Forensic Science International 165 (2007) 71–77 www.elsevier.com/locate/forsciint

The application of capillary electrophoresis for enantiomeric separation of N,N-dimethylamphetamine and its related analogs: Intelligence study on N,N-dimethylamphetamine samples in crystalline and tablet forms§ Wing-Sze Lee *, Man-Fai Chan, Wai-Ming Tam, Mei-Yuen Hung Forensic Science Division, Government Laboratory, Homantin Government Offices, Hong Kong, China Received 4 August 2005; received in revised form 23 February 2006; accepted 3 March 2006 Available online 17 April 2006

Abstract This paper reports a new capillary zone electrophoresis method for the simultaneous chiral determination of the enantiomers of N,N- dimethylamphetamine (DMA), (MA), (E), (PE) and methylephedrine (Me-E). In this study, a number of electophoretic parameters were examined and optimized including the choice of chiral selectors, the use of short-chain tetraalk- ylammonium cations, the effect of chiral selector concentration, buffer concentration, applied voltage and capillary temperature. Heptakis(2,6-di- O-methyl)-b-cyclodextrin (DM-b-CD) and tetrabutylammonium (TBA) being the best chiral selector and buffer cation, respectively, the optimized electrophoretic conditions were found to be: 20 mM DM-b-CD, 50 mM TBAPO4 at pH 2.5, applied voltage at 30 kV and temperature at 25 8C. Under the optimized conditions, all analytes were well separated with resolution factors between 3.3 and 24.0 achieved. Using as an internal standard, the intra-day (n = 8) precisions for the relative migration times and peak areas of all analytes were below 1.09%, while the inter- day precisions (n = 12, 6 days) for the relative migration times and peak areas of all analytes were under 3.77%. This method has been applied to the measurement of the enantiomeric purities of the seized DMA samples. The result of the measurement could provide important clues about the possible synthetic pathways of these samples. # 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: N,N-Dimethylamphetamine; Capillary electrophoresis; Chiral determination

1. Introduction c). Since the crystalline forms of DMA and MA (‘ICE’) show similar physical appearance, DMA may wrongly be sold to Methamphetamine (MA), a powerful of the abusers as MA. Most of the seized DMA samples were central nervous system, is one of the most popular abused crystalline solids that were either in the pure form or mixed in Hong Kong. The crystalline form of its hydrochloride salt is with MA. Apart from its presence in crystalline solid samples, more commonly known as ‘ICE’. A few hundred cases were DMA has also been found in tablets, sold as ‘Ecstasy’ tablets. reported in the past years (Fig. 1a). N,N-Dimethylampheta- Like ‘Ecstasy’, these tablets were of different colours and mine (DMA), the N-methylated analog of the MA, is well markings (as shown in Fig. 2) and were usually mixed with known of producing behavioral effects that are generally various abused drugs including 3,4-methylenedioxyme- comparable to those of MA but with reduced potency [1–3]. thamphetamine (MDMA), 3,4-methylenedioxyamphetamine Cases related to DMA have increased in Hong Kong in the past (MDA), MA and . few years (from 2 cases in 1998 to 58 cases in 2004, Fig. 1band Both MA and DMA are controlled substances under the Dangerous Drugs Ordinance in Hong Kong. The recent increase in the abuse of DMA in Hong Kong is of great concern to law enforcement departments. Since only a few studies on DMA have been reported in the literature [4–7] as compared to its analog, MA, a detailed study on DMA is therefore important for obtaining valuable information for intelligence purpose. 72 W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77

