An overview of capillary electrophoresis: Pharmaceutical ...

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J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
1
2
An overview of capillary electrophoresis: Pharmaceutical,
biopharmaceutical and biotechnology applications
Bhupinder Singh Sekhon
PCTE Institute of Pharmacy
, Near Baddowal Cantt. (Ludhiana) -142021, India.
Email:sekhon224@yahoo.com
Received: November 08, 201
1;
Accepted: November 30, 201
1
ABSTRACT
Capillary electrophoresis (CE) is an establishing separation technique of choice effective for a wide spectrum of
analytes, ranging from small inorganic ions to DNA macromolecules as it provides reliable data, requires
minimal sample preparation and offers a high degree of automation. CE is an alternative to more traditional
methods such as gel electrophoresis and liquid chromatography and is employed to detect both high and low
affinity molecular interactions, and separation of both charged and non-charged molecules. CE technique covers
different modes that essentially rely on a high voltage electric field being applied over a solution which is held in
a capillary tube.
In CE, the separation is based on charge, size and frictional force and it offers fast separations
with exceptional efficiency
. CE has pr
oved to be an efficient and versatile appr
oach for
physicochemical
characterization of bioactive molecules and resolution for charged substances such as biomolecules, low molecular
weight basic or acidic drugs and ions. CE is now an established technique in several areas of analysis and is
capable of detecting millimolar to nanomolar binding interactions and offers the benefits of a powerful and
proven technology applied to drug discovery screening on a wide variety of targets such as enzymes, membrane
receptor domains, structural proteins, nucleic acid complexes, bioactive peptides, protein-protein interactions
and antibodies. CE can also determine chiral purity in pharmaceuticals and can be successfully used to support
aspects of early drug discovery and drug development testing, analysis of protein-based pharmaceuticals and
routine quality control of marketed pharmaceuticals. CE has found application in the analysis of complex
carbohydrates and in the increased associated activities of CE-MS techniques in the biotechnology and
biopharmaceutical industry
.
CE-fr
ontal analysis pr
ovided a method on the interaction of drugs with plasma
pr
oteins.
V
arious applications of hyphenated capilliary electrphor
esis techniques include characterization of
quantum dots and quantum dots-conjugated biological molecules. Immunoaffinity capillary electrophoresis has
been reported as a versatile tool for determining protein biomarkers in inflammatory processes and for total
Immunoglobulin E quantification in serum. CE technology has been valuable for the comprehensive
characterization of macr
omolecules, used both as biologics and in pr
oteomic study
, and to analyze and characterize
therapeutic pr
oteins in biotechnology
.
Keywords:
Capillary electrophoresis, glycoproteins, chiral separation, quality control, pharmaceutical, biopharmaceutical,
biotechnology applications.
INTRODUCTION
Electrophoresis is the migration of ions or solutes
under the influence of an electric field. Capillary
electrophoresis (CE) is electrophoresis performed in a
capillary tube and is the most efficient separation technique
available for the analysis of both large and small molecules
1-3
. CE is an analytical technique that separates ions based
on their electrophoretic mobility with the use of an applied
voltage. The electrophoretic mobility is dependent upon
the char
ge of the molecule, the viscosity
, and the atom’
s
radius.
The rate at which the particle moves is directly
proportional to the applied electric field—the greater the
field strength, the fast the mobility
. In CE, the thin
dimensions of the capillaries greatly increased the surface
to volume ratio, which eliminated overheating by high
voltages.
The potential of CE as a high-resolution
analytical separation technique was first described in 1981
using glass capillary column and aqueous buffer to
separate charged compounds
4
. CE technology employs
narrow-bore capillaries to perform electrophoresis in free
solution or nonconductive medium, such as a gel and can
detect both high and low affinity molecular interactions
covering both large and small molecules
5-7
. The typical
voltages employed in the region of 10-30 kV across a
capillary with a bore diameter of about 25-100mu give
rise to operational currents in the region of 10-100
microamps.
A
brief history of CE is given in
T
able 1.
J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
1
3
Y
ear
Discovery event
1967
First high voltage CE system (with rotating 3 mm internal diameter capillaries.
1981
CE was
first described in 1981 by Jorgenson and Lukacs. These researchers demonstrated highly efficient
electrophoresis separations of peptide by performing electrophoresis in narrow bore capillaries filled with buffer,
normally in the range from 25 to 100 µm of internal diameter
.
1983
The first paper on capillary gel electrophoresis was published in the 80s by Hjertén.
1984
Isoelectric focussing was introduced in capillary format
1984
Micellar electrokinetic chromatography
, a hybrid of electrophoresis and chromatography was introduced by
T
erabe.
1985
Anionic micelles were used as a pseudostationary phase to separate neutral compounds.
1987
Karger and co-workers addressed the application area of protein separations in polyacrylamide gels in the presence
of sodium dodecyl sulphate (SDS).
1987-
The first automated CE instrument was introduced commercially under the name Microphoretic 1000 by
1989
Microphoretic Systems (Sunnyvale, CA, USA) in 1987. Second by
Applied Biosystems (Foster City
, CA) in
1988. In 1989, Beckman Instruments introduced the first fully automated capillary electrophoresis instrument (P/
ACE™ 2000)
1987
Capillary electrochromatography (CEC) in open tubular columns and detailed theoretical analysis of
electrochromatography and technique development.
1988
Kar
ger

s
group shows DNA
separations of single strandedoligonucleotides with gel-filled capillaries.
1989
Bob Brownlee and coworkers introduced the first commercial instrument with on column UV/VIS detection,
automatic injection and computerized data analysis for rapid, high-resolution CE separation.
1989
Beckman Coulter (then Beckman Instruments) introduced the first fully automated capillary electrophoresis
system — the P/ACE™ 2000.
1990-
Karger s group shows DNA separations with sieving polymers on DNA restriction fragments.
1993
1990
V
arious workers presented the reports of capillary array electrophoresis for DNA
sequencing.
1991
The capillary column packed with particulate stationary phase, which generally consists of inorganic particles
(silica beads) allows high loading capacity
.
1991
Grossman expands work with sieving polymers.
1991
Everaerts demonstrated the use of isotachophoresis in open tubular fused silica capalliaries.
1992
Mathies
et al.
developed the approach of multiplexing the separation of DNA sequencing fragments by means of
the use of arrays of capillaries. This was a significant and necessary development in capillary based DNA sequencing,
leading to a 96-column format as the method of choice for production-scale work.
1994
The potential of CEC in the analysis of mixtures relevant to the pharmaceutical industry was realized by Smith and
Evans.
1995
The most important CE techniques and their use for the analysis and characterization of proteins and DNA.
1996
Rapid and automated genetic typing using capillary electrophoresis for the analysis of short tandem repeats
(STRs).
2001
Conditions for typing PCR-amplified short tandem repeat (STR) loci by capillary electrophoresis were investigated
using the
ABI Prism 310 Genetic
Analyzer
.
2004
High-resolution CE provides an alternative for rapid identification of
Mycobacterium
species.
2005
An overview over quantitative measurements performed by CE-MS.
2008
CE coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates
to biological molecules.
2009
Papers detailing the use of affinity capillary electrophoresis in examining binding parameters between biological
species.
2011
Beckman Coulter introduced the CESI 8000 High Performance Separation-ESI Module with OptiMS technology
consisting of the first commercial CESI sprayer combined with new capillary electrophoresis instrumentation
specifically designed for mass spectrometry (MS). CESI is the integration of CE and ESI by a dynamic process
Reference
8
9
10
11
12
13
14,15
16
17,18
19
20
21
22-24
25-28
29
30
31
32
33
34
35
36
37
38
39
40
41
T
able. 1.
A
brief history of CE.
J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
1
4
within a single device;
Agilent
T
echnologies Launches
Automated Electrophoresis System to
Accelerate Sample
and Library QC for Next-Generation Sequencing
2011
Dr
. Stellan Hjertén, the “Father” of Capillary Electrophoresis, received the 201
1 award at CASSS’

Annual
A
ward
Dinner on November 3, 201
1 in Oakland, California. Dr
. Hjertén has done pioneering work in many branches of
separation science, introducing agarose gels for electrophoresis and polyacrylamide gels for chromatography and
using them for chromatography and electrophoresis.
2011
Rogers
et al
. demonstrated the use of microstructured fibres for capilliary zone electrophoresis, taking advantage
of their relatively high surface-to-volume ratio and the small individual size of each channel to effect highly efficient
separations, particularly for dye-labelled peptides.
42
43
Advantages over HPLC
Qualities of CE such as high efficiency of separation,
short analysis time, remarkably low injection volume, the
possibility of chiral separation without the need of special
expensive columns, ease of operation and a variety of
detection systems have facilitated acceptance of this
technology
44
. One of the major advantages of CE over
other separation technique is the ability to separate both
charged and non-charged molecules. CE offers several
advantages over high-pressure or high-performance liquid
chromatography (HPLC).
These include simplicity
, rapid
analysis, automation, ruggedness, different mechanisms
for selectivity
, and low cost
45
. Separation mode in HPLC
is determined by column stationary phase and mobile
phase, however
, separation mode in CE is determined
only by buffer and one type of capillary can be used for
different modes of separations.

