Determination of origin of ephedrine used as precursor for illicit methamphetamine by carbon and nitrogen stable isotope ratio analysis

Article abstract of DOI:10.1021/ac035417c

The sale of ephedrine, one of the precursors of methamphetamine, is strictly controlled and monitored in various countries to prevent the production of illicit methamphetamine. There are three kinds of production scheme for ephedrine manufacture, and it is very useful for precursor control to investigate the origin of ephedrine used for the synthesis of illicit methamphetamine. By means of stable isotope ratio mass spectrometry (IR-MS), we investigated the origin of ephedrine based on the δ¹³C and δ¹⁵N values. The various origins of ephedrine (biosynthetic, semisynthetic, or synthetic) could be discriminated clearly by using these values. The δ¹⁵N values of synthetic ephedrine were more negative than those of ephedrine from other sources. By the repeated distillation of methylamine in our laboratory, we confirmed that this could be due to isotope separation during distillation for the purification of methylamine used for ephedrine synthesis. The values for ephedrine used as the precursor were well-correlated with those for methamphetamine synthesized from it. This drug characterization analysis should be useful to illuminate the origin of the precursors used for clandestine methamphetamine and to trace the diversion of medicinal ephedrine for illicit manufacture of methamphetamine.

Full text of DOI:10.1021/ac035417c

Anal. Chem. 2004, 76, 4233-4236
Determination of Origin of Ephedrine Used as
Precursor for Illicit Methamphetamine by Carbon
and Nitrogen Stable Isotope Ratio Analysis
Naoki Kurashima,† Yukiko Makino,‡ Setsuko Sekita,§ Yasuteru Urano, and Tetsuo Nagano*,
Central Customs Laboratory, Ministry of Finance, 6-3-5, Kashiwanoha, Kashiwa-shi, Chiba 277-0882, Japan, Narcotic
Control Department, Kanto-Shin’etsu Regional Bureau of Health and Welfare, Ministry of Health, Labor and Welfare,
2-4-14, Nakameguro, Meguro-ku, Tokyo 153-0061, Japan, Tsukuba Medical Plant Research Station, National Institute of
Health Sciences, Ministry of Health, Labor and Welfare, 1, Hachimandai, Tsukuba-shi, Ibaraki 305-0843, Japan, and
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ratio analyses have also been used for examination of legitimate
items of commerce.5,6 The natural isotopic variation of organic
products in plants is fixed during biochemical synthesis, and the
observed isotopic variations reflect differences in metabolic
processes or environmental conditions during growth. For ex-
ample, the carbon stable isotope ratio (δ13C) values of plants grown
under low-humidity or water-stressed conditions are more positive
than those of plants grown under conditions of high humidity or
high soil water.7 Variations in plant nitrogen stable isotope ratio
(δ15N) values of 10‰ or more occur and represent a record of
soil and microbial N2 status.8 Several reports have shown that δ13C
and δ15N values for cocaine and heroin are valuable to trace their
geographical origin.9-11 Ephedrine, one of the precursors of
methamphetamine, is produced in substantial amounts for medical
purposes. Commercial ephedrine is produced by (a) extraction
from ephedra plant, namely biosynthesis; (b) fully chemical
synthesis; and (c) semisynthesis, as shown in Figure 1. Originally,
ephedrine was commonly produced from ephedra plant, but
recently, large amounts have been produced by synthesis or
semisynthesis. On the other hand, illicit ephedrine is still generally
thought to be produced from ephedra plants.
The sale of ephedrine, one of the precursors of metham-
phetamine, is strictly controlled and monitored in various
countries to prevent the production of illicit methamphet-
amine. There are three kinds of production scheme for
ephedrine manufacture, and it is very useful for precursor
control to investigate the origin of ephedrine used for the
synthesis of illicit methamphetamine. By means of stable
isotope ratio mass spectrometry (IR-MS), we investigated
the origin of ephedrine based on the δ13C and δ15N values.
