Capillary Electrophoresis: Clinical Applications☆

David S Hage, University of Nebraska, Lincoln, NE, United States

© 2018 Elsevier Inc. All rights reserved.

This is an update of Z.K. Shihabi, Capillary Electrophoresis | Clinical Applications, In Encyclopedia of Analytical Science (Second Edition), edited by Paul
Worsfold, Alan Townshend and Colin Poole, Elsevier, Oxford, 2005, Pages 381–392, ISBN 9780123693976.

Introduction 1
Proteins and Peptides 1
Amino Acids 2
Hemoglobin 3
Enzymes 3
Immunoglobulins and CE Immunoassays 4
Hormones 4
Therapeutic Drug Monitoring 6
Drug Screening 7
DNA Testing 7
Other Clinical Applications 8
References 9
Further Reading 13

Introduction

Capillary electrophoresis (CE) is a technique that can be utilized for the separation and quantification of a wide variety of analytes.
This method makes use of the differential rates of migration of these compounds through a narrow bore capillary and in the
presence of a high electric field.1 Some advantages of CE versus traditional electrophoresis include its small sample size require-
ment, rapid analysis times, and ease of automation. Other advantages of CE when compared to traditional electrophoresis include
the many detection methods and separation modes that can be used in this method.2 All of these features have made CE appealing
for measuring analytes in complex matrices, as are often encountered in clinical samples.2,3 This article will examine the various
applications for which CE has been explored and used in clinical testing.

Proteins and Peptides

The interest in protein analysis for clinical testing by electrophoresis dates to the late 1930s, when electrophoresis was first
developed.4 CE and traditional electrophoresis are valuable for this type of separation because proteins (and peptides) are
composed of amino acids, which can carry both positive and negative charges.2,5,6 An additional feature that needs to be considered
in CE is that proteins may have some binding to the capillary wall, which can distort their peak shape and size. To prevent this effect,
buffer additives, salts, or coated capillaries are often used in these separations to minimize such binding.1
One area of clinical testing in which CE has been employed is to profile serum proteins.5,7,8 There are many proteins in serum;
however, these are generally separated by agarose gel electrophoresis (AGE) into 5–12 major bands that are used for clinical
diagnosis.5 AGE can be time-consuming but is used routinely to detect such disorders as renal failure, infections, and monoclonal
gammopathies.3–5 This same type of separation can be accomplished by capillary zone electrophoresis in approximately 2–10 min
versus the 1–2 h that are often needed for AGE (see Fig. 1).2,5
A similar approach can be used to profile proteins in urine.9,10 This type of analysis can be used to detect Bence-Jones protein for
the diagnosis of multiple myeloma or to distinguish between glomerular proteinuria and tubular proteinuria. One issue with
examining proteins in urine is that these are often present at
10–100 times lower concentrations than proteins in serum. In
addition, urine contains numerous compounds that absorb UV light, which can interfere with the detection of these proteins.
Although some urine samples can be analyzed directly, most require concentration or pretreatment before their analysis by CE.9–11
Proteins in cerebrospinal fluid (CSF) can examined by CE to help characterize disorders in the central nervous system.12–16 For
instance, detection of the oligoclonal bands in the gamma globulin region can be used as an indicator of multiple sclerosis.16
Because proteins in CSF occur at around 100-times lower concentrations than in serum, preconcentration is again needed for this
type of sample.

☆
Change History: May 2018. David S Hage revised and rewrote the text of all sections, including the addition of new sections on the analysis of hormones and
immunoglobulins and the use of capillary electrophoresis in immunoassays. The list of references was updated and expanded, Figs. 2 and 3 were added, and the
remaining figures were modified.

Encyclopedia of Analytical Science, 3rd Edition https://doi.org/10.1016/B978-0-12-409547-2.14433-6 1

2 Capillary Electrophoresis: Clinical Applications

A
(A) Agarose gel electrophoresis

g a2
T a1
M C

(B) Capillary electrophoresis

A

C
T a2 a1 a 1 a 1
a2

6.0 8.0
Time (min)
Fig. 1 Protein profiling for a serum sample from of a patient with a small monoclonal band as determined by (A) agarose gel electrophoresis and (B) CE. Symbols:
A, albumin; C, complement; I, internal standard; M, monoclonal band; T, transferrin; X, marker; a1, a1-globulin; a2, a2-globulin; and g, g-globulins. CE conditions:
30 cm  50 mm I.D. capillary; applied voltage, 9 kV; detection wavelength, 214 nm; separation buffer, 7 g/L boric acid, 7 g/L sodium carbonate, and 5 g/L
polyethylene glycol 8000 in water. The time scale on the lower axis is for the CE method.