CE also uses aqueous
rather than organic solvents and is thus environmentally
friendlier than HPLC
46-48
.
V
arious separation modes of CE
The differential movement for migration of ions by
attraction or repulsion in an electric field separate the
components of a mixture using an electric field v = Eq / f,
where v = velocity of molecule E = electric field, q = net
charge of molecule, and f = friction coefficient. CE is an
umbrella term for many different methods. The process
of capillary electrophoresis is actually a generic term and
depending on the types of capillary and electrolytes used,
the technology of CE can be segmented into several
separation techniques. Examples of these include:
i
)
Capillary zone electrophoresis (CZE)
i
i
)
Nonaqueous capillary electrophoresis (NACE)
iii)
Capillary gel electrophoresis (CGE)
i
v
)
Capilliary electrokinetic chromatography (CEKC)
/Capillary electrochromatography (CEC)
v
)
Micellar electrokinetic chromatography (MEKC)
v
i
)
Microemulsion electrokinetic chromatography
(MEEKC)
vii)
Capillary isoelectric focusing (CIEF)
viii)
Capillary isotachophoresis (CITP)
i
x
)
Pressurized capillary electrochromatography
(pCEC)
x
)
Affinity capillary electrophoresis (ACE)
x
i
)
Imunoaffinity capillary electrophoresis (IACE)
xii)
Nano capillary electrophoresis (NCE)
xiii)
Microchip-based capillary electrophoresis
(Microchip-based CE)
xiv)
Microfluidic capillary electrophoresis (MFCE)
i)
Capillary zone electrophoresis (CZE)
In CZE, analytes move in the electroosmotic flow
but separate into bands due to differences in their
electrophoretic mobilities (ì). Differences in
µ
results each
analyte’
s overall migration velocity slightly dif
ferent, and
difference in migration velocity leads to separation.
Electrophoretic mobilities are roughly a function of analyte
charge and frictional and size differences. CZE is a
separation technique based on charge: mass ratio and is
the most commonly used mode of separation in CE.
Cations with the largest charge-to-mass ratios separate
out first, followed by cations with reduced ratios, neutral
species, anions with smaller charge-to-mass ratios, and
finally anions with greater ratios. This technique is able
to separate both cations and anions in the same run under
high electroosmotic flow (EOF) conditions. The separation
in CZE relies on a constant field strength across the
capillary and the pH of the buffer solution, and then
J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
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5
ultimately on the differences in analytes’ electrophoretic
mobilities that result in different velocities of migration of
ionic species
49,50
. In CZE, different types of chiral
selectors including cyclodextrins, oligo- and
polysaccharides, crown ethers, macrocyclic antibiotics,
ligand exchange systems and proteins are employed
51,52
.
Protein isoforms with small pI differences were rapidly
analyzed using CZE with new dynamic coatings
53
. The
Sebia CAPILLAR
YS™ CDT
system proved a simple
and reliable method to screen for Congenital Disorders
of Glycosylation in pediatric and adult patients with an
unexplained clinical syndrome
54
. CZA method was
successfully applied to determination of sparfloxacin in
pharmaceutical tablets
55
.
i
i
)
Nonaqueous capillary electrophoresis (NACE)
Nonaqueous capillary electrophoresis (NACE) is a
process similar to CZA which is useful in the separation
of compounds that are insoluble in water as it relies mainly
on the use of organic solvents. The viscosity and dielectric
constants of organic solvents affect both sample ion
mobility and the level of electro osmotic flow
. NACE is
undergoing rapid development
56
as the use of non-aqueous
medium allows additional selectivity options in methods
development
57
. Chiral separations of pharmaceutical
racemic amines were achieved by NACE using various
cyclodextrins
58-61
. Researchers demonstrated UV
- and
mass-spectrometric detection of enantiomeric amines
resolved by NACE
62
.
A
simple enantioselective method
based on CE using cyclodextrins as chiral selector was
found to be appropriate for controlling pharmaceutical
formulations containing isradipine enantiomers
63
. The
NACE proved as the best CE technique out of CZE,
MEKC and MEEKC employed for the determination of
impurities in bromazepam, as its low solubility in water
was easily overcome
64
. NACE has been widely used in
pharmaceutical and the recent advances in the applications
of NACE were reported
65
.
iii)
Capillary gel electrophoresis (CGE)
Capillary gel electrophoresis (CGE) ability to separate
molecules accurately and consistently relies on a constant
field strength across the capillary and the pH of the buffer
solution, and then ultimately on the charge/mass ratio of
the molecules. CGE separation is based on the difference
in solute size as the particles migrate through the gel.
CGE
is used for the size and shape-based separation of
biological macromolecules such as oligonucleotides, DNA
restriction fragments and proteins
66-70
.
An overview of
uncommon replaceable matrices (gels)
containing
saccharides, newly developed acrylamide-based gels and
thermoadjustable viscosity polymers, namely triblock
copolymers and grafted polyacrylamide for CGE were
reported
71
. Researchers have described the development
of a method that uses CGE to analyze mixtures of inorganic
polyphosphate (P
i
)
n
. Resolution of (P
i
)
n
on the basis of n,
the number of residues of dehydrated phosphate, is
accomplished by CGE using capillaries filled with solutions
of poly(N,N-dimethylacrylamide) to exploit sieving
properties of low-viscosity solutions of PDMA and indirect
detection by the UV absorbance of a chromophore,
terephthalate, added to the running buffer
72
. Researchers
developed a picoliter-scale partial translational
spontaneous injection approach for high-speed protein
separation under sodium dodecyl sulphate (SDS)-CGE
mode. In this context, a high-speed CE system for protein
separation based on a short capillary and slotted-vial array
has the advantages of simple structure, ease of building
without the requirement of microfabricated devices,
convenient operation, and low cost. Five fluorescein
isothiocyanate labeled proteins including myoglobin, egg
albumin, bovine serum albumin, phosphorylase b, and
myosin were separated within 60s with an effective
separation length of 1.5
cm. Moreover
, the separation
speed and separation efficiency of the present system
were comparable to those of most microchip-based
capillary electrophoresis systems for protein separation
73
.
CGE was used for the rapid separation of SDS-
protein complexes according to their molecular masses
and a linear relationship between migration time and log
molecular masses was found
74
. The application of a SDS-
CGE method for the analysis of triple 2/6/7 and double-
layered 2/6 rotavirus-like particles , candidate vaccines
against rotavirus infection has been reported. SDS-CGE
analysis of rotavirus-like particles resulted in peaks that
could be attributed to the viral proteins (VP2, VP6 and
VP7) according to their apparent molecular mass
75
.
Researches described microchannel-based SDS-CGE of
proteins and demonstrated its speed and high resolution
76
.
SDS-CGE is not widely accepted in proteomic research
primarily due to the difficulties in identifying the well-
resolved proteins. Poly(tetrafluoroethylene) membranes
are excellent materials for collecting SDS-CGE-separated
J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
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proteins. Scientists demonstrated the SDS washing bound
to the collected proteins and identified these proteins on
membrane with MALDI-T
OF-MS
77
.
A
capillary sodium
dodecyl sulfate gel electrophoresis (cSDS-CGE) method
for the analysis of monoclonal antibody (mAb1) under
reduced and non-reduced conditions was found to be
linear
, accurate, and precise in the range of 0.25–3.0 mg/
mL protein concentration
78,79
.
CGE can be used as an accurate and high throughput
diagnostic procedure for mutational status in the CEBP
A
gene identifying not only the same mutational frequency
as in the published reports but also the
T
AD2C
polymorphism in addition
80
.
iv) Capillary electrokinetic chromatography
(CEKC)/ Capillary electrochromatography (CEC)
CE technique with development of CEKC and
recently CEC are separation technique based on a
combination of liquid chromatographic and electrophoretic
separation methods. CEC offers both the efficiency of
CE and the selectivity and sample capacity of packed
capillary HPLC. CEC capillaries accommodate relatively
large amounts of sample due to high surface area of these
packing materials, thereby
, making detection a simpler
task than it is in CE
81
. Both CEKC and CEC enable
separation of neutral analytes by partition between two
phases. In CEKC, stationary phase is a moving pseudo
stationary phase, however
, a fixed stationary phase in
capillary column in case of CEC
82
.
Capillary electrochromatography (CEC)
CEC is a separation technique which combines liquid
chromatography and CE. It can be divided into three parts:
i) CEC open-tubular capillary electrochromatography
(OT
-CEC) in which the stationary phase is immobilized
on the inner walls of the capillary
, ii) CEC with packed
columns, and iii) CEC with monolith
82
. The separation
mechanism is a packed column similar to
chromatography
.
Although CEC and HPLC are very
similar
, the main dif
ference is the flat flow velocity profile
generated by the electroosmotic flow in CEC, in
comparison to the pressure driven parabolic flow velocity
profile in HPLC. Using CE capillaries packed with LC
stationary phases, CEC offers the load ability and
selectivity of LC and the high efficiency of CE
83-87
. CEC
is used to concentrate samples prior to separation by CZE
and have been used for pharmaceutical applications
including analysis of steroid isomers
88,89
.
The fast separation of chiral
!
-blockers on a cellulose
tris(3,5-dimethylphenylcarbamate) (CDMPC)-modified
zirconia monolithic column by CEC was reported. The
porous zirconia monolithic capillary column was prepared
by using the sol-gel technology and then zirconia surface
modified with CDMPC
90
. The separation of acidic and
basic compounds on monolithic columns demonstrated
CEC separation protocol.
An on-line concentration
technique in CEC was reported.
As a result of the
coexistence of stationary phase and electric field in a CEC
column, it is possible to employ chromatographic zone
sharpening and field-amplified sample stacking effects
simultaneously to improve CEC detection sensitivity
91
.
The determination of drug partition in membrane
phospholipids
92
and the recent applications in
enantioselective analysis by means of CEC were reported
93
.
v) Micellar electrokinetic capillary chromatography
(MEKC)
In MEKC, the secondary phase is a micellar dispersed
phase in the capillary
.
The separation principle of MEKC
is based on a differential partition between the micelle
and the solvent and this principle can be employed with
charged or neutral solutes and may involve stationary or
mobile micelles. MEKC has great utility in separating
mixtures that contain both ionic and neutral species, and
has become valuable in the separation of very hydrophobic
pharmaceuticals from their very polar metabolites. In
MEKC, hydrophobic molecules will spend the majority
of their time in the micelle, while hydrophilic molecules
will migrate quicker through the solvent. MEKC utilizes
surfactant above critical micelle concentration as pseudo
stationary phase and can separate neutral as well as
charged analytes as well as separating both charged and
neutral enantiomers with high ef
ficiency
, selectivity and
flexibility
94-96
. The selectivity of MEKC can be controlled
by the choice of surfactant and also by the addition of
modifiers to the buf
fer
.
MEKC can be employed to separate both charged
and neutral molecules, individually or simultaneously
,
including chiral compounds and benefits from high peak
efficiency due to electroosmotic flow in the separation
capillary
, compounded with lar
ge variety of synthetic
J Pharm Educ Res V
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surfactants, organic modifiers, temperature and variable
separation voltage
97
. The partial filling micellar
electrokinetic chromatography (PF-MEKC) technique
was reported for comprehensive profiling of metabolites
involved in mammalian steroid metabolism
98
.
Developments in micellar electrokinetic chromatography
(MEKC) have been reported
99,100
.
vi) Microemulsion Electrokinetic Chromatography
(MEEKC)
Electrokinetic chromatography is a family of
electrophoresis separation techniques which include
electroosmosis, electrophoresis and chromatography and
the separation is based on a combination of electrophoresis
and interactions of the analytes with additives such as
surfactants that form a dispersed secondary phase moving
at a different velocity
101
, also called a pseudo stationary
phase or separation carrier
102-105
.
A
key example of this
was cyclodextrin-mediated electrokinetic chromatography
where the differential interaction of enantiomers with the
cyclodextrins allowed for the separation of chiral
compounds
106,107
. This approach to enantiomer analysis
has made significant impact on the pharmaceutical
industry’
s approach to assessing drugs containing
enantiomers. Methods for correcting enantioselectivities
measurement in the presence of chiral impurities in the
chiral microemulsion were reported
108
. The effect of
cosurfactant identity on microemulsion size, elution range,
retention factor
, enantioselectivity
,
methylene selectivity
,
ef
ficiency
, and resolution in chiral microemulsion
formulations was reported and efficiency and resolution
values varied with different cosurfactants
109
.
MEEKC is an electrodriven separation technique in
which separations are typically achieved using oil-in-water
microemulsions, which are composed of nanometre-sized
oil droplets suspended in an aqueous buf
fer
.
The droplets
are stabilised by a surfactant and a cosurfactant. The
microemulsion droplets are usually formed by sonicating
immicible heptane or octane with water
. SDS addition at
relatively high concentrations stabilized the emulsion which
facilitated the separation of both aqueous and water-
insoluble compounds. Compared to MEKC, the presence
of a water-immiscible oil phase in the microemulsion
droplets of MEEKC gives rise to some special properties,
such as enhanced solubilization capacity and enlarged
migration window
, which could allow for the improved
separation of various hydrophobic and hydrophilic
compounds, with reduced sample pretreatment steps,
unique selectivities and/or higher ef
ficiencies.
Applications
of MEEKC are reported with emphases placed on the
discussion of MEEKC in the separation of chiral
molecules and highly hydrophobic substances, as well as
in the determination of partition coefficients, followed by
a survey of applications of MEEKC in the analysis of
pharmaceuticals and chiral separation
110-112
. Online
sample concentration, suppressed electroosmosis
MEEKC, chiral separations, MEEKC-MS, MEEKC-ICP-
MS and ME structure characterisation were also reported
113,114
. MEEKC is used effectively by the pharmaceutical
industry as generic methodology to analyze a broad
spectrum of pharmaceuticals
115
as well as drug partition
in microemulsions
116
.
vii) Capillary isoelectric focusing (CIEF)
CIEF is a separation technique based on isoelectric
point

