Structure Assignment of Seized Products Containing Cathinone Derivatives Using High Resolution Analytical Techniques

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Article Structure Assignment of Seized Products Containing Cathinone Derivatives Using High Resolution Analytical Techniques

João L. Gonçalves 1,* , Vera L. Alves 1 , Joselin Aguiar 1, Maria J. Caldeira 2, Helena M. Teixeira 3,4 and José S. Câmara 1,5,*

1 CQM—Centro de Química da Madeira, Campus Universitário da Penteada, Universidade da Madeira, 9020-105 Funchal, Portugal; [email protected] (V.L.A.); [email protected] (J.A.) 2 Laboratório de Polícia Cientíﬁca da Polícia Judiciária, Novo edifício-sede da Polícia Judiciária, Rua Gomes Freire, 1169-007 Lisboa, Portugal; [email protected] 3 Instituto Nacional de Medicina Legal e Ciências Forenses, I.P., Polo das Ciências de Saúde (Polo III), Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; [email protected] 4 Faculdade de Medicina da Universidade de Coimbra, Azinhaga de Santa Comba, Celas, 3000-548 Coimbra, Portugal 5 Faculdade de Ciências Exatas e da Engenharia, Campus da Penteada, Universidade da Madeira, 9020-105 Funchal, Portugal * Correspondence: [email protected] (J.L.G.); [email protected] (J.S.C.)

Abstract: The innovation of the new psychoactive substances (NPS) market requires the rapid identification of new substances that can be a risk to public health, in order to reduce the damage from their use. Twelve seized products suspected to contain illicit substances were analyzed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), gas chromatography coupled to mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). Synthetic Citation: Gonçalves, J.L.; Alves, V.L.; cathinones (SCat) were found in all products, either as a single component or in mixtures. Infrared Aguiar, J.; Caldeira, M.J.; Teixeira, spectra of all products were consistent with the molecular structure of SCat, showing an intense H.M.; Câmara, J.S. Structure absorption band at 1700–1674 cm−1, corresponding to the carbonyl stretching, a medium/strong Assignment of Seized Products −1 Containing Cathinone Derivatives peak at 1605–1580 cm , indicating stretching vibrations in the aromatic ring (C=C) and bands with −1 Using High Resolution Analytical relative low intensity at frequencies near 2700–2400 cm , corresponding to an amine salt. It was 0 Techniques. Metabolites 2021, 11, 144. possible to identify a total of eight cathinone derivatives by GC-MS and NMR analysis: 4 -methyl-α- https://doi.org/10.3390/ pyrrolidinohexanophenone (MPHP), α-pyrrolidinohexanophenone (α-PHP), 3-fluoromethcathinone metabo11030144 (3-FMC), methedrone, methylone, buphedrone, N-ethylcathinone, and pentedrone. Among the adulterants found in these samples, caffeine was the most frequently detected substance, followed by Academic Editor: Cornelius Hess ethylphenidate. These results highlight the prevalence of SCat in seized materials of the Portuguese market. Reference standards are usually required for confirmation, but when reference materials Received: 1 February 2021 are not available, the combination of complementary techniques is fundamental for a rapid and an Accepted: 24 February 2021 unequivocal identification of such substances. Published: 27 February 2021

Keywords: chemical characterization; new psychoactive substances; synthetic cathinones; FTIR; Publisher’s Note: MDPI stays neutral GC-MS; NMR with regard to jurisdictional claims in published maps and institutional afﬁl- iations.

1. Introduction In the last two decades, a tremendous change in the illicit drug market has become evident, with the emergence of a “new generation” of psychoactive substances [1]. Mimick- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ing the effects of illicit drugs, and sometimes being traded alongside them, these NPS have This article is an open access article introduced many challenges to legal systems, as well as emergency and drug treatment distributed under the terms and services [2]. Among these substances, SCat rapidly gained popularity, especially among conditions of the Creative Commons young people, and currently constitute the second largest group of NPS monitored in Attribution (CC BY) license (https:// Europe, with almost 140 identiﬁed substances so far [3,4]. creativecommons.org/licenses/by/ Natural cathinone, which is the major active component of the Khat plant (Catha 4.0/). edulis Forsk), has been used as a prototype for the development of numerous synthetic

Metabolites 2021, 11, 144. https://doi.org/10.3390/metabo11030144 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, x FOR PEER REVIEW 2 of 21

among young people, and currently constitute the second largest group of NPS monitored Metabolites 2021, 11, 144 2 of 19 in Europe, with almost 140 identified substances so far [3,4]. Natural cathinone, which is the major active component of the Khat plant (Catha edulis Forsk), has been used as a prototype for the development of numerous synthetic deriv- derivativesatives [5,6]. [In5,6 general,]. In general, functional functional group group substitutions substitutions in the in cathinone the cathinone occur occur in three in three dis- distincttinct regions regions of the of themolecule, molecule, namely namely the theamino amino group, group, the alkyl the alkyl side sidechain, chain, and andthe aro- the aromaticmatic ring ring (Figure (Figure 1),1 ),which which has has led led to to a amult multitudeitude of of derivatives derivatives to the streetstreet andand cybercyber drug markets [[7,8].7,8].

Figure 1. General chemical structure of SCat and some representative examples of functional group substituents on the aromatic Figure 1. General chemical structure of SCat and some representative examples of functional group substituents on the ring, the alkyl side chain and the amino group. aromatic ring, the alkyl side chain and the amino group.

SCat can be found on the market in products sold as “bath salts” or “plant feeders”, in different forms,forms, includingincluding tablets,tablets, capsules,capsules, crystalscrystals oror powderspowders [[1,9].1,9]. TheseThese substancessubstances are usuallyusually takentaken orally,orally, either either by by swallowing swallowing capsules capsules or or ‘bombing’, ‘bombing’, where where the the powder powder is wrappedis wrapped in in a cigarettea cigarette paper paper and and subsequently subsequently swallowed swallowed [ 10[10,11].,11]. Inhalation Inhalation isis anotheranother common mode mode of of administration administration of of these these substances, substances, whereas whereas gingival gingival delivery, delivery, intrave- intra- venousnous or orintramuscular intramuscular injection injection and and rectal rectal administration administration are are less less common common routes routes [11,12]. [11,12]. Similarly toto otherother stimulant stimulant drugs drugs like like cocaine cocaine or or 3,4-methylenedioxymethamphetamine 3,4-methylenedioxymethampheta- (MDMA),mine (MDMA), SCat interactSCat interact with monoaminewith monoamine membrane membrane transporters, transporters, leading leading to an increaseto an in- increase serotonin in serotonin concentrations concentrations (5-HT), dopamine (5-HT), dopamine (DA) and noradrenaline(DA) and noradrenaline (NA) [8,10,13 (NA)]. In general,[8,10,13]. these In general, substances these createsubstances a feeling create of a euphoria, feeling of alertness,euphoria, empathy,alertness, andempathy, increased and libido,increased being libido, highly being sought highly after sought as an after alternative as an alternative to traditional to traditional drugs of drugs abuse of [ 14abuse,15]. However,[14,15]. However, the consumption the consumption of SCat canof SCat lead can to alead varied to a andvaried unpredictable and unpredictable number number of side effects,of side andeffects, in manyand in cases, many are cases, linked are to linked aggression to aggression and violent and behavior violent behavior [16,17]. Despite[16,17]. theirDespite relatively their relatively short time short on the time clandestine on the clandestine market, SCat market, have SCat been have responsible been responsible for several casesfor several of acute cases poisonings of acute poisonings and fatal overdoses and fatal overdoses [18–21]. Acute [18–21]. intoxication Acute intoxication with SCat with can produceSCat can aproduce wide range a wide of symptoms,range of symptoms, including including psychiatric psychiatric disturbances disturbances (panic attacks, (panic hallucinations,attacks, hallucinations, paranoid paranoid psychosis, psychosis, and suicidal and ideations), suicidal ideations), sympathomimetic sympathomimetic toxidrome (agitation,toxidrome (agitation, increased aggression,increased aggression, hypertension, hypertension, tachycardia, tachycardia, chest pain, chest cardiac pain, cardiac arrest), seizures,arrest), seizures, acid-base acid-base imbalance imbalance and multi-organ and multi-organ failure [1 failure,4,22,23 [1,4,22,23].]. In general, In thegeneral, most fre-the quentmost frequent cause of cause intoxication of intoxication and fatalities and fatali is relatedties is to related the unknowledge to the unknowledge about the about product the content and its purity, or even with the concentration of the psychoactive substance(s) in the administered dose [24]. Up to now, many efforts and some progress has been made to control the sale and distribution of these substances. From temporary banning orders to consumer legislation, Metabolites 2021, 11, 144 3 of 19

governments are using a host of tools in an attempt to curtail the free sale and distribution of these products [2]. In Portugal, the commercialization of SCat has been outlawed since 2013, after the introduction of the Decree-Law no. 54/2013, of 17 April, which defines the legal framework for the prevention and protection against the advertising and commerce of the NPS and forbids the production, importation, exportation, publicity, distribution, possession and selling of 159 NPS, including 34 SCat [25]. Despite measures that have helped to reduce the supply of NPS, by closing down the so-called “smartshops” (retail establishments specialized in selling psychoactive substances and related parapherna- lia), SCat continue to appear in the drug market and its abuse still represents a public health issue. The ever-evolving nature of these substances has created many analytical challenges for forensic and chemistry laboratories, due to the large number of potential compounds to be investigated, and for which there are no available reference standards [26,27]. Although it is possible to routinely analyze the already identified substances, using appropriate instrumentation, such as liquid or gas chromatography coupled to mass spectrometry (LC-MS and GC-MS), the identification of unknown substances generally requires a structural elucidation before being included in routine methods [28]. Typically, this process requires the use of NMR spectroscopy to identify the chemical structure of the compound. However, the structural similarities of some substances, such as the SCat, may offer analytical challenges, which make the analysis particularly difficult. In such situations, the selection of analytical techniques can be challenging, since each technique has its own advantages that may be more or less suitable, depending on the situations. This highlights the importance of using complementary analytical techniques to confirm the identity of a substance [28]. In this context, the present study describes and discusses the analytical assays performed to identify the components of 12 products seized in Portugal, suspected to contain SCat. Structure elucidation of compounds was carried out by ATR-FTIR, GC-MS and NMR spectroscopy.

2. Results and Discussion The identiﬁcation of products containing NPS is a challenging task, as the present substances do not generally correspond to the label, and can contain a wide range of impurities and/or adulterants [23]. The products were presented in brightly colored packaging or, occasionally, in transparent plastic bags with the trade name, the supposed composition, the product quantity along with the warning “not for human consumption”. Most products (83%) were in powder form, and only two products were presented in the crystal (product 2) and tablets (product 10) forms (Table1). The predominant color of the powders and tablets were white, whereas the crystal had a brownish color. In relation to the composition indicated on the packaging, all had “ketones” in the list of ingredients, presumably indicative of the presence of Scat, except products 1 and 2. In addition, most powder products also referred the presence of caffeine and glucose. The composition indicated on the packaging of the tablets was more elaborated, with the information of the active principle and of other excipients related to the production of the tablets.

Table 1. List of provided samples with the information about their chemical composition indicated on the product label.