stimulating effect than their corresponding counterparts, L-MA and L-DMA. By measuring the enantiomeric excesses of the seized samples, intrinsic characteristics of these samples can be observed and analysed. In addition to this, the enantiomeric purities of the seized drugs can provide clues for predicting possible synthetic routes. Identification of racemic DMA or MA indicates that these samples may be derived from reductive amination of an achiral precursor 1-phenyl-2-propanone (P2P). On the other hand, optically pure DMA or MA may have come from stereospecific reduction of the enantiopure b-hydroxyphe- nethylamines (i.e. ephedrine (E), pseudoephedrine (PE), methylephedrine (Me-E) and methylpseudoephedrine (Me-PE)). A number of analytical methods have been employed for the separation of chiral compounds. Both GC and HPLC are commonly used methods; however, they do have limitations. For example, GC analysis usually requires tedious derivatiza- tion with chiral reagents while HPLC analysis involves the use of expensive chiral columns/mobile phase. In contrast, capillary electrophoresis is relatively simple, of low cost and has very high separation efficiency. In fact, previous study in our laboratory has successfully demonstrated the use of CE for enantiomeric separation of DL-MA and its related compounds (i.e. DL-E and DL-PE) [8]. In this study, a new CE method for the simultaneous chiral separation of DL-DMA, DL-MA, DL- ephedrine, DL-PE and DL-Me-E was developed. The method has subsequently been applied to analyse seized DMA samples in the crystalline and the tablet form.

Fig. 1. Seizures of: (a) MAÁHCl (‘‘Ice’’) in Hong Kong (1998–2004); (b) DMA (crystal form) in Hong Kong (1998–2004); (c) DMA (tablet/capsule) in Hong 2. Materials and methods Kong (1998–2004). 2.1. Chemical Both MA and DMA have a chiral carbon centre at the a-position which is known to be affecting their potency. For Anhydrous sodium dihydrogen orthophosphate (from BDH, example, D-MA and D-DMA are known to have stronger Poole, England), tetrabutylammonium hydroxide 40 wt%

