Forensic
Science
Forensic
ELSEVIER
Science
77 (1996)
International
211
~22’)
Capillary electrophoresis: principles and applications in
illicit drug analysis’
F. Tagliaro”“.‘,
Received
12 June
1995:
revision
S. Turrina”,
received
F.P. Smith”
8 September
1995:
accepted
4 October
1995
Abstract
Capillary electrophoresis, which appeared in the early 198Os, is now rapidly expanding into
many scientific disciplines. including analytical chemistry. biotechnology and biomedical and
pharmaceutical
sciences. In capillary electrophoresis.
electrokinetic
separations are carried
out in tiny capillaries
at high voltages (lo--30 kV). thus obtaining high efficiencies (,li > 105)
and excellent mass sensitivities (down to 10 Ix- 10 “I moles). The main features of capillary
electrophoresis are: versatility of application (from inorganic ions to large DNA fragments),
use of different separation modes with different selectivity. extremely low demands on sample
volume, negligible running costs, possibility of interfacing with different detection systems,
ruggedness and simplicity of instrumentation.
Capillary electrophoresis
applications
in
forensic sciences have appeared only recently. but are now rapidly growing, particularly in
forensic toxicology.
The present paper briefly describes the basic principles of capillary
electrophoresis, from both the instrumental and analytical points of view. Furthermore.
the
main applications in the analysis of illicit,controlled
drugs in both illicit preparations and
biological samples are presented and discussed
(43 references).
It is concluded
that
the
particular separation mechanism and the high complementarity
of this technique to chro
matography makes capillary electrophoresis
a new pow-erful tool of investigation in the
hands
of
forensic
toxicologists.
* Corresponding
author.
’ A first version
was presented
National
Drug
Testing
Laboratories
as plenary
from
lecture
at the UNDCP
the countries
participating
Laboratory
Project’,
Thessaloniki.
3
‘At
present.
visiting
professor
in
Alabama
at Birmingham,
Birmingham.
7 October,
1994.
the Graduate
Program
AL.
USA.
037Y-0738’96~Sl5.00
Science
SSDl
0379.0738(95)01863-E
12 1996
Elsekier
Ireland
Ltd.
in
All
consultative
in the
Forensic
rights
reswed
Meeting
‘Regional
Science.
The
of Heads
01
Balkan
Route
University
of
212
F. Tagliaro
et al. / Forensic
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77 (1996)
21 I-229
Keywords: Capillary electrophoresis;Forensic toxicology; Illicit drugs; Analysis; Seizures;
Biological samples
1. Introduction
Capillary Electrophoresis (CE) (or high-performance
capillary electrophoresis
(HPCE)), probably, is the most rapidly expanding analytical technique appeared in
the recent years. In fact, as it is depicted in Fig. 1, the papers appeared in the
leading international journals have rapidly increased, as well as books, volumes
[l - lo] and symposia dedicated to this emerging technique.
In CE, electrokinetic separations are carried out in tiny capillaries at high
voltages (lo-30 kV), thus obtaining high efficiencies (N > 105) and excellent mass
sensitivities (down to 10 - 18- 10 - *’ moles).
The main features of CE could be summarized as follows:
- versatility of application (from inorganic ions to large DNA fragments);
- modes of separation with different selectivity can be applied with the same
hardware;
- extremely low demands upon sample volume (few nl are injected);
- minimum need of solvents (running buffer) and low cost of other consumables
(capillaries);
- possibility of interfacing with different detection systems;
- ruggedness and simplicity of the basic instrumentation.
100
Cl
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
years
Fig. 1. Papers on CE quoted yearly in Chemical Abstracts in the years 1883-1993.
Fig.
2. Scheme
of a capillary
electropherograph
2. Instrumentution
The basic equipment (Fig. 2), consists of an injection system, a capillary (in most
instances made of fused silica, generally, 20&100 pm internal diameter (i.d.),
20-100 cm long), a high voltage source (operating within lo-30 kV), electrodes,
electrode jars and detector(s).
2. I. Samplr
injection
Because of the miniaturization of the separation compartment (the capillary),
only minute, but well controlled volumes of sample should be injected, if high
efficiency in separations is to be attained. Modern, automated instruments do
assure in real conditions injection reproducibility better than 2”/0. Manually operated CE systemsmay be less reproducible. but still acceptably precise with the use
of appropriate internal standards.
In general, two injection techniques can be used: hydrodynamic injection and electrokinetic injection. The two differ essentially becausethe former is non-specific (what
is injected represents the composition of the sample exactly), while the latter is selective, depending on ion mobility, charge and concentration of the ions in the sample.
The simplest way to introduce nanoliter amounts of sample solution into the
capillary is by gravity, raising the injection end of the capillary for a fixed time.
Also, hydrodynamic injection can be accomplished by application of pressure for a
fixed time onto the sample vial in which the injection end of the capillary is dipped,
or by application of vacuum at the opposite end.
In order to allow injection by means of standard high performance liquid
chromatography (HPLC) syringes, a split-flow injector for CE has also been
developed, in which the liquid injected is split between the capillary and a split-vent.
