δ13C, δ15N and δ2H isotope ratio mass spectrometry of ephedrine and pseudoephedrine: application to methylamphetamine profiling

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2009; 23: 2003–2010

) DOI: 10.1002/rcm.4109
Published online in Wiley InterScience (www.interscience.wiley.com
d13C, d15N and d2H isotope ratio mass spectrometry of

ephedrine and pseudoephedrine: application to

methylamphetamine profiling

Michael Collins1*, Adam T. Cawley1, Aaron C. Heagney1, Luke Kissane1,

James Robertson2 and Helen Salouros1,3

1National Measurement Institute, Australian Forensic Drug Laboratory, 1 Suakin Street, Pymble, Sydney, Australia2Australian Federal Police, Weston, ACT, Australia3University of Sydney, School of Chemistry, Sydney, Australia

Received 16 February 2009; Revised 5 May 2009; Accepted 5 May 2009

*Correspo1 SuakinE-mail: m

Conventional chemical profiling of methylamphetamine has been used for many years to determine

the synthetic route employed and where possible to identify the precursor chemicals used. In this

study stable isotope ratio analysis was investigated as a means of determining the origin of the

methylamphetamine precursors, ephedrine and pseudoephedrine. Ephedrine and pseudoephedrine

may be prepared industrially by several routes. Results are presented for the stable isotope ratios of

carbon (d13C), nitrogen (d15N) and hydrogen (d2H) measured in methylamphetamine samples

synthesized from ephedrine and pseudoephedrine of known provenance. It is clear from the results

that measurement of the d13C, d15N and d2H stable isotope ratios by elemental analyzer/thermal

conversion isotope ratio mass spectrometry (EA/TC-IRMS) in high-purity methylamphetamine

samples will allow determination of the synthetic source of the ephedrine or pseudoephedrine

precursor as being either of a natural, semi-synthetic, or fully synthetic origin. Copyright # 2009

Commonwealth of Australia. Published by John Wiley & Sons, Ltd.

CH3

H

NHCH3

CH3

H

NHCH3

Methylamphetamine (Fig. 1) is a widely abused drug within

Australia.1 It appears to have beenfirst synthesized in 1893 by

Nagai2 and since then many synthetic routes, employing a

variety of precursor molecules, have been described.3,4 For

many years the domestic manufacture of methylampheta-

mine in small clandestine laboratories has dominated supply

of this drug inAustralia.1,5However, since 1995 thenumberof

border level detections of methylamphetamine smuggling

has increased. Between 1995 and 2008 the total weight of

methylamphetamine seized at the Australian border

increased, peaking in 2002 at 400 kg.5 Law enforcement

agencies such as the Australian Federal Police (AFP) have

successfully intervened to disrupt drug trafficking operations

across the Australian border. The National Measurement

Institute (NMI) collaborates with the AFP in the provision of

the Australian Illicit Drug Intelligence Program (AIDIP). Part

of the role of the AIDIP is to undertake chemical profiling of

methylamphetaminewith a goal of determining the synthetic

route employed in the production of seized material and the

identity of precursor chemicals and reagents. Such infor-

mation can assist national and international agencies in

monitoring the diversion of legitimate industrial chemicals

for illegitimate purposes. Most clandestine production

employs (1R,2S)-(�)-ephedrine, (1S,2S)-(þ)-pseudoephe-

drine or phenyl-2-propanone (P2P) as the immediate

precursors and all procedures employ a reduction step of

ndence to: M. Collins, National Measurement Institute,Street, Pymble, Sydney, [email protected]

Copyright # 2

some kind.6–15 These common synthetic routes involving

ephedrine and pseudoephedrine are shown in Fig. 2. Ephe-

drine and pseudoephedrine are produced in large quantities

formedical use by threemainprocesses shown inFig. 3. These

involve (1) extraction from the ephedra plant, (2) a semi-

synthetic procedure whereby pyruvic acid is fermented with

benzaldehyde in the presence of pyruvate decarboxylase to

give 1-hydroxy-phenyl-2-propanone which on reductive

amination gives ephedrine,16 and (3) a fully synthetic product

produced from 1-phenyl-1-propanone.

The major chemical profiling techniques employed in

forensic drug laboratories today focus on organic impurity

profiling,17,18 chiral profiling,19–23 elemental analysis,24 and

determination of adulterants and diluents.23,25 These techniques

are used to determine the synthetic pathway and precursors

used and to assist law enforcement agencies in establishing links

between seizures. The measurement of stable isotope ratios of

carbon and other elements, using isotope ratio mass spectrom-

etry (IRMS), is a more recent profiling technique and it has been

successfully employed to complement conventional chemical

S-(+)-Methylamphetamine R-(-)-Methylamphetamine

Figure 1. Structures of S-(þ)-methylamphetamine (left) and

R-(�)-methylamphetamine (right).

