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Received: 18 February 2014 Revised: 8 May 2014 Accepted: 14 June 2014 Published online in Wiley Online Library: 28 July 2014
Keywords: child protection; external contamination; hair analysis; tetrahydrocannabinol; tetrahydrocannabinolic acid A
Introduction
* Correspondence to: V. Auwrter, Institute of Forensic Medicine, Forensic
Analysis of hair samples for drugs of abuse is applied in various
Toxicology Department, University Medical Center Freiburg, Albertstrae 9,
settings today and has become a standard method for certain 79104 Freiburg, Germany. E-mail: volker.auwaerter@uniklinik-freiburg.de
issues,[1] three of the most common being child protection
cases,[24] abstinence control programs,[57] and workplace drug a Institute of Forensic Medicine, Forensic Toxicology Department, University
testing.[8] Considering the severe consequences positive or nega- Medical Center Freiburg, Albertstrae 9, 79104, Freiburg, Germany
tive test results may have for the person concerned, inter- b Hermann Staudinger Graduate School, University of Freiburg, Hebelstrae 27,
pretation has to be carried out with utmost care. In particular, 79104, Freiburg, Germany
external contamination should be excluded or discussed ade-
quately. In the case of hair analysis for cannabinoids this is partic- c Institute of Legal Medicine, Charit-University Medicine Berlin, Turmstrae 21,
Building N, 10559, Berlin, Germany
ularly difcult, since the drug is quite often handled extensively
prior to consumption (e.g. when preparing a joint) and smoke d State Ofce of Criminal Investigation Baden-Wrttemberg, Taubenheimerstrae
349
causes a further source of external contamination. Further 85, 70372, Stuttgart, Germany
Drug Test. Analysis 2015, 7, 349357 Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis B. Moosmann et al.
aggravating the interpretation of THC positive hair results is analyzed using methanolic extraction and liquid chromatography-
the circumstance that quite often the oxidative metabolite tandem mass spectrometry (LC-MS/MS) analysis as well as applying
9-carboxy-11-nor-9-tetrahydrocannabinol (THC-COOH), which alkaline hydrolysis and headspace-solid phase microextraction-
would conrm a body passage, cannot be detected despite gas chromatography-mass spectrometry (HS-SPME-GC-MS) or
considerable 9-tetrahydrocannabinol (THC) concentrations GC-MS analysis, respectively.
and application of highly sensitive analytical methods.[916]
A previous study has shown that 9-tetrahydrocannabinolic
acid A (THCA-A, structure in Figure 1), the biogenetic non psycho- Materials and methods
active precursor of THC, could potentially serve as a marker for an
Materials
external hair contamination, since it is not incorporated into hair
through the bloodstream after repeated oral intake of high doses Acetonitrile and methanol (MeOH) (both gradient grade) were
THCA-A.[17] However, in many forensic hair samples analyzed in obtained from J.T. Baker (Deventer, the Netherlands) and Th.
our laboratory, THCA-A was detected in concentrations ex- Geyer (Renningen, Germany), n-hexane (p.a., ACS) was purchased
ceeding that of THC (own unpublished data and Auwrter from Merck (Darmstadt, Germany). Formic acid (ROTIPURAN
et al.[17]). These observations lead to the assumption that THCA- 98%, p.a.) and petroleum ether (ROTIPURAN 40-60C) were
A in hair results from external contamination exclusively. Addi- obtained from Carl Roth (Karlsruhe, Germany) and acetone as
tionally, also parts of the detected THC and cannabinol (CBN) well as ethyl acetate (both p.a., ACS) from Sigma Aldrich
may originate from such sources. Based on further experiments (Steinheim, Germany). Lecithin (egg phosphatidylcholine, as a
it could be ruled out with a high certainty that relevant amounts solubilizer for better stability of processed samples) was supplied
of THCA-A are transferred through side-stream marihuana smoke, by Lipoid (Ludwigshafen, Germany). THC, CBN (1 mg/mL each),
as the smoke only contains negligible amounts of THCA-A.[18] and THC-D3 (0.1 mg/mL) were obtained from Cerilliant (Round
One possible way of an external contamination of hair could Rock, TX, USA). THCA-A and CBN-D3 (0.1 mg/mL) were purchased
also be transfer through contaminated hands[19] as it has already from Lipomed (Arlesheim, Switzerland). THCA-A was dissolved in
been shown in a contamination experiment for heroin.[20] Given MeOH (0.1 mg/mL). THCA-A-D3 was synthesized according to
the high lipophilicity of cannabinoids, it seems plausible that Roth et al.[26] leading to a mixture of THCA-A-D3 and THC-D3
during the handling of raw cannabis plant material or hashish - (>90% / ~9%). Deionized water was prepared using a cartridge
as it occurs when e.g. rolling a joint or preparing hash-cookies deionizer from Memtech (Moorenweis, Germany). Blank hair
a transfer to the ngers and later to the own or the childrens hair was provided by volunteers and tested for the absence of canna-
might occur. Such a contamination may take place for instance binoids prior to use.