Glycoproteins are important for cell recognition, receptor interactions, and the immune response.6 Transferrin isoforms were
among the first glycoproteins to be separated by CE.17 Other glycoproteins that have been examined by CE include ribonuclease,
ovalbumin, tissue plasminogen, human chorionic gonadotropin, and alpha1-acid glycoprotein.18–21 Borate buffers, as well as
additives that can affect the surface charge of the capillary (e.g., alkylammonium salts, diaminobutane, and formic acid) are often
important in these separations.18,22,164
CE has also been utilized to examine peptides in clinical and pharmaceutical samples. For example, glutathione in blood has
been analyzed by CE.23 Peptide analysis by CE has been used for the quality control of such compounds in biopharmaceuticals.
Examples include the analysis of endorphins, insulin peptides, aprotinin, and substance P by CE.24 In addition, CE can be used in
peptide mapping by coupling this method with detection based on fluorescence or mass spectrometry.1,25

Amino Acids

Amino acid analysis is used in clinical chemistry for various reasons. First, this type of analysis may be used to examine specific types
of amino acids, as might be used in the detection of inborn errors of metabolism like phenylketonuria or maple syrup urine
disease.6 Examples of amino acids that have been analyzed for this purpose by CE are tyrosine, proline, and phenylalanine.26–28
A second application of amino acid analysis is to examine a protein hydrolysate and/or amino acid mixture, as might be used to
determine the structure of a protein or its nutritional value. This approach, which has frequently been carried out by CE, requires an
analysis of all
20 amino acids that may be present in a protein.26–28
The analysis of amino acids poses several challenges. Because most amino acids lack a strong chromophore, they may require
derivatization (e.g., with a fluorescent tag) for detection and analysis by CE. Also, many amino acids are similar in structure and may
be difficult to separate, and samples such as serum can contain many compounds that may interfere in their analysis (e.g., peptides
 Capillary Electrophoresis: Clinical Applications 3

or uncommon amino acids). Both capillary zone electrophoresis and micellar electrokinetic capillary chromatography (MEKC)
have been explored for use in the separation of amino acids. Running buffer additives such as urea, cyclodextrin, and tetrabutyl
ammonium salts have been used in this work to improve the separation of amino acids.26–28

Hemoglobin

CE has also been used in the field of hematology, which concerns diseases that occur in blood or with the components that form
blood.6 Hemoglobin (Hb) is one blood component that has frequently been examined by electrophoresis and CE.29–35 Hb is a
tetrameric protein with four heme groups and is responsible for transporting oxygen in blood.6 This protein can occur in several
variants. Some of these variants are harmless; however, others are associated with severe anemia, a decreased capacity to carry
oxygen, or an altered shape for red blood cells. The most common variants of Hb are A, F, S, and C.6,30,32
Hb variants can be difficult to separate by capillary zone electrophoresis because of their small differences in charge and similar
isoelectric points (pI).36–39 A good separation of Hb variants by CE can be obtained by using a high buffer concentration, a narrow
bore capillary, a minimum volume of sample, and a low applied voltage. Tris, tricine, and arginine buffers at pH 8–8.4 give a good
separation in such a method. The globin chains, which are useful for investigating thalassemias, have been analyzed by capillary
zone electrophoresis in phosphate buffer at either pH 11.8 or pH 2.5–4.5 after acetone precipitation. Tryptic digests of globin chains
have also been analyzed by capillary zone electrophoresis. An alternative form of CE that can be used to separate many Hb variants
is capillary isoelectric focusing (cIEF).40,41 In addition to the common Hb variants, this method can be used to examine HbA1c (i.e.,
an indicator for long-term glucose control in diabetes), Hb-G Philadelphia, HbA2, and Hb Bart’s.40–42