Fig. 2. DMA tablets seized in Hong Kong. W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77 73 solution in water (from Aldrich, Milwankee, WI, USA), Table 1 tetramethylammonium hydroxide 25 wt% solution in water Other electrophoretic conditions tested in chiral separation of 10 analytes (from Aldrich), b-cyclodextrin (b-CD) (from Sigma, St. (i) Chiral selector concentrationa: 15, 20 and 25 mM Louis, MO, USA), heptakis(2,6-di-O-methyl)-b-cyclodextrin (ii) Buffer concentrationb: 25, 50 and 75 mM c (DM-b-CD) (from Sigma), heptakis(2,3,6-tri-O-methyl)-b- (iii) Applied voltage : 20, 25 and 30 kV (iv) Temperatured: 20, 25 and 30 8C cyclodextrin (TM-b-CD) (from Fluka, Buchs, Switzerland), a hydroxypropyl-b-cyclodextrin (HP-b-CD) (from Aldrich) Conditions: [buffer] = 50 mM, applied potential at 30 kVand temperature at and phosphoric acid (from Eastman Kodak Company, 25 8C. b Conditions: [chiral selector] = 20 mM, applied potential at 30 kV and Rochester, NY, USA) were purchased and used as received. temperature at 25 8C. Authentic standards: DL-E, DL-PE, DL-Me-E and DL-MA, were c Conditions: [buffer] = 50 mM, [chiral selector] = 20 mM and temperature purchased from Sigma. D-DMA and L-DMA were prepared in at 25 8C. d moderate yields from D-MA and L-MA, respectively, Conditions: [buffer] = 50 mM, [chiral selector] = 20 mM and applied according to literature procedures [9,10]. Deionized water potential at 30 kV. from a Millipore Milli-Q System was used for buffer and standard solution preparations. All other chemicals used were temperature at 25 8C. Detection was performed at a fixed analytical reagent grade. wavelength of 200 nm for all measurements. All injections were accomplished by applying pressure at 50 mbar for 4 s. 2.2. Instrumentation 3. Results and discussion A capillary electrophoresis system (Hewlett-Packard3D CE system, Germany) equipped with a photodiode array UV 3.1. Choices of chiral selector detector was employed. An untreated fused silica capillary of 50 mm inside diameter (i.d.) and 65 cm in length from HP was The previously reported CE conditions [8] (i.e. electrolyte used for all the experiments. The capillary was conditioned consisting of 15 mM b-CD and 300 mM NaH2PO4 at pH 2.5, before use by successively washing for 30 min with 1 M NaOH, applied potential at 20 kV and with the temperature at 30 8C) 10 min 0.1 M NaOH and 10 min water, followed by a 30 min were applied for chiral separation of DL-E, DL-PE, DL-Me-E, flushing with the run buffer. The capillary was also flushed with DL-MA and DL-DMA. Results showed that all analytes were the run buffer for 5 min between injections. poorly separated (Fig. 3).Asaresult,anumberofchiral selectors were experimented in order to improve the 2.3. Standard and sample preparations separations of all enantiomers. In this study, four different cyclodextrins (i.e. b-CD, DM-b-CD, TM-b-CD and HP-b- Drug standards DL-E, DL-PE, DL-Me-E, DL-MA and DL-DMA CD) were chosen since cyclodextrin is well documented to be were dissolved directly in water (ca. 0.1 mg/ml) in the presence one of the most useful chiral selectors for separation of chiral of 0.1 mg/ml of phentermine as an internal standard. Samples drugs and other compounds in capillary electrophoresis [11]. containing not less than 0.2 mg/ml of DMA and MA were Twenty millimolars of each of the CD was added to 50 mM weighed and dissolved directly in water in the presence of NaH2PO4 at pH 2.5 and results are shown in Fig. 4.Noneofthe 0.1 mg/ml of phentermine as the internal standard. CDs could separate 10 enantiomers simultaneously. Results also indicated that the introduction of an alkyl group at the 2.4. Electrophoretic conditions C-2 position of the b-CD (i.e. DM-b-CD and HP-b-CD) generally improved the separation efficiency for all enantio- In this study, a number of electrophoretic conditions were mers. Out of the four CDs tested, DM-b-CD gave the best examined and optimized. The most suitable chiral selector result. The presence of both the methoxy and hydroxyl groups and buffer cation were first studied and selected. Thus, four on the secondary rim of the CD could probably contribute to chiral selectors (i.e. b-CD, DM-b-CD, TM-b-CD and HP-b- its success in separation. On the other hand, chiral separation CD) were tested in the phosphate buffer with sodium or TMA or TBA as the cation. (The study was carried out at the following experimental conditions: 20 mM chiral selector, 50 mM phosphate buffer at pH 2.5 with an uncoated capillary (64.5 cm  50 mm), applied potential at 30 kV and with the temperature at 25 8C.) Using the best chiral selector and buffer cation, other electrophoretic conditions such as concentration of chiral selector, concentration of buffer, applied voltage and capillary temperature were thenvaried and optimized. Detailed Fig. 3. Electropherograms of 10 enantiomers: (a) L-methylephedrine; (b) L- conditions are summarized in Table 1. After optimization, the pseudoephedrine; (c) D-ephedrine; (d) D-methylephedrine; (e) L-ephedrine; (f) D-pseudoephedrine; (g) L-methamphetamine; (h) L-dimethylamphetamine; (i) best conditions have been found to be: 20 mM DM-b-CD, D-methamphetamine; (j) D-dimethylamphetamine. Conditions: 15 mM b-CD,

50 mM TBAPO4 at pH 2.5 with an uncoated capillary 300 mM NaH2PO4 buffer at pH 2.5 with an uncoated capillary (64.5 cm  50 mm), applied potential at 30 kV and with the (64.5 cm  50 mM), applied potential at 20 kV and temperature at 30 8C. 74 W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77

and TBA) as buffer cations was studied in the presence of b- CD, DM-b-CD, TM-b-CD or HP-b-CD as the chiral selectors. Results have been summarized in Figs. 5 and 6. When comparing TMA or TBA with sodium, it was found that the migration times for each analyte increased and the overall separation was also improved. However, in TMAPO4 buffer, unstable and noisy baseline was obtained and also all analytes were not completely separated. As a result, the best cation was found to be TBA. Again, amongst the CDs investigated, DM-b-CD was identified to be the most effective resolving agent for all analytes allowing the 10 enantiomers to be well separated when a TBA was used as buffer cation.

