Common methods for the chiral determination of amphetamine and related compounds II. Capillary electrophoresis and nuclear magnetic resonance

Trends in Analytical Chemistry, Vol. 31, 2012

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Common methods for the chiral determination of amphetamine and related compounds II. Capillary electrophoresis and nuclear magnetic resonance Justyna Małgorzata Płotka, Calum Morrison, Marek Biziuk Amphetamine (AM) group and related ring-substituted substances are generally synthetic compounds, belonging to one of the most heavily abused drug groups in recent years. Some compounds in this class also originate from plants. Analysis of the enantiomers of AM-type compounds and metabolites is extremely important for a number of scientific disciplines. From studies of biological activity or mechanisms through determination of precursor molecules in a criminal investigation all use analytical procedures. This article reviews capillary electrophoresis and nuclear magnetic resonance as among the most common, useful methods for the chiral determination of AMs and AM-derived designer drugs in different matrices, including blood, hair, urine, and medicaments. Papers published in the last 15 years were considered including commonly used types of chiral derivatization reagent and chiral stationary phase. Tables summarize basic information about conditions and reference data of each procedure. Other methods (e.g., gas chromatography, liquid chromatography and high-performance liquid chromatography, and thin-layer chromatography) were described in Part I [Trends Anal. Chem. 30 (2011) 1139]. ª 2011 Published by Elsevier Ltd. Keywords: Amphetamine; Capillary electrophoresis (CE); Chiral derivatization; Chiral determination; Chiral selector; Chiral stationary phase; Enantioseparation; Microchip; Nuclear magnetic resonance; Stimulant

1. Introduction Amphetamine (AM)-group substances [primarily AM, methamphetamine (MAM) and methcathinone) and ecstasy-group substances (e.g., MDMA, MDA, DOM, and EP; Fig. 1), including synthetic and natural substances, show a trend of increasing abuse in recent years. These drugs are potent in stimulating the central nervous system (CNS) [1]. The effects are diverse and are classified into the three main classes: emphatogenic, hallucinogenic and psychoanaleptic. AM-group stimulants have many effects on the body (e.g., mood elevation, induced euphoria, increased alertness and energy, reduced fatigue, decreased appetite, increased movement and

speech, and a sense of increased personal power and prowess) [2]. The popularity of AM-group substances can be attributed to several reasons: (1) first, strong ‘‘positive’’ effects on the body; (2) second, laboratories used for synthesis of AM-type compounds can clandestinely operate anywhere and can be relocated easily as the risk of detection increases; (3) AM-type compounds can be synthesized from a variety of precursor chemicals using many different methods; and, (4) if traditional precursors are unavailable, replacements are easily found [3].

0165-9936/$ - see front matter ª 2011 Published by Elsevier Ltd. doi:10.1016/j.trac.2011.06.021

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Justyna Małgorzata Płotka*, Marek Biziuk Department of Analytical Chemistry, Chemical Faculty, Gdan´sk University of Technology, Narutowicza 11/ 12, 80-233 Gdan´sk, Poland Calum Morrison School of Science, Faculty of Science and Technology, University of the West of Scotland, Paisley PA1 2BE, Scotland, United Kingdom

*

Corresponding author. Tel.: +48 58 347 21 10; Fax: +48 58 347 26 94. E-mail: plotkajustyna@gmail. com

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Trends in Analytical Chemistry, Vol. 31, 2012

Figure 1. Amphetamine-type stimulants: amphetamine (AM); 2,5-dimethoxy-4-methylamphetamine (DOM); ephedrine (EP); p-hydroxymethamphetamine (p-HMAM); methamphetamine (MAM); 3,4-methylenedioxyamphetamine (MDA); 3,4-methylenedioxy-N-ethylamphetamine (MDEA); 3,4-methylenedioxymethamphetamine (MDMA); norephedrine (NEP); norpseudoephedrine (NPEP); pseudoephedrine (PEP).

Fig. 2 shows the number of reported laboratories (all sizes) for AM-type compounds by region, 1999–2008 [3]. Nowadays, abuse of AM-type compounds is a serious social problem, so their analysis is important, especially in forensic science. In recent years, the analysis of AMs has been extended to include separation and quantitation of enantiomers. This is important, since these drugs are chiral compounds and the range of effects on the body depends on the enantiomeric composition. Within an enantiomeric pair, there may be differences in pharmacological activity and rates of metabolism [4]. For example, it is well known that the S-(+)-enantiomer exhibits greater pharmacological potency than the corresponding R-( )-enantiomer and is about five times as active as the R-( )-enantiomer [5,6]. Generally, enantiomeric analysis of AMs has fundamental significance in forensic science laboratories, because characterization of enantiomeric composition of AMs may supply valuable information about the conditions and the chemicals used in the clandestine laboratories, which can provide information on the original source of the sample [7]. For example, l-deprenyl (selegiline) is a medicine used for ParkinsonÕs disease and is metabolized to the l-isomers of AM and MAM. For that reason, chiral information is needed to discriminate AM and MAM abuse from deprenyl use [8]. 24

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Recently proposed methods for the determination and enantiomeric separation of AM-type compound include gas chromatography (GC), liquid chromatography (LC), thin-layer chromatography (TLC) and capillary electrophoresis (CE) as well as spectroscopic methods [e.g., nuclear magnetic resonance (NMR)] [9,10]. GC and LC were traditionally the methods of choice. However, CE and NMR also provide many advantages. This article presents an overview of the strategies used for the enantiomeric separation of AM-type compounds using CE and NMR techniques. We briefly discuss the potential advantages and limitations of the different approaches. Also, we compare the different methods in terms of separation efficiency, sensitivity, and time of analysis, or potential for automation.

2. Determination using CE and NMR Several LC, GC and TLC methods are available for the enantiomeric characterization and composition determination of AM-type compounds. These methods usually use a chiral column or chiral derivatizing reagents, which greatly enhances them. The derivatization affords greater selectivity and sensitivity in enantiomeric separation. These methods were described in Part I [Trends Anal. Chem. 30 (2011) 1139].

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Figure 2. Number of reported amphetamine-type compound laboratories, by region, 1999–2008.