Sample Product Description Appearance Quantity (g) Composition Indicated on the Label Number Name 1 Unknown n.a. 1 White powder 1 n.a. 1 2 Flakka n.a. 1 Brownish crystal 5 n.a. 1 3 Bloom Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 4 Bloom Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 5 Bloom Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 6 Bloom Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 7 Bloom Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 8 Charlie Plant feeder Light yellow powder 1 100% Ketones 9 Bliss Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose MetabolitesMetabolites 20212021,, 1111,, 144x FOR PEER REVIEW 44 of of 1921

Sample Num- Product Table 1. Cont. Description Appearance Quantity (g) Composition Indicated on the Label ber Name Sample Product 1 1 1 Unknown Description n.a. White Appearance powder Quantity 1 (g) Composition Indicatedn.a. on the Label Number2 NameFlakka n.a. 1 Brownish crystal 5 n.a. 1 3 Bloom Plant feeder White powder 1 Per Pill:94 % 120 Ketones, mg lactose, 5 % caffeine, 20 mg magnesium 1 % glucose 4 Bloom Plant feeder White powder 1 stearate,94 100% Ketones, mg corn 5 starch,% caffeine, 160 mg1 % ketones,glucose 10 Bliss Plant feeder White tablets 5 tablets 5 Bloom Plant feeder White powder 1 50 mg94 calcium% Ketones, stearate, 5 % caffeine, 4 mg E142, 1 % glucose 6 mg 6 Bloom Plant feeder White powder 1 94 % Ketones,E132, 205 % mg caffeine, E124 1 % glucose 11 Blast Plant feeder White powder 1 89% Ketones, 10% caffeine, 1% glucose 7 Bloom Plant feeder White powder 1 94 % Ketones, 5 % caffeine, 1 % glucose 12 Kick Plant feeder White powder 1 94% Ketones, 5% caffeine, 1% glucose 8 Charlie Plant feeder Light yellow powder 1 100 % Ketones 9 Bliss Plant feeder White powder1 n.a.: not available. 1 94 % Ketones, 5 % caffeine, 1 % glucose Per Pill: 120 mg lactose, 20 mg magnesium stearate, 100 10 Bliss Plant2.1. feeder ATR-FTIR AnalysisWhite tablets 5 tablets mg corn starch, 160 mg ketones, 50 mg calcium stearate, 4 mg E142, 6 mg E132, 20 mg E124 11 Blast Plant feederThe infrared White spectra powder of all seized1 materials were89 consistent % Ketones, 10 with % caffein thee, molecular 1 % glucose struc- 12 Kick Plantture feeder of SCat (FigureWhite S1 powder Supplementary1 Material). All94 spectra % Ketones, showed 5 % caffeine, a prominent 1 % glucose absorption band around 1700–16741 n.a.: not cm available.−1 that corresponds to the carbonyl group stretching (C=O). The intensity of this band exhibited variation among the samples, but waswas thethe majormajor peak in in many many cases. cases. Bands Bands observed observed at 3063–3024 at 3063–3024 cm−1 cmand− 12952–2711and 2952–2711 cm−1 can cm be− 1assignedcan be assignedto aryl C–H to aryland C–Halkyl and C–H alkyl stretch C–H vibration, stretch vibration, respectively. respectively. On the other On the hand, other the hand, me- thedium/strong medium/strong band observed band observed at 1605–1580 at 1605–1580 cm−1 can cm indicate−1 can indicate stretch stretcharomatic aromatic ring vibra- ring vibrationstions (C=C), (C=C), while while a set aof set bands of bands in the in range the range of 2700–2400 of 2700–2400 cm− cm1 can−1 canbe assigned be assigned to the to theamine amine salt salt[28,29]. [28, 29Figure]. Figure 2 describes2 describes an example an example of the of theinfrared infrared bands bands identified identiﬁed in one in oneseized seized product product (product (product 8). 8).

100

80 3063 cm-1 Aromatic 75 C-H stretch 2474 cm-1 70 Amine salt 65 %T 2932-2715 cm-1 60 Aliphatic C-H stretch -1 55 1683 cm C=O stretch 50 1597 cm-1 Aromatic 45 C=C stretch

40 697 cm-1 out of 35 plane C-H bend 31 4000 3500 3000 2500 2000 1500 1000 600 cm-1

FigureFigure 2.2. FTIR spectrum ofof productproduct 88 (“Charlie”).(“Charlie”).

For absorptions belowbelow 15001500 cmcm−−1,, the the differences differences between between samples werewere moremore signiﬁ-signif- cant.icant.A Astrong strong band band observed observed in in products products 9 9 and and 10 10 at at 1259 1259 cm cm−−11 dominates the IR spectrum and couldcould indicate a C–O–C stretchingstretching [[30,31].30,31]. InIn general,general, thethe out-of-planeout-of-plane C–HC–H bendingbending bands in the region ofof 675–900675–900 cmcm−−11areare used used to to differentiate differentiate between between substi substitutedtuted aromatic compounds [[32,33].32,33]. For monosubstituted benzenebenzene rings, out-of-plane C–HC–H bendingbending bandsbands fall between 770–730 770–730 cm cm−−1 1andand 710–690 710–690 cm cm−1 −and1 and areare often often the themost most intense intense bands bands in the in thespectra spectra [32,34]. [32, 34In]. this In thissense, sense, products products 2–8 and 2–8 12 and showed 12 showed a pattern a pattern consistent consistent with these with thesecharacteristics, characteristics, indicating indicating that we that may we be may facing be facing compounds compounds with withmonosubstituted monosubstituted aro- aromaticmatic rings. rings. It is It important is important to note to note that that "Bloom" "Bloom" products products (products (products 3–7) 3–7) had had identical identical IR IRspectra, spectra, which which may may indicate indicate that that they they have have the the same same chemical chemical composition. composition.

2.2. GC-MS Analysis Methanolic solutions of the seized materials were directly analyzed by GC-MS, resulting in different chromatographic proﬁles (Figure S2, Supplementary Information). The EI mass spectra for most compounds were consistent with the fragmentation pattern of Metabolites 2021, 11, x FOR PEER REVIEW 5 of 21

2.2. GC-MS Analysis Metabolites 2021, 11, 144 5 of 19 Methanolic solutions of the seized materials were directly analyzed by GC-MS, resulting in different chromatographic profiles (Figure S2, supplementary information). The EI mass spectra for most compounds were consistent with the fragmentation pattern of SCat, showing the presence of iminium cations as the base peak and small or absent in- tensitiestensities ofof thethe molecularmolecular ionsions (Figure(Figure3 3).). TableTable2 2shows shows the the identiﬁed identified compounds, compounds, their their molecular formulas, retention time, the base peakpeak andand otherother characteristiccharacteristic ionsions forfor thethe identiﬁedidentified compounds.compounds.

FigureFigure 3. EI 3. massEI mass spectra spectra of (A of) (MPHP,A) MPHP, (B) (αB-PHP,) α-PHP, (C) ( CN)-ethylcathinone,N-ethylcathinone, (D) ( Dbuphedrone,) buphedrone, (E) ( Emethedrone,) methedrone, (F) ( Fmethylone,) methylone, (G) 3- (G) 3-FMC and (H) pentedrone foundFMC in and seized (H products.) pentedrone found in seized products.

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Table 2. Active substances detected by GC-MS, with the respective retention times (RT), molecular formula (MF), molecular Tableweight 2. Active(MW), substancesbase peak and detected other by characteristic GC-MS, with ions. the respective retention times (RT), molecular formula (MF), molecular Peakweight (MW), base peak and other characteristicDirect Analysis ions. Derivatization with TFAA Compound Name No. RT (min) MF MW Ions (m/z) RT (min) MF MW Ions (m/z) Direct Analysis Derivatization with TFAA 1 MPHP 1 18.654 C17H25NO 259 140, 91, 119, 41 - - - - Peak No. Compound Name RT 2 α-PHP 1 17.950RT (min) C17H25NO MF 245 MW140, 77, Ions 96, (m 105/z) - MF - MW - Ions - (m/z) (min) 3 N-Ethylcathinone 10.658 C11H15NO 177 72, 44, 77, 105 12.173 C13H14F3NO2 273 168, 105, 140, 77 1 1 MPHP 18.654 C17H25NO 259 140, 91, 119, 41 - - - - 4 Buphedrone1 10.785 C11H15NO 177 72, 77, 44, 105 11.813 C13H14F3NO2 273 168, 105, 77, 110 2 α-PHP 17.950 C17H25NO 245 140, 77, 96, 105 - - - - 11 15 2 13 14 3 3 5 3 MethedroneN-Ethylcathinone 14.820 10.658 C H CNO11H15 NO193 177 58, 135,72, 44, 77, 77, 92 105 16.096 12.173 C C13HH14FF3NONO2 273289 135168, ,77, 105, 154, 140, 92 77 6 4Ethylphenidate Buphedrone 17.357 10.785 C14H19 CNO11H152 NO233 177 84, 91,72, 77,56, 44, 164 105 18.538 11.813 C C1317HH1420FF33NONO2 3 273343 180168, ,164; 105, 67; 77, 11055 5 Methedrone 14.820 C11H15NO2 193 58, 135, 77, 92 16.096 C13H14F3NO3 289 135, 77, 154, 92 7 Caffeine 1 17.775 C8H10N4O2 194 194, 109, 67, 55 - - - - 6 Ethylphenidate 17.357 C14H19NO2 233 84, 91, 56, 164 18.538 C17H20F3NO3 343 180, 164, 67, 55 1 8 7 MethyloneCaffeine 16.31417.775 C11H C138NOH103N 4O2 207 194 58, 149,194, 109,65, 121 67, 55 17.239 - C13H12 -F3NO4 303 - 149, 154, 121, - 110 9 83-FMC Methylone 10.007 16.314 C10H12 CFNO11H13 NO3 181 207 58, 5895,, 149, 75, 65,123 121 10.837 17.239 C C1312HH1211FF34NONO4 2 303277 154149, ,110, 154, 123, 121, 11095 9 3-FMC 10.007 C10H12FNO 181 58, 95, 75, 123 10.837 C12H11F4NO2 277 154, 110, 123, 95 10 Isopentedrone 11.516 C12H17NO 191 120, 42, 118, 91 13.239 C14H16F3NO2 287 110, 216, 182, 140 10 Isopentedrone 11.516 C12H17NO 191 120, 42, 118, 91 13.239 C14H16F3NO2 287 110, 216, 182, 140 1111 Pentedrone Pentedrone 12.083 12.083 C12H17 CNO12H17 NO191 191 86, 44,86, 44,77, 77, 105 105 13.098 13.098 C C1414HH1616FF33NONO2 2 287 182182, 140,, 140, 105, 105, 77 77 1 Derivatization1 Derivatization did did not not occur; occur; BoldBold numbers: numbers: base base peak peak ion. ion.

