Microsolv.net2

Electrophoresis 2004, 25, 1580–1591 Ira S. Lurie
Capillary electrophoresis analysis of a wide variety
Patrick A. Hays
Kimberly Parker
of seized drugs using the same capillary with
dynamic coatings
Special Testing andResearch Laboratory,U.S. Drug Enforcement Capillary electrophoresis methodology is presented for the routine analysis of a wide variety of seized drugs using the same capillary with dynamic coatings and multiple run buffers. The types of exhibits analyzed using diode array UV detection include phe-nethylamines, cocaine, oxycodone, heroin, lysergic acid diethylamide (LSD), opium,hallucinogenic mushrooms, and g-hydroxybutyrate-g-butyrolactone (GHB-GBL). Bothqualitative and quantitative analyses are achieved using run buffers that contain addi-tives that provide for secondary equilibrium and/or dynamic coating of the capillary.
Dynamic coating of the capillary surface is accomplished by rapid flushes of 0.1 Nsodium hydroxide, water, buffer containing polycation coating reagent, and a buffercontaining a polyanionic coating reagent (with or without cyclodextrin(s)) or a micellecoating reagent. Dynamic coating with a polyanionic coating reagent is used for theanalysis of moderately basic seized drugs and adulterants. The use of cyclodextrin inthe run buffer not only allows for chiral analysis but also greatly enhances separationselectivity for achiral solutes. A capillary dynamically coated with a micelle allows forthe analysis of neutral, acidic, and weakly basic drugs (GHB, GBL and neutral, acidic,and weakly basic adulterants). Dynamic coating, which gives rise to a relatively highand robust electroosmotic flow at pH , 7, allows for rapid, precise and reproducibleseparations. For a wide variety of drugs, excellent linearity and migration time precisionand good peak area precision (external and internal standard) is obtained. Quantitativeresults for synthetic mixtures are in good agreement with actual values. Screening foradulterants is greatly enhanced by the use of automated library searches.
Keywords: Capillary electrophoresis / Dynamically coated capillaries / Seized drugs
1 Introduction
MS [9, 10], nuclear magnetic resonance (NMR) [11, 12],and capillary electrophoresis (CE) [13–18] have been The analysis of seized drugs is important for legal and used for this purpose. Although GC offers the highest re- intelligence purposes. Rapid, precise, and reproducible solving power for achiral solutes, limitations exist for the methodology is required for the quantitative as well as analysis of highly polar (e.g., amphetamines, morphine), qualitative determination of drugs of forensic interest and thermally labile (e.g., lysergic acid diethylamide (LSD), related materials. Gas chromatography (GC) [1, 2], gas psilocybin, g-hydroxybutyrate (GHB)) and nonvolatile chromatography-mass spectrometry (GC-MS) [3, 4], high- solutes (e.g., sugars and polyhydric alcohols) [8]. For performance liquid chromatography (HPLC ) [5–8], HPLC- these solutes, derivatizations and/or prior extractions arerequired. HPLC, which allows for the direct analysis of theabove compounds, inherently lacks resolution. Although Correspondence: Ira Lurie, Special Testing and Research Labo-
NMR identifies compounds in simple mixtures, and can ratory, U.S. Drug Enforcement Administration, Dulles, VA 20166,USA perform quantitation without a primary reference drug E-mail: [email protected]
standard, complex samples can be difficult to identify and Fax: 1703-668-3301
quantitate. CE, which also allows for the direct analysis of Abbreviations: DM-â-CD, dimethyl-b-cyclodextrin; GBL, g-bu-
the above solutes, has significantly greater resolv- tyrolactone; GHB, g-hydroxybutyrate; HP-â-CD, hydroxypropyl-
ing power than HPLC. In addition, for the chiral GC or b-CD; LAMPA, lysergic acid methylpropylamide; LSD, lysergic
HPLC analysis of chiral solutes, expensive chiral columns acid diethylamide; MDA, methylenedioxyamphetamine; MDEA,
(usually specific for a class of compounds) and/or derivati- methylenedioxyethylamphetamine; MDMA, methylenedioxy-
methamphetamine
zation are required [19]. Even with the above, HPLC can  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 suffer from poor resolution and/or long analysis time is highly selective for the analysis of moderately basic [19, 20]. CE, which uses chiral additives such as cyclodex- solutes since most weakly basic, acidic, and neutral com- trins (CDs) in the run buffer, allows for the direct chiral pounds exhibit smaller positive mobilities, negative mobili- determination of seized drugs using conventional capil- ties or no mobilities, respectively. For separations (achiral or chiral) requiring additional selectivity, CDs are added tothe pH 2.5 dynamically coated capillary system [48, 49, 51].