We review in this section CE and NMR procedures for the determination of AM drugs and chiral derivatization processes. 2.1. CE CE may be considered complementary to other analytical techniques (e.g., GC or LC) for enantioseparation [11]. CE is a powerful analytical technique widely applied to chiral analysis in different areas of research (e.g., forensic, clinical, and pharmaceutical). CE is a fast, economical, powerful and effective method. High-resolution separation is also reported [12]. Indirect or direct methods are used for the successful separation of enantiomers. In the indirect method, the two enantiomers strongly react with a chiral selector in order to form two diastereomers, while, in the direct method, the enantiomers weakly interact with the chiral selector. The diastereomers formed have different physico-chemical properties and are therefore resolvable [9]. 2.1.1. Direct separation method The direct separation method can be achieved in three ways. The first, most commonly used is based on adding a chiral selector to the background electrolytes (BGEs). The chiral agent can also be bound to the capillary wall or included in the gel matrix [9]. Direct separation methods have been shown to be attractive for chiral separation using various additives [e.g., cyclodextrins (CDs), crown ethers, proteins, macrocyclic antibiotics, and other chiral selectors] [13].

CDs are the most popular chiral selectors used in CE. Native and neutral CDs are the most frequently used chiral selectors for the chiral analysis of AM and related compounds. Native CDs are cyclic oligosaccharides consisting of six, seven or eight glucopyranose units (named a-, b- and c-CDs, respectively), linked by a-(1,4) bonds. Fig. 3

Figure 3. Structures of b-cyclodextrins (b-CDs). The R group are: natural (-H); carboxymethyl (-CH2COO-); hydroxyethyl (-CH2CH2OH); hydroxypropyl (-(-CH2CH(OH)CH3); and, methyl (-CH3).

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shows the general chemical structure of b-CDs (native R = H). Because all glucopyranose units occur in chair conformation, all primary hydroxyl groups are located on one side of the ring, and the secondary hydroxyl groups on the opposite side. The molecule takes the shape of a truncated cone (toroid). Free electron pairs are directed towards the interior of the molecule, so the interior of the toroid is hydrophobic, while the outer surface is hydrophilic. The hydroxy groups in positions 2, 3 and 6 are available for derivatization, which is needed to obtain new products with altered enantioselective capacity [9]. Table 1 lists the main properties of native CDs. We describe below some examples of AM-type compounds whose enantiomers were resolved by CE with CDs as chiral selectors, and they are summarized in Table 2. Also, we mention other chiral selectors in the section below. 2.1.1.1. Application to enantioseparation of AM-type compounds. As previously mentioned, the determination of enantiomeric AM and MAM is important [e.g., in urine samples in order to distinguish use of the prescription drug l-deprenyl from the illicit use of S-(+)-AM and S-(+)-MAM]. The products of metabolized l-deprenyl are R-( )-AM and R-( )-MAM and this knowledge is vital for certain criminal investigations. Analysis of AM and MAM enantiomers, and l-deprenyl in urine was carried out by Heo and co-workers [14]. Illegal and therapeutic drugs were taken into consideration. Optimization of analytical condition was performed by CE using chiral selectors including b-CD, carboxymethyl-b-CD (CM-b-CD) and 2-hydroxypropylb-CD (HP-b-CD). The factors to obtain the best chiral resolution (Rs), sensitivity, wide range of linearity in concentration and separation efficiency were examined. Optimum resolutions were achieved using 100 mM phosphate buffer, pH 2.5, containing 10 mM of CMb-CD. This simple, routine stereospecific analysis of enantiomers AM and MAM has given important information for seizures of illicit MAM. Also, analysis of urine from patients who had taken l-deprenyl was successful.

Thus, this method is reliable for determining the chiral metabolites of therapeutic drug l-deprenyl and illicit MAM in urine. Also, good sensitivity, relevant linearity and reproducibility were found for R-( )-AM, R-( )MAM and S-(+)-AM, and S-(+)-MAM. A rapid, simple method for the analysis of isomers of MAM and related drugs by CE coupling with mass spectrometry (MS) with direct injection of urine was studied by Iio et al. [8]. The electrolyte used was formic acid with ammonium formate containing heptakis-(2, 6-diacetyl-6-sulfato)-b-CD (DAS-b-CD), which was a suitable chiral selector for this method because of its high Rs for AM-related drugs. The proposed method was successful for the chiral analysis of urine sample from MAM and dimethylamphetamine (DMA) addicts and patients under l-deprenyl pharmacotherapy. In other work, Iio et al. [15] developed a method for six AM-related drugs, and chiral selectors [e.g., b-CD, heptakis-(2,6-diacetyl6-sulfato)-b-CD (DAS-b-CD), heptakis-6-sulfato-b-CD (S-b-CD), heptakis-(2,6-di-O-methyl)-b-CD (DM-b-CD) and heptakis-(2,3-dimethyl-6-sulfato)-b-CD (DMS-b-CD)] were taken into consideration. Results are shown in the Table 3. This study also compared BGEs. In the first case, the BGE was 1 M formic acid with MS detection. In the second case, 75 mM tris(hydroxymethyl)-aminomethane (Tris) was used as a BGE and then diode-array UV detection. Using the first set of conditions, the Rs of all analysis compounds, except NEP, were under 1. In the second case (with Tris as a selector), the Rs of all compounds, except AM, were over 1. For urine analysis, the first case was used. All compounds were clearly detected and the reproducibility of this method was good. Unfortunately, RSDs of the migration times and of the peak area were large. The sensitivity of this method was sufficient to detect the concentration of AM and MAM in urine. The proposed method is selective for the simultaneous chiral analysis of the enantiomers mentioned, and could be applicable to the analysis of urine samples. Highly-sulfated c-CD (SU(XIII)-c-CD) is a commercially-available anionic CD and may be used as a chiral selector in CE for the separation of AM compounds.

Table 1. Properties of native cyclodextrins [9] Properties

Molecular weight Number of glucopyranose [aD25] Solubility [g/ 100 ml in water] Depth [nm] Cavity diameter [aD25], optical rotation.

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CD type a

b

c

972 6 +150.5 14.5 0.78 0.47–0.6

1135 7 +162.0 1.85 0.78 0.8

1297 8 +177.4 23.2 0.78 1.0

Direct method

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Sample matrix

Chiral selector

Capillary

Apparatus

AM MAM EP PEP NEP MDA p-HMAM AM MAM DMA EP NEP MEP PEP AM MAM

U

DAS-b-CD

Fused-silica capillary 50 lm i. d. · 100 cm (GL Science)

Agilent CE (Agilent Technologies)

Formic acid/ ammonium formate 10:0.2 v/v + DAS-b-CD pH 2

MS

0.01 lg/mL

[8]

U

DAS- b -CD

Fused-silica capillary 50 lm i. d. · 100 cm (GL Science)

Agilent CE (Agilent Technologies)

1 M formic acid + 0.85 mM DASb –CD pH 1.7

ESI/MS

0.01 lg/mL

[8]

U

b-CD CM-bCD HP-b-CD

HP 3D CE (HewlettPackard)

Phosphate buffer + b-CDs pH 2.5

UV

0.01 lg/mL

[14]

