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Article Enantioseparation and Determination of and Its Metabolites by Capillary Electrophoresis Using Cyclodextrins as Chiral Selectors

Pavel Rezankaˇ 1,* , Denisa Macková 1, Radek Jurok 2,3, Michal Himl 3 and Martin Kuchaˇr 2 1 Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic; [email protected] 2 Department of Chemistry of Natural Compounds, Forensic Laboratory of Biologically Active Substances, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic; [email protected] (R.J.); [email protected] (M.K.) 3 Department of Organic Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic; [email protected] * Correspondence: [email protected]

 Academic Editor: Estrella Núñez Delicado  Received: 25 May 2020; Accepted: 19 June 2020; Published: 23 June 2020 Abstract: Mephedrone, a psychoactive compound derived from , is widely used as a designer . The determination of mephedrone and its metabolites is important for understanding its possible use in medicine. In this work, a method of capillary electrophoresis for the chiral separation of mephedrone and its metabolites was developed. Carboxymethylated β-cyclodextrin was selected as the most effective chiral selector from seven tested cyclodextrin derivates. Based on the simplex method, the optimal composition of the background electrolyte was determined: at pH 2.75 and 7.5 mmol L 1 carboxymethylated β-cyclodextrin the highest total resolution of a mixture of analytes · − was achieved. For mephedrone and its metabolites, calibration curves were constructed in a calibration range from 0.2 to 5 mmol L 1; limits of detection, limits of quantification, precision, and repeatability · − were calculated, and according to Mandel’s fitting test, the linear calibration ranges were determined.

Keywords: capillary electrophoresis; chiral separation; cyclodextrin; mephedrone; metabolites

1. Introduction Mephedrone, a psychostimulant drug first synthesized in the 1920s, is classified as a synthetic cathinone [1]. According to the European Monitoring Centre for and Drug [2], synthetic , together with synthetic , are the most abundant substances in the group of so-called new psychoactive substances. In most cases, these drugs have entactogenic effects; however, the biological activity of such substances is difficult to predict and some of these artificially prepared substances have been shown to have potential therapeutic uses [3], for example, is used to treat and cessation [4] and diethylpropion is prescribed as an anti-obesity drug [5]. On the other hand, has been prescribed for the treatment of chronic obesity and lethargy but has been withdrawn for its abuse by patients [4,6]. These new psychoactive substances can also mimic prescription therapeutic psychoactive drugs; for example, , modafinil, and , and hence are more available even for healthy people because they are available on the Internet [7]. So, an intensive study of these compounds is needed. The most commonly used techniques for isolating new psychoactive substances from biological samples are liquid-to-liquid extraction (LLE) and solid-phase extraction (SPE) [8]. LLE is a simple method, but its problems are easy contamination and matrix influence. On the contrary, the SPE is very selective but time-consuming [9]. A popular method is the QuEChERS method (quick, easy, cheap,