and it relies on a graduated pH buffer gradient to
provide an isoelectric point where the net charge on the
molecule is zero. In CIEF
, the molecules migrate under
the influence of the electric field, so long as they are
charged, in a pH gradient generated by ampholytes having
pI values in a wide range (polyaminocarboxylic acids)
and dissolved in the separation buffer
117
. CIEF

is used
for the separation of amphoteric substances such as
proteins, peptides, amino acids, and pharmaceuticals in
polymer matrices as well as free solutions. CIEF is an
interesting technique for the characterization of
proteins
118
.
On-line capillary isoelectric focusing-mass
spectrometry (CIEF-MS) enabled determination of
concentrations of peptides and proteins using angiotensin
II and human tetrasialo-transferrin as the model samples.
CIEF-MS employing 1% Pharmalyte 3-10 and a sheath
liquid containing water/methanol/acetic acid (50/49/1)
resolved angiotensin I and II (5 microM each,
DeltapI=0.2) at an Rs value of 2.29. Human tetrasialo-
transferrin concentration was also determined by CIEF-
MS
119
.
Besides performing conventional CIEF
, a method
termed as pH gradient driven electrophoresis separated
ampholytic compounds with isoelectric points (pIs) beyond
the pH gradient besides performing conventional CIEF
and the proteins such as lysozyme, cytochrome C, and
J Pharm Educ Res V
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8
pepsin with pIs higher than 10 or below 3 were separated
in a pH gradient provided by Pharmalyte (pH 3-10)
120
.
CIEF with an open tubular immobilized pH gradient. CIEF
and CZE coupling with LIF detection to create an
ultrasensitive 2-D separation method for proteins were
reported
121
. Researchers have reported a multiplexed
capillary electrophoresis system employing an array of
32 capillaries with a micromachined sheath-flow cuvette
as the detection chamber
.
The sample streams were
simultaneously excited with a 473-nm laser beam, and
the fluorescence emission was imaged on a CCD camera
with a pair of doublet achromat lens. The instrument
produced mass detection limits of 380 +/- 120 yoctomoles
for fluorescein in zone electrophoresis. Capillary isoelectric
focusing of fluorescent standards produced peaks with
an average width of 0.0029 +/- 0.0008 pH. Capillary
coating stability limits the reproducibility of the analysis
122
. Using biologically relevant HIS-tag and FLAG-tag
purifed protein complexes, researchers have demonstrated
the separations of protein complex isoforms of the
mammalian target of rapamycin- complex(mTORC1 and
2) and the subcomplexes and different phosphorylation
states of the Dam 1 complex using native capillary
isoelectric focusing
123
.
viii) Capillary Isotachophoresis (CITP)
Capillary isotachophoresis (CITP)

is isotachophoresis
performed in a capillary
. In CITP
, a sample is inserted
between a leading electrolyte and a trailing (or terminating)
electrolyte without electroosmotic flow
.
The leading
electrolyte has a higher mobility and the trailing electrolyte
has a lower mobility than ions in the sample zone.
Separation in CITP relies on differences in the velocities
of analyte ions within the sample zone
124
.

CITP is
primarily used as a concentration technique by assembling
specific molecules into small focused zones
125
. Cations
and anions cannot be separated at the same time using
CITP
. CITP
in cationic regime of the separation with
conductometric detection has been used for the separation
and determination of promethazine hydrochloride in
commercial mass-produced pharmaceutical preparations
126
. CITP is an effective way to analyze serum lipoprotein
127
. Determination of neomycin trisulphate in a dosage
form (Neox and Neosol) was carried out by CITP with
conductometric detection
128
.
Cyclodextrin-mediated CITP in cationic regime of the
separation was developed for the separation and
quantitation of alkylamine, antihistamine, dimethindene
and pheniramine enantiomers in various pharmaceutical
preparations such as capsules, oral drops, gel, granulated
powder
.
The minimal sample pretreatment and low
running costs make the proposed CITP method a good
alternative to commonly used analytical methods (CZE,
HPLC)
129
. CITP with conductimetric detection has been
used for separating and determining bopindolol in
commercial mass-produced pharmaceutical preparations
130
.
(ix) Pressurized capillary electrochromatography
(pCEC)
Pressurized capillary electrochromatography (pCEC)
is a separation technology in which the retention
mechanism is based on both chromatographic partition
and electrophoresis. pCEC successfully makes use of
columns with small particles of 1.5µm, and dramatically
enhanced the ef
ficiency
, speed, peak capacity
,
reproducibility and sensitivity
, compared to traditional
HPLC and CE. CEC technology is widely applied in
various fields, including pharmaceutical sciences
131,132
.
pCEC has several advantages over HPLC: i) high
separation efficiency and resolution using a column packed
with extremely small particles, ii) high selectivity with a
double separation mechanism, iii) high speed with driven
force of both pressure and electroosmotic flow
, iv)
quantitative sample injection with rotary type valve, and
v) gradient solvent elution with binary solvent delivery
. It
has been widely applied in various fields, including
pharmaceuticals
133,134
.
A
comprehensive survey of
development of CEC and pCEC, including the
development of instrumentation, capillary columns and
stationary phase as well as CEC and pCEC applications
in biotechnology and pharmaceutical analysis were
reported
135
.
x)
Affinity capillary electr
ophor
esis (ACE)
Afûnity capillary electrophoresis (ACE) is an
electrophoretic mode that takes advantage of the speciûc
interactions of receptors, antibodies, or ligands with the
analyte. In contrast to other CE modes,
ACE is not
dedicated to general analysis, but rather is focused on
measuring molecular interactions of the solute with
speciûc receptors
136-138
. This method has been
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successfully used to examine protein-drug, protein-DNA,
peptide-carbohydrate, peptide-peptide/glycopeptide,
DNA-dye, carbohydrate-drug, antigen-antibody
, enzyme-
substrate, enzyme-inhibitor
, and other bimolecular non-
covalent interactions
139-142
.
An experimental design has
been reported for the determination of binding constants
within a couple of hours using capillary arrays or
miniaturized systems
143
.
Micellar afûnity capillary electrophoresis (MACE) is
a fast, manageable, and reliable method for afûnity studies
with weak binding ligands. In MACE, the alteration of
the ionic mobility as a factor of the tenside concentration
in the background electrolyte solution is a measure of the
strength of interaction, which may be evaluated graphically
144
. Multiple-injection affinity capillary electrophoresis
(MIACE) is a versatile analytical technique to probe
bimolecular noncovalent interactions and to estimate
binding constants between receptors and ligands.
Scientists demonstrated the use of MIACE and several
variations to MIACE to determine binding constants
between the glycopeptide antibiotics [vancomycin,
ristocetin, and teicoplanin from
Str
eptomyces orientalis,
Nocar
dia lurida, and
Actinoplanes teichomyceticus
respectively] and D-Ala-D-Ala terminus peptides
145
.
xi) Immunoaffinity capillary electrophoresis (IACE)
IACE combines the advantages of both immunoassay
and capillary electrophoresis, such as high specificity
, high
separation ef
ficiency
, rapid and low consumption
146
. IACE
technology is rapidly emerging as the most promising
method for the analysis of low-abundance biomarkers and
involves a three-step procedure: (i) bioselective adsorption
and (ii) subsequent recovery of compounds from an
immobilized affinity ligand followed by (iii) separation of
the enriched compounds
147
. IACE has been employed
for the secretion of pro-inflammatory cytokines
148
and
for

