Separation of enantiomers and positional isomers of novel psychoactive substances in solid samples by chromatographic and electrophoretic techniques – A selective review

Journal of Chromatography A 1624 (2020) 461256

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Separation of enantiomers and positional isomers of novel
psychoactive substances in solid samples by chromatographic and
electrophoretic techniques – A selective review
Martin G. Schmid∗, Johannes S. Hägele
Department of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 1, 8010 Graz, Austria

a r t i c l e

i n f o

Article history:
Received 28 February 2020
Revised 13 May 2020
Accepted 15 May 2020
Available online 21 May 2020
Keywords:
Enantioseparation
Novel psychoactive substances
Solid samples
HPLC
Capillary electrophoresis
GC

a b s t r a c t

Novel Psychoactive Substances (NPS) represent an alternative to established illicit drugs. They are traded
via the internet and exhibit small alterations in their chemical structure to circumvent law, however,
their psychotropic effects are comparable. There is still poor knowledge about side effects and health
risks. By the end of 2018, 730 NPS were reported to EMCDDA (European Monitoring Centre for Drugs and
Drug Addiction). Among different compound classes, many NPS are chiral and few publications deal with
the different pharmacological and toxicological properties of their pure enantiomers. Therefore, analytical
method development concerning enantioseparation of NPS is of great interest. Chiral separation protocols
of established illicit drugs have been transferred for NPS, selected examples are given as well. Different methods for enantioseparation of NPS comprising mainly stimulating drugs such as cathinones, pyrovalerones, amphetamines, ketamines, (2-aminopropyl) benzofuranes, phenidines, phenidates, morpholines and thiophenes are reviewed. Moreover, chiral resolution of some cannabinomimetics by HPLC is
presented. Chromatographic and electrophoretic techniques such as GC, HPLC, SFC, CE and CEC are discussed and in some cases compared. Mainly, solid samples either purchased from internet vendors, seized
by police or collected from patients in hospitals are subject to analysis. Chiral selectors used for HPLC are
listed in a Table. It was shown that particularly stimulating drugs are traded as racemic mixtures, which
is not the case with cannabinomimetics. Mainly, HPLC and CE were used for enantioseparation of NPS.
© 2020 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license.
( />
M. G. Schmid:
Johannes S. Hägele:
• Selective review about enantioseparation of Novel Psychoactive
Substances.
• Approaches for chiral GC, HPLC, SFC, CE and CEC are presented.
• Comprehensive Tables for chiral methods are given for GC,
HPLC, SFC, CE and CEC.
1. Introduction
Consumption behavior of illicit drugs changed considerably during the last ten years because of a disadvantageous situation for
drug consumers: During the first decade of the 21st century, the
quality of well-established illicit drugs such as cocaine was poor.
As a consequence, particularly drug consumers being open for
∗

Corresponding author.

E-mail address: (M.G. Schmid).

new experience watched out for cheap and effective alternatives.
At this time the triumphal introduction of so called novel psychoactive substances (NPS) started worldwide: As a first generation, synthetic cathinones emerged. These stimulating compounds
structurally related to amphetamines were synthesized in huge
amounts in clandestine labs mainly in China prior to worldwide
shipping [1]. Upon arrival e. g. in Europe, they were traded in
headshops and via the internet. Although they exhibit effects related to illicit drugs, they were not prohibited because of lacking restriction by a drug-law. The most impressive representative
of this compound class was mephedrone (nicknames: drone, MCAT, White Magic and meow meow), the 4-methyl derivative of Nmethcathinone (4-MMC) coming up in 2003 and spreading worldwide within a short period of time. This compound was scheduled
in different countries and finally by a general prohibition in the EU
in 2010.
In the sequel, dozens of structurally slightly altered cathinones entered the drug market as following NPS generations, accompanied by other compound classes such as empathogenic 2-

/>0021-9673/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. ( />

2

M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

aminopropyl benzofurans (“Benzofuries”), synthetic tryptamines,
phenethylamines, synthetic cannabinoids, arylamines or piperazines. To date, any classic illicit drug can be replaced by NPS.
To disguise their potential harm, NPS are generally sold as “Legal Highs”, “Research Compounds”, “Leisure Drugs”, “Plant food”,
“Bird’s cage cleaners” or “Room odorizers”.
In the recent 8 years, 730 not scheduled synthetic new compounds were reported to the European Monitoring Centre for
Drugs and Drug Addiction (EMCDDA) [1]. The World Wide Web
still remains the main distributor of NPS because of the ease to
purchase drugs via the internet, regardless of taking use of the
Clearnet or the Darknet. Comprehensive information about each
compound regarding its application, onset and desired effects are
spread e. g. via drug fora, social networks and YouTube channels and even Smartphone Apps are available [2]. In 2017, EMCDDA classified NPS in four potential dangerous categories: synthetic cannabinoids, synthetic stimulants, new opioids and new antidepressants [1]. Since these compounds are completely new because of their design to circumvent law, there is few knowledge