AM MAM EP PEP NEP NPEP MDA MDMA AM MAM MDA MDMA MDEA EP NEP PEP NPEP EP PEP MEP MDMA AM-S

MAM

SU(XIII)- c -CD

Fused-silica capillary 50 lm i. d. · 65 cm (Polymicro Technologies) Fused-silica capillary 50 lm i. d. · 32.5 cm (Polymicro Technologies)

Agilent CE system G1600A (Agilent Technologies)

Phosphate buffer + 20% SU(XIII)c –CD pH 2.6

UV-DAD

0.01 lg/mL

[16]

S

SU(XIII)- c -CD

Fused-silica capillary 50 lm i. d. · 56 cm (Polymicro Technologies)

Agilent CE (Agilent Technologies)

1 M formic acid + 2.5 Mm SU(XIII)- c -CD

ESI/MS MS/MS

0.01-0.02 lg/mL

[16]

T

Fused-silica capillary 50 lm i. d. · 65 cm (Hewlett-Packard)

HP 3D CE (HewlettPackard)

Phosphate buffer + sodium/ TMA/TBA + b-CDs pH 2.5

UV

< 3.00 lg/mL

[18]

S

b-CD DM-b-CD HP-b-CD TM-b-CD RM B

Isco model 3850 (Isco)

-

[20]

S

RM B

UV

-

[21]

AM

S

Dextran 20

Phosphate buffer/ 2-propanol 70:30 v/v + RM B pH 7 Phosphate buffer/ 2-propanol 60:40 v/v + RM B pH 7 TEA phosphate buffer (50 mM) + CS pH 3

UV

EP

Fused-silica capillary 50 lm i. d. · 65 cm (Isco) Fused-silica capillary 50 lm i. d. · 57.5 cm (Quadrex Corp.) Fused-silica capillary 50 lm i. d. · 50 cm

UV

-

[24]

Quanta 4000 CE (Waters) Biofocus 2000 CE (BioRad Laboratories)

Electrolytes

Detection

LOD

Ref.

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Analyte(s)

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Table 2. Capillary electrophoresis – direct and indirect separation methods for chiral separations of amphetamine-type compounds

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Table 2. (continued)

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Direct method Analyte(s) MDA MDMA MDEA EP PEP NEP NPEP EP PEP NEP PEP

AM MAM MDA MDMAMDEA NE AM MAM MDA MDMA MDEA NE

Sample matrix

Chiral selector

Capillary

Apparatus

Electrolytes

Detection

LOD

Ref.

Dextran 70 Pullulan l-Leu

Fused-silica capillary 50 lm i. d. · 43 cm (Yongnian Optical Fiber Factory)

Bio-Rad 3000 CE (BioRad Laboratories)

Phosphate buffer/ 2-propanol 80:20 v/v + l-Leu pH 9

UV

< 0.01 mg/mL

[26]

S

Fused-silica capillary 50 lm i. d. · 64 cm (Polymicro Technologies) Fused-silica capillary 50 lm i. d. · 70 cm (Polymicro Technologies) Fused-silica capillary 50 lm i. d. · 64.5 cm (Composite Metal Service)

Agilent CE (Agilent Technologies)

TEA+water+ Phosphate acid + CS pH 2-3

UV

325 ng/mL

[27]

Agilent CE (Agilent Technologies)

TEA+water+ HCOOH + NH4OAc + CS pH 2-3

MS

325 ng/mL

[27]

P

p-L-SUCLS p-L-SUCILS p-L-SUCVS p-L-SUCLS p-L-SUCILS p-L-SUCVS SU(XIII)- c -CD

Agilent HP 3D CE (Agilent Technology)

100 mM Tris-phosphate buffer pH 2.5

UV

-

[33]

P

SU(XIII)- c -CD

Fused-silica capillary 50 lm i. d. · 64.5 cm (Composite Metal Service)

Agilent HP 3D CE (Agilent Technology)

30 mM ammonium formate buffer pH 2.5 + formic acid + SU(XIII)- c -CD

MS

-

[33]

MarfeyÕs reagent

Fused-silica capillary 50 lm i. d. · 48.5 cm (Hewlett-Packard)

HP 3D CE (HewlettPackard)

Sodium borate + methanol +CS

UV

-

[28]

FITC

Fused-silica capillary 75 lm i. d. · 60 cm (Beckman)

Beckman P/ACE MDQ (Beckman)

20 mM borate + sodium hydroxyl pH 12

DAD-LIF

0.2 ng/mL

[30]

U

Indirect method AM D MAM HAM MAM MDMA MDEA DMA DMMA BDMA AM U MAM EP PEP B MDA MDMA

AM, Amphetamine; AMS Amphetamine sulfate; b-CD, b-cyclodextrin; CM-b-CD, Carboxymethyl-b-cyclodextrin; DAS-b-CD, Heptakis-(2,6-diacetyl-6-sulfato)-b-cyclodextrin; DM-b-CD, Heptakis(2,6-di-O-methyl)-b-cyclodextrin; DMS- b-CD, Heptakis-(2,6-dimethyl-6-sulfato)-b-cyclodextrin; EP, Ephedrine; Eph, Ephedra; HP-b-CD, 2-hydroxypropyl-b-cyclodextrin; l-Leu, lleucine; MAM, Methamphetamine; MDA, 3,4-methylenedioxyamphetamine; MDEA, 3,4-methylenedioxyethylamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; MS, Mass Spectrometry; NEP, Norephedrine; NPEP, Norpseudoephedrine; P, Plasma; p-HMAM, p-hydroxymethamphetamine; p-L-SUCILS, Polysodium N-undecenoyl-L-isoleucine sulfate; p-L-SUCLS, Polysodium N-undecenoyl-L-leucine sulfate; p-L-SUCVS, Polysodium N-undecenoyl-L-valine sulfate; PEP, Pseudoephedrine; RM B, Rifamycin B; S, Standard; SU(XIII)- c –CD, Sulfated ccyclodextrin; T, Tablet; U, Urine.

