Determination of the chiral status of different novel psychoactive substance classes by capillary electrophoresis and β-cyclodextrin derivatives

Article abstract of DOI:10.1002/chir.23268

Besides the abuse of well-known illicit drugs, consumers discovered new synthetic compounds with similar effects but minor alterations in their chemical structure. Originally, these so-called novel psychoactive substances (NPS) have been created to circumvent law of prosecution because of illicit drug abuse. During the past decade, such compounds came up in generations, the most popular compound was a synthetic cathinone derivative named mephedrone. Cathinones are structurally related to amphetamines; to date, more than 120 completely new derivatives have been synthesized and are traded via the Internet. Cathinones possess a chiral center; however, only little is known about the pharmacology of their enantiomers. However, NPS comprise further chiral compound classes such as amphetamine derivatives, ketamines, 2-(aminopropyl)benzofurans, and phenidines. In continuation of our project, a cheap and easy-to-perform chiral capillary zone electrophoresis method for enantioseparation of cathinones presented previously was extended to the aforementioned compound classes. Enantioresolution was achieved by simply adding native β-cyclodextrin, acetyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, or carboxymethyl-β-cyclodextrin as chiral selector additives to the background electrolyte. Fifty-one chiral NPS served as analytes mainly purchased from online vendors via the Internet. Using 10 mM of the aforementioned β-cyclodextrins in a 10 mM sodium phosphate buffer (pH 2.5), overall, 50 of 51 NPS were resolved. However, chiral separation ability of the selectors differed depending on the analyte. Additionally, simultaneous enantioseparations, the determination of enantiomeric migration orders of selected analytes, and a repeatability study were performed successfully. It was proven that all separated NPS were traded as racemic mixtures.

Full text of DOI:10.1002/chir.23268

Received: 6 February 2020
DOI: 10.1002/chir.23268
Revised: 19 June 2020
Accepted: 1 July 2020
R E G U L A R A R T I C L E
Determination of the chiral status of different novel
psychoactive substance classes by capillary electrophoresis
and β-cyclodextrin derivatives
Johannes S. Hägele | Eva-Maria Hubner | Martin G. Schmid
Department of Pharmaceutical Chemistry,
Abstract
Institute of Pharmaceutical Sciences,
University of Graz, Graz, Austria
Besides the abuse of well-known illicit drugs, consumers discovered new syn-
thetic compounds with similar effects but minor alterations in their chemical
structure. Originally, these so-called novel psychoactive substances (NPS) have
been created to circumvent law of prosecution because of illicit drug abuse. Dur-
ing the past decade, such compounds came up in generations, the most popular
compound was a synthetic cathinone derivative named mephedrone. Cathinones
are structurally related to amphetamines; to date, more than 120 completely new
derivatives have been synthesized and are traded via the Internet. Cathinones
possess a chiral center; however, only little is known about the pharmacology of
their enantiomers. However, NPS comprise further chiral compound classes
such as amphetamine derivatives, ketamines, 2-(aminopropyl)benzofurans, and
phenidines. In continuation of our project, a cheap and easy-to-perform chiral
capillary zone electrophoresis method for enantioseparation of cathinones pres-
ented previously was extended to the aforementioned compound classes.
Enantioresolution was achieved by simply adding native β-cyclodextrin, acetyl-
β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, or carboxymethyl-β-cyclodextrin
as chiral selector additives to the background electrolyte. Fifty-one chiral NPS
served as analytes mainly purchased from online vendors via the Internet. Using
10 mM of the aforementioned β-cyclodextrins in a 10 mM sodium phosphate
buffer (pH 2.5), overall, 50 of 51 NPS were resolved. However, chiral separation
ability of the selectors differed depending on the analyte. Additionally, simulta-
neous enantioseparations, the determination of enantiomeric migration orders
of selected analytes, and a repeatability study were performed successfully. It
was proven that all separated NPS were traded as racemic mixtures.
Correspondence
Martin G. Schmid, Department of
Pharmaceutical Chemistry, Institute of
Pharmaceutical Sciences, University of
Graz, Universitätsplatz 1, A-8010 Graz,
Austria.
Email: martin.schmid@uni-graz.at
K E Y W O R D S
2-hydroxypropyl-β-cyclodextrin, acetyl-β-cyclodextrin, capillary electrophoresis, carboxymethyl-
β-cyclodextrin, native β-cyclodextrin, novel psychoactive substances
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2020 The Authors. Chirality published by Wiley Periodicals LLC.
Chirality. 2020;1–17.
wileyonlinelibrary.com/journal/chir
1