when cannabis consuming parents, relatives or acquaintances
sweep their hands through the hair of a young child.
Sampling of hair
Hair analysis for cannabinoids is further complicated by
the fact that quite often alkaline hydrolysis is used as the Hair samples from children (n = 41; age 7 months12 years), teen-
method of sample preparation,[17,2124] leading to decarbox- agers (n = 4; age 1317 years) and adults (n = 34; age: 1859
ylation of THCA-A and therefore articially elevating the THC years) were collected between 2011 and 2013 on the order of
concentration. the Ofces of Social Services at the Senate of the Hanseatic City
Regarding child protection cases, the latest report from the of Bremen or at the Municipal Administrations of Bremerhaven
European Monitoring Centre for Drugs and Drug Addiction in the frame of a previously described project.[3] All the hair sam-
(EMCDDA) states that about 10% of clients entering treatment ples were taken from the posterior vertex region of the head as
for drug problems in 2010 lived with children.[25] It is often help- close to the scalp as possible with a remaining hair length at
ful to evaluate whether the children are directly exposed to drugs the scalp of approximately 12 mm and stored in the dark at
in order to evaluate health risks. In cases of positive ndings in room temperature until analysis. The study was performed ac-
children hair it has to be carefully differentiated whether (1) the cording to the Helsinki ethical principles for medical research in-
drug was administered to the child for example for sedating it, volving human subjects of the Word Medical Association. Hair
(2) the drug was consumed in the presence of the child leading sampling and analysis was performed either on the basis of a
to passive uptake by the child, or (3) the drug was consumed in written informed consent or on decision of the local family court.
the absence of the child. All three scenarios bear different health In case of children the informed consent was signed by the par-
risks for the child and probably require different approaches from ents or caregivers. Emphasis was put on anonymous investiga-
a child-care perspective. tion in order to maintain condence with the families. The
In order to evaluate based on the presence of THCA-A, if the samples were anonymized immediately after collection and only
positive cannabinoid results found in children hair in a child the age of the individual was recognizable by the laboratory.
protection project[3] are caused by external contamination, hair sam- Only for some of the samples the connections between children
ples from 41 children and 34 cannabis-consuming parents were and adults were disclosed for extended interpretation.
Sample preparation
The sample preparation of the initial forensic analysis is described
in a previous paper[3] (Method 3). In this study, excessive hair
left over from this forensic investigation with a positive THC
result was reanalyzed. Thirty-two hair samples were shorter
9
Figure 1. Formation of -tetrahydrocannabinol (THC) by decarboxylation than 7.0 cm and therefore processed without segmentation,
350
9
of -tetrahydrocannabinolic acid A (THCA-A). 36 hair samples were segmented into one proximal segment
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Drug Testing
Cannabinoid ndings in children hair what do they really tell us? and Analysis
of 6 cm length and the distal segment discarded. A further 11 hair Table 1. MRM transitions and corresponding voltages applied in
samples were not segmented despite a hair length between 7 and LC-MS/MS analysis (Method 1). (Q1) m/z of the precursor ion, (Q3)
20 cm as only a limited sample amount was still available. The m/z of the fragment ion, (DP) declustering potential, (CE) collision
energy, (CXP) collision cell exit potential.
samples or individual segments were washed by shaking for four
minutes with 4 mL water and twice with 4 mL acetone. Finally, the Analyte Q1, amu Q3, amu dwell DP, V CE, V CXP, V
hair was left to dry for 24 h. time, ms
hydrolyzed at 95C for 10 min. For liquid-liquid extraction (LLE) phase microextraction (HS-SPME) with N,O-bis(trimethylsilyl)
Drug Test. Analysis 2015, 7, 349357 Copyright 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta
Drug Testing
and Analysis B. Moosmann et al.
triuoroacetamide (BSTFA) as derivatization agent and gas chro- evidence about the incorporation mechanism into hair as a basis
matography mass spectrometry in selected ion monitoring mode. for interpretation was expected.