Enzymes

Enzymes are proteins with catalytic activity.6,43 Some enzymes tend to occur in certain tissues or organs and may appear in the
circulation when damage occurs to these parts of the body. This feature makes many enzymes useful as biomarkers for events that
lead to such injuries (e.g., damage to the heart or liver). The levels of enzymes in cells may also be altered in some diseases
(e.g., leukemia).6,44
Enzymes that are to be used in clinical tests can be measured by CE directly, such as through absorbance measurements for
enzymes with relatively high concentrations, or by their ability to catalyze the conversion of a substrate into a product.44–49 An
enzyme’s catalytic activity is usually measured in CE by50 mixing and incubating the enzyme with the substrate before injection into
the capillary or51 incubation of the enzyme with a substrate within the capillary (see Fig. 2).48 Other approaches, such as

(A) (B) 3
3 1
2 4

5
5
2

1
Relative fluorescence

(C) 4 3 (D) 3
4

5 2
2
5
1 1

0 2 4 6 0 2 4 6
Time (min)
Fig. 2 Determination of lactate dehydrogenase (LDH) isoenzymes (peaks 1–5) in various samples by using CE with LIF detection after incubation of the isoenzymes
with a substrate (NADþ) in the running buffer. These results are for (A) a standard LDH mixture, (B) lysed normal lymphocytes, and (C) lysed T-type or (D) lysed
B-type lymphoblastic leukemia cells. Adapted with permission from Xue, Q., Yeung, E. S. Determination of Lactate Dehydrogenase Isoenzymes in Single
Lymphocytes from Normal and Leukemia Cell Lines. J. Chromatogr. B. 1996, 677, 233–240; © Elsevier.
4 Capillary Electrophoresis: Clinical Applications

postcapillary reactions, are also possible.44,45,47,49 Some advantages of using CE to measure enzymes are its small sample
requirements and good detection limits (e.g., allowing enzyme assays to be conducted even on single cells), as well as the ability
of CE to simultaneously determine and compare the activities of isoenzymes.44,46,48
A number of enzymes have been measured in clinical samples by CE. For instance, CE with laser-induced fluorescence (LIF) has
been used to measure isoenzymes of lactate dehydrogenase in single human erythrocytes and lymphocytes (see Fig. 2). CE with
electrochemical detection has been used to measure the total content of lactate dehydrogenase in human erythrocytes.44,48,52
Aminotransferase and alanine aminotransferase have been measured by CE with electrochemiluminescence detection to study the
effects of ethanol on HepG2 hepatoma cells and to distinguish alcoholic from nonalcoholic liver disease.45 The isoenzymes of
alkaline phosphatase have been measured in mouse bone marrow fibroblast cells by using CE with on-capillary incubation and
electrochemical detection.46 The isoenzymes of amylase have been measured in saliva by using cIEF with whole column imaging
and direct absorbance detection.49 Additional enzymes that have been examined by CE are chloramphenicol acetyl transferase,
glutathione peroxidase, and cathepsin D.53–55

Immunoglobulins and CE Immunoassays

Various CE methods have been developed for the detection of immunoglobulins, which are glycoproteins that are produced by the
immune system to bind to a given target, or antigen.43 Variations in the levels of immunoglobulins may occur during autoimmune
diseases, infections, immunodeficiency, or liver disease. Multiple myeloma, Waldenstrom’s disease, and light chain disease can also
cause increased levels of these agents in the form of “paraproteins” or “myeloma proteins”.6,43 Myeloma proteins have traditionally
been detected by using immunofixation, which is a time-consuming method that uses gel electrophoresis. CE can be modified to
perform immunofixation by reacting serum proteins with specific antibodies that are bound to a solid matrix. The sample is assayed
before and after binding to this support, with the difference between the two results representing the myeloma protein that is
present.
Cryoglobulins are special immunoglobulins that reversibly precipitate from serum at cold temperatures. Cryoglobulins can be
monoclonal immunoglobulins, a mixture of polyclonal and monoclonal immunoglobulins, or a mixture of polyclonal immuno-
globulins. Cryoglobulins are associated with several immune-type disorders, viral infections, glomerulonephritis, peripheral
neuropathy, and diffuse vasculitis. These agents can be detected by precipitating an aliquot of serum at 4 C, centrifuging, and
dissolving the precipitate in a buffer followed by CE under the same conditions as used for serum proteins.56
Immunoglobulins that bind to a given antigen are known as antibodies. Antibodies can be used in CE to bind and analyze both
immunoglobulins and other types of compounds. The combination of antibodies as binding agents with CE is often called a “CE
immunoassay”.57 One way antibodies can be used as binding agents is by attaching them to a support for the extraction of a given
target or group of related targets prior to the analysis of these captured agents, or the agents remaining in the sample, by CE. For
instance, this is the approach that has been used to combine immunofixation with CE. Such a method may involve using
immobilized antibodies that are separate from the CE system (i.e., off-line immunoextraction) or part of the CE system (i.e.,
on-line immunoextraction).57,58
It is also possible to combine antibodies with CE for the indirect detection of an analyte. One approach for doing this is a
competitive CE immunoassay, in which a labeled analog of the desired target analyte is added to the sample to compete with the
target in binding to a limited amount of antibodies. This method is illustrated in Fig. 3A.57 The amount of labeled analog that is
bound to the antibodies or that remains free in solution is then measured after CE has been utilized to separate these fractions. In a
noncompetitive CE immunoassay, as is shown in Fig. 3B, the antibody is labeled. An excess of the labeled antibody is mixed with
the analyte and the antibody-analyte complexes are separated by CE from any excess nonbound labeled antibodies. The amount of
the labeled antibody-analyte complex is then measured to determine how much of the analyte was present in the sample.57,59,60
A CE immunoassay can be carried out in a sandwich format by using an immobilized antibody in the capillary to capture the
analyte and a labeled antibody that is later passed through the capillary to form a complex with the captured analyte.57 In this case,
the amount of labeled antibody that binds to the capillary is measured as it is later released, making it possible to determine who
much analyte was present in the sample. The various types of CE immunoassays (i.e., immunoextraction, competitive, noncompe-
titive, and sandwich formats) have been used to measure various immunoglobulins in clinical samples, such as immunoglobulin G,
immunoglobulin E, and immunoglobulin A.59–61 The same methods have been used for many other types of compounds of clinical
interest, as will be discussed in the following sections.

