Chiral separation of cathinone and amphetamine derivatives by HPLC/UV using sulfated β-cyclodextrin as chiral mobile phase additive

Article abstract of DOI:10.1002/chir.22341

In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated β-cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated β-cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP-8e column. Copyright

Full text of DOI:10.1002/chir.22341

CHIRALITY (2014)
Chiral Separation of Cathinone and Amphetamine Derivatives by
HPLC/UV Using Sulfated ß-Cyclodextrin as Chiral Mobile
Phase Additive
1
MAGDALENA TASCHWER,1 YVONNE SEIDL,1 STEFAN MOHR,1,2 AND MARTIN G. SCHMID
*
1Department of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz, Graz, Austria
2Research Center Pharmaceutical Engineering, Graz, Austria
ABSTRACT
In the last years the identification of new legal and illegal highs has become a huge
challenge for the police and prosecution authorities. In an analytical context, only a few analytical
methods are available to identify these new substances. Moreover, many of these recreational
drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency.
Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mix-
tures. The goal of this research work was to develop an inexpensive method for the chiral separa-
tion of cathinones and amphetamines. This should help to discover if the substances are sold as
racemic mixtures and give further information about their quality as well as their origin. Chiral sep-
aration of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various In-
ternet shops, is presented. A LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm served as the stationary
phase. The chiral mobile phase consisted of methanol, water, and sulfated ß-cyclodextrin. Measure-
ments were performed under isocratic conditions in reversed phase mode using UV detection. Four
model compounds of the two substance classes were used to optimize the mobile phase. Under fi-
nal conditions (methanol:water 2.5:97.5 + 2% sulfated ß-cyclodextrin) enantiomers of amphetamine
and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or
partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the appli-
cation of this method is limited for cathinone analogs. Additionally, the results were compared with
an RP-8e column. Chirality 00:000–000, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: cathinone; amphetamine; chiral separation; sulfated ß-cyclodextrin
INTRODUCTION
activity, metabolic, and pharmacokinetic characteristics, one
of the enantiomers might show a stronger effect, while the
second one might exhibit a weaker or even toxic effect. The
S-(À)-enantiomer of methcathinone and the S-(+)-enantiomer
of amphetamine, for example, show more stimulating effects
than their R-(+)- and R-(À)-antipodes.2–4
In the last years the spectrum of legal and illegal drugs has
increased enormously. Further derivatives or even new
unknown substances entered the market nearly every week.
According to the EU drug market report of the European
Monitoring Centre for Drugs and Drug Addiction, 73 new
psychoactive substances showed up in 2012, 49 in 2011, and
41 in 2010. Since 2005, more than 200 new compounds have
been reported in the EU.1 4-Methylmethcathinone, better
known as mephedrone, is the most popular compound traded
via the Internet as plant fertilizer or bath salt in the last years.
As it was a legal alternative to the amphetamines or ecstasy, it
caused much hype in Europe. In 2009, the hype was at its
peak and the authorities were forced to react. Since the 3rd
of December 2010 there has been an EU-wide prohibition of
mephedrone. As a consequence, the drug market replaced
them with further psychoactive cathinone derivatives to
circumvent the law. In Austria, e.g., the New Psychoactive
Substances Act and the New Psychoactive Substances Regu-
lation was introduced in January 2012. This law regulates
the distribution or trade of these new compounds.
The new drugs are synthesized in clandestine laboratories
and are sold worldwide on the Internet as research com-
pounds, bath salts, room odorizers, or fertilizers for plants.
The progressively worldwide black market of these legal
and illegal highs represents a big problem for the police and
prosecution authorities. Some of these new drugs are chiral
amphetamine and cathinone derivatives. Syntheses of these
drugs are sometimes simple and cheap, but not enantiopure.
As the enantiomers can differ in their pharmacological
© 2014 Wiley Periodicals, Inc.
The drug market has changed completely in recent years.
In order to circumvent the law, illegal drugs are replaced by
legal alternatives instead. Amphetamines, for example, are
replaced by legal cathinones, cannabis by legal incense, and
LSD by legal phenetylamines or legal tryptamines. In contrast
to the well-known illegal drugs such as heroin and cocaine,
the new substances do not have common names. They are
called NRG-1 or synthacaine, for instance. Besides the
problem that the addicted do not know what they consume,
there is additionally the risk of unknown impurities. Recently,
drug dealers publish product analyses by mass spectroscopy
(MS) and nuclear magnetic resonance (NMR) on their Internet
pages in order to prove the purity of the offered substances.
