This article was downloaded by: [69.23.67.

103] On: 02 July 2013, At: 00:14 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Australian Journal of Forensic Sciences

Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tajf20

Long term stability of cannabis resin and cannabis extracts

Christian Lindholst
a a

Aarhus University, Department of Forensic Medicine, Aarhus, Denmark Published online: 05 Jul 2010.

To cite this article: Christian Lindholst (2010): Long term stability of cannabis resin and cannabis extracts, Australian Journal of Forensic Sciences, 42:3, 181-190 To link to this article: http://dx.doi.org/10.1080/00450610903258144

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Australian Journal of Forensic Sciences Vol. 42, No. 3, September 2010, 181190

Long term stability of cannabis resin and cannabis extracts

Christian Lindholst*
Aarhus University, Department of Forensic Medicine, Aarhus, Denmark

Downloaded by [69.23.67.103] at 00:14 02 July 2013

The aim of the present study was to investigate the stability of cannabinoids in cannabis resin slabs and cannabis extracts upon long-term storage. The levels of tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and cannabigerol (CBG) on both neutral and acidic form were measured at room temperature, 48C and 7208C for up to 4 years. Acidic THC degrades exponentially via decarboxylation with concentration halve-lives of approximately 330 and 462 days in daylight and darkness, respectively. The degradation of neutral THC seems to occur somewhat slower. When cannabinoids were stored in extracted form at room temperature the degradation rate of acidic THC increased signicantly relative to resin material with concentration halve-lives of 35 and 91 days in daylight and darkness, respectively. Once cannabis material is extracted into organic solvents, care should be taken to avoid the inuence of sunlight. Keywords: forensic science; stability study; cannabis; THC; resin; extracts

Introduction Forensic examination of cannabis plant material (marihuana) and cannabis resin often includes qualitative as well as quantitative determinations of the sample cannabinoid content. Many cannabinoids have been reported in the literature1, with cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabinol (CBN) as some of the most frequently studied species. All three types of cannabinoids exist as both carboxylic acid derivatives as well as neutral compounds. The relative composition of cannabinoids in a given sample may provide useful forensic information such as ripening stage, relative potency and maybe even indications about the geographic origin26. However, knowledge about the prior sample history as well as correct sample handling in the analytical laboratory is often essential if information about cannabinoid composition is intended for subsequent forensic use. The stability of dierent cannabinoids is namely shown to be highly dependent on the storage form and storage conditions. When cannabinoids are extracted from herbal material or resin into organic solvents both the temperature and light exposure is shown to inuence their stability7 11 . It is, however, important to distinguish between the degradation of neutral and acidic species. Short-term stability studies show that neutral cannabinoids are stable in extracts stored in darkness for up to 15 days7. However, when exposed to light a

*Email: cl@forensic.au.dk
ISSN 0045-0618 print/ISSN 1834-562X online 2010 Australian Academy of Forensic Sciences DOI: 10.1080/00450610903258144 http://www.informaworld.com

182

C. Lindholst

Downloaded by [69.23.67.103] at 00:14 02 July 2013

signicant decrease in the neutral cannabinoid species can be observed. In contrast, the decarboxylation of acidic cannabinoids occur in both daylight and darkness and seems to be a temperature dependent process7,8. A number of data exist on the eect of using dierent solvents for cannabis extraction and subsequent storage. One study concludes that CBD is stable in ethanol but unstable when dissolved in chloroform9. Other studies support this nding and conclude that chloroform extracts may only be stored temporarily10. Both acidic and neutral cannabinoids seem to be more unstable in chloroform or light petroleum extracts than in methanol or methanol: chloroform (9:1) extracts7. A few studies have focused on the stability of cannabinoids in cannabis resin slabs and herbal material stored under various conditions. The levels of total CBD and THC in pulverised resin slabs seem to decrease exponentially upon storage at room temperature for a prolonged period of time12. At the same time, an initial increase in the total CBN level followed by a slow decrease has been observed. The exponential decrease in total CBD and THC levels occur in both daylight and darkness, although the concentration half-lives are shorter in light-exposed material. In other studies using cannabis resin as whole slabs or as powder, the exposure to light has a signicant eect on the degradation of THC8. Upon storage in darkness at room temperature for one year, no THC loss has been detected. However, when the material was exposed to light, a marked decrease in THC levels could be observed. One of the more recent publications on cannabinoid stability describes the thermal conversion of THC acid into neutral THC13. At 1401608C a maximum of 70% THC acid was converted into neutral THC. In a controlled smoking experiment only about 30% of the THC acid was recovered as neutral THC indicating a signicant thermal decomposition of the cannabinoids at such elevated temperatures13. With a few exceptions, the majority of the literature describing cannabinoid stability in hashish, marihuana and cannabis extracts dates back to the period from 1970 to 1980. The present paper is therefore an updated and more extensive study of the stability of specic cannabinoids in whole slabs and extracted resins at various storage conditions. The experimental data has furthermore been collected during a 24 year period providing a long-term perspective. In order to address the issue of both neutral and acidic cannabinoid stability, measurements on both GC-FID and HPLC-DAD have been applied. Materials and methods Sample collection In the present experiment, a total of seven slabs of cannabis resin containing relatively high amounts of THC have been selected for the study of cannabinoid stability. The slabs originated from two dierent seizures of illicit cannabis resin made by the Danish police. From one seizure, six slabs were selected for the cannabis resin stability study, whereas one slab was selected from another seizure for the cannabis extract stability study. The resin samples were described as to weight, size and colour to ensure that homogeneous material was obtained. Preparation of cannabis resin slabs In the cannabis resin stability study, six slabs of equal size, appearance and cannabinoid content were selected and divided into three groups of two slabs. The