determining protein biomarkers in inflammatory
processes
149
.
xii) Nano capillary electrophoresis (NCE)
NCE is employed especially in proteomics and
genomics and it has gained increasing importance
150-153
.
Further
, NCE is a suitable technique for samples that may
be difficult to separate by Nano liquid chromatography
as the principles of separation are entirely different.
Moreover
, lower detection limits of NCE lead to the
possibility of separating and characterizing small quantities
of materials, and the enzymatic reactions for analytical
purposes can be conducted within the capillary
.
Furthermore, detection of drugs at low concentration can
be achieved by NCE
154
. The analyses of proteins and
nucleic acids at low levels have been described using chip-
based nano-liquid chromatography and nano-capillary
electrophoresis in genomics and proteomics
155,156
.
A nanocapillary electrophoretic electrochemical
(Nano-CEEC) chip with amperometric detection for
directly capturing and analysing zeptomole-level (30–75
zeptomoles ) detection of catecholamines, including
dopamine and norepinephrine (noradrenaline) that are
released from coupled single PC-12 cells in an integrated
fashion has been developed. This Nano-CEEC chip
integrated a polydimethylsiloxane microchannel for cell
sampling and biomolecule separation and a silicon dioxide
nanochannel for sample pre-concentration and
amperometric detection. The cell-capture voltage ranges
from 0.1 to 1.5 V with a frequency of 1–10 kHz for PC-
12 cells, and the single cell-capture efficiency was
optimized by varying the duration of the applied field.
All
of the processes, from cell sampling to neurotransmitter
detection were completed within 15 min
157
.
xiii) Microchip-based capillary electrophoresis
(Microchip-based CE)
Microchip-based CE system is one of the techniques
that have been developed in the area of separation on a
microchip
158-161
.
Analysis of human serum proteins
162
,
monoclonal antibodies
163
, determination of lactate
dehydrogenase isoenzymes
164
, pharmaceutical
applications and clinical analysis applications were
reported
165,166
.
A
new method to combine chip-PCR
and microchip-based capillary electrophoresis was
developed and reported for detection of SARS virus
167
.
xiv) Microfluidic capillary electrophoresis (MFCE)
In practice, microfluidic systems are based on the
principles of CE.
A
universal conductivity detector was
presented that allowed detection of charged species down
to the
µ
M level.
Additionally
, powderblasting was
presented as a novel technique for direct etching of
microfluidic networks. The performance of powderblasted
devices with integrated conductivity detection was
illustrated by the separation of lithium, sodium, and
potassium ions and that of fumaric, malic, and citric acid
168
.
A microfluidic device was described in which an
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electrospray interface to a mass spectrometer was
integrated with a capillary electrophoresis channel, an
injector and a protein digestion bed on a monolithic
substrate. This chip provided a convenient platform for
automated sample processing in proteomics applications
169
. Microfluidic assays have been developed for three
important drug target classes using the Mobility-Shift
Assay kinetic format on the LC3000 platform.
Assay
development and optimization was simplified and
accelerated by the ability to follow the reactions in real-
time. Actual
K
m
and K
i
measurements were also derived
by the kinetic measurement of initial rates of product
formation, and reaction linearity was directly monitored
170
.
A
novel MFCE device featuring a double-T
-form
injection system and an expansion chamber located at
the inlet of the separation channel utilized a double-L
injection technique and combines the expansion chamber
to minimize the sample leakage effect and to deliver a
high-quality sample plug into the separation channel so
that the detection performance of the device was
enhanced. This MFCE device has demonstrated a sound
potential for future use in high-quality
, high-throughput
chemical analysis applications
171
.
A fabrication process for producing monolithic
sampling probes on glass chips, with tip diameters of a
few hundred micrometers was developed, using simple
tools including a glass cutter and a bench drill. Microfluidic
chips with probes fabricated by this approach were
coupled to a linearly moving slotted-vial array sample
presentation system for performing continuous sample
introduction in the chip-based CE system. The
performance of the system was demonstrated in the
separation of fluorescein isothiocyanate-labeled amino
acids with LIF detection, by continuously introducing a
train of dif
ferent samples without interruption
172
. A

chiral
separation model of gel electrochromatography in a
polydimethylsiloxane microfluidic device for amino acids
has been reported. In this context, six pairs of fluorescein
isothiocyanate-labeled dansyl amino acids were separated
in a 36-mm effectual separation channel in less than 120
sec
173
.
A
simple, rapid MFCE system with a continuous
sample introduction interface has been described for
analysis of two commercial pharmaceutical preparations
containing trimethoprim, sulfadiazine, and
sulfamethoxazole with UV detection at 214 nm
174
.
Detection systems
CE is a growing technique that is continuously
developing with improved detectors
175
. Separation by CE
can be detected by several detection devices which include
UV
or UV
-V
is absorbance, light-emitting diode, laser
-
induced fluorescence
176
, mass spectrometry
177
, surface
enhanced Raman spectroscopy (SERS)
178
, fluorescence
179
, chemiluminescence
180,181
, contactless conductivity
182,183
, amperometry
184-186
, radiochemical
187
, and NMR
188,189
.
A
high power UV
light-emitting diode for
fluorescence detection in CE separations of proteins, and
peptides, that have been derivatized with UV
-excited
fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-
mercaptoethanol has been reported
190
. Indirect UV
detection is widely used for detecting solutes having no
chromophores such as metal ions or inorganic anions, for
example chloride, sulphate and nitrate
191
. This type of
determination is popular in the water and pharmaceutical
industries. Low UV wavelengths (ca 190-200 nm) are
also used to detect simple compounds such as organic
acids
192
.
A
simultaneous LIF
, coaxial thermal lens spectroscopy
(TLS) and retro-reflected beam interference detection
for CE, has been described. In its optical scheme, a diode-
pump solid-state (DPSS) laser was employed as the pump
laser in both LIF detection and coaxial TLS detection,
and a He-Ne laser was utilized as the probe laser in coaxial
TLS detection and RBI detection. LIF and coaxial TLS
detection owned high sensitivity
, and retro-reflected beam
interference detection indicated versatile property
, based
on which the reported detection achieved a sensitive and
universal detection for CE
193
. Microchip microemulsion
electrokinetic chromatography with indirect fluorimetric
detection provided an accurate method for estimating log
P octanol/water values for neutral and basic compounds
and also a means for analyzing compounds that are non-
fluorescent
194
. Microchip capillary electrophoresis,
coupled with indirect fluorescence detection was
investigated for estimating the pK(a) values of non-
fluorescent compounds including several of
pharmaceutical importance with pK(a) values from 10.3
to 4.6
195
. The different approaches for constructing
carbon nanotubes, nanoparticles, and nanorods-based
detectors for conventional CE and microchip
electrophoresis have been reported. In this context, the
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enhanced detection performance of nanomaterial-based
detectors, such as higher sensitivity
, improved limits of
detection, and higher peak capacity
, along with various
biomedical applications were described
196
.
Hyphenated capilliary electrphoresis techniques
The combination of the powerful technique of mass
spectrometry (MS) with CE was first reported by
Olivares et al.
197
, Smith et al.
198
and Chamberlain
199
.
CE-ESI-MS represents a promising hyphenated
microseparation platform in metabolomics and offers a
convenient format for the separation of complex mixtures
of cationic, anionic, and/or zwitterionic metabolites, as well
as their isobaric/isomeric ions without complicated sample
handling
200
. The CE coupling to sensitive and selective
MS detection allows the qualitative and quantitative
analysis of even complex samples. Inductively coupled
plasma mass spectrometry (ICP-MS) and electrospray
ionization time-of-flight mass spectrometry (ESI-T
oF-MS)
coupled to CE are complementary tools that are used in
the field of speciation. Structural information can be
obtained by means of ESI-T
oF-MS, while ICP-MS is
ideally suited to quantify elements even in very low
concentration ranges
201
. The complementary application
of CE-ICP-MS and CE-ESI-MS detection techniques to
the structural and functional characterisation of metal-
binding proteins and their structural metal-binding moieties
was reported
202
. The instrumentation and the integration
of mass spectroscopy and CE for the analysis of small
oligonucleotides and DNA-ligand interactions have been
reported
203,204
.
A growing number of CE-MS applications make use
of capillaries where the internal wall is modified with
surface coating agents. In CE-MS, capillary coatings are
used to prevent analyte adsorption and to provide
appropriate conditions for CE-MS interfacing.