Molecules 2020, 25, 2879; doi:10.3390/molecules25122879 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 12 healthy people because they are available on the Internet [7]. So, an intensive study of these compounds is needed. The most commonly used techniques for isolating new psychoactive substances from biological samples are liquid-to-liquid extraction (LLE) and solid-phase extraction (SPE) [8]. LLE is a simple Molecules 2020, 25, 2879 2 of 12 method, but its problems are easy contamination and matrix influence. On the contrary, the SPE is very selective but time-consuming [9]. A popular method is the QuEChERS method (quick, easy, echeap,ffective, effective, rugged, rugged, and safe) and modified safe) modified in 2012 in for 2012 the for extraction the extraction of psychoactive of psychoactive substances substances from biologicalfrom biological samples samples [10]. [10]. Simple colorimetric colorimetric tests tests are used are for used rapid for orientation rapid orientationdetection of detectiondrugs. However, of drugs. the However,identification the identificationis very indicative,is very detecting indicative, only detecting the presence only theof presencecertain structural of certain motifs structural [11]. motifsHowever, [11 ].immunologicalHowever, immunological methods that methods are commethat arercially commercially available are available more arecommonly more commonly used for usedrapidfor drug rapid detection. drug detection. However, However, there is no there sufficiently is no suffi effectiveciently e ffimmunoassayective immunoassay on the market on the marketfor the fordetection the detection of cathinones of cathinones that does that not does show not cross-reactivity show cross-reactivity between between analogs analogs [9,12,13]. [9, 12,13]. Of the advanced chromatographic techniques, gas and liquid chromatography with a mass spectrometer (GC-MS (GC-MS and and LC-MS) LC-MS) ar aree the the most most suitable suitable for for the the analysis analysis of ofsynthetic synthetic cathinones. cathinones. In Ingeneral, general the, the GC-MS GC-MS technique technique is used is used more more for drug for drug analysis, analysis, mainly mainly due to due shorter to shorter elution elution times times[14–19]. [14 Chiral–19]. Chiral separation separation is mo isst most often often performed performed in inan anachiral achiral environment environment after after the the previous conversion of enantiomers to diastereoisomers [20], [20], which, however, brings complications for the quantificationquantification itself [[21].21]. It should be also notednoted thatthat cathinonescathinones areare thermodegradablethermodegradable substancessubstances and due due to to the the thermal thermal conditions conditions necessary necessary for sepa for separationration by GC, by their GC, partial their partial decomposition decomposition occurs occurs[5]. Therefore, [5]. Therefore, LC-MS LC-MS[22–24] [and22– 24LC-UV] and LC-UV[25,26] usin [25,26g a] usingchiral astationary chiral stationary or mobile or phase mobile is phaseoften isused. often Rapid used. separation Rapid separation of cathinones of cathinones is also poss is alsoible possible by supercritical by supercritical fluid chromatography fluid chromatography (SFC) (SFC)[27,28]. [ 27Carnes,28]. etCarnes al. compared et al. compared the separation the separation of cathinones of cathinones by ultrahigh by efficiency ultrahigh SFC effi ciency(UHPSFC), SFC (UHPSFC),ultrahigh efficiencyultrahigh LC effi (UHPLC)ciency LC with (UHPLC) a non-pola withr acolumn, non-polar UHPLC column, with UHPLC a hydrophilic with a column, hydrophilic and column,GC with anda weakly GC with polara weaklycolumn.polar The column.combination The of combination GC and UHPSFC, of GC and which UHPSFC, is the fastest which of is the fastesttested methods, of the tested seems methods, to be ideal seems [29]. to be ideal [29]. The firstfirst enantioseparationenantioseparation of cathinone derivativesderivatives by capillary electrophoresis (CE) was performed byby MohrMohr etet al. in 2012 using sulfated β-cyclodextrin-cyclodextrin as a chiral selector [30]. [30]. As As with with HPLC, HPLC, CE-UV andand CE-MS CE-MS techniques techniques are usedare used [31– 33[31–33].]. For example, For example, to study to the study incorporation the incorporation of cathinones of intocathinones hair, a into CE methodhair, a CE was method developed was indeveloped which extraction in which from extraction hair at from an elevated hair at temperaturean elevated andtemperature pressure and into pressure ammonium into hydroxide ammonium solution hydroxid wase first solution performed. was first Subsequently, performed. theSubsequently, extract was concentratedthe extract was on aconcentrated solid phase thaton a was solid part phase of the that CE capillarywas part (inline of the couplingCE capillary of SPE-CE), (inline andcoupling analysis of wasSPE-CE), performed and analysis using various was performed cyclodextrins using as various chiral selectorscyclodextrins [34]. as chiral selectors [34]. Mephedrone is referred to as a catecholaminecatecholamine reuptakereuptake inhibitor [35–37], [35–37], but it also positively aaffectsffects the release of into into the the synapt synapticic cleft cleft [13,38–41]. [13,38–41]. Theref Therefore,ore, it itseems seems to to have have a amixed mixed effecteffect [42–44]. [42–44 ].In In both both cases, cases, the the result result is isan an increase increase in inthe the concentration concentration of ofcatecholamines catecholamines in inthe the synaptic synaptic cleft, cleft, resulting resulting in in the the typical typical acti actionon of psychostimulants. Acute Acute intoxication with mephedrone, usually in combination with other drugs and , has already resulted in hundreds of deaths across Europe [[9].9]. Mephedrone has a chiral center on the α carboncarbon and and exists exists in in two two enantiomeric enantiomeric forms forms (Figure (Figure 11).). Both enantiomers have a similar affinity affinity for dopaminergic transporters, but ( S)-mephedrone is about 50 timestimes moremore potentpotent asas aa serotonergicserotonergic mediator.mediator. Nevertheless,Nevertheless, the (R)-enantiomer is primarily responsible for the euphoric eeffectsffects [[45].45].

Figure 1. Structures of ( S)- and ( R)-mephedrone.

In phase I I biotransformation, biotransformation, mephedrone mephedrone ca cann undergo undergo several several reactions: reactions: (i) (i)oxidative oxidative N- Ndemethylation,-demethylation, (ii) (ii) oxidation oxidation of ofthe the 4-methyl 4-methyl group, group, (iii) (iii) ωω-oxidation-oxidation at at the the 3 3′ 0position,position, andand (iv) reduction of the carbonyl groupgroup (Figure(Figure2 2))[ 46[46].].

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Figure 2. SchemeScheme of of phase phase I biotransformat I biotransformationion of ofmephedrone: mephedrone: (i) oxidative (i) oxidative N-demethylation,N-demethylation, (ii) (ii)oxidation oxidation of the of 4-methyl the 4-methyl group, group, (iii) ω (iii)-oxidationω-oxidation at the 3 at′ position, the 30 position, and (iv) andreduction (iv) reduction of the carbonyl of the carbonylgroup. group.

