Successful use of a novel lux i-Amylose-1 chiral column for enantioseparation of “legal highs” by HPLC

Article abstract of DOI:10.1002/chir.23135

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as “Legal Highs”. These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010–2018. The CSP, namely Lux 5?μm i-Amylose-1, LC Column 250 x 4.6?mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245?nm and 230?nm, injection volume was 10?μl and flow rate was 1?ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.

Full text of DOI:10.1002/chir.23135

Received: 23 July 2019
DOI: 10.1002/chir.23135
Revised: 24 August 2019
Accepted: 3 September 2019
R E G U L A R A R T I C L E
Successful use of a novel lux® i‐Amylose‐1 chiral column for
enantioseparation of “legal highs” by HPLC
Kian Kadkhodaei | Marlene Kadisch | Martin G. Schmid
Department of Pharmaceutical Chemistry,
Abstract
Institute of Pharmaceutical Sciences,
Bath salts, fumigations, cleaners and air fresheners, behind these terms sub-
stances are hidden, which count as “Legal Highs”. These fancy names are
used to pretend Legal Highs as harmless compounds, to circumvent legal reg-
ulations for marketing as well as to increase the sales. Besides classic illicit
drugs of synthetic origin such as amphetamines, cocaine and MDMA, the
trade of these compounds, also known as new psychoactive substances
(NPS), is not uncommon today. In many countries, NPS are still not subject
to drug control. Among them, there are stimulants such as new amphet-
amine derivatives or cathinones, which possess a chiral centre. Little is
known about the fact that the two possible enantiomers may differ in their
pharmacological effect. The aim of this study was to test a novel HPLC col-
umn for the enantioseparation of a set of 112 NPS coming from different
chemical groups and collected by internet purchases during the years 2010–
2018. The CSP, namely Lux® 5 μm i‐Amylose‐1, LC Column 250 x 4.6 mm,
was run in normal phase mode under isocratic conditions, UV detection
was performed at 245 nm and 230 nm, injection volume was 10 μl and flow
rate was 1 ml/min. With a mobile phase consisting of n‐hexane/isopropanol/
diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers
successfully, for 37 of them baseline resolution was achieved. After increase
of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were
baseline separated. It was found that all separated NPS are traded as racemic
compounds.
Austria
Correspondence
Dr. 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
KEYWORDS
Cathinones, chiral separation, HPLC‐UV, lux i‐Amylose‐1, NPS, Phenylcarbamate
1 | INTRODUCTION
new compounds of different compound classes showed
up. The so called first generation of NPS came on the
During the past 10 years, a significant change in syn-
thetic drug consumption has taken effect. Besides the
classic and well known illicit stimulants such as
amphetamine (Speed), MDMA (Ecstasy) and cocaine,
market within the years 2007–10 including the well‐
known mephedrone (4‐methyl‐methcathinone). Since
these compounds were sold via the internet, no longer
contact to dealers was required. Over time and due to
Chirality. 2019;1–11.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

2
KADKHODAEI ET AL.
the prohibition of mephedrone in several countries, the
second generation of NPS evolved. Since then, names
like AB‐FUBINACA, 4‐MEC and 6‐ABP were irreplace-
able in the drug scene.1,2 In general, their advantage
was a high purity grade, low price and the fact that
these compounds were not scheduled because of their
novelty. Simply, classic chemical structures were slightly
altered with the goal not to count as prohibited drugs.
In the age of the Internet and the ever‐increasing-
influence of social media platforms, to date,
these substances are advertised and distributed via
YouTube channels, drug forums and specially developed
smartphone applications. Buyers and sellers are able to
buy and sell anonymously due to new payment options,
so‐called cryptocurrencies such as bitcoins.3 As a conse-
quence, consumption of NPS increased considerably by
young people worldwide.
a dichlorophenyl derivative of cellulose, was used in nor-
mal phase mode for the chiral separation of NPS.5 Our
group has been working for some time on the enantio-
meric separation of NPS. Satisfactory results were
obtained not only by HPLC with chiral and non‐chiral
columns, but also by means of CE using β‐cyclodextrin
derivatives as chiral selector added to the mobile
phase.5,6,12,20-23
Mainly, the aim of the present contribution was to
establish an isocratic HPLC enantioseparation method
with the presented novel Lux i‐Amylose‐1 column,
avoiding work with gradient steps. Moreover, the method
should be suitable for a lot of these NPS and also poten-
tially for NPS upcoming during the next years.
2 | MATERIALS AND METHODS
2.1 | Chromatographic conditions
Every year a report on the progress and development
of the used drugs is published by the European Monitor-
ing Centre for Drugs and Drug Addiction (EMCDDA).
NPS are definitely a health problem for society. By the
end of 2017, more than 670 NPS were reported to the
EMCDDA for the first time. As examples for synthetic
stimulants, cathinones, amphetamines, benzofurans,
ketamines, morpholines, quinazolines, phenidines,
phenidates and thiophenes count as NPS; also the afore-
mentioned substance classes possess a chiral centre. The
fact that the S‐isomer of amphetamine for example, is
more potent than the R‐isomer and more effective on
ingestion suggests that even chemically similar sub-
stances can exert such a response to the human body. In
2017, Philogene‐Khalid et al. reported the different effects
of the enantiomers of mephedrone. It turned out that the
Analyses were performed using an Agilent 1260 Series Liq-
uid Chromatograph (Agilent Technologies, Waldbronn,
Germany), equipped with an autosampler and a VWL
detector. UV‐data were collected at 230 nm and 254 nm.
