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Sassafras oils as precursors for the production of synthetic drugs: Profiling via
MEKC-UVD

Conference Paper · April 2005

DOI: 10.13140/2.1.4698.5285

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XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 198

Sassafras oils as precursors for the

production of synthetic drugs: Profiling
via MEKC-UVD
C. Huhn, M. Pütz, R. Dahlenburg, U. Pyell

Abstract
The still high demand for Ecstasy among drug users in Germany encourages its
clandestine production. The surveillance of the chemicals used for the synthesis of
mainly MDMA (3,4-methylenedioxymethamphetamine) as active substance is a major
issue to break down supply chains and identify suppliers. One of the most important
precursors for MDMA is safrole, the major compound (up to 95%) found in the essential
oils of sassafras albidum, cinnamomum camphora and ocotea pretiosa.
A micellar electrokinetic chromatography (MEKC) method was developed for
the separation of their hydrophobic constituents, such as safrole, eugenol, methyleugenol,
α-asarone and trans-anethole. The run buffer consisted of borate (c = 7.5 mmol L-1),
sodium dodecyl sulfate (c = 60 mmol L-1), urea (c = 4 mol L-1), CaCl2 (c = 0.5 mmol L-1)
at pH 9.2 and 20% (v/v) acetonitrile. Detection limits, linear range and repeatability were
studied. The constituents of several sassafras oils from clandestine laboratories were
identified and determined. The safrole content was found to be 60-95%; minor
compounds detected were mainly eugenol and methyleugenol. These as well as traces of
non-identified substances resulted in a fingerprint region with a clear recognition of two
different patterns. The comparison with electropherograms from defined plant material
enabled the classification according to their biological sources.

1. Introduction
The demand for Ecstasy tablets containing amphetamine-type-stimulants
(ATS) among drug users in Germany is still high. In 2004, for the first time since
2001, the number of Ecstasy seizures as well as the total amount of seized Ecstasy
tablets in Germany increased significantly compared to the previous year [1]. In
literature more and more hints are given for the negative impacts of frequent
consumption of Ecstasy on cognitive skills (e.g. memory), psychobiological
functions such as sleep and appetite as well as a noticeable psychological impact
concerning for example lethargy and depression [9]. In 2004 more than 90% of the
Ecstasy tablets seized in Germany contained only one ATS as active substance, in
95% of these mono preparations the active substance was MDMA (3,4-methylene-
dioxymethamphetamine) [1].
In addition to seizures of Ecstasy tablets, the surveillance of the
chemicals (precursors) used for the synthesis of mainly MDMA (e.g. safrole) is a
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 199

major issue to break down supply chains and to reduce drug traffic. Safrole can
easily be converted to piperonylmethylketone (PMK), both are the predominant
precursors used for the clandestine production of MDMA. Therefore, safrole,
isosafrole, PMK and piperonal are classified as category 1 precursors for MDMA
and other methylenedioxy-substituted ATS as defined by the German precursor
monitoring act („Grundstoffüberwachungsgesetz“, GÜG). The access to MDMA
via safrole is fast-forward as shown in Figure 1.

O O

O O
safrole isosafrole

O O

NH O
O O

PMK
MDMA

Fig. 1: Reaction scheme from safrole to MDMA

Safrole can be found in the essential oils of many plants. Economically

interesting concentrations are reported mainly for the tree plants cinnamomum
camphora, sassafras albidum and ocotea pretiosa (ocotea cymbarum). The
essential oils of both, sassafras albidum and ocotea pretiosa, are traded as
“sassafras oil”, the latter one often referred to as “Brazilian sassafras oil”.
Sassafras albidum is indigenous to Atlantic North America, Northern Mexico and
Taiwan, ocotea pretiosa is mostly found in Brazil [3]. Seized sassafras oils are
often linked to Asian countries.
The quantification of safrole has so far been dominated by GC and
HPLC techniques [2,4,7,10]. The aim of this work was to quantify safrole together
with minor compounds present in these oils to enable batch-to-batch comparisons
of samples from clandestine laboratories and to provide hints for the herbal origin
of these oils. We concentrate on the aromatic constituents described in literature
[5,7]. Their structures are shown in Figure 2.
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 200

O O O

O
O O O
piperonal isosafrole safrole

HO HO

O O
isoeugenol methyleugenol eugenol

O O
HO

O
O

O
thymol
myristicin asarone
O
O

O NC

trans-anethole methyl 4-cyanobenzoate (IS)

Fig. 2: Structures of analytes and internal standard (IS)

We used micellar electrokinetic chromatography (MEKC) in combination

with UV absorbance detection for the determination of these compounds in the
essential oils. This method is known to provide high separation efficiency for the
separation of structurally closely related compounds as well as a high tolerance
towards compounds being present in concentrations several orders of magnitude
higher than the analyte. The high safrole content of up to 95% found in the
essential oils of sassafras albidum and ocotea pretiosa reveals an excess of this
substance compared to the concentration of minor constituents of up to 1000:1.