1 The major major metabolites metabolites of of mephedrone mephedrone (1; (4-methyl; 4-methyl ) methcathinone) are 4-methylcathinone are 4-methylcathinone (2; 4- 2 3 (MC); 4-MC)and 4-hydroxymethyl and 4-hydroxymethyl methcathinone methcathinone (3; 4-OH-MMC). ( ; These, 4-OH-MMC). together with These, 4-methylephedrine together with 4-methylephedrine(4; 4-ME), 4-carboxym (4;ethylcathinone 4-ME), 4-carboxymethylcathinone (6; 4-CMC), 4-methylnorephedrine (6; 4-CMC), 4-methylnorephedrine (7; 4-MNE), and 7 ω (normephedrone-; 4-MNE), andω-carboxylic normephedrone- acid (3′-OOH-4-MC),-carboxylic were acid identified (30-OOH-4-MC), in the urine were of users identified by ultra- in thehigh-performance urine of users liquid by chromatography ultra-high-performance‒ liquid chromatography-mass (UHPLC-MS)[47]. Other spectrometry metabolites, (UHPLC-MS)namely 4-carboxyephedrine [47]. Other metabolites(5; 4-CE), 4-MNE, namely (7), 4-hydroxymethylcathinone 4-carboxyephedrine (5; (8 4-CE),; 4-OH-MC) 4-MNE [17], ( 74-), 4-hydroxymethylcathinonecarboxynorephedrine (9; 4-CNE), (8; 4-OH-MC) 4-carboxycathinone [17], 4-carboxynorephedrine (4-CC), and 4-hydroxymethylnorephedrine (9; 4-CNE), 4-carboxycathinone (4- (4-CC),OH-MNE)and [46] 4-hydroxymethylnorephedrine were found in the urine of rats(4-OH-MNE) by GC-MS [46 and] were LC-MS. found in the urine of rats by GC-MS and LC-MS.The aim of this work was to find the optimal conditions for the separation of mephedrone and its metabolitesThe aimof by this capillary work electrophoresis was to find the (for optimal details conditionsabout this method, for the separationsee, for example, of mephedrone Bernardo- andBermejo its metabolites et al. [48]), by using capillary cyclod electrophoresisextrins (CDs) (for[49] detailsas chiral about selectors. this method, The optimization see, for example, of the Bernardo-Bermejobackground electrolyte et al. [(BGE)48]), using was carried cyclodextrins out by (CDs)using the [49] simplex as chiral procedure, selectors. Thewhich optimization was thoroughly of the backgrounddescribed by electrolyte Catai and (BGE)Carrilho was [50]. carried Mephedrone out by using and the all simplex its available procedure, metabolites, which wasselected thoroughly on the describedbasis of current by Catai knowledge and Carrilho on the [50 ].biotransformatio Mephedrone andn of all mephedrone, its available metabolites, are chiral substances selected on that the basismay ofundergo current different knowledge metabolic on the biotransformation pathways and thus of mephedrone, have different are chiral biological substances effects that for may individual undergo dienantiomers.fferent metabolic pathways and thus have different biological effects for individual enantiomers. 2. Results and Discussion 2. Results and Discussion 2.1. Cyclodextrin Optimization 2.1. Cyclodextrin Optimization The first optimization step was to select a suitable CD. Based on our previous experience The first optimization step was to select a suitable CD. Based on our previous experience with1 with CDs [51], analyses were performed in the presence of CDs at a concentration of 10 mmol L− , CDs [51], analyses were1 performed in the presence of CDs at a concentration of 10 mmol·L1 −1, either· either in a 50 mmol L− phosphate buffer at pH 2.5 (Figure3) or in a 50 mmol L− acetate buffer in a 50 mmol·L−1 phosphate· buffer at pH 2.5 (Figure 3) or in a 50 mmol·L−1 acetate· buffer at pH 5 (Figure 4). From the measured electropherograms, it was concluded that the separation proceeds

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Molecules 2020, 25, x FOR PEER REVIEW 4 of 12 Molecules 2020, 25, x FOR PEER REVIEW 4 of 12 at pH 5 (Figure4). From the measured electropherograms, it was concluded that the separation better in the phosphate buffer at a lower pH value. From the selected CDs (Figure 5), i.e., β-CD, proceedsbetter in better the phosphate in the phosphate buffer at a bu lowerffer atpH a va lowerlue. From pH value.the selected From CDs the (Figure selected 5), i.e., CDs β-CD, (Figure 5), carboxymethylated β-cyclodextrin (CM-β-CD), heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin i.e.,carboxymethylatedβ-CD, carboxymethylated β-cyclodextrinβ-cyclodextrin (CM- (CM-β-CD),β -CD),heptakis(2,3,6-tri- heptakis(2,3,6-tri-O-methyl)-O-methyl)-β-cyclodextrinβ-cyclodextrin (Me-β-CD), 2-hydroxypropylated β-cyclodextrin (HP-β-CD), sulfated β-cyclodextrin (S-β-CD), γ-CD, (Me-(Me-β-CD),β-CD), 2-hydroxypropylated 2-hydroxypropylatedβ β-cyclodextrin-cyclodextrin (HP- (HP-β-CD),β-CD), sulfated sulfated β-cyclodextrinβ-cyclodextrin (S-β-CD), (S-β γ-CD),-CD, γ-CD, and carboxymethylated γ-cyclodextrin (CM-γ-CD), CM-β-CD, which is often used for the separation and carboxymethylatedγ γ-cyclodextrin (CM-γγ-CD), CM-ββ-CD, which is often used for the separation andof carboxymethylated basic nitrogenous subs-cyclodextrintances [52,53], (CM- seems-CD), to CM-be the-CD, most which suitable. is often This usedmay forbe thedue separationto the of of basic nitrogenous substances [52,53], seems to be the most suitable. This may be due to the basicinteraction nitrogenous of the substances carboxyl group [52,53 of], CM- seemsβ-CD to bewith the the most NH suitable.group of the This analyte may beand due also to due the to interaction the interaction of the carboxyl group of CM-β-CD with the NH group of the analyte and also due to the of theappropriate carboxyl size group of the of β CM--CDβ cavity.-CD with The inappropriateness the NH group of of the S-β analyte-CD may and be due also to due too tomany the appropriate appropriate size of the β-CD cavity. The inappropriateness of S-β-CD may be due to too many sulfate sizegroups of the β(7–11)-CD cavity.compared The to inappropriateness three carboxylic groups of S-β of-CD CM- mayβ-CD, be see due Section to too 3.1. many In sulfateits presence, groups 18 (7–11) groups (7–11) compared to three carboxylic groups of CM-β-CD, see Section 3.1. In its presence, 18 diastereomers were separated within 30 min, βso it was used in all the other work. The advantage of compareddiastereomers to three were carboxylic separated groups within of30 CM-min, so-CD, it was see used Section in all 3.1the. other In its work. presence, The advantage 18 diastereomers of CM-β-CD over β-CD is its higher solubility in water (50 mg·mL−1 compared to 19 mg·mL−1). Thus, in wereCM- separatedβ-CD over within β-CD 30is its min, higher so itsolubility was used in inwater all the(50 mg·mL other work.−1 compared Theadvantage to 19 mg·mL of−1). CM- Thus,β-CD in over the search for optimal separation conditions, it 1is possible to achieve higher1 concentrations if β-CDthe is search its higher for optimal solubility separation in water conditions, (50 mg mL it− iscompared possible to to achieve 19 mg mLhigher− ). Thus,concentrations in the search if for necessary. · · optimalnecessary. separation conditions, it is possible to achieve higher concentrations if necessary.