Experiments were carried out at ambient temperature
under isocratic conditions with a flow rate of 1 ml/min
and an injection volume of 10 μl.
Data evaluation was performed using a ChemStation
for LC 3D Systems Rev. C. 01.07 SR2 [255] (Agilent Tech-
nologies, Waldbronn, Germany) software.
A Lux® i‐Amylose‐1, 250 x 4.6 mm, 5 μm from
Phenomenex® (Aschaffenburg, Germany) served as sta-
tionary phase.
R‐isomer has
a
positive effect on the reward
system making its abuse understandable.4 Generally, in
the light of so many NPS coming up every year, there is
an urgent need to develop chiral separation methods of
NPS as well as achiral methods to distinguish between
NPS, particularly these of slight difference in chemical
structure, e. g. positional isomers.
2.2 | Chemicals and solutions
Isopropanol (IPA) and n‐hexane were purchased from
VWR (Darmstadt, Germany). Diethylamine (DEA) was
obtained from Sigma‐Aldrich (St. Louis, MO, USA).
In
literature,
various
chromatographic
and electrophoretic methods are presented which can be
used by suitable separation techniques. Successful
attempts were achieved in both chiral and achiral mode,
with high performance liquid chromatography,5-7 gas
chromatography8-10 and capillary electrophoresis.11-13
Not only powders (e. g. seized by police) are used
as samples, meanwhile it is also possible to detect NPS
in biological matrices, such as blood,14,15 urine16,17 and
hair.18,19
2.3 | All chemicals were of analytical
grade
Tested samples were either purchased at different internet
vendor shops from 2010–2018, synthesized for scientific
purposes in our lab or seized by Austrian police. To ensure
the identity of the samples they were checked by GC–MS
and if necessary by NMR, prior the investigation.
Regarding HPLC, different chiral stationary phases
(CSPs) were chosen for successful enantioseparation of
NPS. Recently, a Lux i‐cellulose‐5 column, consisting of
As
a
mobile phase either n‐hexane/IPA/DEA
(90:10:0.1) or if necessary n‐ hexane/IPA/DEA (99:1:0.1)
were used. The solutions were degassed under vacuum.

KADKHODAEI ET AL.
3
TABLE 1 Chemical structures of the NPS investigated in this study
A0: All R = H
A1: R1 = Br
4‐Bromoamphetamine (4‐BA)
A2: R2 = cl
2‐Chloroamphetamine (2‐CA)
A3: R1 = cl; R2 = R4 = OCH3
A4: R1 = F
2,5‐Dimethoxy‐4‐chloroamphetamine (DOC)
4‐Fluoroamphetamine (4‐FA)
A5: R2 = F
2‐Fluoroamphetamine (2‐FA)
A6: R1 = Br; R6 = CH3
A7: R1 = cl; R6 = CH3
A8: R2 = F; R6 = CH3
A9: R1 = F; R6 = CH3
4‐Bromomethamphetamine (4‐BMA)
4‐Chloromethamphetamine (4‐CMA)
2‐Fluoromethamphetamine (2‐FMA)
4‐Fluoromethamphetamine (4‐FMA)
A10: R3 = F; R6 = C2H5
A11: R1 = F; R6 = C2H5
Benzedrone (4‐MBC)
3‐Fluoroethamphetamine (3‐FEA)
4‐Fluoroethamphetamine (4‐FEA)
4‐Chlorophenyl‐2‐(isopropylamino)propan‐1‐on (4‐ClC)
B0: R1 = CH3; R2 = OH
N‐(1‐(2,3‐Dihydrobenzo[b][1,4]dioxin‐6‐yl)propan‐2‐yl)‐N‐
methylhydroxylamin (EFLEA)
C0: All R = H
C1: R2 = C3H6NH2
C2: R3 = C3H6NH2
C3: R2 = C5H11NH
C4: R2 = C4H9NH
5‐(2‐Aminopropyl)benzofurane (5‐APB)
6‐(2‐Aminopropyl)benzofurane (6‐APB)
1‐(Benzofuran‐5‐yl)‐N‐ethylpropan‐2‐amine (5‐EAPB)
1‐(Benzofuran‐5‐yl)‐N‐methylpropan‐2‐amine (5‐MAPB)
D0: R2 = C5H11NH
1‐(Benzofuran‐6‐yl)‐N‐ethylpropan‐2‐amine (6‐EAPB)
1‐(Benzofuran‐5‐yl)‐N‐(2‐methoxybenzyl)propan‐2‐amine (N‐MOB‐5‐APB)
E0: All R = H
E1: R1 = C3H6NH2
E2: R2 = C3H6NH2
1‐(2,3‐Dihydro‐1‐benzofuran‐5‐yl)propan‐2‐mine (5‐APDB)
1‐(2,3‐Dihydro‐1‐benzofuran‐6‐yl)propan‐2‐mine (6‐APDB)
F0: All R = H
F1: R1 = CH3; R3 = CH3
F2: R1 = CH3; R4 = CH3
F3: R1 = CH3; R5 = CH3
F4: R1 = CH3; R3 = R4 = CH3
F5: R1 = CH3; R3 = OCH3
F6: R1 = CH3; R4 = OCH3
F7: R1 = CH3; R5 = cl
F8: R1 = CH3; R4 = cl
F9: R1 = CH3; R3 = cl
F10: R1 = CH3; R3 = C2H5
4‐Methylmethcathinone (4‐MMC)
3‐Methylmethcathinone (3‐MMC)
2‐Methylmethcathinone (2‐MMC)
3,4‐Dimethylmethcathinone (3,4‐DMMC)
4‐Methoxymethcathinone (Methedrone)
3‐Methoxymethcathinone (3‐MeOMC)
2‐Chloromethcathinone (2‐CMC)
3‐Chloromethcathinone (3‐CMC)
4‐Chloromethcathinone (4‐CMC)
4‐Ethylmethcathinone (4‐EMC)
F11: R1 = R3 = CH3; R6 = OCH3 Mexedrone
F12: R1 = CH3; R3 = F
F13: R1 = CH3; R4 = F
F14: R1 = CH3; R5 = F
F15: R1 = CH3; R3 = Br
4‐Fluoromethcathinone (4‐FMC)
3‐Fluoromethcathinone (3‐FMC)
2‐Fluoromethcathinone (2‐FMC)
4‐Bromomethcathinone (4‐BMC)
(Continues)

4
KADKHODAEI ET AL.