about their pharmacological properties. Regarding their chemical
structure, there are hundreds of NPS possessing a chiral centre and
it is well known that nearly all of them are traded as racemic mixtures. Additionally, several NPS exist as positional isomers, mostly
bearing side chains on a phenyl ring in ortho-, meta or para- constitution. This means e. g. that the compound mephedrone comprises 6 different forms: 3 positional forms with two enantiomers
each (Fig. 1).
After successful elucidation of a novel chiral compound, this
fact of chirality implies the further question, to which enantiomer
the drug effect of each single compound is related. For this reason, it is of great importance and interest to conduct pharmacological and toxicological studies of structure-activity relationship
as well as chiral analytical method development with respect to
enantiomeric separation and isolation of NPS enantiomers.
Up to now, examples of different potencies or effects of individual illicit drug enantiomers reported in literature are given for
example for mephedrone [3], amphetamine (Speed) [4], methamphetamine (Crystal Meth) [5] or methcathinone [6,7]. Achiral and
chiral determination of seized NPS as internet products was published as a book chapter [8]. Enantioselectivity and chiral resolution of synthetic cathinones as a huge substance class of chiral NPS
was recently reviewed by Silva et al. [9].
This article is intended to give a survey of the progress of
method development to resolve enantiomers of NPS using various
chromatographic and electrophoretic techniques such as GC, HPLC,
SFC (supercritical fluid chromatography), CE and CEC (capillary
electrochromatography). The presented applications are mainly devoted to solid samples either purchased from internet vendors,
seized by police or collected from patients in hospitals. In general, samples are available in a variety of different forms, including powders, liquids, tablets, nasal sprays and even sprayed on
papers as known from lysergic acid diethylamide tickets. Also,
NPS may require a low dose only because of their high potency
[10].

2.1. Enantioseparation of novel psychoactive substances by gas
chromatography

2. Enantioseparation of novel psychoactive substances by
chromatographic and electrophoretic techniques

2.2. Enantioseparation of novel psychoactive substances by HPLC

In the following Chapters, different approaches for separation
of chiral NPS are reviewed. Regarding chromatographic separation
techniques, HPLC has been used more frequently than GC. Due to
its simplified use, also SFC has been used for this purpose. As electrophoretic methods, a broad spectrum of CE data has been published and a further publication dealing with CEC has appeared.

Gas chromatography is known as a fast and reliable analytical technique. To date, only two publications have come up showing the successful use of a GC-capillary coated with a chiral selector representing the direct chiral separation attempt of NPS
enantiomers [11,12]. More frequently, indirect chiral separation
has been applied successfully by means of chiral derivatization
reagents. However, in this case, an additional sample preparation
step is necessary. Herein, the enantiomeric pair of the NPS is transferred to diastereomers and can be separated on conventional achiral GC-capillaries, which are less expensive and more temperature
resistant compared to chiral GC-capillaries.
Before NPS emerged, an indirect chiral separation method
was published by Le Belle et al. [13] in 1995: They applied
(R)-(+)-α -methoxy-α -(trifluoromethyl)phenylacetic acid (MTPA) as
a chiral derivatization reagent for classic illicit drugs such
as N-methamphetamine, methcathinone, ephedrine and pseudoephedrine. All compounds were separated on a common
DB-5 column simultaneously within 15 min. As an alternative, trifluoracetyl-L-prolyl chloride (L-TPC) turned out as a useful chiral derivatization reagent to separate amphetamine, Nmethamphetamine, 3,4-methylene-dioxy-amphetamine (MDA) and
3,4-methylene-dioxy-methamphetamine (MDMA) [14]. In both
cases, an MS-unit served as detection unit. In 2011, Drake et al.
presented the direct separation mode by means of a γ -CD (cyclodextrin) chiral column (Chiraldex G-PN). This phase sized 30
m × 0.25 mm (coating film thickness 0.12 μm) and incorporated a phase consisting of a 2,6-di-O-pentyl-3-propionyl derivative of γ -cyclodextrin. N-methamphetamine, ephedrine and pseudoephedrine were derivatized with trifluoro acetic anhydride as
achiral reagent prior to analysis [12]. One year later, successful indirect separation of a broad spectrum of NPS was published by
Mohr et al. [15]. They derivatized 14 amphetamine derivatives and
18 cathinone derivatives by means of L-TPC and separated them on
a common 30 m HP-5-MS achiral capillary. Fig. 2 shows a simultaneous GC-MS measurement of six cathinones after chiral derivatization. Later, this method was adapted by Alremeithi et al. for
successful simultaneous determination of 14 cathinone-like NPS
in urine and plasma [16]. Physiological samples were spiked with
nikethamide as internal standard prior to solid phase extraction

and measurement. Weiß et al. synthesized 8 amphetamine derivatives with the background that they could emerge in future on
the drug scene [17]. Analytes were derivatized with MTPA or (1R)(−)-menthylchloroformate prior to analysis on a HP-5MS column.
Shortly later, chlor-methamphetamine showed up in an Ecstasy
pill instead of MDMA in Vienna for the first time [18]. Recently,
fresh samples of Catha edulis were checked for their cathinone content by Dhabbah [19]. After derivatization by menthyl chloroformate, the two cathinone enantiomers were quantified. It turned
out that S-cathinone, the stronger psychoactive stereoisomer, exhibited an increasing concentration from lower to upper stems of
the plant. Interestingly, both enantiomers are present in all parts of
freshly harvested Khat plants in varying nonracemic ratios [19]. An
overview of approaches given in this Chapter is listed in Table 1.

Since HPLC is used very frequently for enantioseparation of
drugs, natural compounds, pesticides etc., it is obviously a good
choice for NPS chirality studies as well. There is a much broader
spectrum of chiral stationary phases available for HPLC rather than
for GC. Before the broad emerge of the first generation of NPS took
place, a strategy for enantioresolution of amphetamine and related
compounds was proposed applicable for aqueous solutions, urine


M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

3

Fig. 1. Chemical structures of the six possible isomeric forms of methyl-methcathinone.