FigureFigure 3. Electropherograms3. Electropherograms ofof thethe analyzed mixture mixture of ofthe the studied studied analytes analytes 1–9 (Figure 1–9 (Figure 2) obtained2) obtained Figure 3. Electropherograms of the analyzed mixture of the studied analytes 1–9 (Figure 2) obtained at different CDs used; fused-silica capillary: od/id = 375/75 μm,µ total/effective length: 58.5/50.0 cm; at diatff differenterent CDs CDs used; used; fused-silica fused-silica capillary:capillary: od/id od/id = =375/75375/ 75μm, m,total/effective total/effective length: length: 58.5/50.0 58.5 cm;/50.0 cm; BGE: 50 mmol·L1−1 phosphate buffer, pH 2.5, 10 mmol·L−1 CD;1 voltage: 20 kV (−20 kV for S-β-CD); BGE:BGE: 50 mmol50 mmol·LL− −1phosphate phosphate bubuffer,ffer, pH pH 2.5, 2.5, 10 10 mmol·L mmol−L1 −CD;CD; voltage: voltage: 20 kV 20 (− kV20 (kV20 for kV S-β for-CD); S-β -CD); temperature:· 25 °C. · − temperature:temperature: 25 25◦C. °C.

Figure 4. Electropherograms of the analyzed mixture of the studied analytes 1–9 (Figure2) obtained at different CDs used; fused-silica capillary: od/id = 375/75 µm, total/effective length: 58.5/50.0 cm; BGE: 1 1 50 mmol L− acetate buffer, pH 5, 10 mmol L− CD; voltage: 20 kV; temperature: 25 ◦C. · · Molecules 2020, 25, x FOR PEER REVIEW 5 of 12

Figure 4. Electropherograms of the analyzed mixture of the studied analytes 1–9 (Figure 2) obtained Molecules 2020at ,different25, 2879 CDs used; fused-silica capillary: od/id = 375/75 μm, total/effective length: 58.5/50.0 cm; 5 of 12 BGE: 50 mmol·L−1 acetate buffer, pH 5, 10 mmol·L−1 CD; voltage: 20 kV; temperature: 25 °C.

FigureFigure 5. 5.Structures Structures of of CDs CDs derivativesderivatives used used as as chiral chiral selectors. selectors.

