ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 8, с. 805-810
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
TRANSFORMATIONS OF TETRAHYDROCANNABINOL, TETRAHYDROCANNABINOLS ACID AND CANNABINOL DURING THEIR EXTRACTION FROM CANNABIS SATIVA L. © 2015 D. Wianowska*, 1, A. L. Dawidowicz*, M. Kowalczyk**
Chromatographic Methods Department, Faculty of Chemistry, Maria Curie Sklodowska University 20-031 Lublin, Pl. Marii Curie Sklodowskiej 3, Poland 1E-mail: dorota.wianowska@poczta.umcs.lublin.pl **Forensic Laboratory of Regional Police Headquarters in Lublin 20-019 Lublin, Narutowicza street 73, Poland Received 08.10.2013; in final form 24.01.2015
The presented study discusses the difference in the results of estimation of A9-tetrahydrocannabinol (THC), tetrahydrocannabinol acid (THCA) and cannabinol (CBN) amount in Cannabis sativa L., using long-lasting liquid extraction in the Soxhlet apparatus and pressurized liquid extraction (PLE) as sample preparation methods in the analytical procedure. THC amounts extracted in the PLE process using n-hexane or methanol are very similar. THCA amounts extracted in the PLE process using n-hexane are generally greater than those extracted in the Soxhlet apparatus but smaller when methanol is applied in the PLE process. The obtained results evidently show that the mutual transformations of THCA, THC and CBN occur not only during Soxhlet extraction but also during short-lasting PLE. In the last case, however, they are small. The intensity of the transformation depends on the applied extractant type.
Keywords: cannabis, cannabinol, pressurized liquid extraction, A9 -tetrahydrocannabinol, tetrahydrocannab-inol acid.
DOI: 10.7868/S0044450215080216
Marihuana (cannabis) is one of the most popular intoxicants. In many countries its possessing is interdicted. Cannabis contains more than 60 cannabinoids [1, 2]. It is well known that A9-THC is the strongest bioactive component responsible for the psychotropic properties of cannabis. Its activity is 3—4 times stronger when inhaled. Apart from A9-THC, cannabidiol and CBN are also recognized as other main cannabinoids. In current opinion, these two components intensify the effects of A9-THC, although they themselves do not show intoxicant activity [3, 4]. As results from detailed investigations [2, 5—7], A9-THC is formed in cannabis from THCA. This conversion occurs also in the cut plant. During its thermal and/or light exposition THCA undergoes decarboxylation, transforming itself into psy-choactive A9-THC [2, 7—10]. For this reason the consumption of cannabis products is usually preceded by their thermal treatment (smoking, vaporizing, boiling of extracts, cooking of cakes with cannabis addition, etc.). The final product of THCA and A9-THC transformation is CBN [9, 11, 12]. Longer storage and drying of cannabis also result in the transformation of A9-THC into CBN [7-10].
The generally accepted gas chromatography (GC) procedures of A9-THC determination in cannabis, performed to estimate its psychotropic ability, involve thermal treatment of the plant in order to decarboxy-late THCA and form A9-THC. In the course of this process the CBN concentration increases. It should be stressed that these transformations are not fully controlled. Usually a part of THCA remains in the thermally treated plant [6, 9]. This is why the GC procedures examining the quality of cannabis in the context of its intoxicant activity should take into consideration the necessity of estimating the concentration of all three key components in the examined material: THCA, A9-THC and CBN.
Due to the character and complexity of plant material, the analytical examination of plant constituents involves the application of the sample preparation procedure to fully isolate the analyzed substances from the plant matrix. Simple solvent extraction such as Soxhlet extraction, maceration, and extraction under reflux are generally applied as sample preparation procedures for the determination of A9-THC in cannabis. Although these methods are relatively simple, they suffer from such shortcomings as a long extraction
time, a relatively high solvent consumption, and often unsatisfactory reproducibility. In the face of temperature transformation of THCA to A9-THC and CBN, the application of these methods for cannabis analysis is even more disputable.
In the attempt to improve the extraction process, also for the isolation of analytes from plant material, and to reduce or eliminate the mentioned drawbacks, innovative extraction methods such as ultrasonic-assisted solvent extraction (UASE), supercritical fluid extraction and pressurized liquid extraction have been recently developed and introduced. PLE has been shown to have significant advantages over the competing techniques. For example, unlike UASE, PLE does not require the filtration step, since the matrix components that are not dissolved in the extraction solvent are retained inside the sample extraction cell. This is very convenient for the automation and on-line coupling of the extraction and separation techniques [13]. Moreover, variations of temperature and pressure during the PLE process influence the solubility behavior of the compounds. Furthermore, PLE, due to high pressure of the extraction process, allows using an ex-tractant at a temperature above its normal boiling point and, in consequence, removing the analytes efficiently and quickly from various matrices [14—17].
