Jacob Bell
New Member
Gisela Skopp,1* Lucia Po¨tsch,2 and Martin Mauden1
(1 Institute of Legal Medicine, University of Heidelberg,
Vossstrasse 2, 69115 Heidelberg, Germany;
2 Institute of Legal Medicine, University of Mainz, Am
Pulverturm 3, 55131 Mainz, Germany; * author for correspondence:
fax 49-6221-565252, e-mail gisela_skopp@
med.uni-heidelberg.de)
It has been well recognized that hashish and marihuana
lose potency during storage because of a decrease in the
content of tetrahydrocannabinol (THC), which is the
major psychoactive constituent of cannabis (1 ). The effect
of oxygen on stored plant and resin materials or solutions
of pure cannabinoids seems much less significant than
that of higher temperatures ($37 °C) or light (2—7 ). A few
data are available on the stability of THC and major
metabolites in blood (8—11). However, the stability of
cannabinoids in the hair shaft has not been addressed,
although scalp hair represents one of the most exposed
parts of the body. Therefore, a study was performed to
elucidate whether cannabinoids such as THC, cannabinol
(CBN), or cannabidiol (CBD), which usually are determined
from hair samples, would exhibit similar instability
in this particular biological matrix when exposed to
solar radiation.
Clipped hair bundles (n 5 11) suggested to be cannabis
positive were washed twice with dichloromethane (10 mL each time for 10 min) and divided into four strands each.
Two of the strands were kept in the dark at ambient
temperature. Under these storage conditions, the analytes
and their concentration remained unchanged as ascertained
in a preliminary experiment (data not shown). The
other two strands were stored outside in quartz glass vials
(Pyrex) and exposed to natural sunlight for 10 weeks from
August 1, 1999 to October 10, 1999 (114 m above sea level,
40° 300 degree of latitude).
CBN is suggested to be produced from THC by oxidation
and might, therefore, be used as a degradation
marker of THC. To monitor CBN production from THC
and to test its stability in the presence of active oxygen
species, an accelerated degradation experiment (40 °C;
observation period, 3.5 h) was performed by adding
hydrogen peroxide (;300 mL/L; 40 mL of Roth) to 2 mL
of a solution of THC in ethanol (0.1 g/L; Sigma). Aliquots
(1 mL) were injected directly into a gas chromatograph
equipped with a flame ionization detector (Shimadzu).
The cannabinoid content in the hair samples was determined
by gas chromatography—mass spectrometry. Each
strand was pulverized in a ball mill, and ;50 mg of the
hair powder was weighed; 25 ng of THC-d3 (Radian
International) was added as internal standard, and the
sample was further processed according to Cirimele et al.
(12 ). A 1-mL aliquot of the extract was injected into a gas
chromatography—mass spectrometry system [HP 6890 gas
chromatograph and HP 6890 mass spectrometer, both
from Hewlett Packard; column, CP-Sil 5, 12.5 m 3 0.53
mm (i.d.), from Chrompack]. Detection was by ionization
in the electron impact mode (70 eV), with the scan mode
set at single-ion monitoring (THC, m/z 299, 314, 271;
THC-d3, m/z 302, 317, 274; CBN, m/z 295, 310, 238; CBD,
m/z 231, 246, 314). Measurements were taken twice from
each strand. The THC concentrations determined from a
particular sample differed by at least 15% and are given as
mean values (n 5 4; Table 1). Deuterated standards for CBN and CDB are not yet commercially available; hence,
these compounds were not quantified, and triplicate
measurements of six drug-free hair samples were used to
estimate their particular detection limits (mean 1 3 SD).
The limit of detection was 0.05 ng/mg of hair for all
analytes.
Upon gross inspection, all hair samples had faded
slightly in color during the 10 weeks of exposure. The
results obtained from the particular strands kept in the
dark and those exposed to global solar radiation are
summarized in Table 1. The original concentrations were
within the ranges reported for Caucasian hair in the
literature (13 ). Exposure to light produced marked decreases
in THC, CBN, and CBD concentrations. THC was
detectable in small amounts in only 3 of the 11 samples,
and CBN and CBD could still be found in 3 and 1 of 8 and
3 formerly positive samples, respectively. Obviously, the
decrease in cannabinoid content was not connected to the
hair color. Initially, THC might have been converted to
CBN, which could be further degraded in the presence of
an oxidizing agent as already indicated by the stress test.