However, neither the addicted understand the information,
nor can they check its correctness. Therefore, there is a need
for development of analytical methods for identification of these
new drugs. Since to our knowledge no rapid tests to identify
*Correspondence to: Martin Schmid, Department of Pharmaceutical Chemis-
try, Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz,
Universitätsplatz 1, A-8010 Graz, Austria. E-mail: martin.schmid@uni-graz.at
Received for publication 13 February 2014; Accepted 15 April 2014
DOI: 10.1002/chir.22341
Published online in Wiley Online Library
(wileyonlinelibrary.com).

TASCHWER ET AL.
these new substances are available, it is reasonable to use
chromatographic methods.
Therefore, the aim of this work was to develop an inexpensive
and easy to perform method for the chiral separation of
cathinone and amphetamine derivatives using a common
reversed-phase column under isocratic conditions.
A further problem of these legal and illegal highs is that
there are only few data available about the pharmacology,
toxicology, and the long-term damage they cause. Most of
the information about the effects and side effects are based
on progress reports of users, which are discussed on Internet
forums.
In the literature, there are some articles dealing with the de-
termination and chiral separation of cathinone, amphetamine,
and related substances by high-performance liquid chromatog-
raphy (HPLC),5–8 capillary electrophoresis (CE),9–12 and gas
chromatography (GC).13–15
MATERIALS AND METHODS
Chromatographic Conditions
Chiral separation experiments were carried out with an HP Hewlett
Packard (Corvallis, OR) Series II; 1090; Liquid Chromatograph, equipped with
an autosampler and a diode array detector. Ultraviolet (UV)-detection was
performed at 220, 240, and 270 nm. The measurements were carried out
Nhujak’s group reported on the chiral separation by CE
using a dual cyclodextrin system consisting of ß-cyclodextrin
and dimethyl-ß-cyclodextrin; the improved enantioseparation
of amphetamine and drug enantiomers could be achieved
with a single chiral selector.16
TABLE 2. Chemical structures of the tested amphetamine
analogs
For HPLC, chiral stationary phases based on crown ether
showed very successful separation results; however, they
are limited by the presence of a primary amine group at the
analyte.17–19
Compound
R1
R2
R3
R4
Previously, our group succeeded in resolving 19 of 25
cathinone derivatives into their enantiomers using the
CHIRALPAK AS-H column as stationary phase.20 Another suc-
cess was the chiral separation of cathinone derivatives by CE
using sulfated ß-cyclodextrin as an added chiral selector.21 As
these results of the enantioseparation were satisfactory, the
attempt was to use sulfated ß-cyclodextrin as a chiral additive
in the mobile phase to achieve enantioseparation with HPLC.
Amphetamine
H
H
H
H
H
H
H
CH3
CH3
CH3
H
3-Fluoroamphetamine (3-FA)
4-Fluoroamphetamine (4-FA)
4-Fluoromethamphetamine
(4-FMA)
N-Methamphetamine
3,4-Methylendioxymethamphetamine
(MDMA)
3-F
4-F
4-F
CH3 CH3
H
H
CH3 CH3
CH3 CH3
H
3,4-
methylendioxy
TABLE 1. Chemical structure of the tested cathinone derivatives
Compound
R1
R2
R3
R4
Benzedrone (4-MBC)
4-Bromomethcathinone (4-BMC)
Buphedrone
H
H
H
H
CH3
H
H
H
H
H
H
H
H
Benzyl
CH3
CH3
CH3
CH3
CH3
C2H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C3H7
CH3
CH3
CH3
C3H7
C3H7
C3H7
C3H7
CH3
4-CH3
4-Br
H
3,4-methylendioxy
3,4-methylendioxy
3,4- dimethyl
H
4-C2H5
3,4-methylendioxy
2-F
3-F
4-F
4-CH3
4-OCH3
4-CH3
4-CH3
3,4-methylendioxy
Butylone
N,N-Dimethylbutylone
3,4-Dimethylmethcathinone (3,4-DMMC)
Ethylbuphedrone
4-Ethylmethcathinone (4-EMC)
Ethylone
2-Fluoromethcathinone (2-FMC)
3-Fluoromethcathinone (3-FMC)
4-Fluoromethcathinone (4-FMC)
Mephedrone (4-MMC)
Methedrone
4-Methyl-alpha-Pyrrolidinopropiophenone (MPPP)
4-Methylbuphedrone
Methylendioxypyrovalerone (MDPV)
4-Methylethcathinone (4-MEC)
3-Methylmethcathinone (3-MMC)
Methylone
Naphyrone
Pentedrone
H
pyrrolidinyl
CH3
pyrrolidinyl
H
H
H
H
C2H5
CH3
CH3
4-CH3
3-CH3
3,4-methylendioxy
β-naphtyl instead of phenyl
H
3,4-methylendioxy
H
H
pyrrolidinyl
H
H
CH3
CH3
pyrrolidinyl
pyrrolidinyl
Pentylone
α-Pyrrolidinopentiophenone (α-PVP)
α-Pyrrolidinopropiophenone (α-PPP)
Chirality DOI 10.1002/chir

CHIRAL SEPARATION OF CATHINONE AND AMPHETAMINE DERIVATIVES
under isocratic conditions at ambient temperature with a flowrate of 1 ml/min
and an injection volume of 10 μl. Data were collected with Chemstation Rev. A.