This paper studies the difference in the estimation of A9-tetrahydrocannabinol, tetrahydrocannabinol acid and cannabinol amount in Cannabis sativa L. using long-lasting liquid extraction in the Soxhlet apparatus and pressurized liquid extraction as sample preparation methods in the analytical procedure. The experiments were performed applying two types of extractants, methanol and n-hexane.
EXPERIMENTAL
Materials and chemicals. Cannabis plants (Cannabis sativa L.) were obtained by courtesy of the Lublin Provincial Police Headquarters (after previous consent of Provincial Court) on the basis of cooperation agreement. All material was confiscated by police in the course of various crime investigations. Only female flower tops were used in this study. It is worth mentioning that the concentrations of THC, THCA and CBN in cannabis are different. To increase the reliability of the obtained results, the plant material used in these experiments was a mixture of a few cannabis samples. A sufficiently large representative sample of the plant material was ground and sieved to obtain the particle size of 0.2—0.3 mm. Precisely weighed portions of the material were used for extractions.
THC, THCA and CBN standards were purchased from THC Pharm GmbH (LGC Standards, Poland). Acetonitrile (HPLC grade), methanol (HPLC grade) and n-hexane (analytical grade) were purchased from Polish Chemical Plant (POCh, Gliwice, Poland). 4A molecular sieve and the silylating agent (1% solution of
trimethylchlorosilane, TMCS, in N,O-bis(trimethylsi-lyl)trifluoroacetamide, BSTFA) were obtained from Sig-ma-Aldrich (Seelze, Germany).
Methods. Pressurized liquid extraction. PLE was performed with a Dionex ASE 200 instrument (Dion-ex, Sunnyvale, CA, USA). Samples of cannabis (0.3 g) were accurately weighed and mixed with sand. They were then placed into a 22 mL stainless steel extraction cell with a cellulose filter at the bottom end. The sample cells were then closed to finger tightness and placed into the carousel of the ASE 200 system. n-Hexane or methanol were used as extraction solvents. Extractions were carried out at 25, 50, 75, 100, 125 and 150°C at operating pressure of 40 bar. Extractions were performed for 5, 10, 15 or 20 min. After the extraction process, the extraction cell content was flushed with the same solvent in the amount equal to 60% of the extraction cell volume and purged for 60 s by applying pressurized nitrogen (at 150 psi). The obtained extract was transferred to a 50 mL volumetric flask, which was subsequently filled up to its volume with the extraction solvent and immediately subjected to the further step of analytical procedure. All the extraction procedures were repeated 5 times.
Soxhlet extraction. Exhaustive extractions in the Soxhlet apparatus were performed using 2.0 g of ground dry plant matrices. Precisely weighed samples were transferred to a paper thimble. The loaded thimble was inserted into a 100 mL Soxhlet extractor. Extractions were performed for 1, 2 or 3 h with 75 mL of n-hex-ane or methanol, resulting in more than 8 extraction cycles per hour. After cooling to room temperature, the obtained extract was transferred to a 100 mL volumetric flask, which was subsequently filled up to its volume with the extraction solvent and immediately subjected to the further step of analytical procedure. The Soxhlet extractions were repeated five times.
Derivatization. The obtained extract (400 ^L) was evaporated to dryness using a vacuum rotary evaporator. To the residue, 100 ^L of the silylating agent (BSTFA + TMCS) was added and incubated for 15 min at ambient temperature. After silylation, the obtained mixture was dissolved in the dried acetonitrile (500 ^L) and subjected to GC analysis. Acetonitrile was dried by shaking with 4A molecular sieve and stored over the sieve.
Chromatographic analysis. Quantification of extracts was performed using a gas chromatograph with a flame ionization detector model GC-17A (Shimadzu, Kyoto, Japan); 1 ^L of the sample was injected by the AOC—20i type autosampler into a ZB5 fused-silica capillary column (30 m x 0.32 mm i.d., 0.50 ^m film thickness, Phenomenex, USA). The following temperature program was applied: a linear temperature increase from 170 to 260°C at the rate of 15 grad/min; isothermal separation at 260°C for 10 min, and finally linear temperature increase up to 280°C at the rate of 5 grad/min. Peaks identification was carried out by comparing the GC retention indices with those for the
>
иТ
0.08
0.06
0.04
0.02
0
_ (a
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.