.d.
Total 7.89 13.09 3.26 9.57 2.82 14.38 9.67
Aldehydes
508 2.31 Propanal n.d. 1.04 0.00 n.d. n.d. 0.97 0.02 0.62 0.10 n.d.
574 3.23 2-Methyl-2-propenal n.d. n.d. n.d. n.d. n.d. n.d. n.d.
643 3.74 2-Methyl-butanal n.d. n.d. n.d. n.d. n.d. n.d. n.d.
643 3.83 3-Methyl-butanal n.d. n.d. n.d. n.d. n.d. n.d. n.d.
785 10.12 Hexanal 6.35 0.40 2.53 0.01 10.51 0.36 7.04 0.29 3.18 0.36 2.60 0.18 1.80 0.12
905 15.05 Heptanal n.d. 0.63 0.03 0.46 0.02 n.d. n.d. n.d. n.d.
814 16.24 2-Hexenal 1.23 0.06 1.41 0.03 1.02 0.02 0.53 0.75 11.50 0.40 0.82 0.04 n.d.
1005 18.74 Octanal n.d. 1.92 0.07 n.d. 1.93 0.32 n.d. n.d. n.d.
913 19.72 2-Heptenal 1.17 0.05 1.72 0.09 5.21 0.57 8.29 0.77 1.54 0.00 5.44 0.12 1.11 0.02
1104 21.67 Nonanal n.d. 1.20 0.14 n.d. n.d. n.d. n.d. n.d.
1013 22.53 2-Octenal 1.49 0.02 n.d. n.d. 0.95 0.00 n.d. n.d. n.d.
921 24.01 2,4-Heptadienal 8.05 0.11 n.d. 2.31 0.19 1.37 0.20 n.d. 1.24 0.02 n.d.
982 24.77 Benzaldehyde 1.45 0.18 n.d. 1.90 1.67 1.55 0.31 0.43 0.10 8.46 3.09 n.d.
1174 28.04 3,7-Dimethyl-2,6-octadienal n.d. n.d. n.d. n.d. n.d. 1.30 0.07 n.d.
Total 19.73 10.43 21.41 21.66 17.61 20.48 2.92
487 2.63 Acetic acid-methyl ester n.d. n.d. n.d. n.d. n.d. n.d. n.d.
586 3.33 Acetic acid-ethyl ester n.d. n.d. n.d. n.d. n.d. n.d. n.d.
686 5.35 Acetic acid-propyl ester n.d. n.d. n.d. n.d. n.d. n.d. n.d.
721 6.77 Acetic acid-2-methyl-propyl ester n.d. n.d. n.d. n.d. n.d. n.d. n.d.
785 9.76 Acetic acid-buthyl ester n.d. n.d. n.d. n.d. n.d. n.d. n.d.
820 12.27 1-Butanol-3-methyl acetate n.d. n.d. n.d. n.d. n.d. n.d. n.d.
992 19.62 3-Hexen-1-ol-acetate n.d. 0.66 0.01 n.d. n.d. 0.82 0.08 n.d. n.d.
1183 22.24 Butanoic acid-hexyl ester n.d. 0.53 0.04 n.d. n.d. n.d. n.d. n.d.
Total n.d. 1.19 n.d. n.d. 0.82 n.d. n.d.
Ketones
455 2.50 2-Propanone 0.63 0.04 2.20 0.24 0.54 0.05 2.11 0.02 0.78 0.15 2.41 0.45 n.d.
1161 13.19 1-(1,3-dimethyl-3-cyclohexen-1-yl)-Ethanone n.d. n.d. n.d. 2.75 0.05 n.d. 0.89 0.05 n.d.
853 14.89 2-Heptanone n.d. 2.70 0.03 1.05 0.04 1.32 0.02 n.d. 1.45 0.36 n.d.
952 18.57 2-Octanone n.d. n.d. 5.08 0.42 0.55 0.03 n.d. n.d. n.d.
960 19.99 6-Octen-2-one n.d. n.d. 0.80 0.04 n.d. n.d. n.d. n.d.
987 20.17 6-Methyl-5 hepten-2 one 0.73 0.10 1.65 0.06 0.53 0.03 6.00 0.58 0.76 0.04 6.50 0.28 n.d.
960 21.96 3-Octen-2-one n.d. n.d. 0.57 0.05 n.d. n.d. n.d. n.d.
962 22.51 Ketone n.d. n.d. n.d. n.d. n.d. 0.71 0.01 n.d.
968 24.67 3,5-Octadien-2-one 0.59 0.07 n.d. 1.44 0.17 1.44 0.13 n.d. 1.39 0.20 n.d.
Total 1.94 6.55 10.01 14.16 1.54 13.35 n.d.
Terpenes
939 6.67 α-Pinene 1.35 0.06 120.21 0.54 4.27 0.25 147.07 1.23 n.d. 94.47 3.96 2.27 0.07
932 7.03 α-Thujene n.d. 2.86 0.13 n.d. 9.40 0.18 n.d. 2.16 0.11 n.d.
961 8.67 Camphene n.d. 1.69 0.15 n.d. 3.04 0.02 n.d. 7.45 0.26 n.d.
989 10.75 β-Pinene 0.70 0.00 37.67 1.33 1.30 0.14 33.28 0.36 n.d. 21.91 0.34 0.87 0.01
985 11.66 Sabinene n.d. 1.64 0.13 n.d. 0.71 0.05 n.d. n.d. n.d.
879 11.74 2,4(10)-Thujadien n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1017 12.99 δ-3-Carene n.d. 48.70 4.87 n.d. 3.82 0.10 n.d. 6.63 0.99 n.d.
1015 13.81 α-Phellandrene n.d. 7.37 0.11 n.d. 2.59 0.40 n.d. 3.70 0.60 n.d.
991 14.24 β-Myrcene 13.88 2.66 429.64 5.70 10.12 0.33 344.78 5.22 n.d. 74.69 5.07 10.65 0.30
1026 14.44 α-Terpinene n.d. 4.29 0.26 n.d. 2.35 0.19 n.d. 2.95 0.49 n.d.
1038 15.31 Limonene 2.38 0.50 20.56 0.29 2.40 0.15 50.09 1.95 n.d. 23.56 1.63 5.53 0.46
1045 15.49 Eucalyptol n.d. 3.13 0.02 0.41 0.06 15.41 0.72 0.56 0.09 17.91 0.72 n.d.
946 15.61 β-Phellandrene n.d. 20.96 0.14 n.d. 7.16 0.27 n.d. 13.75 0.51 n.d.
976 17.05 Cis-ocimene n.d. 19.76 0.13 n.d. 5.45 0.45 n.d. 7.22 0.43 n.d.
1066 17.15 γ-Terpinene n.d. 4.77 0.03 n.d. 7.17 0.43 n.d. 4.59 0.24 n.d.
Terpenes