009 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.

CH3

NHCH3

OH

CH3

NHCH3

Cl

CH3

NHCH3

SOCl 2

Pd/BaSO4 /H

2

('Emde' Synthesis)

Li or Na, Liquid NH3

(Birch reduction)

HI, Red Phosphorus ('Nagai' route)

I2, H3O2P ('Hypo' route)

I2, Red Phosphorus, Water ('Moscow' route)

Figure 2. Potential synthetic routes for the manufacture of

methylamphetamine from ephedrine or pseudoephedrine.

2004 M. Collins et al.

profiling in determining the geographical origin of cultivated

drugs suchas cocaine26,27 andheroin,27–31 andsynthetic route for

drugs such asmethylamphetamine32–34 and ‘ecstasy’.35–43 IRMS

alsohas thepotential tobeauseful technique inestablishing links

between drug seizures. Kurashima et al.32 and Makino et al.33

have described the determination of carbon and nitrogen stable

isotope ratios for ephedrine and pseudoephedrine and the

methylamphetamine synthesized in their laboratory from

these precursors. They demonstrated that, at least in the

case of ephedrine, the different synthetic origins could be

discriminated on the basis of these isotope ratios and that

different batches of methylamphetamine could also be dis-

criminated even when the purity was so high that conventional

organic impurity profiles were unhelpful.

In this paper we describe work done at the NMI extending

theworkofMakino et al.33 to include the stable isotope ratios of

hydrogen,aswellas thoseof carbonandnitrogen, inephedrine

and pseudoephedrine from different origins. Results will be

presented for the stable isotope ratios measured in methy-

lamphetamine samples synthesized at the NMI from these

precursors via a number of common synthetic routes. The

precursor chemicals include ephedrine hydrochloride

obtained from natural ephedra extracts and ephedrine hydro-

chloride and pseudoephedrine hydrochloride certified by the

supplier as being of semi-synthetic origin, i.e. produced by the

fermentation process employing benzaldehyde. Benzal-

dehyde samples were purchased from a number of suppliers

for d2H measurements and included a sample certified by

Sigma-Aldrich as having been extracted from Cinnamomum

cassia blume and being a natural or food grade material.

EXPERIMENTAL

Reagents and chemicalsAnalytical grade chloroform, dichloromethane and sodium

acetate (anhydrous) were obtained from Biolab (Mulgrave,

Copyright # 2009 Commonwealth of Australia. Published by John Wiley & S

VIC, Australia). Formic acid, hydrochloric acid (36%),

hypophosphorous acid (50%), glacial acetic acid, sodium

hydroxide pellets and iodine were obtained from UNIVAR

Ajax Finechem (Seven Hills, NSW, Australia). Benzene and

thionyl chloride were obtained from Riedel-deHaen (Seelze,

Germany). Hydriodic acid was purchased from BDH

Chemicals (Poole, UK). Benzaldehyde (puriss standard for

GC, Product #09143, �99.5%), benzaldehyde (puriss stan-

dard, Product #12010, �99%), benzaldehyde (Product

#418099, purified by re-distillation, �99.5%), benzaldehyde

(Product #W212717-100G-K, food grade product of China,

extracted from Cinnamomum cassia blume, classed as natural

or ‘Kosher’ product [d2H¼�129%, d13C¼�28.1%], �98%)

and benzaldehyde (Product #B1334-2G, Reagent Plus,�99%)

were all obtained from Sigma-Aldrich (Castle Hill, NSW,

Australia). Benzaldehyde for synthesis (Product

#8.01756.0100, �99%), isopropanol and diethyl ether were

obtained from Merck (Kilsyth, VIC, Australia). An ephedra

extract, ‘Power Plus Super Ephedra 850’ was obtained from

Prices Power International Inc. (Newport, VA, USA). (1S,2S)-

(þ)-Pseudoephedrine hydrochloride (Product No. E2750,

Lot. No. 125K1410, certified semi-synthetic origin, �99%),

(1R,2S)-(�)-ephedrine hydrochloride (Product No. 862231,

Batch 10620PH, certified semi-synthetic origin, �99%) and

(1S,2S)-(þ)-pseudoephedrine hydrochloride (Product No.

E2750, Batch 59H1089, �99%) were obtained from Sigma-

Aldrich. (1R,2S)-(�)-Ephedrine hydrochloride (Product No.

45285, Batch 350635/1, �99.7%) was obtained from Fluka

(Steinheim, Germany). ‘Sudafed’ tablets were obtained from

Wellcome Australia Ltd. Tris(hydroxymethyl)amino-

methane (99.9%), nonadecane (99%), ammonium bicarbon-

ate, palladium 5wt.% on barium sulphate reduced, platinum

(IV) oxide, phenylbenzylamine and red phosphorus were

obtained from Sigma-Aldrich. All reagents were used

without further purification. A certified reference material

of methylamphetamine hydrochloride was obtained from

the reference collection of the NMI. The internal standard

solution for gas chromatography/flame ionization detection

(GC/FID) was prepared by dissolving the appropriate

amount of phenylbenzylamine in chloroform to give a final

concentration of approximately 800mg/mL.

