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.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
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
NovelPsychoactiveSubstances(NPS)representanalternativetoestablishedillicitdrugs.Theyaretraded viathe internetand exhibitsmall alterationsintheir chemical structureto circumventlaw,however, theirpsychotropiceffects arecomparablẹThereis stillpoor knowledge aboutside effects and health risks.Bytheendof2018,730NPSwerereportedtoEMCĐĂEuropeanMonitoringCentreforDrugsand DrugAđiction).Amongdifferentcompoundclasses,manyNPSarechiralandfewpublicationsdealwith thedifferentpharmacologicalandtoxicologicalpropertiesoftheirpureenantiomers.Therefore,analytical methođevelopmentconcerningenantioseparationofNPSisofgreatinterest.Chiralseparationprotocols
ofestablishedillicit drugshave beentransferred forNPS, selectedexamplesaregivenas well Differ-entmethodsforenantioseparationofNPScomprisingmainlystimulatingdrugssuchascathinones, py-rovalerones,amphetamines,ketamines,(2-aminopropyl)benzofuranes,phenidines, phenidates, morpho-linesand thiophenesarereviewed.Moreover,chiralresolutionofsomecannabinomimetics byHPLCis presented.ChromatographicandelectrophoretictechniquessuchasGC,HPLC,SFC,CEandCECare dis-cussedandinsomecasescompared.Mainly,solidsampleseitherpurchasedfrominternetvendors,seized
bypoliceorcollectedfrompatientsinhospitalsaresubjecttoanalysis.ChiralselectorsusedforHPLCare listedinaTablẹItwasshownthatparticularlystimulatingdrugsaretradedasracemicmixtures,which
isnotthecasewithcannabinomimetics.Mainly,HPLCandCEwereusedforenantioseparationofNPS
© 2020TheAuthors.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBY-NC-NDlicensẹ
(http://creativecommons.org/licenses/by-nc-nd/4.0/ )
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
dur-ing 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 ađress: martin.schmid@uni-graz.at (M.G Schmid)
new experience watched out for cheap and effective alternatives.
At this time the triumphal introduction of so called novel psy-choactive substances (NPS) started worldwide: As a first genera-tion, 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 ẹ g in Europe, they were traded in headshops and via the internet Although they exhibit effects re-lated to illicit drugs, they were not prohibited because of lack-ing restriction by a drug-law The most impressive representative
of this compound class was mephedrone (nicknames: drone, M-CAT, White Magic and meow meow), the 4-methyl derivative of N-methcathinone (4-MMC) coming up in 2003 and spreading world-wide within a short period of timẹ 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 cathi-nones entered the drug market as following NPS generations, ac-companied by other compound classes such as empathogenic 2-https://doịorg/10.1016/j.chromạ2020.461256
0021-9673/© 2020 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND licensẹ ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
Trang 22 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256
aminopropyl benzofurans (“Benzofuries”), synthetic tryptamines,
phenethylamines, synthetic cannabinoids, arylamines or
piper-azines To date, any classic illicit drug can be replaced by NPS.
To disguise their potential harm, NPS are generally sold as
“Le-gal Highs”, “Research Compounds”, “Leisure Drugs”, “Plant food”,
“Bird’s cage cleaners” or “Room odorizers”.
In the recent 8 years, 730 not scheduled synthetic new
com-pounds 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
chan-nels and even Smartphone Apps are available [2] In 2017,
EM-CDDA classified NPS in four potential dangerous categories:
syn-thetic cannabinoids, synthetic stimulants, new opioids and new
an-tidepressants [1] Since these compounds are completely new
be-cause 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
mix-tures Additionally, several NPS exist as positional isomers, mostly
bearing side chains on a phenyl ring in ortho-, meta or para-
con-stitution This means e g that the compound mephedrone
com-prises 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
rea-son, it is of great importance and interest to conduct
pharmaco-logical 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
indi-vidual illicit drug enantiomers reported in literature are given for
example for mephedrone [3] , amphetamine (Speed) [4] ,
metham-phetamine (Crystal Meth) [5] or methcathinone [ 6 , 7 ] Achiral and
chiral determination of seized NPS as internet products was
pub-lished as a book chapter [8] Enantioselectivity and chiral
resolu-tion 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
de-voted to solid samples either purchased from internet vendors,
seized by police or collected from patients in hospitals In
gen-eral, samples are available in a variety of different forms,
includ-ing 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 Enantioseparation of novel psychoactive substances by
chromatographic and electrophoretic techniques
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
elec-trophoretic methods, a broad spectrum of CE data has been
pub-lished and a further publication dealing with CEC has appeared.