The method was validated according to international guidelines
with resulting LOQs of 0.01 ng/mg for all three compounds.
Extraction with methanol and LC-MS/MS (Method 1)
A summary of the results obtained by applying Method 1
Analysis of marihuana and hashish samples
(methanol extraction, LC-MS/MS) can be found in Table 2, the
For marihuana samples, a representative amount was homoge- complete data are shown in the supplementary Table S1.
nized using a knife mill and in case of the hashish samples, two THCA-A could be detected in all but one hair sample and
cores were drilled and the drilling chips were homogenized in a ranged from 6.5 to 4700 pg/mg (median: 223 pg/mg). In 77
mortar using liquid nitrogen. Afterwards, 10 mL internal standard out of the 78 THCA-A positive cases, the concentration of
solution (0.5 mg/mL tribenzylamine in n-heptane) were added THCA-A was higher than the concentration of THC (median of
into a glass vial containing 120 mg of hashish homogenate or THCA-A/THC 4.2) and in 14 cases no THC could be detected
80400 mg of marihuana homogenate, respectively, and despite the presence of THCA-A (median of the THCA-A con-
sonicated at 50 C for 20 min. After the rst 10 min the sample centrations in these particular samples 63 pg/mg).
was shaken manually and additionally vortexed at the end of The results of a comparison between active consumers (adults,
the 20 min period. After deposition of the sediment, 500 L of age 1859 years) and passive exposure (children, age 12 years)
the supernatant were transferred into a GC vial and 200 l MSTFA are shown in Table 2 and Figure 2. The four teenagers (age
as well as 30 L pyridine were added. The closed vial was placed 1317 years) were excluded since the kind of contact with
in an oven at 70C for 20 min to perform silylation. Finally, the cannabis is not clear. It can be seen from the mean values,
samples were left to rest for 4 h at room temperature and after- the medians and the box and whiskers plots that the concen-
wards 1 L was injected into the gas-chromatography-ame ion- trations of THCA-A as well as THC are clearly lower in hair of
ization detector (GC-FID) system (Focus GC or Trace GC Ultra, the children than in hair of the adult consumers. However,
both Thermo Scientic, Dreieich, Germany). The GC-FID condi- there is no signicant difference in the concentration ratio
tions were as follows: Split injection mode; column: BPX-5, SGE THCA-A/THC between both groups if the specic relationship
(15 m x 0.32 mm ID, 0.25 m lm thickness); injection port between child and caregiver remains unconsidered in this
temperature: 250 C; detector (FID) port temperature 300 C; carrier overall statistics. This is conrmed by analysis of variance
gas: helium; ow rate: 1.5 mL/min; oven temperature: initially 200C (ANOVA) with = 0.000 for both THCA-A and THC (signicantly
for 2 min, increased to 235 C at 2 C/min, to 280 C at 50 C/min, different) and = 0.212 for THCA-A/THC (not signicantly