In this experiment, THC decreased to 77% of its initial
concentration 10 min after the experiment started. The
THC concentration then decreased more slowly, to 68% of
the starting concentration after 3.5 h. CBN was formed
and reached peak concentrations after 1.5 h (12%, relative
to THC), but its concentration continuously declined to
4% (relative to THC) after 3.5 h.
This study clearly demonstrated that cannabinoids usually
measured in hair analysis are more affected by solar
radiation than other drugs of abuse detected in hair, such
as 6-acetylmorphine, dihydrocodeine, or cocaine (14 ).
CBN is thought to be a chemical degradation product of
THC, and the compound concomitantly appeared during
storage in plant materials or in solution, whereas the THC
concentration decreased (1, 7). However, in keratinized
hair exposed to sunlight, CBN was obviously further
degraded. A review of the literature on the photodegradation
of THC in solution and the present stress test
tentatively support the suggestion of a light-induced
radical reaction as the underlying mechanism (6 ). Melanin
is a photoreactive biopolymer. At visible and long
ultraviolet (UV) wavelengths, it releases most of its energy
as heat, whereas at short UV wavelengths, reactive
intermediates may be formed (15 ). Both eumelanin and
pheomelanin have the ability to generate active oxygen
species, such as O2 . , HO . , and H2O2, in various amounts
(16, 17). These reactive species have been shown to degrade
both THC and CBN as demonstrated by the hydrogen
peroxide experiment. In addition to the deleterious
effect of sunlight on the stability of cannabinoid constituents
in hair, the weathering of hair, which damages the
hair fiber at the ultrastructural level, may cause additional
changes in drug concentrations in hair (18—20).
The present findings may have implications on both
sample collection and the interpretation of analytical
results in hair analysis. In general, only a sample below
the top hair should be collected, and information should
be acquired on the hair donor's outdoor activities as well as exposure to artificial UV light (such as visits in the
solarium). The presence of fading in single hairs of the
strand or its ends should also be noted. Because of the
rapid decrease in cannabinoid constituents in hair exposed
to UV radiation, the result from a particular sample
might be rather of a qualitative nature, and a negative
finding may not conclusively indicate that there was no
cannabis use. At present, it seems a real challenge to
identify degradation products of THC, CBN, and CBD as
markers for a positive cannabis test in hair. Such work is
in progress.
References
1. Harvey DJ. Stability of cannabinoids in dried samples of cannabis dating
from around 1896—1905. J Ethnopharmacol 1990;28:117—28.
2. Fairbairn JW, Liebmann JA, Rowan MG. The stability of cannabis and its
preparation on storage. J Pharm Pharmacol 1976;28:1—7.
3. Liskow B. Marihuana deterioration. JAMA 1970;214:1709.
4. Turner CE, Hadley KW, Fettermann PS, Doorenboos NJ, Quimby MW, Waller
C. Constituents of Cannabis sativa L. IV. Stability of cannabinoids in stored
plant material. J Pharm Sci 1973;62:1601—5.
5. Garrett ER, Tsau J. Stability of tetrahydrocannabinols I. J Pharm Sci
1974;63:1563—74.
6. Bonucelli CM. Stable solutions for marijuana analysis. J Pharm Sci 1979;
68:262—3.
7. Razdan RK, Puttick AJ, Zitko BA, Handrick GR. Hashish VI. Conversion of
(2)-1(6)-tetrahydrocannabinol to (2)-1(7)-tetrahydrocannabinol. Stability of
(2)-1- and (2)-1(6)-tetrahydrocannabinols. Experientia 1972;28:121—2.
8. Wong AS, Orbanosky MW, Reeve VC, Beede JD. Stability of D-9-tetrahydrocannabinol
in stored blood and serum. In: Hawks R, ed. Analysis of
cannabinoids. NIDA Res Monogr 1982;42:119—24.
9. Johnson R, Jennison TA, Peat MA, Foltz RL. Stability of D9-tetrahydrocannabinol
(THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in blood and
plasma. J Anal Toxicol 1984;5:202—4.