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Table 3. Resolutions of enantiomers of AM, MAM, DMA, EP, MEP NEP [15] Analyte1

b-CD and DM-b-CD2 (3.10 mM)

S-b-CD2 (2.5 mM)

DMS-b-CD2 (10 mM)

DAS-b-CD2 (0.85 mM)

b-CD and DM-b-CD3 (3.10 mM)

AM MAM DMA EP MEP NEP

<1 <1 <1 <1 <1 1.1

1.8 2.3 <1 <1 <1 Not separated

<1 <1 <1 1.7 1.7 1.0

2.1 2.9 4.7 4.4 7.2 2.9

<1 1.2 2.1 1.4 2.0 1.4

AM, Amphetamine; b-CD, b-cyclodextrin; DAS-b-CD, Heptakis-(2,6-diacetyl-6-sulfato)-b-cyclodextrin; DMA, Dimethylamphetamine; DM-bCD, Heptakis(2,6-di-O-methyl)-b-cyclodextrin; DMS- b-CD, Heptakis-(2,6-dimethyl-6-sulfato)-b-cyclodextrin; EP, Ephedrine; MAM, Methamphetamine; MEP, Methylephedrine; MS, Mass Spectrometry; NEP, Norephedrine; S-b-CD, Hptakis-6-sulfato-b-cyclodextrin, U, Urine. 1 5 lg/mL each. 2 The background electrolyte, 1 M formic acid; detector MS. 3 The background electrolyte, 0.75 mM Tris buffer; detector DAD.

Iwata et al. [16] examined this type of chiral selector for the analysis of seized MAM. In this study, the simultaneous chiral separation of nine AM compounds by reversed-polarity CE with UV detection using SU(XIII)-c-CD was investigated. Two types of injection were compared (hydrodynamic and electrokinetic). For impurity analysis, RP-CE-UV with electrokinetic injection was employed. Analysis was successful. In other work, Iwata et al. [17] used RP-CE-ESI+MS and tandem MS (MS2) for simultaneous chiral separation of nine AM-type stimulants (NEP, NPEP, EP, PEP, AM, MAM, MDA, MDMA, and MDEA). Using SU(XIII)-c-CD as a chiral selector, the nine AM-type compounds were completely separated within 50 min. The limit of detection (LOD) of MS2 was 10 times more sensitive than for single MS. Also, using this method, seized d-MAM hydrochloride samples were analyzed. Impurities originating in the precursor (e.g., l-ephedrine and d-pseudoephedrine) were detected and quantified. Due to the high resolving power for the enantiomers, the high efficiency for separation of AMs and the low frequency of cleaning the instrument, these methods could be proposed for the analysis of seized MAM samples. A new capillary zone electrophoresis method for the simultaneous chiral determination of the enantiomers of MDA, MAM, EP, PEP and MEP was carried out by Lee et al. [18] and a number of electophoretic parameters were examined and optimized. b-CD, DM-b-CD, HP-b-CD, TM-b-CD were examined as chiral selectors. The results of using of tetraalkylammonium (TAA) and tetrabutylammonium (TBA) as a cation were compared. DM-b-CD and 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 25C. The main advantages of this method are sensitivity and precision, which are sufficiently good for

application in the chiral analysis of seized MDA samples (both tablet and crystalline forms). Also, uncharged b-CD polymer (EP-b-CD) has been applied to enantioseparation by CE. This polymer comprises b-CD cross-linked with epichlorohydrin and the application was by Ingelse [13]. Different basic compounds, including EP, NEP, MAM, and selegiline were selected for the electrophoretic experiments. Several parameters were examined. The resolution and the selectivity were influenced by the concentration of chiral selector (the mobility of the analytes decreased as the concentration of EP-b-CD increased). Addition of organic modifiers to the BGE was also examined and deleterious effects of these organic modifiers on the resolution were reported. Temperature was taken into consideration and had a negative effect on resolution (except EP, for which resolution increased as temperature increased). Looking at the examples above, we can conclude that CDs are popular chiral selectors in CE and provide good Rs of AM-type compounds. The big advantages of the CDs are that the solubility and the selectivity can be improved by derivatization. Neutral CDs and modified CDs (charged CDs) are known. The use of modified CDs in chiral separation by CE can increase resolution in comparison with neutral CDs. Charged CDs also provide much greater flexibility in optimizing separation conditions. The separation efficiency and resolution depend on a number of parameters, including pH, the concentration of the chiral selector or the addition of organic modifier. Macrocyclic antibiotics are another type of chiral selector used for enantioseparation in CE. Although CDs and their derivatives are still the most popular selectors, in the past few years, interest in antibiotics has been continuously increasing [19]. Antibiotics exhibit a variety of interactions (inclusion, electrostatic, and hydrogen bond), which enable high resolution with a wider

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range of analytes (acidic, basic, or neutral) to be achieved. Rifamycin B (RM B, Fig. 4) is most commonly used chiral selector for the determination and enantioseparation of AM-type compounds. RM B comprises carboxylic and hydroxyl groups that are ionizable and can exist as a dibasic acid. Thus, it seems to be well suited for separating positively-charged analytes. This antibiotic is amphiphilic and a strong UV-absorber. Rifamycin B was examined as a chiral selector by Ward et al. [20] for the enantiomeric resolution of AM sulfate. The optimized electrophoretic conditions were found to be: 25 mM rifamycin B in 0.1 M 2-propanol/phosphate buffer at pH 7.0 (30:70 v/v). Rifamycin B was also used for the enantioseparation of ephedrine by Armstrong et al. [21]. 25 mM rifamycin B was added to the 0.1 M 2-propanol/ phosphate buffer at pH 7.0 (40:60 v/v). By adjusting the solution pH, ionic strength, concentration of the rifamycin B, organic modifier type and concentration, buffer type and capacity, the migration times, enantioselectivity and electrophoretic mobilities were controlled and optimized in both studies. They found that enantioresolution of analytes depended on pH and increased with pH up to the value of 7; thereafter resolution decreased with increasing pH, due to the charge on the analyte and the chiral selector. No enantioselectivity was observed in the organic modifier used in the run buffer. Also, the concentration of rifamycin B was examined and, in higher concentrates of this chiral selector, enantioresolution increased. However, this caused slightly increased migration times due to the small decrease in electrophoretic mobility. The applicability of RM B in the Rs of examined analytes is satisfactory. The measurement showed 0.1% of one enantiomer in the presence of the other and can be considered an advantage of this chiral selector, so it can be applied in assaying enantiomeric purity. While the variety of commercially-available chiral selectors has continually increased, few chiral selectors have had as immediate and significant an impact as macrocyclic antibiotics. The majority of enantiomeric separations in CE can be performed successfully with any of the macrocyclic antibiotics. The advantages include