2
HÄGELE ET AL.
1 | INTRODUCTION
is added to the electrolyte. Successful use of cyclodextrins
(CDs), macrocyclic antibiotics, or chiral crown ethers as
chiral selectors has been shown. Particularly native CDs
and diverse substituted derivatives turned out to be used
successfully for this purpose. As chiral separation princi-
ple, formation of inclusion complexes and additional
interactions of the moieties of its derivatives have to be
taken into account. Different complex stability constants
of the CD–analyte complexes and consequently differing
electrophoretic mobilities are responsible for chiral
discrimination.
The goal of this study was the continuation of our
project to extent a cheap and easy-to-perform chiral CE
method for enantioseparation of cathinones presented
previously to enantioseparation of amphetamine deriva-
tives, ketamines, 2-(aminopropyl)benzofurans, and phe-
nidines.27 Four different β-CDs, namely, native β-CD,
acetyl-β-CD, 2-hydroxypropyl-β-CD, and carboxymethyl-
β-CD, served as chiral selectors. Additionally, this
method should give information about the enantiomeric
status of real-life samples and possibly the origin of the
substances.
Novel psychoactive substances (NPS) gained an enor-
mous popularity during the past decade. As the main rea-
son for the fast and easy distribution of NPS, the
technological progress of the Internet can be referred.
The substances are mainly synthesized in China or other
Asian countries, and they can be purchased from diverse
online vendors titulated as “birdcage cleaners,” “plant
food,” or “research chemicals” via the World Wide Web.
Their labels, for example, “Not for human consumption”
and doubtful purity and identity data online, guarantee
low risk of prosecution for online vendors. Dubious infor-
mation about ways of consumption and the effectiveness
of the compounds consumers gain, for example, via
YouTube channels or drug fora.1,2 The United Nations
Office on Drugs and Crime (UNODC) reported an
increase of NPS in 111 countries worldwide by the end of
2017. However, Asia, Europe, and North America have
the lead in the number of substances reported.3 The sec-
ond important institution for European concerns, the
European Monitoring Centre for Drugs and Drug Addic-
tion (EMCDDA), reported in their annual review mean-
while more than 730 different NPS derivatives and give a
special emphasize to four main categories: synthetic can-
nabinoids, stimulants, opioids, and antidepressants.2 NPS
are mainly synthesized by more or less simple modifica-
tions of molecular structures of well-known classic syn-
thetic illicit drugs. Lead substances like, for example,
native amphetamine, are therefore chemically modified
to bypass national and international drug controls. Con-
sumers replace prohibited illegal compounds with this
legal “imitations.” A huge number of them possess a chi-
ral center yielding in two possible enantiomeric forms.
Their pharmacological effects like, that is, their potencies
and effects, may differ as it is well known from diverse
active pharmaceutical ingredients. Referred examples of
lead substances showing different effects are, for exam-
ple, methcathinone, mephedrone, amphetamine, and
methamphetamine.4–7 The fact of a potentially different
pharmacological behavior of NPS makes chiral method
development indispensable. A further goal is the develop-
ment of chiral separation methods to check the enantio-
meric status of real-life samples. Up to now, some articles
report enantiomeric separations of NPS. Separation tech-
niques like high-performance liquid chromatography
(HPLC),8–20 capillary electrophoresis (CE),21–31 gas chro-
matography (GC),32–36 and supercritical fluid chromatog-
raphy (SFC)37–41 are cited in literature. These separation
techniques are used to check the compounds as solid
samples or in biological matrices.42–45 Among them, CE
turned out to be a cheap, easy-to-perform, and reliable
separation technique. Advantageously, the chiral selector
2 | MATERIALS AND METHODS
2.1 | Chemicals and solutions
Native β-CD and 2-hydroxypropyl-β-CD (degree of substi-
tution: 0.6) were from Fluka Chemika AG (Buchs, Swit-
zerland). Acetyl-β-CD (degree of substitution: 1.0) and
carboxymethyl-β-CD (degree of substitution: 0.5) were
purchased from Wacker-Chemie GmbH (Salzburg, Aus-
tria). Sodium phosphate and diluted phosphoric acid
were bought from Merck KGaA (Darmstadt, Germany).
Milli-Q-Water (HiPerSolv CHROMANORM) was from
VWR International (Vienna, Austria). All reagents were
of analytical grade.
NPS were mostly not commercially available from
official suppliers due to their novelty. As a consequence,
they were bought from diverse online stores like, for
example, www.purechemicals.net, www.Get-RC.to or
www.rc-supply.to. Additionally, some analytes represent
real-life samples seized by Austrian police or were syn-
thesized in microscale amounts in our laboratory. Pure
enantiomers were prepared via a semipreparative HPLC
method (unpublished results) in our laboratory in milli-
gram scale for scientific purposes.
All analytes were characterized by GS-electron impact
mass spectrometry (GC–MS) and, if necessary, nuclear
magnetic resonance (NMR) prior to experiments.
BGEs were prepared by dissolving 10 mM of β-CD or
β-CD derivative, 10 mM sodium phosphate adjusted with

