Electrophoresis 1998, 19, 2777-2790
Review
Franqois Couderc' Drug analysis by capillary electrophoresis and laser-induced
Elizabeth CaussC2 fluorescence
Christophe Ba~le'>~
This review briefly presents the different laser-induced fluorescence detectors,
'Universitt! Paul Sabatier, outlines the different dyes used to derivatize molecules which are used with
Laboratoire de Biologie capillary electrophoresisflaser-induced fluorescence (CE-LIF), and provides an
MoICculaire Eucaryotes, overview and current status of CE-LIF in drug analysis.
Toulouse Cedex, France
2CHU Rangueil, Biochimie,
Toulouse, France
3Zeta Technology,
Ramonville St. Agne, France
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . 2777
2 Laser-induced fluorescence detector . . . 2777
2.1 Principles . . . . . . . . . . . . . . . . . . . . . . 2777
2.2 Instrumentation . . . . . . . . . . . . . . . . . . 2778
2.2.1 Beckman LIF detector . . . . . . . . . . . . . 2778
2.2.2 Zeta LIF detector . . . . . . . . . . . . . . . . 2778
2.2.3 Perkin Elmer LIF detector . . . . . . . . . . 2778
2.2.4 Bio-Rad detector . . . . . . . . . . . . . . . . . 2778
2.3 Derivatization and useful laser . . . . . . . 2778
2.4 Sensitivity performance . . . . . . . . . . . . 2778
3 CE-LIF and drug analysis . . . . . . . . . . 2780
3.1 Direct analysis of fluorescent drugs by
CE-LIF . . . . . . . . . . . . . . . . . . . . . . . . 2780
3.1.1 Visible wavelength excitation . . . . . . . . 2780
3.1.1.1 Anticancer drugs . . . . . . . . . . . . . . . . . 2780
3.1.1.1.1 Free anthracyclines and metabolites . . . 2780
3.1.1.1.2 Doxorubicine immunoconjugate . . . . . . 2781
3.1.1.2 Antineoplastic drugs . . . . . . . . . . . . . . 278 1
3.1.2 UV wavelength excitation . . . . . . . . . . 278 1
3.1.2.1 Use of a 325 nm He-Cd laser . . . . . . . . 2781
3.1.2.1.1 Anticancer drugs . . . . . . . . . . . . . . . . . 278 1
3.1.2.1.1.1 Methothrexate . . . . . . . . . . . . . . . . . . . 278 1
3.1.2.1.1.2 Cis-platin and carboplatin . . . . . . . . . . 278 1
3.1.2.1.1.3 6-Mercaptopurine . . . . . . . . . . . . . . . . 2782
3.1.2.1.2 Vitamins . . . . . . . . . . . . . . . . . . . . . . . 2782
3.1.2.1.3 Hypnotic drugs . . . . . . . . . . . . . . . . . . 2782
3.1.2.1.4 Anti-inflammatory drug . . . . . . . . . . . . 2783
3.1.2.2 Other near-UV wavelengths . . . . . . . . . 2783
3.1.2.3 Low-UV laser wavelength . . . . . . . . . . 2783
3.1.2.3.1 Alzheimer disease drug . . . . . . . . . . . . 2783
3.1.2.3.2 Quality control of biopharmaceuticals . . 2784
3.1.2.3.3 Insulin . . . . . . . . . . . . . . . . . . . . . . . . 2784
3.1.2.3.4 Impurities of illicit heroin . . . . . . . . . . 2784
3.2 Immunoassay using CE-LIF . . . . . . . . . 2784
3.2.1 Competitive immunoassay . . . . . . . . . . 2784
Correspondence:Dr. F. Couderc, Universitk Paul Sabatier, Laboratoire de
Biologie MoMculaire Eucaryotes, 118 Route de Narbone, F-31062
Toulouse Cedex, France (Tel: +33-5-6117-5499; Fax: +33-5-6133-5886;
E-mail: couderc@ipbs.fr)
Abbreviations: CZE, capillary zone electrophoresis; IST, salicylate;
LOD, limit of detection; mAb, monoclonal antibody; NAP, naproxen
Keywords: Capillary electrophoresis/ Drug / Laser-induced fluorescence I
Review
0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998
Drug analysis by CE-LIF 2777
3.2.2 Noncompetitive assay . . . . . . . . . . . . . 2785
3.2.3 Applications . . . . . . . . . . . . . . . . . . . . 2785
3.2.3.1 Insulin quantitation . . . . . . . . . . . . . . . 2785
3.2.3.2 Human growth hormone . . . . . . . . . . . 2785
3.2.3.3 Morphine, phenyclidine . . . . . . . . . . . . 2786
3.2.3.4 Digoxin . . . . . . . . . . . . . . . . . . . . . . . 2786
3.2.3.5 Theophylline, ethosuximide, paracetamol,
salicylate, and quinine . . . . . . . . . . . . . 2787
3.2.3.6 Vitamin A . . . . . . . . . . . . . . . . . . . . . 2787
4 Conclusion . . . . . . . . . . . . . . . . . . . . . 2788
5 References . . . . . . . . . . . . . . . . . . . . . 2788
1 Introduction
During the past 10 years, instrumentation for electrokinetic
separations in fused-silica capillaries, known as capillary
electrophoresis (CE) has become available and its use in
clinical and pharmaceutical laboratories has begun to be
explored intensively [l-51. Most of the studies concerning
drugs have been completed with ultraviolethisible (UV-
Vis) direct or indirect absorption. Due to the need in
sensitivity, drug monitoring in body fluids necessitates the
use of laser-induced fluorescence (LIF) as a detector.
Although there are nearly a dozen companies manufactur-
ing electrokinetic capillary instrumentation [6], only four
are able to sell LIF detectors, whose price is twice that of
UV-Vis diode array detectors. Moreover, if laser-induced
fluorecence detection is a sensitive method, the lack of
natural native fluorescence of most molecules implies a
need to tag them with fluorescent dyes. These limitations
explain the small amount of literature in drug analysis using
CE-LIF. The purpose of this review is to present the
different optical arrangements of laser-induced fluorescence
detectors and to provide a brief overview and current status
of CE-LIF in drug analysis.
2 Laser-induced fluorescence detector
2.1 Principles
Conventional fluorescence has been successfully applied in
conventional high performance liquid chromato raph
yielding detection limits ranging from lo-" to 10- l molli y
L [7]. The sensitivity can be enhanced considerably by
increasing the power of the monochromatic light source,
since the fluorescence signal observed is proportional to the
0173-0835/98/1617-2777 $17.50+.50/0
2778 F. Couderc, E. Causse and C . Bayle
applied excitation power. For this purpose laser systems are
useful because the light source provides extremely intense
collimated light beams of highly monochromatic radiations
with output powers up to several watts. In laser-induced
fluorescence detection systems, excitation power may be
increased four to six orders of magnitude compared to
conventional fluorescence detection, resulting in a much
better signal-to-noise ratio, although background noise
signals may be elevated as well as a result of intensified
scattering of excitation radiation. Various laboratories have
developed different LIF detectors in the past 15 years. They
were first used with HPLC and transposed for CE. Several
authors have described the different arrangements used for
CE analysis [8]; for a detailed review on LIF detector
optical arrangements see Nouadje et al. [9]. Only four
companies sell the LIF detector: Beckman Instruments
(Fullertown CA, USA) on the PACE system, Zeta
Technology (Modular instrument), (Ramonville, France),
Perkin Elmer (Norwalk, CT, USA) and Bio-Rad (Hercules,
CA, USA) on the CE instrument [6]. The present review
addresses the optical arrangements of the four commercially
available instruments.
2.2 Instrumentation
2.2.1 Beckman LIF detector
The Beckman LIF detector is dedicated to the CE PACE
instrument. An optical fiber ended by a small on-end fiber
lens delivers the laser beam to the capillary and, to improve
sensitivity, a parabolic mirror reflects the fluorescence to
the photomultiplier tube (PMT). Not all of the laser power
is concentrated in the inner diameter of the capillary. An
appropriate filter kit minimizes the background noise. No
adjustment of the laser beam in front of the capillary is
allowed. This detector is used with several laser wave-
lengths, from 647 to 325 nm.
2.2.2 Zeta LIF detector
This detector is based on the colinear arrangement of an epi-
illumination microscope. The laser beam is deviated by a
dichroic mirror and focused on the inner diameter of the
capillary using a long focus distance lens and a small
sapphire ball lens. It illuminates the inner diameter of the
capillary and improves the illuminated volume. Moreover,
the very high numerical aperture of the 2 mm ball lens
provides a large amount of fluorescence which is partic-
ularly useful to increase the sensitivity of the detection [lo].
The colinear arrangement is also useful to minimize the
Raman background noise. The position of the laser beam
into the capillary can be easily adjusted by the user to
improve the sensitivity. Most of the laser wavelengths have
been used with this detector (647-325 nm).
2.2.3 Perkin Elmer LIF detector
This detector is dedicated to a capillary DNA analyzer or
sequencer (ABI PRISM, Perkin-Elmer, Norwalk, CT,
USA). It has a colinear arrangement. The 488 and 514 nm
wavelengths of an argon laser are colimated via a ball lens
on the capillary. A dichroic mirror and a filter kit make it
possible to obtain fluorescence without reflections of the
Electrophoresis 1998, 19, 2111-2190
laser wavelengths. A monochromator and a 64 X 152
charge-coupled device (CCD) camera, cooled by two Peltier
devices, record the fluorescence spectra along the analysis.
When using different fluorotides for DNA sequencing, the
emission spectra are used to identify the different tags. To
our knowledge, there is no limit of detection (LOD) in the
literature.
2.2.4 Bio-Rad detector
This two-excitation wavelength LIF detector, mainly used
for nucleic acids analysis, uses two laser tubes : an argon-
ion laser (488 nm) and an He-Ne laser (647 nm). The laser
beams are focused on two optical fibers which illuminate
the capillary. The fluorescence is collected orthogonally
and the fluorescence induced by the different lasers can be
recorded.
2.3 Derivatization and useful laser
The main limitation of capillary electrophoresis is the
unfavorable concentration detection limits frequently ob-
tained. Although the mass detection limits reported can be
withn the pmol to fmol range, the concentration detection
limits are normally significantly higher because the
injection and, consequently, the detection volumes are in
the nL range. This means that, in order to obtain the same
sensitivity as in liquid chromatography (LC), special
detection techniques, such as LIF, must be used to detect
the analytes better. It is often useful to derivatize the
molecules. Pre- and postseparation derivatizations have
been described previously in the literature for different
functional groups [ll]. Table 1 summarizes the different
tags used. One of the major problems of molecule labeling
is the reaction yield of the functional group. If reactions are
performed with concentrations around lo4 M, there are no
labeling problems. If derivatization is performed with a
concentration less than lop7 M, some authors found that
derivatization (e.g. for amino acids with FITC or dichlor-
otriazinylaminofluoresceine (DTAF)) was then not quanti-
tative [23]. On the other hand, Robert et al. [33]
demonstrated that they were able to derivatize amino acids
and catechol amines at concentrations of lo-'' M in 1 pL of
rat microdialysis using naphthalene dicardoxalyldehyde
(NDA). In conclusion, functional group labeling is a
problem that may be difficult to solve. Moreover,
fluorescence yield of the fluorophore can be reduced after
derivatization.
2.4 Sensitivity performance
The LIF pre- or postcolumn derivatization is often carried
out to detect various biological molecules. Unfortunately,
these methods can result in a number of problems when
detecting proteins and peptides. Because derivatization can
cause multiple labeling of analytes with more than one
derivatization site, as is the case with most proteins and
peptides, native fluorescence is then advantageous. A
varying number of derivatized sites will cause different
electrophoretic mobilities for the same analyte, resulting in
multiple peaks. Using native fluorescence alleviates this
problem of multiple labeling. With an excitation at 275 nm,
Electrophoresis 1998, 19, 2777-2790 Drug analysis by CE-LIF 2779

