Author: S. A. ROSS, M. A. ELSOHLY
Creation Date: 1999/12/01
CBN and D9-THC concentration ratio as an indicator of the age of stored marijuana samples*
S. A. ROSS
National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy, School of Pharmacy, University of Mississippi, Oxford, Mississippi, United States of America
M. A. ELSOHLY
National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences, Department of Pharmaceutics, School of Pharmacy, University of Mississippi, Oxford, Mississippi, United States of America
The concentration of D9-tetrahydrocannabinol (THC) and cannabinol (CBN) in cannabis plant material (marijuana) of different varieties stored at room temperature (20-22o Celsius (C)) over a four-year period was determined. The percentage loss of THC was proportional to the storage time. On average, the concentration of THC in the plant material decreased by 16.6% ±7.4 of its original value after one year and 26.8% ±7.3, 34.5% ±7.6 and 41.4% ±6.5 after two, three and four years, respectively. A relationship between the concentration ratio of CBN to THC and the storage time was developed and could serve as a guide in determining the approximate age of a given marijuana sample stored at room temperature.
The stability of (-)-D9-tetrahydrocannabinol (D9-THC) has been the subject of several investigations (1-13). In 1970, Liskow (1) reported that marijuana deteriorates during storage at room temperature because of the loss of D9-THC at a rate of 3 to 5 per cent a month. Shoyama and others (3) were able to isolate cannabinolic acid (CBNA) from stored hemp but not from fresh hemp, and concluded that conversion of tetrahydrocannabinolic acid (THCA) to CBNA was effected by ultraviolet light and by storage and heat. The same conclusion was reached by Turner and others (4, 5), who reported that THC disappeared at a rate of 3.83, 5.38 and 6.92 per cent per year over two years when stored at -18o, 4o, and 22o C, respectively. The loss of THC was essentially complete at 37o C and 50o C. Fairbairn and others (6) reported that carefully prepared herbal or resin cannabis products are reasonably stable for one to two years if stored in the dark at room temperature.
Razdan and others (2) found thatD9-THC is much less stable than D8-THC and is converted mainly to CBN. The degradation of D9-THC to CBN in the plant material on storage was also proposed by Waller and others (7), Razdan and others (8), El-Kheir and others (9), Hanus and others (10) and Yotoriyana and others (11). Although CBN is the major observed decomposition product of THC, it could not account for the decrease in the concentration of THC over a period of time when the latter is kept under conditions suitable for decomposition (12). Turner and ElSohly (13) addressed this problem and proposed a possible pathway for the decomposition of THC to CBN which involvesformation of epoxy and hydroxylated intermediates. These include 9,10-dihydroxy-D6a(10a)-THC (racemic mixture) and 8,9-dihydroxy- D6a(10a)-THC (racemic mixture). They found that these intermediates could be detected only by gas chromatography as their trimethyl silyl (TMS) derivatives. They also indicated that these compounds were susceptible to heat and acid and that the final product was CBN.
In the present report, the change in the level of THC and CBN in stored marijuana was studied over a four-year period. THC and CBN were analysed annually in marijuana stored at room temperature and a correlation was developed between the ratio of CBN to THC and the age of the plant material. The empirical correlation could be used to estimate the age of a given marijuana sample.
The plant material used in the study was grown at the University of Mississippi medicinal plant garden. Mature plants were harvested and dried in a drying barn. The temperature was set initially at 50o C and was then increased at 2.5o C per hour until 70o C was reached. Under those conditions, dryness was complete within 6-8 hours. The dried materials were then coarsely manicured, packed in closed barrels and stored in an air-conditioned vault.
Plants used in the study were grown from seeds of Colombian, Jamaican, Mexican or hybrid varieties.
The dried plant material was stored in closed barrels in the dark in a secured air-conditioned vault. Room temperature fluctuated slightly over time but generally remained between 20o and 22o C. Samples were obtained annually from stored material for cannabinoid analysis.
The method used for analysis has been previously described by Ross and others (14). Briefly, each dried sample was manicured by passing through a metal sieve (number 14). One hundred milligrams (mg) of each sample was weighed and extracted with 3 millilitres (ml) of extraction solution (internal standard), which was a mixture of 100 mg of 4-androstene-3,17-dione, 10 ml of chloroform and 90 ml of methanol. After the samples were allowed to stand for one hour, the extracts were separately removed from each flask and transferred into screw-cap vials, from which aliquots were transferred into 2-ml gas-chromatography vials.