0903 (Agilent Technologies, Waldbronn, Germany) software.
A LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, from Merck (Darmstadt
Germany) and a LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, from
Macherey-Nagel (Düren, Germany) served as stationary phases.
Based on previous results of our group, a LiChrospher 100
RP-18e, 250 x 4 mm, 5 μm was chosen as the stationary phase.
According to the information by the manufacturer, a LiChrospher
TABLE 5. Chiral separation results of a set of 25 cathinone
derivatives using an RP-18e column
Chemicals and Solutions
Compound
t1 (min)
t2 (min)
α
Rs
As the compounds (benzedrone, 4-BMC, buphedrone, butylone, 3,4-DMMC,
N,N-dimethylbutylone, 4-EMC, ethylone, ethylbuphedrone, 2-FMC, 3-FMC, 4-
FMC, methedrone, MDMC, MDPV, 4-methylbuphedrone, 4-MEC, MPPP,
4-MMC, 3-MMC, naphyrone, pentedrone, pentylone, α-PPP, α-PVP, amphet-
amine, 3-FA, 4-FA, 4-FMA, MDMA, methamphetamine) were not available
at official suppliers, they were purchased from various Internet shops over
the last years. In Tables 1 and 2 the abbreviations of the compounds are listed.
The identity of these substances was proven by GC-MS and if neces-
sary by NMR.
Benzedrone
4-BMC
Buphedrone
Butylone
3,4-DMMC
N,N-Dimethylbutylone
4-EMC
57.81
39.42
27.23
24.35
73.31
37.02
100.24
44.37
17.29
10.00
14.15
15.09
57.75
82.04
n.d.
35.79
6.13
73.64
15.70
9.63
53.36
n.d.
22.07
104.22
104.37
61.37
40.16
28.87
26.03
83.99
45.08
103.94
47.00
-
11.05
14.62
16.31
61.16
93.53
n.d.
-
7.40
80.04
-
-
-
n.d.
24.02
120.58
-
1.064
1.020
1.065
1.075
1.149
1.229
1.038
1.062
-
1.108
1.033
1.092
1.061
1.143
-
0.51
0.20
0.78
0.73
0.95
2.73
0.54
0.84
0
1.26
0.40
0.96
0.51
1.61
-
Ethylbuphedrone
Ethylone
Methanol and sulfated ß-cyclodextrin (degree of substitution: 7–11
mol*molÀ1) were from Sigma-Aldrich (St. Louis, MO). Nanopure water
was produced in our lab.
2-FMC*
3-FMC
4-FMC
4-MEC
4-Methylbuphedrone
MDPV
All chemicals were of analytical grade.
The mobile phase was prepared by mixing methanol and water in the
required ratios prior to adding 2% sulfated ß-cyclodextrin. To accelerate
the dissolving process of the cyclodextrin, a magnetic stirrer was used.
Then the solution was degassed with helium for 2 min and filtered
through a 0.45-μm pore size cellulose filter (Carl Roth, Karlsruhe,
Germany). For adjusting the pH a diluted sulfuric acid was used.