Synthetic chemistry

Synthesis of chloroephedrine and chloropseudoephedrinePseudoephedrine hydrochloride (4 g) was slowly added to a

solution of chloroform (6mL) and thionyl chloride (4mL) at

ice-bath temperature and the mixture was stirred for several

hours. Diethyl ether was added causing precipitation of a

white solid which was filtered and washed with diethyl

ether/chloroform (50:50). The solid was air-dried to give 4 g

(91% relative yield) of chloroephedrine hydrochloride.

Chloropseudoephedrine was prepared using the same

procedure and using ephedrine hydrochloride (4 g).

Synthesis of methylamphetamine via the ‘Emde’ routeSodium acetate (0.8 g) was dissolved in water (11mL) in a

Parr hydrogenation vessel and the mixture made neutral

with acetic acid. Palladium on barium sulfate (1 g) and

chloroephedrine hydrochloride (2 g), obtained as above were

ons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2003–2010

DOI: 10.1002/rcm

CH3

O

OH

CH3

OH

NHCH3

CH3

NHCH3

CH3

Br

CH3

O O O

Ephedra

&BenzaldehydeacidPyruvic

fermentation CH3NH2

PtO(i) 2/H2Al/Hg(ii)NaBH(iii) 4NaBH(iv) 3CN

either

Br2 CH3NH2

NaBH4

(A)

(B)

(C)

Ephedrine

Figure 3. (A) Preparation of ephedrine from the ephedra plant; (B) semi-synthetic synthesis of

ephedrine; and (C) fully synthetic route to ephedrine.

Methylamphetamine profiling by IRMS 2005

added and the flask attached to a Parr hydrogenation

apparatus. Air was removed by vacuum and the flask

flushed with hydrogen several times, charged to a pressure

of 30 psi with hydrogen and mechanically shaken until

uptake of hydrogen ceased. The catalyst was filtered and

washed with water and the combined reaction mixture and

aqueous washings were basified with dilute sodium

hydroxide solution and extracted with dichloromethane.

The dichloromethane was removed using a rotary evapor-

ator leaving an oil (1.4 g). The oil was dissolved in cold

isopropanol, acidified with concentrated hydrochloric acid

and diethyl ether was added, resulting in precipitation. The

solid was filtered, washed with a mixture of isopropanol and

diethyl ether and dried to give 1.5 g (88%) of white crystals,

identified as methylamphetamine by comparison of GC

retention time and mass spectrum with a certified reference

standard of methylamphetamine hydrochloride. The purity

of the methylamphetamine hydrochloride was determined

by GC/FID.

Synthesis of methylamphetamine via the ‘Nagai’ routePseudoephedrine hydrochloride (2 g), red phosphorus (0.7 g)

and hydriodic acid (4.8mL) were refluxed for 24 h and

allowed to cool. The reaction mixture was filtered, diluted

with water and basified with dilute sodium hydroxide

solution. The mixture was extracted with dichloromethane

which was removed by rotary evaporation leaving an oil.

The oil was dissolved in cold isopropanol, acidified with

concentrated hydrochloric acid and diethyl ether was added,

resulting in precipitation. The solid was filtered, washed

with a mixture of isopropanol and diethyl ether and dried to

give 1.4 g (77%) of white crystals, identified as methyl-

amphetamine by comparison of GC retention time and mass

spectrum with a certified reference standard of methyl-


amphetamine hydrochloride. The purity of the methyl-

amphetamine hydrochloride was determined by GC/FID.

Synthesis of methylamphetamine via the ‘Moscow’ routePseudoephedrine hydrochloride (2 g), red phosphorus

(0.6 g), iodine (4 g) and water were refluxed for 24 h and

allowed to cool. The reaction mixture was filtered, diluted

with water and then basified with dilute sodium hydroxide

solution. The mixture was extracted with dichloromethane

which was removed by rotary evaporation leaving an oil.

The oil was dissolved in cold isopropanol, acidified with


resulting in precipitation. The solid was filtered, washed

with a mixture of isopropanol and diethyl ether and dried to

give 1.4 g (77%) of white crystals, identified as methyl-

amphetamine by comparison of GC retention time and mass

spectrum with a certified reference standard of methylam-

phetamine hydrochloride. The purity of the methylamphet-

amine hydrochloride was determined by GC/FID.