1000 17.24 Terpene 0.87 0.01 1.88 0.14 n.d. n.d. n.d. 1.30 0.17 n.d.
1029 17.60 β-Ocimene 4.44 0.07 217.33 2.36 2.42 0.28 40.93 2.46 0.88 0.01 24.18 1.39 2.91 0.27
1034 18.01 p-Cymene n.d. 10.17 0.39 0.66 0.05 43.02 2.52 0.66 0.27 13.51 0.89 n.d.
1094 18.43 α-Terpinolene 3.55 0.19 249.23 0.57 0.76 0.07 52.02 3.15 n.d. 16.01 1.06 1.00 0.07
1177 22.74 Para-cymenyl 1.81 0.11 47.64 0.55 0.56 0.20 7.84 0.51 1.07 0.02 7.16 0.93 n.d.
1136 23.01 Terpene n.d. 1.67 0.11 n.d. 0.53 0.06 n.d. n.d. n.d.
1083 23.35 4,8-Epoxy-p-menth-1-ene n.d. 5.65 0.01 n.d. 2.49 0.35 n.d. n.d. n.d.
1164 23.48 Linalool oxide 0.70 0.01 1.37 0.40 n.d. 2.96 0.25 n.d. n.d. n.d.
1221 23.68 α-Ylangene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1082 25.29 β-Linalool n.d. 6.35 0.07 n.d. 3.98 0.45 n.d. 4.90 0.14 n.d.
1494 25.75 γ-Caryophyllene 8.60 1.21 1.63 0.09 0.57 0.00 5.30 0.59 1.23 0.05 8.86 1.07 n.d.
1430 26.03 α-Bergamotene 6.78 1.09 1.06 0.24 0.53 0.03 11.82 1.57 2.94 0.11 19.89 3.59 n.d.
1456 26.12 α-Guaiene n.d. n.d. n.d. n.d. n.d. 1.08 0.19 n.d.
1494 26.23 Trans-caryophyllene 78.99 10.43 39.18 2.69 4.72 0.14 92.75 9.99 15.78 0.51 228.18 33.12 1.62 0.28
1209 26.40 4-Terpineol n.d. 2.07 0.13 n.d. 3.74 0.68 n.d. 2.46 0.32 n.d.
1440 26.59 Sesquiterpene n.d. n.d. n.d. 2.50 0.02 n.d. 2.80 1.76 n.d.
1386 27.21 Sesquiterpene 1.73 0.24 n.d. n.d. 6.03 0.75 1.56 0.03 7.09 1.10 n.d.
1131 27.47 Trans-pinocarveol n.d. 2.74 0.03 n.d. 8.40 0.80 0.85 0.10 2.41 0.17 n.d.
1482 27.73 α-Humulene 31.19 5.31 7.35 0.58 1.28 0.04 27.08 3.69 5.27 0.05 68.14 12.59 n.d.
1189 28.13 1,8-Menthadien-4-ol 2.13 0.13 15.23 1.04 1.44 0.81 12.22 1.05 2.44 0.08 19.06 0.12 n.d.
1209 28.32 α-Terpineol n.d. 2.13 0.19 n.d. 3.26 0.03 0.76 0.12 3.59 1.20 n.d.
1189 28.40 Borneol n.d. n.d. n.d. 1.13 0.09 n.d. n.d. n.d.
1490 28.65 δ-Guaiene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1519 28.72 β-Selinene 6.81 1.46 0.90 0.15 n.d. 8.70 1.17 3.03 0.03 13.72 2.84 n.d.
1522 28.82 α-Selinene 3.53 0.59 1.05 0.12 n.d. 5.44 0.74 1.81 0.05 9.48 1.82 n.d.
1474 28.98 Sesquiterpene 1.60 0.24 n.d. n.d. 1.01 0.15 0.58 0.04 2.13 0.41 n.d.
1190 29.03 Carvone n.d. n.d. n.d. n.d. n.d. n.d. 0.79 0.14
1507 29.86 Selina-3,7(11)-diene 5.95 1.25 1.88 0.33 n.d. n.d. 1.86 0.03 6.99 1.18 n.d.
1191 30.13 Myrtenol n.d. n.d. n.d. 1.23 0.16 n.d. n.d. n.d.
1284 31.17 Cuminol 1.51 0.26 8.56 1.41 n.d. 3.13 0.07 0.97 0.11 6.46 1.79 n.d.
1322 33.41 Humulene oxide 1.71 0.47 n.d. n.d. 1.54 0.32 0.92 0.05 2.44 0.37 n.d.
1419 34.11 Sesquiterpene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1392 34.86 Eugenol 0.76 0.15 1.18 0.14 n.d. n.d. n.d. n.d. n.d.
Total 180.97 1349.52 31.43 981.37 43.15 752.82 25.64
Miscellaneous
906 11.03 3,3,6-Trimethyl-1,5-heptadiene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
907 12.72 1,3-Dimethyl-benzene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1040 16.87 2-Pentyl-furan 3.01 0.27 n.d. 0.56 0.04 n.d. n.d. 1.26 0.12 n.d.
1020 18.31 1,2,4,-Trimethyl-benzene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
894 19.54 2,5-Dimethyl-pyrazine n.d. n.d. n.d. n.d. n.d. n.d. n.d.
891 19.74 2,6-Dimethyl-pyrazine n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1176 20.72 1,4-Bis (1-methylethyl)-benzene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
985 21.89 2,6-Dimethyl-2,6-octadiene n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1081 22.10 Diethyl carbitol n.d. n.d. n.d. n.d. n.d. n.d. 0.91 0.23
1039 22.36 1,3,5-Trimethylenecycloheptane n.d. 1.54 0.00 n.d. n.d. n.d. 1.15 0.06 n.d.
986 28.45 5-Ethyldihydro-2(3H)-furanone 0.82 0.04 5.86 1.44 0.73 0.19 1.26 0.10 n.d. 5.40 1.36 n.d.
1190 30.85 1-Methoxy-4(1-propenyl)-benzene n.d. n.d. n.d. n.d. n.d. n.d. n.d.