2. Experimental

2.1 Chemicals
Methanol, eugenol, safrole, thymol, piperonal and asarone were from
Fluka, Buchs, CH; urea and sodium dodecyl sulfate (SDS) were from Roth,
Karlsruhe, D; sodium tetraborate, methyl 4-cyanobenzoate, CaCl2, isosafrole, and
Triton X 100 were from Merck, Darmstadt, D; acetonitrile from J.T.Baker,
Deventer, NL; methyleugenol and trans-anethole were from Aldrich, St. Louis,
USA; myristicin was from Sigma, Steinheim, D; Samples: sassafras albidum oil
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 201

was bought from Aldrich – Flavors & Fragrances, Milwaukee, US, ocotea
pretiosa oil was purchased from Aromalandia, Bela Horizonte, BR, a young
camphor tree (cinnamomum camphora) was imported from Taiwan via Pflanzen-
versand Röpke, Seesen, D; seized sassafras oil samples were provided by the
Bundeskriminalamt.

2.2 Instrumental
A PrinCE CE instrument (Prince Technologies BV, Emmen, NL) was
used. The sample injection was done hydrodynamically at 50 mbar for 3 s. Fused
silica capillaries from Polymicro Technologies LLC (Phoenix, AZ, USA) were
used with an inner diameter of 50 µm and an outer diameter of 363 µm. The
length was set to 53.5/75 cm. New capillaries were conditioned by flushing them
first with NaOH solution (0.1 mol L-1) for 10 minutes and subsequently with run
buffer for 10 minutes. A rinsing step with run buffer for 1 minute was used for
cleaning the capillary between runs. A voltage of +30 kV was used for separation.
UV detection was accomplished with a Spectra 100, Thermo Separation Products,
San José, US, at 240 nm with a sampling rate of 4 Hz. For data acquisition the
software Weinanalytik HPLC Mono from Planum GmbH, Kirchhain, D was used.
Origin 6.0 Professional software served for data analysis.

2.3 MEKC-conditions
Separation buffer: 7.5 mmol/L sodium tetraborate, 60 mmol/L SDS, 4 mol/L urea,
0.5 mmol/L CaCl2, 20% (v/v) acetonitrile, pH 9.2.
Injection solution: For solubility enhancement we used Triton X 100 in the
injection solution. For all measurements 750 µL of the aqueous Triton X 100
solution (25 g L-1) were mixed with 250 µL of the methanolic sample solution to
yield a total volume of 1000 µL injection solution.
Sample preparation: The oil samples were diluted by 1:100, 1:1000 and 1:10 000
to yield the described injection solutions. Steam distillation of the herbal samples
of cinnamomum camphora was done by mixing 20 g dried and ground sample
with 200 mL water. Steam produced by a steam generator was lead through the
sample until the distillate remained clear. The collected distillate was extracted
with diethyl ether followed by the removal of the solvent.

3. Results and discussion

The MEKC method development of this study will be published
elsewhere. We here present the results concerning the forensic background.

3.1 Separation
The ten analytes selected and the internal standard were baseline sepa-
rated at a high voltage of 30 kV within 12 minutes. In general, compounds with
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 202

more polar keto- and phenolic hydroxy-substituents show lower distribution

coefficients concerning the micellar pseudo-stationary phase and the aqueous
phase and are less retarded. The detection limits are in the range of 0.2-6.9 mg L-1.
Calibration curves were linear with correlation coefficients higher than 0.99. Plate
numbers were in the range of 50 000 to 260 000, with lower values for the later
eluted compounds.