Numerous methods using uncoated capillaries have been The use of dynamically coated capillaries in the normal reported for the routine CE analysis (achiral and/or chiral) polarity MEKC mode allows for the analysis of neutral, of seized drugs including phenethylamines [21–32], co- acidic, and weakly basic solutes over a wide pH range, caine [33–35], propoxyphene [36], heroin [25, 27, 37–39], even at low pH. For dynamically coated MEKC, several LSD [27], opium [40–44], psilocybin [45, 46], and GHB-g- approaches have been reported including using dextran butyrolactone (GBL) [47]. These methods involve capillary sulfate [52], polyvinylsulfonate [53] or SDS [54] as the an- zone electrophoresis (CZE) [24, 26–32, 34, 36, 39, 41, 44– ionic coating with sodium dodecyl sulfate (SDS) in the run 46] with or without secondary equilibrium, micellar elec- buffer. At pH  6.5, excellent selectivity has been demon- trokinetic chromatography (MEKC) [22, 23, 33, 35, 37, strated for the determination of neutral, acidic, and weakly 38, 40, 42, 47], and electrokinetic chromatography (ECC) basic adulterants present in heroin in the presence of mod- [21, 43]. For methods using CZE, MEKC or ECC, separate erately basic compounds [49]. At this pH range, most basic capillaries would be recommended. “Memory effects” solutes are significantly ionized and will ion-pair with SDS lead to nonreproducible separations unless tedious re- (either on stationary phase or run buffer) and therefore will conditioning steps are carried out. Therefore, for a foren- migrate after the acidic and neutral solutes.
sic lab wishing to perform CE analysis on a wide varietyof seized drugs, either multiple instruments, or a single In this report, the routine analysis of a wide variety of seized instrument with multiple capillaries would be recom- drugs using a single dynamically coated capillary with run mended. In the latter case, the types of unattended anal- multiple buffers is demonstrated. A dual coating procedure ysis would be limited, the complexity of operation would is used consisting of initial coating with a proprietary poly- increase and the frequent swapping of capillaries greatly meric cation followed by coating with either a proprietary increases the chances of broken capillaries.
polymeric anion (with or without CD(s)) or SDS.
Dynamically coated capillaries provide significant im-provement in separation times, precision and selectivity.
2 Materials and methods
Dynamically coated capillaries, using an initial coatingwith a polymeric cation and subsequent coating with a pol- 2.1 Chemicals
ymeric anion or micelle, offer faster separation times,improved precision, and increased selectivity for the analy- Drug standards were obtained from the reference collec- sis of basic, acidic, and neutral drugs of forensic interest.
