Forensic Electrochemistry Applied to the Sensing of New Psychoactive

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Forensic Electrochemistry Applied to the Sensing of New Psychoactive Substances: Electroanalytical Sensing of Synthetic Cathinones and Analytical Validation in the Quantification of Seized Street Samples Jamie P. Smith,† Jonathan P. Metters,† Osama I. G. Khreit,‡ Oliver B. Sutcliffe,*,† and Craig E. Banks*,† †

Faculty of Science and Engineering, School of Chemistry and the Environment, Division of Chemistry and Environmental Science, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, Lancashire, U.K. ‡ Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, U.K. S Supporting Information *

ABSTRACT: The electrochemical sensing of new psychoactive substance(s) (NPSs), synthetic cathinone derivatives also termed “legal highs”, are explored with the use of metallic modified screen-printed electrochemical sensors (SPES). It is found that no significant electrochemical enhancement is evident with the use of either in situ bismuth or mercury film modified SPES compared to the bare underlying electrode substrate. In fact, the direct electrochemical reduction of the cathinone derivatives mephedrone (4-methylmethcathinone; 4-MMC) and 4′-methyl-N-ethylcathinone (4-methylethcathinone; 4-MEC) is found to be possible for the first time, without heavy metal catalysis, giving rise to useful voltammetric electroanalytical signatures in model aqueous buffer solutions. This novel electroanalytical methodology is validated toward the determination of cathinone derivatives (4-MMC and 4-MEC) in three seized street samples that are independently analyzed with high-performance liquid chromatography (HPLC) wherein excellent agreement between the two analytical protocols is found. Such an approach provides a validated laboratory tool for the quantification of synthetic cathinone derivatives and holds potential for the basis of a portable analytical sensor for the determination of synthetic cathinone derivatives in seized street samples. he term “legal highs” or the standardized scientific term, “new psychoactive substance(s)” (NPSs), is typically used to refer to substances that are not deemed controlled by a nation’s drug legislation, such as the misuse of drugs act in the U.K., but mimic the effects of common illicit materials (for example, methamphetamine and cannabis). It is possible NPSs have negative side effects on health, but these may not be fully recognized because they are relatively new materials and as a result understudied; as such, their long-term health risks are also not always understood.1 Sold at head shops (a shop typically specializing in drug paraphernalia) and on the Internet, NPSs are typically given nondescript aliases such as research chemicals, plant food, bath salts, exotic incenses, etc. with the instructions in similar vein to “not for human consumption” or “not tested for hazards or toxicity” in order to bypass legislative control. “Legal high” products often have brand names, such as “Benzo Fury”, NRG1, and NRG-2, although their name may not be descriptive of the actual contents; mephedrone has been found in products marketed as naphyrone in the U.K. even after it was made a controlled substance in 2010.2 Derived from cathinone, an organic stimulant found in Khat (Catha edulis, a plant found on the Arabian Peninsula and east Africa), substituted cathinones are an amphetamine-like cheap alternative to the phenethylamine class of psychoactives (e.g.,

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© 2014 American Chemical Society

amphetamine and methamphetamine). Mephedrone (4-methylmethcathinone; 4-MMC) and 3,4-methylenedioxypyrovalerone (MDPV) are the most prominent synthetic cathinones found within “legal high” products with 4-MMC more common in Europe and MDPV more abused in the United States;3 Scheme 1 overviews the chemical structures of these compounds. Mephedrone in particular is currently a “hot topic” considering its use has been linked to several deaths worldwide.4,5 There has been a tightening of the legislation regarding synthetic cathinone derivatives globally; for example, they are illegal in the U.K. as well as the United States, Germany, Norway, Sweden, The Netherlands, Finland, Romania, Republic of Ireland, Denmark, Canada, and Israel.6,7 Despite their controlled status, cathinone derivatives are still prevalent in many “legal high” products;2,8,9 hence, the development of methods for their detection and quantification is still timely and urgently required. As published by a number of groups, a range of chromatographic techniques including high-performance liquid chromatography (HPLC) and gas chromatography/mass Received: August 11, 2014 Accepted: August 27, 2014 Published: August 27, 2014 9985