3.3. Effects of chiral selector concentration, buffer concentration, capillary temperature and applied voltage

From the above results, the best buffer and chiral selector were found to be TBAPO4 and DM-b-CD, respectively. Thus, CE conditions were optimized using TBAPO4 buffer containing DM-b-CD as chiral selector. First, the concentration of DM-b- CD was examined by varying from 15 to 25 mM and results are shown in Fig. 7. The separation between L-MA and L-DMAwas poor at low concentration (i.e. 15 mM DM-b-CD). In this case, the number of chiral selector molecules may be insufficient for

Fig. 4. Electropherograms of 10 enantiomers: (a) L-pseudoephedrine; (b) D- ephedrine; (c) D-methylephedrine; (d) L-ephedrine; (e) L-methylephedrine; (f) D-pseudoephedrine; (g) L-methamphetamine; (h) L-dimethylamphetamine; (i) D-methamphetamine; (j) D-dimethylamphetamine. Conditions: 20 mM chiral selector—(i) b-CD; (ii) DM-b-CD; (iii) TM-b-CD; (iv) HP-b-CD, 50 mM

NaH2PO4 buffer at pH 2.5 with an uncoated capillary (64.5 cm  50 mM), applied potential at 30 kV and temperature at 25 8C. of all analytes was lost when both the C-2 and C-3 hydroxyl groups of the b-CD are alkylated (i.e. TM-b-CD). It is because no hydroxyl group is available in TM-b-CD for forming hydrogen bonding with the analytes and hydrogen bonding plays an important role in the chiral discrimination process by CD.

3.2. Use of short-chain tetraalkylammonium cations in buffer

It is known that reversal of electroosmotic flow (EOF) (i.e. counteracting the migration of the solute enantiomers) caused a general increase in migration time, which in turn improved the resolution of analytes [12]. The use of tetraa- lkylammonium(TAA)asthecationinthebuffersolutionis one of the ways that causes a reverse of the EOF [13].A number of studies reported that short-chain TAA cations such as tetramethylammonium (TMA) and tetrabutylammo- Fig. 5. Electropherograms of 10 enantiomers: (a) L-pseudoephedrine; (b) D- nium (TBA) are more effective than those with long-chain ephedrine; (c) D-methylephedrine; (d) L-ephedrine; (e) L-methylephedrine; (f) [13,14]. TAA with short chain lengths are relatively more D-pseudoephedrine; (g) L-methamphetamine; (h) L-dimethylamphetamine; (i) D-methamphetamine; (j) D-dimethylamphetamine. Conditions: 20 mM chiral hydrophilic and less likely to occupy the hydrophobic cavity selector—(i) b-CD; (ii) DM-b-CD; (iii) TM-b-CD; (iv) HP-b-CD, 50 mM of the CDs rendering less competition with the analytes. In TMAPO4 buffer at pH 2.5 with an uncoated capillary (64.5 cm  50 mM), this research, the use of two short-chain TAA cations (TMA applied potential at 30 kV and temperature at 25 8C. W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77 75

Fig. 7. Effect of DM-b-CD concentration on separation of 10 enantiomers: (a) L-pseudoephedrine; (b) L-ephedrine; (c) L-methylephedrine; (d) D-ephedrine; (e) D-methylephedrine; (f) D-pseudoephedrine; (g) L-methamphetamine; (h) L- dimethylamphetamine; (i) D-methamphetamine; (j) D-dimethylamphetamine. Conditions: [DM-b-CD]—(i) 15 mM; (ii) 20 mM; (iii) 25 mM, 50 mM

TBAPO4 buffer at pH 2.5 with an uncoated capillary (64.5cm  50 mM), Fig. 6. Electropherograms of 10 enantiomers: (a) L-pseudoephedrine; (b) D- applied potential at 20 kV and temperature at 30 8C. ephedrine; (c) D-methylephedrine; (d) L-ephedrine; (e) L-methylephedrine; (f) D-pseudoephedrine; (g) L-methamphetamine; (h) L-dimethylamphetamine; (i) D-methamphetamine; (j) D-dimethylamphetamine. Conditions: 20 mM chiral selector—(i) b-CD; (ii) DM-b-CD; (iii) TM-b-CD; (iv) HP-b-CD, 50 mM