HÄGELE ET AL.
3
TABLE 1 Psychoactive compounds and their chemical structure investigated in this study
A
A0: All R = H
A1: R1 = Br
A2: R2 = Cl
Amphetamine (( )-1-Phenylpropan-2-amine)
4-Bromoamphetamine (4-BA, ( )-1-(4-Bromophenyl)propan-2-amine)
2-Chloroamphetamine (2-CA, ( )-1-(2-Chlorophenyl)propan-2-amine)
2,5-Dimethoxy-4-chloroamphetamine (DOC, ( )-1-(4-Chloro-2,5-dimethoxyphenyl)propan-2-amine)
A3: R1 = Cl;
R2 = R4 = OCH3
A4: R1 = F
4-Fluoroamphetamine (4-FA, ( )-1-(4-Fluorophenyl)propan-2-amine)
3-Fluoroamphetamine (3-FA, ( )-1-(3-Fluorophenyl)propan-2-amine)
2-Fluoroamphetamine (2-FA, ( )-1-(2-Fluorophenyl)propan-2-amine)
4-Nitroamphetamine (4-NA, ( )-1-(4-Nitrophenyl)propan-2-amine)
A5: R3 = F
A6: R2 = F
A7: R1 = NO2
A8: R1 = SH
4-Methylthioamphetamine (MTA, ( )-1-[4-(Methylsulfanyl)phenyl]propan-2-amine)
2,5-Dimethoxyamphetamine (2,5-DMA, ( )-1-(2,5-Dimethoxyphenyl)propan-2-amine)
3,4-Dimethoxyamphetamine (3,4-DMA, ( )-1-(3,4-Dimethoxyphenyl)propan-2-amine)
4-Methoxyamphetamine (( )-1-(4-Methoxyphenyl)propan-2-amine)
A9: R2 = R4 = OCH3
A10: R1 = R4 = OCH3
A11: R1 = OCH3
A12: R1 = Br;
4-Bromo-2,5-dimethoxyamphetamine (DOB, ( )-4-Bromo-2,5-dimethoxyamphetamine)
R2 = R4 = OCH3
A13: R6 = C2H5
N-Ethylamphetamine (( )-N-Ethyl-1-phenylpropan-2-amine)
A14: R6 = C3H6Cl
A15: R6 = CH3
Mefenorex (( )-3-Chloro-N-(1-methyl-2-phenylethyl)propan-1-amine)
N-Methamphetamine (( )-N,α-dimethylphenethylamine)
A16: R1 = Br; R6 = CH3
A17: R2 = Cl; R6 = CH3
A18: R1 = Cl; R6 = CH3
A19: R2 = F; R6 = CH3
A20: R1 = F; R6 = CH3
B
4-Bromomethamphetamine (4-BMA, ( )-1-(4-Bromophenyl)-N-methylpropan-2-amine)
2-Chloromethamphetamine (2-CMA, ( )-1-(2-Chlorophenyl)-N-methylpropan-2-amine)
4-Chloromethamphetamine (4-CMA, ( )-1-(4-Chlorophenyl)-N-methylpropan-2-amine)
2-Fluoromethamphetamine (2-FMA, ( )-1-(2-Fluorophenyl)-N-methylpropan-2-amine)
4-Fluoromethamphetamine (4-FMA, ( )-1-(4-Fluorophenyl)-N-methylpropan-2-amine)
N-Methyl-3,4-methylendioxy-amphetamine (MDMA, ( )-1-(Benzo [1,3]
dioxol-5-yl)-N-methyl-propan-2-amine)
C
( )-N-(1-(2,3-Dihydro-benzo[b][1,4]dioxin-6-yl)propan-2-yl)-N-methylhydroxylamine (EFLEA)
D
(Continues)

4
HÄGELE ET AL.
TABLE 1 (Continued)
D0: All R = H
D1: R1 = C3H6NH2
D2: R2 = C3H6NH2
D3: R3 = C3H6NH2
D4: R4 = C3H6NH2
D5: R2 = C5H11NH
D6: R3 = C5H11NH
D7: R2 = C4H9NH
E
4-(2-Aminopropyl)-benzofurane (4-APB, ( )-1-(1-Benzofuran-4-yl)propan-2-amine)
5-(2-Aminopropyl)-benzofurane (5-APB, ( )-1-(1-Benzofuran-5-yl)propan-2-amine)
6-(2-Aminopropyl)-benzofurane (6-APB, ( )-1-(1-Benzofuran-6-yl)propan-2-amine)
7-(2-Aminopropyl)-benzofurane (7-APB, ( )-1-(1-Benzofuran-7-yl)propan-2-amine)
( )-1-(Benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB)
( )-(1-(Benzofuran-6-yl)-N-ethylpropan-2-amine) (6-EAPB)
( )-1-(Benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB)
( )-1-(Benzofuran-5-yl)-N-(2-methoxybenzyl)-propan-2-amine (N-MOB-5-APB)
F
F0: All R = H
F1: R1 = C3H6NH2
F2: R2 = C3H6NH2
G
( )-1-(2,3-Dihydro-1-benzofuran-5-yl)propan-2-amine (5-APDB)
( )-1-(2,3-Dihydro-1-benzofuran-6-yl)propan-2-amine (6-APDB)
G0: All R = H
G1: R2 = Cl
Ketamine (( )-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone)
N-Ethyl-ketamine (( )-2-(2-Chlorophenyl)-2-(ethylamino)cyclohexanone)
2-Oxo-PCE (( )-2-(Ethylamino)-2-phenylcyclohexan-1-one)
G2: R1 = CH3; R2 = Cl
G3: R1 = CH3
G4: R2 = F
4-Fluoroketamine (( )-2-(2-Fluorophenyl)-2-(methylamino)cyclohexanone)
2-MeO-Ketamine (( )-2-(2-Methoxyphenyl)-2-(methylamino)cyclohexanone)
Methoxetamine (( )-2-(3-Methoxyphenyl)-2-(ethylamino)cyclohexanone)
G5: R2 = OCH3
G6: R1 = CH3; R3 = OCH3
H

