Electrophoresis 2000, 21, 28912898 2891

Giulia Manetto1 Field-amplified sample stacking capillary zone

Franco Tagliaro2
Federica Crivellente1
Vincenzo L. Pascali2
Mario Marigo1
1 The present paper describes the methodological optimization and validation of capil-
Department of Public
Medicine and Health,
Section of Forensic Medicine,
University of Verona,
Verona, Italy
2
Institute of Forensic
Medicine, Catholic University
of the Sacred Heart,
Rome, Italy
electrophoresis applied to the analysis of opiate
drugs in hair
lary zone electrophoresis (CZE) for the determination of major opiates (morphine,
codeine, 6-monoacetylmorphine, acetylcodeine, heroin) in hair samples by using a
field-amplified sample stacking injection before the separation in a binary running
buffer (0.1 M sodium phosphate, pH 2.5, with 40% ethylene glycol). The applied poten-
tial was 20 kV, at 25oC. Detection was by UV absorption at the fixed wavelength of
214 nm or by recording the full spectrum between 190400 nm, thus improving the an-
alytical selectivity and identification power of CZE. Hair samples were liquid/liquid
extracted; dried extracts, reconstituted with a low-conductivity solvent (0.1 mM phos-
phoric acid, with 80% 1-propanol), were injected by electromigration at 10 kV for 99 s,
after a 0.5 mm plug of water. Under the described conditions, the limit of detection (with
a signal-to-noise ratio of 3) in hair extracts was 100 pg/mL for codeine, 75 pg/mL for
morphine and 6-monoacetylmorphine (6-MAM), 150 pg/mL for ethylmorphine, and
0.75 ng/mL for acetylcodeine and heroin. The precision of the method was validated
for standards in pure solution by using internal standardization, providing for intraday
and day-to-day assays, in terms of migration times, relative standard deviation (RSD)
values 0.2%, and in terms of peak areas, RSD values <5.71%.

Keywords: Capillary electrophoresis / Field-amplified sample stacking / Abused drugs / Hair

analysis EL 4107

1 Introduction Since the end of the 1970s, toxicological hair analysis has
increasingly been used to investigate drug abuse histories
In the determination of forensic drugs in biological sam- and is now widely accepted as a tool to monitor past,

ples a major analytical challenge is the limit of detection
and quantitation [1] because of the low concentration lev-
els, which may be of analytical interest. Moreover, addi-
tional problems come from the high and variable content
of proteins, lipids and ions which may interfere with drug
determination [2, 3]. Therefore, the analysis of biological
fluids generally requires preliminary sample clean-up and
preconcentration steps [4, 5]. Capillary electrophoresis
(CE) is a relatively new analytical technique, which has
been applied to many aspects of drug analysis [6]. As is
well known, although very high mass sensitivity detection
is easily obtained, CE, particularly when applied with UV
absorbance detection, suffers from limited concentration
sensitivity due to the short optical pathlength inherent in
the in-capillary detection.
Correspondence: Dr. Giulia Manetto, Department of Public
Medicine and Health, Section of Forensic Medicine, Policlinico
Borgo Roma, 37134 Verona, Italy
E-mail: giumnt@borgoroma.univr.it
Fax: +39-045-505-259
Abbreviations: FASS, field-amplified sample stacking; IS, inter-
nal standard; 6-MAM, 6-monoacetylmorphine
CE and CEC
chronic exposure to drugs [7, 8]. Unfortunately, drug and
drug metabolite concentration in the hair matrix is low (in
the order of fractions of nanogram per milligram of hair)
and, for aesthetic reasons, the amount of hair sample
which can be collected is limited. In hair analysis, CE has
become a valuable tool because its separation principles
are completely different from those of gas and liquid chro-
matography, which are at present the most used tech-
niques. Furthermore, CE analysis is characterized by
minimal sample consumption, which is important in order
to preserve hair sample for further confirmation analyses.
In our experience [9], capillary zone electrophoresis
(CZE), carried out in borate buffer at pH 9.2, allowed mor-
phine and cocaine detection in hair extracts, but the assay
sensitivity was moderate, i.e., insufficient for use in real
cases of chronic but moderate drug abuse.
Enhanced limits of detection in CZE have been reported
by using on-column concentration techniques like field-
amplified sample stacking (FASS), which is based on a
mismatch between the electric conductivity of the sample
and that of the running buffer [1013]. If the sample con-
ductivity is lower than that of the background electrolyte

 WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/1414-2891 $17.50+.50/0

2892 G. Manetto et al. Electrophoresis 2000, 21, 28912898

(BGE), the higher electric field and consequently the high-
er migration velocities of the analytes result in an injected
sample plug in comparison with the separation buffer, and
this produces a sharpening of the analyte zone at the
boundary with the BGE. A preliminary separation using a
sample stacking method coupled with CZE separation in
plain aqueous medium resulted in about 100-fold in-
creased sensitivity, thus allowing UV spectral identifica-
tion and quantitation of abused drugs in hair samples
down to low ng/mg concentration levels [14]. The present
study describes a further optimization of a head-column
FASS approach in a binary buffer system, aimed at the
high efficiency separation and high sensitivity detection of
a mixture of commonly available opiates (i.e., methadone,
morphine, codeine, 6-monoacetylmorphine (6-MAM ace-
tylcodeine, heroin). Also, a preliminary application of the
optimized CZE conditions to hair analysis is reported as
well as the use of diode array UV detection in order to
improve analytical selectivity and the identification power
of CZE.
with a diode array UV detector. Uncoated fused-silica
capillaries (47 cm total length, 40 cm effective length,
75 mm ID) from Composite Metal Services (The Chase,
Hallow, Worcestershire, UK) were used throughout.
2.3 Electrophoretic conditions
The electrophoretic separations were carried out using a
running buffer composed of 0.1 M sodium phosphate
adjusted to pH 2.5. Binary running buffers were com-
posed of aqueous phosphate solution (pH 2.5) supple-
mented with ethylene glycol at percentages ranging be-
tween 2560% v/v. A constant voltage of 20 kV was
applied with the anode at the injection end. UV absor-
bance detection took place at the fixed wavelength of 214
nm (filter UV detector) or by recording the full spectrum
between 190 and 400 nm with the diode array detector.
The temperature of the capillary was kept constant at
25oC. Conditioning of fresh capillaries was effected by
rinsing with 1 M NaOH (4 min), 0.1 M NaOH (4 min), H2O

2 Materials and methods

2.1 Chemicals and standards

Salts used for buffer preparation were of analytical rea-
gent grade and the organic solvents 1-propanol and ethyl-
ene glycol were of HPLC grade. All chemicals were pur-
chased from Carlo Erba (Milan, Italy). Standards of ana-
lyzed drugs (morphine, codeine, methadone, ethylmor-
phine, 6-MAM, acetylcodeine, heroin, nalorphine) were
provided by Salars (Como, Italy). Stock solutions of each
standard were prepared in methanol at a concentration of
1 mg/mL and stored at 20oC. Working standards were
prepared daily by dilution of stock solutions at suitable
concentrations in suitable solvents. The running buffer
was prepared from sodium dihydrogen phosphate disso-
lution in water or water/ethylene glycol mixtures and the
pH was adjusted with 4 M phosphoric acid. For conven-
ience, the pH value given for the binary systems refers to
that of an aqueous buffer with the same phosphate com-
position. Commercially available ready-to-use tubes
(Toxi-Tubes A) for liquid-liquid extraction of basic com-
pounds from biological samples were supplied by Marion
(Irvine, CA, USA).

2.2 Instrumentation Figure 1. Typical electropherogram of the separation of

A capillary electropherograph P/ACE 5500 (Beckman
Coulter, Fullerton, CA, USA) fitted with a filter UV absor-
bance detector was used in the first part of the work. Con-
trol of the instrumentation, data acquisition and analysis
was performed with the software P/ACE Station release
1.0 (Beckman Coulter). On-line spectral analysis of peaks
was performed with the same electropherograph fitted
1, methadone; 2, morphine; 3, codeine; 4, 6-MAM; with
nalorphine as IS (later replaced with ethylmorphine,
because of mediocre resolution from 6-MAM), at the con-
centration of 5 mg/mL. Conditions: injection, pressure
application 0.5 psi for 5 s; capillary, uncoated fused silica,
47 cm 75 m ID; buffer, 0.1 M phosphate, pH 2.5; poten-
tial 15 kV; detection, UV absorbance at 214 nm. For other
conditions see Section 3.1.
Electrophoresis 2000, 21, 28912898 FASS in toxicological hair analysis 2893