Table 1. Fluorescent markers used for different chemical functions with CE-LIF“’
~ ~

Function Reagent Compound Limit of Excitation References

detection wavelength
(LOW laser tube

Amines
AEOC Amino acids 0.11150 amol 351 nm, Ar+
Benzoin Arginine-containing 270 amol 325 nm, He-Cd
peptides
CBQCA Amino acids 9 zmol 488 nm, Ar+
Peptides 4-70 amol 488 nm, Ar+
Amino sugars 240 am01 488 nm, Ar+
CTSP Amino acids 1-7 am01 663 nm, Diode
DCC Amino acids, small 0.1 amol Red diode
peptides
DCCS Amino acids 100 amol 415 nm, Diode
DNSH Toxins 325 nm, He-Cd
DTAF Amino acids amol 488 nm, Ar+
FITC Amino acids 9zmol-lam01 488 nm, Ar+
Peptides 488 nm, Ar’
Fluorescamine Proteins 280 nm
Peptides 365 nm, Ai’
Amino acids 24 fmol 390 nm
Toxines
FMOC Amino acids 0.8 fmol, 0.2 amol 351 nm, Ar+
NBD-F Amino acids 488 nm, Ar+
Dipeptides 70 amol 488 nm, Ar+
NDA Amino acids 12 am01 442 nm, He-Cd
Catechol amines 0.5 amol 442 nm, He-Cd
Jeffamine polymers 442 nm, He-Cd
OPA Toxins 0.1 amol 325 nm, He-Cd
Amino acids 80 am01 325 nm, He-Cd
TRIC Amino acids 1 zmol 543 nm. He-Ne
Keto or aldehyde of carbohydrates
AC SugarsPolysaccharides 488 nm, Ar’
AHNS Monosaccharides 325 nm, He-Cd
3-ANDA Oligosaccharides 325 nm, He-Cd
5-ANSA Monosaccharides 325 nm, He-Cd
ANTS Oligosaccharides 325 nm, He-Cd
AP Mono/oligosaccharides 325 nm, He-Cd
APTS Mono/oligosaccharides 2 pmol 488nm, Ar+
AQ Charged 325 nm, He-Cd
oligosaccharide
CBQCA Mono/oligosaccharides 0.5 amol 488 nm, Ar+
TRSE Monosaccharides 17 zmol 514 nm
Carbonyl
MPD Peptides 390 amol 488 nm, Arf [15]
Carboxyl
AAF Phenoxy acid 3 amol 488 nm, Arf [481
herbicides
7-ANDA Phenoxy acid am01 325 nm, He-Cd [49]
herbicides
PDAM Dicarboxylic acid 488 nm, Ar+ [50]
MF Short and long 488 nm, Ar+ [51]
fatty acids
Sulfiydryl
Cys 5.3aIA 670 nm, diode [52]
Cys 5.4aIA 635 nm, diode [52]
MBrB Glutathione amol 355 nm, Ar+ [53]
NDA Glutathione am01 458 nm, Ar+ [54]
OPA 325 nm, He-Cd [55]

a) AAF, 5-(aminoacetamido)fluorescein; AC, 2-aminoacridone; ACE, affinity capillary electrophoresis;