HÄGELE ET AL.
5
TABLE 1 (Continued)
Diphenidine (( )-1-(1,2-Diphenylethyl)piperidine)
I
Ephenidine (( )-N-Ethyl-1,2-diphenylethylamine)
J
Methoxphenidine (( )-2-Methoxy-1-(1,2-Diphenylethyl)piperidine)
K
K1: R1 = H
K2: R1 = CH3
L
Thiopropamine (( )-1-(Thiophen-2-yl)-2-aminopropane)
Methiopropamine (( )-1-(Thiophen-2-yl)-2-methylaminopropane)
Thiothinone (( )-2-(Methylamino)-1-(thiophen-2-yl)propan-1-one)
M
N
( )-5-(2-Aminopropyl)-indole (5-API)
Mephtetramine (MTTA, (( )-2-(Methylaminomethyl)-3,4-dihydro-2H-naphthalen-1-one))
O
(Continues)

6
HÄGELE ET AL.
diluted phosphoric acid in Milli-Q-Water (pH 2.5). Prior
to the chiral separation studies, solutions were degassed
by ultrasonification and filtered through a 0.45-μm pore
size nylon filter (Carl Roth, Karlsruhe, Germany).
were also filtered through a 0.45-μm pore size filter (Carl
Roth, Karlsruhe, Germany).
3 | RESULTS AND DISCUSSION
2.2 | Instrumentation
All NPS and pure NPS enantiomers comprising different
compound classes were collected since 2010. They were
purchased via the Internet, produced by chemical synthe-
sis and by semipreparative methods (unpublished
results), or were seized by Austrian police. The chemical
structures of the analyzed substances are given in
Table 1.
For CE measurements, a fully automated 3DCE system
(Agilent Technologies, Waldbronn, Germany) equipped
with a diode array detector was used. All experiments
were carried out at ambient temperature (25ꢀC). CE was
performed in 50 μm ID-fused silica capillaries
(MicroQuartz, Munich, Germany) with a total length of
68.5 cm and an effective length of 60 cm. UV absorption
was measured at 209 nm. Before and after each measure-
ment, the capillary was flushed with 0.2 M sodium
hydroxide, water, and BGE, respectively. All samples
were injected by applying a pressure of 10 mbar * 5 s on
the inlet vial.
Based on the work of Merola et al.22 and a further
method optimization of our group,27 10 mM β-CD in a
10 mM sodium phosphate buffer (pH 2.5) was found out
to be appropriate as final BGE. A voltage of 30 kV to the
cathode was applied during the chiral separation experi-
ments. The electrolyte conditions of native β-CD were
additionally
transferred
to
the
derivatives
2-hydroxypropyl-β-CD, acetyl-β-CD, and carboxymethyl-
β-CD. However, the applied voltages were adjusted to
29 kV to the cathode for 2-hydroxypropyl-β-CD and
acetyl-β-CD and 22 kV to the cathode for carboxymethyl-
β-CD to create a stable current under the fastest possible
separation conditions. A scheme of all used CDs is given
in Figure 1.
2.3 | Sample preparation
Because the samples consisted mainly of hydrochloric
acid salts, each sample was dissolved in Milli-Q-Water in
a concentration of 1.0 mg/ml. To accelerate the dissolving
processes, the samples were given in an ultrasonic bath
for 1 min before filtration. After ultrasonification, they
Using the stated conditions, a set of 51 NPS including
23 amphetamine derivatives, 10 2-(aminopropyl)
TABLE 1 (Continued)
( )-6,7-Methylendioxy-2-aminotetraline (MDAT)
P
α-Pyrrolidinopentiothiophenone (α-PVT, ( )-2-(Pyrrolidin-1-yl)-1-(thiophen-2-yl)pentan-1-one)
Q
Q1: R1 = H
Methaqualone (( )-3-(2-Methylphenyl)-2-methylquinazolin-4-one)
Ethaqualone (( )-3-(2-Ethylphenyl)-2-methylquinazolin-4-one)
Q2: R1 = CH3

HÄGELE ET AL.
7
FIGURE 1 Chemical structures applied
β-cyclodextrin derivatives (already published in
Hägele et al.27)
benzofurans, six ketamine derivatives, three phenidines,
and nine other NPS was tested. Overall, 50 of the
51 analytes were partially or baseline separated by at least
one of the different CD-electrolytes. Measurements did
not exceed 48 min. A complete overview of all chiral
separation data is shown in Tables 2–6 in detail. An
electropherogram of a single chiral separation of ( )-
1-(benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB)
using the chiral selector carboxymethyl-β-CD is given
in Figure 2. Chiral separation data within the different
compound classes were satisfactory. All substances
except one, namely, MDAT (6,7-methylenedioxy-
2-aminotetraline), were resolved in their enantiomers
partially or completely.
showed the best chiral separation results regarding reso-
lution in combination with slightly extended migration
times.
Regarding the analyzed ketamine derivatives shown
in Table 4, resolution ranged from 0.6 to 5.6. All keta-
mine derivatives were resolved in their enantiomers with
at least one of the chosen CDs within 22 min. Only
acetyl-β-CD was able to separate all analytes and there-
fore turned out to be the most potent chiral selector for
In Table 2, all chiral separation results of the tested
amphetamine derivatives are given. All substances were
chirally discriminated with at least one chiral selector
within 33 min. The majority of the analytes could be
detected within a migration time less than 25 min. Reso-
lution factors ranged from 0.5 to 7.2. Overall,
carboxymethyl-β-CD gave the best chiral separation data
regarding chromatographic resolution. However, analysis
times using this chiral selector were longer than using
the other CD derivatives. Additionally, for some analytes
like, for example, DOB and DOC, only with acetyl-β-CD
chiral separations were observed. A potential reason for
this observation could be a higher affinity of their molec-
ular structure to the acetyl moiety of the CD derivative.
Table 3 shows all separation data of the tested
2-(aminopropyl)benzofuran derivatives. Again, all deriva-
tives out of this analyte group could be chirally separated
by at least one of the chosen CDs within 48 min. Mainly,
the analytes were detected within 15 min. Resolution for
the separated 2-(aminopropyl)benzofuran enantiomers
varied from 0.5 to 6.4. Again, carboxymethyl-β-CD
FIGURE 2 Single chiral separation of 5-MAPB. Conditions:
10 mM carboxymethyl-β-cyclodextrin, 10 mM sodium phosphate,
pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC,
applied voltage: 22 kV to cathode, injection: 10 mbar for 5 s,
sample: 1 mg/ml in water