separation efficiency, tremendous enantioselectivity, and short analysis times. In time, these should become as established as the CDs and find use in routine analytical practice. Polysaccharides are the next class of chiral mobilephase modifiers used for enantioseparation. Two types of polysaccharides are used: neutral and ionic. Polysaccharides comprise D-glucose units that are linked by a-(1-4), a-(1-6), b-(1-3) and b-(1-4). The conformation of a-(1-4) polysaccharides in the presence of buffer salt and analytes is changed and has an impact on enantioselectivity. In a-(1-6)-bonded polysaccharides, the helical structure cannot be formed, and pore size will affect enantiorecognition. In other ionic polysaccharides, electrostatic interactions play an important role [22]. Electrically-neutral polysaccharides are used for the enantioseparation of acidic or basic compounds, while charged polysaccharides are using where electrically neutral enantiomeric compounds can be separated as well as charged variants [23]. Gotti et al. [24] examined linear, neutral polysaccharides [e.g., Dextrin 20 (D20), Dextran 70 (D70) and Pullulan] as chiral mobile-phase modifiers for resolution of centrally-acting basic drugs of pharmaceutical and toxicological interest, including AM, MDA, MDMA, and MDAE. Table 4 shows the characteristics of these sugars. Due to the basic nature of the analytes, an acidic running buffer was used (pH 3.0 TEA phosphate). Dextran 70 was better than Dextran 20 as chiral selector for several reasons. First, separations were generally obtained in a shorter analysis time. Second, enantiomeric separations were achieved for all the drugs (except MDEA). This advantage indicated good general suitability of these chiral buffer additives for basic racemates.

Table 4. Characteristics of Dextrin 20, Dextrin 70 and Pullulan Polysaccharide

Characteristics

Dextrin 20 High dextrose equivalent maltodextrin Low molecular mass a-(1,4)-linked D-glucose Dextrin 70

OH

OH

H 3CCOO

OH

O OH NH

MeO

Pullulan

O O

O

O

COOH

Figure 4. Structure of Rifamycin B.

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Low equivalent dextrose polysaccharide Higher viscosity of D70 solution compared to those of D20 a-(1,6)-linked D-glucose

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High molecular mass D-glucose units linked by a-(1-4) and a(1-6) bonds High viscosity

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Background containing proportions of D20 lower than 15% did not give enantiomeric resolution of analytes. In BGEs containing low amounts of Pullulan, enantioselectivity was poor and migration slow. This resulted in high viscosity of the resulting BGE. In summary, D70 was found to be good chiral mobilephase modifier and to be more widely applicable to enantiomeric resolution of AM-type compounds. The reported data suggested that there is a difference in mechanism of enantiorecognition between Dextrin 70 and Dextrin 20. D20, based on a-(1-4) linked D-glucose, can define a helical arrangement to simulate, while D70, comprising D-glucose units linked by a-(1-6) bonds, resembles a network able to provide a complexion-sizing mechanism. This knowledge can be used in the future analysis and investigations. Linear polysaccharides are attractive resolving agents because of the low UV absorption of these sugar derivatives and also because of the high efficiency of the separations achieved. The use of proteins as chiral selectors for enantioseparation of AM-type compounds was also reported. These natural polymeric compounds are usually added to the BGE. Proteins comprise several opticallyactive amino acids, whose characteristic feature is the isoprotic and the isoelectric point, pI. The proteins can be positively charged at pH values under their isoelectric point and negatively charged at pH values above their isoelectric point. Thus, stereoselective interaction between protein as chiral selector and analyte strongly depends on the pH of the BGEs, so pH is a very important operating parameter for optimizing chiral selectivity [9]. Among the many proteins (e.g., a1-acid glycoprotein, avidin, pepsin or human albumin) used as chiral selectors in CE, bovine serum albumin (BSA) is most widely applied to the enantioresolution of AMs. For example, BSA was used as chiral selector for enantioseparation of ephedrine-type compounds by Ye et al. [25]. The method developed can be applied to the analysis of ephedra-plant extracts that contain the four test drugs (EP, PEP, NEP, and NPEP). The use of proteins as chiral mobile-phase modifiers for the enantioresolution of AM-type drugs presents some advantages, including the possibility of changing the charge, which depends on the pH of the BGE; protein charges provide electrophoretic mobility for separation. The optimization of chiral selectivity is easily achieved due to changes in pH of the BGE, which is an important operating parameter. Using proteins as chiral selectors, it is also possible to separate both uncharged and charged species. However, the adsorption of proteins onto the CE column would cause zone broadening, low reproducibility and low-efficiency effects [11]. Leucine (Leu), another common chiral selector used for separation and determination of ephedrine enantiomers, was investigated by Gu and co-workers [26].

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These studies examined several parameters (e.g., l-leucine and buffer concentration, buffer pH and organic modifier), which affected the chiral separation. The optimized electrophoretic conditions were found to be: pH 9, 20 mM phosphate buffer, 10 mM leucine and using 20% 2-propanol as organic modifier. This method was recommended for further investigation of ephedrine enantiomers in Ephedra and related preparations. Micellar electrokinetic chromatography (MEKC) with chiral surfactants as selectors is also used for the Rs of AM drugs. Micelles have been used as chiral selectors in CE for the determination of AM-type compounds by Rizvi et al. [27]. Here, three amino acids (L-leucinol, L-isoleucinol and L-valinol), as sulfated chiral surfactants, were synthesized, polymerized, and then used as chiral selectors in MEKC for resolution of 10 phenylethylamines (PEAs), including ephedrine, pseudoephedrine and norephedrine. In general, the enantiomers of this type of PEA were best resolved using polysodium N-undecenoyl-Lleucine sulfate (p-L-SUCLS) or p-L-SUCLS under acidic pH, whereas under moderately acidic to neutral pH conditions, polysodium N-undecenoyl-L-isoleucine sulfate (p-L-SUCILS) and polysodium N-undecenoyl-L-valine sulfate (p-L-SUCVS) seemed to provide the maximum Rs. 2.1.2. Indirect separation method The indirect separation method in CE is not as common as the direct separation method. In the indirect separation method, two enantiomers react with a chiral selector, before the electrophoretic run. A couple of diastereomers with different physico-chemical properties are formed and can be easily separated by CE. The indirect separation method in CE has some requirements, including: (1) high purity of chiral reagents; (2) reacting groups (amino and carboxylic); and, (3) the two enantiomers should react at the same rate. These requirements and other disadvantages (e.g., the derivatization process is time consuming) mean that few applications are available for CE [9]. Many chiral derivatizing reagents used in the indirect method are being tested for the forensic applications, especially for biological sample and drug analysis. Some applications are shown in Table 3. 2.1.2.1. Application of indirect CE to the separation of AMtype compounds. Examples of the use of the indirect separation method using CE in drug analysis were given by Cladrowa-Runge et al. [28], who separated nine AMtype compounds. The enantiomers were derivatized to diastereomers by MarfeyÕs reagent followed by separation using MEKC with non-chiral SDS micelles in alkaline solutions. Separations of enantiomers were successful particularly for ring-substituted AMs (MDEA and MDA). These compounds could be separated with good selectivity coefficients. Baseline enantioresolution http://www.elsevier.com/locate/trac