In CE-MS,
separation is achieved through channels etched on the
surface of the capillary (connected to an external high-
voltage power supply) which delivers sample to ESI-
MS.
It is an automatable approach, with great sensitivity
.
An overview of the various capillary coating strategies
used in CE-MS was reported
205
. MEKC coupled to
ESI-MS method shows good potential for the detection
and structure elucidation of minor impurities in drug
substances
206
.
A capillary electrophoresis mass spectrometry (CE-
MS) interface utilizing a flow-through microvial is used
to ensure the electric continuity and supply the catholyte
and mobilizer solutions during the CIEF and mobilization
process. The flow-through microvial provided a stable
chemical environment and improved the ionization
efficiency without significantly diluting the analyte. The
CE-MS interface facilitated the transfer of the mobilized
CIEF effluent to the site of electrospray ionization, and
the gaseous ions can be detected directly by a mass
spectrometer
. It also allows for complete focusing and
mobilization processes to be performed automatically in
programmed sequences with commercial CE systems.
T
wo dif
ferent strategies, using either a part of the capillary
or the flow-through microvial of the CE-MS interface as
the catholyte reservoir for bare fused silica capillaries or
neutral coated capillaries, respectively
, were developed
for automated CIEF-electrospray ionization (ESI)-MS.
Reasonable separation efficiency was achieved using
proper concentration of carrier ampholytes and suitable
strategies of electroosmotic/electrophoretic mobilization
207
.
A highly efficient and reliable membrane-assisted
capillary isoelectric focusing (MA-CIEF) system coupled
with MALDI-FTMS for the analysis of complex
neuropeptide mixtures was reported. The new interface
consisted of two membrane-coated joints made near each
end of the capillary for applying high voltage, while the
capillary ends were placed in the two reservoirs which
were filled with anolyte (acid) and catholyte (base) to
provide pH difference. Optimizations of CIEF conditions
and comparison with conventional CIEF were carried out
by using bovine serum albumin (BSA) tryptic peptides. It
was shown that the MA-CIEF could provide more
efficient, reliable and faster separation with improved
sequence coverage when coupled to MALDI-FTMS.
Analyses of orcokinin family neuropeptides from crabs
Cancer borealis and Callinectes sapidus brain extracts
have been conducted using the established MA-CIEF/
MALDI-FTMS platform. Increased number of
neuropeptides was observed with significantly enhanced
MS signal in comparison with direct analysis by MALDI-
FTMS. The results highlighted the potential of MA-CIEF
as an efficient fractionation tool for coupling to MALDI
MS for neuropeptide analysis
208
.
An orthogonal method of coupling MEKC and MS
utilized atmospheric pressure chemical ionization (APCI)
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and atmospheric pressure photoionization (APPI)
209
. In
these methods the ionization efficiency has been shown
to be unaf
fected by the surfactant SDS using both
APCI
and APPI
210,21
1
.
The
APCI-MS set-up enabled detection
of basic, neutral, and acidic compounds, whereas apolar
and ionic compounds could not be detected. The analysis
of a mixture of basic compounds and a steroid using
volatile and nonvolatile background electrolyte further
demonstrated the feasibility of CE-APCIMS
212
. The
successful coupling of CZE, NACE, MEKC and CEC
with MS detection provided an efficient and sensitive
analytical tool for the separation, quantitation, and
identification of numerous pharmaceutical and therapeutic
compounds. Chip-based microdevices were also reported
regarding fabrication methods, designs and MS interfacing
213
.
A
microchip-based capillary electrophoresis system
was interfaced with a microwave induced plasma optical
emission spectrometry (MIP-OES) to provide copper
species separation capabilities
214
. The use of
nanoparticles as pseudostationary phases´s, in combination
with continuous full filling (CFF) and an orthogonal ESI-
interface, has shown to be a good way to separate neutral
analytes without affecting the electrospray process or
contaminate the ion source
215,216
.
The improvements in the separation as well as the
coupling of CE with MS have been reported
217-220
.
Reproducible investigations of the interaction between a
novel Ru-based anticancer agent indazolium [trans-
tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) with
the proteins (human serum albumin (HSA) and transferrin
) as well with human plasma, human serum and HSA-
and IgG-depleted human serum was reported to shed light
on the fate of the drug upon intravenous application
221
.
The coupling of Ru(bpy)
3
2+
-based
electrochemiluminescence detection with CE was
reported for the simultaneous determination of proline
(association with chronic uremia ) and pipemidic acid (used
in the treatment of Gram-negative urinary tract infections
) in human urine
222
.
A
pCEC coupled with on-column
chemiluminescence detection was reported for direct
determination of amino acids, which was based on the
principle of an enhanced effect of Cu(II)–amino acid
complexes on the chemiluminescence reaction between
luminol and hydrogen peroxide in alkaline solution
223
.
CE applications in pharmaceutical analysis
CE is a powerful analytical technique which is
increasing in utility in the pharmaceutical industry
. It is
used as an alternative or complementary technique to
HPLC due to its high ef
ficiency
, speed of analysis,
reduction in solvent and sample consumption, and low
operating cost compared to HPLC methodology
224
. CE
is used for routine, particularly for analyzing serum
proteins and disease markers in many hospitals and clinics.
CE technique has also dramatically increased throughput
for DNA profiling in criminal investigations and CE data
have been shown to be credible evidence in law courts,
and forensic testing laboratories have published validated
procedures. Pharmaceutical companies make extensive
use of CE, in particular for chiral separations, and the
technique is widely accepted by regulatory authorities such
as the US Food and Drug
Administration. CE has been
widely adopted for analyzing biomolecules such as DNA
and proteins by CE gel, steroids by MECC
225
.
Applications of CE, CEC and their derived techniques
to assay active pharmaceutical ingredients (APIs), drug
impurity testing, chiral drug separation, determination of
APIs in biological fluids
226-235
and therapeutic drug
monitoring have been reported
236
,
237
. CE has contributed
immensely to better understanding of affinity interactions
between cyclodextrins and chiral drugs
238
. CE methods
were reported for assessing drug stability
, separating
metallodrugs and their metabolites, characterizing related
metal-bioligand species, metabolomic and proteomic
analysis focussed on metal-based drugs
239
.
CE assay was developed for the stability evaluation
of tramadol enantiomers in commercial tablets using
maltodextrin as chiral selector and best separation for
the tramadol enantiomers was achieved on an uncoated
fused-silica capillary at 20°C using borate buffer (50
mM, pH 10.2) containing 10% m/v maltodextrin and using
UV detector at 214 nm. The range of quantitation for
both enantiomers was 5–100
µ
g/mL (
R
>0.996)
240
.
Examples of mechanistic aspects of capillary
electromigration enantioseparations and applications of
chiral pharmaceutical and biomedical analysis were
reported
241
. CE applicability to the routine quality control
was successfully demonstrated for the determination of
bromhexine and amoxicillin, and pyrimethamine and
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sulfadoxine in several pharmaceutical formulations
242,243
.
The pharmaceutical analysis applications used for various
modes of capillary electrophoresis have been reported
244-248
. CE is commonly used in the analysis of small
molecules
249
. Proteomic analysis with CE-MS coupling
permits fast and accurate identification and differentiation
of polypeptide patterns in the urine of patients with IgA
nephropathy
, allowing dif
ferentiation from healthy controls
and, probably
, other renal diseases
250
. CZE was used for
the analysis of water-soluble vitamins in corns, in which
on-line concentration methods, namely field-enhanced
sample stacking and dynamic pH junction, were utilized
to improve the detection sensitivity
251
.
Since different drug metabolite enantiomers are
formed in many metabolic pathways, the advantage of
CE over HPLC is considered better for chiral separation
without the need of special expensive columns. The
sample preparation, separation and detection modes
involved in CE drug metabolism studies, and distinction
between drug metabolism analysis
in vivo
and
in vitr
o
has been reported
252
.

In pharmaceutical industry
, CE has
been used to support all of stages from chemical synthesis
to compound selection for drug development and quality
control. In this context, various applications include:
physicochemical profiling; pK
a
screening, Log p screening,
enzyme activity screening, chiral discovery and screening,
metabolomics, immunoassay
, SDS-protein sizing,
oligonucleotide quality control, genetic analysis,
quantitation of gene expression, viral load quantitation,
genotyping, automated DNA sequencing and

DNA-
protein interactions
253
. Recently
, researchers have
developed a CZE method of analysing antimalarial drugs
based on sulfadoxine and pyrimethamine and analysis of
different tablet formulations has shown a good agreement
with the liquid chromatography method described in the
United States Pharmacopoeia
254
.
Due to inexpensive quality control as well as projecting
CE as a powerful alternative to HPLC, CE approach has
been progressively introduced in the pharmaceutical
industry
255-258
. CE has been used in the analysis and
small-scale separation of PEGylated proteins
259
. The
CZE method for the simultaneous analysis of omeprazole
and lansoprazole was achieved and this method was
applied to determine the quality of commercial capsules
260
. Suxamethonium was determined by CE coupled with
attenuated total internal reflectance infrared
microspectroscopy (FT
-IR)
261
. Some applications of
CEC in the analysis of pharmaceuticals products have
been reported
262-265
.
The mixed buffer of 20 mM phosphate/10 mM citrate
containing 5 mM methyl-
!
-cyclodextrin or 5 mM
carboxymethyl-
!
-cyclodextrin as a chiral selector showed
superior separation ability compared with single component
buffers in CE separation of sibutramine enantiomers. This
method was applied successfully to the enantiomeric
determination of (
R
)- and (
S
)-sibutramine in commercial
pharmaceutical formulations. Further
, the resolution of the
sibutramine enantiomers was attributable mainly to the
dif
ference in stability constants
266
.
A
simple method based
on capillary electrophoresis with a capacitively coupled
contactless conductivity detector (CE-C
4
D) was found
suitable for controlling pharmaceutical formulations
containing suxamethonium and degradation products
267
.
The affinity of drugs to both to excipients and to
vehicle systems is of utmost importance in the
development and optimizations of pharmaceutical
formulations.
ACE finds application for characterization
of ion pairing of hydrophilic drug with lipophilic counter
ions. Results showed that lipophilic counter ions as
absorption enhancer via ion pairing improved the oral
bioavailability of hydrophilic drugs such as cefpirome. In
addition,
ACE was used to optimize the solubilization
capacity for lipophilic drugs of colloidal formulations such
as mixed micelles and microemulsions. Furthermore,
ACE
was applied to determine the potential toxic labile iron in
parenteral iron formulations using off-capillary and on
capillary complexation with EDT
A