The various origins of ephedrine (biosynthetic, semisyn-
thetic, or synthetic) could be discriminated clearly by
using these values. The δ15N values of synthetic ephedrine
were more negative than those of ephedrine from other
sources. By the repeated distillation of methylamine in
our laboratory, we confirmed that this could be due to
isotope separation during distillation for the purification
of methylamine used for ephedrine synthesis. The values
for ephedrine used as the precursor were well-correlated
with those for methamphetamine synthesized from it. This
drug characterization analysis should be useful to il-
luminate the origin of the precursors used for clandestine
methamphetamine and to trace the diversion of medicinal
ephedrine for illicit manufacture of methamphetamine.
The abuse of amphetamine-type stimulants (ATS) is an increas-
ing problem. In Asia, including Japan, the abuse of methamphet-
amine itself is the most serious problem. Trade in precursors of
methamphetamine is strictly controlled and monitored in various
countries, since methamphetamine can easily be clandestinely
manufactured from its precursors. To prevent the production of
illicit methamphetamine, it is important not only to control and
monitor the trade in ephedrine, but also to evaluate the origin of
Isotope ratio analyses at natural abundance levels have been
used to establish the environmental source or the geographic
origin of various biological and nonbiological materials.1-4 Isotope
* To whom correspondence should be addressed. Phone: +81-3-5841-4850.
Fax: +81-3-5841-4855. E-mail: tlong@mol.f.u-tokyo.ac.jp.
† Central Customs Laboratory, Ministry of Finance.
‡ Narcotic Control Department, Kanto-Shin’etsu Regional Bureau of Health
and Welfare, Ministry of Health, Labor and Welfare.
§ Tsukuba Medical Plant Research Station, National Institute of Health
Sciences, Ministry of Health, Labor and Welfare.
(5) Krueger, D. A.; Krueger, H. W. J. Agric. Food Chem. 1 9 8 3 , 31, 1265-
1268.
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2662-2666.
Graduate School of Pharmaceutical Sciences, The University of Tokyo.
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1325-1331.
(7) Farquhar, G. D.; Ehleringer, J. R.; Hubick, K. T. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 1 9 8 9 , 40, 503-537.
(8) Wada, E.; Yoneyama, T.; Minagawa, M.; Ando, T.; Fry, B. D. Stable Isotopes
in the Biosphere; Kyoto University Press: Kyoto, 1995; p 192.
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Lamotte, A. Forensic Sci. Int. 1 9 9 7 , 85, 113-125.
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Cult. Stud., Kyushu Univ. 2 0 0 0 , 6, 37-45.
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Lanord, C. F.; Giard, D.; Narvaez, D.; Rondeau, B. Science 2 0 0 0 , 287, 631-
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10.1021/ac035417c CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/29/2004
Analytical Chemistry, Vol. 76, No. 14, July 15, 2004 4233

Figure 1. Production schemes of ephedrine: (a) bromination of propiophenone followed by amination, (b) fermentation of sugar followed by
amination, and (c) extraction from ephedra plant.
ephedrine used as a precursor of seized methamphetamine by
scientific analyses. It is possible to identify the precursor or the
method of synthesis of methamphetamine by analyzing impurities
in methamphetamine.12-16 Analyses of impurities by HPLC have
enabled identification of the kind of ephedrine used for the
synthesis of seized methamphetamine,17 but these methods are
not sufficient to determine the origin of the ephedrine.
Chemically, no differences are observed among ephedrine
samples produced by different methods. Nevertheless, the δ13C
Figure 2. Synthetic pathways of methamphetamine from ephedrine
in this study.
and δ15N values differ. Furthermore, the isotope ratio of biosyn-
thetic ephedrine will be different depending upon the environment-
al conditions during growth of the ephedra plants. In this paper,
we report the discrimination of the source of ephedrine on the
basis of the δ13C and δ15N values using IR-MS, and we describe
the relationship of the δ13C and δ15N values of the precursor
ephedrine and those of methamphetamine synthesized from it.