tion of the Drug Enforcement Administration Special Test- Phenethylamines and cocaine exhibits have been analyzed ing and Research Laboratory (Dulles, VA, USA). CElixir using this dual coating procedure with a polymeric anion Reagent A (pH 2.5), CElixir Reagent B (pH 2.5), 50 mM [12]. Opium preparations [48] and heroin samples [49] phosphate-borate (pH 6.5), 50 mM phosphate (pH 6.5), have been analyzed using this latter coating methodology and 0.1 N sodium hydroxide were acquired from Micro- with CD(s) added to the buffer containing the polymeric Solv Technology (Eatontown, NJ, USA). Hydroxypropyl- anion. The same methodology can be used for LSD exhib- b-cyclodextrin (HP-b-CD) dimethyl-b-cyclodextrin (DM- its [48]. A coating procedure developed by Chevigne and b-CD), and sodium dodecyl sulfate (SDS) were obtained Janssens [50] is the basis for above separations. This from Sigma (St. Louis, MO, USA). Sodium phosphate methodology, carried out during every run, consists of a (monobasic), phosphoric acid, and sodium hydroxide two-step process whereby the capillary (after base hydro- were reagent grade. HPLC-grade methanol was acquired lysis) is first coated with a polycation (an initiator), then from Burdick and Jackson (Muskegon, MI, USA). Deion- coated with a polyanion (an accelerator). The run buffer ized and high purity water (HPLC-grade water) were ob- contains the latter coating reagent. This process gives rise tained from a Millipore Synergy 185 water system (Bed- to a highly precise EOF over a wide pH range and to a capillary surface with more favorable kinetics. For basicsolutes, it is desirable to perform dynamic coating at a low 2.2 Instrumentation and procedures
pH such as 2.5. At this pH most basic solutes (moderatelybasic compounds) are fully ionized (pKa . 5) and therefore An Agilent Model HP3DCE Capillary Electrophoresis Sys- their mobilities will not change with small differences tem equipped with a diode array detector (Waldbronn, in run buffer pH. In addition, at pH 2.5 this CE system Germany) was used for CE experiments. Prior to first  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 use, new bare silica capillaries were conditioned following 2.3 Preparation of standards and samples
the same procedure used for regular analysis using CElixirReagent B (pH 2.5). The capillaries were first flushed with The preparation of the internal standard, standard and sample solutions is shown in Tables 1 and 2, respec- N sodium hydroxide for 1 min, then water for 1 min, followed by CElixir Reagent A for 1 min, and finally run tively. Unless indicated otherwise, samples are sonicated buffer for 2 min. For conditioning new dynamically coated for 15 min. For the above standard and sample prepara- capillaries for use with run buffers containing SDS, the Table 1. Preparation of internal standards
capillaries were first flushed with 0.1 N sodium hydroxidefor 1 min, then water for 1 min, followed by CElixir Re- agent A for 1 min, then either 50 mM phosphate-borate (pH 6.5) or 50 mM phosphate (pH 6.5) for 1 min, and finally the run buffer for 6 min. For subsequent injections only2 min flushes with run buffer were required. When switch- ing between a CE method using CElixir Regent B and a method using SDS, the new capillary coating procedure (for an SDS run buffer) was employed for the first injec- tion. For overnight or prolonged storage, the capillary was flushed with water for 10 min and then stored with the inlet and outlet dipped in water. The method usedeither 2.0 mL CE glass vials or 1.0 mL CE polypropylene a) 75 mM sodium phosphate monobasic, adjusted to pH vials as electrolyte reservoirs. When using glass vials, 2.6 with phosphoric acid and diluted 1:20 with HPLC- grade water; alternatively, injection solvent concen- trate (which can be purchased from MicroSolv) diluted vials, run buffer, standard and sample vials were filled with 1000 mL of liquid (for 0.1 N sodium hydroxide add b) 1:11 mixture of methanol and dilution solvent 1 500 mL to polypropylene vial). For polypropylene vials, c) 50 mM sodium phosphate monobasic, adjusted to pH waste vials were filled with 250 mL of water, while all 6.5 with sodium hydroxide and diluted 1:10 with others were filled to 500 mL of liquid.