Table 1. Peak resolutions (R) and migration times (T) of

the sample mixture (methadone, morphine,
codeine, 6-MAM) at different volume fractions
of ethylene glycol in 0.1 M, pH 2.5 phosphate
buffer.
Ethylene 1 2 3 4
glycol (%)
0 T 13.86 13.99 14.13 14.69
R 0 0.96 0.94 3.87
25 T 20.68 21.1 21.53 22.78
R 0 2.16 2.17 4.14
40 T 23.83 25.74 25.12 26.98
R - 6.41 2.94 5.21
50 T 26.17 29.29 28.48 30.61
R - 10.16 3.27 5.24
60 T 37.99 42.81 42.94 44.28
R - 22.89 0.95 5.41

(4 min) and running buffer (10 min) by applying a positive

high pressure (20 psi) at the injection end. Pressurized
(0.5 psi for 510 s) injections or FASS were carried out
using conditions which will be described in the specific
section. Between runs, reversed washes with 0.1 M NaOH
(1 min) and running buffer (3 min) were carried out.

2.4 Sample preparation Figure 2. Typical electropherogram of the separation of

Hair samples (50100 mg) were collected from the scalp
of the vertex posterior of the head, by cutting with scissors
as close to the skin as possible. Blank hair was obtained
from well-known subjects abstinent from any drugs for the
prior six months. When needed, blanks were spiked with
suitable amounts of drugs to mimic concentrations usually
found in real users (0.510 ng/mg). Positive hair samples
from real drug users were preliminarily analyzed by our
routine methods based on radioimmunoassay screening
and HPLC confirmation [15]. Sample treatment was sim-
ilar to the procedure adopted for HPLC [15]. In short, hair
samples were first washed with an aqueous solution of
0.3% Tween-20 (20 mL, two times), in order to remove
the lipids and potential contaminants present on the sur-
face. Then hairs were cut into small fragments and in-
cubated overnight in 1 mL of 0.1 M HCl at 45oC. Finally,
the incubation mixture was neutralized with equimolar
amounts of NaOH and extracted into organic phase with
Toxi-Tubes A. The organic layer was evaporated under a
stream of air and the dried residue was reconstituted in a
suitable amount of a specific sample solvent as described
as follows.
2.5 Head-column FASS
In method development, different sample matrices were
tested, by varying the percentage of 1-propanol (ranging
from 60 to 90%) in 0.1 mM H3PO4. Pure standard mixtures
1, methadone; 2, codeine; 3, morphine; 4, 6-MAM. Condi-
tions: injection, pressure application 0.5 psi for 5 s, capil-
lary, uncoated fused silica, 47 cm 75 mm ID; buffer,
0.1 M phosphate, pH 2.5, 40% ethylene glycol; potential,
20 kV; detection, UV absorbance at 214 nm. For other
conditions see Section 3.1.
were prepared at the fixed concentration of 0.1 mg/mL for
each analyte. FASS instrumental conditions were chosen
on the basis of a method reported by Zhang and Thor-
mann [10] and can be summarized as follows: first, the
injection end of the capillary was dipped into water for 5 s
without applying pressure (external rinse step), then a
plug of water was loaded for 1 s at 0.5 psi and the sample
was injected electrokinetically for 99 s at 10 kV. In order
to increase the sample loading with no loss in column effi-
ciency and resolution, the dried hair extracts were recon-
stituted with 0.2 mL of the low-conductivity sample solvent
and injected using the FASS protocol described above.
2.6 Separation parameters
To monitor the electroosmotic flow (EOF), benzyl alcohol,
as a neutral marker, was injected at a concentration of
30 mg/mL. Resolution (R) was calculated according to the
equation R=[2(t2t1)]/(W1+W2), where t1 and t2 are the
migration times of the two adjacent peaks, and W1 and
W2 are the peak widths at the baseline. Efficiency,
2894 G. Manetto et al.
expressed as number of theoretical plates, was calcu-
lated as N=16 (t/W)2, where t is the migration time and W
is the peak width at peak base. The length of the water
plug injected by pressure application was calculated
according to the following equation: L=Pr2t /8hLt, where P
is the pressure difference between the capillary ends, r is
the capillary radius, h is the viscosity of the buffer, and Lt
is the total length of the capillary. The viscosity of buffers