Hormones

Hormones are chemicals that are produced by endocrine glands and cause a specific type of cell, tissue or organ to produce a
response or regulate a given function of the body.6,43 The hypothalamus gland produces several hormones that are of interest in
clinical chemistry, which include thyrotropin-releasing hormone (TRH), somatostatin, and gonadotropin-releasing hormone
(GnRH).6 An on-line immunoextraction method has been used with CE and mass spectrometry or absorbance detection to measure
GnRH in serum and urine.62 CE with absorbance detection has been utilized to examine somatostatin and TRH in plasma.63
 Capillary Electrophoresis: Clinical Applications 5

Fig. 3 General schemes for (A) a competitive CE immunoassay and (B) a noncompetitive CE immunoassay. Adapted with permission from Moser, A. C.;
Willicott, C. W.; Hage, D. S. Clinical Applications of Capillary Electrophoresis Based Immunoassays. Electrophoresis 2014, 35, 937–955; © Wiley.

Many hormones that are produced by the pituitary gland have been analyzed by using CE. Examples are follicle-stimulating
hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and vasopressin (or antidiuretic hormone).6,43
Competitive and noncompetitive CE immunoassays have been used with chemiluminescence to measure FSH and LH in
serum.64–66 TSH has been determined in blood by using a noncompetitive CE immunoassay with LIF detection.67 Vasopressin in
plasma has been measured by using CE and absorbance detection, and a CE competitive immunoassay with LIF detection has been
employed to measure vasopressin in CSF.63,68
Hormones that are secreted by the pancreas and thyroid gland have been measured by CE. Insulin and glucagon are hormones
that are produced by the islets of Langerhans in the pancreas.6,43 Competitive CE immunoassays with fluorescent detection have
been used to measure the secretion of both these hormones.69,70 This general approach has also been utilized to analyze insulin,
glucagon, and islet amyloid polypeptide.71 Thyroxine (also known as tetraiodothyronine or T4) is the major hormone secreted by
the thyroid gland and has been examined by using both competitive and noncompetitive CE immunoassays in clinical
samples.72–75 Microextraction has been combined with CE for the simultaneous determination of T4 and several related compounds
in serum.76
Estrogens and testosterone are steroid hormones that have been examined by a variety of CE methods. A competitive CE
immunoassay was created to analyze estradiol in serum,77 and off-line immunoextraction has been employed with MEKC to
analyze estradiol, estrone, and estriol in urine.78 CE has further been combined with time-of-flight mass spectrometry for the
6 Capillary Electrophoresis: Clinical Applications

analysis of estrogens and their glucuronide or sulfate metabolites in serum.79 Solid-phase extraction and MEKC with a partial filling
approach have been utilized to measure testosterone, androstenedione, and epitestosterone in urine,80 while off-line immunoex-
traction has been combined with MEKC to measure testosterone and epitestosterone in urine.81,82 A competitive CE immunoassay
has been used to determine testosterone in urine.83
Catecholamines such as norepinephrine, epinephrine, and dopamine are important hormones and neurotransmitters in
humans.6,43 These compounds have been measured in urine by CE with absorbance or fluorescence detection.84–88 CE with
electrochemical or chemiluminescence detection has also been used to measure catecholamines in urine.52,89–91 In addition, CE
and mass spectrometry have been combined for the determination of catecholamines and related metabolites in urine.92,93

Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) uses the analysis of drugs in the circulation to maximize therapeutic effects and minimize side
effects.43 Many CE methods have been developed for drug analysis by using capillary zone electrophoresis or MEKC. Nonaqueous
CE has been used in some cases for drugs that have low solubilities in water. Various sample pretreatment methods have combined
with these methods to improve the detection limits or selectively. Some of these methods have included the use of sample stacking,
as a means to concentrate drugs and other compounds prior to their analysis by CE (see Fig. 4), and pretreatment techniques such as
solid-phase extraction and liquid–liquid extraction. Detection in these CE methods have ranged from absorbance detection to mass
spectrometry.
Many classes of drugs have been examined by CE, including antibiotics such as amikacin, cefepime, and tobramycin. Tobra-
mycin has been measured in serum by using CE and absorbance detection after derivatization and solid-phase extraction.94
Amikacin has been determined by CE and conductivity detection in bronchial epithelial lining fluid.95 Cefepime has been measured
in serum by employing MEKC with absorbance detection and protein precipitation.96–98 The antiepileptic drug lamotrigine has
been determined in serum and plasma by using CE with absorbance detection or mass spectrometry after protein precipitation.99,100
Various CE methods have been reported for psychoactive drugs. Indirect absorbance detection has been used in CE for the
analysis of lithium in serum,101 while CE with direct absorbance detection has been employed for measuring aripiprazole in
plasma.102 MEKC with absorbance detection and injection based on sweeping has been utilized for determining paliperidone in
plasma.103 Nonaqueous CE with absorbance detection has been used to determine various antidepressants in plasma,104 and field-
amplified sample injection has been combined with CE and absorbance detection for the analysis of threo-methylphenidate in
saliva.105
Several other types of drugs have been measured in clinical samples by CE. Antifungal drugs (e.g., voriconazole, itraconazole,
and posaconazole) have been determined in serum and plasma by using sample extraction followed by MEKC and absorbance
detection, with the possible inclusion of sample injection based on sweeping.106–108 The chemotherapy agent methotrexate has
been measured in blood or plasma by using CE and absorbance detection with sample stacking and solid-phase extraction.109,110
Imatinib, another chemotherapy agent, has been examined in plasma by means of CE combined with mass spectrometry.111 The
immunosuppressant mycophenolic acid has been determined in serum by using CE and absorbance detection.112 Isoniazid (i.e., an

0.010

I F
Absorbance, 280 nm

0.005

0.000

0.0 2.0 4.0 6.0

Time (min)
Fig. 4 Analysis of fenofibric acid in serum by CE with the use of sample stacking for injection. Iohexol (I) was used as an internal standard. CE conditions:
25 cm  50 mm I.D. capillary; applied voltage, 7 kV; sample size, 13% of capillary volume; separation buffer, 7 g/L boric acid, 7 g/L sodium carbonate, and 5 g/L
polyethylene glycol 8000 in water.
 Capillary Electrophoresis: Clinical Applications 7

agent used to treat tuberculosis) and methadone (i.e., a synthetic opioid) have been assayed in serum or plasma by combining
injection that used cation-selective exhaustive injection–sweeping with MEKC and absorbance detection.113,114
About 40% of the most common drugs contain at least one chiral center.115,116 The different chiral forms of a drug may exhibit
differences in their biological effects, binding to proteins, or metabolism, which makes the separation and analysis of these forms
important in clinical and pharmaceutical testing. This type of separation can be accomplished in CE by adding a chiral binding
agent to the running buffer and that has different interactions with the chiral forms of a drug.115–118 This method is sometimes
known as affinity capillary electrophoresis.118 Examples of binding agents that have been used for this purpose are cyclodextrins,
heparin, proteins, and some antibiotics.115–118 Along with using a chiral agent in the running buffer, the chiral agent may also be
placed onto a support in the capillary. This second approach is a form of capillary electrochromatography.