AEOC, 2-(9-anthryl)ethylchloroformate; A H N S , 4-amino-5-hydroxynaphthalene-2,7-sulfonic
acid; 3-ANDA,
3-aminonaphthalene-2,7-disulfonicacid; 7-ANDA, 7-aminonaphthalene-1,3-disulfonic acid; 5-ANSA, 2-
aminonaphthalene-1-sulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonicacid; AP, 2-aminopyridine;
APTS, 9-aminopyrene-l,4,6-trisulfonate;AQ, 6-aminoquinoline; CBQCA, 3-(4-carboxybenzoyl)-2-quinoline
carboxaldehyde; CTSP, 9-cyano-N,N,N’-triethyl-N’-(5-succinimidy~oxycarbonylpentyl)pyronin;DCC, dicarbo-
cyanine reagent; DCCS, 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester; DNSH, 5-(dimethyl-
amino)naphthalene-1-sulfonehydrazine; DTAF, dichlorotriazinylaminofluorescein; FMOC, 9-fluorenyl-methyl
chloroformate; HGH, human growth hormone; MBrB, monobromobimane; MF, 5-bromomethym-fluorescein;
MPD, 4-methoxy-l,2-phenylenediamine;NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiazole; NDA, naphthalene
dicardoxaldehyde; OPA, o-phthaldialdehyde; PDAM, 1-pyrenyldiazomethane;PMT, photo multiplier tube;
TFUC, tetramethylrhodamine isothiocyanate; TRSE, 5-carboxytetramethylrhodamine
2780 F. Couderc, E. CaussC and C. Bayle Electrophoresis 1998, 19, 2777-2790

therefore, tyrosine and tryptophane can be detected without use of CE-LIF with chemically labeled drugs. Actually,
any need of chemical derivatization. With a KrF laser other most of the work on drugs using CE was done with CE and
workers [56, 571 used the 248 nm wavelength to im rove UV detection [3, 51.
the detection of tryptophane (7 X M, 7 X 10- M).
Po
Polycyclic aromatic hydrocarbons were also detected in
such a way [58, 591. The limitation in this field is the much 3.1 Direct analysis of fluorescent drugs by CE-LIF
higher cost of a UV laser compared to a conventional
visible laser. The powerful diode lasers and the improve- 3.1.1 Visible wavelength excitation
ment of frequency doubling or tripling will result in
inexpensive UV lasers in the next few months and, 3.1.1.1 Anticancer drugs
consequently, in the increased use of LIF detection.
3.1.1.1.1 Free anthracyclines and metabolites

3 CE-LIF' and drug analysis
The two main strategies to detect and quantitate drugs with
CE-LIF are direct analysis of fluorescent drugs or immuno-
assays using labeled antibodies. Of the large amount of
work published on labeling reactions, only few relate to the
One of the first studies on drugs using an argon-ion laser for
CE-LIF analysis was performed on anthracyclines. Rhein-
houd et al. [60] analyzed anthracycline in plasma using CE-
LIF with the 488 nm wavelength of an argon-ion laser.
Doxorubicin, daunorubicin and epirubicin fluoresce natu-
rally at 488 nm. The sensitivity obtained is 0.5-1 ng/mL.

1 ) KO

I I

Figure 1. (A) Distribution of possible conjugated species from monoclonal antibody

(mAb) BR96-doxorubicine immunoconjugateupon denaturation. (B) SDS-capillary gel
electrophoretic separation of mAb BR96 and mAb BR96-doxorubicin immunoconjugate
with laser-induced fluorescence detection. (a) mAb BR96-Dox immunoconjugateunder
denaturation conditions; (b) mAb BR96-Dox immunoconjugate under denaturation and on ion mn X O

reduced conditions; (c) mAb BR96 under denaturation and reduced conditions [64]. nme (rntn)
Eiectruphuresis 1998, 19, 2111-2790
The author presents an electropherogram of these anthracy-
clines from an extract of plasma sample taken 120 min after
administration to a patient treated intravenously with
epirubicin. Daunorubicin was used as an internal standard.
Hempel G. et al. [61] looked for doxorubicinol, a metabolite
of doxorubicine. Doxorubicinol was monitored over 8 h in
patients' plasma treated with doxorubicin. In the same
manner the authors [62] proposed the quantitation of
idarubicin and its metabolite idarubicinol in plasma. The
small sample volume of 100 pL is of particular advantage
for studies in pediatric oncology. The reproducibility of the
method has been shown to be sufficient for drug monitoring
or pharmacokinetic studies. The limit of quantification for
idarubicin in plasma is 0.5 ng/mL [62].
3.1.1.1.2 Doxorubicine immunoconjugate
The potential usefulness of monoclonal antibody conjugated
with antitumor drug provides a highly selective vehicle for
drug delivery to the tumor site [63]. Recognition of the
target cell and the consequent increase in the local
concentration of drug allows for site-directed killing of
the cell. Monoclonal antibodies possess many potential sites
for drug attachment. In an ideal situation, the immunocon-
w[ Syringe Pump #2
A Transfer
Ljne A
Syringe Pump X I Interface
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r-----l
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A
Time (seconds)
Figure 2. (A) Scheme of the on-line microdialysis/capillaryelectrophore-
sis system. (B) Typical electropherogramsof blood dialysates using the on-
line microdialysidCE system. Samples: (A) to a blank prior to dosing:
(B) 10 min after a 4 mgkg i.p. dose of SR 4233. Peak 1, SR 4233: 2, SR
4317 (main metabolite of SR 4233) [65].
Drug analysis by CE-LIF 2781
jugate would carry the maximum number of drugs per
molecule of antibody while retaining immunoreactivity and
specificity. Liu et al. [64] presented an analysis of
monoclonal antibody chimeric BR96-doxorubicine immu-
noconjugate by SDS-capillary gel electrophoresis and laser-
induced fluorescence. The authors used these separation and
detection means for the analysis of monoclonal antibody
chimeric BR 96 and the corresponding immunoconjugate
prepared between BR 96 and the anticancer drug doxo-
rubicin. These molecules were studied in their native,
denatured and reduced status. Six peaks were identified
following separation of the denatured bioconjugated
molecule. As expected, they corresponded to all possible
conjugated species. Analysis of the resulting fingerprint
maps indicated that the light, heavy, and light-heavy chain
conjugates were the predominant species. Figure 1 presents
the possible conjugated species from the immunoconjugated
species upon denaturation and the separation of the different
denaturated species [64].
3.1.1.2 Antineoplastic drug
In a study on coupled microdialysis CE, Hogan et al. [65]
monitored the antineoplastic drug SR 4233 in free-moving
rats using the 442 nm He-Cd laser. Figure 2A shows the
design of the on-line microdialysisicapillary electrophoresis
system. Typical electropherograms of blood dialysates
showed SR 4233 baseline-resolved from its main metabolite
within a 60 s run time (Fig. 2B). The CE-LIF procedure
permitted pharmacokinetic studies.
3.1.2 UV wavelength excitation
3.1.2.1 Use of 325 nm He-Cd laser
3.1.2.1.1 Anticancer drugs
3.1.2.1.1.1 Methothrexate
This was the first measurement using CE-LIF and a UV
wavelength. Roach et aE. [66] demonstrated that they could
quantitate methotrexate and 7-hydroxy-methotrexate in
clinical sam les. The detection limits were 5 X lo-'' M
and 2 X 10-g M, respectively. The extraction efficiency for
the drug and its metabolite from serum was around 85%
using Sep-Pak cartridge. Methotrexate and its metabolite
were oxidized with potassium permanganate to obtain
detectable fluorescent molecules.
3.1.2.1.1.2 Cis-platin and carboplatin
The micellar electrokinetic capillary chromatographic
separation of a dansylated mixture of normal nucleotides
and platinated cross-link adducts of d(pGpG) and d(pApG)
was realized by Sharma et al. [67]. CELIF was able to
detect 1 adduct/104 normal nucleotides/mg DNA by
fluorescence post-labeling assay. The enrichment of the
adduct, prior to dansylation, enhanced the detection limit to
1 adduct/107 normal nucleotides/mg DNA. Calf thymus
DNA reacted in v i m with cis-platin and carboplatin with
total input drug/nucleotide ratios of 0.05 and 0.5, respec-
tively. For 2 h 2780 human ovarian carcinoma cultured cells
were exposed to 25 mM cis-platin. The cells were incubated
2782 F. Couderc, E. Causd and C. Bayle Electrophoresis 1998, 19, 2111-2790