Table 2. Preparation of standard and sample solutions
a) See Table 1b) 0.1 mg/mL standard in methanol (sample extracted mechanically shaken for 30 min); combine 1.0 mL methanol so- lution with 1.0 mL IS solution and 10.0 mL of dilution solvent 1 c) 0.025 mg/mL each of standard morphine HCl, codeine HCl, thebaine base, noscapine base, and papaverine HCl dissolved in dilution solvent 2. For opium sample, weigh 100 mg opium into 50 mL volumetric flask, add 25 mL methanol, sonicate30 min at 50–607C, dilute to volume with dilution solvent 1. For opium latex, after thoroughly mixing, weigh 250 mg into 100mL volumetric flask, add 50 mL methanol, vortex 1 min, dilute to volume with dilution solvent 1. For both sample types,pipette 200 mL of sample into a 2.0 mL vial and combine with 1.0 mL of tetracaine HCl (0.03 mg/mL with dilution solvent 1) d) 0.5 mg/mL and 0.6 mg/mL standard psilocin and psilocybin, respectively, in methanol (sonicated for 5 min). For Psilocybe mushroom exhibits, psilocybin concentration in methanol (after sonicating for 50 min) should equal approximately thestandard concentration. Combine 1.0 mL of standard and sample solution with 11.0 mL of dilution solvent 1  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 tions, filter approximately 500 mL or 1.0 mL of solution 3 Results and discussion
with 0.5 mm nylon filter (SRI) into either a 1.0 mL CE poly-propylene plastic vial, or a 2.0 mL CE glass vial, respec- For the analysis of seized drugs, dynamically coated capillaries have been used in both the CZE mode [12, 48,49] (moderately basic solutes) and MEKC mode [49](weakly basic, acidic, and neutral). In order for a single 2.4 Capillary electrophoretic conditions
capillary to be used for a wide variety of seized drugs,these coating procedures should be compatible. Five All experiments were carried out with either a 50 mm ID injections of moderately basic solutes (phenethylamines) 32 cm (24 cm to the detector) fused-silica capillary followed by five injections of weakly basic, acidic, and obtained from Polymicro Technologies (Phoenix, AZ, neutral compounds (acetaminophen, theophylline, caf- USA) or a 50 mm ID 33 cm (24.5 cm to the detector) feine, aspirin, salicylic acid, antipyrene, phenobarbital, pre-made capillary (Agilent) operated at 157C. 50 mbar phenacetin, and benzocaine) and subsequently five in- pressure injections of 2–10 s durations were used jections of moderately basic solutes were performed.
followed by a 35 mbar pressure injection of water for Both systems are highly compatible and precise (run- 1 s. For electrophoresis, an initial 0.5 min linear volt- to-run migration time RSDs  0.12%,  0.61%, and age ramp from 0 V to the final voltage was used for  0.04%, respectively, for the three experiments). For most analyses (heroin analysis 1.0 min ramp). All run CZE analysis, a dual coating procedure is required be- buffers (which can be purchased from MicroSolv) tween every injection for good CE performance. How- were filtered into a 22 mL Teflon PVA vial (Cole Parmer) ever, for MEKC separations a dual coating procedure using a 0.5 mm nylon filter (SRI) and refiltered weekly.
was only required for the first separation. For subse- CE conditions for the various analyses are given in quent injections using this technique, the capillary was Table 3. CE conditions for the analysis of a wide variety of seized drugs using dynamically coated
by co-injection100 mbar?s sample 130 mbar ?s standard a) % w/vb) pH adjusted using phosphoric acid  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 3.1 Quantitative analysis of phenethylamines,
droxide is used instead of 1 N sodium hydroxide to improve cocaine, and oxycodone and identification
longevity of the capillary. Figures of merit for amphetamine, of moderately basic adulterants
methamphetamine, methylenedioxyamphetamine (MDA),methylenedioxymethamphetamine (MDMA), methylene- For the quantitative analysis of phenethylamine and co- dioxyethylamphetamine (MDEA), cocaine, and oxycodone caine exhibits, updated coating methodology and sample are shown in Table 4. Excellent linearity, good run-to-run preparation procedures over reported methodology [12] relative area precision and good quantitative accuracy are are presented (see Section 2.2). The same conditioning obtained for these solutes. Relative migration time data for steps used between injections are now used for a new cap- phenethylamines and related compounds (internal stand- illary. It is not necessary, as previously reported [12], to use a ard, impurities and adulterants) and cocaine, oxycodone longer base wash for the first injection on a new capillary and related compounds are shown in Tables 5 and 6, than for subsequent injections. In addition, 0.1 N sodium hy- respectively. Detection at 235 nm provides increased se- Table 4. Figures of merita) for seized drugs using dynamically coated capillary systems
a) Linearity and accuracy data for amphetamine, methamphetamine, MDA, MDMA, MDEA, and b) For basic solutes analysis was performed on known mixtures of the seized drug and mannitol, inositol or lactose. For GHB and GBL analysis was performed on Gatorade spiked with knownamount of seized drug.