Drug Screening

A clinical laboratory may also seek to identify a drug of abuse or to identify a drug that may have been taken as an intentional or
accidental overdose.43 This type of analysis can be done by using various modes of CE, including CE immunoassays.57 For instance,
a competitive CE immunoassay with LIF detection has been used to determine several metabolites of acetylsalicylic acid (i.e.,
aspirin) in urine for detecting this drug in overdose cases.119 The analysis of acetaminophen and its metabolites in urine for a similar
purpose has been carried out by using CE with absorbance detection or mass spectrometry.120
Several potential drugs of abuse have been examined by CE. For instance, amphetamine, methamphetamine, and related
compounds have been analyzed in urine by using CE with detection based on absorbance, LIF, mass spectrometry, or electrochem-
ical properties.22,121–126 In addition, affinity CE has been employed to separate the chiral forms for many compounds in this class
by using cyclodextrins as running buffer additives.127–130 Cocaine and its metabolites have been examined in urine by using CE and
mass spectrometry with sample stacking.73,131 In addition, several CE methods have been reported for the determination of opiates
in urine, such as the use of CE with mass spectrometry, absorbance, or LIF detection and a competitive CE immunoassay with LIF
detection.50,51,132–135

DNA Testing

DNA testing is another area in which CE has been employed for clinical analysis.3,136–141 DNA is composed of two chains of
nucleotides with complementary base pairs (bp) that make up a double-stranded helix. Because DNA consists of long strands, it
needs to be cut into smaller fragments by special enzymes (i.e., nucleases) before its analysis. These fragments can be characterized
in terms of the number of bases they contain or their base sequences. Because these fragments are present at low concentrations, they
are typically amplified before their analysis by using the polymerase chain reaction (PCR).3
DNA molecules all carry essentially the same charge per mass, so they cannot be separated by electrophoresis simply based on
this property. However, a separation can be achieved by using their differences in size.3 Agarose gels have been used in traditional
electrophoresis to separate double-stranded DNA (dsDNA) fragments after digestion for mapping, while slab polyacrylamide gel
electrophoresis has been used to separate single-stranded DNA (ssDNA) fragments for sequencing. Initially, CE methods for DNA
separations attempted to mimic polyacrylamide gel electrophoresis by using the same gels but now using migration times to
characterize the size of DNA fragments. However, these gel-filled capillaries had several practical problems with regards to the short
lifetime of the gels in the capillary and difficulties with air bubbles. Solutions of uncrossed polymers such as polyacrylamide,
cellulose derivatives, poly(vinylpyrrolidone), poly(ethylene oxide), and dextrans have become popular alternatives to gels for
separating DNA fragments (see Fig. 5).142 For DNA sequencing, enzymatic synthesis of the purified template is performed in the
presence of deoxynucleotides and fluorescent labeled dideoxynucleotides that will terminate the DNA strand at specific nucleotides.
These conditions result in the production of fragments with different lengths and with terminating labels that can be used in CE to
determine the sequence of the original DNA.3
CE has been used extensively for fragment size analysis and DNA sequencing.3 dsDNAs that have been obtained after PCR
amplification have been utilized in CE to identify bacteria and viruses.138,143 This type of sequence analysis can also be used to
monitor changes in the DNA of a virus (e.g., HIV),138 which can allow a better match of a treatment with the genetic makeup of the
infectious agent. Diagnosis of an inherited disease or cancer often involves looking for unknown point mutations or known
mutations in several genes, as can lead to single nucleotide polymorphisms (SNPs). Heteroduplex analysis (HA) and single-strand
conformation polymorphism (SSCP) have been used for this purpose in electrophoresis with slab gels and in CE. In SSCP, the
strand is amplified through PCR and denatured through heat and the addition of formamide. The separated strands adopt folded
structures that are determined by their sequences and which can be examined by CE based on their different electrophoretic
mobilities. In HA, the PCR-amplified DNA of allelic fragments are denatured and re-annealed to give a mixture of four duplexes
(i.e., two homoduplexes and two heteroduplexes) in heterozygote samples, where the heteroduplexes generally move more slowly
in a gel than the homoduplexes. These methods have been applied to the detection of BRCA1 and BRCA2 mutations, p53, and
fragile X.144–146
8 Capillary Electrophoresis: Clinical Applications

Fig. 5 Separation of eleven DNA fragments by CE using an untreated capillary. Fragment sizes: (1) 72 bp, (2) 118 bp, (3) 194 bp, (4) 234 bp, (5) 271 bp, (6) 281
(bp), (7) 301 bp, (8) 603 bp, (9) 872 bp, (10) 1078 bp, and (11) 1353 bp. Adapted with permission from Shihabi, Z. K. (1999). Capillary Electrophoresis of Double-
Stranded DNA in an Untreated Capillary. J. Chromatogr. A 1999, 853, 349–354; © Elsevier.