268
.
The results obtained with the PEGylated liposomes
demonstrated that CE as a useful tool for the
characterization of liposomal drug formulations such as
oxaliplatin (anti-cancer agent)
269
.
A
method based on
CE-ICP-MS and interface units, was developed for
ascertaining possible metabolic transformations of metal-
based drugs. In this context, using a novel anticancer
gallium compound, it was demonstrated that the drug
remains intact in the simulated intestine juice. On the
contrary
, it under
goes a marked change in speciation
(mostly
, due to binding to transferrin) in human serum
270
.
The copper (II) complex of a modified cyclodextrin,
namely 6-mono-deoxy-6-[4-(2-aminoethyl) imidazolyl]-
!
-
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CD was investigated in ligand exchange capillary
electrophoresis (LECE) using ESI-MS as the detection
device. This hyphenated method potential was compared
with the results previously obtained with optical detection
by studying the chiral resolution of tryptophan racemate.
It was observed that chiral separation conditions
compatible with LECE-ESI-MS could be achieved based
on the figures of merit obtained by LECE-UV
.
The values
of detection limit obtained by LECE-ESI-MS were
significantly better than those obtained by LECE-UV
271
.
V
arious CE modes were applied to dif
ferent areas of
pharmaceutical analysis such as enantiomer separation,
analysis of small molecules such as amino acids or drug
counter-ions, pharmaceutical assay and physiochemical
measurements such as log P and pK
a
of compounds
272-
275
.
In order to automate the key tasks in large-scale
nucleic acid CE analysis (including the profile alignment),
researchers reported a computational method called high-
throughput robust analysis for capillary electrophoresis
(HiTRACE). In this context, HiTRACE quantitation of
the largest datasets was achieved in 3–12 min as compared
to 7–10 h of manual intervention using prior approaches
276
.
An automated CZE system utilizing a capillary
cartridge having a plurality of capillary tubes was reported
277
.
Macrocyclic antibiotics (ansamycins and the
glycopeptides) and macrolides are used as chiral selectors
in CE for enantioseparation applications. Clarithromycin
lactobionate, belonging to the group of macrolide
antibiotics, allowed excellent separation of the enantiomers
of metoprolol, atenolol, propranolol, bisoprolol, esmolol,
ritodrine, and amlodipine, as well as partial
enantioresolution of labetalol and nefopam at the buffer
pH range of 7.3–7.5 using 12.5

mM
borax
buffer
with
50% v/v methanol, 60 mM clarithromycin lactobionate,
and 20
kV
applied voltage
278
. Chiral CE helps better
understanding of mechanisms of enantioselective
intermolecular interactions, and the examples include chiral
pharmaceuticals in bulk and formulations in presence of
(sometimes also achiral) impurities, chiral drugs with
multiple centres of chirality
, chiral drugs and metabolites
in complex biomedical matrices such as urine, plasma,
etc
279
.
Drug-protein interactions between the platinum-based
anticancer drug (oxaliplatin) with human serum albumin
(HSA) in aqueous solution at physiological pH with drug
concentrations of 10 to 100 microM and a constant
concentration of HSA (5.0 x 10
-5
M) were studied using
CIEF with whole column imaging detection. The altered
CIEF profile demonstrated the possible conformation
change due to the binding of the drug. Further
, a significant
protein’
s pI shift for higher HSA-oxaliplatin incubation
ratios was observed. Furthermore, spectroscopic evidence
showed that oxaliplatin caused the fluorescence quenching
of HSA by formation of HSA-oxaliplatin complex. In
addition, the quenching rate constants K(q) at three
different temperatures indicated the presence of static
quenching mechanism in the interactions of oxaliplatin with
HSA
280
.
A simple ampholyte-free CIEF free-flow
electrophoresis design establishing a pH gradient spanning
from 2.3 to 8.9 was reported and the separation of proteins
such as
!
-Lactoglobulin, Hemoglobin, Myoglobin and
Cytochrome
C
was confirmed by CIEF with pI markers
281
. CIEF has been proved as a high-resolution technique
for protein and peptide separation in pharmaceutical
industries for the analysis and characterization of, for
example, isoforms of glycoproteins, point mutations in
hemoglobin, and peptide mapping. Further
, hyphenation
to MS and chip based CIEF (microfabrication) have
shown promise and CIEF kits and specific recipes /
application notes are available from vendors of CE
equipment, as are a vast amount of publications and
handbooks of CE published over recent years
282
.
The results of a CE microfluidic device with a novel
injection structure with a 45° U-shaped injector and an
expansion chamber at the inlet of the separation channel
significantly prevented sample leakage and transports
suitable sample plugs into the separation channel enhancing
the detection performance
283
. The development and
application of the electrophoretic and microchip
technologies for the analysis of outer membrane protein
was reported
284,285
. Pharmaceutical analysis focused
on the detection of anions and cations in the course of
stoichiometric analysis was achieved by various CE
techniques
286
. CE-MS was applied to the chiral
separation of baclofen using sulfobutylether-
!
-cyclodextrin
chiral selector in partial filling counter current mode, and
on-line UV detection was simultaneously used. The
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cyclodextrin showed stereoselective complexation
towards baclofen enantiomers, thereby
, allowing chiral
resolution at low concentration. Complete enantiomeric
separation was obtained by using 0.25 M formic acid
background electrolyte containing 1.75 mM of chiral
selector and water/methanol (30:70, v/v) 3% formic acid
as sheath liquid
287
. The separation and determination of
amoxicillin, ampicillin, sulfamethoxazole, and
sulfacetamide was achieved by MEKC method using UV
detection at 210 nm
288
. Stereoselective methods
employing capillary electrophoresis for chiral drug
metabolism studies were also reported
289
.
MEKC method with diode-array detection for the
simultaneous and short-time analysis of six statin drugs
(lovastatin, simvastatin, pravastatin, fluvastatin,
atorvastatin and rosuvastatin) in pharmaceutical samples
was reported using ketoprofen as an internal standard.
Optimized conditions were found to be a 25 mM borate
buffer pH 9.5 with 25
mM sodium dodecyl sulphate and
10% methanol added as an or
ganic modifier
, an applied
voltage of 23
kV and a separation temperature of 30
°C.
The linearity of the detector response for each statin was
within the concentration range from 10 to 100
µ
g mL
”1
with a correlation coefficient greater than 0.9994
290
.
MEEKC method has been developed which
separated a range of nine steroids and was validated for
the determination of 17â-estradiol content, a hormone
steroid, in transdermal patches.
A
microemulsion containing
ethyl acetate, butan-1-ol, sodium dodecyl sulfate, 15%
(
v/v
) acetonitrile and 12
mmol L
”1
sodium tetraborate
aqueous buffer at pH 9.2 was used with direct UV
detection at 200
nm. The method was validated for the
determination of 17â-estradiol content, a hormone steroid,
in transdermal patches
291
.
A chiral microemulsion electrokinetic chromatography
method has been developed for the separation of the
enantiomers of the phenethylamines ephedrine,
N
-
methylephedrine, norephedrine, pseudoephedrine,
adrenaline (epinephrine), 2-amino-1-phenylethanol,
diethylnorephedrine, and 2-(dibutylamino)-1-phenyl-1-
propanol, respectively
.
Additionally
, the developed method
was successfully applied to the related substances analysis
of noradrenaline, adrenaline, dipivefrine, ephedrine and
pseudoephedrine monographed in the European
Pharmacopoeia
292
. The methodological and instrumental
improvements for enhancing sensitivity in chiral analysis
by CE were reported
293
. Off-line and on-line sample
treatment techniques, on-line sample preconcentration
strategies based on electrophoretic and chromatographic
principles, and alternative detection systems to the widely
employed UV/V
is detection in CE are the most relevant
approaches discussed for improving sensitivity
. Microchip
technologies can open up great possibilities to achieve
sensitive and fast enantiomeric separations
294
. Sulfated
cyclodextrins (HS-CDs) were used as selectors for chiral
resolution of seven basic and two zwitterionic drugs. The
electrophoretic conditions for the stereoselective analysis
of drugs were in the carrier mode with 25 mM sodium
phosphate buffer containing 1.25% w/v of each HS-CD
at pH 2.5 with an applied voltage of +15 kV and rapid
enantioresolution of all drugs were achieved under these
conditions
295
.
A bromoacetate-substituted
!
-cyclodextrin
(Br-
!
-CD) was successfully bound to the 3-
aminopropylmethyldimethoxysilane-modified capillary
column prepared by microwave irradiation, as a chiral
stationary phase for open tubular capillary
electrochromatography
. Compared with conventional
synthesis, the microwave-assisted process significantly
decreased the preparation time of the stationary phase
from 16
h to 40
min. Baseline chiral separation of 1-
phenyl-1,2-ethanediol was achieved using the Br-
!
-CD
modified column
296
. The applications of CE and
microchip CE with conductivity detector C
4
D in
pharmaceutical and biological analysis have been reported
297
. CZE with a capacitively coupled contactless
conductivity detector (CE-C
4
D) was successfully applied
to the simultaneous determination of atenolol and amiloride
in different pharmaceutical tablet formulations
298
.
Determination of ethambutol , a first-line drug against
tuberculosis, was achieved using CE based method with
capacitively coupled contactless conductivity detection
299
. The determination of ciclopirox olamine in
pharmaceutical formulations using CE with capacitively
coupled contactless conductivity detection was reported
in an alkaline and acidic medium.
A
linear working range
from 2.64 to 264 g/mL in sodium hydroxide electrolyte as
well as low detection limit (0.39 g/mL) and a good
repeatability (RSD = 3.4% for 264 g/mL ciclopirox solution
(n = 10)) were achieved. Olamine was determined in its
cationic form when acetic acid was used as the electrolyte
J Pharm Educ Res V
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16
solution. The results obtained include a linear range from
26.4 to 184.8 g/mL and a detection limit of 2.6 g/mL
olamine
300
.
Micro-electrodialysis and CE were combined for rapid
pretreatment and subsequent determination of inorganic
cations in biological samples as such, and this hyphenation
method greatly improved the analytical performance of
the latter as the adsorption of high molecular weight
compounds present in real samples on the inner capillary
wall was eliminated
301
. CE and CE-based analytical
tools have great potential in the determination of lipids
302
and were employed to study interactions between analytes
and biomimetic lipid vesicles
303
.
Most NACE applications carried out for the analysis
of compounds of pharmaceutical interest include (i)
analysis of drugs and related substances, (ii) analysis of
chiral substances, (iii) analysis of phytochemical extracts
and (iv) analysis of drugs in biological fluids
304
.
Researchers reported the ingredients in acne formulations
(salicylic acid, cloramphenicol and resorcinol in presence
of azulene) by CZE
305
. CE has been used for analysis of
various classes of antifungal compounds such as
miconazole and 5-fluorocytosine
306
. Developments in
chiral separation using CZE, EKC, and CEC were
reported
307
. Researchers have reported on a novel closed-
loop system to identify ionic species with similar
electrophoretic mobilities unlike many conventional CE
systems. In this method, the sample undergoes separation
to travel back and forth along the short channel multiple
times, and moreover
, it was independent of migration time
308
. MEKC has been used for analysis of water-soluble
neutral compounds, weak acids and bases
309,310
.
A simple, rapid microfluidic capillary electrophoresis
system with a continuous sample introduction interface
has been applied to the analysis of two commercial
pharmaceutical preparations containing trimethoprim,
sulfadiazine, and sulfamthoxazole with UV detection at
214 nm, achieving baseline separation within 2.5 min
311
.
CE significance as a complementary biophysical tool for
protein characterization has been reported
312
. Lamivudine,
didanosine, and saquinavir were simultaneously
determined by CZE
313
. MEKC was used for
determination of stavudine, didanosine, saquinavir (mixture
A) and stavudine, didanosine, efavirenz (mixture B), the
anti-HIV drug mixtures containing zidovudine /didanosine/
nevirapine (mixture
A) and zidovudine/didanosine/ritonavir
(mixture B) were quantitated in human serum samples
314-316
. Researchers demonstrated the potential of CE as
a routine analytical separation technique for the analysis
of non chiral, chiral and impurities in pharmaceuticals
formulation of fluoroquinolones and primaquine
317
. A
sweeping CE was established for the determination trace
levels of abused drugs in addict’
s urine
318
. The
analysis
of anxiolytic drugs, including barbiturates and
benzodiazepines, by CE with bare and coated capillaries
was also reported
319,320
. The determination of ã-
hydroxybutyric acid in urine and serum samples with (CE-
C
4
D was reported
321
. MEKC analysis of a group of
date-rape drugs by permitted the analysis of uncharged
molecules by providing a secondary separation through
the addition of a surfactant that formed into micelles
322
.
ACE found applications in the identification of
antimicrobial compounds that bound the novel tar
g
et
Y
i
hA
[an essential gene of unknown function in both
Escherichia coli
and
Bacillus subtilis
conserved in
bacteria and represents an attractive target for the
discovery of new antibiotics].
Y
ihA
encoded a putative
GTP-binding protein and screened a small-molecule library
of 44,000 compounds initially identified 1
15 binders, of
which 76% were confirmed. Furthermore, the
ACE assay
distinguished diverse compounds that possessed drug-like
properties and antimicrobial activity against drug-resistant
clinical isolates. Such data validated
ACE as a valuable
tool for the fast, efficient detection of specific binding
molecules that possess biological activity
323
.
Researchers have demonstrated development of
electrophoretically mediated micro analysis for screening
protein tyrosine phosphatase inhibitors in natural extracts
324,325
. The determination of the physicochemical and
thermodynamic parameters of drug compounds offering
strategies to explore and characterize interactions between
drugs, drug vehicles, and biological membranes to facilitate
developments in controlled drug delivery was achieved
using ACE
326
.
Regulatory requirements limit the amount of the host
cell DNA that can be present in vaccines or human
therapeutics. The viral quantitative capillary
electrophoresis was successfully applied to quantify the
oncolytic vaccinia virus in the range of 10
6
to 10
12
ivp/
mL. In this context, researchers separated intact virus
J Pharm Educ Res V
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1
17
particles and the residual DNA, and measured the level
of virus contamination with DNA impurities using CE
method, and intercalating
YOYO-1 dye was used to detect
the encapsulated and free DNA by laser-induced
fluorescence
327
.