and plasma samples given in comprehensive Tables [20]. For chiral
resolution of NPS by HPLC, mainly chiral columns were used with
different chiral selectors.
In 1997, Aboul Enein’s group employed a chiral (S)-18-crown6-ether phase for the enantioseparation of four phenylalkylamines, namely cathinone, amphetamine, norephedrine and norphenylephrine [21]. Another crown ether HPLC column based
on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector found application by Hyun’s group for chiral resolution

of methoxyphenamine (2-methoxy-N-methamphetamine) and its
analogues using polar-organic conditions [22]. Chiral discrimination is proposed to take place because of the interaction of the
protonated secondary amino group of the analyte and oxygens and
carboxylic groups of the chiral selector as well as enantioselective
inclusion complexation. Obviously, the use of crown ethers as chiral selector is not restricted to primary amines.
Reports about enantioseparation of NPS came up in 2012:
Perera et al. presented a screening approach for chiral resolution of mephedrone and related cathinones of the first generation available at this time [23]. HPLC columns with packed
5 μm particles bearing different selectors were tested by different chromatographic modes. Generally, these chromatographic
modes are dependent on the chiral columns. The most frequently
used are reversed-phase mode, meaning the use of aqueous solutions with methanol or acetonitrile, polar-organic or polar-ionic

mode based on mixtures of rather hydrophilic organic compounds
without water or the normal-phase mode with non-polar solvents. While the use of mobile phases in reversed-phase mode
was not successful, mephedrone was resolved on a Whelk-O1
column with a mobile phase consisting of isopropanol-hexanetrifluoroacetic acid-triethylamine. In the same year, Mohr et al. employed a Chiralpak AS-H column comprising amylose tris [(S)-α methylbenzylcarbamate] as chiral selector coated on 5 μm silica
particles [24]. Polysaccharides contain a helical structure with hydrophobic character available for hydrophobic parts of molecules.
Further binding forces of hydrophilic moieties of the analytes such
as hydrogen bondings and dipole-dipole interactions with the chiral selector can be considered. Again, the normal phase modus
was chosen and enantioseparation was shown for 20 cathinone
derivatives including mephedrone purchased on internet platforms
in comparison with 3 amphetamine analogues. Interestingly, resolution power was poor for amphetamines compared to their
cathinone analogues, amphetamine itself was not separated. This
might be due to the lack of the beta-keto group in amphetamine
molecules being responsible for further interactions. Up to 5 cathinones were resolved in their enantiomers in one run and enantiomer elution order was shown for the parent compound methcathinone [24]. In the sequel, other CSPs were used as follow:
Silva’s group presented enantioseparation of further 14 NPS samples purchased in Portuguese smart shops prior to closing of these


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M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

Fig. 2. Total ion chromatogram (TIC) of the simultaneous chiral separation of (1) cathinone, (2) ethcathinone, (3) mephedrone, (4) 4-methylbuphedrone, (5) methe- drone
and (6) methylone all as L-TPC derivatives by indirect GC. Reprinted from [15] with permission.
Table 1
Overview of approaches for enantioseparation of NPS by GC.
Brand name of
column

Mode
TM

Astec Chiraldex
G-PN
Astec ChiraldexTM
G-PN
DB-5, J and
W-Scientific

Direct
Direct

Chiral selector/derivatization reagent

Separated NPS

Ref.

2,6-Di-O-pentyl-3-propionyl

Amphetamine, methamphetamine, ephedrine and

pseudoephedrine
Methamphetamine, ephedrine and pseudoephedrine

[11]

N-Methamphetamine, methcathinone, ephedrine and
pseudoephedrine

[13]

Amphetamine, N-methamphetamine, MDA and MDMA

[14]

Amphetamine derivatives and cathinone derivatives
Cathinones in urine and plasma
Amphetamine derivatives

[15]
[16]
[17]

Cathinone enantiomers in parts of freshly harvested Khat
plants
Clephedrone (4-chloro-methcathinone)
DL-4662

[18]

γ -cyclodextrin

2,6-Di-O-pentyl-3-propionyl

Indirect

γ -cyclodextrin
(R)-(+)-α -Methoxy-α -

Indirect

(trifluoromethyl)phenylacetic
acid
L-TPC

DB-17, J and
W-Scientific
HP-5-MS
HP-5-MS
HP-5MS

Indirect
Indirect
Indirect

HP-5 MS

Indirect

L-TPC
L-TPC

MTPA or
(1R)-(−)-menthylchloroformate
(1R)-(−)-Menthylchloroformate

HP-5-MS
HP-5-MS

Indirect
Indirect

L-TPC
L-TPC

[12]

[59]
[60]


M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

shops because of prohibition [25]. 3,4-Methylenedioxypyrovalerone
(MDPV), a NPS likely introducing neuroadaptive changes and behavioral effects [26] as well as further 8 cathinones were tested
by different CSPs such as Chiralpak AS-H, (S,S)-Whelk-O 1, LPhenylglycine, Chirobiotic T and selfmade columns. In general,
polysaccharide based phases turned out to be superior. The group
achieved enantioseparations on a (S,S)-Whelk-O 1 column as well,
however, connected with retention times up to one hour. Furthermore, toxicity studies with pure enantiomers of MDPV collected on
a semipreparative column were carried out coming to the conclusion that no MDPV enantioselectivity for its toxicity was revealed
in this chosen cellular in vitro model [25].
Several CSPs based on derivatives of polysaccharides such as