Other Clinical Applications

Many organic and inorganic ions are of interest in clinical testing and have been examined by CE.43 Cations and anions can often be
analyzed in the same run and can be separated by CE using a simple running buffer or a buffer that contains a chelating agent.
Inorganic ions in serum are important in maintaining osmotic pressure and pH. Common inorganic ions such as Naþ, Kþ, Ca2þ are
usually measured by using ion-selective electrodes in clinical laboratories; however, other ions such as nitrite and nitrate can be
easily determined by CE (see Fig. 6).147 Some organic acids are important in detecting inborn errors of metabolism, infection, and
metabolic disorders. Many of these compounds have been determined by CE directly or indirectly through the addition of a UV
absorbing compound into the running buffer. For example, oxalate, pyruvate, lactate, malonate, maleate, succinate, and other
organic ions have been measured by CE in urine,148 and lactate has been examined in CSF by CE.149

Fig. 6 Analysis of nitrite (Ni) and nitrate (Na) by CE in (A) urine or (B) cerebrospinal fluid. Bromide (Br) was used as an internal standard. Adapted with permission
from Friedberg, M. A.; Hinsdale, M. E.; Shihabi, Z. K. Analysis of Nitrate in Biological Fluids by Capillary Electrophoresis. J. Chromatogr. A. 1997, 781, 491–496; ©
Elsevier.
 Capillary Electrophoresis: Clinical Applications 9

Simple carbohydrates are important in the diagnosis of diabetes and inborn errors of metabolism.6 These compounds pose
several problems in their analysis by CE, such as their poor absorbance of light in the UV range.150,151 Indirect detection of
carbohydrates can be accomplished by using buffers in CE with ions that contain chromophores. Another problem is that simple
sugars are not ionized unless the pH is above 12. These sugars can be separated by capillary zone electrophoresis when using buffers
that contain sodium hydroxide or lithium hydroxide, with riboflavin added for indirect UV detection. Carbohydrates can also be
derivatized with fluorescent reagents or complexed with an agent such as borate.150,151
Tumor biomarkers are substances that are used to detect or monitor a certain form of cancer.43 Many types of compounds can be
used as tumor biomarkers and have been examined by CE for this purpose. For instance, CE has been used to measure enzymes such
as lactate dehydrogenase and alkaline phosphatase as tumor biomarkers.44,46–48 Prostate-specific antigen, which is used to detect
prostate cancer, has been measured in serum by a noncompetitive immunoassay based on CE with electrochemical detection.152
Also, immunoextraction has been used with CE and absorbance detection for analyzing the various isomers of this glycoprotein in
seminal plasma.153 Tumor biomarkers that are oncofetal antigens, such as carcinoembryonic antigen and a-fetoprotein, have been
examined by using noncompetitive CE immunoassays based on LIF, chemiluminescence, or electrochemical detection.152,154–157
The same is true for tumor biomarkers that are able to bind to certain types of antibodies, such as cancer antigen 125 and cancer
antigen 15-3.111,158,159 Human chorionic gonadotropin, which can have elevated levels when it is produced by tumors of the ovary
or testes, has also been measured by using a noncompetitive CE immunoassay with electrochemical detection.152

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Further Reading
Elhamili, A.; Bergquist, J. A Method for Quantitative Analysis of an Anticancer Drug in Human Plasma With CE-ESI-TOF-MS. Electrophoresis 2011, 32, 1778–1785.
Greene, D. N.; Vaughn, C. P.; Crews, B. O.; Agarwal, A. M. Advances in Detection of Hemoglobinopathies. Clin. Chim. Acta 2015, 439, 50–57.
Li, X. M.; Zhang, F.; Zhang, S. S. Capillary Electrophoresis Enzyme Immunoassay for Alpha-Fetoprotein and Thyroxine in Human Serum With Electrochemical Detection. J. Sep. Sci.
2008, 31, 336–340.