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Scheme 1. Synthetic Cathinone-Derived New Psychoactive Substances (NPSs)

and revealed voltammetric profiles that would undoubtedly interfere with the response of substituted cathinones if applied to a real street sample; as such this prior work cannot be easily applied to seized street samples. In this paper we report an improvement on earlier proof-ofconcept work38 utilizing a different, novel, electrochemical approach. Additionally inspired by Krishnaiah et al.39 as well as other reports that electroanalytical signals have been improved through the use of bismuth-modified electrodes (though not yet applied to the sensing of NPSs);44 the use of in situ formed mercury and bismuth film modified graphite screen-printed electrodes is explored for the first time toward the sensing of the substituted cathinones, namely, 4-MMC and 4-MEC. While no significant improvements are observed using these film modified electrodes, the direct electrochemical reduction is found to be possible for the first time. A novel electrochemical sensing protocol is proposed utilizing disposable graphite screen-printed electrodes and offers a low-cost, single-shot, disposable yet highly reproducible and reliable sensing platform for a potential portable sensing approach for the detection of NPSs. Adulterants that are typically found in street samples are also electrochemically characterized for their potential interference in the simultaneous sensing of 4-MMC and 4-MEC and found to have no interference unlike that of prior work.38 This new electrochemical protocol as a tool is validated in seized street samples which are independently validated with liquid chromatography/ mass spectrometry (LC−MS) and HPLC showing excellent agreement and providing validation that our electroanalytical approach can be used for the quantification of the synthetic cathinone products in seized street samples.

spectrometry (GC-MS) have been employed for the detection of substituted cathinones.2,5,9−28 The first fully validated HPLC method for the quantification of mephedrone11 was reported by Santali et al. where limits of detection and quantification were reported to be 0.1 and 0.3 μg mL−1, respectively. Khreit et al. further refined this method enabling the detection of both mephedrone and two novel derivatives, 4-MEC (4-methyl-Nethylcathinone) and 4-MBC (4-methyl-N-benzylcathinone), in seized samples of NRG-2. In this case, the limits of detection and quantification were reported as 0.03 and 0.08 μg mL−1 for 4-MEC and 0.05 and 0.14 μg mL−1 for 4-MBC both in their pure form and in the presence of common adulterants such as caffeine and benzocaine.2,9 Recently, direct analysis using real-time mass spectrometry (DART-MS) has been utilized to characterize the multitude of new and emerging NPSs.29 Solid synthetic cathinone samples (2-FMC, 2-MEC, 2-FEC, and 2-EEC) were sampled directly without pretreatment, and positive ion mass spectra were acquired.29 Another approach reported by Mabbott et al.30,31 utilized surface-enhanced Raman spectroscopy (SERS) and reported the quantification of 4-MMC in model aqueous solutions with a limit of detection of 1.6 μg mL−1.31 Electrochemistry is an advantageous analytical tool that is adaptable to an in-the-field device, in light of its portability, and can exhibit sensitivity and selectivity toward many target analytes.32−38 There is only one study in the literature reporting the electrochemical reduction of 4-MMC using a dropping mercury electrode (DME) by Krishnaiah et al.,39 detailing an analytical range of 2.7 × 10−4 to 1.8 μg mL−1 with a detection limit of 2.2 × 10−3 μg mL−1. While yielding favorable analytical responses, a problem does arise with the use of a DME since mercury is widely reported as a harmful chemical and banned in numerous countries.40−43 Additionally, there is a need to translate a method like this into that of one which could be used as an in-the-field sensor and potentially be applied to seized samples; consequently, this prior work is unlikely to be adopted as either a laboratory tool or in-the-field sensor and is yet to be properly applied and validated in seized street samples. Our previous work38 reported the first electrochemical method for the sensing of cathinone substitutes, methcathinone, mephedrone (4-MMC), and 4′-methyl-N-ethylcathinone (4-MEC), which were analyzed with a scope to provide a potential on-the-spot analytical screening tool with graphite screen-printed electrodes.38 It was demonstrated for the first time that the electroanalytical oxidation of methcathinone, 4MMC, and 4-MEC is possible with accessible linear ranges found to correspond to 16−200 μg mL−1 for methcathinone (at pH 12) and 16−350 μg mL−1 for both mephedrone and 4MEC in pH 2, with limits of detection (3σ) found to correspond to 44.5, 39.8, and 84.2 μg mL−1, respectively, in model/buffer conditions. The effect of adulterants that are commonly incorporated into cathinone “legal high” explored