A chromatograph, model 5880A, equipped with an automatic liquid sampler, model 7673, was used under the following conditions: (a) column: DB-1, 15 m × 0.25 mm, with 0.25 µm film thickness; (b) temperature: initial, 170o C for 1 minute then programmed to 250o C at the rate of 10o C/min; (c) injector temperature: 240o C; (d) detector temperature: 260o C; (e) carrier gas: helium at approximately 1 ml/min; and (f) detector: flame ionization detector with hydrogen flow rate of 30 ml/min and air flow rate of 300 ml/min. Each sample was analysed in duplicate and the average percentage for THC and CBN was calculated. The results are summarized in tables 1-4.
Table 1. Concentration of THC and CBN in marijuana samples stored
for one year at room temperature
a The varieties and year of cultivation were as follows: CJAF-93: cultivated Jamaican variety, female, 1993; CK1X-93: cultivated hybrid, mixture, 1993; and CMEF-93: cultivated Mexican variety, female, 1993.
Table 2. Concentration of THC and CBN in marijuana samples stored
for two years at room temperature
a The varieties and years of cultivation were as follows: CJAF-91: cultivated Jamaican variety, female, 1991; CMEF-91: cultivated Mexican variety, female, 1991; CCOF-91: cultivated Colombian variety, female, 1991; CK1X-93: cultivated hybrid, mixture, 1993.
Table 3. Concentration of THC and CBN in marijuana samples stored for three years at room temperature
a The varieties and years of cultivation were as follows: CJAF-91: cultivated Jamaican variety, female, 1991; CMEF-91: cultivated Mexican variety, female, 1991; CCOF-91: cultivated Colombian variety, female, 1991.
Table 4. Concentration of THC and CBN in marijuana samples
stored for four years at room temperature
a The varieties and years of cultivation were as follows: CCOF-91: cultivated Colombian variety, female, 1991; CMEF-91: cultivated Mexican variety, female, 1991; CK1F-91 cultivated hybrid, female, 1991; CJAF-91: cultivated Jamaican variety, female, 1991; CMEF-91: cultivated Mexican variety, female, 1991.
Results and discussion
Determination of the age of a marijuana sample is often required in forensic work, and to date there is no reported method to estimate it.Since D9-THC is known to oxidize to CBN over time, the presence of CBN in a marijuana sample indicates that the sample is not fresh. It is presumed that the higher the amount of CBN, the older the sample. In an effort to correlate between the amount of CBN and THC as they relate to the age of marijuana samples, the present study was carried out.
The dried marijuana samples produced from different cannabis crops of different varieties were stored at room temperature and were analysed shortly after harvesting (time 0) and yearly thereafter for up to four years. Tables 1-4 show the concentration of both THC and CBN at time 0 and after one, two, three and four years of storage, respectively. The percentage loss of THC in each case is also presented along with the amount of CBN relative to THC (percentage) after each storage period.
Figure I shows the relationship between the percentage loss of THC and the storage time. Although it is clear that there was a wide variation in the percentage loss at each data point (standard deviation of 6.5-7.6 per cent), the percentage loss of THC was proportional to the storage time. On the average, the concentration of THC in the plant material decreased by 16.6% ±7.4 of its original value after one year, 26.8% ±7.3 after two years, 34.5% ±7.6 after three years and 41.4% ±6.5 after four years.
Figure I. Relationship between the percentage loss of THC
and years of storage
Several attempts were made to develop a relationship between the time of storage and the concentration of THC and CBN. It was found that the percentage ratio of CBN to THC at the time of analysis was most predictive of the age of the plant material. Tables 1-4 show the values for all samples stored for one, two, three and four years, respectively. On average, the percentage ratio of CBN to THC was found to be 2.5 ±0.9, 6.7 ±1.4, 9.4 ±1.7 and 14.2 ±1.2 for samples stored for one, two, three and four years respectively, as reflected in figure II.
A number of observations were made. First, not all of the THC converts directly to CBN, suggesting other intermediates in that process as previously described by Turner and ElSohly (13). Secondly, CBN does not exist in the freshly and carefully dried marijuana, supporting previous reports (3, 4). Thirdly, the degradation of THC appears to proceed at a higher rate for the first year than subsequent years and levels off after two years to a rate of loss of approximately 7 per cent per year. Therefore, studies carried out with "old" material could feasibly report a 7 per cent loss per year. It is believed that the percentage loss in THC content is also a function of the initial THC concentration. The higher the concentration of THC, the faster the degradation over the first one or two years. That could account for the high variability in the percentage loss in THC over time in the samples represented by several varieties used in the present study.
The results reported above show that it is feasible to determine the age of a given marijuana sample on the basis of its THC and CBN contents, assuming that storage was carried out at room temperature. It is evident from figure II that samples with a ratio of CBN to THC of less than 0.013 are less than six months old, and those with a ratio of between 0.04 and 0.08 are between one and two years old. Figure II could be used to estimate the age of a given sample on the basis of the concentration of CBN and THC.
Figure II. Relationship between the percentage ratio of
CBN to THC and years of storage
It should be emphasized, however, that variations from the experimental conditions described in the present report should be considered in the interpretation of the analytical results.