10. Christophersen AS. Tetrahydrocannabinol stability in whole blood: plastic
versus glass containers. J Anal Toxicol 1986;10:129—31.
11. Moody DE, Monti KM, Spanbauer AC. Long-term stability of abused drugs
and antiabuse chemotherapeutical agents stored at 220 °C. J Anal Toxicol
1999;23:535—40.
12. Cirimele V, Sachs H, Kintz P, Mangin P. Testing human hair for cannabis. III.
Rapid screening procedure for the simultaneous identification of D9-
tetrahydrocannabinol, cannabinol, and cannabidiol. J Anal Toxicol 1996;20:
13—6.
13. Sachs H, Kintz P. Testing for drugs in hair. Critical review of chromatographic
procedures since 1992. J Chromatogr B 1998;713:147—61.
14. Skopp G, Po¨tsch L, Mo¨ller MR. Zum Suchtmittelnachweis in Haaren. V.
Auswirkung von Sonne, Regen und Wind auf den Drogengehalt in Kopfhaaren
von Drogenkonsumenten–ein Pilotprojekt. Rechtsmedizin 1997;7:
176—9.
15. Forest SE, Simon JD. Wavelength-dependent photoacoustic calorimetry
study of melanin. Photochem Photobiol 1998;68:296—8.
16. Chedekel MR, Smith SK, Post PW, Pokora A, Vessell DL. Photodestruction
of pheomelanin: role of oxygen. Proc Natl Acad Sci U S A 1978;75:
5395—9.
17. Persad S, Menon IA, Habermann HF. Comparison of the effects of UV-visible
irradiation and melanin-hematoporphyrin complexes from human black and
red hair. Photochem Photobiol 1983;37:63—8.
18. Robbins CR. Chemical and physical behavior of hair. Berlin: Springer,
1992:108—13.
19. Braida D, Dubief C, Lang G. Photoageing of the hair fiber and photoprotection.
Skin Pharmacol 1994;7:73—7.
20. Po¨tsch L, Skopp G, Becker J. Ultrastructural alterations and environmental
conditions influence the opiate concentrations in hair of drug addicts. Int J
Legal Med 1995;107:301—5.
Source: Stability of Cannabinoids in Hair Samples Exposed to Sunlight
(1 Institute of Legal Medicine, University of Heidelberg,
Vossstrasse 2, 69115 Heidelberg, Germany;
2 Institute of Legal Medicine, University of Mainz, Am
Pulverturm 3, 55131 Mainz, Germany; * author for correspondence:
fax 49-6221-565252, e-mail gisela_skopp@
med.uni-heidelberg.de)
It has been well recognized that hashish and marihuana
lose potency during storage because of a decrease in the
content of tetrahydrocannabinol (THC), which is the
major psychoactive constituent of cannabis (1 ). The effect
of oxygen on stored plant and resin materials or solutions
of pure cannabinoids seems much less significant than
that of higher temperatures ($37 °C) or light (2—7 ). A few
data are available on the stability of THC and major
metabolites in blood (8—11). However, the stability of
cannabinoids in the hair shaft has not been addressed,
although scalp hair represents one of the most exposed
parts of the body. Therefore, a study was performed to
elucidate whether cannabinoids such as THC, cannabinol
(CBN), or cannabidiol (CBD), which usually are determined
from hair samples, would exhibit similar instability
in this particular biological matrix when exposed to
solar radiation.
Clipped hair bundles (n 5 11) suggested to be cannabis
positive were washed twice with dichloromethane (10 mL each time for 10 min) and divided into four strands each.
Two of the strands were kept in the dark at ambient
temperature. Under these storage conditions, the analytes
and their concentration remained unchanged as ascertained
in a preliminary experiment (data not shown). The
other two strands were stored outside in quartz glass vials
(Pyrex) and exposed to natural sunlight for 10 weeks from
August 1, 1999 to October 10, 1999 (114 m above sea level,
40° 300 degree of latitude).
CBN is suggested to be produced from THC by oxidation
and might, therefore, be used as a degradation
marker of THC. To monitor CBN production from THC
and to test its stability in the presence of active oxygen
species, an accelerated degradation experiment (40 °C;
observation period, 3.5 h) was performed by adding
hydrogen peroxide (;300 mL/L; 40 mL of Roth) to 2 mL
of a solution of THC in ethanol (0.1 g/L; Sigma). Aliquots
(1 mL) were injected directly into a gas chromatograph
equipped with a flame ionization detector (Shimadzu).