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The percent volume of the sample introduced in the capillary (usually - l/1000)
depends on the difference in hydrodynamic resistance between the ‘capillary port’
and the ‘vent port’, which depends on the relative differences in length and diameter
of the connected tubings (i.e. the capillary and the waste).
In electrokinetic injection, the sample is introduced into the capillary by applying
a voltage (in general, lower than that used in the separation), while the injection
end is dipped into the sample. Thus, the molecules contained in the sample are
injected by electromigration
as well as transportation with the electroosmotic flow
(see later). Consequently, both ion mobility and electroosmotic flow influence the
amount of the analytes injected. In conclusion, although theoretically superior in
terms of selectivity, electrokinetic injection is dependent on factors often difficult to
keep under control.
2.2. Capillary
Typical dimensions of capillaries for CE are id. 20-100 pm and lengths ranging
from 20-100 cm. The capillary is the compartment where separation and, in
general, detection occur. Consequently, the capillary should be chemically and
physically resistant, inexpensive, UV transparent, precisely produced with narrow
internal diameters, not prone to adsorb solutes and, in order to allow dissipation of
the Joule heating, should have a high thermal conductivity.
Fused silica capillaries, resembling those currently used in capillary gas chromatography, can be commercially found with i.d. values ranging from less than 10
pm to more than 200 pm and meet almost all the above mentioned requirements,
except that, in some instances, they tend to adsorb the analytes.
CE capillaries can be uncoated, internally coated or filled with gels. In order to
give them good flexibility, they are externally protected with polyimide. Where the
capillary needs to be optically transparent, in order to allow photometric detection,
the external coating is burned off, leaving a highly UV transparent, but fragile,
wall.
The conditions of the internal surface of the capillary (in the case of fused silica,
the ionization of the acidic silanol groups) are extremely important to achieve the
best performances from CE. The simplest way to obtain a ‘reproducibly clean’ inner
capillary surface is washing the capillary with a strong base solution (e.g. 1.0-0.1
M NaOH), in order to dissolve a thin film of silica from the wall. Washing must be
followed by a thorough rinse with the run buffer: the choice of pH, ionic strength
and composition of buffer is, of course, crucial. Alternatively, the capillary wall can
be coated with ‘modifiers’, which can be physically adsorbed, chemically bound or
simply added to the buffer. The most common coatings are polyacrylamide, PEG,
cellulose, PVA, amino acids, proteins, amines, surfactants, etc.
2.3. Power supply
High voltage power supplies, in commercial CE instrumentation,
generally
deliver up to 30 kV and currents up to 200-300 PA. Because of the direction of the
F. Tagliaro
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77 (1996)
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215
electroosmotic flow, the common polarity is with the anode at the injection end of
the capillary and the cathode close to the detector; however, under particular
experimental conditions the polarity is reversed.
CE separations are usually carried out at constant potential, but constant current
can be used too and, sometimes, this is preferable, because the system tends to
compensate for temperature changes. Constant power operation is typical of
isoelectric focusing.
2.4. Detection
Detection in CE has to face two challenging problems, namely small amounts of
analytes injected and tiny peak volumes (in the order of nanolitres).
Up to the present, I-IV-visible detectors are by far the most widely adopted. In
order to maintain the high efficiency of separation and to avoid loss of sensitivity
due to peak dilution, detection is carried out directly ‘in-column’,
through a
window obtained by burning (or scraping) off the polyimide external coating of the
capillary. However, the short i.d. of the capillary (i.e. the detection cell path-length)
limits the concentration sensitivity to lo-“- 10e6 M and the linear range.
A promising way to increase the path length (up to few mm) is to use a Z-shaped
capillary, in which detection is by axial illumination.
This, reportedly, improves the
sensitivity by a factor of ten or more, with minimal decrease of resolution (see Ref.
[l] pp. 61-69). Alternatively, the path length can be increased by, a ‘bubble cell’
design of the capillary [ll].
Improved selectivity in absorbance detection is achieved by photodiode array or
multiwavelength detectors, analogous to HPLC, which, with only a moderate loss
of sensitivity, allow on-line recording of UV(-visible) spectra of the separated zones.
This is important for peak purity evaluation and for investigation of peak identity,
on the basis of the spectral analysis of the peaks.
Lamp-based fluorescence in-column detection encounters difficulties in focusing
enough excitation energy into the capillary and in efficiently collecting the emitted
radiation. This limits the theoretical sensitivity that fluorescence detection could
provide; on the other hand, in terms of selectivity, the advantage of fluorescence
over UV absorbance is maintained.
Laser-induced fluorescence, allowing high energy excitation, is more sensitive
(reportedly 10 - ’ up to 10 - I2 M) but instrumentally more complex and limited in
the excitation wavelengths available from the current types of laser sources (325 nm
for He-Cd and 488 nm for Ar ion lasers). Since a minority of analytes are naturally
fluorescent, pre-column derivatization is often applied, primarily according to the
procedures well established for HPLC. Post-column derivatization, although feasible, is basically more complex, due to the addition and mixing of reagents after the
separation, which tend to cause band spreading incompatible with the tiny volumes
of the separated zones.
Electrochemical detection, in particular conductivity and amperometric detection,
have been applied in CE with encouraging results (see Ref. [l] pp. 108-121).