As reported by Zuba [35], [35], the α-cleavage process is a key feature of the mass spectral fragmentation ofof SCat.SCat. ThisThis process process results results in in the the formation formation of theof the iminium iminium ion ion by fragmen-by frag- mentationtation of the of C–Cthe C–C bond bond between between the αtheand α andβ carbon β carbon atoms atoms under under EI conditionsEI conditions (Figure (Figure4). 4).In theIn the case case of cathinonesof cathinones with with a straight-chained a straight-chained aliphatic aliphatic side side ring, ring, the structuralthe structural formula for- + mulaof the of base the iminiumbase iminium ion is ion represented is represented by C bynH C2n+2nHN2n+2N(n+ (=n 1,= 1, 2, 2,... …),), resultingresulting inin aa basebase peak of mass-to-charge ratio ((mm//zz)) 44, 44, 58, 58, 72, 72, 86, 86, 100 100 and and so so on, on, depending on the number of carbon atoms contained in thethe iminiumiminium ionion [[35,36].35,36]. NN-Ethylcathinone, buphedrone, + pentedrone, methedrone,methedrone, methylonemethylone andand 3-FMC3-FMC are are SCat SCat that that produce produce typical typical C nCHnH2n+22n+2NN+ iminium ions. ions. For For cathinones cathinones with with a pyrrolidine a pyrrolidine ring ring in the in the side side chain, chain, such such as MPHP as MPHP and αand-PHP,α-PHP, fragmentation fragmentation leads leads to the to formation the formation of characteristic of characteristic ions ionsrepresented represented by the by + + structuralthe structural formula formula CnH C2nnNH+ 2n(nN = 5,(n 6,…),= 5, 6, which... ), whichcorresponds corresponds to R3CH=N to R3+CH=N(C4H8) species(C4H8) [35].species The [35 consequent]. The consequent fragmentation fragmentation of the pyrrolidine of the pyrrolidine ring leads ring to leadsthe formation to the formation of char- acteristicof characteristic ions m/ ionsz 70, m55,/z 4270, and 55, 41 42 [35,37]. and 41 [35,37].

R2 R R O O 2 O 2 N -e- N -e- N R R4 R1 R4 R1 R4 1 R3 R3 R3

R2 R2 O O O + N H N + R R4 R4 R1 1 R4 R3 R3 Acylium ion not observable Iminium ion not observable

-CO Strait chain cathinones Pyrrolidine cathinones C H N+ (n =1,2,…) C H N+ (n =5,6,…) + n 2n+2 n 2n R4 m/ z 44, 58, 72, 86, 100, ... m/ z 84, 98, 112, 126, 140, ...

Figure 4. General mass spectra fragmentation pattern of SCat under EI conditions (adapted(adapted from ZubaZuba [[35]).35]).

Another commoncommon reactionreaction observed observed in in SCat SCat under under EI EI condition condition is theis the formation formation of the of theacylium acylium ion. ion. In general, In general, this processthis process takes takes place place at the at same the same location location as for as the for formation the for- mationof the iminium of the iminium ion and ion involves and involves the dissociation the dissociation of the of C αthe–C Cβα–Cbond,β bond, leaving leaving behind behind an anacylium acylium ion ion [36 [36].]. The The consequent consequent loss loss of of carbon carbon monoxide monoxide (CO) (CO) fromfrom thethe acyliumacylium ion results in in the the formation formation of of the the phenyl phenyl cation cation,, as as indicated indicated in inFigure Figure 4. 4The. The phenyl phenyl (m/ (zm 77),/z methylphenyl77), methylphenyl (m/z (91),m/ zand91), fluorophenyl and ﬂuorophenyl (m/z 95) (m /cationsz 95) cations are some are representative some representative exam- plesexamples of ions of ionsproduced produced by the by theloss loss of ofcarb carbonon monoxide monoxide from from the the corresponding corresponding acylium ions at m/z 105 (benzoyl ion), 119 (methylbenzoyl ion) and 123 (ﬂuorobenzoyl ion). Al-

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thoughions at these m/z 105 fragmentation (benzoyl ion), patterns 119 (methylbenzoyl in the mass spectraion) and provide 123 (fluorobenzoyl structural informationion). Alt- abouthough the these elemental fragmentation composition, patterns they in do the not mass differentiate spectra provide structural structural isomers information with different substitutionabout the elemental patterns composition, on the aromatic they ring. do not differentiate structural isomers with differ- entUnfortunately, substitution patterns the EI on method the aromatic is often ring. limited to differentiate structurally similar cathinones.Unfortunately, The similar fragmentation the EI method patterns is often in limit someed ofto thesedifferentiate compounds, structurally associated similar with thecathinones. absence of The molecular similar fragmentation ion peak, may patterns produce inambiguous some of these mass compounds, spectra [38 associated]. However, thewith differentiation the absence of molecular these compounds ion peak, can may be produce achieved ambiguous using an alternativemass spectra approach, [38]. How- such asever, chemical the differentiation derivatization, of which these cancompounds represent can a straightforward be achieved using and an cost-effective alternative wayap- to improveproach, thesuch capability as chemical of compoundderivatization, identification which can represent [39,40]. a straightforward and cost- effective way to improve the capability of compound identification [39,40]. TFAA is one of the most widely used derivatizing agents, known to react with the TFAA is one of the most widely used derivatizing agents, known to react with the primary and secondary amine groups of the amphetamine-type stimulants [41,42]. Deriva- primary and secondary amine groups of the amphetamine-type stimulants [41,42]. Deriv- tives are formed via acylation, where the amine hydrogen is removed and replaced with atives are formed via acylation, where the amine hydrogen is removed and replaced with a trifluoroacyl group [43]. Not all substituted cathinones can be directly derivatized us- a trifluoroacyl group [43]. Not all substituted cathinones can be directly derivatized using ingTFAA, TFAA, which which requires, requires, at least, at least, one onehydrogen hydrogen atom atomin the in amino the amino group groupor an active or an hy- active hydrogendrogen atom atom from from other other function functionalal groups groups (e.g. (e.g., –OH, –OH, –COOH). –COOH). AfterAfter derivatization derivatization and and knowing knowing the the molecular molecular weight weight and ions and resulting ions resulting from frag- from fragmentation,mentation, it was it was possible possible to toidentify identify the the chemical chemical structures structures of the of theformed formed compounds. compounds. FigureFigure S3 S3 (Supplementary (supplementary Information)information) shows shows the the typical typical GC-MS GC-MS chromatograms chromatograms of the of the seizedseized products products afterafter derivatizationderivatization with with TFAA, TFAA, and and Figure Figure 55 describes describes the the respective respective massmass spectra spectra of of SCat SCat afterafter derivatization.derivatization. Table Table 22 showsshows the the identified identified TFAA TFAA derivative derivative compounds,compounds, their their molecular molecular formulas, formulas, retention retention time, time, thethe basebase peakpeak andand otherother characteristiccharacter- ionsistic for ions the for identified the identified compounds. compounds.

Figure 5. EI mass spectra of (A) buphedrone (B) N-ethylcathinone (C) methedrone (D) methylone (E) 3-FMC and (F) pentedrone after derivatization with TFAA.

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Derivatization of SCat with TFAA produced a better chromatographic resolution and Figure 5. EI mass spectraincreased of (A) buphedrone mass spectral(B) N-ethylcathinone abundances (C) methedrone for molecular (D) methylone and fragment (E) 3-FMC ions.and (F) Beforepentedrone derivatization, after derivatization with TFAA. products containing N-ethylcathinone and buphedrone (products 3–8) had a poor resolution, and theDerivatization fragmentation of SCat patternswith TFAA observed produced ona better the masschromatographic spectra of resolution both compounds and were very similar.increased Aftermass spectral derivatization, abundances these for molecu SCatlar showed and fragment better ions. chromatographic Before derivatiza- separation, with larger,tion, products narrower, containing and N more-ethylcathinone symmetric andchromatographic buphedrone (products peaks, 3–8) hadand a poor the mass frag- mentationresolution, pattern and ofthe both fragmentation compounds patterns were observed readily on distinguished the mass spectra by of the both different com- relative pounds were very similar. After derivatization, these SCat showed better chromato- abundancegraphic of separation, two common with larger, fragment narrower, ions and (m/ morez 140 symmetric and 110). chromatographic In general, SCat peaks, with a methyl substituentand the onmass the fragmentation nitrogen atom, pattern such of both as compounds buphedrone, were have readily a characteristicdistinguished by ion the at m/z 110 + ([CH3different–N≡C–CF relative3] ).abundance This cation of two may common be formed fragment from ions ( them/z 140 decomposition and 110). In general, reaction of the TFAASCat imine with specie a methyl at m substituen/z 168 (Figuret on the 6nitrogenA). N-Ethylcathinone atom, such as buphedrone, with an have ethyl a substituentcharac- in the teristic ion at m/z 110 ([CH3–N≡C–CF3]+). This cation may be formed from the decomposi- + amino group produced analogous cation at m/z 124 corresponding to [C2H5–N≡C–CF3] tion reaction of the TFAA imine specie at m/z 168 (Figure 6A). N-Ethylcathinone with an (Figureethyl6B), substituent with low in the relative amino abundancegroup produced (2%). analogous In addition, cation at m both/z 124 compounds corresponding showed a characteristicto [C2H5–N ion≡C–CF at m3]+/ (Figurez 140. 6B), This with cation low relative is more abundance abundant (2%). in InN addition,-ethylcathinone both com- (29%) than in buphedronepounds showed (4%) a and characteristic is probably ion originatedat m/z 140. fromThis cation a rearrangement is more abundant of the in ethyl N- group of the methylcathinone/z 168 cation (29%) to lose than ethylene. in buphedrone (4%) and is probably originated from a rearrangement of the ethyl group of the m/z 168 cation to lose ethylene.

Figure 6. ProbableFigure 6. Probable fragmentation fragmentation pathways pathways of of TFAA TFAAderivatives derivatives of, of, (A () Abuphedrone,) buphedrone, and (B and) N-ethylcathinone. (B) N-ethylcathinone. We can conclude, based on these results and considering the number of potential Wecompounds can conclude, to be investigated, based on associated these results to theand lack consideringof reference standards the number and the of exist- potential com- poundsence to of be a wide investigated, diversity of associated isomers with to identical the lack fragmentation of reference patterns standards under and EI condi- the existence of a widetions, diversity that the ofderivatization isomers with with identical TFAA showed fragmentation to be an effective patterns strategy under to confirm EI conditions, the that the derivatizationidentity of these withsubstances, TFAA allowing showed us to to obtain be an more effective specific strategy structural toinformation confirm of the identity of thesethese substances, compounds. Thus, allowing through us the to GC-MS obtain analys moreis,specific it was possible structural to identify information a total of of these compounds. Thus, through the GC-MS analysis, it was possible to identify a total of 11 different substances, belonging, the most of them, to the class of SCat. MPHP was identified as the main compound in product 1, while α-PHP was the main component in product 2. Both substances showed characteristic mass spectral fragmentation pattern with SCat containing a pyrrolidine ring in the side chain. Methylone was identified as the main component in product 10, and together with caffeine in product 9. “Bloom” products (products 3–7) had a similar chromatographic profile and identical chemical composition, which means that they probably belong to the same batch. N-Ethylcathinone, buphedrone, Metabolites 2021, 11, 144 9 of 19

methedrone, caffeine and ethylphenidate were the identified substances in these samples. It was also confirmed by GC–MS that products 8, 11 and 12 contained N-ethylcathinone, making this SCat the most frequently detected psychoactive substance (67% of the total analyzed products). On the other hand, 3-FMC and pentedrone were the main compounds in products 11 and 12, respectively. Also present, in product 12, was an isomeric impurity identified as isopentedrone (1-methylamino-1-phenylpentan-2-one), a by-product of the pentedrone synthesis, in which the amino moiety and keto group changed their position in the molecule. In relation to the adulterant compounds found in these products, caffeine was the most frequently detected substance (67% of the total analyzed products), followed by ethylphenidate (50% of the total analyzed products).

