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.
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 (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 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: 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 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- https://doi.org/10.1016/j.chroma.2020.461256 0021-9673/© 2020 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 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 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 different forms: 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 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 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 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 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 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 Fig 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 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 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 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 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 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256 Fig 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 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 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 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 shows both enantiomeric and regioisomeric separation of the 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 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- phenes and 2-aminopropyl benzofurans including real-life samples [30]; 51 of them were resolved within The simultaneous chiral separation of seven different cathinone derivatives on this column is given in Fig 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 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 Fig 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, chiral representatives were resolved successfully by means of a Astec Cyclobond I 20 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 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, 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 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256 Fig 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, m, mobile phase: ACN: isopropanol: DEA: FA (100%) (95: 5: 0.1: 0.1), ambient temperature, flow: ml/min, UV: 254 nm, injection: 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 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 Fig Simultaneous chiral separation of seven different cathinone derivatives by chiral HPLC Conditions: Column: Trefoil® CEL1 2.5 μm, 150 × 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: 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 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 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 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256 Fig Simultaneous chiral separation of four different ketamine derivatives by chiral HPLC Conditions: Column: Luxđ AMP m, 150 ì 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: μl (Unpublished results) M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256 Fig 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., 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: μl Reprinted from [42] with permission 10 M.G Schmid and J.S Hägele / Journal of Chromatography A 1624 (2020) 461256 Table 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 μm Cyclobond I 2000 Chiralpak® AS Composition of the chiral selector is not provided by the vendor Native β -cyclodextrin Amylose tris [(S)-α -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 NP-mode: Chiralpak ADRH, Lux® Cellulose 1, Lux® Cellulose Lux® Cellulose PO-mode: Lux® Cellulose 2, Chiralcel ODRH, Lux® Cellulose 4, Sepapak [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 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 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, and 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 [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 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 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 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 Additionally, with respect to regioselective discrimination, separation of 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 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 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 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 s (Unpublished results) there might be potential for further success in this field All approaches by CEC are summarized in Table 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 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 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 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 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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]... -cyclodextrin Amphetamine and methamphetamine Amphetamine, methamphetamine, methcathinone and ephedrine derivatives Phenethylamines and impurities Cathinone derivatives [46] [47] Amphetamine derivatives