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EXPERIMENTAL SECTION All chemicals used were of analytical grade and were used as received without any further purification from Sigma-Aldrich (Gillingham, U.K.). All solutions were prepared with deionized water of resistively no less than 18.2 Ω cm. All solutions (unless stated otherwise) were vigorously degassed with nitrogen to remove oxygen prior to analysis. The synthetic cathinone hydrochloride (or hydrobromide) salts were prepared at the University of Strathclyde prior to the legislative change on April 16, 2010 using the methods reported in previous work.38 The synthesis of the five target compounds (4-MMC, 4-MEC, 4MBC, 4-FMC, and MDPV) were achieved using a modification of the previously reported methods9,12,29 from the prerequisite α-bromoketones, as stable, colorless to off-white powders after recrystallization from acetone. To ensure the authenticity of the materials utilized in this study the synthesized samples were fully structurally characterized, and the purity was confirmed by elemental analysis (>99.5% in all cases).9,11,28,38 Voltammetric measurements were carried out using a μAutolabIII (Eco Chemie, The Netherlands) potentiostat/ galvanostat and controlled by Autolab GPES software version 9986

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deposition of mercury and bismuth metal occur through the application of the cathodic scan with stripping potentials at ∼+0.1 V and ∼−0.3 V (vs Ag/AgCl), respectively, which is in agreement with current literature.49 The in situ formed mercury and bismuth metal modified SPEs were tested toward the electrochemical detection of the “legal high” constituent 4-MMC; Figure 1 shows the voltammetric profiles of each as well as a typical calibration plot with additions of 4-MMC made over the range of 100− 400 μg mL−1. The limit of detection (3σ) was determined to correspond to 28.61 μg mL−1 (Ip/A = −0.06 A/μg mL−1 − 6.89 A; R2 = 0.99; N = 6), 15.22 μg mL−1 (Ip/A = −0.07 A/μg

4.9 for Windows XP. Experiments were performed using boron-doped diamond, glassy carbon, and screen-printed graphite macroelectrodes; both the boron-doped diamond and glassy carbon electrodes have a 3 mm diameter working area. Screen-printed graphite macroelectrodes (denoted as SPEs herein) which have a 3 mm diameter working electrode were fabricated in-house with appropriate stencil designs using a DEK 248 screen-printing machine (DEK, Weymouth, U.K.). For the fabrication of the screen-printed sensors, first, a carbongraphite ink formulation (product code C2000802P2; Gwent Electronic Materials Ltd., U.K.) used previously was screenprinted onto a polyester (Autostat, 250 μm thickness) flexible film (denoted throughout as standard-SPE). This layer was cured in a fan oven at 60 °C for 30 min. Next a silver/silver chloride reference electrode was included by screen-printing Ag/AgCl paste (product code C2040308D2; Gwent Electronic Materials Ltd., U.K.) onto the polyester substrates. Finally, a dielectric paste (product code D2070423D5; Gwent Electronic Materials Ltd., U.K.) was then printed onto the polyester substrate to cover the connections. After curing at 60 °C for 30 min the screen-printed electrodes are ready to be used. Shown in Supporting Information Scheme S1 is the entire graphite screen-printed electrodes (GSPE) fabricated as described above and a scanning electron micrograph (SEM) of the electrode surface. The reproducibility of the batch of screen-printed sensors were found to correspond to 0.76% relative standard deviation (RSD) using the Ru(NH3)2+/3+ redox probe in 1 M KCl. The heterogeneous rate constant, ko for the Ru(NH3)2+/3+ redox probe in 1 M KCl was found to correspond to 3.36 × 10−3 cm s−1. Note that a new SPE was utilized for each experiment performed, including during concentration studies. Four street samples of NRG-2, obtained from independent Internet vendors, were received as white crystalline powders in clear zip-lock bags. LC−MS and HPLC were performed independently to quantify the chemical composition of the NRG-2 samples. Details of the experimental protocol for both LC−MS and HPLC methods can be found in the Supporting Information.

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RESULTS AND DISCUSSION First considered, in light of the inspiring work by Krishnaiah et al.39 who reported electrochemical measurements of 4-MMC over the concentration range of 2.7 × 10−4 to 1.8 μg mL−1 with a reported detection limit of 2.2 × 10−3 μg mL−1 utilizing a DME, an in situ formed mercury surface using GSPEs was used in order to try and provide a potential alternative to the DME. Exploring in Situ Mercury and Bismuth Film Modified GSPEs. To accomplish this, mercury was formed in situ through the addition of a mercury(II) salt into a pH 4.3 model acetate buffer, as reported previously in the literature.45−47 In this approach cyclic voltammetry is utilized where the potential is swept cathodically to electrochemically reduce the mercury(II) ions to mercury metal upon the GSPE surface. Supporting Information Figure S1 shows the typical electrochemical signatures. The use of in situ formed bismuth-modified electrode surfaces was also explored since this has been reported to be a “green” alternative to mercury.48,49 Similar to that of the in situ formed mercury metallic surface, a bismuth(III) salt was utilized and consequently electrochemically reduced to form bismuth metal on the GSPE surface. As shown in Supporting Information Figure S1, the (independent) electrochemical reduction of both metals on GSPE using the aforementioned conditions is visible where the electrochemical