The cannabinoid content in the hair samples was determined
by gas chromatography—mass spectrometry. Each
strand was pulverized in a ball mill, and ;50 mg of the
hair powder was weighed; 25 ng of THC-d3 (Radian
International) was added as internal standard, and the
sample was further processed according to Cirimele et al.
(12 ). A 1-mL aliquot of the extract was injected into a gas
chromatography—mass spectrometry system [HP 6890 gas
chromatograph and HP 6890 mass spectrometer, both
from Hewlett Packard; column, CP-Sil 5, 12.5 m 3 0.53
mm (i.d.), from Chrompack]. Detection was by ionization
in the electron impact mode (70 eV), with the scan mode
set at single-ion monitoring (THC, m/z 299, 314, 271;
THC-d3, m/z 302, 317, 274; CBN, m/z 295, 310, 238; CBD,
m/z 231, 246, 314). Measurements were taken twice from
each strand. The THC concentrations determined from a
particular sample differed by at least 15% and are given as
mean values (n 5 4; Table 1). Deuterated standards for CBN and CDB are not yet commercially available; hence,
these compounds were not quantified, and triplicate
measurements of six drug-free hair samples were used to
estimate their particular detection limits (mean 1 3 SD).
The limit of detection was 0.05 ng/mg of hair for all
analytes.
Upon gross inspection, all hair samples had faded
slightly in color during the 10 weeks of exposure. The
results obtained from the particular strands kept in the
dark and those exposed to global solar radiation are
summarized in Table 1. The original concentrations were
within the ranges reported for Caucasian hair in the
literature (13 ). Exposure to light produced marked decreases
in THC, CBN, and CBD concentrations. THC was
detectable in small amounts in only 3 of the 11 samples,
and CBN and CBD could still be found in 3 and 1 of 8 and
3 formerly positive samples, respectively. Obviously, the
decrease in cannabinoid content was not connected to the
hair color. Initially, THC might have been converted to
CBN, which could be further degraded in the presence of
an oxidizing agent as already indicated by the stress test.
In this experiment, THC decreased to 77% of its initial
concentration 10 min after the experiment started. The
THC concentration then decreased more slowly, to 68% of
the starting concentration after 3.5 h. CBN was formed
and reached peak concentrations after 1.5 h (12%, relative
to THC), but its concentration continuously declined to
4% (relative to THC) after 3.5 h.
This study clearly demonstrated that cannabinoids usually
measured in hair analysis are more affected by solar
radiation than other drugs of abuse detected in hair, such
as 6-acetylmorphine, dihydrocodeine, or cocaine (14 ).
CBN is thought to be a chemical degradation product of
THC, and the compound concomitantly appeared during
storage in plant materials or in solution, whereas the THC
concentration decreased (1, 7). However, in keratinized
hair exposed to sunlight, CBN was obviously further
degraded. A review of the literature on the photodegradation
of THC in solution and the present stress test
tentatively support the suggestion of a light-induced
radical reaction as the underlying mechanism (6 ). Melanin
is a photoreactive biopolymer. At visible and long
ultraviolet (UV) wavelengths, it releases most of its energy
as heat, whereas at short UV wavelengths, reactive
intermediates may be formed (15 ). Both eumelanin and
pheomelanin have the ability to generate active oxygen
species, such as O2 . , HO . , and H2O2, in various amounts
(16, 17). These reactive species have been shown to degrade
both THC and CBN as demonstrated by the hydrogen
peroxide experiment. In addition to the deleterious
effect of sunlight on the stability of cannabinoid constituents
in hair, the weathering of hair, which damages the
hair fiber at the ultrastructural level, may cause additional
changes in drug concentrations in hair (18—20).
The present findings may have implications on both
sample collection and the interpretation of analytical
results in hair analysis. In general, only a sample below
the top hair should be collected, and information should
be acquired on the hair donor's outdoor activities as well as exposure to artificial UV light (such as visits in the
solarium). The presence of fading in single hairs of the
strand or its ends should also be noted. Because of the
rapid decrease in cannabinoid constituents in hair exposed
to UV radiation, the result from a particular sample
might be rather of a qualitative nature, and a negative
finding may not conclusively indicate that there was no
cannabis use. At present, it seems a real challenge to
identify degradation products of THC, CBN, and CBD as
markers for a positive cannabis test in hair. Such work is
in progress.