8
HÄGELE ET AL.
TABLE 2 Chiral separation data of 23 amphetamine and methamphetamine derivatives
Compound
t1 (min)
8.42
t2 (min)
8.49
n.d.
α
Rs
0.7
-
Chiral selector
β-CD
Applied voltage (kV)
+30
Amphetamine
1.009
-
11.28
7.63
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
7.70
17.30
11.25
n.d.
1.009
1.029
1.009
-
0.8
3.7
0.8
-
+29
16.81
11.16
14.43
10.49
25.09
9.22
+22
4-Bromoamphetamine
2-Chloroamphetamine
2-Fluoroamphetamine
3-Fluoroamphetamine
4-Fluoroamphetamine
4-Nitroamphetamine
MTA
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
10.59
25.67
9.50
10.28
8.44
18.18
8.94
n.d.
1.009
1.023
1.030
1.018
1.024
1.056
1.012
-
0.9
2.4
2.6
0.6
1.4
5.7
1.0
-
+29
+22
+30
10.09
8.24
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
17.21
8.83
+22
+30
10.59
7.80
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
7.88
16.50
8.98
11.60
7.68
15.91
8.59
10.13
7.90
17.44
n.d.
1.010
1.041
1.011
1.007
1.009
1.024
1.010
1.008
1.009
1.023
-
0.7
3.8
1.2
0.6
0.8
2.3
0.7
1.4
0.9
3.8
-
+29
15.85
8.88
+22
+30
11.52
7.62
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
15.53
8.51
+22
+30
10.05
7.83
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
17.04
8.63
+22
+30
13.38
8.04
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
n.d.
-
-
+29
18.17
12.60
14.33
10.95
27.27
8.99
18.33
n.d.
1.009
-
1.3
-
+22
+30
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
n.d.
-
-
+29
27.71
9.07
n.d.
1.016
1.009
-
1.2
0.8
-
+22
2,5-DMA
+30
10.45
8.28
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
n.d.
-
-
+29
18.97
9.25
19.34
n.d.
1.020
-
3.1
-
+22
DOB
+30
9.13
9.19
n.d.
1.007
-
0.8
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
8.03
+29
15.54
8.44
n.d.
-
-
+22
DOC
n.d.
-
-
+30
9.80
9.86
n.d.
1.007
-
0.6
-
Acetyl β-CD
HP-β-CD
+29
8.30
+29

HÄGELE ET AL.
9
TABLE 2 (Continued)
Compound
t1 (min)
t2 (min)
α
Rs
Chiral selector
Applied voltage (kV)
15.17
n.d.
-
-
CM-β-CD
+22
3,4-DMA
7.56
8.56
n.d.
n.d.
-
-
β-CD
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
7.71
n.d.
-
-
13.14
10.50
12.78
8.88
13.23
10.58
n.d.
1.007
1.008
-
1.0
0.7
-
4-MeO-amphetamine
N-Methamphetamine
4-BMA
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
8.96
1.009
1.030
1.012
1.013
1.018
1.031
1.009
-
0.7
2.3
0.9
0.9
1.4
4.8
0.7
-
21.54
8.91
22.19
9.02
12.38
8.07
12.55
8.22
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
15.99
12.12
15.27
11.24
26.56
9.33
16.49
12.24
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
11.39
26.96
9.64
1.014
1.015
1.033
1.035
1.034
1.073
1.008
-
1.0
1.5
2.2
1.2
2.1
6.0
0.9
-
2-CMA
11.00
8.58
11.38
8.87
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
18.84
9.62
20.22
9.69
4-CMA
13.55
9.26
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
9.34
1.009
1.028
1.025
1.013
1.023
1.059
1.009
1.017
1.008
1.020
1.013
-
0.6
2.0
1.8
0.5
1.6
5.5
0.8
1.1
0.8
1.9
0.9
-
23.20
9.14
23.84
9.37
2-FMA
11.21
8.18
11.35
8.36
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
18.12
8.61
19.19
8.68
4-FMA
12.64
7.78
12.86
7.85
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
15.42
11.02
14.67
10.57
25.66
9.74
15.73
11.16
n.d.
MDMA
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
10.83
26.45
9.85
1.024
1.031
1.011
1.014
1.019
1.032
1.010
1.014
2.2
2.9
0.8
0.9
1.3
4.2
0.8
1.0
N-Ethylamphetamine
Mefenorex
13.24
9.30
13.42
9.48
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
17.64
9.99
18.20
10.10
14.35
14.16
Acetyl β-CD
(Continues)