Figure 1. (A) Voltammetric responses of acetate buffer in the presence of 4-MMC (solid line) using the in situ formed mercury(II) (dashed line) and bismuth(III) film modified SPEs (dotted line) recorded in a pH 4.3 acetate buffer. Scan rate: 50 mV s−1 (vs Ag/AgCl). (B) Typical calibration resulting from the analysis of the voltammetric signatures obtained in panel A in the form of a plot of peak height against 4MMC concentration using GSPEs in a pH 4.3 acetate buffer (triangles) and with the in situ film formed mercury (squares) and bismuth (circles) over a linear range of 100−400 μg mL−1. Note: a new GSPE was utilized with each scan/addition. Scan rate: 50 mV s−1 (vs Ag/AgCl). 9987

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mL−1 − 1.95 A; R2 = 0.99; N = 6), and 38.45 μg mL−1 (Ip/A = −0.05 A/μg mL−1 − 7.031 A; R2 = 0.99; N = 6) for 4-MMC, 4MMC with the in situ formed bismuth film, and 4-MMC with the in situ formed mercury film, respectively. It is indicative from this study that the in situ mercury and bismuth film modified SPEs do not improve the electrochemical signal with respect to the sensing of 4-MMC. It must also be noted that 4MMC is able to be electrochemically reduced directly (Figure 1) in pH 4.3 acetate buffer solution exhibiting a wave at ∼−1.4 V (vs Ag/AgCl); this is shown for clarity in Supporting Information Figure S2 where the electrochemical signature of 4-MMC is clearly visible Direct Electrochemical Reduction of NPSs 4-MMC and 4-MEC. Attention was now solely turned to improving the electrochemical performance of 4-MMC where the electrochemical reduction of 4-MMC in acetate buffer solutions was explored with a range of commercially available carbon-based electrodes. Evident from the voltammetric profiles, as depicted in Figure 2, comparable electrochemical reduction over-

in intensity, justifying the use of pH 4.3 acetate buffer solution for optimum sensing conditions. A plot of peak potential Ep/V versus pH was constructed where a gradient of 0.033 V was observed (E/V = −0.033 V − 1.30 E/pH R2 = 0.87). This value is indicative of an electrochemical process involving double the number of electrons over that of the protons (30 mV per pH unit at 25 °C) as described from the following equation (eq 1) where E0f is the formal potential, n is the number of electrons transferred in the rate-determining step, R is the ideal gas constant, T is temperature in Kelvin, and F is the Faraday constant. 0 Ef,eff = Ef0(A /B) − 2.303

mRT pH nF

(1)