Australian Journal of Forensic Sciences

183

groups were subsequently stored under the following conditions: room temperature (20228C) with daylight exposure, room temperature without light exposure (in a light proof container) and at 7208C without light exposure. At the start of the experiment, four samples were taken from each slab in order to measure the initial cannabinoid concentration. In the remaining part of the experiment only two samples were taken from each slab (four samples per group) during sampling. Each sample consisted of a 10 mm section through the entire slab produced by means of a power drill. The cross-sectional sampling technique ensured a homogeneous mixture of both surface and core material. Sampling was performed in duplicate at two dierent locations on the slab. Sampling was continued for almost 4 years (1416 days) at regular intervals, although more frequently during the rst year of storage. Preparation of cannabis extracts For the purpose of the cannabis extract stability study, one slab of cannabis resin was selected. A total of 8 g of material was removed for homogenisation and subsequent extraction in 800 ml methanol:chloroform (9:1). After an extraction period of 2 hours, stirring of the extracts stopped so that suspended material could precipitate. The supernatant was transferred to 8 6 100 ml Erlenmeyer glass asks equipped with conical glass plugs to avoid evaporation of the solvents. The asks were split into four groups of two extracts and stored under the following conditions: room temperature (20228C) with daylight exposure (transparent ask), room temperature without light exposure (dark brown ask), 48C without light exposure (dark brown ask) and 7208C without light exposure (dark brown ask). The storage period lasted for nearly 2 years (703 days) during which regular sampling occurred. At the time of sampling, 200 ml of extract was removed from each ask and analysed as described in the following sections. Extraction procedures Subsequent to sampling the resin material was homogenised in a lab homogeniser (IKA, A11 basic) and 100 mg of the homogenised material was transferred to glass test tubes and extracted twice using 2 6 5 ml methanol/chloroform (9:1) for a period of 1 hour. Following each of the two extractions, suspended material was pelleted in a low speed centrifugation (3000 rpm for 5 minutes) and the supernatant was transferred to a new glass test tube wrapped in tin foil. Samples of 200 ml of the combined extracts were analysed within the same day using GC and HPLC. All samples were analysed in duplicate. Quantication Following extraction, the samples were subjected to both gas chromatographic (GCFID) and liquid chromatographic (HPLC-DAD) analysis. All sample extracts originating from the resin stability study were analysed within the same day to avoid decomposition of the cannabinoids. The combination of GC and HPLC analysis was used in order to determine the concentrations of both neutral and acidic cannabinoids. Due to decarboxylation of the acidic cannabinoids upon injection, the GC method quantied the total amount of cannabinoids in the samples (both acidic and neutral species). The HPLC analyses, however, resolved both acidic and