280 C for 4 min. different).
However, the comparison within families leads to another
Statistical evaluations result. For this evaluation only a subset of 10 child-adult pairs
living in the same household could be included (Table 3), as
Statistical analysis was performed using SPSS v19.0. Analysis information about familiar connections was not disclosed in all
comparing drug concentration and concentration ratios uti- cases for data protection reasons. In these 9 families it can be
lized analysis of variance (ANOVA) to determine whether or observed that in 9 of the 10 hair samples of children the ratio
not signicant differences existed in cannabinoid concentra- THCA-A/THC is either higher than the ratio measured in the
tions and concentration ratios between children and adults samples from the cannabis consuming adults (n = 6), or only
hair and between hair and marihuana or hashish. Signicance THCA-A was detectable in the hair samples of the children
was attributed to differences that attained a value of lower (n = 3). Analysis of variance (ANOVA) showed a signicant
than or equal to 0.05. difference between the two groups regarding the THCA-A/THC
ratios ( = 0.042; in case of absence of THC, the minimum ratio
based on the LOD of the method was used)
Results
In this study, hair samples from children and cannabis consuming Cannabinoids and cannabinoid ratios in marihuana and hashish
parents were analyzed by three different methods: In case of the consuming parents, no information was available
- Method 1: Extraction with methanol and LC-MS/MS. With this regarding the type of cannabis products used. As the investiga-
method no decarboxylation of THCA-A to THC occurs and the tion of conscated plant material shows a huge variation in the
original concentrations of THC and THCA-A are measured. THCA-A to THC ratios between different marihuana samples
- Method 2: Alkaline hydrolysis, liquid-liquid extraction, derivati- and especially between marihuana and hashish samples (Table 4),
zation and GC-MS. With this method, THCA-A was included in the composition of the used plant material should have a strong
analysis and the extent of transformation can be checked. effect on the ratios found in hair. The comparison of the data
- Method 3 (Initial forensic analysis): Alkaline hydrolysis, liquid- in Tables 2 and 4 and of the box an whiskers plots in Figure
liquid extraction and derivative headspace solid phase 3 show that the THCA-A/THC ratios in hair (all samples, mean
microextraction with gas chromatography-mass spectrometry 6.7, median 4.2) are in the range between hashish (mean 2.8,
(HS-SPME-GC-MS). With this method, THCA-A was not measured median 2.1) and marihuana (mean 11.0, median 8.3). Statistical
but should be largely transformed to THC, as observed in the comparison by ANOVA resulted in signicant differences for
studies conducted by Dussy et al.[28] and Auwrter et al.[17] hair samples and marihuana ( = 0.004 and F = 8.249), and
for hair samples and hashish ( = 0.000 and F = 35.321). It
From the comparison between non-consuming children and can be seen from Figure 3 that the majority of the hair sam-
352
adults and between the results of the three methods further ples fall in the 2575 percentile range of the marihuana
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Cannabinoid ndings in children hair what do they really tell us? and Analysis
Table 2. Cannabinoid concentrations in hair measured by Method 1 and statistical comparison between children and adults. Samples of adoles-
cents (age 13 to 17 years, n = 4) were neither counted as adults nor as children.
All samples (n = 79) THCA-A, pg/mg THC, pg/mg CBN, pg/mg Ratio (n = 64*) THCA-A: THC
Adults (n = 34) THCA-A, pg/mg THC, pg/mg CBN, pg/mg Ratio (n = 33*) THCA-A:THC
Mean 961 214 89 5.7
Median 556 127 44 4.1
SD 1096 271 157 5.4
Range 464700 01200 0740 0.929
Comparison children vs. THCA-A, pg/mg THC, pg/mg CBN, pg/mg Ratio THCA-A:THC
adults by ANOVA
0.000 0.000 0.212
F 18.593 18.413 1.591
Different Yes Yes No
Figure 2. Box and whiskers plots of THCA-A and THC concentration as well as THCA-A to THC ratios in hair compared between adults and children
(n = 34 adults and 41 children), * and o indicate outliers.
samples whereas there is almost no overlap between the 2575 an essential part of THCA-A remains undecomposed. Generally,
percentile ranges of hair and hashish samples. this can be an essential source of error in determination of THC
in hair by alkaline hydrolysis.
If THCA-A would be quantitatively transformed by decarboxyl-
Alkaline hair hydrolysis and GC-MS (Method 2) ation to THC (Figure 1), and with consideration of the different
A subset of the washed specimens, where enough material was molecular masses, the expected total THC concentration can be
available as a left-over from Method 1 (30 samples), was analyzed calculated according to Eqn (1).
applying Method 2 in order to examine to which extent THCA-A
is transformed to THC under alkaline hydrolysis conditions. The THCtotal 314:47 x THCA-A= 358:47 THC (1)
summarized results are shown in Table 5 and in detail in the sup-
plementary Table S2. It can be seen that despite 10 min treat- The comparison in Table 5 and the Supplementary Table S2
353
ment in 1 N NaOH at 95 C and heating during derivatization shows that in 24 out of 30 hair samples these total THC
Drug Test. Analysis 2015, 7, 349357 Copyright 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta
Drug Testing
and Analysis B. Moosmann et al.