Synthesis of methylamphetamine via the ‘Hypo’ routePseudoephedrine hydrochloride (1g), iodine (1g) and

hypophosphorous acid (1g)were refluxed for 4h and allowed

to cool. Water was added to the reaction mixture which was

then basified with dilute sodium hydroxide solution and the

whole extracted with dichloromethane. The dichloromethane

was removed by rotary evaporation leaving an oil (0.7 g). The

oil was dissolved in cold isopropanol, acidified with


resulting in precipitation. The solid was filtered, washed with

a mixture of isopropanol and diethyl ether and dried to give

0.8 g (88%) ofwhite crystals, identified asmethylamphetamine

by comparison of GC retention time and mass spectrum

with a certified reference standard of methylamphetamine


DOI: 10.1002/rcm


hydrochloride. The purity of the methylamphetamine hydro-

chloride was determined by GC/FID.

Ephedrine hydrochloride from ephedra extract samplesThe combined contents of 20 capsules of ‘Super Ephedra 850’

were dissolved in 150mL of 0.1M hydrochloric acid solution.

The solution was washed with toluene (2� 150mL) and the

aqueous portion was made alkaline with saturated sodium

carbonate solution. The alkaline solution was extracted twice

with diethyl ether and the combined extracts were dried over

anhydrous sodium sulfate. The solvent was removed under

vacuum and the resulting yellow oil was dissolved in 12mL

isopropyl alcohol and acidified with concentrated hydro-

chloric acid. Diethyl ether (12mL) was added, causing

precipitation of an off-white solid. The solid was filtered,

washed with diethyl ether and allowed to air dry giving

0.45 g ofwhite crystals whichwere identified as ephedrine by

GC/MS. Identification was made by comparison of retention

time andmass spectrumwith a certified reference material of

ephedrine hydrochloride.

Sample identificationThe identity of the methylamphetamine was verified by

GC/MS using the method of Anderson et al.44,45 The

methylamphetamine purity was determined using GC/

FID, using phenylbenzylamine as internal standard and a

multiple point calibration standard using a certified

reference material of methylamphetamine hydrochloride.

Sample preparationThe sample (25mg) was accurately weighed into a 25mL

volumetric flask and in water to give a solution of

approximately 1mg/mL concentration. The solution

(3mL) was accurately transferred into a screw-capped vial

and the aqueous solution was basified with concentrated

ammonia solution. Chloroform (3mL) was added and

the mixture was shaken for 15min and centrifuged for

5min at 2500 rpm. The chloroform layer was passed through

sodium sulfate and 100mL was transferred to a GC vial.

Phenylbenzylamine (100mL) internal standard solution and

chloroform (800mL) were added.

Instrument parametersQuantification by GCwas performed using an Agilent 7890A

gas chromatograph fitted with a flame ionization detector

(FID) (Agilent, Forest Hill, Victoria, Australia). A HP-5

column (0.32mm i.d.� 30m, 0.5mm; Biolab, Mulgrave,

Victoria, Australia) was used. Helium (1.2mL/min) was

used as carrier gas in the constant flow mode (head pressure

12 psi). The injection port temperature was 2408C and the FID

temperature was 2808C. The oven temperature was pro-

grammed from 508C (1min) initially rising by 208C/min to

2708C (3min). Injections (1mL) were made in splitless mode.

A 990mL injection port liner with glass wool packing was

employed for all injections. The percentage of methyl-

amphetamine in the free base form was calculated using

the Agilent ChemStation software to create a five-point

calibration curve. The methylamphetamine hydrochloride

purity (as %m/m) is determined directly from this curve and

the input data.


Stable isotope ratio mass spectrometry

Sample preparationApproximately 0.8mg of drug material was weighed into

tin foil capsules (3.3mm� 5mm; IVA Analysentechnik,

Meerbusch Germany) for d13C/d15N analysis and 0.2mg

into silver foil capsules for d2H analysis.

Isotopic calibration and quality control of EA/TC-IRMSmeasurementsA Flash elemental analyzer (EA) 1112, with dual combustion

and thermal conversion (TC) capabilities, connected to a

ConFlo IV interface and Delta V Plus mass spectrometer (all

from ThermoScientific, Bremen, Germany) was used to

determine d15N, d13C and d2H values by continuous flow for

samples synthesized in this laboratory. Data was acquired

using ISODAT 2.5 software (ThermoScientific). Prior to

sequence acquisitions, zero enrichments were performed

using each of the three reference gases. The standard

deviation of nine 20-s gas pulses was determined to be less

than 0.1% for N2 and CO2, and 0.5% for H2.