2.3. NMR Analysis In order to complement the FTIR and GC-MS results, NMR analyzes were performed for a correct structural elucidation. The 1H and 13C NMR spectra of all substances were registered at 400 and 100 MHz, respectively. Signals assignments were based on chemical shifts (δ, ppm) of 1H and 13C, on the multiplicity patterns of proton resonances depicted by the J couplings (Hz), and on data of homonuclear 1H-1H COSY and heteronuclear 1H-13C HMBC and HSQC. The NMR experiments and the signals assignments were made for all compounds and presented in Tables3 and4. For product 11, which contains a fluoromethcathinone, a 19F NMR spectrum (376.5 MHz) was also recorded and the respective signal assignment was presented in Table4. For pyrovalerone derivatives, namely MPHP and α-PHP, identified in products 1, and 2, respectively, the NMR spectra (Figures S4–S8, Supporting Information) showed many similarities, but also some differences that allowed unequivocal identification of both compounds. MPHP is a 1,4-disubstituted aromatic compound with a symmetric distribution of protons on the aromatic ring, and for this reason, the 1H NMR signal of the aromatic protons exhibits a characteristic splitting pattern constituted by two doublets at 7.96 ppm (H-20/H-60) and 7.47 ppm (H-30/H-50). The methyl group attached to the para position of the benzene ring (H-70) produced a large singlet at 2.46 ppm, which was also observed by Westphal et al. [44]. In contrast to MPHP, the 1H NMR spectrum of α-PHP showed a typical phenyl pattern at 7.65 ppm (meta, appears as a 2H triplet), 7.81 ppm (para, appears as a 1H triplet), and 8.06 ppm (ortho, appears as a 2H doublet), which is consistent with the values reported in the literature [45,46]. The methine proton (H-2) of both pyrovalerone derivatives showed a triplet signal at around 5.3 ppm, and the protons of the alkyl side chain were found between 2.18 ppm (H-3) and 0.76 ppm (H-6). The multiplets of the methylene protons H-100 and H-4” of the pyrrolidine-ring appeared as separated signals, centered at 3.07 ppm (1H from H-4”), 3.36 ppm (1H from H-100) and 3.74 ppm (two overlapped protons; 1H from H-4” and 1H from H-100), while the proton signals of H-200 and H-3” appeared between 2.01 and 2.30 ppm and were partially overlap with the methylene protons H-3 of the alkyl side chain. Due to their structural similarities, it was also possible to observe several common characteristics in the 13C NMR spectra, including the signals for the carbon of the carbonyl group (C-1) around 198 ppm, the chiral carbon C-2 at 70 ppm and the carbons of the aliphatic side chain C-3, C-4, C-5 and C-6 between 13.2 ppm and 30.2 ppm. The carbons C-1” and C-4” of pyrrolidine moiety resonate at 52.6 ppm and 55.8 ppm, respectively, while C-2” and C-3” signals are partially overlap at 23.3 ppm, in both compounds. The six aromatic carbons appearing as four signals for these pyrovalerone derivatives showed some differences, thus indicating chemical and structural differences between compounds. By the attachment of a substituent group to the benzene ring, the electronic density and consequently the chemical shifts of the carbon atoms will increase or decrease depending on the electronic nature of the substituent and its position in the aromatic ring [47]. In this sense, the presence of a methyl group in the para position of the aromatic ring of MPHP leads to a downfield shift of 12 ppm of the C-40 resonance in comparison with α-PHP. In addition, the carbon of the methyl group C-70 attached to the aromatic ring produced a signal at around 21 ppm, which is in agreement with the results obtained by Westphal et al. [44]. Metabolites 2021Metabolites, 11, x FOR 2021 PEERMetabolites, 11, REVIEWx FOR 2021 PEER ,Metabolites 11 REVIEW, x FOR PEER2021 , 11REVIEW, x FOR PEER REVIEW 10 of 21 10 of 21 10 of 21 10 of 21

atives showedatives some showed differences,atives some showed differences, thusatives some indica showed differences, thusting indicachemical some ting thusdifferences, and chemicalindica strutingctural thus and chemical indicadifferencesstructuralting and chemicaldifferences be-structural and differencesbe- structural be- differences between compounds.tween compounds. By tweenthe attachment compounds. By thetween attachment of a Bycompounds.subs thetituent attachmentof a subs group By tituentthe ofto attachment athe subsgroup benzenetituent to ofthe ring, agroup benzenesubs thetituent to elec- the ring, benzenegroup the elec- to ring, the benzene the elec- ring, the electronic densitytronic and density consequentlytronic and density consequently thetronic andchemical consequentlydensity the sh chemicalifts and of consequently thethe sh carbonchemicalifts of theat theomssh carbonifts chemical will of attheincreaseoms carbonsh iftswill or of increase at theoms carbon will or increase atoms willor increase or decrease dependingdecrease depending ondecrease the electronic ondepending thedecrease nature electronic on dependingof the the nature electronic substituent ofon the the nature substituent andelectronic ofits theposition naturesubstituentand its in of positionthe the aro-and substituent itsin theposition aro- and in its the position aro- in the aromatic ring matic[47]. In ring this [47].matic sense, In ring thisthe [47].presencesense,matic In thethisring of presence sense,a[47]. methyl In the ofthis group presencea sense,methyl in theof group apresencepara methyl inposition the groupof para a ofmethyl position inthe the grouppara of positionthe in the paraof the position of the aromatic ringaromatic of MPHP ringaromatic leads of MPHP to ringa downfield leadsaromatic of MPHP to a downfieldshiftring leads of MPHPto12 a ppmshift downfield leads ofof the12 to ppm C-4’ shifta downfield ofresonance of the 12 C-4’ppm shift inresonance of com- ofthe 12 C-4’ ppm in resonance com- of the C-4’ in resonancecom- in comparison withparison α-PHP. with Inparison addition,α-PHP. with In the addition, parisonα -PHP.carbon withIn theof addition, the carbon α-PHP. methyl ofthe In thegroup addition,carbon me thylC-7’ of the grouptheattached carbon me C-7’thyl to of attachedgroup the the aro- me C-7’ thylto attachedthe group aro- C-7’ to the attached aro- to the aromatic ring maticproduced ring aproducedmatic signal ring at a aroundproduced signalmatic 21 atring aroundppm,a signalproduced which 21 at ppm,around isa signalin which agreement 21 atppm, isaround in which agreementwith 21 theisppm, in results withagreement which the is results in with agreement the results with the results obtained byobtained Westphal, byobtained etWestphal, al. [44]. by et Westphal,obtained al. [44]. by et Westphal,al. [44]. et al. [44]. Metabolites 2021, 11, 144 10 of 19 Table 3. NMRTable assignments 3. NMRTable assignments of SCat 3. NMR constituted Tableassignmentsof SCat 3. by constitutedNMR a pyrrolidineof assignments SCat by constituted a ripyrrolidineng of in SCat the by constitutedsidea ri pyrrolidineng chain in the or byside ari amethoxyng pyrrolidinechain in the or or sidea methoxya rimethylenodioxy chainng in orthe or a aside methoxy methylenodioxy chain or or a a methylenodioxy methoxy or a methylenodioxy group attachedgroup to theattached aromaticgroup to the ring.attached aromatic group to thering. attached aromatic to ring. the aromatic ring. Table 3. NMR assignments of SCat constituted by a pyrrolidine ring in the side chain or a methoxy or a methylenodioxy group attached to the aromatic ring.

O O O O 2' H 2' H 2' H 2' H 2 2 2 2 3' 1' N3' 1' N 1' N 1' N 1 1 3' 1 3' 1 1'' 1'' 1'' 1'' 7' 4' 7' 4' 7' 4' 7' 4' 6' 6' 6' 6' O 3O 3 O 3 O 3 5' 5' 5' 5'