RI a : retention index calculated on a Rtx-Wax (30 m × 0.25 mm × 0.25 μm f.t.); RT b : retention time (min); Average c : mean value (n = 3); Data are expressed in μg/g SD d : Standard deviatio ; n.d.: not detected.

2.2. Volatile Fingerprint: Terpene Profile and Secondary Lipid’s Oxidation Products

Terpenes and cannabinoids share biosynthetic pathways and, in fact, cannabinoids are terpenophenolic compounds. In Cannabis plants, terpenes are secreted and stored together with cannabinoids in glandular trichomes. Considering this fact also in relation to recent evidence of the synergic action of terpenes and cannabinoids (“entourage effect”), a comprehensive survey of terpenes is fundamental for the evaluation of cannabis oil preparations as dietary supplement with therapeutic applications. Complete data concerning the terpenes profile are summarized and reported in Table 2 . Overall, up to 110 volatile compounds composed the volatile fingerprint, including 48 terpenes that are further divided into classes as presented in Figure 2 . The sample Oil_6 contained an extremely high amount of terpenes compared with all other samples. α-Pinene, β-myrcene and limonene are the most concentrated terpenes in this preparation, which points toward the extremely efficient extraction method applied. Samples 5, 12 and 14 contained a distinct number of various terpenes, although in far lower concentration compared to sample Oil_6. Apparently, these formulations were obtained by an extraction process able to preserve naturally occurring terpenes profile from initial Cannabis sativa plants, as their terpene profile is in accordance with those already published in literature [12,21,22,23,24,35]. Similarly, Bedrolite oil extract (Oil_1) contains various terpene structures, reflecting the initial plant profiling. A particular profile is observed for the sample Oil_4 that showed a different terpene fingerprint compared to the other oils as it predominantly contains monoterpene subclass molecules. It has previously been demonstrated how the preparation method used for the production of cannabis extracts is able to affect the presence of different terpenes [18,19], and this is most probably reason of such a specific terpene profile found in Oil_4.

Terpenes classes quantified in CBD based oils preparations (expressed as μg/g IS equivalents) (a) Mono/di/tri Terpenes (b) sesquiterpenes and oxygenated terpenes.

β-Myrcene and limonene accompanied by β-ocimene and trans-caryophyllene were found in all samples, but in a much reduced amounts compared to sample 6, and their concentration differed greatly from sample to sample. α-Pinene and β-pinene were identified in the majority of samples, excluding samples Oil_3 and Oil_13. This remains unclear considering that the two pinenes are quite balanced within the different Cannabis varieties representing around the 10% of the terpenes group and not exceeding 15–20% [20]. The occurrence of α-terpinolene in all samples (except Oil_2 and Oil_13), might be important as this compound was suggested as a genetic marker for distinguishing two important gene pools for breeding low-THC varieties [35,36]. Sample Oil_14 was particularly rich in trans-caryophyllene followed by α-humulene. The dominance of those two sesquiterpenes over the other terpenes detected in this preparation may indicate the geographic provenience of the starting Cannabis sativa material, and as a matter of fact, the producer specified the mountain region where the plant was cultivated.

It is also important to notice that sample Oil_15 was almost completely deprived of a terpene fraction aside from the traces of the main three mentioned above. This might indicate an inefficient/inadequate processing of the starting materials or even artificial addition of CBD to the oil matrix, as some essential cannabinoids were also missing ( Table 1 ). In addition, extremely low terpene content was established for samples 11 and 13, while the remaining samples had total terpene contents in the range between 174 and 1367 μg/g, with pronounced variation in composition from sample to sample. Nevertheless, qualitative and quantitative differences observed in the chemical profiles of terpene fractions are conditioned by many factors such as: hemp variety, cultivation and environmental conditions harvest time and post-harvest conditions, storage and drying of raw plants, extraction procedure applied, matrix used and finally storage of the oil formulation.

Besides hydrocarbon terpenes, oxygenated terpenoids such as linalool and α-terpineol, were found in some preparations ( Table 2 , Figure 2 ) with a notably high concentration again in sample Oil_6. Those compounds correspond to secondary photooxidation products of the initial terpenes. In the presence of light and singlet oxygen, terpenes are also known to undergo photooxidation leading to the formation of allylic hydroperoxides [35].

In addition to the terpene compound profiles that accounted for more than 90% of the detected volatile constituents of the oils, it was possible to note the presence of other organic compounds commonly found in natural extracts such as esters, alcohols, aldehydes and ketones ( Table 2 ). Only a few low chain esters could actually be identified, with ethyl acetate dominating in sample Oil_3. Its presence is most likely to be due to the preparation method and could also be considered indicative of potential adulteration.