Sample sequences for d15N and d13C analysis were

bracketed by triplicates of l-alanine reference material

(d15NAir¼�1.1%, d13CVPDB¼�19.9%) kindly provided by

Dr Yukiko Makino from the Narcotic Control Department,

Kanto-Shinetsu Regional Bureau of Health and Welfare,

Ministry of Health, Labour and Welfare, Japan. System

performance was verified by repeat analysis (n¼ 28) of a

high-purity (94.9% as base hydrochloride form) methyl-

amphetamine quality control (d15NAir¼þ3.1%, d13CVPDB¼�28.0%) every five samples. The d13C values, reported as per

mille (%) differences from the Vienna Pee Dee Belemnite

(VPDB) international standard, were measured relative to

high-purity (>99.5%) CO2 gas (BOC Gases, Sydney, Aus-

tralia; d13C¼�6.8%) that was calibrated against NBS 19

(Environmental Isotopes Pty. Ltd., Sydney, Australia). Ultra-

high-purity (>99.99%) N2 gas (BOC Gases) was calibrated

internally using acetanilide certified reference standard

(d15NAir¼þ1.2� 0.1%) purchased from Indiana University

and verified using the l-alanine reference material. The

precision of the d15N and d13C measurements was deter-

mined to be 0.4% and 0.2%, respectively.

The d2H values, reported as per mille (%) differences from

the Vienna Standard Mean Ocean Water (VSMOW) inter-

national standard, were measured relative to ultra-high-

purity (>99.99%) H2 gas (BOC Gases; d2H¼�340%)

calibrated against certified reference materials; namely,

C36 n-alkane (hexatriacontane; d2HVSMOW¼�247� 1%),

phenanthrene (d2HVSMOW¼�84� 1%) and icosanoic acid

methyl ester (d2HVSMOW¼þ76� 1%) obtained from Indiana

University. Subsequent repeat analysis (n¼ 21) of a high-

purity methylamphetamine sample assigned the d2H value

of this laboratory quality control, analyzed every five

samples within each sequence, to be �195� 2%. The Hþ3

factor (4.66 ppm/nA) was determined at weekly intervals

from reference H2 gas pulses with the signal size linearly

incremented. The precision of the d2H measurements as

monitored by standards and laboratory controls was

determined to be 2%.


DOI: 10.1002/rcm


d13C and d15N analysis by EA/IRMSDaily jump calibrations enabled a change in continuous

monitoring of m/z 28 and 29 to m/z 44, 45 and 46 for the

determination of d15N and d13C in the same analysis. A folded

tin foil capsule containing no sample material was the first

and final analysis performed in a sequence to demonstrate

that the system was void of contamination. A typical

sequence comprised 10 samples run in triplicate, preceded

and followed by a triplicate analysis of acetanilide standard.

Crimped tin capsules containing sample material were

introduced into a ThermoScientific No Blank chamber and

pressurized with high-purity oxygen gas (BOC Gases) to

exclude the contribution of nitrogen from ambient air. The

sample entered the combustion furnace operated at 9808Cwhere it burns exothermically in a stream (250mL/min, 3 s)

of high-purity (>99.5%) oxygen. The oxidized sample was

reduced in situ in the presence of copper before water was

removed from the resultant gas stream using a trap filled

with magnesium perchlorate. A post-reactor GC column,

operated at 358C, separated evolved N2 and CO2. The ultra-

high-purity (>99.99%) helium (BOC Gases) pressure was set

to 100 kPa to enable a run time of 650 s. Two N2 and CO2

reference gas pulses were applied prior to the sample N2

signal and following the sample CO2 signal, respectively.

d2H analysis by TC-IRMSSilver foil capsules containing sample material were

introduced into the TC reactor consisting of an alumina

ceramic outer containing a glassy carbon tube with glassy

carbon granulate and silver wool packing. The TC furnace

was operated at 14508C and the post-reactor GC column at

858C. The helium pressure was set to 330 kPa to enable a run

time of 300 s. A typical sequence comprised 10 samples run in

triplicate, preceded and followed by a triplicate analysis of

C36 n-alkane hexatriacontane and icosanoic acid methyl

ester standards.

Figure 4. Multivariate representation of d13C, d15N and d2H values for methylamphe-

tamine samples synthesized from known ephedrine/pseudoephedrine.


Graphical softwareThe multivariate plot depicted in Fig. 4 was produced using

SigmaPlot for Windows Version 11.0# (Systat Software Inc.,

San Jose, CA, USA).