However, a problem still not fully resolved is the need to separate the high potential
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compartment of the capillary from the detection compartment. This insulation has
been obtained by means of a porous conductive joint inserted before the detection
end of the capillary, but no commercial devices are at present available.
Conductimetric detection is also possible, e.g. by applying two electrodes into the
capillary through a perpendicularly laser drilled hole. A CE instrument fitted with
a conductimetric detector has recently become commercially available.
A peculiar detection mode, suitable for measurement of ionic compounds which
neither absorb UV radiation nor are fluorescent or electrochemically
active, is
‘indirect detection’ (see Ref. [l] pp. 121-1260). The analytes are detected as ‘holes’
of UV absorbance (or fluorescence or electrochemical activity) of the background
electrolyte, which contains a UV absorbing (or fluorescent or electrochemically
active) ionic additive. This additive is displaced, to maintain the electroneutrality,
from the zones occupied by the analytes of interest, thus producing ‘negative’ peaks
at the detector.
Indirect detection is ‘universal’ (but often too non-specific) and does not require
instrumental hardware changes. Drawbacks are sensitivity, which is l-2 orders of
magnitude lower than with the corresponding ‘direct’ detection mode, and the
narrower range of linearity.
A variety of other detection techniques have been applied in CE including: laser
based thermo-optical
detection, refractive index detection, radioisotope detection
and notably, mass spectrometric (MS) detection, also in the configuration MS-MS.
Because of the importance of information provided by MS techniques in almost
all analytical environments, many efforts have been spent in the development of
interfaces between CE and MS (see Ref. [l] pp. 131- 145).
The electro-spray interface seems the most suitable for CE. The extremely low
flow rate of CE is easily handled by this interface, in which also a sheath flow can
be added in order to allow the composition of the electrospray liquid to be
independent of CZE buffer (often with high ionic strength). Also, ion-spray/atmospheric pressure ionization and continuous flow fast-atom bombardment have been
coupled to CE with success.
3. Separation techniques
Highly efficient separations can be performed in CE using different modes, such
as capillary zone electrophoresis (CZE), micellar electrokinetic chromatography
(MEKC or MECC), capillary electrochromatography
(CEC), capillary isoelectric
focusing (CIEF), capillary gel electrophoresis (CGE) and capillary isotachophoresis
(CITP). It is noticeable that the separation modes can be switched with no or
minimal changes in the instrumental hardware.
3.1. Capillary
zone electrophoresis
(CZE)
The theory of electrophoretic separations finds its most direct application in
CZE, but most of its fundamental
principles operate also in the other CE
separation modes.
In CZE the ionic analytes are separated according to a plain electrophoretic
principle, on the basis of the differences in their electrophoretic mobilities, resulting
in different migration velocities.
The migration velocity (c) of a charged particle in an electric field depends on its
electrophoretic mobility (1~~) and on the applied electric field (E)
in which
,LL,is described
by the following
equation:
p, = 4
67rtp
where y = ion charge, r = ion radius, ~1 = solution viscosity.
From the above equation, it is clear that molecular dimensions and charge are
the main factors influencing ion mobility. For weak acids and bases, it is obvious
that the charge is dependent on the individual pK values and on the pH of the
running buffer.
An additional fundamental factor operating in CZE (as well as in other CE
modes) is electroosmosis.
This phenomenon, causing an electrically driven flow of
liquid in the capillary, occurs in any electrophoretic
separation, but, while in slab
gel electrophoresis
it is a disturbing factor, in CE it is responsible for important
aspects of the separation process.
Electroosmosis
originates from the charges present on the inner surface of the
capillary resulting, in the case of fused silica capillaries, from the dissociation of
silanols (SO ~ ) occurring at pH values > 2. These negative charges at the wall
attract cations of the buffer, which create a double-layer and a potential difference
(zeta potential, i). The application of a voltage difference along the capillary,
causes the migration of these cations toward the cathode, which osmotically drag
water in the same direction.
The linear velocity of the electroosmotic
flow is described by the following
equation:
i:
1' ,.<, =
~
4Jlt1
i
“E
where: ;: = dielectric constant; i = zeta potential, q = solution viscosity.
In short, electroosmotic
flow, in most cases, is directed from the anodic to the
cathodic end of the capillary; it can be empirically measured by injecting a neutral
marker (e.g. acetone) and determining its migration time.
A specific feature of electroosmotic flow is that, being generated at the walls of
the capillary. its flow profile is almost flat, without the parabolic shape typical of
the pressure generated laminar flow, as it occurs in capillary liquid chromatography
(Fig. 3). It is clear that this characteristic is important in limiting the broadening of
the zones during their migration.
Considering
that a ‘normal’ set up of the CE instrument
necessitates that
injection is at the anodic end and detection is close to the cathodic end, and that the
electroosmotic
flow velocity is greater than the electrophoretic
velocity of most
analytes, it is clear that in CE cations, neutral species and anions can be detected
in the same run.
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The actual migration velocity of cations and anions will result from the algebraic
summation of the individual electrophoretic mobilities (in the same direction of the
electroosmotic flow or in the opposite, respectively) and the electroosmotic flow. All
neutral species will migrate together at the same velocity as the electroosmotic flow:
this means that non-ionic compounds, could not be separated, reaching the detector
in a single zone.