c) Area of solute/area of internal standardd) Corrected area (area/migration time) of solute/corrected area of internal standarde) Corrected area lectivity (de-creased sensitivity) for cocaine analysis.
to-run precision is obtained for the phenethylamines (RSD Screening for basic adulterants is facilitated by the use  0.12%) and propoxyphene (RSDs  0.04%). Enantio- mer identification is facilitated by peak enhancementaccomplished by subsequentially co-injecting a mixtureof standard and sample. Relative migration data for phe- 3.2 Identification of enantiomers of
nethylamine and propoxyphene enantiomers is pre- phenethylamines and propoxyphene
The identification of enantiomers of solutes such as phen-ethylamines (controlled and noncontrolled) and propoxy- 3.3 Quantitative analysis of LSD
phene is easily accomplished by adding the chiral selec-tor HP-b-CD to CElixir Reagent B (pH 2.5). As shown d-LSD and d-lysergic acid methylpropylamide (d-LAMPA) in Fig. 1, an excellent simultaneous separation of six comigrate, while d-LSD and d-iso-LSD are resolved using racemic phenethylamines is obtained in , 4 min. d,l-Pro- CElixir Reagent B (pH 2.5). The addition of a mixture of poxyphene is baseline-resolved in , 5 min. Excellent run- CDs to the CElixir Reagent B (pH 2.5) run buffer provided,  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 Table 5. Relative migration times for phenethylamines
Table 7. Relative migration times for phenethylamine and
and related compounds using a dynamically propoxyphene enantiomers using a dynamically Table 6. Relative migration times for cocaine, oxyco-
done, and related compounds using a dynami-cally coated capillary system Figure 1. Dynamically coated CE separations using a
33 cm (24.5 cm to the detector window)650 mm ID fused-silica capillary. Solute concentrations were approx- imately 0.05 mg/mL with CE conditions as described in Section 2. Electropherogram of a standard mixture of(a) l-amphetamine, (b) d-amphetamine, (c) l-methamphet-amine, (d) d-methamphetamine, (e) l- or d-n-butylamphet-amine, (f) l- or d-n-butylamphetamine, (g) l- or d-MDA, via secondary equilibria, the selectivity to fully resolve the (h) l- or d-MDA, (i) l- or d-MDMA, (j) l- or d-MDMA, (k) l- or above solutes (Fig. 2). It is unclear as to why LAMPA (not present in actual LSD samples) exhibits a broader peakthan LSD. Figures of merit for LSD are shown in Table 4.
Excellent linearity, good run-to-run area precision and LSD and tetracaine (IS) (RSD  0.76%) [48]. Relative excellent quantitative accuracy are obtained for these migration time data for LSD and related compounds is solutes. Good run-to-run precision is also obtained for  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 Excellent run-to-run migration time precision is obtainedfor these solutes (RSD  0.12%) [48]. The same CE con-ditions are applicable for special analyses, i.e., the quan-titation of opium preparations [48]. Identification of themajor opium alkaloids is facilitated by the use of auto-mated library searches.
3.5 Quantitative analysis of heroin and
identification of moderately basic
adulterants
Figure 2. Electropherogram of a standard mixture of
(a) d-LSD, (b) d-LAMPA, (c) d-iso-LSD, and (d) tetracaine
Methodology has been previously reported for the quanti- (internal standard). A 32 cm (23.5 cm to the detector win- tative analysis of heroin, basic impurities, and basic adul- dow)6 50 mm ID fused-silica capillary was used. Solute terants using dynamically coated capillaries [49]. It was concentrations were approximately 0.008 mg/mL with necessary to add dimethyl-b-cyclodextrin (DM)-b-CD to CE conditions as described in Section 2.