2.2. BGE2.2. BGE Optimization Optimization TheThe enantioselective enantioselective separation separation of of ionicionic analytesanalytes with with ionic ionic CDs CDs is controlled is controlled by at by least at leasttwo two mechanisms.mechanisms. The The first first is is the the interaction interaction ofof thethe hy hydrophobicdrophobic cavity cavity of ofthe the CD CD derivative derivative with withthe the analyte;analyte; the secondthe second is the is the electrostatic electrostatic interaction interaction between the the ionic ionic groups groups of the of theCD CDderivative derivative and and the analyte [54]. The approximate value of the dissociation constant pKa CM-β-CD is 3.0–3.5 [55]. the analyte [54]. The approximate value of the dissociation constant pKa CM-β-CD is 3.0–3.5 [55]. Cathinones are basic drugs and have high values of dissociation constants; for example, the pKa of Cathinones are basic drugs and have high values of dissociation constants; for example, the pKa of mephedrone is 8.77 [56], for methylated cathinones it is 8.4–9.5 [57], for phenylethylamine it is 10.3 mephedrone is 8.77 [56], for methylated cathinones it is 8.4–9.5 [57], for phenylethylamine it is 10.3 [58], [58], and for it is 9.9 [59]. In addition to pH, the separation is also affected by the and forconcentration methamphetamine of cyclodextrin, it is 9.9which [59 affects]. In additionthe amount to of pH, complexed the separation analyte and is also thus a ffaffectsected its by the concentrationoverall electrophoretic of cyclodextrin, mobility which [49]. affects The theseparation amount efficiency of complexed can be analyte increased and at thus higher affects voltages, its overall electrophoreticbut this also mobilityincreases the [49 ].temperature The separation difference effi ciencyin the middle can be and increased at the edge at of higher the capillary, voltages, whichbut this also increasesleads to a thedecrease temperature in efficiency. difference Based on in theour middleexperience and with atthe a similar edge ofBGE the [51], capillary, we havewhich chosen leads a to a decreasevoltage in (see effi Sectionciency. 3.3) Based such onthat our the experiencecurrent is around with 50 a similarμA. In addition BGE [51 to], voltage, we have the chosen currenta also voltage (see Sectiondepends 3.3 on) suchthe ionic that strength the current of the is BGE. around Again, 50 weµA. used In addition our previous to voltage, experience the here, current and also in most depends on thecases ionic used strength a buffer of with the BGE.a concentration Again, we of used 50 mmol·L our previous−1. experience here, and in most cases used Thus, the optimization step was to find1 a suitable concentration of CM-β-CD and a suitable pH a buffer with a concentration of 50 mmol L− . of BGE. In order to monitor both parameters· simultaneously, the simplex method was used to find Thus, the optimization step was to find a suitable concentration of CM-β-CD and a suitable pH the optimal conditions [60]. The first step was to construct an initial simplex. The first two points of BGE.correspond In order to to the monitor conditions: both (1) parameters pH 2.5 and 5 simultaneously, mmolv·L−1 CM-β-CD; the simplex (2) pH 3.5 method and 5 mmol·L was used−1 CM- to find the optimalβ-CD (Figure conditions 6A). The [60 ].third The point first was step calculated was to construct on the basis an initialof the properties simplex. Theof an first equilateral two points 1 1 correspondtriangle,Molecules 2020 to i.e., the, 25pH, x conditions: 3FOR = 2.5 PEER + 0.5·1 REVIEW = (1) 3, pHwhere 2.5 2.5 and corresponds 5 mmolv toL the− CM-first pointβ-CD; and (2) value pH 1 3.5 to andthe length 5 mmol6 of of12 L− · · CM-βthe-CD simplex (Figure edge6A). on The the third pH pointaxis, and was c calculated3 = 5 + √3⁄2·5.774 on the = basis 10 mmol·L of the− properties1, where 5 ofis anthe equilateralinitial simplex forms points (3), (4), and (5), to proceed in two directions, namely to point (6) and point (6′), triangle,concentration i.e., pH3 and= 2.5 5.774+ 0.5 is the1 = length3, where of the 2.5 simplex corresponds edge corresponding to the first pointto the andc-axis, value which 1 has to the been length chosenwhich gives so that a smallthe CM- response.β-CD· concentration Points (3) and is (4) an alsointeger. provide the maximum value1 of the criterion Rkr. of the simplex edge on the pH axis, and c3 = 5 + √3⁄2 5.774 = 10 mmol L− , where 5 is the initial Point (3) was chosen as the point meeting the optimal conditions,· as in the second· −1 case the analysis concentrationA mixture and 5.774 of analytes is the length1–9 (Figure of the 2) simplexwith a concentration edge corresponding of 1 mmol·L to the wasc-axis, measured which twice has been underwas extended each pair to of moreconditions. than Based100 min. on theThe resoluti simplexon ofprocedure the peaks is of summarized the individual in enantiomers, Figure 6B. The the chosenresulting so that optimum the CM- wasβ-CD found concentration at pH 2.75 and is an 7.5 integer. mmol·L−1 CM-β-CD (Figure 7). average criterion Rkr was calculated (see above), which served as a compared response for the simplex procedure. Simplex followed the rules of the equilateral simplex procedure. After calculating the coordinates of point (8), the simplex looped. Thus, the chiral separation proceeded best under the initial conditions chosen. The simplex procedure is summarized in Figure 6A. A half-simplex was designed to more accurately determine the optimal chiral separation conditions (Figure 6B). The first point corresponds to the first determined optimal conditions: (1) pH 2.5 and 5 mmol·L-1 CM-β-CD. The other two points correspond to: (2) pH2 = 2.5 − 0.5·0.5 = 2.25 and c2 = 5 + √3⁄2·2.887 = 7.5; (3) pH3 = 2.5 + 0.5·0.5 = 2.75 and c3 = c2, where the values 0.5 and 2.887 correspond to half the sizes of the edges of the first simplex. Furthermore, the simplex proceeded according to the rules of the equilateral simplex procedure. Since points (3) and (4) provide the same response, Rkr = 12.5, it is possible in a situation where the

FigureFigure 6. Simplex 6. Simplex procedures procedures in optimization in optimization of separation of separation conditions; conditions; (A) full-simplex;(A) full-simplex; (B) ( half-simplex;B) half- μ 1 fused-silicasimplex; capillary: fused-silica od capillary:/id = 375 od/id/75 µ =m, 375/75 total /effm,ective total/effective length: 58.5length:/50.0 58.5/50.0 cm; BGE: cm; 50BGE: mmol 50 L− mmol·L−1 phosphate buffer (46.61 mmol·L−1 HCl at pH 1.5), variable CM-β-CD concentration, variable · phosphate buffer (46.6 mmol L− HCl at pH 1.5), variable CM-β-CD concentration, variable pH; voltage: pH; voltage: 20 kV (10 kV· for pH 1.5); temperature: 25 °C. 20 kV (10 kV for pH 1.5); temperature: 25 ◦C.