Position

Posi- Posi- Posi- Posi- tion tion MPHPtion tion α-PHP Methylone Methedrone 13C(δ/ppm) MPHP1H(δ /ppm,MPHP protons, MPHP13C( αδ-PHP/ppm) MPHPα -PHP1H(δ /ppm,α-PHP protons, Methylone 13α -PHPC(Methylone δ/ppm) Methylone 1H( Methedroneδ/ppm, Methylone protons, Methedrone Methedrone13C(δ/ppm) Methedrone1H(δ/ppm, protons, 13C multiplicity1H 13(δC/ppm, pro-a,1H coupling (13δ/ppm,C pro-13C1H (δ/ppm, 13C1H ( pro-δ13/ppm,C 1H pro-( δ1/ppm,H (δmultiplicity13/ppm, Cpro- pro-13C1 H (aδ, /ppm,13C1H 13(pro-δC/ppm, 1H ( pro-δ/ppm,1H (δ13/ppm,C pro- 13 Cpro-multiplicity 1H (δ/ppm,131HC 13(δC /ppm,pro- a, coupling1H pro- (1δH/ppm, (13δC/ppm, pro- pro-1H (δ/ppm,13C pro- 1H (multiplicityδ/ppm, pro- a, (δ/ppm) tons,(δ/ppm) constants)multiplic- tons,(δ/ppm) multiplic- (δ/ppm)tons,( δ/ppm) multiplic-tons,(δ/ppm) multiplic-tons, tons,couplingmultiplic-(δ/ppm) multiplic-( δ constants)/ppm)tons, (δ multiplic-/ppm)tons,(δ/ppm) multiplic-tons, multiplic-tons,(δ/ppm) multiplic-(δ/ppm) tons, (δ /ppm)constants)multiplic-(tons,δ/ppm) multiplic- tons,tons,( δmultiplic-/ppm) multiplic- tons,(δ/ppm) multiplic- couplingtons, multiplic- constants) 1 197.9itya, coupling - itya, coupling itya, coupling 198.4ity 1, coupling itya, couplingity 1, coupling - ity 1, couplingitya, coupling ity 195.9 1, coupling itya, coupling itya, couplingitya -, coupling itya, ity couplinga, coupling 196.4itya, coupling itya, coupling - 2 69.9 5.27,constants) 1H, t, J = 5.06constants) Hz constants) 70.0constants) constants)5.31, constants) 1H, t, J = 5.06constants) Hzconstants) constants) 59.9 constants) 5.06–5.01,constants)constants) 1H, q,constants) constants) 59.9constants) 5.11–5.05,constants) 1H, q, 1 197.91 197.91 - 197.9 - 1 198.4 197.9 - 198.4 - - 198.4 - 195.9 198.4 - 195.9 - - 195.9 - 196.4 195.9J = - 196.4 7.23 -Hz 196.4 - - 196.4 - J = - 7.21 Hz 3 30.2 2.18–2.01, 2H, m 30.0 2.18–2.01, 2H, m 16.3 1.64, 3H, d, J = 7.24 Hz 16.2 1.64, 3H, d, J = 7.24 Hz 2 69.92 5.27,69.9 21H, t, J =5.27, 69.9 1H, 2 t, 70.0J = 5.27, 69.9 1H, 5.31, t, 70.0 J =1H, 5.27, t, J = 5.31,1H, 70.0 t, 1H, J = t, 59.9J = 5.31, 1H, 70.0 5.06–5.01, t, 59.9 J = 5.31, 1H, 5.06–5.01, 1H,q, 59.9 t, J = 59.91H, q, 5.06–5.01, 59.9 5.11–5.05,59.9 1H, q, 1H, 5.06–5.01, 5.11–5.05, q, J59.9 1H, 1H, q, q, 5.11–5.05, J 59.9 1H, q, 5.11–5.05,J 1H, q, J 4 25.8 1.28–1.22, 1H, m 25.3 1.28–1.18, 1H, m ---- 1.15–1.11,5.06 Hz 1H,5.06 m Hz 5.06 Hz 5.06 Hz 5.061.15–1.11, Hz5.06 Hz 1H, m5.06 HzJ = 7.23 Hz5.06 JHz = 7.23 Hz J = 7.23 =Hz 7.21 HzJ = 7.23= 7.21 Hz Hz = 7.21 Hz = 7.21 Hz 53 22.430.23 2.18–2.01, 1.28–1.21,30.23 2H,2.18–2.01, 2H,m 30.2 m 3 2H, 30.0 m2.18–2.01, 30.2 22.3 2.18–2.01, 2H,30.0 m 2.18–2.01, 2H, 2.18–2.01, 1.28–1.18, 30.02H, m 2H, 16.3 2H, 2.18–2.01, m 30.0 1.64, 16.32H, 3H, 2.18–2.01, d, -J = 1.64, 16.3 2H,3H, 16.2 d, J = 1.64, 16.33H, 1.64,16.2 d, - 3H, J = d, 1.64, J = 1.64, 3H,16.2 3H,d, J d,= J = 1.64, -16.2 3H, d, J = 1.64, 3H, d, - J = 6 13.3 0.76, 3H, t, J = 6.92 Hz 13.2m 0.76, 3H,m t, J = 7.02 Hzm 7.24 Hz -m 7.24 Hz 7.24 Hz7.24 - Hz 7.247.24 Hz Hz -7.24 Hz 7.24 Hz - 0 1 4 131.625.84 1.28–1.22,25.84 1H, -1.28–1.22, m 25.8 4 1H, 25.3 m1.28–1.22, 25.8 134.1 1.28–1.18, 1H,25.3 m 1.28–1.22, 1H, 1.28–1.18, 25.31H, m -1H, - 1.28–1.18, 25.3 1H,- - 1.28–1.18, 127.3 -1H, ------125.8 - - - - 20 129.7 7.96, 2H, d, J = 8.16 Hz 129.5 8.06, 2H, d, J = 7.52 Hz 108.5 7.47, 1H, d, J = 1.64 Hz 132.2 8.04, 2H, at, 1.15–1.11, 1H,1.15–1.11, m 1H, m1.15–1.11, 1H, mm1.15–1.11, 1H,m m m m J = 8.84 Hz 30 130.6 7.47, 2H, d, J = 8.08 Hz 129.91.15–1.11, 1H, 7.65,1.15–1.11, 2H, t, 1H,J = 1.15–1.11, 7.82 Hz 1H, 1.15–1.11, 148.9 1H, - 115.1 7.14, 2H, d, J = 8.96 Hz 40 148.4 - 136.3m 7.81, 1H,m t, J = 7.46 Hzm 154.1m - 165.4 - 0 5 5 130.622.45 7.47,1.28–1.21,22.4 2H,5 d, 2H,J 1.28–1.21,= m 8.08 22.4 Hz 5 2H, 22.3 m1.28–1.21, 22.4 129.9 1.28–1.18, 2H, 22.3 m 1.28–1.21, 2H, 7.65, 1.28–1.18, 22.32H, m t, 2H,J =- 1.28–1.18,7.82 Hz22.3 2H,- - 1.28–1.18, 109.1 -2H, - - 7.04, 1H,- - d, - J = - 8.28 Hz - - - 115.1 - - 7.14, 2H, - d, J = 8.96 Hz 0 6 129.7 7.96, 2H, d, J = 8.16 Hz 129.5 m 8.06, 2H,m d, J = 7.52 Hzm 127.0m 7.70–7.68, 1H, dd, J = 8.32, 132.2 8.04, 2H, at, 1.72 Hz J = 8.84 Hz 6 13.36 0.76,13.3 63H, t, J =0.76, 13.3 3H, 6 t, 13.2J = 0.76, 13.3 3H, 0.76, t, 13.2 J =3H, 0.76, t, J = 0.76,3H, 13.2 t, 3H, J = t, J -= 0.76, 3H, 13.2 t, J- = - 0.76, 3H, t,- J -= ------70 21.6 2.46, 3H, s - - 103.2 6.14, 2H, s 56.3 3.48, 3H, s 6.92 Hz 6.92 Hz 7.02 Hz 7.02 Hz 100 52.6 3.80–3.69, 1H, m 6.9252.6 Hz 6.92 3.80–3.70, Hz 1H, m7.02 Hz 31.57.02 Hz 2.82, 3H, s 31.6 2.83, 3H, s 1’ 131.61’ 131.63.39–3.32,1’ - 1H,131.6 m - 1’ 134.1 131.6 - 134.1 - 3.40–3.32,- 134.1 - 127.3 1H, m 134.1 - 127.3 - - 127.3 - 125.8 127.3 - 125.8 - 125.8 - - 125.8 - - 200 2’ 23.3129.72’ 7.96,129.7 2.18–2.01, 2’2H, d, J 2H,=7.96, 129.7 m 2H, 2’ d, 129.5 J = 7.96, 129.7 2H, 23.3 8.06, d, 129.5 J 2H,= 7.96, d, J = 8.06,2H, 2.18–2.01, 129.5 d, 2H, J = d, 108.5 J 2H,= 8.06, m 129.52H, 7.47, d, 108.5 J =1H, 8.06, d, -J 2H,= 7.47, 108.5 d, 1H, J =132.2 d, J = 7.47, 108.5 1H, 8.04,132.2 d, - 2H,J = at, 7.47, J 8.04,= 1H,132.2 2H, d, J at,= J = 8.04, -132.2 2H, at, J = 8.04, 2H, at, - J = 00 3 23.4 2.30–2.23,8.16 Hz 1H,8.16 m Hz 8.1623.4 Hz 7.52 Hz 8.16 2.27–2.21, Hz7.52 Hz 1H, m7.52 Hz 1.64 Hz 7.52---- Hz1.64 Hz 1.64 Hz8.84 Hz 1.648.84 Hz Hz 8.84 Hz 8.84 Hz 3’ 130.63’ 7.47, 130.62.18–2.01, 3’2H, d, J 1H,= 7.47, 130.6 m 2H, 3’ d, 129.9 J = 7.47, 130.6 2H, 7.65, d, 129.9 J =2H, 7.47, t, J = 2H, 7.65,2.18–2.01, 129.9 d, 2H, J = t, 148.9 J = 1H, 7.65, m 129.92H, t, 148.9 J = - 7.65, 2H, 148.9 t, J -= 115.1 148.9 7.14, - 115.1 2H, d, J = 7.14, 115.1 - 2H, d, J = 7.14, 115.1 2H, d, J = 7.14, 2H, d, J = 400 55.8 3.80–3.69, 1H, m 55.8 3.80–3.70, 1H, m ---- 8.08 Hz 8.08 Hz 8.08 Hz 7.82 Hz 8.08 Hz7.82 Hz 7.82 Hz 7.82 Hz 8.96 Hz 8.96 Hz 8.96 Hz 8.96 Hz 3.10–3.04, 1H, m 3.12–3.06, 1H, m 4’ 148.44’ 148.44’ - 148.4 - 4’ 136.3 148.4 - 7.81, 136.3 1H, t, J = 7.81,- 136.3 1H, t,154.1 J = 7.81, 136.31H, t,154.1 J = - 7.81, 1H,154.1 t, J -= 165.4 154.1 - 165.4 - 165.4 - - 165.4 - - a abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, at = apparent triplet, dd = doublet of doublets. J = coupling constant. 7.46 Hz 7.46 Hz 7.46 Hz 7.46 Hz 5’ 130.65’ 7.47,130.6 5’2H, d, J =7.47, 130.6 2H, 5’ d, 129.9 J = 7.47, 130.6 2H, 7.65, d, 129.9 J =2H, 7.47, t, J = 2H, 7.65, 129.9 d, 2H, J = t, 109.1 J = 7.65, 129.92H, 7.04, t, 109.1 J = 1H, 7.65, d, J =2H, 7.04, 109.1 t, 1H, J = 115.1 d, J = 7.04, 109.1 1H, 7.14,115.1 d, 2H,J = d, 7.04, J = 7.14, 1H,115.1 2H,d, J d,= J = 7.14,115.1 2H, d, J = 7.14, 2H, d, J = 8.08 Hz 8.08 Hz 8.08 Hz 7.82 Hz 8.08 Hz7.82 Hz 7.82 Hz 8.28 Hz 7.82 Hz8.28 Hz 8.28 Hz8.96 Hz 8.288.96 Hz Hz 8.96 Hz 8.96 Hz 6’ 129.76’ 7.96, 129.7 6’2H, d, J = 7.96, 129.7 2H, 6’ d, 129.5 J = 7.96, 129.7 2H, 8.06, d, 129.5 J 2H,= 7.96, d, J = 8.06,2H, 129.5 d, 2H, J = d,127.0 J = 8.06, 129.52H, 7.70–7.68,d,127.0 J = 8.06, 1H, 2H, 7.70–7.68, 127.0 d, J =132.2 1H, 7.70–7.68,127.0 8.04,132.2 1H, 2H, at, 7.70–7.68, J 8.04,= 132.2 2H, 1H, at, J = 8.04, 132.2 2H, at, J = 8.04, 2H, at, J = 8.16 Hz 8.16 Hz 8.16 Hz 7.52 Hz 8.16 Hz7.52 Hz 7.52 dd,Hz J = 8.32, 1.727.52dd, Hz J = 8.32, 1.72dd, J = 8.32,8.84 1.72 Hz dd, J = 8.32,8.84 1.72Hz 8.84 Hz 8.84 Hz Hz Hz Hz Hz 7’ 21.67’ 21.62.46,7’ 3H, s 2.46,21.6 3H,7’ s - 2.46,21.6 3H, s - - 2.46, 3H, - s - 103.2 - - 103.2 6.14, 2H, s - 6.14,103.2 2H, 56.3 s 6.14, 103.2 2H, 56.3 3.48, s 3H, s 6.14, 3.48, 56.3 2H, 3H,s s 3.48, 56.3 3H, s 3.48, 3H, s 1’’ 52.61’’ 3.80–3.69,52.61’’ 1H,3.80–3.69, m 52.6 1’’ 1H, 52.6 m3.80–3.69, 52.6 3.80–3.70, 1H,52.6 m 3.80–3.69, 1H, 3.80–3.70, 52.61H, m 1H, 31.5 3.80–3.70, 52.6 31.51H, 2.82, 3H, 3.80–3.70, s 2.82, 31.5 1H, 3H, 31.6 s 2.82, 31.5 3H, 31.6 2.83, s 3H, s 2.82, 2.83, 31.6 3H, 3H,s s 2.83, 31.6 3H, s 2.83, 3H, s 3.39–3.32, 1H,3.39–3.32, m 1H, m3.39–3.32, 1H, mm3.39–3.32, 1H,m m m m

Metabolites 2021, 11, x FOR PEER REVIEW 11 of 21

Metabolites Metabolites2021, 11, x 2021FOR, PEER11, x FOR REVIEW PEER Metabolites REVIEW 2021, 11, x FOR PEER REVIEW 11 of 21 11 of 21 11 of 21