10
HÄGELE ET AL.
TABLE 2 (Continued)
Compound
t1 (min)
t2 (min)
α
Rs
Chiral selector
Applied voltage (kV)
10.04
10.22
1.018
1.6
HP-β-CD
+29
21.47
13.19
16.10
14.42
30.53
22.27
n.d.
1.037
-
3.7
-
CM-β-CD
β-CD
+22
+30
+29
+29
+22
EFLEA
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
14.61
32.38
1.013
1.061
1.0
7.2
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.
ketamines under the stated conditions. Again, the reason
for this observation might be a stronger interaction of the
analyte enantiomers with the acetyl moieties of this CD
derivative.
Furthermore, phenidine derivatives were tested
(Table 5). They also serve as NPS and are available at
different internet vendors. Again, all compounds
were chirally discriminated within 34 min by at least
one chiral selector. Mainly, the substances were
detected within 16 min. Resolution factors ranged from
0.9 to 2.4.
2-aminotetraline (MDAT) were separated within 30 min.
Resolution factors ranged from 0.5 to 6.4. Carboxymethyl-
β-CD turned out to be superior as chiral selector.
In addition to the single chiral separation experi-
ments, attempts of simultaneous enantioseparations were
carried out successfully. An example of a simultaneous
chiral separation is shown in Figure 3. Five of the six
investigated ketamine derivatives were resolved in one
single measurement by acetyl-β-CD as chiral selector
additive.
Besides ketamine being abused as hallucinogenic,
derivatives have entered the NPS market. For example,
methoxetamine is available via internet platforms
since 2009.
Table 6 shows the chiral separation results of diverse
subcategories of NPS, including, for example, thiophene
derivatives. All substances except 6,7-methylenedioxy-
FIGURE 3 Simultaneous enantioseparation of five different ketamine derivatives. Conditions: 10 mM acetyl-β-cyclodextrin, 10 mM
sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, applied voltage: 29 kV to cathode, injection: 10 mbar
for 5 s, sample: 1 mg/ml in water

HÄGELE ET AL.
11
TABLE 3 Chiral separation results of a set of 10 benzofuran derivatives
Compound
t1 (min)
7.79
t2 (min)
7.85
α
Rs
0.7
0.9
0.8
1.9
0.7
-
Chiral selector
β-CD
Applied voltage (kV)
+30
4-APB
1.008
1.011
1.009
1.017
1.009
-
9.61
9.71
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
8.13
8.21
+29
12.50
12.77
15.68
12.62
13.45
9.79
12.72
12.88
n.d.
+22
5-APB
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
12.85
13.66
9.89
1.018
1.016
1.011
1.009
1.015
1.016
1.013
-
1.6
3.3
0.8
0.8
1.4
3.4
0.9
-
+29
+22
5-APDB
5-EAPB
5-MAPB
N-MOB-5-APB
6-APB
+30
12.60
10.00
13.95
11.46
15.50
12.40
14.65
10.83
14.89
11.49
14.32
12.81
15.72
14.03
40.56
11.52
15.71
13.13
14.41
11.32
15.23
11.81
26.00
13.37
16.06
12.71
47.24
7.67
12.72
10.15
14.18
11.61
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
+22
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
12.65
14.90
11.00
15.04
11.75
14.56
n.d.
1.020
1.017
1.016
1.010
1.022
1.017
-
1.2
6.4
0.9
0.7
1.5
3.5
-
+29
+22
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
+22
+30
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
n.d.
-
-
+29
40.84
11.63
16.07
13.35
14.65
11.43
n.d.
1.007
1.009
1.023
1.016
1.017
1.009
-
0.8
0.8
2.1
2.2
3.5
0.5
-
+22
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
+29
+22
6-APDB
6-EAPB
7-APB
+30
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
12.05
26.74
n.d.
1.021
1.028
-
1.4
2.5
-
+29
+22
+30
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
+29
12.93
48.00
n.d.
1.018
1.016
-
1.2
1.2
-
+29
+22
+30
9.41
9.47
1.006
-
0.5
-
Acetyl β-CD
HP-β-CD
CM-β-CD
+29
8.28
n.d.
+29
13.77
13.98
1.015
1.4
+22
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.