While the exact electrochemical mechanism is unknown, the electrochemical process likely involves one proton and two electrons; future work will be focused to elucidate this. The analytical response of the promised sensing methodology was re-explored; note that earlier the solutions comprised metal salts for the in situ formation of the mercury and bismuth film modified electrode. To this end the analytical performance of 4-MMC using GSPEs in model pH 4.3 acetate buffer solution was explored with additions made over the linear range of 0.00−200.00 μg mL−1 (Ip/A = −0.11 A/μg mL−1 − 6.82 A; R2 = 0.95; N = 10). The limit of detection (3σ) was found to correspond to 11.80 μg mL−1 when compared to the value reported earlier of 28.61 μg mL−1 using GSPEs. Now attention was turned to another substituted cathinone commonly found in street samples of 4-MEC. The effect of the electrochemical reduction signal of 4-MEC as a function of pH over the range of 2−12 was investigated. As with 4-MMC, the reduction peak for 4-MEC (∼−1.4 V) is observed to shift to more negative potentials with increasing pH. A plot of Ep/(V) pH has a gradient of 0.029 mV again indicating a one-proton and two-electron process (according to eq 1). Similarly the peak potential decreased with increasing scan rate with the plot of peak height against the square root of scan rate was linear (Ip/A = 108.6 A (V s−1)−0.5 − 0.55 A; R2 = 0.98) and therefore, again, a diffusional process. With additions of 4-MEC into pH 4.3 acetate buffer using GSPEs, the corresponding calibration plot demonstrated a linear response (Ip/A = −0.07 A/μg mL−1 − 10.04 A; R2 = 0.93; N = 10) over the linear range of 0.00− 200.00 μg mL−1; the limit of detection (3σ) was found to correspond to 11.60 μg mL−1. Typically, street samples containing cathinones are “cut” with adulterants such as caffeine and benzocaine. With this in mind, both caffeine and benzocaine’s effect on the response in pH 4.3 acetate buffer was tested. Note: benzocaine will not dissolve in pH 4.3 acetate buffer and requires 20% methanol to dissolve. Visible from Figure 3 is the adulterant’s effect on the electrochemical response; caffeine shows little interference around the reduction overpotential for the substituted cathinones (∼+1.45 V vs Ag/AgCl); however, benzocaine has a considerable effect on the entire voltammetric waveform. Considering methanol is required to dissolve benzocaine, simply dissolving samples into aqueous buffer solution without alcohol serves as a simple pretreatment as the benzocaine is insoluble and can just be filtered off and the remaining constituents can be electroanalytically analyzed; this become evident when applied into the sensing of NPSs in real samples (see below). The limits of detection reported herein are an improvement on the values reported in our earlier work38 (13.2 μg mL−1 for

Figure 2. Cyclic voltammetric profiles recorded using boron-doped (dotted), glassy carbon (dashed line), and GSPEs (solid line) in 90.91 μg mL−1 4-MMC, pH 4.3 acetate buffer. Scan rate: 50 mV s−1 (vs Ag/ AgCl).

potentials are observed using glassy carbon and GSPEs. A more intense voltammetric peak is achieved with the GSPEs while no wave is visible for boron-doped diamond in the proposed scan range and is likely outside the accessible voltammetric window; these observations suggest the electrochemical process is dependent on the density of the edge-plane sites.50 This has been shown to be the case for a multitude of electroactive analytes but not yet for NPSs. Next, the effect of scan rate upon the electrochemical reduction of 200 μg mL−1 4-MMC in a pH 4.3 acetate buffer solution was investigated. A plot of peak height against the square root of scan rate was found to be linear indicating a diffusional process (Ip/A = 76.9 A (V s−1)−0.5 − 0.18 A; R2 = 0.93) with the peak potential observed to progressively become negative with increasing scan rate suggesting the process is electrochemically irreversible. Attention was turned to exploring the effect of the electrochemical reduction signal as a function of pH over the range of 2−12 where the reduction peak is observed to shift to more negative potentials with increasing pH. The reduction peaks at pHs greater than 6, while having larger peak currents, have large overpotentials, which is not desirable, and for pHs lower than 4 the peak current is low 9988

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MBC) min, respectively (see Figure 4), with a slight peak tailing (asymmetry factor; As ∼ 1.2−1.7) observed in each case.

Figure 4. Typical chromatogram of a mixture containing (a) (±)-4′fluoromethcathinone hydrobromide (4-FMC, 10 μg mL−1), (b) caffeine (10 μg mL−1), (c) (±)-4′-methylmethcathinone hydrochloride (4-MMC, 10 μg mL−1), (d) (±)-4′-methyl-N-ethylcathinone hydrobromide (4-MEC, 10 μg mL−1), (e) (±)-3′,4′-methylenedioxypyrovalerone hydrochloride (MDPV, 10 μg mL−1), (f) benzocaine (10 μg mL−1), and (g) (±)-4′-methyl-N-benzylcathinone hydrobromide (4-MBC, 10 μg mL−1) obtained using an ACE C18 column (150 mm × 4.6 mm i.d.; particle size, 3 μm); mobile phase, methanol/ 10 mM ammonium formate (pH 3.5) (see the Supporting Information for gradient elution program); detector wavelength, 264 nm; t0 (2.2 min) was determined from the tR of a solution of uracil (5 μg mL−1); peak (h) at tR = 12.3 min is a system peak.