2.1 Enantioseparation of novel psychoactive substances by gas chromatography
Gas chromatography is known as a fast and reliable analyti-cal technique To date, only two publications have come up show-ing the successful use of a GC-capillary coated with a chiral se-lector 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 trans-ferred to diastereomers and can be separated on conventional achi-ral 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 pseu-doephedrine All compounds were separated on a common DB-5 column simultaneously within 15 min As an alterna-tive, trifluoracetyl-L-prolyl chloride (L-TPC) turned out as a use-ful chiral derivatization reagent to separate amphetamine, N-methamphetamine, 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 (cy-clodextrin) chiral column (Chiraldex G-PN) This phase sized 30
m × 0.25 mm (coating film thickness 0.12 μ m) and incorpo-rated a phase consisting of a 2,6-di-O-pentyl-3-propionyl deriva-tive of γ -cyclodextrin N-methamphetamine, ephedrine and pseu-doephedrine were derivatized with trifluoro acetic anhydride as achiral reagent prior to analysis [12] One year later, successful in-direct 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 simul-taneous GC-MS measurement of six cathinones after chiral deriva-tization 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 deriva-tives 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 con-tent by Dhabbah [19] After derivatization by menthyl chlorofor-mate, the two cathinone enantiomers were quantified It turned out that S-cathinone, the stronger psychoactive stereoisomer, ex-hibited 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
2.2 Enantioseparation of novel psychoactive substances by HPLC
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
Trang 3Fig 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-crown-6-ether phase for the enantioseparation of four
phenylalky-lamines, namely cathinone, amphetamine, norephedrine and
nor-phenylephrine [21] Another crown ether HPLC column based
on ( + )-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral
se-lector found application by Hyun’s group for chiral resolution
of methoxyphenamine (2-methoxy-N-methamphetamine) and its
analogues using polar-organic conditions [22] Chiral
discrimina-tion 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
chi-ral 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
resolu-tion of mephedrone and related cathinones of the first
gener-ation available at this time [23] HPLC columns with packed
5 μm particles bearing different selectors were tested by
dif-ferent 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
so-lutions 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 sol-vents 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-hexane-trifluoroacetic acid-triethylamine In the same year, Mohr et al em-ployed 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 hy-drophobic 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 chi-ral 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, res-olution 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 cathi-nones were resolved in their enantiomers in one run and enan-tiomer elution order was shown for the parent compound meth-cathinone [24] In the sequel, other CSPs were used as follow: Silva’s group presented enantioseparation of further 14 NPS sam-ples purchased in Portuguese smart shops prior to closing of these
Trang 44 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
Astec Chiraldex TM
G-PN
Direct 2,6-Di-O-pentyl-3-propionyl
γ-cyclodextrin
Amphetamine, methamphetamine, ephedrine and pseudoephedrine
[11] Astec Chiraldex TM
G-PN
Direct 2,6-Di-O-pentyl-3-propionyl
γ-cyclodextrin
Methamphetamine, ephedrine and pseudoephedrine [12] DB-5, J and
W-Scientific Indirect (R)-( + )- α-Methoxy- α-
(trifluoromethyl)phenylacetic acid
N-Methamphetamine, methcathinone, ephedrine and
DB-17, J and
W-Scientific
HP-5 MS Indirect (1R)-( −)-Menthylchloroformate Cathinone enantiomers in parts of freshly harvested Khat
plants
[18]
Trang 5shops because of prohibition [25] 3,4-Methylenedioxypyrovalerone
(MDPV), a NPS likely introducing neuroadaptive changes and
be-havioral effects [26] as well as further 8 cathinones were tested
by different CSPs such as Chiralpak AS-H, (S,S)-Whelk-O 1,
L-Phenylglycine, 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
Further-more, toxicity studies with pure enantiomers of MDPV collected on
a semipreparative column were carried out coming to the
conclu-sion 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
abil-ity 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®