amylose or cellulose were tested in the sequel for their ability to resolve NPS: Besides the established Chiralpak® columns,
the vendor Phenomenex came up with similar columns named
“Lux®” to compete the market of CSPs. In 2017, a Lux® Cellulose2 column was proven for its enantioseparation ability for 40
NPS from different drug compound classes including cathinones,
amphetamine derivatives, 2-aminopropyl benzofurans, thiophenes,
phenidine and phenidate derivatives [27]. Isocratic conditions and
the polar organic mode with 95% acetonitrile was used not
only for enantioseparation but also for resolution of regioisomers. In terms of slight alteration of the NPS structure to circumvent law, positional isomers of already emerged NPS, particularly cathinones or amphetamines became available. After the
ban of mephedrone (4-methyl-methcathinone) in 2010, its chiral
positional isomers mophedrone (3-methyl-methcathinone, 3-MMC)
and later 2-methyl-methcathinone (2-MMC) appeared on the market and separation methods had to be developed for a clear distinction. The consumption of positional isomers of mephedrone
might be treated very differently in European countries, meaning that e. g. there is no or a milder punishment provided for 3methyl-methcathinone compared to mephedrone (Fig. 1). Since it
is sometimes challenging to indistinguish them by common achiral GC or HPLC, the development of chiral separation methods can
be an additional benefit for this purpose. Fig. 3 shows both enantiomeric and regioisomeric separation of the 6 possible forms of
methyl-methcathinone [27]. A broad spectrum of NPS being cathinones or coming from other compound classes were subject to
chiral separation experiments on further Lux columns: A Lux®
i-Cellulose-5, available since 2016 and subject to 3.5 μm particles was found to be applicable for successful enantioresolution
of 93 out of 102 NPS using normal phase mode [28]. Also in this
field of interest, the general trend to move to smaller CSP particle size was taken into account. Furthermore this study reveals
the effect of different substituents on the phenyl ring of cathinones on enantioseparation, e. g. the comparison of chiral separation of flephedrone (4-fluoromethcathinone), clephedrone (4chloromethcathinone) and brephedrone (4-bromomethcathinone).
Similarly, a Lux® i-Amylose-1 chiral column found application to
be tested for a set of 112 chiral NPS purchased from internet vendors or seized by Austrian police [29]. Both latter columns do not
contain a coated but an immobilized chiral selector, namely cellulose tris(3,5-dichlorophenylcarbamate) [28] or amylose tris(3,5dimethylphenylcarbamate) [29]. They contain a chemical crosslinking between the polysaccharide and silica supports providing robustness against strong solvents and work optimal in normal phase
mode. Both of them showed excellent enantioseparation results as
well as elucidation of positional isomers.
Recently, also HPLC columns with smaller particle size became commercially available: A Waters Acquity UPC2® TrefoilTM
CEL1 2.5 μm column containing 2.5 μm cellulose tris(3,5dimethylphenylcarbamate) was subject to enantioseparation of 78
of 95 NPS of different compound classes, such as cathinones, amphetamines, ketamines, phenidines, phenidates, morpholines, thio-

5

phenes and 2-aminopropyl benzofurans including real-life samples
[30]; 51 of them were resolved within 6 min. The simultaneous
chiral separation of seven different cathinone derivatives on this
column is given in Fig. 4.
Cathinones were also resolved by other chiral separation principles: Wolrab et al. tested structurally different chiral ion-exchange
type stationary phases [31]. Ion-exchange type chiral stationary
phases previously reported were compared with a novel strong
cation-exchange type previously synthesized. The authors achieved
enantioresolution for 14 cathinone derivatives. A commercially
available CSP, namely Lux® AMP 3 μm originally designed for fast
enantioseparation of classic well established drugs such as amphetamine, methamphetamine and MDMA was checked for enantioseparation ability of NPS [32]. The composition of the chiral selector was not provided by the vendor, however, an aqueous ammonium bicarbonate solution adjusted with ammonia to a pH of
11.3 mixed with acetonitrile served as an unusual mobile phase.
Overall, 83 of 95 NPS purchased from different internet vendors or
seized by Austrian police were separated in their enantiomers successfully within 40 min. Fig. 5 shows the simultaneous chiral separation of four different ketamine derivatives on this column. Besides cathinones, new tryptamines are misused as hallucinogenic
NPS. Although the majority of them is achiral, 3 chiral representatives were resolved successfully by means of a Astec Cyclobond I
20 0 0 under reversed-phase conditions [33].
Based on separation experiments in analytical scale, attempts
to collect pure enantiomers in semipreparative scale were made.
Firstly, Silva’s group determined the absolute configuration of two
cathinones, namely pentylone and methylone; for this purpose
enantioresolution of the two NPS was carried out at a multimilligram scale on a semipreparative Chiralpakđ AS (250 ì 10
mm, inner diameter 5 μm particles size) stationary phase under
normal phase conditions with enantiomeric ratios higher than 98%
[34]. Along with theoretical calculations electronic circular dichroism spectroscopy revealed the correct classification of the enantiomers as (+)-(S) and (−)-(R)-pentedrone, and (−)-(S) and (+)(R)-methylone, respectively. Later, Spálovská et al. reported the
structural analysis of the same two cathinones [35]. Besides chiroptical methods, the two NPS were enantioseparated by means
of ChiralArt Amylose-SA provided by YMC Europe under normal
phase mode in order to collect pure enantiomers for further spectroscopy experiments revealing the 3D structures of methylone and
pentylone in solution.
All the aforementioned citations are based on the use of a chiral

HPLC column. An alternative involves the addition of 2% sulfated
ß-cyclodextrin as a chiral selector to the mobile phase and the use
of a RP-18e column [36]. With this approach, the acquisition of a
pricey chiral column can be circumvented. In this study, 17 cathinones were chirally resolved, 3 of them with baseline separation.
Apart of stimulating NPS, also high abuse of cannabinomimetics
as cited by EMCDDA [1] has become a challenging problem. A few
of these compounds coming from different substance classes are
chiral. They can bind to the cannabinoid receptors much stronger
than delta-9-tetrahydrocannabinol does and there is little information to which enantiomer the desired effects are restricted. Also
long term adverse effects lack in knowledge. Their abuse is also
associated with suspected intoxications, severe illness and fatal
cases [37]. Moreover, their nicknames are hard to distinguish and
easy to confuse. EMCDDA reports 11 of such compounds by 2018
[38]. Some cannabinomimetics contain pairs of enantiomers derived from the chiral centre of their amino acid structures. Doi
et al. synthesized both enantiomers of two synthetic cannabinoids,
namely N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)1H-indazole-3-carboxamide (5F-AB-PINACA) and methyl [1-(5fluoropentyl)-1H-indazole-3-carbonyl]-valinate (5F-AMB) prior to
their enantioresolution by HPLC coupled to high-resolution mass