In the presence of the electroosmotic flow, the migration velocity follows the
equation:
where: tie0 and ,u~= mobilities
of EOF and of the analyte, respectively;
V = potential; L = capillary length.
A careful control of electroosmotic flow is necessary for obtaining the best
performances from CE: in fact it is fundamental in MEKC and CEC, often useful
in CZE, but not desirable (or acceptable only if slow) in CGE, CIEF and CITP. To
this aim, the capillary surface is passivated with modifiers physically adsorbed or
chemically bound or added to the buffer (dynamic coating). Most common coatings
are polyacrylamide, PEG, cellulose, surfactants, organic amines, etc.
Since CE is a highly efficient separation technique, any cause of zone broadening
is deleterious. Zone broadening, with resulting loss of efficiency, can be caused by
different factors, among which are worth mentioning: Joule heating, adsorption of
analytes onto the capillary wall, siphoning between the two electrode jars, injection
volume, high conductivity of injected samples (in comparison to that of the running
buffer) and extra column void volumes.
electroosmotic
pressure
Fig. 3. Schematic
capillary.
representation
flow profile
driven flow profile
of the electroosmotic
and the pressure
driven
liquid
flow
profiles
in a
Joule heating can be controlled by using narrow bore capillaries, low conductivity buffers and column thermostating.
Adsorption
of analytes onto the capillary
wall can be decreased by increasing the buffer ionic strength (by competing with the
charges of the wall), working
at low pH ( < 3) (in order to suppress silanol
ionization) or at high pH ( > 9) (in order to suppress the positive charges of
analytes), or using buffer additives or capillary surface coatings. In general, high
buffer concentrations
have been shown to reduce the adsorption of analytes to the
capillary walls.
In CZE, the main factors affecting the separation are buffer pH and composition.
Important characteristics
of the background
buffer are to have a good buffering
capacity at the chosen pH, low conductivity
(in order to allow the use of high
potentials without generating high currents) and low interferences with the detection system (basically, low UV absorbance).
In order to avoid peak distortion.
mobility of buffer ions should be matched with that of analytes. For most purposes.
phosphate, borate, citrate and phosphate/borate
buffers are suitable. Buffer additives can markedly affect selectivity and resolution. Zwitterionic
buffers are sometimes necessary in order to work at high concentrations
without excessive current.
From the principles mentioned above, it is clear that in CZE neutral substances
migrate at the same velocity of the electroosmotic
flow and can not be separated.
A different analytical approach, which applies also to non-ionic compounds, is
represented by MEKC,
which was introduced around the middle of 1980s by
Terabe [12-141. In this system, which is based on chromatography-like
separation
mechanisms, a micellar pseudo-stationary
phase is introduced in the buffer. which
retains the solutes according to the partitioning between the ‘stationary phase’ itself
and the mobile phase (i.e. the running bulfer).
In MEKC,
the flow of mobile phase is generated by electroosmosis,
which
produces a liquid flow, with a ‘plug-like shape’, which maintains the high efficiency
of separation.
The so-called ‘pseudo-stationary’
phase is obtained by addition to the buffer of
a surfactant at supramicellar concentration,
in most cases, sodium dodecyl sulfate
(SDS). The anionic SDS micelles migrate towards the anode, thus reducing the net
mobility of other solutes, depending on the degree of mutual interactions (mainly
partitioning)
they establish. A selectivity criterion based on partitioning of solutes
in the lipophilic core of the micelles is. thus, introduced. This operates with
non-ionic substances in a ‘quasi chromatographic’
mode, the micelles acting as the
stationary phase and the electroosmotic flow of buffer (often containing additives)
the mobile phase. In short, polar molecules will elute faster than hydrophobic’
compounds, within a ‘time window’ determined by the mobility of the electroosmotic flow (to) and that of the micelles (t,,,) (Fig. 4).
For neutral solutes, the only difference from chromatography
is that the ‘stationary phase’ in MEKC has its own migration velocity (t,,), which is measured by
using a water-insoluble
dye (e.g. Sudan III or Sudan IV) completely included in the
micelles.
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.
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pj
.
electroosmotic flow
micelle migration
Fig. 4. Schematic representation of a MEKC electropherogram: t, = time of migration of a compound
excluded from the micelles, simply driven by the electroosmotic flow; t, = time of migration of a
compound fully included in the micelles.
For charged solutes, the migration behaviour is dependent not only on the degree
of micellar solubilization,
but also on the electrophoretic mobility of the ionized
form of the compound in the aqueous phase.
Several surfactants are used in MEKC, but the most popular are: sodium dodecyl
sulphate (SDS), bile salts and hydrophobic-chain
quaternary ammonium salts.
Factors important in the modulation of selectivity are additives of the aqueous
buffer phase, in particular, organic solvents (methanol, isopropanol, acetonitrile)
have been used up to a concentration of 50% in order to reduce both the
hydrophobic interactions between analytes and micelles, similarly to reversed-phase
chromatography, and to improve efficiency.