12
HÄGELE ET AL.
TABLE 4 Chiral separation results of a set of six ketamine derivatives
Compound
t1 (min)
9.95
t2 (min)
10.04
9.81
α
Rs
0.8
0.6
-
Chiral selector
β-CD
Applied voltage (kV)
Ketamine
1.009
1.008
-
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
9.73
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
8.41
n.d.
19.44
10.13
10.21
8.48
n.d.
-
-
N-Ethylketamine
Methoxetamine
2-Oxo-PCE
n.d.
-
-
10.30
n.d.
1.010
-
0.8
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
20.00
10.74
10.37
9.34
20.38
10.87
11.07
9.41
1.019
1.012
1.068
1.007
1.027
1.006
1.054
-
2.1
1.0
4.7
0.6
2.6
0.6
4.5
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
21.47
9.30
22.05
9.35
9.54
10.06
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
8.56
15.46
9.17
15.62
n.d.
1.011
-
1.1
-
2-F-Ketamine
2-MeO-Ketamine
8.87
9.06
1.022
-
1.6
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
8.33
n.d.
14.31
10.86
10.10
9.21
n.d.
-
-
11.06
10.92
9.28
1.018
1.081
1.007
1.028
1.4
5.6
0.7
1.2
Acetyl β-CD
HP-β-CD
CM-β-CD
20.95
21.52
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.
TABLE 5 Chiral separation results of a set of three phenidine derivatives
Compound
t1 (min)
12.49
16.48
16.93
33.38
13.54
15.48
13.74
27.96
7.99
t2 (min)
n.d.
α
Rs
-
Chiral selector
β-CD
Applied voltage (kV)
Diphenidine
-
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
17.32
33.94
n.d.
1.023
1.017
-
2.4
1.5
-
Methoxyphenidine
Ephenidine
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
13.91
28.42
8.05
1.012
1.017
1.008
1.016
1.025
1.022
1.0
1.8
0.9
1.2
1.7
1.9
15.08
12.25
32.31
15.33
12.56
33.00
Acetyl β-CD
HP-β-CD
CM-β-CD
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.

HÄGELE ET AL.
13
TABLE 6 Chiral separation results of a set of nine other psychoactive substances
Compound
t1 (min)
20.63
25.35
17.25
28.04
10.26
19.82
19.33
26.41
9.96
t2 (min)
n.d.
α
Rs
-
Chiral selector
β-CD
Applied voltage (kV)
Metaqualone
-
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
+30
+29
+29
+22
25.77
n.d.
1.017
-
1.7
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
n.d.
-
-
Etaqualone
α-PVT
n.d.
-
-
19.99
n.d.
1.009
-
1.0
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
26.83
10.07
n.d.
1.016
1.011
-
2.6
1.1
-
10.11
8.80
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
n.d.
-
-
19.98
7.62
20.64
7.68
1.033
1.007
-
3.8
0.6
-
Thiothinone
Methiopropamine
Thiopropamine
5-APi
8.96
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
7.08
7.14
1.008
1.012
1.025
1.008
1.009
1.021
1.007
-
0.8
1.4
2.1
0.6
0.7
2.1
0.7
-
12.49
8.10
12.65
8.30
10.37
7.41
10.46
7.48
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
12.72
8.10
12.99
8.16
11.36
7.13
n.d.
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
n.d.
-
-
13.95
12.27
14.58
10.42
26.43
9.51
14.26
12.61
n.d.
1.022
1.027
-
2.2
2.5
-
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
10.70
28.28
9.62
1.027
1.070
1.012
1.015
1.007
1.014
-
1.7
6.4
1.2
1.6
0.5
1.4
-
MTTA
10.65
8.54
10.82
8.60
Acetyl β-CD
HP-β-CD
CM-β-CD
β-CD
18.71
4.72
18.98
n.d.
MDAT
5.36
n.d.
-
-
Acetyl β-CD
HP-β-CD
CM-β-CD
4.77
n.d.
-
-
8.06
n.d.
-
-
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.
Furthermore, enantiomeric migration orders (EMOs)
and enantiomeric purity checks of the analytes amphet-
amine, diphenidine, and methoxyphenidine were
performed. As chiral selectors, carboxymethyl-β-CD
served for amphetamine and methoxyphenidine.
Hydroxypropyl-β-CD served for the substance
diphenidine. Experiments were carried out by spiking
racemic samples with pure enantiomers. In case of

14
HÄGELE ET AL.
FIGURE 4 Enantiomeric migration order (EMO) determination of diphenidine. Conditions: 10 mM hydroxypropyl-β-cyclodextrin,
10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, applied voltage: 22 kV to cathode, injection:
10 mbar for 5 s, sample: 1 mg/ml in water
TABLE 7 Repeatability data including retention time and resolution
Chiral selector
Model substance
Applied voltage (kV)
Repeatability
t1 (min)
t2 (min)
Rs
β-CD
2-FMA
+30
Intraday n = 5
9.20 0.07
RSD = 0.5%
9.43 0.09
RSD = 0.7%
1.8 0.1
RSD = 2.5%
Interday n = 5
Intraday n = 5
Interday n = 5
Intraday n = 5
Interday n = 5
Intraday n = 5
Interday n = 5
9.27 0.13
RSD = 0.9%
9.50 0.16
RSD = 1.1%
1.7 0.2
RSD = 4.9%
Acetyl-β-CD
HP-β-CD
Methoxetamine
Diphenidine
2-CMA
+29
+29
+22
10.55 0.18
RSD = 1.0%
11.26 0.19
RSD = 1.1%
4.6 0.2
RSD = 2.7%
10.59 0.34
RSD = 2.1%
11.31 0.35
RSD = 2.1%
4.5 0.2
RSD = 3.6%
16.98 0.06
RSD = 0.3%
17.41 0.0.10
RSD = 0.5%
2.4 0.2
RSD = 4.7%
17.04 0.14
RSD = 0.7%
17.48 0.16
RSD = 0.9%
2.4 0.2
RSD = 5.1%
CM-β-CD
18.99 0,15
RSD = 0.6%
20.43 0.21
RSD = 0.7%
6.0 0.2
RSD = 2.5%
19.04 0.20
RSD = 0.7%
20.51 0.29
RSD = 1.1%
5.9 0.3
RSD = 3.7%
Note: Conditions: 10 mM chiral selector, 10 mM sodium phosphate, pH 2.5 adjusted with phosphoric acid, cassette temperature: 25ꢀC, injec-
tion: 10 mbar for 5 s, sample: 1 mg/ml in water.
amphetamine, the (−)-enantiomer migrated faster than
its corresponding (+)-enantiomer. In contrast to the
EMO observation of amphetamine, all other tested
substances showed a reversed EMO. In Figure 4, the
EMO determination of diphenidine is shown. Racemic
sample was spiked with its (+)-enantiomer.
Finally, a repeatability study was carried out. For
each chiral selector a representative model compound