RESULTS AND DISCUSSION

Makino et al.33 noted that the d13C values for ephedrine

derived from the ephedra plant ranged from �31.1% to

�26.0% in the samples that they had studied while semi-

synthetic ephedrine samples were more tightly clustered

with d13C values between �24% and �22%. Pseudoephe-

drine, usually produced by the acid isomerization of

ephedrine, was observed to have d13C values ranging from

�27% to �22% for material made from ephedrine of a semi-

synthetic origin while a sample of pseudoephedrine

prepared from ephedra-derived ephedrine had a d13C value

of �29.8%.32–33 In the present study samples of ephedrine

and pseudoephedrine with known provenance, i.e. the

method of manufacture is known to be either semi-synthetic

or derived from the ephedra plant, were analyzed to

determine the d13C, d15N and d2H values (Table 1). Samples

of ephedrine and pseudoephedrine certified by the manu-

facturer as having been produced using the semi-synthetic

process were purchased for isotopic measurements and for

use in synthesis. Commercially available ephedra extract was

used to prepare ephedrine hydrochloride for isotopic

measurements. The d13C and d15N values obtained were

consistent with the observations ofMakino et al.33 It wasmost

interesting to observe that the d2H values obtained for

ephedrine of semi-synthetic origin were highly positive, yet

negative for ephedrine derived from the ephedra plant.

Methylamphetamine was prepared from ephedrine and

pseudoephedrine precursors whose d13C, d15N and d2H ratios

were known to determine if these isotopic ratios are conserved

through to the product methylamphetamine. The conversion


DOI: 10.1002/rcm

Table 1. d13C, d15N and d2H values for ephedrine/pseudoephedrine of known provenance (values reported as the mean of three

consecutive measurements)

d13CVPDB (%) d15NAir (%) d2HVSMOW (%)

(I) (1S,2S)-(þ)-Pseudoephedrine (99%) Sigma-Aldrich Product No. E2750,Batch 125K1410 Country of manufacture: USA Certified semi-synthetic origin

�23.3 þ5.1 þ168

(II) (1R,2S)-(�)-Ephedrine (99.7%) Fluka Product No. 45285, Batch 350635/1 �26.8 þ6.8 �135

(III) (1S,2S)-(þ)-Pseudoephedrine (99%) Sigma-Aldrich Product No. E2750,Batch 59H1089 Certified semi-synthetic origin

�26.2 þ3.6 þ159

(IV) Pseudoephedrine extracted from ‘Sudafed’ tablets Wellcome Australia Ltd. �26.5 þ0.6 þ78

(V) (1R,2S)-(�)-Ephedrine (99%) Sigma-Aldrich Product No. 862231,Batch 10620PH Country of manufacture: India Certified semi-synthetic origin.

�25.6 �0.1 þ171

Ephedrine from ephedra extract ‘Power Plus Super Ephedra 850’ Prices PowerInternational Inc., Newport, VA, USA

�28.6 þ4.2 �170


was effected using the ‘Nagai’ reaction employing hydriodic

acid and red phosphorus to reduce ephedrine and pseudoe-

phedrine to methylamphetamine.2 Two of the variants of the

Nagai reaction were also investigated: the so-called ‘Hypo’

reaction9 in which hypophosphorous acid and iodine are

used, and the ‘Moscow’ reaction46 which employs red

phosphorus, iodine and water to produce hydriodic acid

in situ. The ‘Emde’ reaction,8 which involves the preparation

of the chloroephedrine and chloropseudoephedrine inter-

mediates and subsequent reduction to methylamphetamine

using hydrogen gas and a palladium catalyst, was also

examined. The methylamphetamine produced in each of the

above reactions was purified and the identity confirmed by

GC/MS and comparison with a certified reference standard.

The only difference between the precursor molecules and the

product molecule is the benzylic hydroxyl group in ephedrine

Table 2. d13C, d15N and d2H values for methylamphetamine sy

provenance (values reported as the mean of three consecutive m

Precursor Reaction Purity (%

Pseudoephedrine (I) Nagai (1) 96d13C¼�23.3, d15N¼þ5.1, d2H¼þ168 Nagai (2) 95

Nagai (3) 93Nagai (4) 94Hypo (1) 93Hypo (2) 93Hypo (3) 90Emde 89Moscow 95

Ephedrine (II) Emde (1) 94d13C¼�26.8, d15N¼þ6.8, d2H¼�135 Emde (2) 94Chloropseudoephedrine intermediate Emde (3) 93d13C¼�28.0, d15N¼þ6.8, d2H¼�105 Hypo (1) 93

Pseudoephedrine (III) Nagai 94d13C¼�26.2, d15N¼þ3.6, d2H¼þ159

Pseudoephedrine (IV) Nagai (1) 90(Sudafed tablet extract) Nagai (2) 93d13C¼�26.5, d15N¼þ0.6, d2H¼þ78 Emde (1) 95