TABLE 1 (Continued)
F16: R1 = R6 = CH3
Buphedrone
F17: R1 = R3 = R6 = CH3
F18: R1 = CH3; R6 = C2H5
F19: R1 = C2H5; R5 = cl
F20: R1 = C2H5; R3 = cl
F21: R1 = C2H5; R6 = CH3
F22: R1 = C2H5; R6 = C3H7
F23: R1 = R7 = C2H5
4‐Methylbuphedrone
Pentedrone
3‐Chloroethcathinone (3‐CEC)
4‐Chloroethcathinone (4‐CEC)
N‐ethyl‐buphedrone
N‐ethyl‐hexedrone
Amfepramone
F24: R1 = C2H5; R3 = CH3
F25: R1 = C2H5; R4 = CH3
4‐Methylethcathinone (4‐MEC)
3‐Methylethcathinone (3‐MEC)
F26: R1 = R5 = OCH3; R7 = CH3 DOMC
F27: R1 = R6 = C2H5; R3 = CH3 4‐Methylpentedrone (4‐MPD)
F28: R1 = R6 = C2H5;
N‐ethyl‐Pentedrone
F29: R1 = R6 = C2H5;
R3 = R4 = OCH3
3,4 Dimethoxyphenyl‐2‐ethylaminopentan‐1‐one (DL‐4662)
F30: R1 = R3 = C2H5;
F31: R1 = C2H5
4‐Ethylethcathinone (4‐EEC)
Ethcathinone
F32: R1 = C3H7; R2 = CH3
F33: R1 = C3H7
4‐Methylpropcathinone (4‐MPC)
N‐Propcathinone (NiPP)
F34: R1 = C3H7; R3 = cl
4‐Chlor‐N‐propcathinone (4‐CPRC)
4‐Fluorophenyl‐2‐isopropylaminopentan‐1‐on
F35: R1 = C3H7; R6 = C2H5;
R3 = F
F36: R1 = C4H9; R3 = cl
F37: R3 = CH3
4‐Chlorbutcathinone (4‐CBC)
Nor‐Mephedrone (4‐MC)
G0: All R = H
G1: R1 = CH3
3,4‐Methylendioxymethcathinone (Methylone)
Dimethylone
Butylone
N,N‐dimethylbutylone
Pentylone
Ethylone
G2: R1 = R2 = CH3
G3: R1 = R3 = CH3;
G4: R1 = R2 = R3 = CH3
G5: R1 = CH3; R3 = C2H5
G6: R1 = C2H5
G7: R1 = C2H5; R4 = CH3
G8: R1 = R3 = C2H5
G9: R1 = CH3; R4 = OCH3
5‐Methylethylone (5‐ME)
N‐Ethylpentylone
5‐Methoxymethylone (2‐AIMP)
H0: All R = H
H1: R1 = CH3
H2: R2 = C3H7
5‐PPDI
5‐BPDI
I0: All R = H
I1: R1 = CH3
3,4‐Methylendioxy‐pyrovalerone (MDPV)
MD‐PHP
5‐Dihydrobenzofuran‐pyrovalerone (5‐DBFPV)
Bk‐iVP
TH‐PVP
(Continues)

KADKHODAEI ET AL.