3.2 Standardisation of electropherograms

Alterations in the electroosmotic flow velocity strongly account for
variations in the migration time of an analyte in different MEKC runs. To enhance
run-to-run repeatability, which is highly desirable for batch-to-batch comparison
of seized samples, the time-based x-axis can be transformed into a scale of the
effective electrophoretic mobility. Being a property of the analytes, this parameter
is independent of the EOF velocity. The calculation is accomplished by Equation
1, here using an internal standard whose effective electrophoretic mobility was
calculated from a separate run employing a marker for the EOF velocity. All other
variables can directly be obtained from the set-up or from the actual
electropherogram [8].
Ld Lt (tm − tint )
µ eff = µint + Eq. 1
Vtmtint
µ eff: effective electrophoretic mobility
µ int: µ eff of internal standard
Ld: capillary length to detector
Lt: capillary total length
tm : migration time of analyte
tint: migration time of internal standard
V: applied voltage

Figure 3 shows ten consecutive runs with a sample containing the

selected standard compounds, without an optimisation of rinsing steps between
runs. Repeatability data are given in Table 1 for some analytes. Clearly, the
repeatability of the peak maximum parameter (referring to the x-axis) is greatly
improved by a factor of up to ten on account of the conversion of time-based data
into mobility-based data. However, there is no improvement concerning the
quantitative parameter peak area, although other researchers have reported a slight
improvement via x-axis transformation [8].
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 203

0,20

0,15
absorbance in a.u.

0,10

0,05

0,00

4 6 8 10 12 14 16 18 20

time in min

0,22
0,20
0,18
0,16
absorbance in a.u.

0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
-0,02
-0,00005 0,00000 0,00005 0,00010 0,00015 0,00020

effective electrophoretic mobility in cm²/Vs

Figure 3: Ten consecutive runs of a standard mixture with A: time scale and
B: scale of effective electrophoretic mobility
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 204

Tab. 1: Relative standard deviation in % for selected analytes, derived from ten consecutive runs

% RSD % RSD % RSD % RSD

time area (time) µ eff area (µ eff)
piperonal 1.25 3.09 0.69 3.49
eugenol 1.68 2.01 0.18 2.31
thymol 2.03 8.14 0.23 10.55
safrole 1.93 2.50 0.21 3.47
myristicin 2.75 3.33 0.33 6.08
asarone 2.12 4.09 0.47 5.66
anethole 2.06 5.92 0.55 7.91

It can be concluded that transformation of data into the mobility scale is a

very effective means to improve peak identification and reproducibility of the
method, which is very important regarding screening aspects [6] and suitability for
forensic batch-to-batch comparisons.

3.3 Batch-to-batch comparisons

Figure 4 shows the electropherograms of a seized “sassafras oil” and of a
commercial sample of the essential oil of sassafras albidum. The comparison of
the two samples shows remarkable analogies. The same analytes were detected in
both samples, namely eugenol, methyleugenol, myristicin and isoeugenol, except
that a small amount of anethole is only present in the commercial oil. Even very
small signals of unknown substances were recorded in both electropherograms.
Presumably, the seized oil was also made from sassafras albidum. Most of the
samples from seizures that we investigated show a peak pattern similar to the one
found for the commercial oil of sassafras albidum.
A few samples showed marked differences as shown in Figure 5 for two
seized essential oil samples, one of which is ascribed to sassafras albidum (dashed
line). In the second sample, only safrole and isosafrole were identified. The peak
patterns of the two electropherograms concerning unidentified ingredients differ
strongly.
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 205

IS1

2
0,2
3
absorption in a.u.

0,1
1 4

0,0
8 10 12 14
time in min

Fig. 4: Comparison of a seized oil (solid line) and a commercial oil of sassafras albidum (dashed
line) at a dilution of 1:100, 1: eugenol, 2: methyleugenol, 3: safrole, 4: isosafrole

IS

2
0,15
absorption in a.u.

0,10 4

1
0,05

0,00

6 8 10 12 14 16 18

time in min

Fig. 5: Comparison of two seized “sassafras oils” at a dilution of 1:100,

1: eugenol, 2: methyleugenol, 3: safrole, 4: isosafrole
XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 206

3.4 Principal component analysis

A principal component analysis (PCA) was carried out with the
quantitative results from the MEKC measurements. We excluded some
compounds found in a few samples from the analysis: Isosafrole has been found in
the analysis of the constituents of sassafras albidum by Zubillaga and Maerker
before, but is presumed to be an isomerisation product and not an ingredient of the
essential oil itself [10]; anethole and asarone are photolabile compounds and
might already be degraded in stored samples. Eugenol, methyleugenol and
myristicin were found to be interesting as markers for the herbal origin of the
studied essential oils and were therefore included into the PCA.

samples
2,0 ocotea pretiosa
eu

cinnamomum camphora
ge
no

1,5 sassafras albidum

ol
l

e ugen
l
thy in
1,0 meyristic
m
0,5
?
PC2

0,0

-0,5

-1,0

-1,5
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0

PC1
Fig. 6: Biplot of the PCA results with two principal components

Two principal components were extracted from the covariance matrix.