Table 3. Comparison of THCA-A concentration, THC concentration concentrations calculated according to Eqn (1) for Method 1. It
and the ratio THCA-A/THC in hair obtained by Method 1 between chil- can be seen that the mean and the median of the concentrations
dren and related adult consumers living in the same household of THC (Method 3) and of calculated total THC (Method 1) are in
(households separated by dotted lines).
relatively good agreement. However, focusing on paired values
Case No. Age, years THCA-A, pg/mg THC, pg/mg THCA-A /THC measured in the samples of the same individual indicates a high
variation with the concentration ratio Method 1/Method 3 rang-
16 3 164 37 4.4
ing from 0.11 to 6.21.
66 31 2540 1227 2.1
Similar to Method 1, mean and median of this ratio are higher
11 2 301 70 4.3 than 1.00. However, on closer consideration of the individual
55 24 74 79 0.9 values in Table S3, this is the case only in 38 out of the 68 sam-
29 6 68 0 >6.8* ples. This includes 22 out of 32 children, two of the four teen-
59 26 128 34 3.8 agers and only 14 out of 32 adults. Possible reasons are
19 3 650 38 17.1 incomplete decarboxylation of THCA-A as well as degradation
37 10 73 0 >7.3* of both compounds under the more aggressive conditions.
70 34 613 161 3.8 In the case of CBN, Method 3 led to higher concentrations than
20 3 230 8 29.1 Method 1 in most of the samples (Table S3).
57 19 315 47 6.7
41 12 24 14 1.8
79 59 749 203 3.7 Discussion
22 4 448 40 11.3 A large variety of methods with respect to extraction, derivatiza-
63 29 452 90 5.0 tion, chromatography, and mass spectrometric detection is used
32 7 532 147 3.6 for the analysis of cannabinoids in hair samples in forensic case
71 34 652 260 2.5 work. Although results of prociency tests with spiked hair sam-
12 2 141 0 >14.1* ples indicate suitability of different methodological approaches,
53 23 472 99 4.8 the presence of THCA-A in authentic hair samples complicates
the situation. The presented data show that analytical results of
* Estimated using the LOD for THC (Method 1, 10 pg/mg) hair analysis for cannabinoids strongly depend on the applied
methodology mainly because of artifactual decarboxylation of
THCA-A under alkaline conditions and/or elevated temperatures.
concentrations from Method 1 (methanol extraction; LC-MS/MS)
This can lead to elevated THC concentrations but does not
were higher than the total THC concentration determined by
necessarily lead to a complete decarboxylation of THCA-A.
Method 2 (GC-MS after alkaline hydrolysis and LLE). The differ-
Nevertheless, only the THC concentration is generally used for
ence is more evident for children (mean ratio Method 1/Method
interpretation concerning patterns of cannabis use. Especially
2 1.64) than for adults (1.24). The smaller total concentration in
in cases applying alkaline hydrolysis and HS-SPME-GC-MS
Method 2 can be explained by a loss of both compounds caused
techniques it has to be carefully assessed how digestion time
by alkaline treatment and high GC injection temperature.[17,28]
and temperature inuence the degradation of the analytes
(e.g. degradation of THCA-A to THC, THC to CBN and CBN to
further oxidation products). On the other hand, remaining THCA-A
Alkaline hydrolysis and derivative HS-SPME-GC-MS (Method 3)
would lead to lower measured THC concentrations and could
The results of Method 1 were also compared with the concentra- give rise to false negative results in forensic cases.
tions of THC and CBN obtained in the initial forensic investigation The detection of THCA-A suggests that in almost all cases an
(given in Supplementary Table S3). For this comparison, only external contamination has occurred, since THCA-A is not incor-
samples were included where hair segments of equal lengths porated through the bloodstream in detectable amounts (LOD
were analyzed in both methods (n = 68). A summarized compari- in a previous study 50 pg/mg).[17] Furthermore, as side-stream
son is shown in Table 6 by using again the total THC marihuana smoke seems to play only a minor role as a source
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Drug Testing
Cannabinoid ndings in children hair what do they really tell us? and Analysis
Table 5. Concentrations of THCA-A and THC in hair obtained by Method 2 and comparison of the total THC concentrations calculated by Eqn (1)
between Methods 1 and 2.