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could also be obtained for AM and HAM. However, enantioseparations obtained for two dimethoxy AMs (DAM and DMMA) and 4-AM were insufficient and BDMA was not separated. Thus, the indirect method with MarfeyÕs reagent is not a suitable method of enantioseparation for these compounds. Another disadvantage of this method is the time-consuming derivatization step. The direct method using b-CDs was also examined and compared with results from the indirect separation method. CM-b-CD and native-b-CD were found to be good chiral selectors, which give optimal resolutions within only a few minutes. The time required for the direct method with b-CDs was about the same as that needed for micellar separation. Fluorescein derivatives are also used for labeling of AM-type compounds [e.g., fluorescein isothiocyanate (FITC) or 1-(9-fluorenyl)ethyl chloroformate (FLEC) were recently used for resolution]. A fully validated method using FITC was developed for the determination of illicit AM and ephedrine derivatives in human blood and urine after solid-phase extraction as sample clean-up [29]. The six AM drugs were separated in a 20 mM borate buffer (pH 12) under an applied voltage of 25 kV and a capillary temperature of 25C. A laser-induced fluorescence (LIF) detector was used at kex = 488 nm and kem = 520 nm. These new methods showed excellent sensitivity when compared with more commonly used analytical methods (e.g., GC/MS) [30]. The same chiral selector was used by Ramseier et al. [31]. In this study, FITC-derivatized AM, MAM and MDMA in human urine were analyzed using chip-based and fused-silica capillary instrumentation with LIF detection. Enantioseparations were successful. Another chiral selector, 2,3,4,6-tetra-O-acetyl-b-Dglucopyranosyl isothiocyanate, was used for the enantioseparation of norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, AM and MAM by Lurie [32] using MEKC. Optimum resolutions were achieved using buffer comprising 20% methanol and 80% SDS solution [100 mM SDS, 10 mM phosphate buffer and 10 mM borate (pH 9)]. The procedure was far superior with respect to both resolution and speed of analysis, when compared to HPLC. Generally, this methodology is applicable in forensics. 2.1.3. Detection method for CE. In enantiomeric determination of AM substances, UV absorption is the detection method most widely used for CE. Both UV and diode-array detectors are used. The great advantage is that detection of analytes is possible without decreasing the resolution because they are detected in the capillary. Due to the small amounts of analytes and matrix interference, the selectivity and the sensitivity of this type of detection is insufficient in some cases (mainly biological samples) [10].

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In recent years, CE/UV was replaced by CE/MS. Compared to UV detection, MS has some major benefits. The mass spectrometer enables unambiguous determination of analytes with different mass-to-charge ratios (m/z). MS also enhances sensitivity. Some constraints include the nature of the background, which should be totally volatile to ensure good stability and long-term performance of CE/MS [33]. 2.1.4. Enantioseparation of AM-type compounds by CE and LC/HPLC. CE can be considered complementary to other analytical techniques, as mentioned in Section 2.1. above. In comparison with LC, CE presents many advantages. First, the amounts of sample and separation buffer are much less than used in LC. CE is cheaper because the chiral selectors are usually dissolved in the BGE, while, in LC, expensive chiral columns are required. The biggest advantages of CE compared to LC/HPLC are higher efficiencies and shorter analysis times. Of course, CE has disadvantages when compared to LC, as described in Table 5 [11]. 2.1.5. Microchip techniques for enantioseparation of AMs. Miniaturization plays an important role in the development of analytical chemistry. The first analytical device on a chip was presented by Terry in 1975 [34]. Since then, the idea of the lab-on-a-microchip has become a reality [35]. The microscale chip is attracting increasing interest in chiral separation by several analytical techniques, including CE. Microchip CE was used for the enantioresolution of ephedrine and pseudoephedrine by Schwarz et al. [36]. The direct electrochemical oxidation of these compounds was achieved at a gold electrode at high pH values. Baseline resolution was obtained for pseudoephedrine enantiomers only. Unfortunately, ephedrine separation was not achieved in the conditions studied. The concentration of the chiral selector has an effect on the sensitivity that could be useful in future research. The adaption of CE to microchips has many advantages, including high speed and high resolution [37]. Coupling CE and microchips will become a potentially useful tool for high-throughput chiral analysis in many

Table 5. CE and HPLC- sensitivity and preparative applications Parameter

CE

HPLC

Reproducibility Sensitivity Preparative applications

Low Poor Few

High Good Many

CE, Capillary electrophoresis; HPLC, High-performance liquid chromatography

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areas, including forensic science and the pharmaceutical industry [11]. 2.2. NMR NMR has also proved highly useful for enantiomeric separation of chiral compounds, with observation of 1H, 13 C, or other nuclei. NMR techniques usually complement chromatographic approaches [38]. NMR methods employ both direct and indirect methods. In direct methods, chiral lanthanide shift reagents (LSR) or chiral solving agents (CSA) are commonly used. In indirect methods, a chiral reagent transforms substrate enantiomers into stable diastereomeric derivatives. NMR focuses on observing separate absorption (different chemical shifts) for corresponding nuclei in chiral analytes. This chemical-shift difference may be thought of as an enantiomeric/diastereomeric shift difference [38]. When a chiral lanthanide shift reagent or chiral solvating reagent is used to measure enantiomeric excess of an analyte, the experiment is typically performed by observing analyte nuclei in the presence of the enantiopure-homochiral LSR or CSA. NMR absorption would correspond to nuclei in, respectively, R substrate or S substrate, averaged between the free and the bound complex states. The time-averaged signals seen for R and S analytes result from their different chemical shifts in the diastereomeric bound complexes [38]. Although NMR methods are appropriate for enantiomeric characterization of chiral compounds, there are few literature references to enantiomeric determination of AM-type compounds by this technique. 2.2.1. Application of direct and indirect NMR to separation of AM-type compounds. NMR determination of enantiomers is usually conducted in a chiral environment by adding the chiral solvating agent to NMR solutions of the substances. Different types of chiral solvating reagents for enantioseparation of AMs compounds are reported, but CDs are most commonly used because of their advantages, including the water solubility of the samples and the narrow chemical-shift range of CD. Chiral discrimination by NMR spectroscopy of ephedrine and N-methylephedrine (N-MEP) induced by b-CD, heptakis-(2,3-di-O-acetyl)-b-CD, and heptakis-(6-O-acetyl)b-CD was examined by Holzgrabe et al. [39]. These studies used NMR spectroscopy to compare the interaction between analytes and b-CDs. NMR spectra of the single enantiomers of EP and N-MEP in the presence of all three CDs gave information about the parts of the ligands that interact differently with the host molecules and may be responsible for chiral discrimination. Binding constants were calculated from the changes in the chemical shifts of the signals upon complexation. This