Emde (2) 94Emde (3) 90

Ephedrine (V) Emde 87d13C¼�25.6, d15N¼�0.1, d2H¼þ171 Nagai 95Chloropseudoephedrine intermediate Moscow 95d13C¼�26.7, d15N¼�0.1, d2H¼þ180 Hypo 94


and pseudoephedrine which is replaced by a hydrogen atom

in methylamphetamine. If isotopic fractionation does not

occur due to the reaction kinetics, the d13C and d15N values

might be expected to remain essentially unchanged within

measurement uncertainty while the d2H value should be

affected to some small extent. It is apparent from the results

presented in Table 2 that the d13C and d15N values obtained in

the methylamphetamine product are essentially the same as

the values for the precursor molecules, consistent with the

observations of Makino et al.32–33 This data is presented

graphically in Fig. 4, which shows the d13C, d15N and

d2H values of synthesized methylamphetamine to cluster

according to the precursors used and not the synthetic route

employed. In any case, conventional chemical profiling is

sufficiently mature to easily detect route-specific markers that

distinguish between the Emde reaction, the Birch reduction

nthesized from ephedrine and pseudoephedrine of known

easurements)

Methylamphetamine product

) d13CVPDB (%) d15NAir (%) d2HVSMOW (%)

�23.2 þ5.2 þ134�23.1 þ5.3 þ111�22.9 þ5.2 þ131�23.0 þ5.1 þ128�23.8 þ4.1 þ124�23.2 þ4.3 þ120�23.1 þ4.7 þ114�22.8 þ5.6 þ145�23.4 þ4.7 þ127

�27.2 þ6.6 �134�27.4 þ6.5 �133�27.3 þ6.5 �131�27.7 þ6.3 �132

�26.2 þ4.3 þ120

�25.9 þ2.7 þ65�26.1 þ2.4 þ65�25.7 þ1.7 þ79�25.9 þ1.9 þ81�25.6 þ1.6 þ77

�25.5 �0.1 þ166�25.6 �0.4 þ133�25.4 �0.3 þ142�25.0 �0.2 þ140


DOI: 10.1002/rcm

Table 4. d13C, d15N and d2H values for selected methylam-

phetamine seizures

Sampled13CVPDB

(%)d15NAir

(%)d2HVSMOW

(%)

1 �25.9 þ6.4 �193 Natural origin2 �26.5 �1.5 �1313 �28.6 �1.3 �1154 �26.4 �1.5 �1115 �26.2 �1.5 �1106 �26.4 �1.5 �1097 �26.3 �1.5 �1068 �26.0 �1.3 �919 �24.9 þ8.7 �64

10 �22.1 þ3.0 þ4 Semi-syntheticorigin11 �24.1 þ4.0 þ12

12 �24.3 þ4.6 þ1213 �21.4 þ3.4 þ1814 �23.6 þ4.5 þ2415 �23.0 þ6.3 þ4816 �21.3 þ2.6 þ5217 �23.7 þ7.8 þ5818 �21.2 þ2.6 þ5919 �23.3 þ8.5 þ7020 �21.3 þ11.3 þ7121 �21.3 þ11.4 þ7222 �21.7 þ11.4 þ7523 �21.8 þ9.1 þ7524 �21.0 þ3.1 þ7625 �23.5 þ8.0 þ7926 �21.9 þ8.7 þ8027 �23.7 þ7.8 þ8128 �21.9 þ3.2 þ8529 �23.7 þ9.1 þ8530 �23.4 þ8.6 þ8531 �23.5 þ8.5 þ8832 �23.4 þ8.3 þ90


and theNagai reaction and its variants. The IRMS technique is

sensitive to different sources of precursors and it is this that

makes the technique so useful in discriminating between

batches of methylamphetamine prepared in the same

clandestine facility, by the same synthetic route, but using

different batches of ephedrine and pseudoephedrine or

ephedrine and pseudoephedrine of different synthetic origin.

The d2H values of the product methylamphetamine

demonstrate that deuterium depletion has occurred in some

instances. Interestingly, the d2H values for samples of

ephedrine and pseudoephedrine known to be of a semi-

synthetic origin were positive. The d2H values of methyl-

amphetamine samples believed to have been synthesized

from semi-synthetic ephedrine on the basis ofMakino’s work

were also positive. When d2H values were determined for

benzaldehyde sourced from several chemical suppliers they

were observed to be positive in the range of þ3% to þ552%(Table 3), except for the Fluka puriss #12010 sample (�52%)

and a naturally derived ‘Kosher’ product that had a

d2H value of �125%, in good agreement with the certified

d2H value of �129� 1%.

Studies of benzaldehyde as an adulterant in flavors

demonstrate the utility of d2H values to discriminate

synthetic materials from botanically derived materials.47,48

Enrichments as high as þ720% were found for synthetic

benzaldehyde made from the catalytic oxidation of toluene.

Such extreme enrichments are atypical for synthetic

processes and have only been identified in synthetic

benzaldehyde and cinnamic aldehyde.47,48 Natural forms

of benzaldehyde and cinnamic aldehyde extracted from

apricot kernels have been observed to have d2H values of

�111� 18% and �120� 15%, respectively.47,48 It seems

likely then that ephedrine produced from synthetic benzal-

dehyde and the pseudoephedrine derived from it will have

positive d2H values. Because the positive d2H values of the

semi-synthetic precursors are maintained in the methyl-

amphetamine produced from semi-synthetic ephedrine or

pseudoephedrine, the determination of d2H values may be

valuable in assessing whether methylamphetamine was

produced from a natural or semi-synthetic precursor.

