Long Term Stability of Cannabis Resin

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Long term stability of cannabis resin

and cannabis extractsChristian Lindholst

a

aAarhus University, Department of Forensic Medicine, Aarhus,

DenmarkPublished online: 05 Jul 2010.

To cite this article:Christian Lindholst (2010): Long term stability of cannabis resin and cannabisextracts, Australian Journal of Forensic Sciences, 42:3, 181-190

To link to this article: http://dx.doi.org/10.1080/00450610903258144

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Long term stability of cannabis resin and cannabis extracts

Christian Lindholst*

Aarhus University, Department of Forensic Medicine, Aarhus, Denmark

The aim of the present study was to investigate the stability of cannabinoids incannabis resin slabs and cannabis extracts upon long-term storage. The levels oftetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and canna-bigerol (CBG) on both neutral and acidic form were measured at room temperature,

48C and 7208C for up to 4 years. Acidic THC degrades exponentially viadecarboxylation with concentration halve-lives of approximately 330 and 462 daysin daylight and darkness, respectively. The degradation of neutral THC seems tooccur somewhat slower. When cannabinoids were stored in extracted form at roomtemperature the degradation rate of acidic THC increased significantly relative toresin material with concentration halve-lives of 35 and 91 days in daylight anddarkness, respectively. Once cannabis material is extracted into organic solvents,care should be taken to avoid the influence 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

different 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 influence 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: [email protected]

Australian Journal of Forensic Sciences

Vol. 42, No. 3, September 2010, 181190

ISSN 0045-0618 print/ISSN 1834-562X online

2010 Australian Academy of Forensic Sciences

DOI: 10.1080/00450610903258144

http://www.informaworld.com


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significant 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 effect

of using different 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 finding 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 usingcannabis resin as whole slabs or as powder, the exposure to light has a significant effect

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 significant thermal decomposition of the

cannabinoids at such elevated temperatures13.

With a few exceptions, the majority of the literature describing cannabinoidstability 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 specific 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 different 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 slabsIn 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

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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

different locations on the slab. Sampling was continued for almost 4 years (1416

days) at regular intervals, although more frequently during the first 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 andsubsequent 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 flasks

equipped with conical glass plugs to avoid evaporation of the solvents. The flasks

were split into four groups of two extracts and stored under the following conditions:

room temperature (20228C) with daylight exposure (transparent flask), room

temperature without light exposure (dark brown flask), 48C without light exposure

(dark brown flask) and 7208C without light exposure (dark brown flask). 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 flaskand 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.

Quantification

Following extraction, the samples were subjected to both gas chromatographic (GC-

FID) 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 quantified the total amount of cannabinoids in the samples (both

acidic and neutral species). The HPLC analyses, however, resolved both acidic and

Australian Journal of Forensic Sciences 183


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neutral cannabinoids. Direct quantification 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 unjustified due to a limited

knowledge of cannabinoid behaviour in the GC inlet. Consequently, in the following

figures, 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 flame 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 aninternal 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 film 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-DADThe 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

flow 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

ChromatographyIn the present study both GC and HPLC were applied in order to detect both

neutral and total species of cannabinoids. Two representative chromatograms

184 C. Lindholst


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

components.

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



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Cannabis resin

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 final 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 first 240 days followed by a slower decrease. The decrease inneutral 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 (20228C) with light exposure (a), room temperature without light exposure (b) and 7208Cwithout light exposure (c). (d) Concentrations of total THC, CBN, CBD and CBG in cannabisresin samples stored at room temperature (20228C) with light exposure.

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Cannabis extracts

In the cannabis resin extract study, four different storage conditions were applied

(Figures 3(a)(e)). The figure 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 final level of 1.7% after almost 2 years. When the cannabis

extract is stored at room temperature but without the influence 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

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 (20228

C)with light exposure (a), room temperature without light exposure (b), 48C without lightexposure (c) and 208C without light exposure (d). (e) Concentrations of total THC, CBN andCBD in cannabis extracts stored at room temperature (20228C) with light exposure.



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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 flasks 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 final 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 andcannabis extracts has been studied. In cannabis resin material (hashish slabs) both

daylight and temperature have an influence 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

influenced 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. Thepresence 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 influence 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 profile 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 finding 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 significantly

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 highersusceptibility 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

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days have been reported under similar storage conditions7. The discrepancy between

the results can be explained by the difference in extract sample volume applied in the

two experiments (1.7 ml versus 100 ml). In small flasks/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 finally 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 influence 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 ofcannabinoids should only have minor or no influence 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 influence the cannabinoid

content of the material1,6,16.

Conclusion

Cannabinoid stability in cannabis material is influenced by light, temperature and

possibly also oxygen availability. The stability of acidic and neutral cannabinoids

differs, 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.

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2. Mechoulam R. Marihuana chemistry. Science. 1970;168:11591166.3. Jenkins RW, Patterson DA. The relationship between chemical composition and

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acid content of cannabis products. J Pharm Pharmacol. 1981;33:369372.5. Grlic L. A combined spectrophotometric differentiation of samples of cannabis. Bull

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Long Term Stability of Cannabis Resin