HÄGELE ET AL.
15
3. UNODC. Understanding the synthetic drug market: the NPS
factor; 2018. https://www.unodc.org/documents/scientific/
Global_Smart_Update_2018_Vol.19.pdf
4. Glennon RA, Young R, Martin BR, Dal Cason TA.
Methcathinone (“CAT”): an enantiomeric potency comparison.
Pharmacol Biochem Behav. 1995;50(4):601-606.
5. Gregg RA, Baumann MH, Partilla JS, et al. Stereochemistry of
mephedrone neuropharmacology: enantiomer-specific behav-
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172(3):883-894.
6. Rasmussen LB, Olsen KH, Johansen SS. Chiral separation and
quantification of R/S-amphetamine, R/S-methamphetamine,
R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-
EI-MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;
842(2):136-141.
was chosen. Five intraday and interday measurements
each were performed with satisfactory results. All repeat-
ability data are given in Table 7 in detail.
4 | CONCLUSION
In recent years, the popularity and the number of NPS
have been growing constantly worldwide. A lot of these
compounds are chiral and may potentially differ in their
pharmacological behavior. Therefore, analytical method
development regarding chiral separation methods is of
great interest.
With the presented study, a reliable and cheap chiral
CE method to separate NPS enantiomers out of various
different substance classes was presented. As chiral
selectors, native β-CD and three of its derivatives (acetyl-
β-CD, 2-hydroxypropyl-β-CD, and carboxymethyl-β-CD)
were investigated. With the chosen separation condi-
tions, all in all, 50 of 51 tested NPS were resolved in
their enantiomers within a maximum of 48 min. Resolu-
tion factors with respect to all tested β-CD derivatives
ranged from 0.5 to 7.2. In direct comparison,
carboxymethyl-β-CD was superior to the other investi-
gated β-CDs regarding chromatographic resolution.
Additionally, the presented chiral selectors were found
to be applicable for simultaneous enantioseparations and
to determine enantiomeric elution orders of the studied
NPS classes.
7. Jirovsky D, Lemr K, Sevcik J, Smysl B, Stransky Z. Metham-
phetamine—properties and analytical methods of enantiomer
determination. Forensic Sci Int. 1998;96(1):61-70.
8. Schmid MG. Optical detection of NPS Internet products via
HPLC-DAD systems. Light Forensic Sci Issues Appl. 2017;11:
301-332.
9. Taschwer M, Ebner E, Schmid MG. Test purchase of new
synthetic tryptamines via the Internet: identity check by GC-
MS and separation by HPLC. J Appl Pharm Sci. 2016;6(1):
28-34.
10. Taschwer M, Grascher J, Schmid MG. Development of an
enantioseparation method for novel psychoactive drugs by
HPLC using a Lux® Cellulose-2 column in polar organic phase
mode. Forensic Sci Int. 2017;270:232-240.
11. Taschwer M, Seidl Y, Mohr S, Schmid MG. Chiral separation of
cathinone and amphetamine derivatives by HPLC/UV using
sulfated β-cyclodextrin as chiral mobile phase additive. Chiral-
ity. 2014;26(8):411-418.
As for cathinones, also for the NPS of other com-
pound classes, it was found that they were traded as race-
mic mixtures, which was confirmed also by means of
other chiral selectors in previous studies.8,10–12,14,21,24,32,46
Generally, there are few data, whether the effect of the
enantiomers differs.
In future, the investigated method can be an addi-
tional useful separation technique of further upcoming
NPS derivatives as well as to check the enantiomeric
composition of real-life samples.
12. Kadkhodaei K, Forcher L, Schmid MG. Separation of enantio-
mers of new psychoactive substances by high performance liq-
uid chromatography. J Sep Sci. 2018;41(6):1274-1286.
13. Li L, Lurie IS. Regioisomeric and enantiomeric analyses of
24 designer cathinones and phenethylamines using ultra high
performance liquid chromatography and capillary electropho-
resis with added cyclodextrins. Forensic Sci Int. 2015;254:
148-157.
14. Kadkhodaei K, Kadisch M, Schmid MG. Successful use of a
novel lux® i-Amylose-1 chiral column for enantioseparation of
“legal highs” by HPLC. Chirality. 2019;32(1):42-52. https://doi.
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ORCID
Martin G. Schmid https://orcid.org/0000-0003-0055-
660X
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How to cite this article: Hägele JS, Hubner E-M,
Schmid MG. Determination of the chiral status of
different novel psychoactive substance classes by
capillary electrophoresis and β-cyclodextrin
derivatives. Chirality. 2020;1–17. https://doi.org/10.
1002/chir.23268