3.40–3.32, 1H, 3.40–3.32, 1H,3.40–3.32, 1H, m 3.40–3.32, 1H, 2’’ 23.3 2.18–2.01, 2H, m m 23.3m 2.18–2.01, 2H, m- - - - 2’’ 2’’23.3 23.32.18–2.01, 2H,2.18–2.01, m 2’’2H, m23.3 23.3 23.3 2.18–2.01, 2.18–2.01, 2H, 2.18–2.01, 2H, m 2H, - m23.3 - 2.18–2.01, - 2H, ------3’’ 23.4 2.30–2.23, 1H, m m 23.4m 2.27–2.21, 1H, m- - - - 3’’ 3’’23.4 23.42.30–2.23, 1H,2.30–2.23, m 3’’1H, 2.18–2.01, m23.4 23.4 1H, 23.4 2.27–2.21, m 2.30–2.23, 1H, 2.27–2.21, 1H, m 1H, - m23.4 - 2.27–2.21, - 1H, ------2.18–2.01, 1H,2.18–2.01, m 1H, m 2.18–2.01,m 1H,m m 2.18–2.01, 1H, m 2.18–2.01, 1H,2.18–2.01, 1H, m 2.18–2.01, 1H, 4’’ 55.8 3.80–3.69, 1H, m m 55.8m 3.80–3.70, 1H, m- - - - 4’’ 4’’55.8 55.83.80–3.69, 1H,3.80–3.69, m 4’’1H, 3.10–3.04, m55.8 55.8 1H, 55.8 3.80–3.70, m 3.80–3.69, 1H, 3.80–3.70, 1H, m 1H, - m55.8 - 3.80–3.70, - 1H, ------3.10–3.04, 1H,3.10–3.04, m 1H, m 3.10–3.04,m 1H,m m 3.12–3.06, 1H, m 3.12–3.06, 1H,3.12–3.06, 1H, m 3.12–3.06, 1H, 1 abbreviations: s = singlet, d = doublet,m t = triplet,m q = quartet, m = multiplet, atm = apparent triplet, dd = doublet of doublets. J = 1 abbreviations:1 abbreviations: s = singlet,coupling s =d singlet,= constant.doublet, d 1= abbreviations: tdoublet, = triplet, t q= =triplet, quartet,s = singlet, q = m quartet, = d multiplet, = doublet, m = multiplet, at t = triplet,apparent at q= =apparenttriplet, quartet, dd triplet,m = =doublet multiplet, dd =of doublet doubl at = apparentets. of doublJ = triplet,ets. J = dd = doublet of doublets. J = coupling couplingconstant. constant. coupling constant. For products 9 and 10, the NMR analysis confirmed the presence of methylone in For productsFor products 9both and samples. 10, 9 theand NMR 10,Methylone the Foranalysis NMR products is analysisconfirmeda 1,3,4-trisubstituted 9 and confirmed the10, thepresence NMRthe aromatic presence ofanalysis methylone compound of confirmed methylone in characterized the in presence by of methylone in both samples.both samples. Methyloneone doubleMethylone is a doublet1,3,4-trisubstitutedboth is a atsamples.1,3,4-trisubstituted 7.70 ppm Methylone aromatic (H-6’) and aromatic compoundis twoa 1,3,4-trisubstituted doublets compound characterized at 7.47 characterized (H-2’) aromatic by and compound7.04by ppm (H- characterized by one doubleone doublet double5’) atdoublet on 7.70 1H ppm NMRat 7.70 (H-6’) onespectrum. ppm double and (H-6’) two The doublet anddoublets methylenedioxy two at doublets7.70 at 7.47 ppm (H-2’) groupat (H-6’) 7.47 and attached(H-2’)and 7.04 two andppm to doublets 7.04aromatic (H- ppm at ring(H-7.47 was(H-2’) iden- and 7.04 ppm (H- 5’) on 1H5’) NMR on 1 Hspectrum. NMRtified spectrum. by The the methylenedioxy singlet 5’)The on methylenedioxyat 1H 6.14 NMR ppmgroup spectrum. (H-7’), attached group while The attached to methylenedioxy aromaticthe methine to aromatic ring proton was group ring iden- (H-2) attachedwas located iden- to aromaticbetween ring was identified bytified the singlet by thethe at singlet carbonyl6.14 ppm at 6.14 group(H-7’),tified ppm and bywhile (H-7’), thethe the singletnitrogen while methine atthe atom6.14 methineproton ppm were (H-2) (H-7’), protonobserved located while (H-2) at betweenthe5.06located methine ppm between (appears proton as(H-2) a 1H located between the carbonylthe carbonyl groupquartet), and group the whichnitrogenand thethe is nitrogen inatomcarbonyl agreement we atomre group observed with we andre the observed theat result 5.06 nitrogen sppm atreported 5.06 (appearsatom ppm inwe the(appears reas observedliteraturea 1H as a at[33,48,49]. 1H 5.06 ppm The (appears as a 1H quartet),quartet), which is which insinglet agreement is at in 2.82 agreement with ppmquartet), the integrates withresult which thes reported3 result isprotons in agreements reported in and the belongs literature within the tothe literature the[33,48,49]. result N-methyls reported[33,48,49]. The group in The (H-1’’),the literature while [33,48,49]. The singlet atsinglet 2.82 ppm at 2.82 theintegrates ppmdoublet integrates 3 at protons 1.64singlet ppm3 andprotons at corresponds 2.82belongs ppmand tobelongs integrates the to theN -methylto te the3rminal protons N group-methyl methyl and (H-1’’), group belongs group while (H-1’’), of to the the whilealkyl N-methyl side chain group (H-1’’), while the doubletthe atdoublet 1.64 (H-3)ppm at 1.64 correspondsthat ppm were correspondsthe confirmed todoublet the te rminal tobyat the 1.64the te methylCOSYppmrminal corresponds experiment group methyl of group the to(Figure alkyl the of tethe side7).rminal alkyl In chain the methyl side 13 C chainNMR group spectrum of the alkyl side chain (H-3) that(H-3) were that confirmed werethe signals confirmed by fromthe COSY(H-3) bythe the eleven thatexperiment COSY were carbon experiment confirmed (Figure atoms we7). (Figureby reIn the assigned the COSY 7). 13C In NMR experimentthebased 13 spectrumC on NMR two-dimensional (Figure spectrum 7). In the exper- 13C NMR spectrum the signalsthe from signals theiments from eleven the 1 H-carbon eleven13C HSQC theatoms carbon signals and we atoms reHMBC from assigned we the (Figurere eleven assigned based 8). carbon on The based two-dimensional carb atoms onon two-dimensional signals were assigned corresponding exper- based exper- on to two-dimensionalthe phe- exper- iments 1H-iments13C HSQC 1H-nyl13C and HSQCring HMBC were and (Figureobserved HMBCiments 8).1(FigureH- at The13 108.5C HSQCcarb 8). (C-2’), Theon andsignals carb 109.1 HMBCon corresponding signals(C-5’), (Figure 127.0corresponding 8). (C-6’), Theto the carb 127.3 phe- onto the(C-1’),signals phe- 148.9 corresponding (C-3’) to the phenyl ring nylwere ring observed wereand observed154.1 at 108.5 ppm (C-2’),at (C-4’),nyl 108.5 ring 109.1 while(C-2’), were (C-5’), the 109.1observed methylenedioxy 127.0 (C-5’), (C-6’),at 127.0108.5 127.3 (C-6’),(C-2’), group (C-1’), 127.3109.1(C-7’) 148.9 (C-1’),(C-5’), produced (C-3’) 148.9127.0 a signal(C-3’)(C-6’), at 127.3 around (C-1’), 148.9 (C-3’) and 154.1and ppm 154.1 (C-4’), ppm103 while ppm. (C-4’), the The while methylenedioxy carbonyland the 154.1 methylenedioxy carbon ppm group (C-4’), (C-1) (C-7’) waswhile group found produced the (C-7’) methylenedioxy at 195.9produced a signal ppm, ata and signalaround group the at methine(C-7’) around produced carbon (C-a signal at around Metabolites 2021, 11, 144 103 ppm.103 The ppm. carbonyl The2) and carbonyl carbon the two (C-1) carbon 103methyl was ppm. (C-1) found groups The was at carbonyl found C-1’’195.9 and ppm,at carbon 195.9 C-3 and appeared ppm, (C-1) the methine andwas at the found 59.9, methinecarbon 31.5at 195.9 and(C- carbon ppm,16.3 (C-ppm, and the respec- methine carbon (C- 11 of 19 2) and the2) twoand methylthe tively.two groups methyl C-1’’ groups2) andand C-1’’ C-3the andappearedtwo C-3methyl appeared at groups59.9, 31.5 at C-1’’ 59.9, and and 31.516.3 C-3 andppm, appeared 16.3 respec- ppm, at respec- 59.9, 31.5 and 16.3 ppm, respectively. tively. tively. Table 4. NMR assignments of N-ethylcathinone, buphedrone, pentedrone and 3-FMC found in seized materials. Table 4. NMR assignments of N-ethylcathinone, buphedrone, pentedrone and 3-FMC found in seized materials. Table 4. NMRTable assignments 4. NMR assignments of N-ethylcathinone, ofTable N-ethylcathinone, 4. NMR buphedrone, assignments buphedrone, pentedrone of N-ethylcathinone, pentedrone and 3-FMC and foundbuphedrone, 3-FMC in seizedfound pentedrone materials.in seized materials. and 3-FMC found in seized materials.

Position N-Ethylcathinone Buphedrone Pentedrone 3-Fluoromethcathinone Posi- 13C 1H(δ/ppm, protons, 13C(δ/ppm) 1H(δ/ppm, protons, 13C(δ/ppm) 1H(δ/ppm, protons, 19F(δ/ppm) 13C(δ/ppm) 1H(δ/ppm, protons, Posi- Posi- tion N-EthylcathinonePosi- Buphedrone Pentedrone 3-Fluoromethcathinone (δ/ppm) multiplicity a, multiplicity a, multiplicity a, multiplicity a, coupling N-EthylcathinoneN-Ethylcathinone 13C 1H Buphedrone( δ/ppm,N -Ethylcathinone Buphedrone 13C 1H Pentedrone ( δ/ppm, PentedroneBuphedrone 13C 1H 3-Fluoromethcathinone(δ/ppm, Pentedrone 3-Fluoromethcathinone 19 F 13 C 1 3-FluoromethcathinoneH (δ/ppm, tion tion coupling constants) tion coupling constants) coupling constants) constants) 13C 131HC (δ/ppm,(δ1 H/ppm) (δ/ppm, protons,13C multi-131CH13 C (δ /ppm,11H H(δ (/ppm)(δδ/ppm, /ppm, 13C protons, 1H1313 C C( δ /ppm,11 (HHδ/ppm) ((δδ/ppm, /ppm, 19Fprotons, 13 19CF13 C 1H(δ 1(H/ppm)13δ/ppm,C ( δ/ppm, 1 H(δ /ppm)(δ/ppm, 19 F protons,13 C 1H (δ/ppm, 1 198.2 - 197.8 - 197.8 - 196.9, d, J = 2.15 Hz -