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Fig. 3. Comparison of the chromatograms of the positional isomers (1) 2-MMC, (2) 4-MMC and (3) 3-MMC, respectively, by chiral HPLC. All three analytes were seized by
Austrian Police. Conditions: Column: Luxđ Cellulose-2, 250 ì 4.6 mm, 5 m, mobile phase: ACN: isopropanol: DEA: FA (100%) (95: 5: 0.1: 0.1), ambient temperature, flow:
1 ml/min, UV: 254 nm, injection: 5 μl. Reprinted from [27] with permission.

spectrometry on a Chiralpak AZ-3R in reversed-phase mode [39].
Additionally they examined ten herbal street samples containing 5F-AB-PINACA and one herbal sample containing 5F-AMB. As
a result all samples contained the (S)-enantiomer, but the (R)enantiomer was only detected in two samples showing a ratio of

less than 20%. In contrast to the afore discussed stimulating “Legal Highs” (cathinones etc.), chiral cannabinomimetics obviously
are not traded as racemic mixtures. The authors assume that the
NPS may be synthesized from L-amino acid derivatives due to their
lower cost and larger availability [39]. Another interesting publication reports on enantiospecific synthesis of four indazole-3carboxamides, namely AMB-FUBINACA, AB-FUBINACA, 5F-MDMBPINACA (5F-ADB) and AB-CHMINACA prior to their enantioresolution [40]. It turned out that a Lux® Amylose-1 showed optimal selectivity for the NPS with a terminal methyl ester moiety using the
reverse-phase mode under isocratic conditions, whereas a Lux® iCellulose-5 column separated NPS with a terminal amide moiety.
Moreover, biological activity test was carried out revealing that the
effect of these cannabinomimetics is mainly restricted to the (S)enantiomers. Seized herbal samples were also tested arising the
concern by the authors that significant differences between syn-

thesis batches might be expected due to small but significant differences in synthesis precursor enantiopurity [40]. All chiral separation methods by HPLC are given in Table 2 showing brand names
and chiral selectors of the used columns.
2.3. Enantioseparation of novel psychoactive substances by
supercritical fluid chromatography (SFC)
In a comparable time period similar to the emerge of NPS, SFC
became a popular chromatographic alternative to HPLC. After its
revival some years ago, a few applications in terms of NPS have
been reported. This is most probably also due to the fact that since
some years, modular HPLC systems can be upgraded to SFC equipments rather easily. Additionally, many chiral stationary phases
originally designed for HPLC can be used in SFC without further
modification. Considering faster separation times, SFC is often superior to HPLC.
In 2015, Pauk et al. reported on the resolution of four phenethylamines as well as isomeric separation of eleven cathinones under supercritical or subcritical conditions with carbon dioxide, nitrous oxide and additives as a mobile phase [41]. The chromato-


M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

7

Fig. 4. Simultaneous chiral separation of seven different cathinone derivatives by chiral HPLC. Conditions: Column: Trefoil® CEL1 2.5 μm, 150 × 3 mm, chiral selector:
cellulose tris-(3,5-dimethylphenyl-carbamate), mobile phase: n-hexane / n-butanol / DEA (100:0.3:0.2), ambient temperature, flow: 1.0 mL/min, UV: 230 nm, injection: 1 μL.
(Unpublished results)

graphic unit was coupled to both a diode array detector and a
triple quadrupol mass selective detector. A BEH silica (1.7 μm), a
BEH 2-ethylpyridine (1.7 μm), a CSH Fluoro-Phenyl (1.7 μm) and
a HSS C18SB (1.8 μm) were examined, whereas the first mentioned column proved to be superior. Another approach was presented by Geryk et al. [42] dealing with chiral separation of amphetamines, cathinones and 2-aminopropyl benzofurans. The latter possess empathogenic and stimulating effects similar to amphetamines. Again, carbon dioxide and additives served as mobile
phase, whereas a commercially available ChiralArt Amylose SA containing amylose tris(3,5-dimethylphenylcarbamate) as chiral selector was chosen. With this method, rapid enantioseparations were
achieved. Enantioseparation of mephedrone, brephedrone and flephedrone is given in Fig. 6. Furthermore the macrocyclic antibiotics
Teicoplanin and Vancomycin were tested for enantioselective potential by means of superficially porous particles-packed columns

originally provided for HPLC [43]. Among other analytes, NPS such
as ketamines and synthetic cathinones were resolved and results
obtained for Teicoplanin were compared to those of Vancomycin.
Both columns showed similar enantioselectivity for NPS. All aforementioned approaches are listed in Table 3.
2.4. Enantioseparation of novel psychoactive substances by capillary
electrophoresis
As a separation technique complementary to HPLC analytes are
separated by different migration velocity in an electric field. Many
chiral separation principles applied in HPLC were transferred to CE
successfully.
In chiral CE for chiral resolution of NPS, cyclodextrins are the
most frequently used chiral selectors. Since they are widely UV
transparent, there is no disturbance in detection. Native CDs are
cyclic oligosaccharides built of six (α -CD), seven (β -CD) or eight


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M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256
Fig. 5. Simultaneous chiral separation of four different ketamine derivatives by chiral HPLC. Conditions: Column: Lux® AMP 3 μm, 150 × 4,6 mm, mobile phase: ammonium bicarbonate (5 mM) adjusted to pH 11.3 with conc.
ammonium hydroxide/acetonitrile (70:30), ambient temperature, flow: 0.5 ml/min, UV: 230 nm, injection: 1 μl. (Unpublished results).