and solutions was measured by the CE apparatus [16,
17] according to the equation h=hO t /tO, where hO indi-
cates the tabulated viscosity of a reference liquid (e.g.,
water) and t and tO the measured times to reach the
detector under constant pressure for the studied liquid
and for the reference liquid, respectively. The dielectric
Electrophoresis 2000, 21, 28912898
Figure 3. Typical electropherograms of the separation of
1, methadone; 2, morphine; 3, codeine; 4, 6-MAM; with
ethylmorphine as IS: (a) 1 mg/mL in 0.1 M phosphate/eth-
ylene glycol 60/40 v/v; pressure injection, 0.5 psi for 5 s.
(b) 0.01 mg/mL in 1-propanol / 0.1 mM H3PO4 60/40 v/v;
FASS injection; peak order as in (a). (c) 0.01 mg/mL in
1-propanol / 0.1 mM H3PO4 80/20 v/v; FASS injection,
peak order as in (a). For other conditions see Section 3.2.
constant of binary media is proportional to the volume
fraction of the components [17] and thus could be calcu-
lated from pure solvents (78.54 and 37.7 for water and
ethylene glycol, respectively) according to the following
equation: e=78.5 (1-x)+37.7 x, where x represents the vol-
ume fraction of ethylene glycol.
3 Results and discussion
3.1 Optimization of CZE conditions
It is well known that, because of the limited volume of the
capillary, separation in CE is highly affected by the length
of the injection plug. FASS provides a way to concentrate
Electrophoresis 2000, 21, 28912898
Figure 4. Typical electropherograms of (a) blank human
hair extract; (b) extract from blank human hair spiked
with: 1, codeine; 2, morphine; 3, 6-MAM; IS, (0.2 ng/mg
for each analyte); 4, acetylcodeine; 5, heroin; at the con-
centration of 0.03 ng/mg. Conditions as in Fig. 2. For
other conditions see Section 3.3.
FASS in toxicological hair analysis 2895
Figure 5. Typical electropherogram of a human hair from
a real heroin user after liquid/liquid extraction. Conditions
as in Fig. 2. For other conditions see Section 3.3.
the analytes in a thin zone at the boundary between the
sample plug and the background buffer, allowing the
increase of injection time without sacrificing efficiency.
However, to ensure the best analytical results, stacking
and separation conditions must be matched carefully. To
this aim, the CZE separation conditions of the analytes of
interest represented by closely related opiates were first
optimized by using binary buffers, on the basis of the pre-
vious experiences by Zhang and Thormann [17]. Run buf-
fers with low pH were chosen in order to minimize EOF,
which hinders the stacking phenomena. Under these ana-
lytical conditions, structurally related opiates (morphine,
codeine, methadone, nalorphine, 6-MAM), displaying the
same net positive charge at the running pH, migrate sub-
stantially according to their molecular masses. Figure 1
shows an incomplete separation in plain aqueous 0.1 M
phosphate buffer, pH 2.5, of a mixture (5 mg/mL) of meth-
adone, morphine, codeine, 6-MAM and nalorphine (inci-
dentally introduced as an internal standard) injected by
hydrodynamic mode (0.5 psi for 5 s).
Binary CZE buffers containing various proportions (25-
40-50-60% v/v) of ethylene glycol (boiling point 198.8oC)
in phosphate aqueous solution were then tested to
achieve optimal conditions for analyte resolution. In
agreement with the general electrophoretic theory [18],
2896 G. Manetto et al. Electrophoresis 2000, 21, 28912898

the migration time of the studied cationic analytes gradu-

ally increased with increasing volume fraction of ethylene
glycol, due to the concomitant increase in viscosity of the
separation medium. On the other hand, the addition of
large amounts of an organic solvent provided sharper
peaks and resolution was improved up to 50% ethylene
glycol; above this percentage, separation was severely
spoiled. Also, changes in selectivity were observed (the
migration order of morphine and codeine was reversed at
40% ethylene glycol). On the basis of peak resolution and
analysis time under the different conditions (Table 1), we
adopted a 40% volume fraction of ethylene glycol in phos-
phate buffer as the best compromise to start the optimiza-
tion of FASS.