Classification of ephedrine as being manufactured by a

completely synthetic procedure would rely on d15N analysis

providing depleted values in the order of �10%, as

previously reported by Makino et al.33 Values for

d13C, d15N and d2H obtained from a number of seizures of

high-purity methylamphetamine known not to contain

adulterants or diluents are presented in Table 4. The

d2H values complement the d13C values obtained by Makino

et al.33 for methylamphetamine produced from semi-

Table 3. d13C and d2H values for commercially available benza

consecutive measurements)

Benzaldehyde sample

Sigma purified by re-distillation; Product #418099 (�99.5%)Fluka Puriss Std; Product #09143 (�99.5%)Sigma Reagent Plus; Product #B1334-2G; (�99%)Merck for synthesis; Product #8.01756.0100 (�99%)Fluka Puriss Standard Product #12010 (�99%)Sigma Kosher; Product #W212717-100G-K (�98%) Certified d13C¼�28.1�


synthetic and natural ephedrine and pseudoephedrine,

and in addition they provide a simple classification based

on positive or negative values. For example, samples 10 to 32

(Table 4) have positive d2H values and would be classified as

semi-synthetic products.

Matsumoto et al.49 recently conducted position-specific

isotope analysis using nuclear magnetic resonance (NMR)

spectroscopy to demonstrate deuterium enrichment in ephe-

drine produced synthetically. The high deuterium enrichment

was found to be located primarily at the benzylic position in

ephedrine produced from benzaldehyde which in turn was

synthesized from toluene by catalytic oxidation. This inde-

pendent observation using NMR technology substantiates our

observation, using IRMS, of deuterium enrichment in ephe-

drine produced via the semi-synthetic process.

ldehyde products (values reported as the mean of three

d13CVPDB (%) d2HVSMOW (%)

�26.3 þ552�26.0 þ535�26.2 þ493�28.0 þ3�25.1 �52

0.1%; d2H¼�129� 1% �28.3 �125


DOI: 10.1002/rcm


CONCLUSIONS

Methylamphetamine was synthesized from ephedrine and

pseudoephedrine and the d13C, d15N and d2H isotope ratios

weredetermined.Bothnatural (derivedfromthe ephedraplant)

and semi-synthetic ephedrine and pseudoephedrine were

used for the syntheses. The natural precursors had d2H values

that were negative while the semi-synthetic precursors had

positive d2H values. The methylamphetamine prepared from

the semi-synthetic ephedrine and pseudoephedrine also had

positive d2H values and that prepared from the natural

materials had negative values for d2H. It was demonstrated

that the unusual positive d2H values found in the semi-

synthetic ephedrine and pseudoephedrine, and any methy-

lamphetamine derived from these precursors, could be

attributed to the synthetic benzaldehyde used as a starting

material in the semi-synthetic preparation of ephedrine and

pseudoephedrine. d2H values can therefore be used to

distinguish ephedrine derived from the ephedra plant and

ephedrine prepared via the semi-synthetic route. d2H values

determined in methylamphetamine prepared from either

ephedrine or pseudoephedrine will indicate whether the

ephedrine or pseudoephedrine was derived from ephedra or

semi-synthetic in origin. The use of d2H isotope values

complements the work by Makino et al. with d13C and

d15Nvalues anda combination of d13C, d15Nand d2Hwill allow

a distinction to be made between methylamphetamine

synthesized from either natural, semi-synthetic, or the fully

synthetic ephedrine and pseudoephedrine. In conjunction

with d13C and d15N values, there is potential for d2H values to

provide additional information for tactical comparisons of

methylamphetamine seizures. While the EA/TC-IRMS tech-

nique is ideal for use with high-purity methylamphetamine

seizures it would not be satisfactory for use with seizures that

havebeenadulterated.Furtherwork isplanned toexamine the

GC-C/TC-IRMS determination of d13C, d15N and d2H isotope

ratios in methylamphetamine adulterated with a range of

organic substances.

AcknowledgementsThe authors are grateful to Dr YukikoMakino of the Narcotic

Control Department, Kanto-Shinetsu Regional Bureau of

Health andWelfare, Ministry of Health, Labour andWelfare,

Japan, and to Dr Barbara Remberg, Laboratory and Scientific

Section, United Nations Office on Drugs and Crime, for most

helpful discussions.

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DOI: 10.1002/rcm

Documents

δ13C, δ15N and δ2H isotope ratio mass spectrometry of ephedrine and pseudoephedrine: application to methylamphetamine profiling