2 58.6(δ/ppm) (δprotons,/ppm) 5.22–5.16, multi-protons, 1H, m multi-(δ/ppm)plicity 65.0 (aδ, /ppm)(cou-δ/ppm)protons, protons, protons, 5.22–5.16, multi-(δ /ppm) 1H,multiplic- m((δδ/ppm)/ppm)protons, 64.2 protons,protons, ( δ 5.19,/ppm)multiplicity 1H, ( t,δ(δ/ppm)J/ppm)(=δa/ppm), 5.26 Hz (protons,δ/ppm)protons, protons,(δ/ppm) multiplicity 60.4(δ/ppm)a, protons, 5.14–5.09, 1H, q, plicitya, cou-plicitya, coupling con-multiplic-plicitymultiplic-a, cou- itya, cou-multiplicitymultiplicitya,multiplic- a, coupling multiplicitymultiplicitya, multiplicitya, a, coupling multiplicityJ =a, 7.28 Hz 3 16.1 1.63, 3H, d, J = 7.4 Hz 23.6 2.24–2.05, 2H, m 32.4 2.12–1.95, 2H, m 15.7 1.65, 3H, d, J = 7.28 Hz pling con- pling constants) itya, couplingitya, cou-con- pling con-coupling couplingitya, cou- constants) couplingcoupling coupling constants) coupling 4 - - 7.9 0.89, 3H, t, J = 7.58 Hz 17.6 1.41–1.32, 1H, m -- stants) stants) pling con- plingstants) constants)constants) constants)pling con- 1.29–1.17, 1H, m constants)constants) constants) constants) 5 -1 -198.2 - -stants) stants)197.8 - - 13.4197.8stants) 0.86, 3H, - t, J = 7.28 Hz 196.9, d, -- - 10 132.9 - 133.7 - 136.0 - 134.9, d, J = 6.75 Hz - 20 1 129.5198.21 8.06,198.2 2H, d,- J = 8.20 Hz - 1197.8 129.4198.2197.8 - 8.06, 2H, - - d, 197.8J = 8.20 Hz197.8197.8 - 129.4 - - 8.05, 2H, d,197.8J 196.9,= 7.40 d, Hz 196.9, - d,- J = 2.15- 115.9, d, J = 196.9, 23.0 Hz d, 7.86,- 1H, d, J = 7.76 30 129.9 7.65, 2H, at, J = 7.62 Hz 129.9 7.65, 2H, at, J = 7.62 Hz 129.8 7.64, 2H, t, J J= = 7.82 2.15 Hz J = 2.15− 114.3Hz 162.0, d, J = 244.8J = 2.15 Hz - 0 4 136.0 7.81, 1H,2 at, J = 7.4458.6 Hz 5.22–5.16,136.1 1H, 7.81,65.0 1H, at J = 7.44 5.22–5.16, Hz 133.6 64.2 7.80, 5.19, 1H, 1H, t, Jt, =J Hz 7.48 HzHz 60.4 122.8,5.14–5.09, d, J = 21.4Hz Hz 7.53, 1H, dt, J = 8.30, 2.13Hz 50 2 129.958.62 7.65,5.22–5.16,58.6 2H, at, J 1H,=5.22–5.16, 7.62 Hz 1H,265.0 129.9m 58.665.0 5.22–5.16, 5.22–5.16,7.65, 5.22–5.16, 2H, at, 1H, J 64.2= 7.62 1H, Hz m 5.19, 64.2 65.0 1H, 129.8 t, 5.19,J 5.22–5.16, 1H, t, J 7.64, = 5.26 2H, t,Hz 64.2J = 60.4 7.82 Hz 5.19, 60.45.14–5.09, 1H, t, J 5.14–5.09, 131.8, 1H, d, J =q, 7.94J 60.4= Hz 5.14–5.09, 7.68–7.62, 1H, m 0 6 129.5 8.06, 2H,m d, J = 8.20m Hz 129.4 1H, m 8.06,1H, 2H,m m d, J = 8.20 Hz = 5.26 Hz129.4 = 5.261H, mHz 8.05, 2H, d, J = 7.40 Hz= 5.261H, Hz q, J = 1H, q, J125.6, = d,7.28J = Hz 2.89 Hz1H, 7.78, q, 1H,J = d, J = 9.28 Hz 100 41.9 3.31–3.22, 1H, m 32.2 2.82, 3H, s 32.3 2.81, 3H, s 31.5 2.85, 3H, s 7.28 Hz 7.28 Hz 7.28 Hz 3.22–3.13, 1H, m 200 11.3 1.39, 3H, t, J = 7.32 Hz ------a abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, at = apparent triplet, dt = double triplet, J = coupling constant.

Metabolites 2021, 11, 144 12 of 19

For products 9 and 10, the NMR analysis confirmed the presence of methylone in both samples. Methylone is a 1,3,4-trisubstituted aromatic compound characterized by one double doublet at 7.70 ppm (H-60) and two doublets at 7.47 (H-20) and 7.04 ppm (H-50) on 1H NMR spectrum. The methylenedioxy group attached to aromatic ring was identified by the singlet at 6.14 ppm (H-70), while the methine proton (H-2) located between the carbonyl group and the nitrogen atom were observed at 5.06 ppm (appears as a 1H quartet), which is in agreement with the results reported in the literature [33,48,49]. The singlet at 2.82 ppm integrates 3 protons and belongs to the N-methyl group (H-100), while the doublet at 1.64 ppm corresponds to the terminal methyl group of the alkyl side chain (H-3) that were confirmed by the COSY experiment (Figure7). In the 13C NMR spectrum the signals from the eleven carbon atoms were assigned based on two-dimensional experiments 1H-13C HSQC and HMBC (Figure8). The carbon signals corresponding to the phenyl ring were observed at 108.5 (C-20), 109.1 (C-50), 127.0 (C-60), 127.3 (C-10), 148.9 (C-30) and 154.1 ppm (C-40), while the methylenedioxy group (C-70) produced a signal at around 103 ppm. The Metabolites 2021, 11, x FOR PEERcarbonyl REVIEW carbon (C-1) was found at 195.9 ppm, and the methine carbon (C-2) and the two13 of 21

methyl groups C-1” and C-3 appeared at 59.9, 31.5 and 16.3 ppm, respectively.

1 1 FigureFigure 7. 7.1 H-H-1HH COSY COSY NMR NMR spectrum spectrum of of methylone methylone found found in in product product 10. 10.

Analytical data for products 3–7 revealed the presence of methedrone together with other cathinones, namely N-ethylcathinone and buphedrone. Methedrone is a methcathinone derivative only differing by the presence of a methoxy group in the para position of the aromatic ring. Hydrogens belonging to the methoxy group were identiﬁed in the 1H NMR spectrum as a singlet at 3.48 ppm, while the proton attached to chiral carbon (H-2) was identiﬁed as a quartet at 5.11 ppm, which are in accordance with the results obtained by Zancajo et al. [50]. An apparent triplet at 8.04 ppm (due to overlap with buphedrone and N-ethylcathinone signals) and a doublet at 7.14 ppm, with coupling constants of 8.84 Hz and 8.96 Hz, supported substitution in para position. The methyl groups H-3 and H-100 produce a doublet at 1.64 ppm, and a singlet at 31.6 ppm, respectively.

Metabolites 2021, 11, 144 13 of 19 Metabolites 2021, 11, x FOR PEER REVIEW 14 of 21

Figure 8. NMR spectraFigure 8. of NMR methylone spectra found of methylone in product found 10. (A in) 1productH-13C HSQC 10. (A and) 1H- (13BC) 1HSQCH-13C and HMBC. (B) 1H-13C HMBC.

AnalyticalN-Ethylcathinone data for products and buphedrone 3–7 revealed have the a close presence similar of chemicalmethedrone structure, together and with for otherthis reason,cathinones, the protons namely correspondingN-ethylcathinone to the and aromatic buphedrone. ring wereMethedrone found overlapped. is a methcathi- The noneprotons derivative in the orthoonly differingposition (H-2by the0 and presence H-60) of appeared a methoxy as agroup doublet in the at 8.06para ppmposition with of a 0 0 0 thecoupling aromatic constant ring. Hydrogens of 8.20 Hz, belonging while meta to(H-3 the methoxyand H-5 group) and para were(H-4 identified) protons in the appear 1H NMRas two spectrum apparent as tripletsa singlet at at 7.65 3.48 ppm ppm, and while 7.81 the ppm proton with attached coupling to constants chiral carbon of 7.62 (H-2) Hz wasand identified 7.44 Hz, respectively.as a quartet at As 5.11 achieved ppm, which by Zancajo are in accordance et al. [50], bothwith compoundsthe results obtained showed bythe Zancajo, signal correspondinget al. [50]. An apparent to the protons triplet from at 8.04 chiral ppm carbon (due to atoms overlap (H-2) with overlapped buphedrone and andappeared N-ethylcathinone as a multiplet signals) at around and a 5.19 doublet ppm. at The 7.14N ppm,-ethyl with side coupling chain of Nconstants-ethylcathinone of 8.84 00 Hzyielded and 8.96 two Hz, multiplets supported for methylenesubstitution protons in para (H-1position.) centered The methyl at 3.27 groups ppm and H-3 3.17 and ppm H- 00 1’’and produce one triplet a doublet for the at terminal1.64 ppm, methyl and a groupsinglet H-2at 31.6at ppm, 1.39 ppm, respectively. which are in agreement withN the-Ethylcathinone results obtained and by buphedrone Ku´set al. [51 have]. For a buphedrone close similar the chemicalN-methyl structure, moiety resonates and for thisas areason, largesinglet the protons at 2.82 corresponding ppm, and the to alkyl the aromatic side chain ring presents were found a multiplet overlapped. centered The at protons2.14 ppm in forthe theortho methylene position protons(H-2’ and H-3 H-6’) and aappeared triplet at as 0.89 a doublet ppm for at the 8.06 terminal ppm with methyl a couplinggroup (H-4). constant of 8.20 Hz, while meta (H-3’ and H-5’) and para (H-4’) protons appear

Metabolites 2021, 11, 144 14 of 19

In relation to product 12, the NMR data supported the previous findings of GC-MS and confirmed the presence of pentedrone. As expected, the 1H NMR results obtained for this SCat was very similar to the 1H NMR results for buphedrone, since both substances have similar chemical structures, differing only in the length of the alkyl chain. Pentedrone is constituted by only one more methylene group than buphedrone. The methinic proton of pentedrone was observed at 5.19 ppm and the triplet is more defined than that verified for buphedrone. The two multiplets observed at 1.37 and 1.23 ppm correspond to the diastereotopic protons H-4, while the methylene protons H-3 appeared at 2.04 ppm as a multiplet. The protons corresponding to the terminal methyl group H-5 remained relatively unchanged at around 0.86 ppm. Maheux and Copeland [29] obtained similar results during the chemical characterization of pentedrone and buphedrone in their study. For product 11, the NMR analysis confirmed the presence of 3-FMC. The observed 1H signals, relative to aliphatic portion of the 3-FMC, are similar to those observed for methedrone and methylone. However, the signals pattern observed in the aromatic region was distinct from these previous ones. The two doublets observed at 7.86 ppm and 7.78 ppm corresponded to the protons at ortho position H-20 and H-60, respectively, while the multiplet centred at 7.65 ppm and the double triplet at 7.53 ppm belonged to the protons in the meta (H-50) and para (H-40) positions. Moreover, the doublet splitting of the carbon 0 0 1 atom signals at position 1 to 6 and the spin-spin coupling constants ( JC30-F = 244.8 Hz, 2 2 3 3 4 JC20-F = 23.0 Hz, JC40-F = 21.4 Hz, JC50-F = 7.94 HZ, JC10-F = 6.75 Hz, JC60-F = 2.89 Hz) were characteristic for fluorine–carbon interactions, which supported the assignment of the 30-position for the fluorine atom [52]. On the other hand, the 19F NMR chemical shift value for the 3-FMC was −114.3 ppm, which is close related with the reported in the literature [52]. Regarding to the purity of these products, Figure9 shows the mass fraction (% m/m) of each compound found in the seized products. Some of the analyzed products only present one active principle (e.g., product 1, 2 and 10), while most of them are mixtures of Metabolites 2021, 11, x FOR PEER REVIEW 16 of 21 psychoactive substances with a high number of constituents, as for example products 3–7 (“Bloom”) or product 12 (“Kick”).

FigureFigure 9. Relative 9. Relative proportions proportions of activeof active substances substances detected detected in 12in seized12 seized products. products.