Cellulose-2 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
regioiso-mers In terms of slight alteration of the NPS structure to
cir-cumvent law, positional isomers of already emerged NPS,
partic-ularly 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
mar-ket and separation methods had to be developed for a clear
dis-tinction The consumption of positional isomers of mephedrone
might be treated very differently in European countries,
mean-ing that e g there is no or a milder punishment provided for
3-methyl-methcathinone compared to mephedrone ( Fig 1 ) Since it
is sometimes challenging to indistinguish them by common
achi-ral GC or HPLC, the development of chiral separation methods can
be an additional benefit for this purpose Fig 3 shows both
enan-tiomeric and regioisomeric separation of the 6 possible forms of
methyl-methcathinone [27] A broad spectrum of NPS being
cathi-nones 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 μ m
parti-cles 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
par-ticle size was taken into account Furthermore this study reveals
the effect of different substituents on the phenyl ring of
cathi-nones on enantioseparation, e g the comparison of chiral
sep-aration of flephedrone (4-fluoromethcathinone), clephedrone
(4-chloromethcathinone) 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
ven-dors or seized by Austrian police [29] Both latter columns do not
contain a coated but an immobilized chiral selector, namely
cel-lulose tris(3,5-dichlorophenylcarbamate) [28] or amylose
tris(3,5-dimethylphenylcarbamate) [29] They contain a chemical
crosslink-ing between the polysaccharide and silica supports providing
ro-bustness 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
be-came commercially available: A Waters Acquity UPC2® TrefoilTM
CEL1 2.5 μm column containing 2.5 μ m cellulose
tris(3,5-dimethylphenylcarbamate) was subject to enantioseparation of 78
of 95 NPS of different compound classes, such as cathinones,
am-phetamines, ketamines, phenidines, phenidates, morpholines,
thio-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 princi-ples: 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 am-phetamine, methamphetamine and MDMA was checked for enan-tioseparation ability of NPS [32] The composition of the chiral se-lector was not provided by the vendor, however, an aqueous am-monium 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 suc-cessfully within 40 min Fig 5 shows the simultaneous chiral sep-aration of four different ketamine derivatives on this column Be-sides cathinones, new tryptamines are misused as hallucinogenic NPS Although the majority of them is achiral, 3 chiral representa-tives 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 multi-milligram scale on a semipreparative Chiralpak® AS (250 × 10
mm, inner diameter 5 μ m particles size) stationary phase under normal phase conditions with enantiomeric ratios higher than 98% [34] Along with theoretical calculations electronic circular dichro-ism spectroscopy revealed the correct classification of the enan-tiomers 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 chi-roptical 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 spec-troscopy 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 cathi-nones 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 informa-tion 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 de-rived 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-(5-fluoropentyl)-1H-indazole-3-carbonyl]-valinate (5F-AMB) prior to their enantioresolution by HPLC coupled to high-resolution mass
Trang 66 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256
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: Lux® Cellulose-2, 250 × 4.6 mm, 5 μm, 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
contain-ing 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
“Le-gal 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
pub-lication reports on enantiospecific synthesis of four
indazole-3-carboxamides, namely AMB-FUBINACA, AB-FUBINACA,
5F-MDMB-PINACA (5F-ADB) and AB-CHMINACA prior to their
enantioresolu-tion [40] It turned out that a Lux® Amylose-1 showed optimal
se-lectivity for the NPS with a terminal methyl ester moiety using the
reverse-phase mode under isocratic conditions, whereas a Lux®
i-Cellulose-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 dif-ferences in synthesis precursor enantiopurity [40] All chiral sepa-ration 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 equip-ments 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 su-perior to HPLC.