All samples tested THCA-A Method 2, THC Method 2, Total THC conc. Method 1, Total THC conc. Method 2, Ratio Method 1:
with Method 1 pg/mg pg/mg pg/mg pg/mg Method 2
and Method 2
(n = 30)
Drug Test. Analysis 2015, 7, 349357 Copyright 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta
Drug Testing
and Analysis B. Moosmann et al.
Table 6. Comparison of results obtained by method 1 with the results obtained by Method 3. The total THC concentration for Method 1 was
calculated from THC and THCA-A by Eqn (1).
All samples tested Total THC conc. THC conc. Total THC conc. CBN conc. CBN conc. CBN conc. Method 1:
with Method 1 Method 1, Method 3, Method 1: THC Method 1, Method 3, CBN conc.
and Method 3 pg/mg pg/mg conc. Method 3 pg/mg pg/mg Method 3
(n = 68, including
4 teenagers)
Children (n = 32) Total THC conc. THC conc. Total THC conc. CBN conc. CBN conc. CBN conc. Method
Method 1, pg/mg Method 3, pg/mg Method 1: THC conc. Method 1, Method 3, 1: CBN conc.
Method 3 pg/mg pg/mg Method 3
Mean 183 133 1.92 11.0 34.6 0.27
Median 73.6 56.0 1.73 0.0 20.0 0.24
SD 230 251 1.38 23.1 66.4 0.29
Range 5.7874 111360 0.136.21 0120 0360 00.92
Adults (n = 32) Total THC conc. THC conc. Total THC conc. CBN conc. CBN conc. CBN conc. Method
Method 1, pg/mg Method 3, pg/mg Method 1: THC conc. Method 1, Method 3, 1: CBN conc.
Method 3 pg/mg pg/mg Method 3
Mean 1081 1093 1.10 91.8 110 0.60
Median 648 705 0.92 43.7 75.0 0.51
SD 1223 1018 0.90 162 101 0.68
Range 405195 1004330 0.114.77 0740 0370 02.75
after HPLC clean-up. Forensic Sci. Int. 2000, 107, 239. ples. J. Mass Spectrom. 2012, 47, 604.
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Drug Testing
Cannabinoid ndings in children hair what do they really tell us? and Analysis
[23] T. Nadulski, F. Pragst. Simple and sensitive determination of 9- [27] N. Roth, B. Moosmann, V. Auwrter. Development and validation of an
tetrahydrocannabinol, cannabidiol and cannabinol in hair by LC-MS/MS method for quantication of 9-tetrahydrocannabinolic acid
combined silylation, headspace solid phase microextraction and gas A (THCA-A), THC, CBN and CBD in hair. J. Mass Spectrom. 2013, 48, 227.
chromatography-mass spectrometry. J. Chromatogr. B 2007, 846, 78. [28] F.E. Dussy, C. Hamberg, M. Luginbuhl, T. Schwerzmann, T.A.
[24] F. Musshoff, H.P. Junker, D.W. Lachenmeier, L. Kroener, B. Madea. Fully Briellmann. Isolation of 9-THCA-A from hemp and analytical as-
automated determination of cannabinoids in hair samples using head- pects concerning the determination of 9-THC in cannabis products.
space solid-phase microextraction and gas chromatography-mass Forensic Sci. Int. 2005, 149, 3.
spectrometry. J. Anal. Toxicol. 2002, 26, 554.
[25] European Monitoring Centre for Drugs and Drug Addiction. Preg-
nancy, childcare and the family: Key issues for Europes response to
drugs. Publications Ofce of the European Union, Luxembourg, 2012. Supporting information
[26] N. Roth, A. Wohlfarth, M. Mller, V. Auwrter. Regioselective synthe-
sis of isotopically labeled 9-tetrahydrocannabinolic acid A (THCA-
A-D3) by reaction of 9-tetrahydrocannabinol-D3 with magnesium Additional supporting information may be found in the online
methyl carbonate. Forensic Sci. Int. 2012, 222, 368. version of this article at the publishers web site.
357
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