On the another hand, the detection of aldehydes and ketones suggests the initiation of lipid peroxidation of polyunsaturated fatty acids (PUFA) in the oils used as a matrix, as demonstrated in our previous work concerning the observed trends of these compounds during storage of macerated Cannabis-derived oils [12]. It is well documented that peroxidation of PUFA leads to the formation of a well-defined series of aldehydes and ketones such as nonenal, hexanal and pentanal, 2-heptenal, especially during storage. The rate of formation of lipid oxidation products depends strictly on several factors, among which the most important are the preparation method temperature, fatty acid composition of the oil in which Cannabis extract was dissolved and the storage conditions (storage temperature) as recently demonstrated in a study [12]. These parameters are crucial to define the ultimate characteristics of the final products as evidenced also by the color of the samples ( Figure 3 ). Other volatile decomposition compounds frequently encountered include 2-hexenal, 2-octenal, 2,4-nonadienal, 4,5-dihydroxydecenal [37], some of which also appeared to be present in some of our samples.

Introduction:

Historically, the vast majority of countries have opted for the blanket prohibition of marijuana. But, after decades of stagnant legislation, this has begun to change at pace in recent years as several countries and US states have adopted, or are poised to adopt, more liberal approaches to regulation of the drug. These have ranged from the experimental – as seen with the nationwide legalization of cannabis in Uruguay – to the tentative – as in Canada which introduced laws permitting strictly regulated medicinal use over 15 years ago. Currently over half of all US states permit medicinal use of the drug and eight, as well as Washington DC, have opted to allow some recreational use for adults under state law.

These legislative changes have been driven by a marked shift in public attitudes towards the legal status of cannabis. For example, in the US, Gallup polls have shown an increase in support for marijuana legalization from 25% in 1996 to 60% in 2016. A 2016 survey of 1000 Canadians found that 70% were either supportive or somewhat supportive of marijuana legalization. And the figures are even higher when voters are asked about medicinal cannabis use specifically.

But moving away from the strict prohibition of cannabis bound in the laws of most countries is far from straightforward. When legislators shift away from this model, there are multiple alternatives to consider and there is no simple black and white, legal or illegal, stance available. Regulators must decide whether to opt for legalization or decriminalization, whether the laws apply to all forms of marijuana (for example dry marijuana or oils and extracts), what quantities and age limits are restricted, and how the laws apply separately to the growth, possession and sales of the drug. Even countries like Uruguay, which have opted for widespread legalization, must decide on a commercial model for legal sales of the drug.

This year’s Pittcon, taking place in Chicago from 5-9 March, 2017, will be host to a number of symposia that will get to grips with the issues emerging from the rapidly developing cannabis industry from a medical, legal, social and analytical point-of-view. This includes talks covering the current state of research into cannabis and derivatives for medical purposes, a look at the social stigma of cannabis and whether this is holding back medical research, as well as a wealth of advances in analytical methods for characterizing cannabis, which will play a role in quality control, regulation and pharmaceutical research.

For example, the session “It’s Legal! Now What?” will look at the challenges facing laboratories in the era of legalized cannabis. What approaches are available for analyzing cannabis samples? How can we stay ahead in the detection of contaminants and adulterants? How can we develop and enforce laboratory standards to ensure medicinal cannabis is safe for patients? This symposium will feature speakers from the Colorado Department of Public Health and Department of Agriculture, one of the few US states to allow recreational cannabis use, and the Association of Commercial Cannabis Laboratories.

And for an update on the use of cannabis for medical purposes, Tracy Ryan from Cannakids, a company that provides cannabinoid-based treatments for children with serious illness, will be speaking during the symposium “Analytical Cannabis II”. Ryan will present anecdotal and clinical trial results of medical cannabis for children and adults, and discuss the range of conditions, such as epilepsy, Crohn’s disease and PTSD, that could stand to benefit from cannabis treatment.

The US Food & Drug Administration has stated that it supports research into the medical use of marijuana, although it has not approved the drug for any indication. During this symposium, Uma Dhanabalan from TotalHealthCareTHC, will ask whether the continued stigma surrounding cannabis is holding back research in this field, and argues that greater awareness and fewer misconceptions will help realize the full potential of cannabis as a medicine.

In the chapters to come, we will be taking a look at some of these topics in greater detail, and what highlights to expect from this year’s Pittcon. This includes analytical methods for characterizing active ingredients and contaminants in cannabis, the development of industry laboratory standards, methods of cannabis extraction, as well as cannabis detection for law enforcement purposes. With major industry players, including Sigma Aldrich, Shimadzu, PerkinElmer, Restek and CEM Analytical, already confirmed for this year’s exhibition, Pittcon 2017 will be a one-stop destination to hear about all the latest developments in cannabis analysis.

References

    1. Boecker K (2016) On D.C.’s one-year anniversary with legalized marijuana, work remains. Available at: https://www.washingtonpost.com/blogs/all-opinions-are-local/wp/2016/02/25/on-d-c-s-one-year-anniversary-with-legalized-marijuana-work-remains/?utm_term=.8fe2654a3dca. Accessed: November 2016.

2. Bostwick JM. Blurred boundaries: the therapeutics and politics of medical marijuana. Mayo Clinic Proceedings 2012; 87: 172-186. doi: 10.1016/j.mayocp.2011.10.003.

5. Caulkins, JP, Kilmer B, Kleiman MAR, et al. Considering Marijuana Legalization: Insights for Vermont and Other Jurisdictions. Santa Monica, CA: RAND Corporation, 2015. http://www.rand.org/pubs/research_reports/RR864.html.

6. Caulkins, JP, Kilmer B, Kleiman MAR, et al. Options and Issues Regarding Marijuana Legalization. Santa Monica, CA: RAND Corporation, 2015. http://www.rand.org/pubs/perspectives/PE149.html.

8. Medical Marijuana.ca. Marijuana Laws. Available at: https://medicalmarijuana.ca/patients/marijuana-laws/. Accessed: November 2016.

