Cannabis sativa L.: Effect of drying time and temperature on cannabinoid profile of stored leaf tissue

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Author: C.B. Ph. D. COFFMAN, W. A. Ph.D. GENTNER
Pages: 67 to 70
Creation Date: 1974/01/01

Cannabis sativa L.: Effect of drying time and temperature on cannabinoid profile of stored leaf tissue

C.B. Ph. D. COFFMAN
W. A. Ph.D. GENTNER
New Herbicide Explorations and Chemonarcocide Unit, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, U.S.A.

Abstract

Leaf tissue of Cannabis sativa L. was subjected to several time-temperature régimes before extraction and analysis for cannabicyclol, cannabidiol, Δ 9tetrahydrocannabinol, and cannabinol. Differences in time-temperature treatments resulted in significant variations in cannabinoid content of leaf tissue. The need for uniformity in tissue-preparation methods is discussed.

Cannabis sativa L. has been used as an intoxicant in the United States for more than seven decades (1). Increased cannabis consumption has resulted in intensified scientific investigations of the narcotic characteristics of the species. Cannabinoids are now being extracted and analysed by laboratories affiliated with universities, forensic chemists, and Federal research agencies.

Cannabinoid analysis involves extraction of plant tissue with an organic solvent, concentration of extract, and subsequent analysis by chromatographic methods. There is little uniformity in methods of plant-tissue preparation before cannabinoid extraction. For example, Carew ( [ 2] ) dried cannabis leaf tissue at 40°C. Lerner ( [ 3] ) and Turner and Hadley ( [ 4] ) used air-dried tissue. Phillips et al. ( [ 5] ) dried tissue for 30 minutes at 85-90°C.

Variation in moisture content of plant tissue is caused by age, leaf location, environment, and other factors. To compare cannabinoid content of plant tissues by weight, moisture content must be made as uniform as possible without destruction of the cannabinoid components. This paper reports the effects of oven-drying C. sativa leaf tissue at several time-temperature periods. Weight loss and variations in cannabinoid content of the heated and unheated leaf tissue samples were compared. This report describes a uniform method of plant-tissue preparation for cannabinoid analysis.

Eight-week-old greenhouse-grown ( [ 6] ) plants of C. sativa L. of Afghan seed ( [ 7] ) were stripped of their leaves. The leaf material was air-dried for 24-48 hours, then ground to pass a 60-mesh sieve, uniformly mixed, and stored in darkness at ambient temperature.

Three subsamples of ground leaf tissue were used for each of the following treatments: 1, 4, 16, and 64 hours at 65°C, 85°C, and 105°C. After exposure to the above time-temperature régimes, the samples were placed in a desiccator containing activated silica gel to equilibrate with ambient temperature. Weighings before and after oven treatments allowed comparisons of the relative weight losses. Check treatments were subjected to the same procedure as the heated treatments, exclusive of heat.

Cannabinoids were extracted and analysed by a modification of the procedure of Turner and Hadley ( [ 4] ). Modifications involved chromatograph temperatures, stationary phase, and method of introduction of internal standard ( [ 8] ). Nitrogen carrier gas was used with a Barber-Coleman ( [ 9] ) model 5005 gas chromatograph ( [ 10] ) equipped with a H-flame ionization detector ( [ 10] ).

The effects of the various time-temperature treatments on the content of cannabicyclol (CCC), cannabidiol (CBD), Δ 9-tetrahydrocannabinol (Δ 9), and cannabinol (CBN) are presented in table 1.

There were no differences in the CCC content of the tissue within the 65 °C treatment (table 1). Decreasing quantities of CCC were associated with the two longer exposure times at 85°C and 105°C. No CCC was detected after 64 hours exposure at 105°C.

A similar pattern of decreasing cannabinoids with increased exposure time at 85 °C and 105 °C was found for CBD (table 1). This response to time and temperature is also illustrated by Δ 9 values.

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CBN values increased with time at 105 oC. This probably reflected the destruction of Δ 9.

Farnsworth indicated ( [ 11] ) that cannabinoid conversion can occur with time in stored material. He stated that conversion was more rapid in tropical areas than in temperate areas, implying heat enhancement of the conversion process.

To increase our confidence in the results of the 16-hours, 65 oC portion of the time-temperature study, a mixture of CCC, CBD, Δ 9, and CBN was prepared ( [ 12] ). A part of this mixture was subjected to 65 oC for 16 hours, while the other portion was refrigerated. Gas chromatograph analysis indicated no cannabinoid degradation by this treatment.