CElixir Reagent B (pH 2.5) not only to resolve solutes fromheroin, but also to improve the separation of the basic Table 8. Relative migration times for LSD and related
impurities. In order to fully resolve these compounds, a compounds using a dynamically coated capil- longer capillary (relative to the capillary length in this pres- ent study) was required (total length, 64 cm) with a higher temperature [49]. For this study since we are only con- cerned with the quantitation of heroin and the screeningof adulterants the shorter capillary is sufficient. Figures of merit for heroin are shown in Table 4. Excellent linearity, excellent run-to-run area precision and quantitative accu- racy are obtained for this solute. Excellent run-to-run migration time precision is also obtained for heroin (RSD 0.07%). Relative migration time data for heroin, moder- ately basic impurities and mostly moderately basic adul-terants (nicotinamide and aminopyrene are weak bases)are shown in Table 9. Screening for adulterants is facili- 3.4 Identification of major alkaloids in opium
tated by the use of automated library searches.
Five major alkaloids in opium, including morphine, papa- Table 9. Relative migration times for heroin and related
verine, codeine, noscapine, and thebaine, are well re- compounds using a dynamically coated capil- solved using the same run buffer used for LSD (Fig. 3).
Figure 3. Electropherogram of a standard mixture of
(a) morphine, (b) papaverine, (c) codeine, (d) noscapine, (e) thebaine, and (f) tetracaine (internal standard). A 32 cm (23.5 cm to the detector window)650 mm ID fused- silica capillary was used. Solute concentrations were ap- proximately 0.02 mg/mL with CE conditions as described  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 Table 9. Continued
bilities. In addition, psilocybin (Psilocybe mushroom spe-cies) migrates near to with significant tailing using CElixir Reagent B (pH 2.5) (Fig. 4A). This is due to the amphoteric nature of psilocybin at pH 2.5 (fully ionized tertiary aminegroup and mostly ionized phosphate group). Lowering the pH to 1.8 partially protonates the psilocybin phosphate group (i.e., imparts a greater D1 charge) and therefore increases the effective mobility of this solute. As a result, psilocybin migrates further from to with an improved peak shape (Fig. 4B). As expected, the relative mobilities of psilocybin and bufotenine, which are also fully protonated at pH 1.8, are similar to their relative mobilities at pH 2.4.
However, due to secondary equilibrium, psilocin and bufotenine are fully resolved after the addition of 50 mM HP-b-CD to the CElixir Reagent B (pH 1.8) buffer with good peak shape for psilocybin (Fig. 4C). Since psilocin and bufotenine have easily distinguishable diode array UVs (Figure 5) and are not found together, the pH 1.8 run buffer which gives shorter migration times for psilocybin is recommended. Excellent run-to-run precision is ob- tained for these solutes (RSD  0.39%).
3.7 Identification of neutral, acidic, and weakly
3.6 Identification of major alkaloids in
basic adulterants
hallucinogenic mushrooms
Methodology has been previously reported for the identi- Psilocin (“Psilocybe mushroom species”) and bufotenine fication of weakly basic, acidic, and neutral adulterants (Bufo toad species) comigrate (Fig. 4A). This is not sur- in heroin using dynamically coated capillaries [49]. This prising since the fully protonated positional isomers psilo- same methodology is applicable to the identification of cin and bufotenine (differing only in position of a phenol these adulterants in phenethylamine and cocaine sam- functional group) would be expected to have similar mo- ples. Relative migration time data for neutral, acidic and Figure 4. Comparison of CE
separations of standard mixture
of (a) psilocin (0.05 mg/mL), (b)
bufotenine (0.04 mg/mL), and
(c)