The results are shown in the biplot in Figure 6. The projection of the loadings of
the original variables (multiplied by the eigenvectors) shows, that the contents of
myristicin and methyleugenol are closely related to each other, whereas the con-
centration of eugenol is nearly independent (90°) from the contents of myristicin
and methyleugenol. The reference oil sample of ocotea pretiosa, marked with a
filled circle (●), is accompanied by two of the seized samples with (identical)
negative specific principal component values very similar to it (surrounded by the
dotted circle). Samples inside the ellipse (with the reference sample (◆) in the
center) are assigned to sassafras albidum. All of these samples exhibit very
similar electropherograms but slightly differ in the relative amount of minor
constituents, mainly methyleugenol and myristicin, as expected from herbal
 XIV. GTFCh-Symposium, 14. – 16. April 2005 in Mosbach 207

products. We are unsure about the samples included in the dashed circle. One is a
sample with a pattern in the electropherogram similar to the samples we assigned
to sassafras albidum, but with higher eugenol content. The other (▲) is a sample
of a steam distillation from a whole young tree of cinnamomum camphora which
might have noticeable differences in the analyte pattern compared to a commercial
product. More samples, especially different reference samples, need to be analysed
to broaden this PCA approach. But these preliminary results show, that an
assignment of samples to their herbal origin is principally possible by this method.

4. Conclusion
With the MEKC method presented here it is possible to determine
aromatic compounds in essential oils in aqueous media. Fast baseline separation
of the analytes is achieved with high separation efficiency. The standardisation of
electropherograms via the effective electrophoretic mobility provides a better peak
identification due to an enhanced repeatability in the x-scale, thus enabling batch-
to-batch comparison of seized safrole-containing essential oils. The classification
of the samples via PCA of quantitative MEKC data allows an assignment of the
herbal origin of the oils which can facilitate the monitoring of trade.

5. References
[1] Bundeslagebild Rauschgift 2004, Bundeskriminalamt
[2] Carlson C, Thompson RD (1997) Liquid chromatographic determination of safrole in
sassafras-derived herbal products. J AOAC Int 80(5): 1023-1028
[3] HagerROM 2004-2005, Springer Verlag, Heidelberg
[4] Heikes DL (1994) SFE with GC and MS determination of safrole and related allybenzenes
in sassafras teas. J Chromatogr Sci 32: 253-258
[5] Hickey MJ (1948) Investigation of the chemical constituents of brazilian sassafras oil. J
Org Chem 13: 443-446
[6] Hudson JC, Malcolm MJ, Golin M (1998) Advancements in forensic toxicology. P/ACE
Setter, The Newsletter for Capillary Electrophoresis 2: 1-5
[7] Kamdem PK, Gage DA (1995) Chemical composition of essential oil from the root bark
of sassafras albidum. Plant Med 61: 574-575
[8] Schmitt-Kopplin P et al. (2001) Quantitative and qualitative precision improvements by
effective mobility-scale data transformation in capillary electrophopresis analysis.
Electrophoresis 22: 77-87
[9] Wartberg L, Petersen KU, Andresen B, Thomasius R (2005) Neuropsychologische Defizi-
te bei Ecstasykonsumenten. Z Neuropsychologie 16(1): 47-55
[10] Zubillaga MP, Maerker G (1990) Determination of safrole and isosafrole in ham by HPLC
and UV detection. J Food Sci 55(4): 1194-1195

C. Huhn, U. Pyell M. Pütz, R. Dahlenburg

Fachbereich Chemie/Analytische Chemie Bundeskriminalamt
Philipps-Universität Marburg Fachbereich KT 34 (Toxikologie)
Hans-Meerwein-Straße Thaerstraße 11
D-35032 Marburg D-65193 Wiesbaden
E-Mail: carolin.huhn@staff.uni-marburg.de michael.puetz@bka.bund.de

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