5
TABLE 1 (Continued)
J0: All R = H
α‐PPP
J1: R1 = CH3
M‐PPP
J2: R1 = CH3; R2 = C2H5
J3: R1 = OCH3; R2 = C2H5
J4: R1 = cl; R2 = C2H5
J5: R1 = F; R2 = C2H5
J6: R2 = C2H5
4‐MPrC
4‐MeO‐α‐PVP
4‐cl‐PVP
4‐F‐PVP
α‐PVP
J7: R1 = F; R2 = C3H7
J8: R1 = F; R2 = C4H9
J9: R2 = C5H11
4‐F‐PHP
4‐F‐PV8
PV9
J10: R2 = C6H13
PV10
J11: R1 = CH3; R2 = C3H7
Naphyrone
4‐Methylphenyl‐2‐pyrrolidin‐1‐yl‐hexan‐1‐on (4‐M‐PHP)
K0: All R = H
K1: R1 = CH3; R2 = F
K2: R1 = CH3
K3: R1 = CH3; R2 = cl
K4: R1 = CH3
2‐F‐ketamine
Deschloroketamine (DXE)
N‐ethyl‐ketamine
2‐Oxo‐PCE
K5: R1 = CH3; R3 = OCH3
Methoxetamine
L0: All R = H
L1: R1 = F
L2: R2 = CH3
3‐Fluorophenmetrazine (3‐FPM)
4‐Methylphenmetrazine (4‐MPM)
Diphenidine
Ephinidine
Methoxphenidine
M0: All R = H
M1: R3 = R4 = cl
M2: R1 = CH3
3,4‐Dichloromethylphenidate (3,4‐CTMP)
Ethylphenidate
Isopropylphenidate (IPPD)
M3: R1 = R2 = CH3
M4: R4 = CH3
4‐methyl‐methylphenidate (4‐me‐TMP)
M5: R1 = CH3; R4 = F
4‐Fluoroethylphenidate (4‐FEPH)
N0: R1 = H
N1: R1 = CH3
Methaqualone
Ethaqualone
Methylnaphtidate (HDMP‐28)
(Continues)

6
KADKHODAEI ET AL.
TABLE 1 (Continued)
Methiopropamine
Thiothinone
α‐Pyrrolidinopentiothiophenone (α‐PVT)
3,4‐Dichloro‐N‐[2‐(dimethylamino)cyclohexyl]‐N‐methylbenzamide (U‐47700)
5‐(2‐Aminopropyl)indol (5‐API)
6,7‐Methylenedioxy‐2‐aminotetralin (MDAT)
out that the amount of hexane was too low. Thus, a com-
position of 90:10:0.1 and later a ratio of 99:1:0.1 was used
for further experiments.
2.4 | Sample preparation
No special preparation was required. Each analyte
(0.5 mg), except amphetamines (1 mg), was dissolved in
1 ml IPA, which turned out to be a common procedure.5
To guarantee a complete dissolution the samples were put
into an ultrasonic bath for 1 min.
Regarding compound classes, synthetic cathinones are
represented as the largest group among the tested NPS.
These synthetic cathinones are related to the primary psy-
choactive alkaloid from the khat shrub Catha edulis.
Meanwhile, however, they are distributed via internet
under terms such as bath salts or room odorizers.25,26
The group of cathinones provided very satisfactory
results, as shown in Table 2.
3 | RESULTS AND DISCUSSION
A general trend revealed that the longer the side chain
of N‐substituents, the worse was the chance of baseline
separation. Methcathinones, with a chlorine atom in
ortho‐ or meta‐ position were not separated successfully.
In contrary, very satisfactory separation results were
obtained for methcathinones substituted in para position
with a halogen atom, e. g. 4‐BMC, 4‐CMC and 4‐FMC.
Figure 1 shows the enantioseparation of these three
substances.
After optimization of the mobile phase to 99:1:0.1, even
better resolution factors were achieved for 35 cathinones.
In addition, because of different retention time patterns it
is possible to distinguish between the three position iso-
mers of positional isomers of mephedrone. A correspond-
ing chromatogram is shown in Figure 2.
In order to continue the current research of our group, a
Lux i‐Amylose‐1 column, released in summer 2017 con-
taining a dimethylphenyl‐derivative of amylose has been
used to study the influence of polysaccharide derivatives
used as chiral stationary phases on enantiorecognition.
This chiral selector was already available earlier as
Chiralpak IA traded by Daicel. In an earlier study, suc-
cessful chiral separation of various NPS was shown by
an i‐Cellulose‐5 column with a pronounced ‐I effect. In
contrast,, the Lux i‐Amylose‐1 column promises a + I
effect that generates hydrogen bonds, dipoles and π‐π
interactions due to its methyl groups to ensure efficient
separation of enantiomers.5,24
This contribution reports on the development and
validation of an analytical procedure for the enantios-
eparation of 112 NPS mainly purchased via the internet
and from various chemical classes, which are shown in
Table 1.
Figure 3 demonstrates the simultaneous enantiose-
paration of n‐ethylbuphedrone, 4‐methylbuphedrone
and buphedrone.
Further important representatives of NPS are subst-
ituted amphetamines. They belong as the cathinones to
the phenylalkylamines and differ in the absence of the
A suggested initial mobile phase consisting of n‐hex-
ane/IPA/DEA (85:15:0.1) was tested to find the appropri-
ate composition by means of model compounds. It turned

KADKHODAEI ET AL.
7
TABLE 2 Separation results of the tested cathinones. Conditions:
Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile phase: N‐hexane/
IPA/DEA (90:10:0.1), *(99:1:0.1), ambient temperature, 254 nm,
flow: 1 mL/min
TABLE 2 (Continued)
Cathinones
Alpha‐PVP*
4‐F‐PVP
t1 (min)
5.04
t2 (min)
5.48
αeff
1.30
‐
Rs
1.29
Cathinones
t1 (min)
t2 (min)
αeff
Rs
3.81
n.d.