TBAPO4 buffer at pH 2.5 with an uncoated capillary (64.5 cm  50 mM), applied potential at 30 kV and temperature at 25 8C. interacting with analytes at this concentration. On the other hand, when high concentration of DM-b-CD was used (i.e. 25 mM), not only the migration time was longer but also some of the analytes (i.e. L-MA, L-DMA and D-DMA) were not well resolved. It may be due to saturation of the run buffer with the DM-b-CD over the analyte. The optimum concentration of DM-b-CD was found to be 20 mM. Next, the concentrations of TBAPO4 rangingfrom25to75mMwerestudied.Asshown in Fig. 8, good separation was not obtained at both low (i.e. 25 mM) and high concentrations (i.e. 75 mM) of TBAPO4.At low concentration, the amount of TBA may not be sufficient to effectively cover the capillary surface. At high concentration, TBA would compete with the analytes for DM-b-CD. The optimal results were achieved with 50 mM TBAPO4.The effect of varying the applied voltage between 20 and 30 kVon the peak resolution was studied. No significant effect on peak resolution was obtained by increasing the applied voltage. High voltage can shorten the analysis time, so 30 kV was selected as the optimal applied voltage. Temperature is one of Fig. 8. Effect of TBAPO4 concentration on separation of 10 enantiomers: (a) L- the important parameters to be considered since it can affect pseudoephedrine; (b) L-ephedrine; (c) L-methylephedrine; (d) D-ephedrine; (e) several parameters such as mobility of analytes, the kinetic and D-methylephedrine; (f) D-pseudoephedrine; (g) L-methamphetamine; (h) L- dimethylamphetamine; (i) D-methamphetamine; (j) D-dimethylamphetamine. thermodynamic of the inclusion-complexation with CDs [11]. Conditions: [TBAPO4]—(i) 25 mM; (ii) 50 mM; (iii) 75 mM, 20 mM DM-b- Capillary temperatures between 20 and 30 8C were tested CD at pH 2.5 with an uncoated capillary (64.5 cm  50 mM), applied potential [8].At208C, no significant improvement in peak resolution at 20 kV and temperature at 30 8C. 76 W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77

Table 2 Table 4 Precisions of intra-day assays (n = 8) for migration times and peak area of Distributions of enantiomers of ÆMA and ÆDMA in 30 crystalline samples crystalline samples No. of sample MA, ee (%) DMA, ee (%) Analyte R.S.D. (%) 7–>98 (D) Relative migration time Relative peak area 5 – 87–95 (D)