3.3. Cupillury isotachophoresis(CITP)
CITP is another important separation mode in CE, which resembles classical
isotachophoresis. Although based on the electrophoretic mobilities of analytes, the
separation principle of this technique is absolutely peculiar. The ionic compounds
migrate in discrete zones at the same velocity between two ionic solutions, one with
the highest mobility (leading electrolyte) the other with the lowest mobility (terminating electrolyte), among all the ions. The different ionic substances migrate in
discrete zones after the leading electrolyte, according to the individual mobilities,
similar to carriages in a train (for a comprehensive presentation, see Ref. [15]).
3.4. Inclusion
conlpkues
~~ Cliird
scpr-rrtions
A field in which CE is already proving to be superior to liquid and gas
chromatography is the separation of enantiomers (seeRef. [l] pp. 257 -269). Chiral
separation can be achieved either in the presence of chirally selective solutes or by
means of chirally selective micelles. The chirally active solutes include cyclodextrins
(CDs), modified CDs, bile salts, crown ethers, Cu(Il)-L-histidine. Cu(II)-aspartame.
etc. Chiral surfdctants forming chirally functionalized micelles have also been used.
The separation is based on the reversible formation of sterically selective inclusion
complexes between the chiral selector and the analyte molecules during electrophoresis. Obviously, in order to allow chiral separation, the migration velocity of
the complexed molecules must be different from that of the ‘free’ drugs. Many
variables can be adjusted to optimize CE chiral separation, among which chiral
selector type and concentration, organic modifier concentration. ionic strength of
the buffer. temperature, applied field strength, etc.
Special mention should be made to CDs [40-421. These oligosaccharides have an
external hydrophilic surface and a hydrophobic cavity. in which they can include
other compounds by hydrophobic interaction. This inclusion mechanism is steritally selective, but also massselective; analytes must fit the size of the cavity, which
changes in dependence of the number of glucose units in their structure (6, 7 and
8, respectively for x-, p- and ;t-CDs). Native CDs are neutral and highly hydrophilic: therefore they migrate at the velocity of the electroosmotic flow. Newly
introduced charged CDs can display a counter migration in relation to the
electroosmotic flow. thus showing a retardation effect on the compounds they
interact with. Neutral CDs, when added to micellar solutions, selectively decrease
the interactions of hydrophobic compounds with the micelles.
Another. alternative possibility for achieving chiral selectivity is the dericatization of the analytes with chiral reagents followed by separation of the resulting
diastereomers.
Among CE separation modes. CEC is that which most resembleschromatography (see Ref. [l] pp. 194- 195). In fact, a chromatographic stationary phase is
contained (mostly packed) in the capillary and interacts with the solutes according
to the usual chromatographic separation mechanisms.The main peculiarity of CEC
is that the flow of the mobile phase (i.e. run buffer) is generated by electroosmosis.
The plug shape of the liquid flow and the absenceof pressurepulsations (usual with
reciprocating pumps) represent clear advantages over the pressure driven flow.
Thus, higher separation efficiency can. in general. be obtained. With chirally
selective packings, efficient chiral separations are accomplished.
However, the current stage of development of CEC is lower than that of the
other CE separation modes and its practical application is still limited.
In the analysis of drugs, CE has mainly been applied as MEKC. CZE and,
sometimes, as CITP and CEC. Other separation modes popular in CE. such as
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capillary gel electrophoresis and capillary isoelectric focusing are suitable for the
separation of biopolymers, but have not yet found application in forensic toxicology.
4. Applications
of CE in forensic toxicology
The analysis of drugs, in both pharmaceutical preparations and body fluids,
represents one of the most rapidly growing application areas of CE. Specific
applications in the field of illicit or controlled drugs have appeared only a few years
ago and are still limited, but tend to increase rapidly. For this reason, the present
review can not be fully updated, but simply gives a glimpse of the potential of this
novel analytical technology, until now fairly esoteric in analytical toxicological
disciplines.
An excellent review on the principles of CE and its use for the analysis of seized
drugs, by IS. Lurie [16], has appeared in a recent book dedicated to the analysis of
illicit and misused drugs, edited by J.A. Adamovics.
To the best of our knowledge, Weinberger and Lurie [17], in 1991, first applied
CE and particularly MEKC, to the analysis of illicit drug substances. The authors
used 50 pm i.d. bare silica capillaries, 25-100 cm in length, and a background
buffer consisting of 85 mM SDS, 8.5 mM phosphate, 8.5 mM borate, at a pH of
8.5, which was added with 15% acetonitrile. The applied voltages were 25-30 kV;
detection was by UV absorption at 210 nm. Under the described conditions, it was
possible to separate with high efficiency, acidic and neutral impurities in illicit
samples of heroin. In general, relative standard deviation (RSD) for migration
times was w 0.5% and for areas and peak heights 448%. However, with the late
peaks (migration time > 40 min) analytical precision was worse, a phenomenon
ascribed to the inconsistent evaporation of the organic modifier from the buffer
reservoirs. As could be expected, better resolution was achieved using a smaller
diameter capillary (25 pm instead of 50 pm i.d.), but the sensitivity was inferior,
because of the reduced optical path length. Also, fluorescence detection (ex.
wavelength 257 nm, em. 400 nm) was tested, achieving higher sensitivity for the
fuorescent analytes (more recently, to the same aim, krypton-fluoride
laser excitation at 248 nm has also been used; see Ref. [43]).