Instruments and IR-Mass Analysis. A stable isotope ratio
mass spectrometer DeltaPlus (ThermoFinnigan, U.S.A.) equipped
with an elemental analyzer Flash EA1112 (ThermoFinnigan,
U.S.A.) was used for the measurements of carbon and nitrogen
isotope ratios of samples. Individual samples (250 µg) wrapped
in tin foil were flash-combusted on an elemental analyzer to afford
CO2, NOx, and H2O in an O2 atmosphere in a quartz reactor packed
with Cr2O3 on alumina and Co3O4/ Ag. The gases were passed
through a copper reactor to reduce NOx to N2. H2O was trapped
with Mg(ClO4)2. Then CO2 and N2 were separated on a GC column
(Porapaq QS), and subjected to IR-MS to obtain the 13C/ 12C and
15N/ 14N ratios. The stable isotope ratios are expressed relative to
the conventional standards, that is, Peedee Belemite for carbon
and atmospheric N2 for nitrogen. The δ value is defined according
to the following equation:
EXPERIMENTAL SECTION
Materials and Chemicals. Thirteen ephedrine hydrochloride
samples were used. Four of them, purchased from Fujiyakuhin
(Saitama, Japan), Maruishi Pharmaceutical (Osaka, Japan) and
Dainippon Pharmaceutical (Osaka, Japan), were medicines pro-
duced by bromination of propiophenone followed by amination18
or by extraction from ephedra plants. Three other samples were
produced by extraction from ephedra plants, and the rest were
produced by fermentation of sugar followed by amination, namely
semisynthesis.19 A sample of medical methamphetamine hydro-
chloride was purchased from Dainippon Pharmaceutical (Osaka,
Japan), and three methamphetamine hydrochloride samples were
synthesized from ephedrine by the Nagai method or the Emde
method, as shown in Figure 2, in our laboratory. Twenty-one illicit
methamphetamine samples seized in Japan were also used.
Methylamine hydrochloride was purchased from Sigma-Aldrich
(MO).
δ (‰) ) (Rsample/ Rstandard - 1) × 1000
where R ) 13C/ 12C or 15N/ 14N. Each sample was measured five
times. The precisions routinely obtained were 0.1‰ or less for
δ13C and 0.2‰ or less for δ15N.
Distillation of Methylamine. Methylamine hydrochloride (10
g) was dissolved in 200 mL of 1 mol/ L NaOH aq. The resulting
free base was distilled by heating. The distillate was conducted
into 1 mol/ L HCl aq, where it was neutralized. The volume of
distilled methylamine was monitored in terms of the pH of the
HCl solution. The early distillate was collected, concentrated, and
recrystallized from acetone. These procedures were repeated three
times.
(12) Lambrechts, M.; Rasmussen, K. E. Bull. Narc. 1 9 8 4 , 36, 47-57.
(13) Cantrell, T. S.; John, B.; Johnson, L.; Allen, A. C. Forensic Sci. Int. 1 9 8 8 ,
39, 39-53.
(14) Skinner, H. F. Forensic Sci. Int. 1 9 9 0 , 48, 123-134.
(15) Tanaka, K.; Ohmori, T.; Inoue, T. Forensic Sci. Int. 1 9 9 2 , 56, 157-165.
(16) Windahl, K. L.; McTigue, M. J.; Pearson, J. R.; Pratt, S. J.; Rowe, J. E.;
Sear, E. M. Forensic Sci. Int. 1 9 9 5 , 76, 97-114.
(17) Makino,Y.; Urano,Y.; Nagano, T. J.Chromatogr., A 2 0 0 2 , 947, 151-154.
(18) E. Merck Chemishe Fabrik. German Patents 469782, 1928, and 472466,
1929.
(19) Hildebrandt, G.; Klavehn, W. U.S. Patent 1956950, 1934. Bockmuhl, N.; Stein,
L.; Ehrhart, G. U.S. Patent 1962476, 1934.