I I
8 16
3.1.2.1.2 Vitamins
Burton et al. [69] proposed the analysis of B6 vitamins
using MEKCLIF. In capillaries of 25-75 pm ID they
separated vitamin B6 and its metabolite. An efficiency as
high as 60000 theoretical platedm is obtained. Pyridoxic
acid, a metabolite of B6, was separated and quantitated in
human urine. Limits of detection were less than a picogram
injected and plates were achieved. This work was also done
by Kurosu et al. [70] using conventional fluorescence to
I
analyze the vitamin B6 group.

3.1.2.1.3 Hypnotic drugs

Zolpidem and zopiclone were analyzed using CE-LIF.
Zolpidem, which is an imidazopyridine molecule, has been
reported to be analyzed with its main metabolite without
extraction from urine. A 10 min separation provides an
LOD of 2 ng/mL [69]. A method has been developed also
for the stereoselective determination of zopliclone (cyclo-
I I pyrrolone drug ) and its main metabolites in urine. In this
I
16 case they are extracted from urine or saliva. The S-(+)-
8 12
enantiomer and its metabolites were always extracted in
higher amounts than R-(-)-enantiomen. Figure 4 [711
c>

-
'
1
I I I
0 12 16

Time (min 1

Figure 3. CELIF analysis of (a) dansylated 1,2 intra-strand cross-link of

d(pGpG) and d(pApG) and post-labeled DNA digests from 5 ng; (b) cis-
platin exposed A2780 human ovarian carcinoma cells and (c) mock-treated
A2780 cells [67].

with drug-free medium for 3 h before harvesting. The

identification of the cross-link adducts in the modified DNA
was confirmed by cochromatography with the authentic
markers. The same 1,Zintra-strand cross-link adducts were
induced by both cis-platin and its second generation drug
carboplatin. Figure 3 [67] shows the CE-LIF analysis of a
dansylated 1,Zintra-strand cross-link of d(pGpG) and l b
d(pApG) and postlabeled DNA digest of cis-platin-exposed
carcinoma cells.
I
3.1.2.1.1.3 6-Mercaptopurine
6-Mercaptopurine and its metabolite were separated and
detected with CE-LIF. The 6-thioguanine nucleotides were
oxidized in order to produce fluorescent species with
potassium permanganate. The limits of detection are around
2 X lo-'' M [68]. The method of extraction proposed by the
authors is simpler than that of the classical studies involving
0

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, I ,

in.00
I ~, ,
u.00
, 1 , ."""
14.00 [mnl
mercury cellulose resins or phenyl mercury adduct Figure 4. Electropherograrns of (a) human urine 6 h after oral
formation. administration of zopiclone and (b) blank urine [72].
Electrophoresis 1998,19, 2111-2790 Drug analysis by CE-LIF 2783

presents the separation graph of these compounds from a
human urine extract [72]. These drugs can be easily
quantitated.
3.1.2.1.4 Anti-inflammatory drug
Soini et al. [73] described a method to determine naxopren
(NAP) in serum. Sample preparation includes a simple
liquid-liquid extraction of 0.25 mL serum aliquots. After
solvent evaporation, samples were dissolved in methanol
and analyzed using CE-LIF. The method provides interfer-
ence-free, linear and highly reproducible results. A
quantitation limit of 3 fmol was achieved. Using warfarin
as internal standard improved the analytical precision
substantially. The CE-LIF method is 30 times more
sensitive than CE-UV detection. Using micellar electro-
kinetic capillary electrophoresis (MEKC), Albrecht et al.
[74] reported the separation of NAP from naproxen
covalently coupled to human serum albumin (NAP-HSA)
or to mannosylated serum albumin (NAP-HSA-MAN) and
the metabolite naproxen-lysine (NAP-LYS). An assay for
selective analysis of the different forms of NAP by direct
plasma injection was canied out with salicylate (IST) as
internal standard and detection by laser-induced fluores-
cence (Fig. 5). MEKC offers the advantage that free and
NAP-HSA-MAN
$ 9.5
N 3.1.2.3.1 Alzheimer disease drug
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ry -0.5
19.5
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covalently bound NAP can be differentiated in one run and
can be accurately monitored in microliter quantities of
plasma. In the same way, NAP was identified in liver and
kidney tissues [75]. Detection limits were 140 X lo-' m o m
of NAP and 7 X lo-' m o m of NAP-HSA [74].
3.1.2.2 Other near-W wavelengths
Pulsed nitrogen laser was used for banned substances in
sport. This type of laser provides enormous energy to be
concentrated into a small sample volume, for example a
modest 2.5 mJ, 10 ns pulse focused to a 75 pm diameter
spot results in an irradiance of 5.6 X 10' Wcm-'. This great
photon flux is capable of detecting a single molecule.
Pulsed lasers require gated detection devices for signal
recovery and accurate timing for synchronization of the
excitation-detection cycles. The triamterene LOD was
reported to be 3.1 mol, acebutolol 8.8 X mol,
bendroflumethiazide 1.8 X mol. Triamterene was
identified in urine [76].
3.1.2.3 Low-W laser wavelength
Only few studies have been performed using low-UV laser
wavelength for the CE-LIF study of drugs, due to the cost of
such a laser tube. Moreover, these studies were performed
on homemade laser detectors. We will summarize the
different applications.
NXX-066 (Fig. 6) [77], which is naturally fluorescent at
La,= 211 nm, has been studied using LIF detection with a
frequency-doubled argon-ion laser delivering a 244 nm