In the same paper, the separation of bulk heroin, heroin impurities, degradation
products and adulterants was demonstrated. The substances migrated in the
following order: morphine, phenobarbital,
6-monoacetylmorphine
(MAM),
3MAM, methaqualone, heroin, acetylcodeine, papaverine, noscapine. The CE separation pattern was different from that of reversed-phase HPLC. A sample of illicit
cocaine was also analyzed, with excellent peak shapes and resolution of benzoylecgonine, cocaine, cis- and trans-cinnamoylcocaine.
In comparison with HPLC, MEKC allowed the resolution of about twice as
many peaks; on the other hand, HPLC was more sensitive. However, using MEKC,
it was still possible to detect heroin impurities down to 0.2%.
To test the performances
of MEKC in the separation of a larger panel of
compounds
of potential forensic interest, a mixture of as many as 18 drugs.
including psilocybin, amphetamines, benzodiazepines, PCP, cannabinoids, etc. was
separated with baseline resolution [ 171.
The application of MEKC for the analysis of illicit heroin and cocaine was also
successfully demonstrated
by Staub and Plaut [18], who used 50 mM SDS in
phosphate/borate
buffer (10 and 15 mM, respectively, pH 7.8) containing 15% of
acetonitrile. In 25 min, paracetamol, caffeine. 6-MAM,
acetylcodeine, procaine,
papaverine, heroin and noscapine were separated in a heroin seizure and paracetamol, benzoylecgonine and cocaine in an illicit cocaine sample. On-line recorded UV
spectra of the peaks helped the identification of the sample components.
A more extensive study of MEKC for the analysis of heroin and amphetamine
seizures has recently been published by Krogh et al. [19]. A 50 cm x 50 pm i.d.
bare silica capillary was used; the running buffer was composed of 25 mM SDS in
phospate/borate
buffer (10 mM for each salt) pH 9, containing 5% acetonitrile.
Detection was by UV absorption at 214 nm. A test mixture of the main alkaloids
found in illicit heroin and heroin adulterants (on the whole, 14 compounds), as well
as a mixture of seven drugs structurally
related to amphetamine were completely
resolved in 15 min. RSD values of 0.5-l .9X and of 0.8992.23’% were obtained for
migration times (relative to crystal violet, the internal standard) in within-day
and
between-day reproducibility
tests, respectively. In quantitative studies, typical standard curves were linear in the range from 0.02 to 0.5 mgjml, with correlation
coefficients from 0.997 to 0.999. Typical RSD values in the analysis of illicit heroin
and amphetamine were in the range 2.0 -4.3%.
Samples could be injected every 13 min; the fused silica capillary was replaced
only after 500 injections to assure good reproducibility.
The authors concluded that
CE is a valuable complement to HPLC and GC in for the analysis of illicit drugs.
Trenerry et al. [20] reported the use of 50 mM cetyltrimethylammonium
bromide
(CTAB),
as micellar agent, as an alternative to SDS. The use of this cationic
micellar agent (50 mM in phosphate/borate
buffer 10 mM, pH 8.6 with 10%
acetonitrile, at - 15 kV (reversed polarity), UV detection at 280 nm) was reported
to give faster separations ( - 15 min) of heroin and related substances, in comparison to SDS. This method was successfully
tested with different illicit heroin
preparations in comparison to HPLC. A good correlation was found between the
two techniques. Precision of MEKC was slightly worse than that of HPLC. but the
resolving power of CE was much higher. allowing the complete resolution even of
very complex heroin seizures.
The same authors [21] reported an application of the above mentioned MEKC
method (only variations in acetonitrile from 10 to 7.5% and detection wavelength
from 280 to 230 nm) for the analysis of cocaine and related substances. Benzoylecgonine, cocaine, cis- and trn/r.s-cinnamoylcocaine
were separated in real
cocaine seizures and the results compared with HPLC and GC. MEKC and GC
correlated pretty well to each other, with similar RSD values, while HPLC gave
slightly higher results (no explanation was gi\:en).
224
F. Tagliaro
et al. / Forensic
Science
International
77 (1996)
21 l-229
According to the authors, these MEKC methods passed inter-laboratory
proficiency tests.
The separation of enantiomers of amphetamine, methamphetamine,
ephedrine,
pseudoephedrine, norephedrine and norpseudoephedrine with application to forensic samples was reported by Lurie [22], as a tool for investigations on the synthetic
methodologies. The author used derivatization with a chiral derivatizing reagent,
2,3,4,6-tetra-0-acetyl-/?-D-glucopyranosyl
isothiocyanate, followed by MEKC of
the resulting diastereomers. The 12 enantiomers of all the above mentioned
phenethylamines were resolved in a single run, using a bare silica capillary (48 cm
x 50 pm i.d.) and a buffer consisting of 20% methanol and 80% SDS solution (100
mM SDS, 10 mM phosphate/borate buffer, pH 9.0).