9. News-medical. New techniques for the extraction & preparation of cannabis oil: an interview with Alison Wake. Available at: http://www.news-medical.net/news/20161024/New-techniques-for-the-extraction-preparation-of-cannabis-oil-an-interview-with-Alison-Wake.aspx. Accessed: November 2016.

10. Office of National Drug Control Policy. Marijuana Resource Center: State Laws Related to Marijuana. Available at: https://www.whitehouse.gov/ondcp/state-laws-related-to-marijuana. Accessed: November 2016.

11. Serrano A (2016). Inside big pharma’s fight to block recreational marijuana. Available at: https://www.theguardian.com/sustainable-business/2016/oct/22/recreational-marijuana-legalization-big-business. Accessed: November 2016.

12. Tahirali J (2016). 7 in 10 Canadians support marijuana legalization: Nanos poll. Available at: http://www.ctvnews.ca/canada/7-in-10-canadians-support-marijuana-legalization-nanos-poll-1.2968953. Accessed: November 2016.

13. US Food & Drug Administration. FDA and Marijuana. Available at: http://www.fda.gov/NewsEvents/PublicHealthFocus/ucm421163.htm. Accessed: November 2016.

Chapter 1 – Active Ingredients in Cannabis

Tetrahydrocannabinol (THC) is the major psychoactive ingredient in cannabis and has consequently received much attention from scientists. However, it has emerged from research that cannabis actually consists in excess of 500 chemical entities and around 60 of these come from the same family as THC – the cannabinoids. Another important family of compounds present in cannabis are terpenes and terpenoids which give cannabis its distinctive flavor and aroma. Both terpenes and other cannabinoids can interact with THC to enhance or antagonize its psychoactive effects.

Potency testing

Verifying the potency of cannabis is key for cannabis testing labs, and can be achieved through LC and GC methods. Restek’s Raptor LC columns can be used alongside any HPLC instrument, accelerating the analysis time without the need for UHPLC equipment. The company says that their high-throughput approach can complete cannabinoid analysis in 3.7 minutes. They have also shown that their Rxi®-35Sil MS column can be used alongside GC equipment to rapidly analyze cannabinoids in a matter of minutes.

Another approach that has the potential to offer cannabis producers access to on-the-spot potency testing is Fourier transform mid-infrared (FT-IR) spectroscopy. A team from PerkinElmer tested the method on intact and ground cannabis bud samples. They showed that the method could accurately quantify the levels of tetrahydrocannabinolic acid which is converted to THC upon heating and, another important cannabinoid, cannabidiolic acid (CBDA) which is converted to cannabidiol. The researchers also demonstrated that FT-IR could detect changes in cannabinoid concentrations according to growing time and conditions, showing that it has the potential to allow cannabis producers to monitor and optimize conditions and determining harvest time.

Steep Hill, also presenting at Pittcon 2017, have developed a cannabis analyzer called the QuantaCann2 that uses near-infrared (NIR) spectroscopy. The company argue that the method, although less versatile, has advantages over other spectral approaches that are destructive, require more sample preparation, greater operator expertise, and the use of solvents. The team that developed the device have shown that it can quantify four major cannabinoids in cannabis – CBDA, cannabidiol, THCA and THC to within 0.7%, 0.4%, 1.3% and 0.6% accuracy, respectively, when compared with HPLC reference spectra.

Terpene profiling

Cannabis contains a complex profile of terpenes which are thought to be responsible for some of the drug’s purported health benefits. As this profile varies between strains, from crop to crop and even plant to plant, it can also be used for quality control purposes. An increased interest in characterizing terpene profiles has followed from the wider legalization of cannabis and is required in some states by law.

Several companies have developed approaches to terpene profiling. For example, Shimadzu, who will be at this year’s Pittcon, created a method using their mass spectrometer with a full evaporation headspace technique (FET) to overcome the fact that plant material does not dissolve in solvent. The Shimadzu team have shown that using a single-phase liquid-gas system they could quantify the presence of terpenes in accordance with Nevada state law in three different strains of cannabis.

Restek have also generated a workflow for separating terpenes using a headspace gas chromatography-flame ionization detection (GC-FID). Like Shimadzu’s approach, the method also uses FET with a single-phase gas system. They have demonstrated the efficacy of the method in characterizing the terpene profile of pelletized hops, as a proxy for cannabis. They used the Shimadzu Rxi®-624Sil MS column, which has a small-bore configuration, and can also be used for analyzing residual solvents in cannabis, using the same setup and technique.

Cannabis component analysis at Pittcon 2017

At Pittcon 2017, Scott Kuzdzal from Shimadzu Scientific Instruments will introduce the symposium ‘Analytical Cannabis II’. In the session ‘Current and Future Analytical Technologies for Cannabis Testing and Research’, Kuzdzal will discuss the many different chemical compounds found in cannabis and some of their reported health benefits. He will also outline why cannabis testing labs are so important for quality control in the era of medical cannabis, including the measurement of terpene concentrations, and the detection of contaminants such as pesticides, heavy metals and mycotoxins. Kuzdzal will also consider how analytical technologies are enhancing quality control testing including in clinical settings.

In an oral session, the conference will also hear from Laura McGregor of Markes International who will describe how two-dimensional gas chromatography couple with time-of-flight mass spectroscopy can aid the analysis of complex plant-based samples, such as cannabis. McGregor will also outline how the approach of tandem ionization could help researchers to keep pace with the emergence of so-called legal highs by boosting researchers’ ability to identify novel structures for which no reference spectra are available.

Also in attendance at this year’s conference will be Sigma-Aldrich who offer a range of solutions for cannabis testing labs. This includes the company’s Ascentis Express columns which can be coupled to any HPLC, UHPLC or LC-MS instrument allowing the characterization of a sample’s active ingredients, including cannabinoids.

Advion will also be at Pittcon 2017 to present their compact mass spectrometer. The device, which is much smaller and priced lower than a standard MS system is designed to increase the accessibility of MS to labs, particularly those with restricted space. Advion have shown that compact MS can be applied to the analysis of cannabinoids alongside thin layer chromatography for qualitative detection of cannabinoids and alongside HPLC for quantitative determination.