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was necessary in order to quantify the complex stabilities. The coupling constants of both species were analyzed, and showed that no significant conformational change occurred upon complexation. Detailed information about the geometry of the complexes was obtained by analysis of Rotating-frame Overhause Effect SpectroscopY (ROESY) spectra of appropriate isomers coupling with the CDs examined. Different intermolecular crosspeaks between the individual isomers of EP and N-MEP were found for native b-CD and its 2,3-diacetylated derivative but not for heptakis-(6-O-acetyl)-b-CD. Analyses of the intramolecular cross-signals of the ligands confirmed that no significant conformational change occurs upon complexation. In other studies [40], the same results and conclusion were obtained with changes in chemical shifts of ligands and hosts upon complexation providing information about the geometry of the complexes and analytes indirectly. Study of the chiral recognition of the enantiomers of EP derivatives with b -CD, heptakis-(2,3-O-diacetyl)-bCD (DIAC) and heptakis-(2,3-O-diacetyl-6-sulfato)-b-CD (HDAS) was also carried out by Hellriegel et al. [41]. CE, UV, MS and NMR techniques were used. The CE findings suggested that the mode of complexation must be different for the various CDs. A mode of chiral recognition was proposed after use of NMR spectroscopy, and, in particular, the complexation-induced shifts (CICSs) of both the ligand and the CD, in addition to plots and rotating frame Overhauser spectroscopy (ROESY), provided the mode of chiral recognition. After the experiment, it was concluded that, in the neutral CDs, 1:1 complexes were formed with EP and N-MEP. These complexes were characterized by the inclusion of the phenyl ring in the cavity and the side chain pointing out of the wider rim. In HDAS, manifold complexes were formed, and, as a result, it was impossible to derive a defined picture of the complexation of both enantiomers. However, it could be concluded that these complexes were characterized by upside-down inclusion of the phenyl ring in the cavity and the side chain pointing out of the narrow rim. In conclusion, comparing the CICS pattern of the various CD derivatives clearly indicates the differences in the complex geometry. In summary, NMRtechnique results obtained by CE gave complete information about the stereochemistry of the compounds analyzed. Moreover, these studies showed that b–CDs examined could be used as chiral solvating agents to resolve enantiomers by NMR. However, to deduce the geometry of ephedrine derivatives with b–CDs requires further study. b-CD, DIAC and HDAS were also tested for their ability to discriminate the enantiomers of EP, PEP, NEP and N-MEP by Wedig et al. [42]. CE and NMR techniques were used. All racemates in presence of b-CD were resolved by CE under optimized conditions (except NEP). Utilizing JobÕs plot by means of UV spectroscopy revealed http://www.elsevier.com/locate/trac

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1:1 complexes formed with b-CD and DIAC. With HDAS, mixed stoichiometry was determined. Inspection of the CICS of both the ligands and the CDs showed that the EP sits deeply in the cavity of b-CD and HDAS. However, with DIAC, the ephedrine is located close to the wider rim of the CD cavity. Thus, comparing the pattern of complexationinduced shifts of the various CD derivatives clearly indicates the differences in the complex geometry. Water-soluble calix[4]resorcinarenes with 3-hydroxyproline and 4-hydroxyproline substituent groups [cis-4hydroxy-d-proline (C4HD), cis-4-hydroxy-l-proline (C4HL), trans-4-hydroxy-l-proline (T4HL), and trans-3hydroxy-l-proline (T3HL)] and tetrasulfonated calix[4]resorcinarene, which contains enantiomerically pure l-prolinylmethyl groups (LPM), were studied as chiral NMR solvating agents with a series of monosubstituted phenyl-containing compounds, including ephedrine hydrochloride, by OÕFarrell et al. [43]. The substrates interact with the calixresorcinarene by inserting the aromatic ring into the cavity. To discriminate enantiomeric composition, the 1H NMR spectrum was used. The N-methyl and C-methyl resonances of ephedrine are shown in Figs. 5 and 6, respectively. The hydroxyproline derivatives (especially T4HL) were more effective at enantiodifferentiation than the corresponding proline derivatives. The hydroxyl group on the proline moieties was probably involved in interactions with the substituent groups of the analyte that are important in creating chiral recognition. The enantiomeric discrimination in the 1H NMR spectrum is large enough to analyze enantiomeric purity. Moreover, water-soluble chiral agents are environmentally friendly, which is desirable from a green

chemistry point of view. The same conclusions were draw by Hagan et al. [44]. Another chiral solving agent used for chiral recognition of AM-type compounds is (R)-(+)- 1, 1Õ-bi-2-naphthol. Examples of the use of NMR with this reagent in analysis of aqueous solutions for separation of some of AM-type compounds were shown by LeBelle et al. [45]. Potassium carbonate was added to aqueous solutions of known composition and extracted twice with CDCl3, evaporated to dryness and redissolved in CDCl3. This solution was used for acquisition of the proton spectrum. (R)-(+)-1, 1Ô-bi-2-naphthol as chiral solvating agent was then added directly to the tube and the spectrum recorded again. Results were compared with chromatograms obtained by GC-FID. Excellent agreement was obtained with the theoretical values using both methods. NMR was shown to be capable of measuring the enantiomeric ratios of mixtures of MAM, EP and PEP. Indirect NMR is not as common as direct NMR and data in the literature about its application is not abundant. An example of the application of indirect NMR analysis following derivatization with chiral derivatizing reagent for the determination of enantiomeric composition of MAM was carried out by Kram and Lurie [46]. The analysis of the AM diastereomers formed by derivatization with 2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl isothiocyanate (GITC) gave variable information for enantiomeric characterization of AMs. The stereospecific chemical shifts of a host of signals from the GITC moiety provided sites for qualitative and quantitative consideration. In summary, few NMR techniques, including ROESY, NOESY and NOE difference, have been used to generate

Figure 5. 1H NMR spectrum of the N-methyl resonance of (a) EP enantiomerically enriched (2/3( )-(1R,2S), 1/3(+)-(1S,2R)) with 40 mM (b) LPM, (c) C4HD, (d) C4HL, (e) T4HL, and (f) T3HL [43].