Applications of capillary electrophoresis
with capacitively coupled contactless conductivity
detection (CE-C4D) in pharmaceutical and biological
analysis were reported
328
.
CE application in biopharmaceutical analysis
CE has demonstrated to be a complementary
alternative to chromatographic techniques in
biopharmaceutical analysis. The complete
characterization of biopharmaceutical drugs such as
erythropoietin and various therapeutic monoclonal
antibodies for glycosylation compositions, IgG purity
, and
impurities for quality control purposes are of utmost
importance. Further
, therapeutic biomolecules have a
highly complex composition and structure. Moreover
, the
products may vary in structure due to the complexity of
cell culture and purification processes
329
. V
arious
modes
of CE offer several possibilities for biopharmaceutical
analysis including glycosylated therapeutic proteins,
monoclonal antibodies, pharmaceutical and
biopharmaceutical impurities
330
. CZE has been used for
proteins as long as there are differences in charge:frictional
drag ratios
331
. The acceleration and rapid success of
Human Genome Project was possible due only to the
introduction of CE-based sequencers
332
. CE-SDS gel
application has become the gold standard for protein purity
and heterogeneity analysis in biopharmaceutical
laboratories and several major biopharmaceutical
companies have adopted CE-SDS as a replacement to
SDS-P
AGE. Denatured proteins can be reduced or left
intact for separation and subsequent analysis
333
. V
arious
modes of CE offer numerous possibilities for
biopharmaceutical analysis
334
. CE- laser-induced
fluorescence detection method has been useful for both
structural characterization and quantitative profiling of N-
linked oligosaccharides derived from recombinant
monoclonal antibodies pharmaceuticals
335
.
CIEF applications related to analysis of
biopharmaceutical compounds and isolated proteins for
metabolomic studies have been reported
336
.

A
novel on-
line 2-D system combining CIEF with pCEC using a micro-
injection valve as the interface was developed for peptide
and protein mapping. Separation effectiveness of this 2-
D system was demonstrated by the analysis of tryptic
digest of BSA
and human red blood cell lysate.
A
theoretical peak capacity of approximately 24 000
achieved for bovine serum albumin digest proved its
promising potential for the application in proteomics
337
.
DNA fragments, SDS protein and macromolecules
analysis has been achieved using CGE
338-342
.
Flow Induced Dispersion
Analysis (FIDA), a general
methodology for assessing non-covalent interactions, has
been used on small molecules, proteins (such as antibodies)
and DNA
343
.