M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

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Fig. 6. Chiral separation of mephedrone, brephedrone and flephedrone by ultra-performance supercritical fluid chromatography. Conditions: Chiral column: CHIRAL ART
Amylose SA (150 mm 3.0 mm i.d., 3 μm); MP (A): CO2/PrOH/ TFA/IPA 90/10/0.05/0.05 (v/v/v/v); MP (B): CO2/PrOH/TFA/IPA 95/5/0.05/0.05 (v/v/v/v); flow rate 2.5 mL min1;
column temperature 35°C; injection volume: 1 μl. Reprinted from [42] with permission.


10

M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

Table 2
Overview of approaches for enantioseparation of NPS by HPLC.
Brand name of column

Chiral selector

Separated NPS

Ref.

Crownpak CR (+)

(S)-18-Crown-6-ether phase

[21]

Chirosil NT-RCA(+)
(S,S)-Whelk-O1 and others

Lux® Cellulose-2

(+)-(18-Crown-6)-2,3,11,12-tetracarboxylic
1-(3,5-Dinitrobenzamido)-1,2,3,4tetrahydrophenanthrene
Amylose tris [(S)-α -methylbenzylcarbamate]
Amylose tris [(S)-α -methylbenzylcarbamate];
1-(3,5-Dinitrobenzamido)-1,2,3,4tetrahydrophenanthrene
Cellulose tris(3-chloro-4-methylphenylcarbamate)

Cathinone, amphetamine, norephedrine and
norphenylephrine
2-Methoxy-N-methamphetamine and analogues
Mephedrone and related cathinones

Lux® i-Cellulose-5
Lux® i-Amylose-1
Acquity UPC2® TrefoilTM CEL1

Cellulose tris(3,5-dichlorophenylcarbamate)
Amylose tris(3,5-dimethylphenylcarbamate)
Cellulose tris(3,5-dimethylphenylcarbamate)

Chiralpak® AS-H
Chiralpak® AS-H; (S,S)-Whelk-O1 and
others

Different ion-exchange CSPs

Lux® AMP 3 μm
Cyclobond I 2000
Chiralpak® AS

Composition of the chiral selector is not provided
by the vendor
Native β -cyclodextrin
Amylose tris [(S)- -methylbenzylcarbamate]

ChiralArt Amylose-SA; Chiralpakđ IA

Amylose tris(3,5-dimethylphenylcarbamate)

RP-18e column

Sulfated ò-cyclodextrin as chiral selector added to
the mobile phase
Amylose tris(3-chloro-4-methylphenylcarbamate)
Amylose tris(3,5-dimethylphenylcarbamate);
Cellulose tris(3,5-dichlorophenylcarbamate)

Chiralpak® AZ-3R
Lux® i-Amylose-1; Lux® i-Cellulose-5
RP-mode:
Chiralcel ODRH, Cellulose 3, Chiralcel
OZH, Lux® Cellulose 2
NP-mode:
Chiralpak ADRH, Lux® Cellulose 1,
Lux® Cellulose 4 Lux® Cellulose 2
PO-mode:

Lux® Cellulose 2, Chiralcel ODRH,
Lux® Cellulose 4, Sepapak 5

[22]
[23]

Mephedrone and other cathinone derivatives
Cathinone derivatives; semipreparative
enantioresolution of MDPV

[24]
[25]

Cathinones, amphetamine derivatives, 2-aminopropyl
benzofurans, thiophenes, phenidine and phenidate
derivatives
Various subclasses of NPS
Various subclasses of NPS
Cathinones, amphetamines, ketamines, phenidines,
phenidates, morpholines, thiophenes and
2-aminopropyl benzofurans
14 Cathinone derivatives
Various subclasses of NPS

[27]

Novel tryptamine derivatives
Semipreparative enantioresolution of pentylone and
methylone
Semipreparative enantioresolution of pentylone and

methylone
Cathinone derivatives

[33]
[34]

5F-AB-PINACA and 5F-AMB
AMB-FUBINACA, AB-FUBINACA, 5F-MDMB-PINACA
(5F-ADB) and AB-CHMINACA

[28]
[29]
[30]

[31]
[32]

[35]
[36]
[39]
[40]
[58]

Cellulose tris(3,5-dimethylphenylcarba-mate),
amylose tris(3,5-dimethylphenyl-carbamate),
amylose tris(5-chloro-2-methylphenylcarbamate)
and cellulose
tris(4-chloro-3-methylphenylcarbamate)

Cathinones, amphetamine derivatives and 6-APB)

Table 3
Overview of approaches for enantioseparation of NPS by SFC.
Brand name of column

Chiral selector

Separated NPS

Ref.

ChiralArt Amylose SA

Amylose tris(3,5-dimethylphenylcarbamate)

[42]

AZYP TeicoShell and
VancoShell
Chiralcel OZH,
Chiralpak ADRH,
Chiralcel ODRH, Lux
Cellulose 4

Teicoplanin and vancomycin

Amphetamines, cathinones and
2-aminopropyl-(benzofurans)
NPS such as ketamines and synthetic cathinones

[43]

Cathinones, amphetamine derivatives and 6-APB

[58]

Cellulose tris(3-chloro-4-methylphenylcarbamate),
Amylose tris(3,5-dimethylphenylcarbamate),
cellulose tris(3,5-dimethylphenylcarbamate)