14.5
NAP-HSA-MAN
E
c
co
co
c?
u) 9.5
(u
0
3
u.
of
4.5 NAP
I

-0.5
0 2 4 6 a
Time (min)
Figure 5. Separation of different naproxen-protein conjugates WAF') after
direct injection of rat plasma supplemented with naproxen-human serum
albumin (NAP-HSA at 50 m&), naproxen-mannosylated human serum
albumin (NAP-HSA-MAN at 125 m&) and salicylate (IST, at 50 mg/L).
Data obtained via injection of calibrators (without plasma) are depicted in Figure 6. CELIF, analysis of NXX-066 at 1 X M (NXX-066 is a
the insets. candidate drug intended for treatment of Alzheimer disease) [77].
2784 F. Couderc, E. Causst and C. Bayle
0 2 4 a a 10
Time (mln)
Figure 7. Electropherogram of purified anti-TAC (28 mg/mL or 180 p~
WI.
wavelength (5 mW). An LOD of 5.1 X M was achieved
and the separation was performed using the isotachophore-
sis principle. The improvement in sensitivity of CE-LIF was
55 times higher than CE-UV.
3.1.2.3.2 Quality control of biopharmaceuticals
The use of living organisms for the manufacture of
pharmaceuticals by recombinant DNA technologies
presents a unique approach and promises to transform
numerous aspects of human health care. Although many
analytical techniques developed in the biological sciences
have been applied to quality control of biopharmaceuticals,
the necessity of accuracy, sensitivity, resolution and speed
appears to be found in using CE-LIF. All the biopharma-
ceuticals studied by Lee et al. [78] can be detected at
subnanomolar level with linear dynamic ranges of at least
three orders of magnitude. CE-LIF, using the 275.4 nm line
of an argon-ion laser, can determine impurities in 'purified'
biopharmaceuticals present in amounts less than 0.01% of
the major component. Figure 7 [78] shows the analysis of
the purified anti-T activating antigen (TAC) protein.
3.1.2.3.3 Insulin
Insulin, intensively studied with imunoassays, was also
analyzed using CE-LIF and direct fluorescence detection
I A
[ I
io io io io SO do
Time (min)
Elecirophoresis 1998,19,2111-2190
with the 275.4 nm argon-ion laser [79, 801. In these studies,
a nonbonded poly(ethy1eneoxide)-coated capillary was used
to achieve the separation. An insulin concentration as low as
73 am01 can then be detected.
3.1.2.3.4 Impurities of illicit heroin
Lurie et al. [81] described the separation and the detection
of acidic and neutral impurities in illicit heroin. Separations
were achieved using charged cyclodextrine-modified
MEKC. Phenanthrene-like heroin impurities exhibit high
native fluorescence under krypton-fluoride laser excitation
(248 nm). The limit of LIF detection for one of these solutes
(acetylthebaol) was 1.8 ng/mL, 500 times more sensitive
12
than UV. Figure 8 [81] is a comparison of UV detection at
260 nm and LIF detection of a refined Southwest Asian
heroin. This methodology is applicable to analysis of both
crude and refined heroin.
3.2 Immunoassay using CE-LIF
The study of antibody-antigen reactions by CE was
demonstrated by Nielsen et al. [82]. Immunoassays have
become the method of choice for a wide variety of clinical
pharmaceutical and chemical analyses owing to their ability
to quantitatively measure small amounts of analyte (anti-
gen) in a complex mixture whose constituents are often at
much higher concentrations [83,84]. The high selectivity of
such an assay is achieved by using antibody reagents
possessing high discriminatory power and high binding
affinity for the analyte of interest even in complicated
matrices. The high sensitivity of immunoassay analysis is
due to the use of specific labels covalently linked to one of
the reactants, antibody or antigens, for detection. The CE
immunoassays which were reported are either in the direct
or the competitive format. These assays are all solution-
phase assays in which all assay components are present in a
dissolved form during incubation. To date, only preliminary
feasibility studies have been presented, with no single CE
assay being completed so that it fulfills the rigorous
analytical standards set for immunoassays.
3.2.1 Competitive immunoassay
Figure 9 [85] illustrates the basics of the assay procedure.
First, a predetermined amount of labeled antigen is added to
Figure 8. Comparison of (A) UV detection
at 260 nm and (B) LIF detection with a
krypton-fluoride laser for CE analysis of a
0 10 20 30 40 50 60 refined Southwest Asian heroin hydrochlor-
Time (min) ide exhibit [81].
Electrophoresis 1998, 19, 2777-2790
antigen
-~
I C. CZE with LIF detection
I Minutes
Figure 9. Competitive immunoassay procedure [85].
the sample containing unlabeled antigen. The sample may
contain either a known amount of antigen in the case of
standard curve generation (calibration) or an unknown
amount in the case of a sample. The amount of labeled
antigen is determined by the desired dynamic range, which
is typically sixfold to tenfold and centered around the
labeled antigen concentration [86]. An internal standard is
added to the sample. After the mixture is thoroughly mixed,
a predetermined limited amount of specific antibody is
added. In the ensuing reaction labeled and unlabeled
antigens compete for the limited amount of antibody, with
the important result that the amount of unbound labeled
antigen increases as the concentration of unlabeled antigen
in the sample increases. The free-solution interaction of
antibody and antigen is usually quite rapid, and CE analysis
can be performed in 1-10 min after the reaction is initiated.
The purpose of the CE analysis is to separate bound from
free labeled antigen so that the area of the free labeled
antigen peak can be determined. In this way, the concen-
tration of unlabeled antigen can be interpolated from the
standard curve of concentration versus area. The standard
curve generated by the assay exhibits a sigmoidal shape,
typical of concentration response after a semi-log trans-
formation. At high concentrations of antigen most of the
labeled antigen is displaced from the antibody binding sites
and the signal of unbound labeled-antigen is large, whereas
the signal of the labeled complex is low. At low
concentrations of antigen the situation is reversed, most of
the labeled antigen is complexed to the antibody and thus
Drug analysis by CE-LIF 2785
the labeled antigen signal is low whereas the labeled
complex signal is large. Only the linear range between the
two extremes is used for quantitation. Determination of the
Concentration is performed by interpolation from the semi-
log curve.
3.2.2 Noncompetitive assay
If antibody determination in a sample is needed, then a
known amount of fluorescently labeled antigens can be
added to the sample. The labeled antigen will form a
complex specifically with the antibody. If there is an excess
of fluorescently labeled antigen, then the formation of
complex will be quantitative and directly dependent on the
amount of antibody present in the mixture. The CE
separation will reveal two zones by LIF detection, one
corresponding to the complex and the other to free
fluorescently labeled antigen. If the complex is stable on
the time scale of separation, then it should be possible to
quantify the amount of antibody in the mixture based on the
amount of complex formed and/or the decrease in the
amount of free fluorescently labeled antigen.
3.2.3 Applications
3.2.3.1 Insulin quantitation
The first study of the CE-LIF assay system was reported by
Schultz and Kennedy [87] for the analysis of insulin. They
compared noncompetitive and competitive immunoassays
using an FITC-labeled insulin and an He-Cd 442 nm
excitation laser. Rapid separations were obtained. The
relative standard deviation was 6.3% for the complex peaks
and 2.9% for the free FITC-insulin peak. The detection limit
for insulin by this competitive assay was about 3 n~ or 420
zmol injected. Figure 10 (A) presents the rapid separation of
a mixture of FITC-insulin and anti-insulin Fab; (B), shows
the calibration curve of competitive assay; and (C) is the
calibration curve for a noncompetitive assay. Approxi-
mately the same results were obtained with both techniques.
Later these authors developed an assay using FITC-
derivatized insulin as a labeled antigen and the Fab
fragment of an anti-insulin monoclonal antibody as the
immunoreagent. Reproducibility and reliability of the assay
were improved when capillary zone electrophoresis (CZE)
capillaries were coated with a neutral hydrophilic polymer.
The assay had an average relative standard deviation of
3.4% and a detection limit of 3 nM or about 6 fg injected.
Using this type of assay, the insulin content of single islets
of Langerhans was determined to be 35 k 4 ng, which is in
good agreement with values found in the literature. An
electropherogram is presented in Fig. 11 [88]. Tao and
Kennedy [89] developed an on-line competitive immuno-
assay for insulin to monitor insulin concentration in a
flowing stream. This system was used for flow injection
analysis to determine the content of single islets of
Langerhans. It could also be used for sensor-like monitoring
~91.
3.2.3.2 Human growth hormone
Proteins have been widely studied using immonoassay CE-
W 1901 or CE-LIF [91, 921. Shimura and Karger [93]
2786 F. Couderc, E. Causse and C. Bayle Electrophoresis 1998, 19, 2111-279O