The application of CE to the glycoform
analysis of glycoproteins and profiling of the N-linked
glycans released from glycoprotein pharmaceuticals, and
applications for structural analysis using CE-MS technique
and glycan profil1ing method for therapeutic antibody were
reported
344
.
Researchers have developed a procedure for the
removal of polysorbate 80 from formulated epoetin alfa,
allowing the material to be analyzed by European
Pharmacopoeia CZE modified method
345
. Practical
aspects of CE devoted to proteins, peptides, and techniques
especially useful in the area of recombinant DNA products
346
, analyses of dosage forms of biotechnology derived
proteins of pharmaceutical importance as well as the
identification and examination of the peptides, obtained
after enzymatic cleavage of proteins by CE have been
reported
347
. CE has been used in the analyses of
Escherichia coli
-derived proteins, glycosylated proteins
derived from mammalian cell cultures, carbohydrate
chains enzymatically removed from the protein, analysis
of the antisense oligonucleotides for the determination of
purity and the analytical studies on the metabolism of these
modified oligonucleotides
348
.
A major advance for CE is its recognition by various
regulatory authorities.
A
general monograph on CE is now
included in the United States Pharmacopoeia (USP
, which
has also been published in the European Pharmacopoeia
(EP) and Japanese Pharmacopoeia
349
. CE methods have
emerged in the official Pharmacopoeial texts. The ICH
Steering Committee, based on the evaluation by the Q4B
Expert
W
orking Group (EWG), recommended that the
analytical procedures described in the official
Pharmacopoeial texts, Ph. Eur
. 2.2.47. Capillary
J Pharm Educ Res V
ol. 2, Issue No. 2, December 201
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18
Electrophoresis, JP General Information 4. Capillary
Electrophoresis, and USP General Information Chapter
<1053> Biotechnology-derived
Articles – Capillary
Electrophoresis,
4
can be used as interchangeable in the
ICH regions.
The key features for method validation of small
molecules using CE in the context of the pharmaceutical
industry have been reported
351,352
. The determination of
K
d
of three drugs namely daunorubicin, pharmorubicin
and idarubicin was reported using CZE with amperometric
detection and the pK
a
values obtained through the method
were in agreement with the values by typical
spectrophotometric method
353
.
A
CZE with amperometric
detection method has been developed for the determination
of idarubicin in human urine
354
, and also for the separation
and determination of sucrose, glucose, and fructose in
Chinese traditional drugs, namely
Astragalus
Membranceus (Fish.) Bge,
Angelica and Codonopsis
pilosula (Franch.) Nannf
355
. The analysis of doxorubicin
(DOX) and daunorubicin (DAU) in human serum using
CZE and mirochip-based CE-LIF detection was validated
with regard to reproducibilities, linearity and limit of
detection. The optimum electrophoretic separation
conditions were 10
mM sodium tetraborate buffer at pH
9.5 with 40% acetonitrile (V/V) and a separation voltage
of 2.1
kV
. DOX and DAU were separated in 60
s under
the optimum separation conditions. Linear relationships
were obtained between the concentration and peak area
in the 1–75
µg mL
-1
range and with the detection limits of
0.3 and 0.2 µg mL
-1
for DOX and DAU, respectively
356,357
. Researchers have reported advances in CE theory
,
instrumentation, and methodologies that are specific to
nucleic acids, proteins and peptides, carbohydrates, lipids,
single cells, and bioparticles, as well as advances in the
use of CE to define functional assays or to investigate
biomolecular interactions
358
.
The applicability of CE-MS was demonstrated on a
separation of glycopeptides from monoclonal antibodies
(mAb) and to identify the glycan modifications. The tryptic
peptide map of the mAb was generated and the
glycopeptide was assigned using accurate mass
measurement. Subsequently
, BioConfirm software was
used to map the glycosylation site on the mAb. The
extremely efficient CE separation and superior mass
accuracy of the Q-TOF platform, combined with the
powerful data processing capabilities of
Agilent
MassHunter and BioConfirm software, enabled
identification of the glycopeptides and glycan moiety
attached to the complex protein. The combination of CE
with Q-TOF MS has proved a valuable tool for peptide
mapping of small quantity biopharmaceuticals
359
. The
potential of ligand-exchange capillary electrophoresis
method in solving major problems, such as the separation
of enantiomers and the determination of biologically active
compounds poorly absorbing in the UV region (sugars,
amines, and amino acids) were reported
360
.
CE applications in biotechnology
CE separation technique is broadly used in the
biotechnology industry for carbohydrate analysis and
significant improvements for the standard CE sample
preparation method of glycan analysis of glycoproteins
by CE-LIF and CE-MS were reported
361-366
.
AdvanCE™ FS platform provided rapid separation
and ample resolution with excellent sensitivity and dynamic
range, to benefit a variety of applications in genomic
research
367
. Several glycoproteins such as fetuin, alpha1
acid glycoprotein, IgG, and transferrin separation was
achieved within only 5 h with the three-step procedure
involving release of glycans, derivatization with Fmoc, and
CE-ESI MS analysis. This method was also applicable
for the analysis of N-glycans derived from monoclonal
antibody pharmaceuticals, as well as from alpha-
fetoprotein (one of the tumor markers of hepatocellular
carcinomas)
368
. Reports on pharmaceuticals and
biotechnology products analyses by CEC were compiled
369
.
A
number of collaborations between various
pharmaceutical companies and regulatory authorities for
the analysis of biomolecules using CE demonstrated the
robustness of CE-SDS across eight different organisations
370
. CE found applications for the separation of
microorganisms as well as the detection and isolation of
Candida albicans
fungus in human blood
371
. The
applications of microchip-based CE to the detection and
separation of DNA fragments in biotechnological and
clinical research were reported
372
.
CE methods showed promise for analysis of
resveratrol and other flavonoid antioxidants (glycosides
and aglycones) in wine
373
.
A
CE-MS technique has been
developed for age estimation of silk textiles based on amino
J Pharm Educ Res V
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19
acid racemization rates ranging in age from several
decades to a few-thousand-years-old. The analysis takes
20 min, consumes only nanoliters of the amino acid
mixture, and provided both amino acid composition profiles
and d/l ratios for 1
1
amino acids
374
. L
ysozyme, bovine
serum albumin and beta-galactosidase were successfully
separated rapidly using a laboratory-made miniaturized
capillary electrophoresis apparatus provided with a
confocal fluorescence spectrometry
375
.
CE based on the principles of frontal analysis was
used to characterize the binding of flavonoids to human
serum albumin at near
-physiological conditions, thereby
,
providing a lot of useful parameters for characterization
of ligand–protein interactions
376
. CE-F
A
was used to
study the interaction of drugs with plasma proteins
377
,
protein binding of basic drugs
378
, protein binding constant,
binding sites of the Strychnos alkaloid-strychnine and
bovine serum albumin
379,380,
. CE has the potential to
become a useful technology to characterize liposomal
systems including liposomal drug formulations and
PEGylated liposomal drug formulation
381-383
. CE provided
better clues about levels of molecular species, enzymatic
processes, and protein expression occurring in a cell at
different time points in the cell life cycle
384
. MEKC CE
method was developed for the quantitative analysis of
cereulide, the emetic toxin produced by
Bacillus
cer
e
us
385
.
The model binding of the glycopeptide antibiotic
teicoplanin from
Actinoplanes teichomyceticus
,
immobilized on magnetic microspheres to d-Ala-d-Ala
terminus peptides was reported using microchip capillary
electrophoresis with continuous frontal analysis. The
binding constants obtained using the technique was
comparable with values reported in the literature
386
.
Researchers have reported the methodology of CE-LIF
immunoassay for the detection of DNA adducts
387
.
The results for the analysis of inorganic ions in whole
blood, specifically lithium demonstrated the versatility of
the CE system for on-site measurement of inorganic ions
388
.
Automated, high-resolution, quantitative, high-
throughput analysis of mono- and oligosaccharides,
produced by enzymatic digestion of cellohexaose (model
substrate) and lignocellulosic biomass, has been
demonstrated using CE in conjunction with a single-step
fluorophore labeling strategy for sensitive laser-induced
fluorescence detection
389
.
Inorganic nanoparticles assisted CE pharmaceutical
applications
CE has seen used in the separation and
characterization of inor
ganic nanoparticles (Ag,
Au,
T
i
O
2
,
Al
2
O
3
, Fe
2
O
3
)
390-394
, quantum dots(QD)
395
, QD-
conjugates with bovine serum albumin and horse radish
peroxidase
396
, and QD-conjugates with
Ulex eur
opaeus
and anti-von
W
illebrand factor
397
. The
immunoassay
application of QD in CE offers considerable advantages.
In this context, quantum dots were conjugated with
antibody and subsequently tested by electrophoretic
separation of free antibody and antibody-antigen complex.
It was observed that antibody was fluorescently labelled
by quantum dots via conjugation procedures and its
electrophoretic characteristics were effectively modified
due to the attachment of quantum dots. The determination
of human IgM by direct CE based immunoassay could
be easily achieved by simply changing the pH value of
separation buf
fer
. Further
, satisfactory separation of
complex from free antibody could be achieved with 20
mM sodium tetraborate as separation buf
fer
, at pH 9.8
398
. CE has also been used for immunoassays involving
hepatitis B, prion protein, alpha-fetoprotein
399-405
, and CE-
characterization of QDs (of differing emission
wavelengths) exclusively conjugated to biotin and
streptavidin
406
. The performance of CE-LIF in
characterizing immunoconjugates of quantum dot-labeled
IgGs suggested that both QDs and CE-LIF can be applied
as a sensitive technique for the detection of biological
molecules
407
. However
, these reports on the use of QDs
in CE-based immunoassays are not satisfactory
, due in
part to a QD-biomolecule conjugate’
s (and
immunoconjuagte’
s) complex char
ge:size ratio. In view
of above, further research is required in its development
as a fast and efficient method for performing
immunoassays. Commercially available QD were
characterized using CE using detection system UV
absorption and LIF
408
. The applications stemming from
the merging of nanotechnology with microfludics in
electrophoresis haves been reported
409
.
Conclusions and perspectives
CE is a well-known automated analytical technique
that separates cationic, anionic and neutral species
covering both large and small molecules by applying
J Pharm Educ Res V
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1
20
voltage across buffer filled capillaries. This technique
allows for minimal organic consumption, fast analysis time,
high degree of resolution and low operating costs. It is
one of the suitable methods for the characterization and
quantiûcation of molecular interactions of bioactive
molecules. CE is employed in quality control of
pharmaceuticals, biopharmaceuticals, and CE analysis
includes purity determination, assays; analysis of metal
ions, simple organic acids, inorganic anions,
c
arbohydrates
and their derivatives, proteins, DNA and trace level
determinations. CE has been increasingly employed for
the separation of pharmaceutical agents and drugs;
pharmaceutical analysis, assay of active pharmaceutical
ingredients, chiral drug separation, detection of impurities
in drugs, analysis of metal and non-metal ions,
c
arbohydrates and their derivatives, proteins, DNA, and
assessment of potency and stability of drugs. The use of
CE is almost routine in many hospitals and clinics,
particularly for analyzing serum proteins and disease
markers. Moreover
, CE technique is widely accepted by
regulatory authorities.
Microchip system providing specific advantages is a
potentially valuable tool for pharmaceutical analysis when
compared to traditional separation methods. MEEKC
technique is capable of separating almost every kind of
drug, including cationic, neutral, and anionic drugs.

The
use of CE for the determination of drugs in biological
matrices and for therapeutic drug monitoring should be
further explored.
Advances in flow injection-capillary
electrophoresis technology have led to enhanced
separation capabilities in the area of pharmaceutical
analysis. In this direction, the fabrication of interfaces
between the flow injection-capillary electrophoresis
components and the microfluidic platform is currently a
focus of researchers.
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