References

    1. Ashton CH. Pharmacology and effects of cannabis: a brief review. British Journal of Psychiatry 2001; 178: 101-106.

3. Restek. Growing analytical solutions for cannabis testing. Available at: http://www.restek.com/pdfs/FFSS2073B-UNV.pdf. Accessed: November 2016.

4. Restek. A Preliminary FET Headspace GC-FID Method for Comprehensive Terpene Profiling in Cannabis. Available at: http://www.restek.com/Technical-Resources/Technical-Library/Foods-Flavors-Fragrances/fff_FFAN2045-UNV. Accessed: November 2016.

5. Shimadzu. Simplified Cannabis Terpene Profiling by GCMS. [data on file].

7. Smith BC, Lewis MA, Mendez J. Optimization of cannabis grows using Fourier transform mid-infrared spectroscopy. Available at: http://www.perkinelmer.co.uk/lab-solutions/resources/docs/APP_Optimization-Cannabis-Fourier-Transform-Mid-Infrared-Spec_012596_01.pdf. Accessed: November 2016.

Chapter 2 – Cannabis Preparation – Extraction Techniques & Residual Solvents

With the market for cannabis growing along with expanding legalization, there is increasing demand for cannabis extract products. This includes oils and waxes that can be used in vaping, drinks, and edibles. For both recreational cannabis users seeking a high and for potential medicinal applications, there is also growing interest in purified forms of cannabis such as cannabinoid isolates.

Methods of extraction

Traditionally butane extraction has been the most commonly used method of cannabis extraction, but it is being used less and less in commercial settings these days due to the risk of explosions from the highly flammable gas. However, the technique has apparently been growing in popularity among illegal cannabis producers who use it to create high-strength butane hash oil (BHO). In the UK, two people have died and 27 people have been injured in the process of generating BHO over the last two years, according to police reports.

In lab settings, the most popular approach is solvent extraction, which requires further purification steps to remove any residual solvents.

Another method, which is more expensive but gaining more widespread use, is supercritical fluids chromatography or CO2 extraction, which uses carbon dioxide as a solvent and therefore doesn’t require as much post-processing.

At this year’s Pittcon, Xiaoning Lu from Sigma-Aldrich will present another technique that could be applied to cannabis extraction – online solid phase extraction (SPE). This method is widely used by labs to isolate analytes from complex matrices but off-line methods are time consuming, labor-intensive and have poor reproducibility.

Lu will present research carried out on thyroid hormone samples in biological matrices using an online-SPE cartridge developed by the company, alongside liquid chromatography/mass spectrometry (LC/MS). He will show how the online cartridge was able to significantly boost the LC/MS response and improve reproducibility, and will also explain how the same cartridge can be applied in the detection of cannabis analytes in blood serum and urine samples.

Removing residual solvents

When solvent-based extraction processes are used, the cannabis extract must undergo further steps to remove any residual solvents, as these can be harmful to human health. It is therefore vital to verify that the solvents have been completely removed and there are a number of chromatographic options available for doing so.

Commonly used is gas chromatography (GC) and static headspace GC can be used to concentrate volatile analytes for analysis and provide rapid identification and quantification of residual solvents. Shimadzu and Sigma-Aldrich, who will both be presenting at this year’s Pittcon 2017 offer such solutions for residual solvent analysis.

Also at Pittcon 2017, Robert Driscoll from Robatel Inc., a Massachusetts-based centrifuge provider, will discuss centrifugal chromatography as an approach to isolating components from organic samples. Driscoll will outline the benefits of fast centrifugal partitioning chromatography – a method that allows components with similar molecular structures to be isolated from a sample – over other techniques available. He will also discuss recent advances in the design of the technology and its use in isolation of cannabis as well as tobacco, opiate derivatives and nutraceuticals.

References

    1. BBC News. Rise in UK explosions linked to super-strength cannabis. Available at: http://www.bbc.co.uk/news/uk-36988316. Accessed: November 2016.

3. Eden Labs LLC. Supercritical CO2 Extraction. Available at: https://www.edenlabs.com/processes/co2-extraction. Accessed: November 2016.

4. News-medical. New techniques for the extraction & preparation of cannabis oil: an interview with Alison Wake. Available at: http://www.news-medical.net/news/20161024/New-techniques-for-the-extraction-preparation-of-cannabis-oil-an-interview-with-Alison-Wake.aspx. Accessed: November 2016.

5. Shimadzu. Cannabis Testing Laboratory Solutions. Available at: http://www.ssi.shimadzu.com/products/literature/life_science/Cannabis_Brochure_v2.pdf. Date accessed: November 2016.

Chapter 3 – Cannabis Testing – Identifying Chemicals and Contaminants

In the United States, although recreational or medical use has been authorized by several states, cannabis is still illegal at the federal level. Consequently, the Food & Drug Administration (FDA) does not recognize cannabis as a regulated product and does not provide any standards for its cultivation and supply. This raises the risk that cannabis products could be contaminated, potentially endangering consumers’ health.

This is particularly pertinent for those accessing medical cannabis who may have compromised immune systems. Fortunately, there are a number of analytical techniques that have been applied in clinical, pharmaceutical, food safety and environmental settings that are equally effective for verifying the absence of chemicals and contaminants in cannabis samples.

At Pittcon 2017, Joshua Crossney from jCanna, Inc., a non-profit organization interested in improving cannabis analytical testing technologies, will discuss how the emerging cannabis testing and research industries are benefiting from collaborating and sharing the knowledge and experience of these other more long-standing industries.

At this year’s conference, you can also hear about some of the specific techniques and the latest methods being developed to assist the provision of safe, uncontaminated products in the new era of legalized cannabis.