(γ -CD) glucopyranose units and three hydroxy groups in position
2, 3 and 6 can undergo derivatization. Hence, a big variety of neutral and charged CDs are commercially available. Chiral recognition
mechanism takes place firstly by inclusion of bulky hydrophobic
groups into the chiral CD cavity and secondly by interactions of
the hydroxyl groups at C2 and C3 with hydrophilic groups of the
NPS [44].
As early as in 1994, about ten years before the hype of NPS
took over, Lurie et. al. reported on enantioseparation of classic illicit drugs, namely amphetamine, methamphetamine, methcathinone and ephedrine derivatives by means of dimethyl-ß-CD
[45]. Later, the same group introduced a mix of different anionic CDs for chiral resolution of phenethylamines and impurities within 9 min [46]. In 2012, Mohr et al. reported on the
application of different CDs for chiral resolution of cathinone
derivatives [47]. It turned out that sulfated ß-CD demonstrated
the best enantioseparation ability for these compounds. One year

later, Burrai et al. published a successful application of the same
chiral selector for the enantioseparation of 13 amphetaminelike designer drugs [48]. Then, sulfobutylether ß-CD was chosen
for the enantioseparation of 16 meanwhile emerged stimulating
NPS, such as cathinones, 2-aminopropyl benzofurans, diphenidine,
ethylphenidate, methiopropamine and thiothinone by CE coupled
with diode array detection [49]. Since 2-aminopropyl benzofurans
(“Benzofuries”) exist in two positional isomeric forms additionally

to their chirality, namely 6-(2-aminopropyl)benzofurans and 5-(2aminopropyl)benzofurans, their discrimination is hardly possible
under achiral conditions. Fig. 7 shows the simultaneous chiral separation of 6-APB (“Benzofury”) and 5-APB by sulfobutylether ß-CD
assisted chiral CE [49].
Besides UV-detection, Mantim et al. [50] presented conductless detection CE4D for this purpose and separated amphetamine,
methamphetamine, ephedrine, pseudoephedrine and norephedrine
as powder samples or urine probes by means of (+)-(18-crown-


M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

11

Fig. 7. Simultaneous chiral separation of 6-APB and 5-APB by CE. Conditions: capillary: 50 μm ID fused silica capillary 78.5 cm (effective length 70 cm); 50 mM ammonium
acetate pH=4.5 containing 10% v/v ACN and 16 mM SBE-CD; UV: 230 nm; applied voltage: 25 kV to cathode; Inj.: by pressure 50 mbar for 5 s. Reprinted from [49] with
permission.

6)-tetracarboxylic acid and/or carboxymethyl-ß-CD as chiral selectors. Furthermore, MS coupling was used in combination with CD
derivatives such as ß-CD and highly sulfated γ -CD for enantioseparation of 12 cathinones by Merola’s group [51].
During the last two years, several NPS were checked by different chiral selectors, such as a single isomer CD, namely heptakis(2,3-di-O-methyl-6-O-carboxymethyl)-ß-CD [52], 2-hydroxyethylß-cyclodextrin [53] and carboxymethyl-β -cyclodextrin [54]. A further chiral separation principle represents the use of chiral crown
ethers as cyclic polyethers which form host-guest complexes with
primary ammonium cations. Hydrogen bonds between the three
amine hydrogens and the oxygens of the macro- cyclic ether as
well as ionic and dipole-dipole interactions are responsible for
enantiorecognition. Since NPS with primary amine structure can
be resolved by (+)-(18-crown-6)-tetracarboxylic acid, a successful
study was presented in 2018 by means of 15 separated NPS [55].
Examples of two resolved NPS are given in Fig. 8. Additionally,
with respect to regioselective discrimination, separation of 3 positional isomers of fluorinated amphetamine was presented [55].
By 2019, more than 200 cathinone derivatives are known to be
traded as NPS. Recently, 58 of them were enantioseparated successfully by 4 different CDs, namely native β -cyclodextrin, acetyl-

β -cyclodextrin, 2-hydroxypropyl-β -cyclodextrin or carboxymethylβ -cyclodextrin [56]. All chiral separation methods of this chapter
are summarized in Table 4.

2.5. Enantioseparation of novel psychoactive substances by capillary
electrochromatography
With this technique, the efficiency of CE is combined with the
selectivity of stationary phases. Under optimal conditions, the analytes migrate along a chiral stationary phase packed in a capillary
driven by an electric field. The advantages of CE such as improved
peak shapes and low electrolyte consumption are used along with
the vast availability of chiral stationary phases. However, capillaries
have to be packed with appropriate chiral particles.
In 2014, Aturki et al. presented the successful application
of a 100 μm ID capillary packed with amylose tris(5-chloro-2methylphenylcarbamate) as chiral stationary phase for chiral separation of NPS [57]. A field-amplified sample injection was applied
in order to obtain a sensitivity improvement. Ten different cathinones were resolved in their enantiomers within a couple of minutes. As seen from the use of polysaccharide columns in HPLC,


12

M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

Fig. 8. Enantioseparation of 2-chlor-amphetamine and 4-APDB by CE using (+)-18-crown-6-tetracarboxylic acid as chiral selector. Conditions: 20 mM (+)-18-crown-6tetracarboxylic acid, 10 mM Tris, 30 mM citric acid, pH 2,1, cassette temperature: 20°C, applied voltage: 30 kV, injection: 10 mbar for 5 s (Unpublished results).

there might be potential for further success in this field. All approaches by CEC are summarized in Table 4.

2.6. Comparative attempts for enantioseparation of novel
psychoactive substances
Besides enantioseparation of NPS using one of the techniques described above, also studies were published showing
different chiral separation techniques in comparison. In 1996,
Armstrong’s group compared the enantioresolution of established

illicit drugs such as amphetamine and methamphetamine by
means of GC, LC and CE [11]. For chiral HPLC, various Cyclobond
and Chiraldex columns were tested, whereas in GC successful enantioresolution was shown on a Chiraldex G-PN column
within 14 min. Hydroxypropyl-ß-CD served as chiral additive
for CE experiments. Regarding NPS, Albals et al. presented a
huge comparative study between CEC, SFC and three liquid chromatographic modes for enantioresolution of 10 NPS comprising
cathinones, amphetamine derivatives and “Benzofury” (6-APB)
[58]. For CEC experiments, four packed polysaccharide-based capillaries containing cellulose tris(3,5-dimethylphenylcarbamate),
amylose
tris(5-chloro-2-methylphenylcarbamate),
amylose