‐
4‐cl‐PVP*
4 M‐PHP
4‐MPrC*
6.54
7.63
1.40
1.17
1.30
1.68
‐
1.82
3.49
1.36
7.10
‐
4‐MC*
55.14
5.95
5.33
5.47
8.44
7.43
7.30
14.91
6.05
6.46
13.11
16.18
5.83
5.88
6.97
4.92
5.01
4.67
15.40
4.67
4.89
6.19
4.59
6.59
5.61
5.68
6.54
8.27
10.87
4.99
9.73
5.95
3.64
4.98
3.59
9.92
9.10
7.26
9.04
69.93
6.83
5.76
6.33
9.82
7.98
n.d.
1.29
1.28
1.17
1.32
1.25
1.12
‐
11.10
5.38
2.87
5.61
6.02
2.86
‐
5.95
6.49
4‐MMC
6.31
7.12
3‐MMC
4‐Meo‐alpha‐PVP*
4F‐PV8
11.03
3.74
16.03
n.d.
2‐MMC
Methedrone
3‐MeoMC
2‐CMC
PV10
3.76
n.d.
‐
‐
Naphyrone*
4F‐PHP
7.85
8.44
1.14
‐
1.68
‐
3.75
n.d.
3‐CMC*
17.81
6.69
7.13
13.65
16.80
6.40
6.75
10.53
5.68
5.82
5.50
19.07
4.90
5.43
7.61
5.34
7.78
7.41
7.42
7.71
10.03
13.66
5.54
11.68
6.48
n.d.
1.26
1.20
1.18
1.06
1.05
1.19
1.28
1.86
1.36
1.37
1.45
1.31
1.13
1.26
1.58
1.41
1.32
1.95
1.88
1.31
1.39
1.39
1.25
1.32
1.17
‐
5.05
4.02
3.92
1.33
1.24
3.62
5.34
16.32
5.57
5.83
6.17
6.22
1.77
4.04
3.26
5.48
6.42
4.12
4.01
6.37
3.42
5.57
4.01
4.41
3.48
‐
PV9
3.81
n.d.
‐
‐
4‐CMC
Methylone*
Dimethylone*
2‐AIMP*
51.57
15.65
61.59
32.09
9.93
54.27
18.16
67.50
35.70
10.81
28.72
32.29
32.49
6.33
1.06
1.21
1.10
1.13
1.14
1.17
1.12
1.12
1.17
1.14
1.87
1.86
1.37
1.11
1.22
1.22
1.13
1.38
1.96
5.27
3.38
3.69
2.31
4.14
3.38
3.47
2.85
2.19
4.47
4.61
5.25
3.51
1.57
1.56
1.30
5.39
4‐BMC
2‐FMC*
3‐FMC*
Butylone*
N,N‐Dimethylbutylone*
Pentylone*
5‐ME*
4‐FMC
4‐EMC
25.17
29.21
29.39
5.82
Mexedrone
Buphedrone
4‐Methylbuphedrone
Pentedrone
3,4‐DMMC*
3‐CEC
Ethylone*
N‐Ethylpentylone
MDPV
5.42
5.79
MD‐PHP*
bK‐iVP*
5.81
7.72
6.80
9.55
4‐CEC
5‐DBFPV*
DOMC*
11.65
60.27
6.34
14.63
66.25
6.98
N‐ethyl‐buphedrone*
4 MPD
5‐PPDI*
N‐ethyl‐pentedrone
N‐ethyl‐hexedrone*
Amfepramone*
DL‐4662
5‐BPDI*
6.37
7.01
TH‐PVP
4.05
4.21
4‐MBC*
11.30
14.22
3‐MEC*
4‐MEC*
beta keto group.27 No satisfying separation results were
obtained with this compound class, which might be due
to the lack of this keto group. In the group of N‐
methamphetamines, the best separation results were
achieved with bromine‐ as well as chlorine‐substituted
compounds. Despite of an increase of the amount of n‐
hexane, for three amphetamines better results were
obtained only (Table 3).
Another compound class of NPS, the (2‐aminopropyl)
benzofurans (nickname: Benzofuries), were first synthe-
sized in the 90s.28 Together with mephedrone, the first
representatives, 6‐APB and 5‐APB entered the NPS mar-
ket. These empathogenic compounds have structural
4‐EEC
Ethcathinone*
4‐MPC
NiPP
4‐CPRC
5.52
3.76
10.80
9.63
7.63
9.50
1.25
1.21
1.14
1.10
1.10
1.08
3.96
1.39
2.41
1.51
1.22
1.35
4‐F‐NPP
4‐ClC*
4‐Chlorbutcathinone*
Alpha‐PPP*
M‐PPP*
(Continues)

8
KADKHODAEI ET AL.
similarities to the well‐known MDMA and MDA but
their 3,4‐methylenedioxyphenyl ring system is replaced
by a benzofuran ring. However, their psychoactive
effects are similar to these substances.29 Compo-
unds containing a secondary amine group were not
resolved into their enantiomers. Probably, there is a
reduced interaction of the secondary amine group with
FIGURE 2 Simultaneous chiral separation of 3‐MMC (a), 2‐
MMC (B), 4‐MMC (C). Conditions: Lux® i‐Amylose‐1. 250 x
4.6 mm, 5 μm, mobile phase: N‐hexane/IPA/DEA (99:1:0.1),
ambient temperature, 254 nm, flow: 1 mL/min
FIGURE 1 Chiral separation of 4‐BMC (a); 4‐CMC (B); 4‐FMC
(C). Conditions: Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile
phase: N‐hexane/IPA/DEA (90:10:0.1), ambient temperature,
254 nm, flow: 1 mL/min
FIGURE
3
Simultaneous
chiral
separation
of
N‐
Ethylbuphedrone (a), 4‐Methylbuphedrone (B), Buphedrone (C).