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Figure 6. 1H NMR spectrum of the C-methyl resonance of (a) EP enantiomerically enriched (2/3( )-(1R,2S), 1/3(+)-(1S,2R)) with 40 mM (b) LPM, (c) C4HD, (d) C4HL, (e) T4HL, and (f) T3HL [43].

binding-interaction maps for AM-type compounds (e.g., ephedrine interacting with a single chiral selector). Several chiral reagents have been developed and used with NMR spectroscopy for determination of enantiomeric purity and assignment of absolute stereochemistry. Usually, these agents are useful in organic solvents. The most widely-studied, broadly applicable water-soluble chiral NMR-shift reagents include native CDs and certain water-soluble CD derivatives. One of the most important advantages of NMR spectroscopy over the other spectrometric techniques is that NMR spectroscopy may, in principle, separate resonance signals for non-covalent diastereomeric complexes between the enantiomers of the selectand and the selector. Because of this, NMR spectroscopy allows application of non-racemic or racemic mixtures of enantiomers for the equilibrium-binding constants of selector-selectand complexes and the stereoselective determination of stoichiometry. Another advantage of NMR spectroscopy is that it offers the opportunity for competitive binding studies, meaning that the interaction between one of the analyte enantiomers with a chiral selector may be carried out in the presence of the other enantiomer. Moreover, NMR spectroscopy also provides a multiple set of data based on one experiment [47].

3. Summary Nowadays, AM-type stimulants are among the most abused drugs. Identification of the enantiomers of these drugs is important, especially for recognition of the source

or usage. Recently, chiral separation of AM and related compounds has been intensively researched with many published papers in this area. Several analytical techniques (e.g., GC, LC, HPLC, CE and NMR) combined with reliable detectors provide good enantiomeric resolution. The analysis of isomers of AM-type compounds can be easily achieved by CE. Indirect and direct methods are used for successful separation. Several chiral selectors have been employed in CE in order to modify the electrophoretic mobilities of the enantiomers selectively. The direct method most commonly uses CDs (native and modified), antibiotics, linear polysaccharides, proteins and chiral micelles. Also, many chiral reagents used in the indirect method have been tested for the forensic applications, especially in biological samples and drugs analysis. Using CE, direct separation is usually preferred. Capillary enantioseparation offers many advantages even over chromatographic techniques (from the molecular recognition point of view) including:  very fast screening of analyte-chiral selector interactions (especially analyte-CD);  high peak efficiency that allows observation of enantioselective effects in selector-selectand interactions that could not be visible by other techniques;  high separation factor due to small thermodynamic selectivity of recognition; and,  very flexible from the point of view of adjusting the enantioseparation factor [47]. Moreover, CE is a fast, economical, powerful, effective method. High-resolution separation is also reported. Each of above advantages is very important in analyzing drugs of abuse, including AM-type compounds.

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In recent years, microfabrication technology using microchip CE offers the possibility of enantioseparation in drug analysis. NMR is another technique used for enantioresolution of AMs. NMR determination of enantiomers is usually conducted in a chiral environment by adding the chiral derivatizing reagent or chiral solvating agent to the NMR solution of the substances [e.g., GITC, (+)-R-+-1,1Õbi-2-naphthol or CDs]. In NMR, direct separation is usually preferred over indirect. NMR is also fast, powerful and effective. One of the most important advantages of NMR is that it provides a multiple set of data based on a single experiment, after which it is possible to obtain all information about the stereochemistry of enantiomers. Although NMR techniques also provide these advantages, data in the literature about their application to enantioseparation of AM-type compounds are still limited. NMR techniques are not therefore as commonly used as CE or other chromatographic techniques, and are usually complementary to chromatographic approaches.

Appendix A. Abbreviations AM B BDMA CD CE CE-b-CD CICS CMPA CM-b-CD CNS C4HD C4HL DAD DAS-b-CD DAM DIAC DMA DMMA d-MTPA DM-b-CD DMS-b-CD DOM EP FITC FLD FS

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Amphetamine Blood 4-bromo-2,5-dimethoxyamphetamine Cyclodextrin Capillary electrophoresis Carboxyethyl-b-cyclodextrin Complexation induced shift Chiral Mobile-Phase-Addictive Carboxymethyl-b-cyclodextrin Central Nervous System cis-4-hydroxy-d-proline cis-4-hydroxy-l-proline Diode array detector Heptakis-(2,6-diacetyl-6-sulfato)b-cyclodextrin 2,5-dimethoxyamphetamine heptakis-(2,3-O-diacetyl)b-cyclodextrin Dimethylamphetamine 2,5-dimethoxymethamphetamine d-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride Heptakis-(2,6-di-O-methyl) -b-cyclodextrin Heptakis-(2,3-dimethyl-6-sulfato) -b-cyclodextrin 2,5-dimethoxy-4-methylamphetamine Ephedrine Fluorescein isothiocyanate Fluorescence detector Forensic sample

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GC GITC

Gas chromatography 2,3,4,6-tetra-O-acetylb-D-glucopyranosyl isothiocyanate H Hair HAM 4-hydroxyamphetamine HDAS heptakis-(2,3-O-diacetyl-6-sulfato)b-cyclodextrin HPLC High performance liquid chromatography HP-b-CD Hydroxypropyl-b-cyclodextrin IR Infrared L Liver Leu Leucine l-FLEC l-(9-fluorenyl)ethyl chloroformate LIF Laser-induced fluorescence LLE Liquid–liquid extraction LPM L-prolinylmethyl groups LSR Lanthanide shift reagents MAM Methamphetamine MBDB Methylenedioxyphenyl-N-methyl2-butanamine MDA 3,4-methylenedioxyamphetamine MDEA 3,4-methylenedioxy-Nethylamphetamine MDMA 3,4-methylenedioxymethamphetamine MEKC Micellar electrokinetic chromatography MS Mass spectrometry M-b-CD Methyl-b-cyclodextrin NAC N-acetyl-cysteine NEP Norephedrine N-MEP N-methylephedrine N-MPEP N-methylpseudoephedrine NMR Nuclear magnetic resonance NP. Normal phase OPA O-phthaldehyde P Plasma PEA Phenylethylamine PEP Pseudoephedrine Ph Pharmaceuticals p-HMAM p-hydroxymethamphetamine PITC Phenyl isothiocyanate p-L-SUCILS polysodium N-undecenoylL-isoleucine sulfate p-L-SUCLS N-undecenoyl-L-leucine sulfate p-L-SUCVS N-undecenoyl-L-valine sulfate RM Rifamycin B ROESY Overhauser spectroscopy RP Reverse phase S Standard solution S-b-CD Heptakis-6-sulfato-b-cyclodextrin SPE Solid-phase extraction SPME Solid-phase microextraction SU(XIII)- c -CD Sulfated c-cyclodextrin T Tablets TAA Tetraalkylammonium

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TBA TLC T3HL T4HL U UVD

Tetrabutylammonium Thin layer chromatography trans-3-hydroxy-l-proline trans-4-hydroxy-l-proline Urine Variable wavelength detector

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