In 2015, Pauk et al reported on the resolution of four phenethy-lamines as well as isomeric separation of eleven cathinones un-der supercritical or subcritical conditions with carbon dioxide, ni-trous oxide and additives as a mobile phase [41] The
Trang 7chromato-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
men-tioned column proved to be superior Another approach was
pre-sented by Geryk et al [42] dealing with chiral separation of
am-phetamines, cathinones and 2-aminopropyl benzofurans The
lat-ter possess empathogenic and stimulating effects similar to
am-phetamines Again, carbon dioxide and additives served as mobile
phase, whereas a commercially available ChiralArt Amylose SA
con-taining amylose tris(3,5-dimethylphenylcarbamate) as chiral
selec-tor was chosen With this method, rapid enantioseparations were
achieved Enantioseparation of mephedrone, brephedrone and
fle-phedrone is given in Fig 6 Furthermore the macrocyclic antibiotics
Teicoplanin and Vancomycin were tested for enantioselective
po-tential 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 afore-mentioned 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
Trang 88
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)
Trang 9Fig 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
Trang 1010 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
norphenylephrine
[21] Chirosil NT-RCA( + ) ( + )-(18-Crown-6)-2,3,11,12-tetracarboxylic 2-Methoxy-N-methamphetamine and analogues [22] (S,S)-Whelk-O1 and others 1-(3,5-Dinitrobenzamido)-1,2,3,4-
tetrahydrophenanthrene
Mephedrone and related cathinones [23] Chiralpak® AS-H Amylose tris [(S)- α-methylbenzylcarbamate] Mephedrone and other cathinone derivatives [24] Chiralpak® AS-H; (S,S)-Whelk-O1 and
1-(3,5-Dinitrobenzamido)-1,2,3,4- tetrahydrophenanthrene
Cathinone derivatives; semipreparative
Lux® Cellulose-2 Cellulose tris(3-chloro-4-methylphenylcarbamate) Cathinones, amphetamine derivatives, 2-aminopropyl
benzofurans, thiophenes, phenidine and phenidate derivatives
[27]
Acquity UPC2® Trefoil TM CEL1 Cellulose tris(3,5-dimethylphenylcarbamate) Cathinones, amphetamines, ketamines, phenidines,
phenidates, morpholines, thiophenes and 2-aminopropyl benzofurans
[30]
Lux® AMP 3 μm Composition of the chiral selector is not provided
by the vendor
Chiralpak® AS Amylose tris [(S)- α-methylbenzylcarbamate] Semipreparative enantioresolution of pentylone and
methylone
[34] ChiralArt Amylose-SA; Chiralpak® IA Amylose tris(3,5-dimethylphenylcarbamate) Semipreparative enantioresolution of pentylone and
methylone
[35] RP-18e column Sulfated ß-cyclodextrin as chiral selector added to
the mobile phase
Lux® i-Amylose-1; Lux® i-Cellulose-5 Amylose tris(3,5-dimethylphenylcarbamate);
Cellulose tris(3,5-dichlorophenylcarbamate)
AMB-FUBINACA, AB-FUBINACA, 5F-MDMB-PINACA (5F-ADB) and AB-CHMINACA
[40] 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
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)
[58]
Table 3
Overview of approaches for enantioseparation of NPS by SFC
ChiralArt Amylose SA Amylose tris(3,5-dimethylphenylcarbamate) Amphetamines, cathinones and
2-aminopropyl-(benzofurans)
[42] AZYP TeicoShell and
VancoShell
Chiralcel OZH,
Chiralpak ADRH,
Chiralcel ODRH, Lux
Cellulose 4
Cellulose tris(3-chloro-4-methylphenylcarbamate), Amylose tris(3,5-dimethylphenylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate)
Cathinones, amphetamine derivatives and 6-APB [58]
( γ -CD) glucopyranose units and three hydroxy groups in position
2, 3 and 6 can undergo derivatization Hence, a big variety of
neu-tral 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
clas-sic illicit drugs, namely amphetamine, methamphetamine,
meth-cathinone and ephedrine derivatives by means of dimethyl-ß-CD
[45] Later, the same group introduced a mix of different
an-ionic CDs for chiral resolution of phenethylamines and
impuri-ties 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 amphetamine-like 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-(2-aminopropyl)benzofurans, their discrimination is hardly possible under achiral conditions Fig 7 shows the simultaneous chiral sep-aration of 6-APB (“Benzofury”) and 5-APB by sulfobutylether ß-CD assisted chiral CE [49]
Besides UV-detection, Mantim et al [50] presented conduct-less detection CE4D for this purpose and separated amphetamine, methamphetamine, ephedrine, pseudoephedrine and norephedrine
as powder samples or urine probes by means of ( +