Conditions: Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile phase:
N‐hexane/IPA/DEA (99:1:0.1), ambient temperature, 254 nm, flow:
1 mL/min

KADKHODAEI ET AL.
9
TABLE 3 Separation results of the tested amphetamines. Condi-
tions: Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile phase: N‐
hexane/IPA/DEA (90:10:0.1), *(99:1:0.1), ambient temperature,
254 nm, #230 nm, flow: 1 mL/min
TABLE 5 Chiral separation results of divers other NPS condi-
tions: Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile phase: N‐
hexane/IPA/DEA (90:10:0.1), *(99:1:0.1) ambient temperature,
254 nm, flow: 1 mL/min
Amphetamines
t1 (min)
t2 (min)
αeff
Rs
Compounds
t1 (min)
t2 (min)
αeff
Rs
2.75
2‐CA
6.28
6.16
24.83
6.15
5.71
7.13
7.15
6.08
4.36
15.03
n.d.
6.64
6.61
28.01
6.62
5.89
7.38
8.54
6.46
4.68
n.d.
1.10
1.13
1.15
1.14
1.06
1.07
1.40
1.12
1.21
‐
2.14
2.59
4.15
2.96
1.07
0.20
4.28
1.49
0.77
‐
2F‐ketamine
N‐ethyl‐ketamine*
2‐Oxo‐PCE
Methoxetamine
DXE
6.70
8.63
4.46
5.20
6.21
16.84
16.20
12.28
10.19
3.59
3.44
3.33
5.04
4.55
4.13
5.82
10.56
9.31
5.64
9.98
7.91
11.36
27.90
6.09
7.15
9.48
1.12
1.16
1.11
1.04
1.27
1.17
1.19
1.60
1.84
‐
4‐BA
2.28
1.38
0.76
5.72
4.00
4.05
15.30
18.69
‐
Doc*#
4.63
2‐FA*
5.29
4‐FA*
7.13
4‐FMA*#
2‐FMA #
4‐BMA*
4‐CMA*
EFLEA*
4‐FEA*
3‐FEA*
4‐MPM
19.05
18.57
18.00
16.36
n.d.
3‐FPM*
Metaqualone
Etaqualone
Ephenidine
Methoxphenidine
Diphenidine
3,4‐CTMP
n.d.
n.d.
‐
‐
n.d.
n.d.
n.d.
‐
‐
5.75
1.32
1.27
‐
4.73
1.91
‐
Ethylphenidate
IPPD
5.02
n.d.
the chiral selector. However, compounds with a primary
amine showed very good separation results, given in
Table 4.
Furthermore, attempts were made to achieve
enantioseparation for different other substance classes
of NPS. These comprises partially legal derivatives of
phenidates, morpholines, thiophenes, phenidines, keta-
mines and quinazolines. Separation results are displayed
in Table 5.
Finally, the intra‐ and interday repeatability by means
of one substance each out of three different compound
classes, was performed. 4‐MMC, 5‐APB and DXE were
used for this purpose. Relative standard deviation
HDMP‐28
6.43
1.20
1.17
1.26
1.10
1.37
1.34
1.55
1.05
‐
3.35
2.04
2.75
0.87
3.58
4.19
12.85
1.64
‐
4‐me‐TMP*
4‐FEPH
11.72
10.77
5.85
Alpha‐PVT
Thiothinone*
Methiopropamine*
U‐47700
12.32
9.35
16.104
29.23
n.d.
5‐API
MDAT
TABLE 4 Separation results of the tested Benzofuries conditions:
Lux® i‐Amylose‐1. 250 x 4.6 mm, 5 μm, mobile phase: N‐hexane/
IPA/DEA (90:10:0.1), *(99:1:0.1) ambient temperature, 254 nm,
flow: 1 mL/min
(RSD) for intraday repeatability of the retention
times was about 0.20% and of the resolution factor was
2.90% or less. The RSD for the interday precision of
the retention times was 0.60% for all substances and less
than 3.30% for the resolution factor. The results of each
test compound are shown in Table 6. Based on
previous work, a comparison between the column used
at the time, a Lux i‐cellulose‐5, and the current Lux i‐
Amylose‐1 can be made as follows:5 Generally Lux i‐Cel-
lulose‐5 column turned out to be superior for
enantioseparation of the chemical groups of (2‐
aminopropyl)benzofurans and amphetamines. However,
for the group of cathinones, including pyrovalerones bet-
ter results were achieved by a Lux i‐ Amylose‐ 1
column.
Benzofuries
t1 (min)
t2 (min)
αeff
Rs
5.05
5‐APB
8.58
10.80
13.48
5.16
9.71
11.36
16.40
n.d.