As veriﬁedAs by verified GC-MS, by caffeine GC-MS, and caffeine ethylphenidate and ethylp werehenidate the main were adulterantsthe main adulterants found found 1 13 in these samples.in these samples. The respective The respective assignments assignments of H andof 1H andC signals 13C signals are described are described in in table S1 (supplementary information). The presence of isopentedrone in product 12 was also confirmed by NMR, being this the minor compound found in this product (2.3%). Addi- tionally, in the same product, NMR analysis allowed the identification of methylamine, which is commonly used in the synthesis of N-methyl cathinones and is not detected in the GC-MS analysis due to its volatility [5]. The presence of methylamine in these products was not surprising, since it was previously reported in other works [5,52]. For product 10, only 31% of the analyzed tablets contained methylone in their composition. In fact, during the preparation of this sample for GC-MS and NMR analysis, an insoluble material was observed and was removed from the solution through filtration, and then analyzed by ATR-FTIR. The results are present in Figure S9 (supporting information) and the substance was identified as microcrystalline cellulose, which probably was used as a binding agent for direct tablet compression or even added to increase the amount of substance (bulking agent) [53].

3. Materials and Methods 3.1. Reagents All used chemicals were of analytical grade. Methanol was obtained from Fisher Chemicals (Loures, Portugal), while ethyl acetate was provided by Riedel-de Haën (Seelze, Germany). Trifluoroacetic anhydride (TFAA, ≥ 99.0%), maleic acid (99.0%), trifluoroacetic acid (TFA, 99.0%) and deuterium oxide (99.9%) were obtained from Sigma- Aldrich (St. Louis, MO, USA), while pure caffeine was purchased from Merck (Darmstadt, Germany).

3.2. Samples Twelve seized products suspected to contain illicit substances were provided by the Forensic Science Laboratory of Portuguese Criminal Police, under a special authorization. The products were presented in different forms, including powders, crystals and tablets, packed in small plastic bags with striking designs or, occasionally, in transparent plastic bags. Some of the products were labelled as “plant feeders” with the indication “not for

Metabolites 2021, 11, 144 15 of 19

Table S1 (Supplementary Information). The presence of isopentedrone in product 12 was also confirmed by NMR, being this the minor compound found in this product (2.3%). Additionally, in the same product, NMR analysis allowed the identification of methylamine, which is commonly used in the synthesis of N-methyl cathinones and is not detected in the GC-MS analysis due to its volatility [5]. The presence of methylamine in these products was not surprising, since it was previously reported in other works [5,52]. For product 10, only 31% of the analyzed tablets contained methylone in their composition. In fact, during the preparation of this sample for GC-MS and NMR analysis, an insoluble material was observed and was removed from the solution through filtration, and then analyzed by ATR-FTIR. The results are present in Figure S9 (Supporting Information) and the substance was identified as microcrystalline cellulose, which probably was used as a binding agent for direct tablet compression or even added to increase the amount of substance (bulking agent) [53].

3. Materials and Methods 3.1. Reagents All used chemicals were of analytical grade. Methanol was obtained from Fisher Chemi- cals (Loures, Portugal), while ethyl acetate was provided by Riedel-de Haën (Seelze, Germany). Trifluoroacetic anhydride (TFAA ≥ 99.0%), maleic acid (99.0%), trifluoroacetic acid (TFA, 99.0%) and deuterium oxide (99.9%) were obtained from Sigma-Aldrich (St. Louis, MO, USA), while pure caffeine was purchased from Merck (Darmstadt, Germany).

3.3. ATR-FTIR Analysis A PerkinElmer® Spectrum Two FTIR Spectrometer (Waltham, MA, USA) equipped with a DuraSamplIR™ diamond ATR unit (Smiths Detection, London, UK) was used for infrared spectroscopy analysis. Approximately 20 mg of powder sample was placed on the small ATR crystal area, and the infrared spectra were collected in the range from 4000 to 600 cm−1, with 32 scans at 4 cm−1 resolution. Spectra were obtained in triplicate and a PerkinElmer Spectrum IR software, version 10.6.0, was used for processing and visualizing the spectra.

3.4. GC-MS Analysis For GC-MS analysis, each sample was homogenized and dissolved in methanol to a final concentration of 1 mg mL−1. Before the GC-MS analysis, each solution was filtered through 0.22 µm polytetrafluoroethylene (PTFE) membrane filters (Millipore, Milford, MA, USA) and 2 µL of sample were directly injected into the GC-MS. Derivatization with TFAA was also performed according to the method developed by Araújo et al. [5] with some modifications. Each sample was prepared to a final concentration of 100 µg mL−1 by dilution of the initial methanolic solution (1 mg mL−1) and was evaporated to dryness under nitrogen flow. Then 100 µL of TFAA/ethyl acetate (1:1, v/v) was added to the dried residue and the incubation was performed at 70 ◦C for 30 min. After cooling to room temperature, the solvent was evaporated to dryness under a nitrogen stream, and the residues were reconstituted in 100 µL of ethyl acetate. A 2 µL aliquot of the resulting solution was injected into the GC-MS system. Metabolites 2021, 11, 144 16 of 19

GC-MS analysis was performed with an Agilent 6890N GC system (Palo Alto, CA, USA) equipped with a 5975 Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA). The samples were separated using an Agilent J&W HP-5 capillary column (30 m × 0.32 mm ID, 0.25 µm film thickness), and helium (Air Liquid, Alges, Portugal) was used as the carrier gas, at a constant flow of 1.3 mL min−1. The injections were performed in split mode, with a ratio of 40:1. The injector port was heated to 250 ◦C and 2 µL of sample was injected in the GC system. The initial column temperature was set to 60 ◦C for 4 min, followed by a temperature ramp of 15 ◦C min−1 to 150 ◦C, held for 5 min, and 20 ◦C min−1 to 290 ◦C held for 10 min. The mass spectrometer was operated in electron impact ionization (EI) mode at 70 eV. For the MS system, the temperatures of the transfer line, quadrupole and ionization source were 250, 150, and 230 ◦C, respectively. The ionization was maintained off during the first 4 min, to avoid solvent overloading. The mass spectra were recorded in range of 40–500 m/z and acquisition was made in Full Scan mode, with a scan rate of 6 scans/s. All mass spectra were compared with NIST 14 MS library and SWGDRUG MS library version 3.4.

3.5. NMR Analysis

For NMR analysis, 10 mg of sample was dissolved in 500 µL of deuterium oxide (D2O) containing maleic acid as the internal standard (5 mg mL−1). The resulting mixture was transferred to a 5 mm NMR tube and the NMR spectra were acquired in a Brucker Avance II 400 MHz Spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at 400.13 MHz for 1H NMR and 100.61 MHz for 13C NMR. Chemical shifts (δ) were expressed as parts per million (ppm) and referenced to the signal of maleic acid (δH = 6.42, δC = 132.16). Coupling constants (J) were reported in units of Hertz (Hz). Structure elucidation by respective assignment of the carbon and proton signals was based on the analysis of NMR spectra obtained by 1D (1H and 13C) and 2D (COSY, HMBC and HSQC) techniques. For product 11, a 19F NMR spectrum (376.5 MHz) was also recorded, and the chemical shifts were referenced to the TFA resonance at −78 ppm. For NMR purity assessment, the 1H signal integration for each compound was cal- culated calibrating for 100 the area of maleic acid resonance peak. The purity of the products was assessed calculating the mass fraction (% m/m) of each compound in the seized products, using the following equation,

IX NIS MX mIS %compound = × × × × PIS (1) IIS NX MIS msample

where IX and IIS are the integrated area of the compound of interest (X) and the internal standard (IS), NX and NIS are the numbers of protons generating the selected signals −1 for integration, MX and MIS are the molecular weights in g mol , msample and mIS are the masses of the sample and the internal standard, respectively, and PIS is the purity of maleic acid.

4. Conclusions The present study describes the identification of 8 SCat as the key constituents found in 12 seized products. In general, the products showed different compositions between them, although very similar for the products labelled with the same name. Among the identified SCat, six of them, methylone, methedrone, N-ethylcathinone, buphedrone, pentedrone and 3-FMC, belong to the “classic cathinone group”, analogues of amphetamines, being considered as the first generation of SCat. Pyrovalerone derivatives, a particular class of SCat, characterized by the presence of a pyrrolidine ring in their chemical structures, were also identified in two distinct products with a high degree of purity. MPHP was found in product 1 with a purity of 89%, while α-PHP was found in product 2 with a purity of 92%. Among the adulterants, caffeine, an active ingredient commonly found in illicit drugs, was the most frequently detected substance (67% of the total analyzed products), followed by Metabolites 2021, 11, 144 17 of 19

ethylphenidate (50% of the total analyzed products). Both substances are probably added to these products, since they are cheap and potentiate the stimulating effect. In relation to the analytical techniques, the combination of ATR-FTIR, GC-MS and NMR techniques proved to be an effective methodology to unequivocally identify the molecular structure of the SCat present in “plant feeders”, even in the absence of certified standards. While ATR-FTIR provided a quick and presumptive identification of the seized drugs without destroying evidence, the GC-MS allowed the precise identification of the substances, being an excellent tool for confirmatory tests. On the other hand, NMR spectroscopy proved to be of great importance for structural elucidation allowing the positional isomers distinction, and the identification of compounds not detected in the GC-MS, such as methylamine.

Supplementary Materials: The following are available online at https://www.mdpi.com/2218-1 989/11/3/144/s1, Figure S1: FTIR spectra of seized products suspected to contain SCat, Figure S2: Typical GC-MS chromatograms of seized products suspected to contain SCat, Figure S3: Typical GC-MS chromatograms of seized products after derivatization with TFAA, Figure S4: 1H NMR spectra of seized products, Figure S5: 13C NMR spectra of seized products, Figure S6: 1H-1H COSY NMR spectra of MPHP and α-PHP found in products 1 and 2, respectively, Figure S7: 1H-13C HSQC and HMBC NMR spectra of MPHP found in product 1, Figure S8: 1H-13C HSQC and HMBC NMR spectra of α-PHP found in product 2, Figure S9: FTIR spectra of the insoluble substance found in product 10, Table S1: 1H and 13C NMR assignments of adulterants found in seized materials. Author Contributions: Conceptualization, J.S.C. and H.M.T.; methodology, J.L.G., V.L.A. and J.A.; investigation, J.L.G., V.L.A. and J.A.; data curation, J.L.G., V.L.A. and J.A.; writing—original draft preparation, J.L.G., V.L.A. and J.A.; resources, M.J.C. and J.S.C.; supervision, H.M.T., J.S.C. and M.J.C.; writing—review and editing, H.M.T. and J.S.C.; project administration, J.S.C.; funding acquisition, J.S.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Fundação para a Ciência e a Tecnologia through Centro de Química da Madeira Base Fund (UIDB/00674/2020, and Programmatic Fund-UIDP/00674/2020) and the PhD fellowships (SFRH/BD/116895/2016 and SFRH/BD/117426/2016) granted to João L. Gonçalves and Vera L. Alves and through Madeira 14–20 Program, project PROEQUIPRAM-Reforço do Investimento em Equipamentos e Infraestruturas Científicas na RAM (M1420–01-0145-FEDER- 000008), and by ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação, through the project M1420-01-0145-FEDER-000005-Centro de Química da Madeira-CQM+ (Madeira 14–20 Program). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Acknowledgments: The authors would like to thank the Forensic Science Laboratory of Portuguese Criminal Police for their collaboration in providing the seized products. Conflicts of Interest: The authors declare no conflict of interest.

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