More recently, Lurie et al. [23] reported the chiral resolution of a number of basic
drugs of forensic interest, namely amphetamine,
methamphetamine,
cathinone,
methcathinone, cathine, cocaine, propoxyphene and various a-hydroxyphenethylamines with the use of neutral and anionic CDs. In this separation scheme,
resolution was optimized by varying the ratio of the neutral/anionic CDs. In fact,
both types of CDs have chiral selectivity, but anionic CDs, due to their negative
charge, display an electrophoretic counter migration and consequently higher
retarding effect on analytes.
Chiral resolution, could be achieved also in CEC, using an enantioselective
stationary phase, thus mimicking HPLC separations, but, reportedly, with higher
efficiency. This approach was adopted by Li and Lloyd [24], who used @,-acid
glycoprotein as stationary phase packed in fused silica capillaries of 50 pm id. for
the separation of the enantiomers of hexobarbital, pentobarbital, P-blockers and
other drugs.
As an alternative to MEKC, Chee and Wan [25], used CZE with 50 mM
phosphate buffer, pH 2.35 in a 75 pm id. 60 cm long bare silica capillary.
Detection was by UV absorption at 214 nm. The authors achieved the separation
of 17 basic drugs in only 11 min, including amphetamine, methamphetamine,
medazepam, lidocaine, diazepam, methaqualone, etc. Under these conditions, according to a plain electrophoretic mechanism of separation, drugs having lower pK,
values and consequently less positive charge, showed higher migration times.
However, the influence of other factors (according to the authors: molecular size,
tendency to interact with the column and ability to form doubly charged species)
hampered a clear correlation between pK, values and migration times.
The migration time RSD values were, in general, less than 1% and peak-area
RSDs were between 1.5 and 4.3%. Poorer reproducibility
was found for analytes
with very slow migration.
The described method was applied to test drug mixtures, but not on real forensic
exhibits. However, it proved suitable for the analysis of biological fluids, after
simple chloroform/isopropanol
(9: 1) extraction. The reported sensitivity was N 0.45
pug/ml, either in blood or urine.
According to the authors, CZE offered advantages over MEKC for drug
screening and, particularly, simple background electrolyte preparation and shorter
analysis times. The main limitation was the inahility to analyze acidic, neutral and
basic drugs all together.
Other research groups active in the field of forensic and clinical toxicology have
focused their attention on the use of CE for the toxicological investigation of
biological fluids.
Wernly and Thormann [26], using MEKC with an aqueous borate/phosphate
buffer, pH 9.1, containing 75 mM SDS, reported the qualitative analysis of many
drugs of abuse and metabolites in urine, including benzoylecgonine,
morphine,
heroin, 6-MAM,
methamphetamine.
codeine, amphetamine, cocaine, methadone,
methaqualone and benzodiazepines.
Urine purification (5 ml) and concentration
was by ‘double mechanism’ (cation exchange and reversed phase) solid-phase
extraction cartridges; the dried extract was then reconstituted with 100 1~1of buffer,
and directly injected, allowing a sensitivity of 100 ng:‘ml. Peak identification was
achieved on the basis of the retention times and of the on-line recorded UV spectra
of the peaks.
CE showed a sensitivity comparable to usual non-isotopic
immunoassays
and
was proposed for confirmation
testing, following usual screenings by enzyme-immunoassays.
Also, MEKC (50 mM SDS in phosphate-borate
buffer, pH 7.8) was used for
rapid and high-resolution
separations of barbiturates, namely barbital, allobarbital,
phenobarbital,
butalbital, thiopental, amobarbital. pentobarbital,
with on-column
multi-wavelength
detection [27]; achieving sensitivities in the range of the low
/Lgg/ml. Urine samples required extraction of barbiturates prior to analysis, but with
human serum barbiturates
eluted in an interference-free
window of the electropherogram, allowing these substances to be determined by direct sample injection
also.
MEKC
with 75 mM SDS in phosphate-borate
buffer, pH 9.1 allowed the
determination
of l l-nor-delta-9-tetrahydrocannabinol-9-carboxylic
acid, the major
metabolite of delta-9-tetrahydrocannabinol
in urine [28]. Using basic hydrolysis of
5 ml of urine, followed by solid phase extraction, it was possible to achieve the
sensitivity of lo-30 rig/ml. Again, the on-line recording of the peak spectrum. by
means of a fast scanning UV detector. gave an additional opportunity for confirmation.
The use of MEKC for determining morphine-3-glucuronide.
the major metabolite of heroin and other opiates, omitting any hydrolysis, was reported by Wernly
for confirmation of
et al. (1993), but the sensitivity. - 1 /lg;rnl, was unsatisfactory
the results of the enzyme-immunoassays
around the NIDA cut-off.
Benzodiazepines are also suitable for CE analysis. as reported by Schafroth et al.
[29]. who determined the major urinary compounds of eight common benzodiazepines
(flunitrazepam.
diazepam.
midazolam.
clonazepam.
bromazepam,
temazepam, oxazepam and lorazepam) by MEKC. using 75 mM SDS in phosphate/borate buffer, pH 9.3. After enzymatic hydrolysis and solid phase extraction
with commercial ‘double mechanism’ cartridges, they achieved a sensitivity better
than with the EMIT”’ immunoassay.