References
1. Harvey DJ. Stability of cannabinoids in dried samples of cannabis dating
from around 1896—1905. J Ethnopharmacol 1990;28:117—28.
2. Fairbairn JW, Liebmann JA, Rowan MG. The stability of cannabis and its
preparation on storage. J Pharm Pharmacol 1976;28:1—7.
3. Liskow B. Marihuana deterioration. JAMA 1970;214:1709.
4. Turner CE, Hadley KW, Fettermann PS, Doorenboos NJ, Quimby MW, Waller
C. Constituents of Cannabis sativa L. IV. Stability of cannabinoids in stored
plant material. J Pharm Sci 1973;62:1601—5.
5. Garrett ER, Tsau J. Stability of tetrahydrocannabinols I. J Pharm Sci
1974;63:1563—74.
6. Bonucelli CM. Stable solutions for marijuana analysis. J Pharm Sci 1979;
68:262—3.
7. Razdan RK, Puttick AJ, Zitko BA, Handrick GR. Hashish VI. Conversion of
(2)-1(6)-tetrahydrocannabinol to (2)-1(7)-tetrahydrocannabinol. Stability of
(2)-1- and (2)-1(6)-tetrahydrocannabinols. Experientia 1972;28:121—2.
8. Wong AS, Orbanosky MW, Reeve VC, Beede JD. Stability of D-9-tetrahydrocannabinol
in stored blood and serum. In: Hawks R, ed. Analysis of
cannabinoids. NIDA Res Monogr 1982;42:119—24.
9. Johnson R, Jennison TA, Peat MA, Foltz RL. Stability of D9-tetrahydrocannabinol
(THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in blood and
plasma. J Anal Toxicol 1984;5:202—4.
10. Christophersen AS. Tetrahydrocannabinol stability in whole blood: plastic
versus glass containers. J Anal Toxicol 1986;10:129—31.
11. Moody DE, Monti KM, Spanbauer AC. Long-term stability of abused drugs
and antiabuse chemotherapeutical agents stored at 220 °C. J Anal Toxicol
1999;23:535—40.
12. Cirimele V, Sachs H, Kintz P, Mangin P. Testing human hair for cannabis. III.
Rapid screening procedure for the simultaneous identification of D9-
tetrahydrocannabinol, cannabinol, and cannabidiol. J Anal Toxicol 1996;20:
13—6.
13. Sachs H, Kintz P. Testing for drugs in hair. Critical review of chromatographic
procedures since 1992. J Chromatogr B 1998;713:147—61.
14. Skopp G, Po¨tsch L, Mo¨ller MR. Zum Suchtmittelnachweis in Haaren. V.
Auswirkung von Sonne, Regen und Wind auf den Drogengehalt in Kopfhaaren
von Drogenkonsumenten–ein Pilotprojekt. Rechtsmedizin 1997;7:
176—9.
15. Forest SE, Simon JD. Wavelength-dependent photoacoustic calorimetry
study of melanin. Photochem Photobiol 1998;68:296—8.
16. Chedekel MR, Smith SK, Post PW, Pokora A, Vessell DL. Photodestruction
of pheomelanin: role of oxygen. Proc Natl Acad Sci U S A 1978;75:
5395—9.
17. Persad S, Menon IA, Habermann HF. Comparison of the effects of UV-visible
irradiation and melanin-hematoporphyrin complexes from human black and
red hair. Photochem Photobiol 1983;37:63—8.
18. Robbins CR. Chemical and physical behavior of hair. Berlin: Springer,
1992:108—13.
19. Braida D, Dubief C, Lang G. Photoageing of the hair fiber and photoprotection.
Skin Pharmacol 1994;7:73—7.
20. Po¨tsch L, Skopp G, Becker J. Ultrastructural alterations and environmental
conditions influence the opiate concentrations in hair of drug addicts. Int J
Legal Med 1995;107:301—5.
Source: Stability of Cannabinoids in Hair Samples Exposed to Sunlight