Downloaded by [69.23.67.103] at 00:14 02 July 2013

184

C. Lindholst

neutral cannabinoids. Direct quantication of the acidic species was, on the other hand, not possible due to a lack of authentic standards. Consequently, THC acid had to be calculated by subtracting the neutral THC as determined by HPLC from the total THC as determined by GC and multiplying by a factor of 1.14 (representing the molecular weight ratio of THC acid and neutral THC)5. A subsequent correction of the total THC measurement, to account for the acidic species, could be argued (so the sum of neutral THC and THC acid would equal total THC). It is, however, the opinion of the authors that such a correction is unjustied due to a limited knowledge of cannabinoid behaviour in the GC inlet. Consequently, in the following gures, the amount of total THC is less than the sum of neutral and acidic THC. GC-FID The GC was a Hewlett Packard model 5890A equipped with a ame ionisation detector (FID), splitless injection mode and automatic injection. The GC was connected to a HP model 3396A integrator. All extracts were added 50 ml of an internal standard (octacosane, C28), evaporated to dryness and subsequently reconstituted in 200 ml of n-heptane. Aliquots of 1 ml were injected on to a SPB-1 column (Supelco), 15 m 6 0.53 mm with a lm thickness of 1.5 mm. The instrumental settings were as follows. Injector temperature: 2758C, detector temperature: 3508C, initial oven temperature: 608C for 1.5 min followed by an increase of 308C/min to 2808C for 12 min. Figure 1(a) shows a chromatogram of a typical cannabis resin sample extracted and analysed by the described method. HPLC-DAD The HPLC system consisted of a Hitachi model 655A-12 pump connected to an automatic injection system (model 655A-40) and a Hitachi model 655A variable UV detector. All samples were detected at a wavelength of 220 nm following separation on a Spherisorb ODS, C-18 column (Phase Sep) 25 cm 6 4.6 mm. The mobile phase consisted of a mixture of 0.02 N sulphuric acid, methanol and acetonitrile (7:8:9) at a ow rate of 1.2 ml/min. Aliquots of 2 ml were used for analysis. The HPLC method was based on the method described by Baker et al.14. Figure 1(b) shows a chromatogram of a typical cannabis resin sample extracted and analysed by the described method. Chemicals The reference drugs 9-THC (D9-tetrahydrocannabinol), CBN (cannabinol), CBD (cannabidiol) and CBG (cannabigerol) were obtained from Makor Chemicals Ltd. (Jerusalem, Israel). Standard solutions of 0.5 mg/ml (9-THC), 0.2 mg/ml (CBD and CBN) and 0.1 mg/ml (CBG) were kept in darkness at 7188C. In the present paper, THC refers to 9-THC. Octacosane (MERCK) was used as an internal standard. Results Chromatography In the present study both GC and HPLC were applied in order to detect both neutral and total species of cannabinoids. Two representative chromatograms

Downloaded by [69.23.67.103] at 00:14 02 July 2013

Australian Journal of Forensic Sciences

185

Downloaded by [69.23.67.103] at 00:14 02 July 2013

Figure 1. (a) GC-FID chromatogram of an extracted cannabis resin sample. Peaks arising from total THC, CBN CBD and CBG together with the internal standard (C28) are identied. (b) HPLC chromatogram of an extracted cannabis resin sample. Peaks arising from neutral THC, CBN, CBD and CBG are identied.

from both types of analysis are presented in Figure 1 to show the separation of the cannabinoic species. Both methods oer acceptable separation of all relevant components.

186 Cannabis resin

C. Lindholst

Downloaded by [69.23.67.103] at 00:14 02 July 2013

The stability of four cannabinoids, THC, CBN, CBD and CBG was studied at room temperature with daylight exposure, at room temperature without light exposure and at 208C without light exposure (Figure 2). The initial concentration of total CBG, CBD, THC and CBN was, on average, 0.4, 3.5, 11.7 and 0.4% in all groups giving a total of 16% cannabinoids in the resin material. In Figures 2(a) (c) the concentration of THC (neutral, acidic and total) and CBN in the three groups is shown as a function of time. The levels of total THC decrease when stored at room temperature to a nal concentration of 12% after almost 4 years. The decrease in total THC is observed in both light-exposed material and material stored in darkness, although the degradation occurs somewhat faster in daylight. This trend is also observed for the acidic THC that degrades in an exponential manner (R2 0.98) in both daylight and darkness with concentration halve-lives of approximately 330 and 462 days, respectively. The level of neutral THC increases during the rst 240 days followed by a slower decrease. The decrease in neutral THC did not seem to occur exponentially. A 78 fold linear increase in the total CBN concentration was observed in the two room temperature groups. All measured cannabinoids, including total CBD and CBG, were stable in darkness at 7208C during the 4 years of storage (Figure 2(c), not all results shown). Only minor reductions in the total CBD and CBG levels could be detected in the light exposed group at room temperature (Figure 2(d)).

Figure 2. Concentrations (% w/w) of total THC, neutral THC (THC), THC acid (THCA) and total CBN in cannabis resin samples during 4 years of storage at room temperature (20 228C) with light exposure (a), room temperature without light exposure (b) and 7208C without light exposure (c). (d) Concentrations of total THC, CBN, CBD and CBG in cannabis resin samples stored at room temperature (20228C) with light exposure.

