Recent advances in capillary electrophoresis and capillary electrochromatography of pollutants

Rania BakryChristian W. HuckMuhammed Najam-ul-HaqMatthias RainerG�nther K. Bonn

Institute of Analytical Chemistryand Radiochemistry, Leopold-Franzens University, Innsbruck,Austria

Review

Recent advances in capillary electrophoresis forbiomarker discovery

The use of non-invasive methods for detecting biomarkers opens a new era inpatient care, since clinical investigators have long been searching for accurate andreproducible measurements of putative biomarkers. There are many factors whichmake this research challenging, beginning with lack of standardization of samplecollection and continuing through the entire analytical procedure. Among thevariety of methods so far used for biomarker screening, capillary electrophoresisrepresents a robust, reliable, and widely used analytical tool. This review will focuson recent applications of CE to the analysis of body fluids and tissues for identifica-tion of biomarkers.

Keywords: Biomarkers / Blood / Cerebrospinal fluid / Tissue / Urine /

Received: August 17, 2006; revised: October 12, 2006; accepted: October 13, 2006

DOI 10.1002/jssc.200600323

1 Introduction

A biomarker is “a characteristic entity that is objectivelymeasured and evaluated as an indicator of a normal bio-logical process, pathogenic process, or pharmacologicalresponse to a therapeutic intervention”. A biomarker canserve as a clinical endpoint, a surrogate endpoint, orboth. A clinical endpoint is a characteristic or a variablethat reflects how a patient feels, functions, or survives. Asurrogate endpoint is a biomarker intended to substitutea clinical endpoint. A surrogate endpoint is expected topredict clinical benefit (or harm, or lack of benefit orharm) based on epidemiological, therapeutic, pathophy-siological, or other scientific evidence [1]. For example,biomarkers as surrogate clinical measures detect earlybiological responses after drug treatment, for analyzingdrug safety and early efficacy of new drugs. Biomarkerscan have both prognostic and diagnostic uses. Forinstance, once the disease is established, these markerscan be used to predict the likely course of disease and tomonitor and assist in the management of disease. Onecan use biomarkers to stratify the diseased populations.Lastly, screening large populations with biomarkers

leads to discrimination of a healthy state from the earlyasymptomatic stage of the disease.

According to the FDA, Guidance for Industry: Pharmaco-genomic Data Submission (2005), a “valid biomarker” is one“that is measured in an analytical test system with well-established performance characteristics and for whichthere is an established framework or body of evidencethat elucidates the physiological, toxicological, pharma-cological, or clinical significance of the test result”. The“known valid biomarker” is differentiated as one “forwhich there is widespread agreement in the medical orscientific community”. The “probable valid biomarker”may not have reached the status of a “known valid bio-marker” because the data elucidating its significancemay have been developed from within a single companyand may not be available for public scrutiny. In addition,biomarkers find many applications in modern biologyand medicine. From pregnancy tests to monitoring cho-lesterol levels, they already impact our daily lives, andthe ongoing hunt for diagnostics and predictors ofresponses promises to affect our future in profoundways. The characteristics and qualities demanded of abiomarker depend on how it will be employed.

Focus in biomarker research is shifting from methodsthat can analyze one marker at a time to so-called profil-ing methods, which allow the simultaneous measure-ment of a range of markers. It is believed that such mar-ker patterns will allow a statistically more stringent dif-ferentiation and a better classification of patient groups(for example, those with cancer and those without). Thisshould improve early detection of disease and alsoreduce the number of false positive or negative results.Better classification of patients will also diminish the

Correspondence: Professor Christian Huck, Institute of Analyti-cal Chemistry and Radiochemistry, Leopold-Franzens Univer-sity, Innrain 52a, 6020 Innsbruck, AustriaE-mail: [email protected]: +43 512 507 2965

Abbreviations: CDT, carbohydrate-deficient transferrin; CSF,cerebrospinal fluid; DAP, 1,4-diaminopropane; FTIR, Fouriertransform infrared spectroscopy; GAGs, glycosaminoglycans;MT, metallothionein; SELDI, surface enhanced laser desorptionionisation

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

192 R. Bakry et al. J. Sep. Sci. 2007, 30, 192 – 201

J. Sep. Sci. 2007, 30, 192 – 201 Electrodriven Separations 193

number of subjects that need to be enrolled in clinicaltrials in order to reach statistically significant levels.Most importantly, it is hoped that a more focused diag-nosis will lead to better therapies as well as to earlierintervention [2]. However, a biomarker need not to becausal or involved mechanistically in the disease process.Once discovered, these biomarkers can be used for manypurposes, including diagnosing disease, monitoringtreatment and recurrence. The challenge, then, is how todiscover and validate these biomarkers in a relativelyefficient manner [3]. To meet this challenge, an analyti-cal technique must separate and detect as many compo-nents as possible. It must be robust to the typical biologi-cal variation in physicochemical properties of the sam-ple. It must perform in the same manner from analysisto analysis. It must be economical in time, effort, andexpense. Body fluids, such as serum, urine, and cere-brospinal fluids, are mostly used to investigate and ana-lyze biomarkers. The advantages of body fluid analysisare less pain for patients when obtaining samples andgreater accessibility for clinical tests. However, the detec-tion of tissue-specific biomarkers in body fluids requiresfirst that the target molecule be secreted, and secondlythat the biomarker be identified as disease tissue-specificfrom a multitude of secreted biomolecules from variouscell types and organs [4]. Srivastava proposed that the bio-marker life cycle depicts the stages in biomarker develop-ment and validation. In the discovery phase, a promisinglead (biomarker) is identified through basic research inanimal models, tissues, or cell cultures. In the develop-ment phase, a biomarker is identified in human speci-mens; an assay for the biomarker is developed and/orrefined; the analytical sensitivity of the assay for the bio-marker is tested; and the clinical sensitivity of the assayis measured to meet specific biological questions. In theevaluation phase, a biomarker is subjected to broad ques-tions to verify the intended use, such as whether the bio-marker is applicable to biological and clinical settings ina variety of conditions. In the application phase, a bio-marker test is field-tested for a specific clinical use, suchas screening of cancer, and for its cost and effectiveness