Figure 2 shows a representative electropherogram of the

baseline separation of the sample mixture, injected by
hydrodynamic mode (0.5 psi for 5 s). CZE was carried out
in 0.1 M phosphate buffer, pH 2.5, containing 40% ethyl-
ene glycol. Under the chosen analytical conditions, the
analytes of interest migrated in the order methadone,
codeine, morphine and 6-MAM. Excellent peak symme-
try, resolution (R between 2.68 and 6.55), and efficiency
(N 100 000) were achieved. The overall analysis time
was about 20 min. The chosen binary running buffer dis-
played a relative viscosity to water (h/hw) of 2.81, a die-
lectric constant of 62.18 and an EOF mobility of
Figure 6. UV spectra of morphine in hair of heroin user
5.9455104 cm2/Vs.
superimposed with the UV spectra of pure morphine.

3.2 FASS in pure solution

Based on a conductivity mismatch between sample solu-
tion and separation buffer, FASS was optimized in terms
of volume fractions of 1-propanol (60-80-90% v/v) in
0.1 mM phosphoric acid as the solvent of the injected ana-
lytes. Moreover, a short water plug was introduced before
the sample to hinder the diffusion of running buffer ions
into the sample zone. Results depicted in Fig. 3 show the
obtained sensitivity improvement, which, for pure solu-
tion, is more than 1000-fold (see peak absorbance and re-
spective concentrations), without loss of efficiency and
resolution. As shown in Fig. 3a, no stacking effect was ob-
tained when the sample (1 mg/mL) was dissolved in
the running buffer and injected by hydrodynamic mode
(0.5 psi for 10 s). On the other hand, stacking occurred
under head-column FASS conditions, with volume frac-
tions of 60 and 80% of 1-propanol, respectively (Fig. 3b
and c, where the concentration was 0.01 mg/mL). No
peaks were observed in the binary media at percentages
of 1-propanol 90 (electropherogram not shown). This is
consistent with the results reported by Thormann [10].
The influence of length of the water plug on stacking was
studied in the range from 0 to 10 s injections. The optimal
water plug was found to be achieved with 1 s injection,
corresponding to a length of about 0.5 mm. A longer
water plug worsened the efficiency of separation, proba-
bly because of local overheating of the injection end of
the capillary with consequent convective zone broad-
ening. Concerning sample solvent composition, the larg-
est stacking efficiency was obtained using 80% 1-propa-
nol (h/hw = 2.53, e = 31.78) in 0.1 mM H3PO4, to enhance
protonation of solutes. These results are in agreement
with the theory of stacking and the current literature [10],
since the lowest conductivity of the sample solution is
reported at 80% 1-propanol in water.
3.3 Application of FASS to hair samples
Because of the low amounts of hair that can be sampled,
for aesthetic reasons, and the moderate concentrations of
analytes present in this matrix, we adopted optimized
stacking conditions to improve the concentration sensitiv-
ity of CZE. Figure 4 shows typical electropherograms
from extracts of (a) blank human hair and (b) from blank
hair spiked with standards of codeine, morphine, ethyl-
morphine (internal standard, IS), and 6-MAM to mimic a
concentration of 0.2 ng/mg of each analyte. Acetylco-
deine and heroin were added at lower concentrations
Electrophoresis 2000, 21, 28912898 FASS in toxicological hair analysis 2897