Pesticides

Cannabis plants are susceptible to a number of bacteria, fungi, yeasts and molds but many pesticides available to try to prevent infestation can themselves be harmful to human health. And while pesticide use on other crops is federally regulated, this is not the case for cannabis. What’s more, no pesticides have even been tested or registered for use on cannabis. As a result, individual states that have legalized cannabis are having to come up with their own regulations for pesticide use and eliminating contamination from the final product.

This year’s Pittcon will feature discussion of a number of techniques now available to help meet the challenge of cannabis quality control. Attending the conference will be Restek, Shimadzu and Sigma Aldrich, who all provide LC-MS and GC-MS solutions for pesticide analysis. There will also be a presentation from Julie Kowalski of Restek who will outline a modified QuEChERS technique – which is already popular for pesticide testing in food and agriculture – using LC-MS/MS to analyze pesticide residues that has so far been applied to over 200 pesticide types.

Jack Henion from Advion will also discuss how mass spectrometry using the company’s compact device could be used as a screening method to test the quality and purity of cannabinoid products and detect the presence of pesticides and other contaminants.

Other analytes and contaminants

Another trace contaminant that can find its way into cannabis products are heavy metals, which are absorbed from the soil into the cannabis plant. Many of these are considered toxic, such as lead, arsenic and mercury. Options for detection include Ultrasonic Nebulizer Inductively Coupled Plasma Optical Emission Spectrometry (USN-ICP-OES) and Mass Spectrometry (ICP-MS) methods, both of which are capable of rapidly analyzing all heavy metals but differ in simplicity and sensitivity.

Moisture content is also another important parameter as excessive moisture can promote mold growth, which can be established using an electronic moisture analyzer like the Shimadzu MOC63u.

Related to this is concern over the presence of mycotoxins, which are toxic metabolites produced by mold. Several of these have been shown to be especially harmful to humans and can be dangerous to immunocompromised patients even at very low concentrations. Therefore, it is critical to have sensitive methods for detecting low levels in cannabis samples. Chromatographic methods available include GC, HPLC and LC-MS/MS.

References

    1. Berry E & Wilcox K (2015). With No U.S. Standards, Pot Pesticide Use Is Rising Public Health Threat. Available at: https://thefern.org/2015/10/with-no-u-s-standards-pot-pesticide-use-is-rising-public-health-threat/. Accessed: November 2016.

3. Restek. Growing analytical solutions for cannabis testing. Available at: http://www.restek.com/pdfs/FFSS2073B-UNV.pdf. Accessed: November 2016.

4. Shimadzu. Cannabis Testing Laboratory Solutions. Available at: http://www.ssi.shimadzu.com/products/literature/life_science/Cannabis_Brochure_v2.pdf. Date accessed: November 2016.

Chapter 4 – Cannabis Standards

The increasing legal status of cannabis has led in turn to a surge in demand for cannabis testing labs. But cannabis testing is very much an emerging industry and there has been considerable confusion surrounding how such labs should operate. For example, some have been uncertain over whether they were able to provide their services legally due to the continued federal prohibition of the drug. And with no federal oversight into cannabis standards, such as cultivation conditions or acceptable levels of residue solvents and pesticides, states have been left to come up with their own rules, sometimes belatedly following legalization. Labs have also had to contend with uncertainty because of the prospect that current standards become obsolete when state lawmakers decide to change the regulations.

At this year’s Pittcon, the symposium “It’s legal! Now what?” will explore some of these emerging issues. Heather Krug from the Colorado Department of Public Health and Environment will detail the challenges the state has faced since legalizing recreational cannabis. This includes issues such as a lack of scientific evidence over the toxicity of cannabis contaminants, systems for monitoring cannabis lab performance and an absence of industry-accepted standards for labs to work to.

The symposium will also hear from Robert Martin, representing the Association of Commercial Cannabis Laboratories, who will outline their efforts to try to establish evidence-based quality measures for cannabis testing. The talk will touch upon issues faced in pesticides, residual solvents and microbiological testing, as well as consider the future of the cannabis testing industry.

Also at Pittcon 2017, Autumn Karcey from Cultivo, Inc. will discuss the optimal conditions for growing cannabis indoors to facilitate medical research into its effects. This includes consideration of factors such as temperature, humidity and room pressure as well as how to minimize the presence of airborne particulates, pests and pathogens.

Barry Schumbmehl from Fritsch Milling and Sizing, Inc. will also discuss how milling and grinding procedures during sample preparation can help to generate uniform, representative samples suitable for analyzing commercially produced cannabis. He will discuss the variability of analytes between crops and even within the same plant and provide data on how potency can therefore differ between that which is stated and as determined from dried flowers or post-milling.

References

    1. Anderson WH. Cannabis testing labs: standards and accreditation. Available at: http://lcb.wa.gov/publications/Marijuana/BOTEC%20reports/2b-Accrediting-Labs-Final-Corrected.pdf. Accessed: November 2016.

2. Berry E & Wilcox K (2015). With No U.S. Standards, Pot Pesticide Use Is Rising Public Health Threat. Available at: https://thefern.org/2015/10/with-no-u-s-standards-pot-pesticide-use-is-rising-public-health-threat/. Accessed: November 2016.

3. News-medical. Creating reference standards for cannabis testing: an interview with Adam Ross. Available at: http://www.news-medical.net/news/20160525/Creating-reference-standards-for-cannabis-testing-an-interview-with-Adam-Ross.aspx. Accessed: November 2016.

4. Restek. Growing analytical solutions for cannabis testing. Available at: http://www.restek.com/pdfs/FFSS2073B-UNV.pdf. Accessed: November 2016.

5. Shimadzu. Cannabis Testing Laboratory Solutions. Available at: http://www.ssi.shimadzu.com/products/literature/life_science/Cannabis_Brochure_v2.pdf. Date accessed: November 2016.

Chapter 5 – Cannabis Detection and Law Enforcement

As we have seen, analytical techniques have been assisting the regulation of legalized cannabis but the fact still remains that the substance is prohibited in most countries, and is still illegal for recreational use in the majority of US states. Detecting illicit cannabis in biological samples and contraband therefore continues to be a priority and advances in analytical science are helping this to be done with greater accessibility, speed and sensitivity.