A
l2 ' 1I
:2
,
5

t
k
0 0.4 0.8 1.2
0 20 40 60 Time (minl
TIME (sec)
0
Figure 11. Competitive assay performed on single islet extracts. The solid
0 electropherogram comes from a sample containing 100 n~ FITC-insulin
m (peak 2), 100 n M Fab and 50 n~ internal standard (peak 1). The dashed
$
Y
electropherogram is from an extract of single islet [MI.
m 0.60
a
n. the analyte as a complex after separation of the excess free

-
-u probe by capillary electrophoresis. As an example, a Fab
a
.--N fragment from a mouse monoclonal antibody of human
m 0.30 growth hormone (HGH) was labeled with tetramethylrhod-
E
L
a
amine-iodoacetamide at a hinge region thiol group (Fig.
a 12A, [93]). Samples were mixed with the purified labeled
antibody fragment, and the associated complex was
0.00
separated by capillary isoelectric focusing with LIF
0.00 100.00 200.00 detection. Methionyl recombinant HGH was determined at
C [insulin], (nM) the detection limit level of 5 X M. Mono- and
dideamidated variants of methionyl recombinant HGH were
1 .oo detected simultaneously with the nondeaminated form of
m
2
Y
the antigen as separate peaks (Fig. 12B).
GI
al
a 3.2.3.3 Morphine, phenyclidine
u 0.50
a,
-N
.-
m
These two drugs were analyzed using a multianalyte drug
assay (competitive assay) in urine using CE-LIF [94].
50 Morphine derivatized with Cys5 and Cys 5.5 (Biological
z Detection Systems, Pittsburg, PA, USA) was used to tag
0.00
phenyclidine. The laser was an He-Ne 632.8 nm; a home-
0.00 100.00 200.00 300.00 made LIF detector was used. Figure 13 [94] shows the
electropherograms for different amounts of the two drugs.
[F(ab)l, (nM) The relative electrophoretic mobilities of Cys 5-morphine
Figure 10. (A) Rapid separation of mixture containing 50 IIM of FITC- and Cys 5.5-phenyclidine are shown in Fig. 13 [94]; Cys 5
insulin and 25 mi of anti-insulin Fab. Peaks 2, 3 and 5 are HTC-insulin. in the mixture acts as a reference peak [95].
Peaks 1 and 4 are due to the formation of a complex of Fab with FITC-
insulin in peaks 2 and 5. (B) Calibration curve for determination of Fab by
the competitive assay. (C) Calibration curve for determination of Fab by
the noncompetitive assay [87].
3.2.3.4 Digoxin

Digoxin was analyzed using CZE. The tracer was labeled

presented a sensitive microscale analytical procedure called with B-phycoerythrin [96, 971. The electropherograms of
affinity probe capillary electrophoresis. One of the two different concentrations of this drug in human serum are
pieces, which can form a biospecific complex is labeled presented in Fig. 14 [96]. A four-point calibration curve
with a fluorescent dye. The affinity probe is used to detect obtained by the authors is also reprinted.
Electrophoresis 1998, 19, 2111-2790
Pepsin
___,
Mcruptocthylamioc
gab'
CootroUcd aridation
Mooothiol Fnb'
Tctramcthylrhodaminc
iodoacctamidc
IR-Fab'
Figure 12. (A) Preparation of labeled antibody fragment. (B) Simultaneous
detection of met-rbGH variants. An artificial mixture of met-rhGH, the
monodeamidated variant (N149D) and the dideamidated variant (N149D,
N152D), each at a concentration of 10 ng/mL, was subjected to affinity
probe capillary electrophoresis [93].
3.2.3.5 Theophylline, ethosuximide, paracetamol,
salicylate, and quinine
For drugs whose concentrations are too low for detection or
that coelute with endogeneous compounds (such as
proteins) coming from a biological sample, it is necessary
before sample injection to add a sample preparation step
which includes drug extraction or removal of proteins by
ultrafiltration or precipitation. Steinmann and Thormann
[95] recently developed a competitive immunoassay for
simultaneous quantitation of theophylline, ethosuximide,
paracetamol, salicylate, and quinine using fluorescent
Drug analysis by CE-LIF 2787
-
2
ai C
1
IC
A A
I
C
2D
A A
0
Figure 13. Electrophergrams demonstrating titration of labeled morphine
and phencyclidine (PCP) with antisera. (A) Peak 1, Cy5.5-F'CP, 2, CyS-
morphine; R, CyS diacid used as internal standard at 10, 10,2 and 2 nmoV
L, respectively. (B) Mixture in A plus antiserum to morphine. Peak C, CyS-
morphme-antibody complex. (C)Mixture in A plus antiserum to PCP.
(D) Mixture in A plus both antisera [94].
tracers, MEKC, and LIF detection. The originality of their
study was the use of commercial tracers from fluorescence
polarization immunoassay kits from Abbott (Abbott Labo-
ratories, Abbott Park, IL,USA). Figure 15 [95] presents the
simultaneous determination of paracetamol and salicylate.
Tracer and antibody reagents for paracetamol and salicylate
were mixed with serum prior to analysis. The electrophero-
gram presented in panel A depicts the data issued from a
mixture containing the zero level calibrators of both reagent
kits. The free paracetamol traced (Tp 8.3 min), the
paracetamol antibody-tracer complex (Cp 10 min), and
the salicylate antibody-tracer complex (Cs 10.5 min) were
monitored as sharp peaks whereas the free salicylate tracer
appeared as a broad double peak after 10.73 min.
Application of the calibrator instead of a blank (B-D) will
further reveal the independent performance of the two
immunoassays.
Beside immunoassay affinity, affinity capillary electro-
phoresis (ACE) was used to quantitate sulfonamide drugs in
body fluids. Tim et al. [98] proposed a direct method to
determine picomolar tight binding sulfonamide drug con-
centrations in human urine and plasma. Fluorescein-labeled
human carbonic anhydrase I1 was used as a fluorescent
reagent to detect such drugs. The assay was performed by
adding a known amount of labeled human carbonic
anhydrase 11 to a sample containing an unknown amount
of target drug. The amount of drug bound to human
carbonic anhydrase I1 is determined by CE-LIF using a
charged competitive ligand that strongly shifts the migra-
tion of human carbonic anhydrase I1 unless it is blocked by
the sulfonamide drug. The dorzolamide detection limit was
16.5 PM in aqueous solutions and 62.5 PM in biological
fluids.
3.2.3.6 Vitamin A
Ma et al. [99] performed a fast microassay of serum vitamin
A. An He-Cd laser set at 325 nm was used for excitation,
2788 F. Couderc, E. CaussB and C. Bayle Electrophoresis 1998, 19, 2171-2190

D-bphyco L Ab
comp1.x

-
.:,F::Kt
A

-0.1

3
4

C
5 0.4
CP CP

-0.1
7.5 9.0 10.5 12.0 7.5 9.0 10.5 12.0

D
Time lrninl Time [mini
I
0 I 2 3 4 5 6 1 9
lime in m ~ n m ~ i
Figure 15. MEKC of salicyclate and paracetamol. Electropherograms
obtained with a mixture containing solution S (antibody preparation) and
solution T (fluorescein labeled drug) and serum calibrator. (A) Zero
calibrator of both drugs; (B) 100pglmL salicylate and 20 pg/mL
B
PO , I
paracetarnol; (C) zero calibrator of salicylate and 20 pg/mL paracetamol,
and (D) 100 pg/mL salicylate and zero calibrator of paracetamol. Ts, free
salicylate tracer; Tp, free paracetamol tracer; Cs, salicylate antibody-tracer
complex; Cp, paracetamol antibody-tracer complex [95].