using various run buffers. A 32cm (23.5 cm to the detectorwindow)650 mm ID fused-silicacapillary was used at 157Cwith a voltage of 10 kV with100 mbar?s injections. (A) An-ionic coating reagent and runbuffer consisting of CElixir Re-agent B (pH 2.5). (B) Anioniccoating reagent and run bufferconsisting of CElixir Reagent B(pH 1.8). (C) Anionic coatingreagent and run buffer consist-ing of CElixir Reagent B (pH 1.8)1 50 mM HP-b-CD.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 Table 10. Relative migration times for weakly basic,
acidic, and neutral solutes using a dynamicallycoated capillary system Figure 5. Diode array UV spectra of (a) psilocin, and
(b) bufotenine obtained using experimental conditions weakly basic adulterants are shown in Table 10. Screen- ing for these adulterants is facilitated by the use of auto- 3.8 Quantitative analysis of GHB and GBL
The separation of GHB and GBL using dynamically coated capillaries is shown in Figure 6. A pH 6.5 run buffer (50 mM phosphate 1 3% SDS) is chosen to minimize the chemical interconversion of GHB and GBL [55]. As demonstrated in Figs. 6A and B, no interconversion is obtained for either solute under these conditions. Further- more, no interconversion occurs even for solutions sitting overnight in the autosampler. This run buffer is used because the latter reagent gives a peak, which can inter- fere with GBL. While GBL, a neutral solute, is retained bythe micelle, the negatively charged SDS aggregate shouldrepel the anionic GHB. The longer migration time of the Figure 6. Electropherograms of (A) standard mixture of
latter solute is probably due to the high mobility of this (a) GBL (7.0 mg/mL), and (b) resorcinol (0.1 mg/mL) (inter- relatively small solute in the anodic direction. The triangu- nal standard) and (B) standard mixture of (b) resorcinol(internal standard) and (c) GHB (3.5 mg/mL). A 32 cm lar GHB peak is caused by electromigration dispersion of (23.5 cm to the detector window)650 mm ID fused-silica the anionic solute present at a relatively high concentra- capillary was used with CE conditions as described in tion (weak extinction coefficient). According to Weinber- ger [56], quantitative results are still maintained as long  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 as peak areas are used and sufficient resolution is de- 4.561024 to 3.661024. However, for the SDS system, re- signed into the separation. This is the case as indicated producible separations were obtained (RSDs of effective by the figures of merit (see Table 4). Excellent linearity, excellent run-to-run area precision, and good quantitative Since different lots of the same CD can vary in both the accuracy are obtained for these solutes. Excellent run- degree of substitution* and the position of substituents, to-run precision is also obtained for these solutes (RSD each time a CD from a new batch is received, test mix-  0.12%). Due to a very weak extinction coefficient, tures are analyzed. For multiple lots of both CDs there 1,4-butanediol was not detected as high as 10.0 mg/mL were no significant changes for most separations tested.
concentration. Unlike GHB, this solute is easily analyzed For a most recent lot of HP-b-CD, d-methamphetamine by GC [55]. Although GBL is also easily analyzed by GC, comigrated with d-pseudoephedrine. Changing the CD this compound can be encountered in combination with concentration from 50 mM to 45 mM resolved these solutes and gave the expected separation for the othercompounds in the test mixtures.
3.9 Reproducibility
Certain test solutes were analyzed by multiple CE sys- 3.10 Applications of the methodology to seized
tems (consecutively, on the same day) over a two-week drugs exhibits
period. d,l-Methamphetamine, d,l-MDMA and d,l-n-bu- Examples of the above methodology for the analysis of tylamphetamine were separated using CElixir Reagent B seized drug samples are shown in Figs. 7–9. These exam- (pH 2.5) and CElixir Reagent B (pH 2.5 ) with 50 mM HP-b- ples include the analysis of an illicit methamphetamine CD. Theophylline and caffeine were resolved by using50 mM phosphate-borate (pH 6.5) with 3% SDS. Althoughno significant changes in the phenethylamine separations * Using electrospray-MS, the manufacturer of DM-b-CD has occurred, the EOF for the SDS system decreased from shown that different lots have different degrees of substitution.