(0.03 ng/mg), solely to confirm heroin use or to deny Table 2. RSDs of absolute and relative migration times
external contamination of hair. The hair blank showed no and peak area of pure standards (intraday:
significant peaks that could interfere with the investigated n=10; day-to-day: n=5)
opioids in the time window from 34 to 40 min. On the RSD% RSD%
other hand, the spiked sample shows well-separated and Compound Migration time Peak area
symmetric peaks of codeine, morphine, ethylmorphine Intraday Day-to-day Intraday Day-to-day
(IS), 6-MAM, acetylcodeine, and heroin, which are clearly
Methadone 0.76 - 14.12 -
distinguishable from the background. The FASS increase
Methadone/IS 0.05 - 3.04 -
in the injected amount of sample allowed the recording of
Codeine 0.79 6.44 13.1 26.98
UV spectra from the peaks, which are overimposable onto Codeine/IS 0.03 0.19 1.15 3.96
the spectra from standards. Figure 5 shows the electro- Morphine 0.80 6.47 11.36 31.02
pherograms of a hair sample from a real user of heroin Morphine/IS 0.02 0.12 0.82 3.98
(containing 0.22 ng/mg of morphine according to a vali- 6-MAM 0.84 6.54 11.22 33.97
dated HPLC method [15]) after direct liquid-liquid extrac- 6-MAM/IS 0.06 0.08 5.71 4.41
tion. On the basis of migration times and UV spectra,
codeine and morphine were identified, at levels of 0.07
and 0.20 ng/mg, respectively. Figure 6 shows the UV on average. The theoretical sensitivity, calculated on
spectra of morphine in the hair of a heroin user superim- the basis of the results from spiked samples, in the
posed with the UV spectra of the pure standard. The back hair extract was 100 pg/mL for codeine, 75 pg/mL for
extraction into 1 mM H3PO4 of the Toxi-Lab organic morphine and 6-MAM, 150 pg/mL for ethylmorphine,
extract gave a slightly cleaner electropherogram, with 750 pg/mL for acetylcodeine and heroin (with a signal-to-
similar quantitative results for codeine and morphine. noise ratio of 3). In terms of drug concentration in hair the
LODs correspond to 20 pg/mg for codeine, 15 pg/mg for
3.4 Validation morphine and 6-MAM, 30 pg/mg for ethylmorphine, and
150 pg/mg for acetylcodeine and heroin, which perfectly
Under optimized conditions, the absolute and relative satisfied the cutoff limits applied in our forensic labora-
within-day relative standard deviations (RSD%) for migra- tory.
tion times and peak areas were determined for ten repli-
cate samples of pure standards at concentrations of
4 Concluding remarks
10 ng/mL. The day-to-day precision was determined over
a period of five days with three replicate samples at con- In recent years, hair analysis for drugs has gained adva-
centrations of 0.1 mg/mL. The typical reproducibility data nces in the forensic toxicology environment, as a tool for
obtained are presented in Table 2. As expected, absolute investigating past, chronic exposure to illicit drugs, as
peak area reproducibility with FASS was poor: day-to-day shown by an increasing body of literature [7, 8]. To this
RSD% values were determined to be between 26.98 and purpose, CE has several unique advantages over current
33.97%, but the area normalization on the basis of the IS techniques (GC, GC-MS,, HPLC) nowadays applied,
(ethylmorphine) allowed us to limit RSDs to values such as small sample size, minimal sample preparation,
4.41%. Absolute intraday RSD values were found to be speed of analysis, and low cost of capillaries and chemi-
in the range between 11.2214.12%, but with internal cals, but its sensitivity in terms of concentration was diffi-
standardization RSD were better than 5.71%. The intra- cult to deal with, considering the minute amounts of drugs
day RSDs of absolute migration times were always <1%, present in hair samples.
while the day-to-day RSDs were <6.54%; for relative
migration times, RSDs were <0.06% and 0.2%, respec- In the present paper, our attention was focused on the
tively. optimization of sample enrichment offered by a FASS
before the separation in a binary CZE buffer, providing
With pure standard solutions, using hydrodynamic injec- excellent resolution power, separation efficiency and ana-
tion (0.5 psi for 10 s; no FASS) the limit of detection lytical precision as the sensitivity was more than enough
(LOD) at 214 nm wavelength (S/N = 3) was 0.15 mg/mL to determine some of the major opioids in the hair sam-
for methadone, 0.12 mg/mL for codeine, 0.10 mg/mL for ples. Furthermore, the substantial increase in sensitivity
morphine, and 0.19 mg/mL for 6-MAM. Under FASS- achieved by sample stacking allowed the recording of UV
optimized conditions the LOD values achieved were: 60 spectra from the peaks of drugs in hair even at the cutoff
pg/mL for methadone, 50 pg/mL for codeine, 40 pg/mL for levels adopted (100 pg/mg) and consequently the spec-
morphine, and 90 pg/mL for 6-MAM. Thus an approxi- tral confirmation of results based on migration times. In
mately 2500-fold increase in sensitivity can be calculated conclusion, CZE coupled to head-column FASS once
2898 G. Manetto et al.
again proved to offer a sensitive, precise, easy, low-cost
and rugged method for toxicological hair analysis, to be
used as a complementary technique for confirmation of
results obtained with traditional methodologies.
This work was co-funded by research grants awarded by
Regione Veneto (# 765/01/97), CARIVERONA and
M.U.R.S.T. #9906404127.
Received March 27, 2000
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