chemical functions exists, very few studies about drugs

have been performed with the derivatization of the
molecule. Only immunoassays and native fluorescence
were used. A promising approach using laser-induced
resonance energy transfer to achieve high sensitivity
detection for drugs has been proposed recently [loll. An
important aspect of this kind of detection is that the analytes
do not need to be fluorescent or made fluorescent. Ideally,
5
0 2 4 6 8 10 12
the acceptor molecule should be soluble in an aqueous
Dig& mncntration.ng/d buffer, have minimal absorbance at the maximum absorp-
tion of the analyte, show fluorescence only in the presence
Figure 14. (A) Electropherogram of CE-LIF of digoxin assay mixture with of the analyte molecule, and be capable of accepting energy
serum calibrators. Peak 1, antigen-antibody complex of the Fab and with high efficiency from a wide range of substances.
digoxin-B phycoerythrin (D-BP); 2, D-PB; 3, unknown fluorescent Terbium and Europium are the ideal elements for fitting the
substance derived from serum. (A) 0.42 nglmL digoxin; (B) 2.72 nglrnL
digoxin; (C) 5.21 ng/mL digoxin; (D) 10.42ng/mL digoxin. (B) Calibration criteria of an ideal acceptor. The authors studied aspirin and
curve for digoxin assay by CE-LIF [97]. its metabolite monitoring Tb3+ luminescence at 547 nm.
They increased the sensitivity of conventional detection by
and the fluorescence of the vitamin A-retinol binding
1000, even if the LOD remains quite high, within the M