Figure 7. Electropherogram of the analyzed mixture of the studied analytes 1–9 (Figure 2) obtained; fused-silica capillary: od/id = 375/75 μm, total/effective length: 58.5/50.0 cm; BGE: 50 mmol·L−1 phosphate buffer, pH 2.75, 7.5 mmol·L−1 CM-β-CD; voltage: 20 kV; temperature: 25 °C.

At pH 2.75, approximately 65–85% of CM-β-CD is in a protonated form. At a low pH, most of the silanol groups on the capillary wall are protonated, leading to low electroosmotic flow, and CM- β-CD carries an approximately one-fold negative charge. At pH 2.75, the groups of all analytes are practically 100% protonated. Metabolites 5, 6, and 9 carry a carboxyl group on the aromatic nucleus in the para position. Upon approximation with , whose pKa is 4.21, it can be assumed that at pH 2.75 the carboxyl group is 4% deprotonated. Thus, it is clear that pH plays an important role in the separation. A lower or higher pH than 2.75 affects the degree of deprotonation of the used CM-β-CD, and thus the separation efficiency. The concentration of CM-β-CD is also an important factor as it affects how much analyte will be complexed with CD. For a given racemic mixture, there is an optimal concentration at which the individual enantiomers will be separated as best as possible [49,61]. This concentration depends on the stability constants of the individual enantiomers and the given chiral selector. In the case of separation of a mixture of racemic substances, as in our case, it is necessary to choose a compromise between these concentrations.

2.3. Calibration

Molecules 2020, 25, x FOR PEER REVIEW 6 of 12 simplex forms points (3), (4), and (5), to proceed in two directions, namely to point (6) and point (6′), whichMolecules gives2020 ,a25 small, 2879 response. Points (3) and (4) also provide the maximum value of the criterion6 R ofkr 12. Point (3) was chosen as the point meeting the optimal conditions, as in the second case the analysis was extended to more than 100 min. The simplex procedure is summarized in Figure 6B. The 1 A mixture of analytes 1–9 (Figure2) with a concentration−1 of 1 mmol L was measured twice resulting optimum was found at pH 2.75 and 7.5 mmol·L CM-β-CD (Figure· 7).− under each pair of conditions. Based on the resolution of the peaks of the individual enantiomers, the average criterion Rkr was calculated (see above), which served as a compared response for the simplex procedure. Simplex followed the rules of the equilateral simplex procedure. After calculating the coordinates of point (8), the simplex looped. Thus, the chiral separation proceeded best under the initial conditions chosen. The simplex procedure is summarized in Figure6A. A half-simplex was designed to more accurately determine the optimal chiral separation conditions (Figure6B). The first point corresponds to the first determined optimal conditions: (1) pH 2.5 and 5 mmol L 1 CM-β-CD. The other two points correspond to: (2) pH = 2.5 0.5 0.5 = 2.25 and c = 5 + · − 2 − · 2 √3⁄2 2.887 = 7.5; (3) pH = 2.5 + 0.5 0.5 = 2.75 and c = c , where the values 0.5 and 2.887 correspond to · 3 · 3 2 half the sizes of the edges of the first simplex. Furthermore, the simplex proceeded according to the rules of the equilateral simplex procedure. Since points (3) and (4) provide the same response, Rkr = 12.5, it is possible in a situation where the simplex forms points (3), (4), and (5), to proceed in two directions, namely to point (6) and point (60), whichFigure gives 6. aSimplex small response.procedures Points in optimization (3) and (4) of also separation provide conditions; the maximum (A) full-simplex; value of the (B criterion) half- Rkr. Pointsimplex; (3) was fused-silica chosen as capillary: the point od/id meeting = 375/75 the optimal μm, total/effective conditions, length: as in the 58.5/50.0 second cm; case BGE: the analysis50 wasmmol·L extended−1 phosphate to more thanbuffer 100 (46.6 min. mmol·L The simplex−1 HCl at procedurepH 1.5), variable is summarized CM-β-CD concentration, in Figure6B. Thevariable resulting optimumpH; voltage: was found 20 kV at(10 pH kV 2.75for pH and 1.5); 7.5 temperature: mmol L 1 CM-25 °C.β -CD (Figure7). · −