tris(3,5-dimethylphenylcarbamate), and cellulose tris(4-chloro3- methylphenylcarbamate) served as chiral selectors. The latter
two phases turned out to be advantageous. Regarding LC, various
chiral columns based on cellulose or amylose derivatives were
checked using SFC and HPLC on normal-phase, reversed-phase and
polar organic mode. The latter mode showed limited separation
success [59]. Also single compounds can be subject to comparative
studies as done for clephedrone (4-chloro-methcathinone) shortly
after its emerge in 2014 [59]. After confirmation of identity by GCMS and NMR, enantioseparation by CE was compared to indirect
GC.
Similarly,
1-(3,4-dimethoxyphenyl)-2-(ethylamino)pentan1-one (nickname DL-4662) was investigated as a brand-new
cathinone derivative by means of chiral HPLC and indirect GC after
derivatization [60].
In 2015, Lurie’s group published a comparative regioisomeric
and enantiomeric study between CE and U-HPLC testing 24
cathinones and phenethylamines [61]. Regarding U-HPLC different
columns provided by Waters Inc., such as BEH-C18, HSS T3, BEH
Phenyl, CSH-Phenyl were tested and CSH-Phenyl turned out to be

optimal for regioisomeric analyses. CE experiments were either
employed with coated capillaries or with CDs as chiral additives
[61]. Additionally seized samples were tested. Later, Carnes et al.


M.G. Schmid and J.S. Hägele / Journal of Chromatography A 1624 (2020) 461256

13

Table 4
Overview of approaches for enantioseparation of NPS by CE and CEC.
Separation
technique

Chiral selector

Separated NPS

Ref.

CE
CE

Hydroxypropyl-ß-cyclodextrin
Dimethyl-ß-cyclodextrin

[11]
[45]

CE

CE

Different sulfobutylether-β - and sulfated-α -cyclodextrins
Native-β -cyclodextrin, carboxymethyl-β -cyclodextrin,
2-hydroxypropyl-β -cyclodextrin, sulfated-β -cyclodextrin and
native γ -cyclodextrin
Sulfated-β -cyclodextrin, carboxymethyl-β -cyclodextrin and
dimethyl-β -cyclodextrin
sulfobutylether-β -cyclodextrin

Amphetamine and methamphetamine
Amphetamine, methamphetamine, methcathinone
and ephedrine derivatives
Phenethylamines and impurities
Cathinone derivatives

[46]
[47]

Amphetamine derivatives

[48]

Cathinones, (2-aminopropyl)-benzofurans,
diphenidine, ethylphenidate, methiopropamine
and thiothinone
Amphetamine, methamphetamine, ephedrine,
pseudoephedrine and norephedrine
12 cathinone derivatives
Ketamine and cathinone derivatives

Methcathinone derivatives
Various NPS
Various NPS
Cathinone and pyrovalerone derivatives

[49]

[51]
[52]
[53]
[54]
[55]
[56]

Cathinone derivatives
Cathinones, amphetamine derivatives and 6-APB

[57]
[58]

Clephedrone (4-chloro-methcathinone)
Cathinones and phenethylamines

[59]
[61]

CE
CE

CE

CE
CE
CE
CE
CE

CEC
CEC

CE

(+)-(18-crown-6)-tetracarboxylic acid and/or
carboxymethyl-ß-cyclodextrin
β -cyclodextrin and highly sulfated-γ -cyclodextrin
Heptakis-(2,3-di-O-methyl-6-O-carboxymethyl)-ß-CD
2-hydroxyethyl-ß-cyclodextrin
Carboxymethyl-β -cyclodextrin
(+)-(18-crown-6)-tetracarboxylic acid
Native ß-cyclodextrin, acetyl-ß-cyclodextrin,
2-hydroxypropyl-ß-cyclodextrin and
carboxymethyl-ß-cyclodextrin
Amylose tris(5-chloro-2-methylphenylcarbamate)
Cellulose tris(3,5-dimethylphenylcarbamate), amylose
tris(3,5-dimethylphenylcarbamate), amylose
tris(5-chloro-2-methylphenylcarbamate) and cellulose
tris(4-chloro-3-methylphenylcarbamate)
Sulfated-β -cyclodextrin
ß-cyclodextrin, (2,6-di-O-methyl)-ß-cyclodextrin and
hydroxypropyl-ß-cyclodextrin

compared separation behavior of 35 cathinones by SFC, U-HPLC
and GC [62]. Although for SFC chiral columns were employed, the
main emphasis of this study was the separation of regioisomers.
3. Conclusion and outlook
Much progress has been done since the emerge of chiral NPS
regarding development of chiral separation methods by different
high performance chromatographic and electrophoretic separation
techniques. Some concepts already presented for amphetaminelike established illicit drugs were successfully transferred to enantioresolution of NPS. Again it has to be emphasized that this selective review refers mainly to the analysis of solid samples being
purchased at internet platforms or recently commercially available
as well as samples seized by police or collected in hospitals.
The worldwide significantly increasing abuse of psychoactive
compounds leads to new issues, such as contamination of wastewater due to clandestine synthesis of drugs or their consumption.
Recently, a publication reported on an indirect chiral GC method
for enantioseparation of illicit amphetamines in wastewater. According to the authors, these studies promote distinguishing between the consumption of prescribed and illicit drugs and support the search for clandestine labs as well as illegal discharge of
sewage [63]. This approach might be also useful in terms of NPS in
future.
Declaration of Competing Interests
I hereby declare that there is no conflict of interest.
CRediT authorship contribution statement
Martin G. Schmid: Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Writing - original draft.
Johannes S. Hägele: Data curation, Resources, Validation, Visualization, Writing - review & editing.

[50]

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