protein complex was monitored. About 8 nL of serum range. The increased popularity of capillary electrophoresis
sample were injected for direct analysis without any sample and of the different LIF detectors commercially available,
preparation. Subfemtomole levels of vitamin A in human with more and more laser wavelengths to be used, will
blood could easily be detected. A virtual relationship was enhance the increased development of drug assays with CE-
obtained for the retinol concentration for 28 serum samples, LIF in the future.
19 dried venous blood samples, and nine capillary dried Received May 18, 1998
blood samples collected on filter paper. The absolute
detection limit for retinol was below 3 p g L [loo].
5 References
[ l ] Thormann, W., Molted, S., Caslavska, J., Scbmutz, A., Electro-
4 Conclusion phoresis 1994, 15, 3-12.
[Z]Thormann, W., Zhang, C. X., Schmutz, A., Ther. Drug Monit. 1996,
In this review the different studies involving the use of CE- 18, 50&520.
LIF for drug analysis were examined. Even though a large __
131 Von Heeren, F.. Thormann, W., Electrophoresis 1997, 18,
amount ofhifferint markers to tag a great number i f 24 15-2426.
Electrophoresis 1998, 19, 2171-2190
[4] Thormann, W. in: Wong, S. H. Y., Sunshine, I. (Eds.), Handbook of
Analytical Therapeutic Drug Monitoring and Toxicology, CRC
Press, Boca Raton 1997, 1-19.
[5] Flurer, C. L., Electrophoresis 1997, 18, 2427-2437.
[6] Gareil, P., Spectra Anal. 1997, 198, 32-37.
[7] Lingeman, H., Underberg, W. J. M., Takadate, A,, Hulshof, A,,
J. Liq. Chromatogr. 1985, 8, 789-874.
[8] Nouadje, G., Amsellem, J., Couderc, B., Verdeguer, P., Couderc, F.,
Progress in HPLC-HPCE, 1997, 5, 49-72.
[9] Nouadje, G., Simton, N., Nertz, M., Couderc, F., Analusis 1996, 24,
360-370.
[lo] Hernandez, L., Escalona, J., Joshi, N., Guzman, N., J. Chromatogr.
1991, 559, 183-196.
[ l l ] Bardelmeijer, H. A., Waterval, J. C. M., Lingeman, H., Van't Hof,
R., Bult, A., Underberg, W. J. M., Electrophoresis 1997, 18,
2214-2227.
[12] Engstriim, A., Anderson, P. E., Josefsson, B., Anal. Chem. 1995,67,
3018-3022,
[13] Cobb, K. A,, Novotny, M. V., Anal. Chem. 1992, 64, 879-886.
Cobb. K. A.. Novotnv. M. V., Anal. Biochem. 1992, 200, 149-155.
Arriaga, E. A,, Zhang, Y., Dovichi, N. J., Anal. Chim. Acta 1995,
299, 319-326.
Liu, J., Cobb, K. A,, Novotny, M., J. Chromatogr. 1990, 519,
189-197.
Liu, J., Hsieh, Y., Wiesler, D., Novotny, M. V., Anal. Chem. 1991,
63, 408412.
Liu, J., Shirota, D., Novotny, M., Anal. Chem. 1991, 63, 413-417.
Fuchigami, T., Imasaka, T., Anal. Chem. Acta 1993,282, 209-213.
Mank, A. J., Yeung, E. S., J. Chromatogr. A 1995, 708, 309-321.
Higashijima, T., Fuchigami, T., Imasaka, T., Ishibashi, N., Anal.
Chem. 1992, 64, 711-714.
Wright, B. W., Ross, G. A,, Smith, R. D., J. Microcol. Sep. 1989,1,
85-89.
Lalljie, S. P. D., Sandra, P., Chromatographia 1995, 40, 519-526.
Cheng, Y. F., Dovichi, N. J., Science 1988, 242, 562-564.
Wu, S., Dovichi, N. J., J. Chromatogr. 1989, 480, 141-155.
Zhao, J. Y., Waldron, K. C., Miller, J., Zhang, J. Z., Harke, H.,
Dovichi, N., J. Chromatogr. 1992, 608, 239-242.
Gump, E, L,, Monnig, C. A,, J. Chromatogr. A 1995, 715, 167-177.
Albin, M., Weinberger, R., Sapp, E., Moring, S., Anal. Chem. 1991,
63, 417422.
Chan, K. C., Janini, G. M., Muschik, G. M., Issaq, H, J.,
J. Chromatogr. 1993, 653, 93-97.
Ruyters, H., van der Wal, S . , J. Liq. Chromatogr. 1994, 17,
1883-1 897.
Beijersten, I., Westerlund, D., J. Chromatogr. A 1995, 716, 389-399.
Nickerson, B., Jorgenson, J. W., J. High Resolut. Chromatogr.
Chromatogr. Com. 1988, 11, 878-881.
Robert, F., Bert, L., Denoroy, L., Renaud, B., Anal. Chem. 1995,67,
1838-1844.
Amankwa, L. N., Scholl, J., Kuhr, W. G., Anal. Chem. 1990, 62,
2189-2 193.
Chan, K. C., Janini, G. M., Muschik, G. M., Issaq, H. J.,
J. Chromafogr. 1993, 622, 269-273.
Zhau, J., Chen, D., Dovichi, N. J., J. Chromatogr. 1992, 608,
117-120.
Greenaway, M., Okafo, G. N., Camilleri, P., Dhanak, D., J. Chem.
SOC.Chem. Commun. 1994, 14, 1691-1692.
Camilleri, P., Harland, G. B., Okafo, G., Anal. Biochem. 1995, 230,
115-122.
Stefansson, M., Novotny, M., J. Am. Chem. SOC. 1993, 115,
11573-1 1580.
Stefansson, M., Novotny, M., Anal. Chem. 1994,66, 11341140.
Chiesa, C., ONeill, R. A,, Electrophoresis 1994, 15, 1132-1 140.
Chen, F. A,, Evangelista. R. A., Anal. Biochem. 1995,230,273-280.
Evangelista, R. A., Lui, M., Chen, F. A,, Anal. Chem. 1995, 67,
2239-2245.
Chiesa, C., H o ~ i t hC.,
, J. Chromatogr. 1993, 645, 337-352.
Liu, J., Shirota, O., Wiesler, D., Novoiny, M., Proc. Natl. Acad. Sci.
USA 1991, 88, 2302-2306.
Liu, J., Shirota, O., Novotny, M., J. Chromatogr. 1991, 559,
223-235.
Zhao, J. Y., Diedrich, P., Zhang, Y., Hindsgaul, 0.. Dovichi, N. J.,
J. Chromatogr B 1994, 657, 307-313.
Drug analysis by CE-LIF 2789
Jung, M., Brumley. W. C., J. Chromatogr. A or B 1995, 717,
299-308.
Mechref, Y., El Rassi, Z., Anal. Chem. 1996,68, 1771-1777.
Schneede, J., Mortensen, J. H., Kvalheim, G., Ueland, P. M.,
J. Chromatogr. A 1994, 669, 185-193.
Zuriguel, V., Causd, E., Bounery, 3. D., Nouadje, G., Simkon, N.,
Nertz, M., Salvayre, R., Couderc, F., J. Chromatogr. A 1997, 781,
233-238.
Mank, A. J. G., Lingeman, H., Gooijer, C., J. High Resol.
Chromatogr. 1994, 17, 797-798.
Hogan, B. L., Yeung, E. S., Anal. Chem. 1992, 64, 2841-2845.
Orwar, O., Fishman, H. A., Ziv, N. E., Scheller, R. H., Zare, R. N.,
Anal. Chem. 1995, 67, 42614268.
Dette, C., Watzig, H., Electrophoresis 1994, 15, 763-768.
Chan, K. C., Janini, G. M., Muschik, G. M., Issaq, H. J.,
J. Chromatogr. A 1995, 718, 203-210,
Chang, H. T., Yeung, E. S., Anal. Chem. 1995, 67, 1079-1083.
Nie, S., Dadoo, R., Zare, R. N., Anal. Chem. 1993, 65, 3571-3575.
Janini, G. M., Muschik, G. M., Issaq, H. J., J. Chromarogr. B 1996,
683, 29-35.
Rheinhoud, N. J., Schroder, E., Tjaden, U. R., Niessen, W. M. A,,
Noever de Brauw, M. C., van der Greef, J., J. Chromatogr. 1990,
516, 147-155.
Hempel, G., Schulze-Westhoff, P., Boos, J., Proceeding of HPCE
98, Orlando, FL, 1998.
Hempel, G., Haberland, S., Schulze-Westhoff, P., Mohling, N.,
Blaschke., G.., Boos. J.. J. Chromatoer. B 1997. 698. 287-292.
[631 Sharkey, R. M., Blumenthal, R. 6, Hansen, H. J., Goldenberg,
D. M., Cancer Res. 1992, 52, 5693-5698.
[64] Liu, J., Abid, S., Lee, M. S . , Anal. Biochem. 1995, 229, 221-228.
[651 Hogan, B. L., Lunte, S. M., Stohaugh, J. F., Lunte, C. E., Anal.
Chem. 1994, 66, 596402.
[661 Roach, M. C., Gozel, P., Zare, R. N., J. Chromatogr. 1988, 426,
129-140.
[671 Sharma, M., Jain, R., Ionescu, E., Slocum, H. K., Anal. Biochem.
1995,228, 307-311.
[68] Rahel, S . R., Trueworthy, R., Stobaugh, J. F., J. High Resolut.
Chromatogr. 1993, 16, 326-327.
[691 Burton, D. E., Sepaniak, M. J., Maskarinec, M. P., J. Chromatogr.
Sci. 1986. 24. 347-351.
Kurosu, Y., Satou, Y., Udagawa, K., Kurioka, S., Vitamins 1992, 66,
91-100.
Hempel, G., Blaschke, G., J. Chromatogr. B 1996, 675, 131-137.
Hempel, G., Blaschke, G., J. Chromatogr. B 1996, 675, 139-146.
Soini, H., Novotny, M. V., Riekkola, M. L., J. Microcol. Sep. 1992,
I, 313-318.
Albrecht, C., Reichen, J., Visser, J., Meijer, D. K. F., Thormann, W.,
Clin. Chim. 1997, 43, 2083-2090.
Alhrecht, C., Thormann, W., J. Chromatogr. A 1998, 802, 115-120.
Gonzalez, E., Montes, R., Laserna, J. J., Anal. Chim. Acra 1993,282,
687-693.
Enlund, A. M., Schmidt, S . , Westlund, D., Proceedings of HPCE 96,
Orlando 1996.
Lee, T. T., Lillard, S. J., Yeung, E. S . , Electrophoresis 1993, 14,
429438.
Tong, W., Yeung, E. S . , J. Chromatogr. B 1996, 685, 35-40.
Tong, W., Yeung, E. S . , J. Chromarogr. B 1997,689, 321-325.
Lurie, I. S., Chan, K. C., Spratley, T. K., Casale, J. F., Issaq, H. J.,
J. Chromarogr. B 1995, 669, 3-13.
Nielsen, R. G., Richard, E. C., Santa, P. F., Sharknas, D. A.,
Sittampalam, G. S . , J. Chromatogr. 1991, 539, 177-185.
Chen, F.-T., Stemberg, J. C., Electrophoresis 1994, 15, 13-21.
Schmalzing, D., Nashabeh, W., Electrophoresis 1997, 18,
21842193.
Prichett, T. J., Evangelista,R. A,, Chen, F.-T., J. Capil. Electrophor.
1995,3, 145-149.
Delaage, R.,Barbet, J., in: Masseyeff, R. F., Albert, W. H., Staines,
N. A. (Eds.), Methods of Immunological Analysis, VCH, New York
1993, I , 493497.
Schultz, N. M., Kennedy, R. T., Anal. Chem. 1993, 65, 3161-3165.
Schultz, N. M., Huang, L., Kennedy, R. T., Anal. Chem. 1995, 67,
924-929.
Tao, L., Kennedy, R. T., Anal. Chem. 1996.68, 3899-3906.
Schmerr, M. J., Goodwin, K. R., Cutlip, R. C., Jenny, A. L.,
J. Microcol. Sep. 1995, 7, 521-527.
2790 F. Couderc, E. Caws6 and C. Bayle Electrophoresis 1998, 19, 2111-2190

[91] Chen, F. T., J. Chromatogr. A 1994, 680,419423.
[92] Reif, 0. W., Lausch, R., Scheper, T., Freitag, R., Anal. Chem. 1994,
66, 40274033.
[93] Shimura, K., Karger, B. L., Anal. Chem. 1994, 66, 9-15.
[94] Chen, F. T., Evangelista, R. A., Clin. Chem. 1994, 40, 1819-1822.
[95] Steinmann, L., Thormann, W., Electrophoresis 1996, 17,
1348-1356.
[96] Chen, F. T., Stemberg, J. C., Elecrrophoresis 1994, IS, 13-21.
[97] Chen, F. T., Pentoney, S . T., J. Chromatogr. A 1994,680,4251130.
1981 Tim, R. C., Kautz, R. A., Karger, B. L., Proceeding of HPCE '98,
Orlando, FL 1998, P519.
1991 Ma, Y., Wu, Z., Fur, H. C., Lamrni-Keefe, C., Craft, N. E.,
J. Chromatogr. 1993, 616, 31-37.
[lo01 Shi, H., Ma, Y., Hurnphey, J. H., Craft, N. E., J. Chromatogr. B
1995, 665, 89-96.
[loll Petersen, J. R., Bissel, M.G ., Mohammad, A. A,, J. Chromatogr. A
1996, 744, 31-44.