FigureFigure 7. 7. ElectropherogramElectropherogram of of the the analyzed analyzed mixture mixture of of the the studied studied analytes analytes 1–9 1–9 (Figure (Figure 2)2 )obtained; obtained; 1 fused-silicafused-silica capillary: capillary: od/id od/id = =375/75375/75 μµm,m, total/effective total/effective length: length: 58.5/50.0 58.5/50.0 cm; cm; BGE: BGE: 50 50 mmol·L mmol L−−1 1 · phosphate buffer, pH 2.75, 7.5 mmol L−1 CM-β-CD; voltage: 20 kV; temperature: 25 C. phosphate buffer, pH 2.75, 7.5 mmol·L· − CM-β-CD; voltage: 20 kV; temperature: 25 °C.◦ β AtAt pH pH 2.75, 2.75, approximately approximately 65–85% ofof CM-CM-β-CD-CD isis inin a a protonated protonated form. form. At At a lowa low pH, pH, most most of theof β thesilanol silanol groups groups on on the the capillary capillary wall wall are are protonated, protonated, leading leading to low to low electroosmotic electroosmotic flow, flow, and CM-and CM--CD βcarries-CD carries an approximately an approximately one-fold one-fold negative negative charge. charge. At pHAt pH 2.75, 2.75, the the amine amine groups groups of allof all analytes analytes are arepractically practically 100% 100% protonated. protonated. Metabolites Metabolites 5, 6, and5, 6, 9 and carry 9 acarry carboxyl a carboxyl group on group the aromatic on the nucleusaromatic in the para position. Upon approximation with benzoic acid, whose pKa is 4.21, it can be assumed that at nucleus in the para position. Upon approximation with benzoic acid, whose pKa is 4.21, it can be assumedpH 2.75 thethat carboxyl at pH 2.75 group the is carboxyl 4% deprotonated. group is 4% Thus, deprotonated. it is clear that Thus, pH playsit is clear an important that pH roleplays in an the β importantseparation. role A in lower the orseparation. higher pH A thanlower 2.75 or ahigherffects thepH degreethan 2.75 of deprotonationaffects the degree of the of useddeprotonation CM- -CD, ofand the thus used the CM- separationβ-CD, and effi thusciency. the separation efficiency. β TheThe concentration concentration of of CM- CM-β-CD-CD is isalso also an animportant important factor factor as it as affects it affects how how much much analyte analyte will willbe complexedbe complexed with with CD. CD. For Fora given a given racemic racemic mixture, mixture, there there is isan an optimal optimal concentration concentration at at which which the the individualindividual enantiomers enantiomers will will be separatedseparated asas bestbest as as possible possible [49 [49,61].,61]. This This concentration concentration depends depends on on the thestability stability constants constants of the of individualthe individual enantiomers enantiomer ands the and given the chiralgiven selector. chiral selector. In the case In ofthe separation case of separationof a mixture of a of mixture racemic of substances, racemic substances, as in our as case, in our it is case, necessary it is necessary to choose to a choose compromise a compromise between betweenthese concentrations. these concentrations.

2.3. Calibration

Molecules 2020, 25, 2879 7 of 12

2.3. Calibration Calibration solutions were measured under the optimized BGE composition, namely at 7.5 mmol L 1 CM-β-CD and pH 2.75. Reduced peak areas (area/migration time) were subjected to the · − Dean–Dixon test to exclude outliers, which is adapted to small datasets with unknown distributions. Furthermore, the regression parameters of the calibration curves were calculated. Based on the test of the significance of the parameter, we assessed whether it is possible to set the intercept a in the regression equation y = bx + a. In the case of analytes 1, 2, 5, and 7, the size of the intercept is insignificant according to the test. Subsequently, the confidence intervals for the intercept a and the slope b of all calibration equations were calculated. If the significance test of the parameter did not allow for ignoring the intercept, the number of degrees of freedom for calculating tcrit was equal to n 2, and in the case of a zero intercept n 1. Ignoring the intercept improved the values of the − − coefficient of determination R2 and thus allowed us to reduce the confidence intervals (Table1).

Table 1. Calibration dependences of separation of analytes 1–7 (Figure2) measured under optimal conditions, where (a L (a)) is the intercept and (b L (b)) is the slope with a confidence interval at the ± ± significance level α = 0.05; R2 is the coefficient of determination.

b L(b) Analyte a L(a) R2 mmol L 1 mmol L 1 · − · − 1 0 32.60 0.27 0.991 ± 2 0 27.76 0.16 0.995 ± 3 5.47 1.31 23.54 1.14 0.807 − ± ± 4 5.07 0.65 19.68 0.29 0.967 − ± ± 5 0 26.14 0.21 0.989 ± 6 3.55 1.07 36.01 0.83 0.959 − ± ± 7 0 21.04 0.20 0.991 ±

According to Mandel’s test, linear concentration ranges were determined and subsequently the LOD and LOQ of individual analytes were calculated (Table2). Precision for all studied analytes was <4.9% and repeatability <4.5%.

Table 2. LOD and LOQ with a linear concentration range of calibration dependences of individual analytes 1–7 (Figure2) according to Mandel’s test.

Linear Concentration Range Analyte 1 LOD mmol L− · LOQ mmol L 1 · − 1 0.35 1.17 5.00 − 2 0.28 0.92 5.00 − 3 0.61 2.02 2.20 − 4 0.53 1.76 5.00 − 5 0.46 1.53 5.00 − 6 0.31 1.04 2.20 − 7 0.21 0.71 2.20 −

3. Materials and Methods

3.1. Chemicals and Materials The following substances were used: ortho-phosphoric acid (50%), β-cyclodextrin (β-CD, 97%), carboxymethylated β-cyclodextrin sodium salt (CM-β-CD, average degree of substitution ~3, 95%), 2-hydroxypropylated β-cyclodextrin (HP-β-CD, average degree of substitution ~0.5–1.3, 95%), sulfated β-cyclodextrin (S-β-CD, average degree of substitution ~7–11, 95%), (99%), thiourea (99%) (all Sigma-Aldrich, Praha, Czech Republic), ultrapure water (Milli-Q grade, Millipore, Molsheim, ), 1 mol L 1 (Tripur, Merck, Darmstadt, Germany), · − Molecules 2020, 25, 2879 8 of 12

(30%)(Suprapur, Merck, Darmstadt, Germany), γ-cyclodextrin (γ-CD, 97%, FUJIFILM Wako, Richmond, VA, USA), carboxymethylated γ-cyclodextrin sodium salt (CM-γ-CD, average degree of substitution ~3–6, 95%, AraChem, Kuala Lumpur, Malaysia), and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (Me-β-CD, 98%, Fluka, Munich, Germany). Psychoactive 1–9 (Figure2) in the form of hydrochloride salts were prepared previously [46]. The purity of the salts studied was determined by UHPLC/MS Agilent 6460 (Waldbronn, Germany) and was higher than 95%.