RESULTS AND DISCUSSION
δ
1 3 C and δ1 5 N Values for Ephedrine. We investigated the
origin of ephedrine on the basis of the δ13C and δ15N values
4234 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

Figure 4. The variation of δ15N values of methylamine with
increasing number of distillations.
Figure 3. Carbon and nitrogen isotope ratios of ephedrine
samples: natural (0), synthesized (2), and semisynthetic (b).
Table 1. The δ13C and δ15N Values of Ephedrines Used
as Precursors of Methamphetamine Synthesis and
Methamphetamine Synthesized from the Ephedrine
obtained by using IR-MS. δ13C and δ15N values for ephedrine
samples are given in Figure 3. The δ13C values for synthetic
medical ephedrine derived from propiophenone were -29.2 to
-28.0‰, and those of biosynthetic ephedrine extracted from
ephedra plants, known as C3-photosynthetic plants, were -31.1
to -26.0‰. Typical values for natural organic compounds from
C3-photosynthetic plants are -28.1 ( 2.5,20 and this range is
essentially the same as that observed for biosynthetic ephedrine.
It is thought that the variations of δ13C values reflect the
differences in humidity conditions of the areas where the source
plants were grown. The δ13C values for ephedrine produced by
fermentation of sugar followed by amination, namely semisynthe-
sis, were -23.1 to -22.0‰. The starting material for semisynthetic
ephedrine is sugar extracted from C4-photosynthetic plants such
as sugarcane, in which typical δ13C values are -13.5(1.5.20
Acetaldehyde generated by sugar fermentation is condensed with
benzaldehyde to form phenylacetyl carbinol. Phenylacetyl carbinol
reacts with methylamine over a Ni catalyst to produce ephedrine.
The δ13C values of acetaldehyde from sugar are more positive
than those of C3-photosynthetic plants or products derived from
petroleum.21 It is supposed that acetaldehyde contributes 2 of the
10 carbon atoms in ephedrine. Thus, the value of δ13C can be
used to differentiate semisynthetic ephedrine from biosynthetic
or synthetic ephedrine (Figure 3), but it does not discriminate
biosynthetic ephedrine from synthetic ephedrine.
On the other hand, δ15N showed remarkable differences: δ15N
values for semisynthetic or biosynthetic ephedrine were 3.8 to
10.6‰, and those for synthetic ephedrine were -10.5 to -10.0‰.
This is because the origins of the ephedrine nitrogen are different.
The source of nitrogen in both synthetic and semisynthetic
ephedrine is methylamine, as indicated in Figure 1, but the source
of nitrogen in the biosynthetic material is nitrate or ammonia in
soil. In the case of synthetic ephedrine, it is considered that isotope
separation occurs during distillation for purification of methy-
lamine used for ephedrine synthesis. Figure 4 shows the variation
of the δ15N values of methylamine during successive distillations
in our laboratory. The δ15N value changed from -5.4 to -7.6‰
after the first distillation, from -7.6 to -9.4‰ after the second
distillation, and from -9.4 to -11.9‰ after the third distillation.
This supports the above hypothesis.
synthetic
pathway
δ13C
(‰)
δ15N
(‰)
compound
methamphetamine
ephedrine (synthetic)
methamphetamine
ephedrine (semisynthetic)
methamphetamine
ephedrine (semisynthetic)
methamphetamine
ephedrine (natural)
methamphetamine
ephedrine (natural)
methamphetamine
ephedrine (natural)
methamphetamine
ephedrine (natural)
Nagai
Nagai
Nagai
Emde
Nagai
Nagai
Nagai
-29.2
-29.2
-23.0
-22.9
-23.1
-23.1
-26.1
-26.0
-29.8
-29.5
-31.4
-31.1
-29.5
-29.2
-11.1
-10.5
9.5
9.5
5.8
6.2
4.4
3.8
3.7
3.9
10.9
10.6
3.9
4.2
Thus, as shown in Figure 3, ephedrine samples prepared in
the three ways could be clearly discriminated by using both δ13C
and δ15N values.