3-MMC
-
0
Mephedrone*
MPPP
1.205
1.089
-
-
-
0.55
0.64
0
Methedrone
Methylone
Naphyrone
α-PVP
α-PPP
Pentedrone
Pentylone
0
Sample Preparation
There was no special sample preparation required; 0.5 mg of each
compound was dissolved in 1 ml nanopure water.
0
-
-
1.069
1.160
-
0.93
3.09
0
RESULTS AND DISCUSSION
In Tables 1 and 2 the structures of the investigated
cathinone and amphetamine derivatives (tabulated by struc-
ture) are given, respectively.
Conditions: Column: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile
phase: methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room tempera-
ture, flow: 1 ml/min, UV: *240 nm, 270 nm, injection: 10 μl.
TABLE 3. Enantioseparation of some test compounds using
10% methanol added to the mobile phase
Compound
t1 (min)
t2 (min)
α
Rs
4-FMC
4-MMC*
3-FA
8,95
13,65
3,64
9,54
—
1,065
—
0,61
—
4,71
2,77
1,290
1,395
2,84
2,31
MDMA*
1,99
Conditions: Column: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile
phase: methanol:water (10:90) + 1% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV:
*240 nm, 270 nm, injection: 10 μl.
TABLE 4. Chiral separation results of a set of 6 amphetamines
using an RP-18e column
Compound
t1 (min)
t2 (min)
α
Rs
Amphetamine
3-FA**
7.68
7.84
7.88
18.59
5.75
10.82
10.02
9.80
9.86
22.04
7.42
13.79
1.404
1.327
1.328
1.206
1.433
1.333
3.30
2.70
2.68
1.98
1.80
2.96
4-FA**
4-FMA**
MDMA*
N-Methamphetamine
Conditions: Column: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile
phase: methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room tempera-
ture, flow: 1 ml/min, UV: 220 nm,
*240 nm,
**270 nm, injection: 10 μl.
Fig. 1. Simultaneous chiral separation of buphedrone, ethylbuphedrone
and 4-methylbuphedrone. Conditions: Column: LiChrospher 100 RP-18e, 250
x 4 mm, 5 μm, mobile phase: methanol:water (2.5:97.5) + 2% sulfated ß-cyclo-
dextrin, room temperature, flow: 1 ml/min, UV: 270 nm, injection: 10 μl.
Chirality DOI 10.1002/chir

TASCHWER ET AL.
TABLE 6. Enantioseparation of some test compounds using an
RP-18e column and 2% methanol added to the mobile phase
100 RP-18e column is suitable for the separation of acidic, neutral,
and weak basic compounds. Due to the endcapping, the column
is especially suitable for basic compounds. As the cathinone and
amphetamine derivatives show a basic character, suitable inter-
actions can be expected. Thus, a mobile phase consisting of
methanol:water (10:90) + 1% sulfated ß-cyclodextrin was tested.
To find the optimal composition of the mobile phase, 3-FA,
MDMA, 4-FMC, 3-MMC, and 4-MMC were used as model com-
pounds of the two substance classes. With the given mobile
phase, retention times were quite short and only amphetamine
derivatives were separated. While 4-FMC was partially sepa-
rated, 3-MMC was not determined and with 4-MMC no separa-
tion was achieved. However, the two amphetamine derivatives
eluted quickly and were baseline separated (Table 3). To reduce
the elution power of the mobile phase the ratio methanol/water
was changed to 2.5:97.5. As expected, the retention times in-
creased but also the separation got worse. In the next step, the
amount of sulfated ß-cyclodextrin was increased to a final con-
centration of 2%. Cyclodextrins represent commonly used chiral
selectors. They are shaped like a truncated cone with a lipophilic
central cavity and a hydrophilic outer surface. They may form in-
clusion complexes with analytes in aqueous solutions. The chiral
Compound
t1 (min)
t2 (min)
α
Rs
Benzedrone
Buphedrone
Butylone
3,4-DMMC
N,N-Dimethylbutylone
4-FMC
4-MEC
Pentedrone
Naphyrone
n.d.
36.80
32.63
n.d.
n.d.
19.51
n.d.
n.d.
38.85
34.76
n.d.
n.d.
21.12
n.d.
—
1.060
1.064
—
—
0.60
0.65
—
—
—
1.083
—
—
0.60
—
n.d.
n.d.
n.d.
n.d.
—
—
—
Conditions: Column: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile
phase: methanol:water (2:98) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV: 270 nm, injection: 10 μl.