Calibration standards were prepared, and the strongly UVabsorbing components (4-FMC, 4-MMC, 4-MEC, 4-MBC, caffeine, and benzocaine) demonstrated a linear response (R2 = 0.999−1) over a 0.5−10.0 μg mL−1 range with excellent repeatability (RSD = 0.014−0.799%; N = 6). The limits of detection for these components were determined as being in the range of 0.03−0.25 μg mL−1. The method was also suitable for the detection and quantification of MDPV which exhibited a weaker UV response. MDPV demonstrated a linear response (R2 = 0.999) over a 2.0−40.0 μg mL−1 range with exceptional repeatability (RSD = 0.026−0.325%; N = 6), and the limit of detection was determined to be 0.12 μg mL−1. The UV-inactive analytes (sucrose, mannitol, and lactose) were shown not to interfere with the seven target analytes. The limits of quantification were determined to be 0.36 (4-FMC), 0.14 (4MMC), 0.09 (4-MEC), 0.36 (MDPV), and 0.41 μg mL−1 (4MBC), respectively, which is comparable to the previously reported method.9 The validation parameters for the seven analytes are summarized in Table S1 reported in the Supporting Information. The four NRG-2 samples obtained from Internet vendors (January 2013) were all purported to be >99% pure and to contain 1 g of NRG-2. The samples were arbitrarily labeled NRG-2-A, NRG-2-B, NRG-2-C, and NRG-2-D. Preliminary LC−MS analysis indicated that all four samples contained two components (NRG-2-A tR = 4.48 min [minor], m/z = 178.1 [M + H]+; tR = 6.47 min [major], m/z = 166.2 [M + H]+; NRG-2-B tR = 2.57 min [minor], m/z = 195.1 [M + H]+; tR = 6.47 min [major], m/z = 166.2 [M + H]+; NRG-2-C tR = 2.57 min [major], m/z = 195.1 [M + H]+; tR = 5.34 min [minor], m/z = 192.2 [M + H]+; NRG-2-D tR = 2.57 min [major], m/z = 195.1 [M + H]+; tR = 4.48 min [minor], m/z = 178.1 [M + H]+) (Table 1).

Figure 3. Effect of common adulterants on the electrochemical response of pH 4.3 acetate buffer using GSPEs: (A) with (dashed line) and without (solid line) the presence of 1000 μg mL−1 benzocaine; (B) with (dashed line) and without (solid line) the presence of 1000 μg mL−1 caffeine. Scan rate: 50 mV s−1.

4-MMC and 36.3 μg mL−1 for 4-MEC) and are sufficient for use in the field as opposed to the lower as the values reported by Krishnaiah et al. which utilizes a DME and is not suitable for use in the field and banned in many countries (see the introductory section). HPLC and LC−MS Analysis of Seized Street Samples. Khreit et al. have reported the utilization of HPLC and LC−MS techniques for the analysis of NRG-2 products.9 The validated HPLC method (which can detect 4-MMC, 4-MEC, and 4MBC) at levels of 0.02 μg mL−1) was expanded and revalidated to screen for these compounds in the presence of 4-FMC, MDPV, caffeine, and benzocaine based on new intelligence received from law enforcement agencies. A gradient elution program was employed (see the Supporting Information), to ensure both optimal detection of the analytes and a rapid analysis time. The five cathinone derivatives eluted at 4.9 (4FMC), 6.6 (4-MMC), 7.2 (4-MEC), 8.4 (MDPV), and 10.8 (49989

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Application of the Proposed Electroanalytical Protocol. With substantial evidence supporting an electroanalytical approach for detecting various substituted cathinones in street samples the viability of the proposed protocol was tested. The “street” samples were reanalyzed using the validated HPLC method at a concentration of 5 μg mL−1. The results (Table 1) confirmed that all the samples only contained two components (NRG-2-A, minor 4-MMC [6.95% w/w, %RSD = 0.01%, N = 3] and major benzocaine [93.87% w/w, %RSD = 0.01%, N = 3]; NRG-2-B, minor caffeine [34.21% w/w, %RSD = 0.07%, N = 3] and major benzocaine [68.77% w/w, %RSD = 0.06%, N = 3]; NRG-2-C, major caffeine [76.03% w/w, %RSD = 0.05%, N = 3] and minor 4-MEC [19.16% w/w, %RSD = 0.36%, N = 3]; NRG-2-D, major caffeine [87.99% w/w, %RSD = 0.08%, N = 3] and minor 4-MMC [11.15% w/w, %RSD = 0.07%, N = 3]). Unlike the NRG-2 samples that were analyzed by Khreit et al., three NRG-2 samples principally contained only benzocaine (NRG-2-A, 93.87% w/w) or caffeine (NRG-2-C and NRG-2-D, 76−88% w/w) in combination with small quantities (