1.20
1.07
1.29
‐
5‐APDB
N‐MOB‐5‐APB*
5‐MAPB
5‐EAPB*
6‐EAPB
2.14
4.88
‐
6.61
6.81
1.07
‐
0.82
‐
4.29
n.d.
6‐APB
8.55
9.68
1.20
1.58
4.92
12.95
6‐APDB
17.63
26.15

10
KADKHODAEI ET AL.
TABLE 6 Repeatability data by means of 4‐MMC, 5‐APB, DXE including retention time and resolution
t1 (min)
t2 (min)
Rs
4‐MMC
Intraday n = 5
Interday n = 5
5‐APB
6.44 0.01, RSD = 0.19%
6.42 0.04, RSD = 0.61%
7.48 0.02, RSD = 0.21%
7.44 0.06, RSD = 0.77%
5.8 0.07, RSD = 1.22%
5.8 0.08, RSD = 1.44%
Intraday n = 5
9.51 0.01, RSD = 0.14%
9.50 0.05, RSD = 0.55%
10.87 0.01, RSD = 0.12%
10.85 0.07, RSD = 0.61%
5.2 0.08, RSD = 1.60%
5.2 0.04, RSD = 0.86%
Interday n = 5
DXE
Intraday n = 5
6.53 0.00, RSD = 0.07%
6.54 0.04, RSD = 0.61%
7.44 0.01, RSD = 0.07%
7.47 0.07, RSD = 0.97%
5.0 0.13, RSD = 2.68%
5.3 0.17, RSD = 3.16%
Interday n = 5
REFERENCES
4 | SUMMARY AND CONCLUSIONS
1. Zawilska JB, Andrzejczak D. Next generation of novel psychoac-
tive substances on the horizon ‐ a complex problem to face. Drug
Alcohol Depend. 2015;157:1‐17.
Referring to the EMCDDA, in average every week one
new NPS gets reported in Europe, meanwhile about 670
of these substances are monitored by 2017.30 Policy
makers face a major challenge to protect public health
and curb the illicit drug market. Through analysis and
determination of the chemical structure and enantio-
meric status, it is possible to avoid imminent dangers
and thus to prevent dangerous side effects of the com-
pounds still easily available via the internet. Since many
NPS are chiral and few is known about the effect of the
pure enantiomers, there is a huge need for development
of enantioseparation methods for NPS.
In this work, it has been found that the column Lux®
i‐Amylose‐1 from Phenomenex® released in 2017 offers a
good possibility to separate a large number of chiral NPS
from different substance classes into their enantiomers
with one method. HPLC‐UV under isocratic conditions
and in normal phase mode turned out to be optimal.
95 out of 112 substances were shown to be traded as
racemic mixtures. Excellent separation results were
achieved with cathinones obtaining high resolution
factors.
2. Brandt SD, Sumnall HR, Measham F, Cole J. Analyses of
second‐generation “legal highs” in the UK: initial findings. Drug
Test Anal. 2010;2(8):377‐382.
3. Miliano C, Margiani G, Fattore L, De Luca M. Sales and adver-
tising channels of new psychoactive substances (NPS): internet,
social networks, and smartphone apps. Brain Sci. 2018;8
(7):123.
4. Philogene‐Khalid HL, Simmons SJ, Nayak S, et al.
Stereoselective differences between the reinforcing and motiva-
tional effects of Cathinone‐derived 4‐Methylmethcathinone
(Mephedrone) in self‐administering rats. ACS Chem Nerosci.
2017;8(12):2648‐2654.
5. Kadkhodaei K, Forcher L, Schmid MG. Separation of enantio-
mers of new psychoactive substances by high‐performance
liquid chromatography. J Sep Sci. 2018;41(6):1274‐1286.
6. Weiß JA, Kadkhodaei K, Schmid MG. Indirect chiral separation
of 8 novel amphetamine derivatives as potential new psychoac-
tive compounds by GC‐MS and HPLC. Science and Justice.
2017;57(1):6‐12.
7. Perera RWH, Abraham I, Gupta S, et al. Screening approach,
optimisation and scale‐up for chiral liquid chromatography of
cathinones. J Chromatogr a. 2012;1269:189‐197.
It is necessary to secure further complementary
methods for the separation of enantiomers of upcoming
NPS of next generations as a further development of
existing methods. The application of newly developed chi-
ral selectors and the analysis of newly launched NPS will
continue to be necessary in the future also to be able to
produce pure enantiomers for clinical studies of these
novel compounds.
8. Weiß JA, Mohr S, Schmid MG. Indirect chiral separation of new
recreational drugs by gas chromatography‐mass spectrometry
using trifluoroacetyl‐L‐prolyl chloride as chiral derivatization
reagent. Chirality. 2015;27(3):211‐215.
9. Mohr S, Weiß JA, Spreitz J, Schmid MG. Chiral separation of
new cathinone‐ and amphetamine‐related designer drugs by
gas chromatography – mass spectrometry using trifluoroacetyl‐
L‐prolyl chloride as chiral derivatization reagent. J Chromatogr
a. 2012;1269:352‐359.