Fig. 2. Comparison of (a) butylone and (b) N,N-dimethylbutylone. Condi-
tions: Column: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile phase:
methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV: 270 nm, injection: 10 μl.
Fig. 3. Determination of the enantiomer elution order by means of amphetamine.
Conditions: Column: LiChrospher 100 RP-18e, 250 x 4mm, 5 μm, mobile phase: meth-
anol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature, flow: 1 ml/min,
UV: 220 nm, injection: 10 μl. A: R-(+)-amphetamine, B: racemic amphetamine.
Chirality DOI 10.1002/chir

CHIRAL SEPARATION OF CATHINONE AND AMPHETAMINE DERIVATIVES
separation mechanism is based on the formation of diastereo-
meric selector-enantiomer complexes, which can be separated
with an achiral stationary phase due to their different physical
and chemical properties. Besides inclusion interactions, dipol–
dipol interactions, hydrogen bondings and hydrophobic
TABLE 8. Chiral separation results of a set of 25 cathinone
derivatives using an RP-8e column
Compound
t1 (min)
t2 (min)
α
Rs
Benzedrone
4-BMC
Buphedrone
Butylone
3,4-DMMC
N,N-Dimethylbutylone
4-EMC
50.33
32.54
25.85
22.34
63.87
36.52
80.65
42.58
15.75
11.27
13.99
14.06
48.51
70.86
47.23
32.12
26.87
63.24
13.57
9.09
53.50
—
1.065
—
0.92
0
27.33
23.88
72.76
44.31
83.64
45.12
—
1.062
1.075
1.143
1.225
1.038
1.062
—
0.93
0.64
1.45
2.67
0.70
0.97
—
Ethylbuphedrone
Ethylone
2-FMC*
—
—
—
—
—
—
3-FMC
4-FMC
4-MEC
4-Methylbuphedrone
MDPV
Fig. 4. Simultaneous chiral separation of 3-FA, 4-FMA, N,N-dimethylbutylone,
3,4-DMMC, pentedrone. Conditions: Column: LiChrospher 100 RP-18e, 250 x 4 mm,
5 μm, mobile phase: methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room
temperature, flow: 1 ml/min, UV: 270 nm, injection: 10 μl.
15.11
51.62
80.83
—
1.086
1.067
1.145
—
0.82
0.84
1.98
—
3-MMC
—
—
—
—
—
—
Mephedrone*
MPPP
68.77
—
—
1.090
—
—
0.92
—
Methedrone
Methylone
Naphyrone
α-PVP
α-PPP
Pentedrone
Pentylone
—
—
n.d.
n.d.
—
—
—
47.44
20.27
85.72
85.03
—
—
22.13
99.03
110.80
—
1.159
1.310
2.97
4.59
Conditions: Column: LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, mobile phase:
methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV:
*240 nm, 270 nm, injection: 10 μl.
Fig. 5. Simultaneous chiral separation of 4-FA and 4-FMA. Conditions: Col-
umn: LiChrospher 100 RP-18e, 250 x 4 mm, 5 μm, mobile phase: methanol:water
(2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature, flow: 1 ml/min, UV:
270 nm, injection: 10 μl.
TABLE 7. Chiral separation results of a set of 6 amphetamines
using an RP-8e column
Compound
t1 (min)
t2 (min)
α
Rs
Amphetamine
3-FA**
8.85
6,86
12.20
16.72
4.51
7.30
8,43
13.90
19.67
5.50
1.336
1.315
1.165
1.199
1.376
1.312
2.38
2,78
2.07
2.31
0.98
2.37
4-FA**
4-FMA**
MDMA*
N-Methamphetamine
7.82
9.67
Conditions: Column: LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, mobile phase:
methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV: 220 nm,
*240 nm,
**270 nm, injection: 10 μl.
Fig. 6. Comparison of the elution of methamphetamine with two different
reversed-phase LiChrospher columns. Conditions: Columns: LiChrospher
100 RP-18e and RP-8e, 250 x 4 mm, 5 μm, mobile phase: methanol:water
(2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature, flow: 1 ml/min,
UV: 220 nm, injection: 10 μl.
Chirality DOI 10.1002/chir

TASCHWER ET AL.
Fig. 9. Enantiomerically pure real life sample: methamphetamine. Condi-
tions: Column: LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, mobile phase:
methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV: 220 nm, injection: 10 μl.