Figure 7. CE analysis of an illicit methamphetamine tablet (same sample vial) using multiple run buffers
with a 32 cm (23.5 cm to the detector window)650 mm ID fused-silica capillary operating at 157C. (A)
Electropherogram with anionic coating reagent and run buffer consisting of CElixir Reagent B (pH 2.5)
with a voltage of 10 kV and 100 mbar?s injection. Identity of peaks: (a) d-methamphetamine, (b) l- or
d-n-butylamphetamine (internal standard), and (c) l- or d-n-butylamphetamine. (B) Electropherogram
with anionic coating reagent and run buffer consisting of CElixir Reagent B (pH 2.5) 1 50 mM HP-b-CD
with a voltage of 20 kV and 100 mbar?s injection. Identities of peaks identical to (A). (C) Electrophero-
gram of co-injection of 100 mbar?s of sample and 35 mbar?s of standard with CE conditions as in (B).
Identity of peaks are same as (A) except for (d) l-amphetamine, (e) d-amphetamine, (f) l-methamphet-
amine, (g) l or d-MDA, (h) l or d-MDA, (i) l or d-MDMA, (j) l or d-MDMA, (k) l or d-MDEA, and (l) l or d-MDEA.
(D) Electropherogram with anionic coating reagent and run buffer consisting of 50 mM phosphate-borate
(pH 6.5) 1 3% SDS with a voltage of 8.5 kV and 100 mbar?s injection. Identity of peak is (m) caffeine.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 1580–1591 tablet (a “Thai Tab”), a seized heroin HCl sample, anLSD exhibit, and a hallucinogenic mushroom. The illicitmethamphetamine tablet is analyzed (same sample in-jection vial) by three of the above CE systems on thesame capillary. The methamphetamine content (24.2%)is first measured (Fig. 7A), followed by a chiral determi-nation for which methamphetamine enantiomers arepresent (Fig. 7B), proceeded by a confirmation of thed-methamphetamine isomer by co-injection (Fig. 7C),and finally an acidic, neutral, and weakly basic adulter-ant screen (Fig. 7D) which indicates the presence of caf-feine (migration time and UV library search). A chiraldetermination without co-injection is necessary in casethe sample also contains a small amount of the other Figure 8. CE analysis of a seized heroin HCl sample
enantiomer, which could be difficult to confirm by co- (same sample vial) using multiple run buffers with a 32 cm (23.5 cm to the detector window)650 mm ID fused-silicacapillary operating at 157C. (A) Electropherogram with an- A brown powder containing heroin HCl is analyzed by ionic coating reagent and run buffer consisting of CElixir two of the above CE systems on the same capillary. The Reagent B (pH 2.5) 1 100 mM DM-b-CD with a voltage of13 kV and 250 mbar?s injection. Identities of peaks: (a) qui- heroin HCl content (27.8%) is first measured and also nine, (b) heroin, (c) O6-monoacetylmorphine, (d) acetyl- library searched (Fig. 8A). The presence of quinine is indi- codeine, (e) morphine, (f) papaverine, and (g) noscapine.
cated by both UV spectra and migration time. The identi- (B) Electropherogram with anionic coating reagent and run fication of basic impurities by both library search and buffer consisting of 50 mM phosphate-borate (pH 6.5) 1 migration time helps to eliminate these commonly occur- 3% SDS with a voltage of 8.5 kV and 100 mbar?s injection.
ring peaks as possible adulterants. An acidic, neutral and weakly basic adulterant screen (Fig. 8B) indicates thepresence of benzocaine (migration time and UV librarysearch). The amount of LSD in a blotter sample (40 mg 4 Concluding remarks
per blotter) (Fig. 9A), and the identification of psilocin andpsilocybin in a mushroom exhibit (Fig. 9B) are determined These data demonstrate that a wide variety of seized drugs by consecutively using two of the above systems, again can be analyzed on a single capillary using dynamic coat- ings generated from the same polymeric cationic coating Figure 9. CE analysis of seized
LSD and psilocybin exhibits
using a 32 cm (23.5 cm to the
detector window)650 mm ID
fused-silica capillary at 157C.
(A) Electropherogram obtained
under conditions as described
in experimental section for LSD.
Identities of peaks are (a) LSD,
and
standard). (B) Electropherogramobtained under conditions asdescribed in experimental sec-tion for hallucinogenic mush-rooms. Identities of peaks are(c) psilocin, and (d) psilocybin.
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 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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