Relationship of δ1 3 C and δ1 5 N Values for Ephedrine and
Methamphetamine. Table 1 summarizes the δ13C and δ15N
values for ephedrine precursors and for methamphetamine synthe-
sized from them. The δ13C and δ15N values for ephedrine were
well-correlated with those for methamphetamine; therefore,
it is possible to know what kind of ephedrine was used as a
precursor by measuring the values of δ13C and δ15N of meth-
amphetamine.
δ
1 3 C and δ1 5 N Values for Methamphetamine. Figure 5
shows δ13C and δ15N values of seized methamphetamine samples
together with those of medical methamphetamine and those of
methamphetamine synthesized from ephedrine in our labora-
tory, as indicated in Table 1. Values of δ13C and δ15N of seized
methamphetamine were in the ranges of -33.1 to -25.5‰
and +3.3 to +7.4‰, respectively. Although δ13C and δ15N each
showed a distribution, the values of illicit samples seized on a
given occasion were all similar. These results indicate that
analysis of δ13C and δ15N can provide a means to distinguish
apparently similar samples seized on different occasions. The δ13C
and δ15N values of a medical methamphetamine sample were
-25.5 and +3.4‰. It can be inferred that the medical meth-
amphetamine and all seized methamphetamine samples examined
in this study were synthesized from ephedrine extracted from
(20) O’Leary, M. H. Phytochemistry. 1 9 8 1 , 20, 553-567.
(21) Stahl, W. J. Chem. Geol. 1 9 7 7 , 20, 121-149.
Analytical Chemistry, Vol. 76, No. 14, July 15, 2004 4235

CONCLUSIONS
It has been shown that the origins of ephedrine can be
discriminated by δ13C and δ15N analysis and that the values of
the precursor ephedrine are reflected in those of methamphet-
amine obtained from it. The more negative δ15N values of synthetic
ephedrine appear to be due to isotope separation during distillation
for purification of methylamine used in ephedrine synthesis. Thus,
δ13C and δ15N analysis of methamphetamine can tell us the origin
of the precursor ephedrine. In addition, this analysis makes it
possible to distinguish methamphetamine samples seized on
different occasions, even if they are difficult to discriminate on
the basis of their impurity profiles. It is impossible to get such
information by other techniques; therefore, this technique will be
very useful for profiling of methamphetamine and monitoring the
sources of ephedrine used for clandestine manufacture of meth-
amphetamine. In the future, it may be possible to establish a
relationship between the sites where ephedra plants are grown
and the δ13C and δ15N values of ephedrine.
Figure 5. Carbon and nitrogen isotope ratios of methamphetamine
samples: seized (O), synthesized from natural ephedrine (0),
synthesized from synthetic ephedrine (2), synthesized from semi-
synthetic ephedrine (b), and commercial methamphetamine (9).
Dotted circles indicate samples that were seized on the same
occasion.
ACKNOWLEDGMENT
ephedra plants, not from synthetic ephedrine. Further, as the
isotope ratios of plants differ depending on their growing
location and conditions, the variations of δ13C and δ15N among
the seized samples must reflect differences in the source of
the ephedra plants. Thus, by using this IR-MS technique, we
can further distinguish for the first time among samples that
do not show marked differences in their impurity profiles.
Therefore, it is possible to obtain a great deal of information
about illicit methamphetamine by measuring the values of δ13C
and δ15N.
We are grateful to Dr. Barbara Remberg and Dr. Howard Stead
(Laboratory and Scientific Section, United Nations Office on Drugs
and Crime, UNODC) for the supply of semisynthetic ephedrine
samples. The present work was supported by a Health Sciences
Research Grant from the Ministry of Health, Labor and Welfare,
Japan.
Received for review December 2, 2003. Accepted April 26,
2004.
AC035417C
4236 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004