One such development is cantilever-enhanced photoacoustic spectroscopy, or CEPAS. At Pittcon 2017, we will hear from Jaakko Lehtinen from Finland-based Gasera Ltd., who were involved in developing this new measurement technique. It can be used in gas phase for detection of volatile organic compounds (VOCs) from drugs or their precursors and in this talk, Lehtinen will discuss how the use of different laser sources can lead to Ppb-level detection of VOCs. He will also outline how photoacoustic spectroscopy is able to distinguish hair samples from cannabis users and non-users, and how THC levels can be determined from cannabis samples using the same set-up.

Pittcon 2017 will be attended by a number of companies whose technologies are providing novel options for drug detection to assist law enforcement.

For example, Advion will be presenting their compact mass spectrometry device. Their team have previously shown how the device, which is intended to make MS more accessible, particularly in labs with limited space, can be used alongside a method called Atmospheric Solids Analysis Probe (ASAP®) combined with atmospheric pressure chemical ionization (APCI) to allow for direct analysis of samples suspected of containing cannabis. The approach was able to produce strong signals for cannabinol, THC/cannabidiolic acid and THC/cannabinol within 20 seconds. Furthermore, they showed that the method could potentially be applied to trace samples from fingertips, as when a person has rolled a cigarette containing cannabis.

Bruker, who will also be presenting at this year’s conference, offer a number of drug testing solutions, including the Toxtyper workflow which facilitates LC-MS drug screening by incorporating a library of over 830 compounds. A team of researchers showed that Toxtyper could be applied to the identification of synthetic cannabinoids. New derivatives of these compounds frequently emerge in slightly modified varieties, sometimes as a deliberate attempt to bypass drug laws, and they can pose a risk to consumer health due to their unknown strength and toxicity. The research team showed using a library of 46 synthetic cannabinoids and nine isotope-labelled analogs, that spiked serum samples could be detected for all substances at concentrations of 0.5 ng/mL or lower.

Bruker also offer an ion mobility spectrometer, called DE-tector, a desktop and portable device, which can detect natural, synthetic, pure and street drugs from samples when present in the low nanogram range.

The company have also shown that it’s Fourier transform-infrared spectrometer, ALPHA, is effective in detecting cutting agents that have been laced into drug samples, and are themselves often illicit and harmful to consumers.

Also at this year’s Pittcon will be Biotage. They have created a range of supported liquid extraction plates and columns called ISOLUTE which can be used in place of traditional liquid-liquid extraction. Not only can the set-up be used to detect cannabinoids in blood and urine samples, but the company have also shown that it can be applied to saliva samples, potentially presenting a quicker and easier way to obtain samples in settings such as traffic accidents or for workplace drug testing.

Thermo Scientific offer a solution for cannabinoid detection in biological samples using their TurboFlow technology – an automated online sample preparation technique – coupled to LC-MS/MS. Their team has shown that the approach is able to quantify THC and its metabolites in spiked blood samples, with a total extraction and analytic runtime of 10.4 minutes. Thermo Scientific say the method, by cutting out sample preparation time, can provide advantages over SPE or liquid-liquid sample preparation.

References

    1. Advion Application Note. Direct sample analysis: Detecting THC/Cannabinoids in contraband by compact mass spectrometry. Available at: https://advion.com/wp-content/uploads/Advion_CMS_ASAP_THC_expanded.pdf. Accessed: November 2016.

3. Bruker Application Note. Comprehensive detection and identification of synthetic cannabinoids using the Toxtyper platform. Available at: https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/Literature/ApplicationNotes/LCMS-99_Toxtyper_SynthCannabinoids_01-2015_eBook.pdf. Accessed: November 2016.

6. Jones R, Williams L, Lodder H, et al. Extraction of ∆9-THC, THCA and 11-nor-9-carboxy-∆9-THC from Oral Fluid using Supported Liquid Extraction (SLE) after collection with the Quantisal, Intercept & Oral-Eze Collection Devices prior to GC/MS Analysis. Available at: http://biotage.com/literature/download/p134_thc_of_tiaft_2015.pdf?ref=http%3A//biotage.com/news/new-poster Accessed: November 2016.

7. Scurati S, Gechtman C, Duretz B, et al. Analysis of THC and THC-COOH in plasma and urine using online extraction LC-MS/MS. Available at: http://apps.thermoscientific.com/media/SID/Europe%20Region/PDF/Thermo_Scientific_Poster_P-23.pdf. Accessed: November 2016.

Conclusion

Against a background of increasingly liberal cannabis laws, regulators and scientists have been trying to keep pace with cannabis as it has gone from a near-universally illicit substance to a burgeoning industry in just a matter of years.

Fortunately, the analytical science industry, drawing on experience in the fields of food, environmental and agricultural science, is rising to the many challenges presented by this revolution.

At Pittcon, taking place in Chicago from 5-9 March, 2017, you can meet the companies at the forefront of innovation in cannabis analysis, regulation and detection. There are also a wealth of talks and symposia during which you can hear directly from the experts who have been involved in developing these technologies and methodologies, and putting them to use in the field.

The many presentations will explore recent developments in cannabis research, law and ethics. The conference will also look to the future, and what it holds for the cannabis testing industry and medicinal uses of the drug. For example, a talk by Kevin Rosenblatt from Integrated Biosource/Cannabis Labs, will consider how medical marijuana fits into another healthcare revolution – personalized medicine. Rosenblatt will look at the potential for genomics, metabolomics and pharmacogenomics to tailor cannabis treatment to patients on an individual basis, and how this could be assisted by analytical techniques like mass spectroscopy.

As this industry tries to make sense of legalized medical and recreational cannabis from an ethical, legal, and analytical perspective, come to Pittcon 2017 to find out where we are now and where we could go next.