3.2. Equipment CE separations were performed with an Agilent CE instrument 7100 (Agilent 3D HPCE, Waldbronn, Germany) equipped with a UV-Vis diode-array detector. A bare fused-silica capillary of 375/75 µm od/id and 58.5/50 cm total/effective length was obtained from Polymicro Technologies (Phoenix, AZ, USA) was used.

3.3. CE Conditions The BGE used for the optimizing of the chiral selector consisted of 50 mmol L 1 sodium phosphate · − buffer, pH 2.5 (50 mmol L 1 orthophosphoric acid adjusted to appropriate pH with 1 mol L 1 NaOH) · − · − or acetate buffer, pH 5.0 (50 mmol L 1 acetic acid adjusted to appropriate pH with 1 mol L 1 NaOH), · − · − and 10 mmol L 1 of the studied CDs (β-CD, γ-CD, CM-β-CD, CM-γ-CD, HP-β-CD, 2-Me-β-CD, · − or S-β-CD). The background electrolyte used for the optimizing of the chiral selector concentration and pH consisted of 46.6 mmol L 1 hydrochloric acid (pH 1.5) or 50 mmol L 1 phosphate buffer (pH 2; · − · − 2.25; 2.5; 2.75; 3; 3.25; 3.5) and 0–10 mmol L 1 of CM-β-CD. · − A new fused-silica capillary was first rinsed with 1 mol L 1 NaOH for 30 min, then with H O for · − 2 30 min. Between the runs, the capillary was rinsed at 99.4 kPa first with 0.1 mol L 1 NaOH for 2 min, · − then with H2O also for 2 min, and finally with the running buffer again for 2 min (for the capillary washing, a different buffer solution than for the subsequent analysis was used). The analytes were injected hydrodynamically at a pressure of 1.5 kPa for 5 s. Separations were performed at 10 kV, 20 kV (anode at the injection capillary end), or 20 kV with a voltage ramp time of 12 s. Detection was carried − out at 207 nm during the optimization step and 258 nm for analytes 1, 2, 3, and 6; 236 nm for analyte 5; and 214 nm for analytes 4 and 7 (i.e., at wavelengths corresponding to the absorption maxima) during the calibration step. The capillary was thermostated at 25 ◦C during the analyses.

3.4. Sample Preparation Individual analytes were dissolved in water at a 20 mmol L 1 concentration. For optimizing the · − chiral selector, the analyte solutions were further mixed and diluted with water to a final concentration of 1 mmol L 1. For calibration, seven different mixtures of analytes 1–7 (Figure2) at concentration · − ranges from 0.2 to 5 mmol L 1 (the sum of all analytes’ concentrations was 10 mmol L 1) were prepared · − · − and each mixture was analyzed five times.

3.5. Simplex Method For optimizing the BGE composition, the two-dimensional simplex method was used [60]. The optimizing parameters were CM-β-CD concentration and electrolyte pH. As an evaluation criterion, Rcr was introduced and calculated as follows. For resolution between two neighborhood peaks i and j, if (Rs,ij) < 0.5 then Rcr,ij = 0, if 0.5 < Rs,ij < 1.5 then Rcr,ij = 0.5, and if Rs,ij > 1.5 then Rcr,ij = 1. The sum of Rcr,ij = Rcr.

4. Conclusions In this work, the composition of the basic electrolyte for the chiral separation of mephedrone and its selected metabolites was optimized. A total of seven cyclodextrin derivatives were selected as potential chiral selectors. From the seven tested cyclodextrins, carboxymethylated β-cyclodextrin Molecules 2020, 25, 2879 9 of 12 was selected as the most suitable. Cyclodextrin concentration and pH were determined based on the simplex method for two parameters. The optimized pH was found to be 2.75 and the concentration of carboxymethylated β-cyclodextrin was 7.5 mmol L 1. A total of nine analytes were present in the · − mixture, and 18 distinguishable peaks were found. Thus, all analytes were separated. Furthermore, the data for the construction of calibration dependences were measured. According to the Dean-Dixon test, outliers were excluded, and regression parameters of calibration lines were calculated. Based on the test of significance of the parameter, the intercept on the y-axis was neglected in some cases. Finally, linear concentration ranges according to Mandel’s test and detection limits, limits of determination, precision, and repeatability were determined.

Author Contributions: Conceptualization, P.R.ˇ and M.K.; methodology, P.R.ˇ and D.M.; validation, D.M.; formal analysis, D.M.; investigation, D.M.; resources, P.R.,ˇ R.J., and M.H.; writing—original draft preparation, P.R.;ˇ writing—review and editing, P.R.ˇ and M.K.; visualization, P.R.ˇ and D.M.; supervision, P.R.ˇ and M.K.; project administration, P.R.;ˇ funding acquisition, P.R.ˇ and M.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Interior of the Czech Republic (Grant number VI20172020056). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–9 (Figure2) are available from the authors.

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