Fig. 7. Simultaneous chiral separation of 3-FA, α-PPP, 4-MEC, 3,4-DMMC.
Conditions: Column: LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, mobile phase:
methanol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature,
flow: 1 ml/min, UV: 270 nm, injection: 10 μl.
The retention times were below 23 min and the resolution
factors ranged from 1.8 (MDMA) to 3.3 (amphetamine).
With this setup, 17 out of 25 cathinone derivatives were
partially or baseline separated (Table 5). Retention times were
between 15 and 120 min and the resolution factors ranged
between 0.20 (4-BMC) and 3.09 (pentedrone). The best results
of the cathinone derivatives were achieved with N,N-
dimethylbutylone, 4-methylbuphedrone, and pentedrone.
Moreover, the side chains of the cathinone derivatives played
interactions are responsible for the separation of the enantio-
mers. The added sulfated ß-cyclodextrin shifts the pH of the
methanol/water mixture to 6.3. During several measurements
a change of the pH was noticed. If the value exceeded 6.3, sep-
aration was lost or even no peak appeared. As a consequence,
the pH was checked daily and adjusted if necessary.
With the final composition of the mobile phase (methanol:
water 2.5:97.5 + 2% sulfated ß-cyclodextrin) the enantiomers
of amphetamine and its derivatives were resolved (Table 4).
Fig. 8. Racemic real life samples: (a) 4-MEC, (b) amphetamine, (c) MDMA. Conditions: Column: LiChrospher 100 RP-8e, 250 x 4 mm, 5 μm, mobile phase: meth-
anol:water (2.5:97.5) + 2% sulfated ß-cyclodextrin, room temperature, flow: 1 ml/min, UV: 220 nm, 240 nm, 270 nm, injection: 10 μl.
Chirality DOI 10.1002/chir

CHIRAL SEPARATION OF CATHINONE AND AMPHETAMINE DERIVATIVES
TABLE 9. Repeatability and reproducibility data including retention time and resolution by means of 3-FA using an RP-18e column
Repeatability
t1 (min)
t2 (min)
Rs
Intraday n = 5
Interday n = 10
7.85 0.02, RSD = 0.19%
7.99 0.14, RSD = 1.76%
9.82 0.02, RSD = 0.17%
10.00 0.20, RSD = 1.99%
2,69 0,17, RSD = 6.39%
3,03 0,39, RSD = 12.76%
Reproducibility
Intraday n = 20
t1 (min)
t2 (min)
Rs
7.79 0.03, RSD = 0.45%
9.71 0.04, RSD = 0.43%
3,54 0,26, RSD = 7.26%
a crucial role regarding resolution. While 4-methylbuphedrone
was baseline separated, buphedrone and ethylbuphedrone
were only partially separated. Obviously, the presence of a
para-methyl group at the phenyl ring improved resolution.
Figure 1 shows that the retention times grew significantly with
the increasing lipophilic character of the compounds. The inclu-
sion of the molecules in the sulfated ß-cyclodextrin is extremely
influenced by the lipophilicity and leads 4-methylbuphedrone
to a satisfactory result. Also, separation of butylone and N,N-
dimethylbutylone can be compared. While the latter is baseline
separated because of the higher lipophilic character, butylone
shows only partial resolution (Fig. 2). Comparing 4-MEC and
its structural isomer 4-EMC, it can be seen that the ethyl-
group at the ring of 4-EMC leads to a better inclusion in the
cyclodextrin and a stronger interaction of the stationary
phase. The retention times are unexpectedly different. De-
spite the long retention time of 4-EMC, only a partial separa-
tion was achieved.
With the present method, ethylone, 2-FMC, 3-MMC,
mephedrone, methylone, naphyrone, and pentylone were
not separated into their enantiomers. MDPV and α-PVP,
which have a similar structure as α-PPP and MPPP, were
not even detected. Among 25 cathinone derivatives, only
three were baseline separated; thus, this method is better
suitable for the enantioseparation of amphetamines.
Furthermore, it was investigated how the concentration of the
organic solvent influences chiral separation. Therefore, a mobile
phase with 2% of methanol was prepared, while the concentration
of the sulfated ß-cyclodextrin remained unchanged. The higher
amount of water resulted in longer retention times, and as a con-
sequence many of the analytes were not determined (Table 6).