L-PE 1.09 1.05 10 >98 (D) >98 (D) L-E 1.07 1.03 4 55–92 (D) >98 (D) L-Me-E 1.06 1.02 2 >98 (L) >98 (D) D-E 1.04 1.00 2 35–41 (L) >98 (D) D-Me-E 1.03 1.00 D-PE 0.95 0.92 L-MA 0.91 0.87 L-DMA 0.90 0.86 3.5. Chiral analysis of crystalline samples of DMA or D-MA 0.88 0.85 mixture of DMA/MA D-DMA 0.85 0.82 Using the optimized CE conditions (see Section 2.4), 30 crystalline samples of DMA (12 as pure samples and 18 as a mixture of DMA/MA samples) were randomly taken from was observed, and also the run time was longer. Although 24 cases for chiral analysis. As shown in Table 4, all DMA higher temperature (30 8C) could shorten the analysis time, samples were D-form predominant (>87% ee). However, MA it led to decease in peak resolution. Thus, temperature of 25 8C samples were found in different enantiomeric forms. Over waschosenastheworkingtemperature. 55% were found to contain optically pure D-MA (>98% ee) and 11% contained optically pure L-MA (>98% ee). The remaining 3.4. Detection limits and reproducibility for standard contained a variable combination of the two enantiomers mixture (varying from À41 to À92% ee based on L-MA). Similar results were also obtained in previous studies [8,15]. One possible Under the optimized CE conditions (see Section 2.4), DL-E, explanation is physical mixing of the two enantiomers in DL-PE, DL-Me-E, DL-MA and DL-DMA were well separated different proportions. In cases where pure L-MA had been with resolution factor of 6.2, 24.0, 5.7, 3.3 and 6.2, detected, repeated run was carried out by spiking D-MA to the respectively, achieved (as shown in Fig. 6ii). The detection test sample for confirmation of its chirality. The results showed limits of all enantiomers, defined as the concentration that that none of them were racemic mixture. This finding indicated produced a signal-to-noise ratio equal to 3, were equal to or that DMA and MA samples were unlikely to be synthesized less than 3 mg/ml. By running a standard solution containing through reductive amination of achiral precursor P2P. Rather, D-MA and L-MA, D-DMA and L-DMA, both enantiomers DMA and MA in samples were more probably prepared from in a ratio of 99:1, optical purity up to 2% ee could be detected. Me-E and PE or E, respectively. In Table 2, it is shown that the intra-day R.S.D. of the relative migration times and relative peak area (n = 8) for all analytes 3.6. Chiral analysis of tablet samples of DMA were less than 1.09 and 1.05%, respectively. As illustrated in Table 3, the inter-day R.S.D. of the relative migration times Apart from the crystalline samples, DMA was also present (n = 12, 6 days) and relative peak area (n =12,6days)were in tablet samples which contained other abused drugs less than 2.42 and 3.77%, respectively. including MDMA, MDA, MA and ketamine. The optimized CE method (see Section 2.4) was applied for chiral analysis of 32 tablet samples of five different markings. Results are Table 3 summarized in Table 5. It has been observed that there was no Precisions of inter-day assays (n = 12, 6 days) for migration times and peak area interference on the separation of the DMA and MA by the of crystalline samples presence of the other drugs (MDMA, MDA and ketamine), as Analyte R.S.D. (%) all of them eluted after DMA and MA. All DMA and MAwere D-form predominant (>93 and >98% ee, respectively). Similar Relative migration time Relative peak area to the crystalline samples, none of these tablet samples were L-PE 2.42 3.15 L-E 2.36 3.67 Table 5 L-Me-E 2.35 3.40 Distributions of enantiomers of ÆMA and ÆDMA in 32 tablet samples D-E 2.31 3.62 No. of sample Logo MA, ee (%) DMA, ee (%) D-Me-E 2.28 3.77 D-PE 2.13 2.10 20 Chilli >98 (D) >98 (D) L-MA 2.05 3.69 5 Heart >98 (D) 93–95 (D) L-DMA 2.03 2.77 5 TOP – >98 (D) D-MA 1.99 2.34 1 Euro – >98 (D) D-DMA 1.92 2.83 1WY–>98 (D) W.-S. Lee et al. / Forensic Science International 165 (2007) 71–77 77 racemic mixture. Similar inference that DMA and MA were tamine: an illicit analog of methamphetamine, Brain Res. 490 (1989) 301– likely to be prepared from reduction of chiral precursor (Me-E, 306. [4] R. Kikura, Y. Nakahara, S. Kojima, Simultaneous determination of E and PE) could be drawn. dimethylamphetamine and its metabolites in rat hair by gas chromato- graphy–mass spectrometry, J. Chromatogr. B 741 (2000) 163–173. 4. Conclusion [5] S. Chinaka, S. Tanaka, N. Takayama, K. Komai, T. Ohshima, U. Ueda, Simultaneous chiral analysis of methamphetamine and related compounds by capillary electrophoresis, J. Chromatogr. B 749 (2000) 111–118. A new CZE method using DM-b-CD as a chiral selector and [6] M. Sato, M. Hida, H. 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