Table 2 shows the average weight loss of cannabis leaf tissue resulting from time-temperature treatments. The general trend was for an increased weight loss as temperature and/or time of exposure increased. No significant differences in weight loss were noted for the 1-hour exposures at 65 oC and 85 oC, or for 4 and 16 hours at 85 °C. Weight loss decreased for the tissue exposed at 65 °C for 64 hours relative to the weight losses for the other time exposures at the same temperature. This portion of the experiment was repeated (in time) with similar results. The precise explanation of this phenomenon has not yet been ascertained. Several hypotheses are proposed: First, exposure at 65 °C for 64 hours may have created a hygroscopic component in the tissue, which had a greater affinity for moisture than the desiccant, thus sorbing moisture while equilibrating at ambient temperature. Further heating at higher temperatures apparently destroyed the hygroscopic characteristics of the unknown moiety. Secondly, an oxidation reaction may have been induced by the particular treatment, resulting in a weight gain. Studies have begun to determine an explanation for this occurrence.

Cannabis: Effect on drying time and temperature on canabinoid profile 69

TABLE

1 CCC, CBD, Δ9, and CBN content in ppm of cannabis leaf tissue subjected to several time-temperature treatments*

 

Times (hours)

Cannabinoid

Temperature° C

 

1

4

16

64

CCC
65   58.3ab 70.0a 60.7ab 61.7ab
  85   51.7ab 55.0ab 48.3b 31.7c
  105   57.3ab 56.7ab 30.0c 0d
 
Control
65.0ab        
CBD
65   733ab 818a 740ab 676bc
  85   733ab 686bc 641bc 618c
  105   721 ab 605c 688bc 480d
 
Control
810a        
Δ9
65   763ab 775ab 695abc 616cd
  85   720ab 690bc 580d 378e
  105   680bc 610cd 445e 147f
 
Control
790a        
CBN
65   345abc 468abc 431abc 395abc
  85   483abc 341abc 531ab 441abc
  105   361abc 321bc 580ab 616a
 
Control
216c        

Values not followed by the same letter (within cannabinoid) are significantly different at the 1 per cent level (13).

TABLE 2

Percentage mean weight loss of cannabis leaf tissue as a result of exposure to time-temperature treatmentsa

 

Times (hours)

Temperature°C

1

4

16

64

65 7.58gb 7.92f 8.16e 7.08h
85 7.39g 9.58cd 9.77c 10.28b
105 9.43d 9.45d 10.46b 11.00a
a

a. Percentage loss compared to weight of air-dried tissue.

b. Values not followed by same letter are different at the 1 per cent level ( [ 13] ).

In summary, various time-temperature treatments have resulted in significant differences between cannabinoid constituents of cannabis leaf tissue. The differences are attributed to: ( a) differences in dry weights resulting from different treatments, and ( b) time-temperature effects on the integrity of specific cannabinoids. These results indicate that valid comparisons of cannabinoid profiles cannot be made without establishing uniformity in tissue preparation. Uniformity could be established by: ( a) appropriate drying methods, or ( b) use of uniform relative-humidity environments for maintenance of tissue at known moisture levels. Sixteen hours at 65 °C was the most appropriate drying treatment used in this investigation.

References and notes

001

"National Commission on Marihuana and Drug Abuse". 1972. Marihuana: A signal of misunderstanding. U.S. Government Printing Office, Washington, D.C.

002

Carew, D. P. 1971. J. Forensic Sciences, 16:1, 87-91.

003

Lerner, P. 1969. Bulletin on Narcotics, XXI, 3, pp. 39-42.

004

Turner, C. E., and K. Hadley. 1973. J. Pharm. Sci. 62:2.

005

Phillips, R., R. Turk, A. Mammo, N. Jain, D. Crim, and R. Forney. 1970. J. Forensic Sci. 15:2, 191-200.

006

"Plants were grown in Maryland during December and January 1972 and 1973". BNDD Analytical Lab. No. PU0084880, Schedule I No. PU0059659.

007

P. I. Number 378939.

008

Inlet temperature was 280°C, column 240°C, and detector 280°C. Internal standard was 4-androstene-3, 17-dione, contained in ethanol solution used to dissolve the concentrated tissue extract before its injection into GC. Three-per cent OV-17 was used instead of 2 per cent OV-17. The support was chromosorb WHP, 100-120 mesh.

009

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.

010

Turner and Hadley used a mass spectrometer which was not used for this study.

011

Farnsworth, N. R. 1969. J. Am. Pharm. Assn. 410-414.

012

Standards obtained from NIMH.

013

Duncan's new multiple-range test.