To check the enantiomer elution order (EEO), both racemic
solutions of amphetamine and R-amphetamine were injected.
As shown in Figure 3, the R-enantiomer elutes first, followed
by the S-enantiomer. The R-enantiomer is responsible for the
main effects such as stimulation, less appetite, and improved
performance on working memory. The determination of the
enantiomer elution order can be used as a purity check.
Moreover, detection of a racemate might be possible evidence
of an illegal origin.
of an RP-18e column resulted in better enantioseparation
compared to an RP-8e column. The separation results of
LiChrospher 100 RP-8e column are shown in Tables 7 and 8.
One exception was pentylone, which was baseline separated,
while with an RP-18e column only one peak appeared. A com-
parison of the effect of the two different reversed-phase
LiChrospher columns on enantioseparation of methamphet-
amine is illustrated in Figure 6. Moreover, simultaneous chiral
separation of 3-FA, α-PPP, 4-MEC, and 3,4-DMMC was performed
with this column (Fig. 7).
Furthermore, this new and easy-to-perform chiral separation
method was tested by means of real-life samples. Amphetamine,
methamphetamine, MDMA, and 4-MEC seized by police in
Austria were analyzed to check the ratios of enantiomers. It was
discovered that amphetamine, MDMA, and 4-MEC were traded
as racemic mixtures (Fig. 8), while methamphetamine was
enantiopure (Fig. 9). This might be a hint that the latter
compound was synthesized illegally, e.g., by reduction of an
enantiopure ephedrine in a clandestine laboratory.
Finally, validation of the new method using the RP-18e col-
umn with regard to the reproducibility and the repeatability of
the retention time as well as the resolution factor was done
with 3-FA as the analyte. The intra- and interday repeatability
of the three parameters was satisfactory. For the intraday
repeatability, a relative standard deviation (RSD) for the
retention times was less than 0.20% and for the resolution fac-
tor about 6.39%. Day-to-day repeatability for the retention
times was less than 2% and for the resolution factor was
12.76%. This high value can be explained because the effi-
ciency of the peaks varied from day to day. In Table 9 the val-
idation data are shown.
Repeatability data were also collected for the RP-8e column
and were found to be similar to those of the RP-18e column.
CONCLUSION
For the majority of the new legal and illegal highs, few
analytical methods for their identification are available. The
interest in and the importance of the development of new chiral
separation methods is increasing, as it is unknown if the enantio-
mers of the new substances differ in their pharmacological activ-
ity, metabolic, and pharmacokinetic characteristics. A simple and
easy-to-perform method for the chiral separation of cathinone
and amphetamine derivatives was introduced using HPLC with
a common RP column and sulfated ß-cyclodextrin added to the
mobile phase under isocratic conditions.
Generally, amphetamines showed less interaction with the
sulfated ß-cyclodextrins and the LiChrospher 100 RP-18e, 250
x 4 mm, 5 μm column than cathinone derivatives. So their reten-
tion times are considerably shorter. While all investigated
amphetamines were baseline separated only, some baseline
separations of the cathinone derivatives to their enantiomers
were achieved.
Besides single determination of analytes, simultaneous
enantioseparation was carried out. An attempt was made to
achieve enantioseparation with a couple of compounds. Chiral
resolution of five substances was feasible. In Figure 4, enan-
tiomers of 3-FA, 4-FMA, N,N-dimethylbutylone, 3,4-DMMC,
and pentedrone were separated simultaneously. The methyl
group of 4-FMA makes the molecule more lipophilic and
leads to a longer retention time in comparison to 4-FA, which
is demonstrated in Figure 5.
A further emphasis of this work was to elucidate the influ-
ence of the RP chain length of the column on enantioresolution.
It was checked if the C8 chains are hydrophobic enough to
obtain the same results as with C18 chains. Generally, the use
Chirality DOI 10.1002/chir

TASCHWER ET AL.
chiral phenethylamines and achiral neutral impurities present in illicit
In addition to the single investigation some simultaneous
chiral separations were carried out. Both RP-18e and RP-8e
columns can be used. A comparison of an RP-18e and an
RP-8e column showed that the chiral separation results
suffered partially from a shorter chain length of the RP of
the stationary phase. Moreover, a validation of the method
through performing intra- and interday repeatability and
reproducibility was shown. Also, the presented method was
shown to be applicable to real-life samples.
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Chirality DOI 10.1002/chir