ORCID
10. Taschwer M, Ebner E, Schmid MG. Test purchase of new syn-
Martin G. Schmid https://orcid.org/0000-0003-0055-660X
thetic tryptamines via the internet: identity check by GC‐MS

KADKHODAEI ET AL.
11
and separation by HPLC. Journal of Applied Pharmaceutical Sci-
ence. 2016;6(1):28‐34.
21. Mohr S, Taschwer M, Schmid MG. Chiral separation of
cathinone derivatives used as recreational drugs by HPLC‐UV
using a CHIRALPAK® AS‐H column as stationary phase. Chiral-
ity. 2012;24(6):486‐492.
11. Cheng W‐C, Lee W‐M, Chan M‐F, Tsui P, Dao K. Enantiomeric
separation of methamphetamine and related analogs by capil-
lary zone electrophoresis: intelligence study in routine
22. Taschwer M, Hofer MG, Schmid MG. Enantioseparation of
benzofurys and other novel psychoactive compounds by CE
and sulfobutylether β‐cyclodextrin as chiral selector added to
the BGE. Electrophoresis. 2014;35(19):2793‐2799.
methamphetamine seizures.
J
Forensic Sci. 2002;47
(6):1248‐1252.
12. Hägele JS, Schmid MG. Enantiomeric separation of novel psy-
choactive substances by capillary electrophoresis using (+)‐18‐
crown‐6‐tetracarboxylic acid as chiral selector. Chirality.
2018;30(8):1019‐1026.
23. 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.
13. Liau AS, Liu JT, Lin LC, et al. Optimization of a simple
method for the chiral separation of methamphetamine
and related compounds in clandestine tablets and urine sam-
ples by β‐cyclodextrin modified capillary electrophoresis: a
complementary method to GC‐MS. Forensic Sci Int. 2003;134
(1):17‐24.
24. Phenomenex. https://www.phenomenex.com/Products/HPLC
Detail/lux.
25. Salomone A, Gazzilli G, Di Corcia D, Gerace E, Vincenti M.
Determination of cathinones and other stimulant, psychedelic,
and dissociative designer drugs in real hair samples. Anal
Bioanal Chem. 2016;408(8):2035‐2042.
14. Vårdal L, Askildsen HM, Gjelstad A, Øiestad EL, Edvardsen
HME, Pedersen‐Bjergaard S. Parallel artificial liquid membrane
extraction of new psychoactive substances in plasma and whole
26. Concheiro M, Anizan S, Ellefsen K, Huestis MA. Simultaneous
quantification of 28 synthetic cathinones and metabolites in
urine by liquid chromatography‐high resolution mass spectrom-
etry. Anal Bioanal Chem. 2013;405(29):9437‐9448.
blood.
J Chromatogr B Analyt Technol Biomed Life Sci.
2017;1048:77‐84.
15. Tomczak E, Woźniak MK, Kata M, Wiergowski M, Szpiech B,
Biziuk M. Blood concentrations of a new psychoactive substance
4‐chloromethcathinone (4‐CMC) determined in 15 forensic
cases. Forensic Toxicology. 2018;36(2):476‐485.
27. Schmid MG. CHAPTER 11 Optical Detection of NPS Internet
Products via HPLC‐DAD Systems: A Selective Review. In: Light
in Forensic Science: Issues and Applications. The Royal Society
of Chemistry. 2018; 301‐332.
16. Kim JY, Suh S, Park J, In MK. Simultaneous determination of
28. Monte AP, Marona‐Lewicka D, Cozzi NV, Nichols DE. Synthesis
amphetamine‐related new psychoactive substances in urine by
and pharmacological examination of Benzofuran, Indan, and
gas chromatography–mass spectrometry.
2018;42(9):605‐616.
J Anal Toxicol.
Tetralin analogues of 3,4‐(Methylenedioxy)amphetamine.
Med Chem. 1993;36(23):3700‐3706.
J
17. Borovcová L, Pauk V, Lemr K. Analysis of new psychoactive
substances in human urine by ultra‐high performance supercrit-
ical fluid and liquid chromatography: validation and
comparison. J Sep Sci. 2018;41(10):2288‐2295.
29. Welter J, Kavanagh P, Meyer MR, Maurer HH. Benzofuran ana-
logues of amphetamine and methamphetamine: studies on the
metabolism and toxicological analysis of 5‐APB and 5‐MAPB
in urine and plasma using GC‐MS and LC‐(HR)‐MSn techniques.
Anal Bioanal Chem. 2015;407(5):1371‐1388.
18. Meyer MR, Maurer HH. Review: LC coupled to low‐ and high‐
resolution mass spectrometry for new psychoactive substance
screening in biological matrices ‐ where do we stand today? Anal
Chim Acta. 2016;927:13‐20.
30. European Drug Report 2018: Trends and Developments.; 2018.
19. Boumba VA, Di Rago M, Peka M, Drummer OH, Gerostamoulos
D. The analysis of 132 novel psychoactive substances in human
hair using a single step extraction by tandem LC/MS. Forensic
Sci Int. 2017;279:192‐202.
How to cite this article: 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;1–11.
https://doi.org/10.1002/chir.23135
20. Mohr S, Pilaj S, Schmid MG. Chiral separation of cathinone
derivatives used as recreational drugs by cyclodextrin‐modified
capillary electrophoresis. Electrophoresis. 2012;33(11):1624‐1630.