Australian Journal of Forensic Sciences Cannabis extracts

187

In the cannabis resin extract study, four dierent storage conditions were applied (Figures 3(a)(e)). The gure presents the amount of neutral, acidic and total THC together with total CBN as a function of time. For the extracts stored in light at room temperature, THC acid decreases exponentially (R2 0.96), with a half-life of 35 days, and becomes undetectable after 140 days of storage. In the same time period (0140 days) neutral THC increases in concentration but starts to decrease slowly thereafter reaching a nal level of 1.7% after almost 2 years. When the cannabis extract is stored at room temperature but without the inuence of light exposure (Figure 3(b)), THC acid still degrades exponentially (R2 0.92) with an approximated half-life of 91 days. The neutral THC, however, seems to be stable, resulting in an initial increase in THC concentration followed by a constant level throughout the study period. The increase in neutral THC coincides with the

Downloaded by [69.23.67.103] at 00:14 02 July 2013

Figure 3. Concentrations (% w/w) of total THC, neutral THC (THC), THC acid (THCA) and total CBN in cannabis extracts during 2 years of storage at room temperature (20228C) with light exposure (a), room temperature without light exposure (b), 48C without light exposure (c) and 208C without light exposure (d). (e) Concentrations of total THC, CBN and CBD in cannabis extracts stored at room temperature (20228C) with light exposure.

188

C. Lindholst

decrease in THC acid as observed in the light-exposed extracts. It should be mentioned that due to a leak in one of the storage asks only a single extract was stored dark at room temperature as opposed to all other storage conditions, where two extracts were used. In the extracts stored dark at 48C, the same pattern is observed as in the room temperature group, although the degradation rate of THC acid is slower (T 4 1000 days). At 7208C the level of all measured species is constant during the entire study period. Figure 3 also displays the level of total CBN at all storage conditions. Despite a minor nal increase in the group stored dark at room temperature, the level of CBN seems to be constant throughout the study. The level of total CBD decreases from 6 to 0.5% in 2 years in the light exposed extract (Figure 3(e)). Discussion Cannabis resin In the present study, the long-term stability of cannabinoids in cannabis resin and cannabis extracts has been studied. In cannabis resin material (hashish slabs) both daylight and temperature have an inuence on cannabinoid stability. At room temperature an exponential decrease in THC acid coincides with a similar increase in neutral THC levels. This shows that decarboxylation is the main reaction by which THC acid degrades. The results also indicate that the decarboxylation reaction is inuenced by, but not dependent on, light exposure since the THC acid half-life increases by 40% when the material is moved from daylight to darkness. Previous short-term studies, although performed with cannabis extracts, support that THC acid does degrade in the absence of light7. In addition, neutral THC degradation was detected at room temperature in both the presence an absence of daylight. The presence of daylight, however, only seemed to result in a minor increase in the degradation rate. Considering the dense colour and structure of a cannabis resin slab it seems likely that light only has an inuence on the cannabinoids present in the surface layer of the material. This may explain the reduced light sensitivity of the resin material. Only a few unclear results on the cannabinoid prole through a resin slab have so far been reported7. At room temperature, an increase in total CBN levels coincides with a decrease in total THC. There is, however, no direct correlation between CBN formation and THC degradation as the increase in total CBN does not correspond to the decrease in total THC. Previous studies suggest that CBN may accumulate in cannabis samples upon extended periods of storage6,15. The present results support this nding and indicate that THC may also degrade into compounds other than CBN. Cannabis extracts When cannabis resin is extracted into organic solvent and stored in daylight at room temperature the degradation of both neutral and acidic THC increases signicantly relative to cannabis resin slabs. Accordingly, the calculated half-life of THC acid in the extract was almost 10 times lower than observed in the resin slabs (*35 days in extract compared to *330 days in resin). In addition, the degradation of neutral THC occurred faster in the extracts. One explanation for this is the higher susceptibility to light when the material is in an extracted form compared to the dense resin plates. In a comparable experiment, THC acid half-lives of only 1213