Degradation of Cannabis and THC: Storage Methods and Mold
Constituents of Cannabis sativa L. XIII: Stability of dosage form prepared by impregnating synthetic (--)-delta 9-trans-tetrahydrocannabinol on placebo Cannabis plant material.
Lewis GS, Turner CE
J Pharm Sci 1978 Jun;67(6):876-8
Synthetic (--)-delta 9-trans-tetrahydrocannabinol impregnated on placebo Cannabis decomposed only 6.3% after being stored for 1 year at --18 degrees. Storage at 5 degrees and room temperature under various conditions led to severe decomposition. The amount of cannabinol observed when (--)-delta 9-trans-tetrahydrocannabinol decomposed indicates that cannabinol is not the only decomposition product.
Stability of Cannabis sativa L. samples and their extracts, on prolonged storage in Delhi.
Narayanaswami K, Golani HC, Bami HL, Dau RD
Bull Narc 1978 Oct-Dec;30(4):57-69
The percentage rate of change into cannabinoids (Cannabidiol [CBD], tetrahydrocannabinol [THC] and cannabinol [CBN]) was higher in cannabis samples than in the extracts. This is probalby due to the decomposition of acids into corresponding neutral cannabinoids under the conditions of storage. Previous claims that CBD content in plant material is relatively constant are not substantiated by our results. There was a 1.0-2.5-fold increase in CBD content in plant material compared with the extracts. However, the fact that there was no appreciable increase in CBD/CBN content in the stored extracts of the same samples supports the view that the step-wise extraction does not bring the acids into the final extract pure delta 9THC decomposed at a rate of 41 per cent per year under tropical storage conditions. The delta 9THC content decreased in the samples and equally in the extracts though 100 per cent conversion of THC to CBN does not take place. The higher CBN content found in extracts than that expected by the conversion THC to CBN is a result of metabolic conversion.
The stability of cannabis and its preparations on storage
Fairbairn JW, Liebmann JA, Rowan MG
J Pharm Pharmacol 1976 Jan;28(1):1-7
Solutions of pure cannabinoids, nine samples of herbal and two of resin cannabis (one freshly prepared) were stored in varying conditions for up to 2 years. Exposure to light (not direct sunlight) was shown to be the greatest single factos in loss of cannabinoids especially in solutions, which should therefore be protected from light during analytical and phytochemical operations. Previous claims that solutions in ethanol were stable have not been substantiated. The effect of temperature, up to 20 degrees, was insignificant but air oxidation did lead to significant losses. These could be reduced if care was taken to minimize damage to the glands which act as "well filled, well closed containers". Loss of tetrahydrocannabinol after exposure to light does not lead to an increase in cannabinol, but air oxidation in the dark does. It is concluded that carefully prepared herbal or resin cannabis or extracts are reasonably stable for 1 to 2 years if stored in the dark at room temperature.
Stability of cannabinoids in dried samples of cannabis dating from around 1896-1905 Harvey DJ
J Ethnopharmacol 1990 Feb;28(1):117-28
Cannabinoids from three samples of cannabis obtained from the Pitt-Rivers Museum, Oxford, and dating from the turn of the century were examined by gas chromatography and mass spectometry for the presence of cannabinoids. Although the samples were from different geographical locations, the profiles of constituent cannabinoids were similar. In common with other aged material, most of the cannabinoid content was present as cannabinol (CBN), the main chemical degradation product of the major psychoactive constituent, delta-9-tetrahydrocannabinol (delta-9-THC). However, a substantial concentration of CBN acid-A was also present; this compound is unstable to heat and readily undergoes decarboxylation to CBN. Methyl and propyl homologues of CBN, together with delta-9-THC and its naturally occurring acid-A were also found at low concentrations in all samples. Intermediates in the formation of CBN from delta-9-THC, previously identified in aged solutions of the drug, were absent or present in only trace concentrations. However, oxidation products involving hydroxylation at the benzylic positions, C-11 and C-1', not seen in solution, were identified in substantial abundance. The results suggest that decomposition of cannabis samples may proceed more slowly than originally thought.
Examination of fungal growth and aflatoxin production on marihuana
Llewellyn GC, O'Rear CE
Mycopathologia 1977 Dec 16;62(2):109-12
Under favorable growth conditions, Aspergillus flavus and A. parasiticus produced aflatoxins on marihuana. Cultures of A. flavus ATCC 15548 produced both aflatoxin B1 (AFB1) and G1 (AFG1). The production of AFG1 was substantially greater than that of AFB1. Cultures of A. flavus NRRL 3251 and A. parasiticus NRRL 2999 produced only AFB1. All natural flora cultures tested negative for aflatoxins. No Aspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 microgram/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.