Due to the limited sensitivity of CE with UV detection, sample preparation and
pre-concentration
is crucial for applications
in biological samples. A stepwise
solid-phase extraction for human urine preliminary
to MEKC
analysis, using
226
F. Tagliaro
et al. / Forensic
Science
International
77 (1996)
21 i-229
commercial disposable cartridges with hydrophobic and ion-exchange interactions,
was described by Wernly and Thormann [30]. This clean-up procedure allowed up
to 50 times concentration of urine samples.
An overview of the strategies for monitoring drugs in body fluids (serum, urine
and saliva) by MEKC, focused on buffer selection and sample preparation procedures, including direct injection, ultrafiltration
and solid-phase extraction, has
appeared recently [3 11.
The comparative use of MEKC, CZE and CITP for the determination of drugs
in body fluids was reported by Caslavska et al. [32]. For MEKC a buffer composed
of 75 mM SDS, in a borate-phosphate buffer, pH 9.1 was employed. CZE analyses
were executed with a 33 mM phosphate buffer, pH 8.3. CITP was performed with
a leader of 10 mM HCl and histidine, pH 6 and a terminator composed of 10 mM
2-(N-morpholino)ethanesulphonic
acid and histidine, pH 6.0. Salicylate, paracetamol and antiepilectics were analyzed in serum and urine. In case of high drug
concentrations, body fluids could be injected directly or with simple pre-treatments,
such as dilution (urine) or ultrafiltration
(serum). However, in cases of drugs at the
low pg/ml level, extraction and concentration were required.
The authors concluded that MEKC and CZE were easier to be applied, whereas
CITP required careful selection of buffers and proved generally less sensitive. Only
with the ITP spike technique, in which baseline-resolved UV absorption peaks are
produced by bracketing the solute with discrete non-absorbing spacers, CITP could
approach the sensitivity of MEKC and CZE.
Hair analysis is gaining increasing popularity in the forensic toxicology environment, as a tool for investigating past, chronic exposure to illicit drugs, as is shown
by an increasing body of literature [33,34]. To this end, CE, in principle, could offer
several advantages over current techniques (GC, GC-MS, HPLC). mainly due to
the requirement of minimal sample. To the best of our knowledge, the few reports
published until now come only from our group.
Initally we adopted CZE separations, because this mode of operation appeared
simpler to be applied and replicated [35,36]. In order to allow rapid reconditioning
of the capillary after the washing cycles with alkali (0.1 M NaOH), a must, after
injection of complex biological extracts, we chose a basic CZE background buffer,
i.e. 50 mM borate, pH 9.2. For the quantitative determination
of cocaine and
morphine, detection was either at 200 nm wavelength (for both substances) or at
the absorbance maxima of each analyte (for cocaine 238 nm; for morphine 214 nm).
Tetracaine and nalorphine were chosen as I.S. for cocaine and morphine, respectively.
Excellent resolution and peak shape was obtained for both the analytes and the
respective I.S. The separations were highly efficient (up to 350 000 theoretical
plates) and repeatable (migration time RSDs: < 1% intra-day, < 3% inter-days),
the determinations accurate and precise (intra-day RSDs in the range 335%). Due
to the tiny volumes of sample injected (few nl) and the moderate concentration
sensitivity of CE with UV detection (in the order of the low pg/ml), the limit of
detection in hair was acceptable ( < 0.2 ng/mg) only if the hair extracts were
reconstituted
with lo--20 1’1, which is almost impracticable in real routine work
situations. More recently [37], the use of sample stacking techniques has allowed the
injection of about ten times larger volumes (i.e. - 60 nl), thus allowing reconstitution of the extracts with as much as 100 ~11of water, without sacrificing efficiency.
In the same paper, MEKC has been applied to hair samples using 0.1 mol/l SDS in
0.025 mol/l borate containing 20% methanol. The sensitivity achieved was only
slightly worse than with CZE, but the selectivity was much higher, due to the
additional ‘reversed-phase
like’ separation mechanism exploited by the SDS micelles.
5. Conclusion
In conclusion, CE has proved to be a new analytical tool suitable for the
investigation of both seized preparations of illicit drugs. as well as as biological
samples.
This technique displays several characteristics,
such as electrophoretic
and/or
chromatography-like
separation mechanisms, negligible consumption
of samples
and reagents, instrumental simplicity and multiple detection modes, which make it
unique on the scene of modern analytical technology. In particular, the possibility
of carrying out separations based on different physical-chemical
mechanisms (CZE.
MEKC and CITP) with the same instrumentation,
provides an interesting possibility of ‘internal’ confirmation
of the results.
The possibility of interfacing with mass spectrometry is an additional feature of
CE crucial in the forensic environment, which is ready to become commercially
available, mainly with electrospray
[38] or atmospheric pressure ionization [39]
interfaces.
Therefore. CE seems to have an important future in forensic toxicology as well
as in other fields of forensic science (e.g. explosive and gunshot residue analysis and
DNA fingerprinting)
and is already ready for use as a complement to the usual.
more consolidated analytical techniques.
Acknowledgements
The authors gratefully acknowledge the assistance of the personnel of the Library
Services of the Hospitals of Verona (Biblioteca Marani) and of the Glaxo Research
Center, Verona, Italy, in obtaining the bibliographic
material needed for this
review.
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