Downloaded by [69.23.67.103] at 00:14 02 July 2013

Australian Journal of Forensic Sciences

189

days have been reported under similar storage conditions7. The discrepancy between the results can be explained by the dierence in extract sample volume applied in the two experiments (1.7 ml versus 100 ml). In small asks/vials the surface to volume ratio is larger resulting in a more extensive light exposure. This will result in faster degradation rates of light sensitive compounds such as neutral and acidic THC. When the extracts were stored in the dark no degradation of neutral THC seemed to occur. In contrast, a build up of neutral THC was observed along with a similar decrease in acidic THC. This observation was made at both room temperature and at 48C, although at a much slower rate, and has also been reported in the literature7. The fact that neutral THC in resin extracts is stable for nearly 2 years is interesting considering that approximately half the neutral THC is degraded when stored under similar conditions as resin plates. One explanation for this could be that oxidation of neutral THC accounts for the decrease in the air-exposed resin plates whereas the extracts are stored virtually oxygen free. Microbial degradation is another explanation although more unlikely due to the low water content in the resin material. Based on the present work, it can be concluded that long-term storage of resin material should be performed in darkness at low temperatures. Seized cannabis material is often stored for several months at police stations or in the forensic laboratories before a case is nally closed and the material destroyed. If re-analysis should be required late in the case history, improper storage of the material may result in inconsistent analytical results. Once cannabis material is extracted into organic solvents, care should be taken to avoid the inuence of sunlight. It is therefore recommended to use light protective containers, such as brown glass vials or test tubes wrapped in tin foil, whenever small sample volumes are prepared. If these precautions are applied in daily laboratory work, the degradation of cannabinoids should only have minor or no inuence on the analytical results. Information about the prehistory of seized cannabis material may also be extracted from analytical data. Knowledge about the degradation pattern of cannabinoids under various storage conditions may to some extent be used in forensic intelligence. For example, if cannabis material is suspected of being stored in a place with elevated temperatures or stored for an extensive time period, the relative cannabinoid ratios may or may not support such theories (e.g. low or high CBD/ THC and CBN/THC ratios). Care should, however, be taken when interpreting analytical data since several factors such as ripening stage, natural biological variation and geographic origin of the plants are known to inuence the cannabinoid content of the material1,6,16. Conclusion Cannabinoid stability in cannabis material is inuenced by light, temperature and possibly also oxygen availability. The stability of acidic and neutral cannabinoids diers, with the acidic species being more susceptible to degradation. Knowledge about cannabinoid stability may ensure correct sample storage, handling and interpretation of analytical results, thereby improving forensic intelligence information. References
1. Turner CE, Elsohly MA, Boeren EG. Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J Nat Prod. 1980;43:169234.

Downloaded by [69.23.67.103] at 00:14 02 July 2013

190

C. Lindholst

Downloaded by [69.23.67.103] at 00:14 02 July 2013

2. Mechoulam R. Marihuana chemistry. Science. 1970;168:11591166. 3. Jenkins RW, Patterson DA. The relationship between chemical composition and geographical origin of cannabis. Forensic Sci. 1973;2:5966. 4. Baker PB, Taylor BJ, Gough TA. The tetrahydrocannabinol and tetrahydrocannabinolic acid content of cannabis products. J Pharm Pharmacol. 1981;33:369372. 5. Grlic L. A combined spectrophotometric dierentiation of samples of cannabis. Bull Narcotics. 1968;20:2530. 6. De Faubert Maunder MJ. The forensic signicance of the age and origin of cannabis. Med Sci Law. 1976;16:7890. 7. Smith RN, Vaughan CG. The decomposition of acidic and neutral cannabinoids in organic solvents. J Pharm Pharmac. 1977;29:286290. 8. Fairbairn JW, Liebmann JA, Rowan MG. The stability of cannabis and its preparations on storage. J Pharm Pharmac. 1976;28:17. 9. Parker JM, Borke ML, Block LH, Cochran TG. Decomposition of cannabidiol in chloroform solution. J Pharm Sci. 1974;63:970971. 10. Turner CE, Henry JT. Stability of synthetic and naturally occurring cannabinoids in chloroform. J Pharm Sci. 1975;64:357359. 11. Turner CE, Hadley KW, Fetterman PS, Doorenbos NJ, Quimby MW, Waller C. Constituents of cannabis sativa L. IV: Stability of cannabinoids in stored plant material. J Pharm Sci. 1973;62:16011605. 12. Martone G, Della Casa E. Analysis of the ageing processes in hashish samples from dierent geographic origin. Forensic Sci Int. 1990;47:147155. 13. Dussy FE, Hamberg C, Luginbu hl M, Schwerzmann T, Briellmann TA. Isolation of D9THC-A from hemp and analytical aspects concerning the determination of D9-THC in cannabis plants. Forensic Sci Int. 2005;149:310. 14. Baker PB, Fowler R, Bagon KR, Gough TA. Determination of the distribution of cannabinoids in cannabis resin using high performance liquid chromatography. J Anal Toxicol. 1980;4:145152. 15. Harvey DJ. Stability of cannabinoids in dried samples of cannabis dating from around 1896-1905. J Ethnopharmacol. 1990;28:117128. 16. Baker PB, Gough TA, Taylor BJ. Illicitly imported cannabis products: Some physical and chemical features indicative of their origin. Bull Narcotics. 1980;32:3140.