in reducing disease burden and mortality due to the dis-ease (Fig. 1) [5]. The use of biomarkers in toxicology isbecoming increasingly important in assessing the healthrisks of exposure to potentially toxic drugs and chemi-cals. Biomarkers are used to measure effects on the cata-bolism after exposure to a toxic compound and theextent of a toxic response that should be easy to measureand quantify with high sensitivity and specificity, andthey should furthermore relate to the biochemicalmechanism of a compound. The ultimate biomarkershould be species-independent and non-invasive andshould predict a toxic effect at realistic doses [6]. Most ofthe recent biomarker research has focused on variousforms of cancer. Modern biomolecular, analytical tech-niques have emerged that promise to improve cancerdiagnosis, classification, prognosis, and follow-up oftherapy and eventually establish possibilities for indivi-dual treatment.

Several modern analytical techniques for screening ofbiomarkers in tissues and biological fluids are utilizedwhich include gas chromatography coupled to massspectrometry [7–12], HPLC coupled or not to MS [13–16],two dimensional gel electrophoresis [17–20], Fouriertransform IR spectroscopy (FTIR) [21,22], surface-enhanced (assisted) laser desorption/ionization-massspectrometry (SELDI-MS) [13–26], material enhancedlaser desorption/ionization [27–29], and nuclear mag-netic resonance [30–32]. Several review articles havebeen published covering applications of the above men-tioned methods [33–39]. Due to the high separation effi-ciency of a great variety of compounds, the small samplesize, and short analysis times, CE has received increasingattention during recent years. In addition, CE is fast,easy, straightforward, can be automated, and gives quan-titative data while providing extremely high separationefficiency in a relatively short time period. Additionally,due to its plug-like flow and minimal diffusion, CE pos-sesses enormous resolving power and large peak capaci-ties. Utilizing any of the many modes of CE enables anal-ysis of chemical species from amino acids, proteins, pep-tides, and nucleic acids to metallic ions, natural pro-ducts, and drugs of abuse [40, 41]. In this review article,we focus on recent applications of capillary electrophor-esis for the screening of biomarkers.

2 Biomarkers in urine

Among the body fluids available, urine appears to haveseveral advantages. Urine is easily accessible in largequantities. Collection of urine samples does not requireany trained medical personnel. In addition, urine shoulddisplay the health status of a limited number of organs,mostly kidney and bladder, which in turn should reducecomplexity. Urine can yield reliable information on a


Figure 1. Life cycle of biomarker. Reprinted from reference[5], with permission.


large series of specific biochemical constituents, includ-ing inorganic ions, organic acids, purines, pyrimidines,amino acids, peptides, and proteins that are expected tochange to a particular disease state. Moreover, the factthat some analytes become significantly more concen-trated in urine than in other fluids makes measurementseasier.

2.1 Proteins and peptides

An “ideal biomarker”, which should display unique char-acteristics (e. g., presence/absence or signal intensity) ineach sample from patients with a certain disease, doesnot exist. Instead, an array of polypeptides that show sig-nificant differences in distribution (both amplitude andpresence/absence) between disease and health have to beconsidered. Using CE-MS it is documented that polypep-tides in urine change significantly during the day, mostlikely as a consequence of physical activity, diet, smok-ing, etc. [42]. As a result, reproducibility at the level of sin-gle polypeptides is limited. Hence, the clinical usefulnessof a single biomarker measurement is imperfect even iftest accuracy and reproducibility are optimal. In con-trast, it is important to analyze as many peptides as possi-ble and to develop methods that can derive diagnosticpower from the patterns of observed peptides. The com-bination of tens or even hundreds of biomarkers to gen-erate distinct diagnostic patterns will make the methodboth more robust and specific. Spurious occurrences ofpeptides can be eliminated without jeopardizing theoverall results and diagnosis. Evidently, combining thesepolypeptides to a signature or pattern results in (i)increased ability to distinguish between disease andhealth and (ii) increased stability, since the absence oruntypical amplitude of single peptides does not result ina significant change of the typical pattern. The highnumber of biomarkers defined for a specific disease alsosuggested that this approach might enable staging, eva-luation of disease progression and the effects of therapy.

The optimal tool for the analysis of a large number ofcomplex biological samples was found to be CE usinguncoated fused silica capillaries, coupled via a sheathliquid interface to a fast and sensitive mass spectrometer[43, 44]. Theodorescu et al. described a CE-MS method forthe identification of single and patterns of polypeptidesspecific for prostate cancer in human urine. 47 urinesamples (21 patients had benign pathology and 26 withprostate cancer) were compared to that of 41 young men(control) without known or suspected clinical prostatecancer. Upon cross-validation of the same samples, sev-eral polypeptides were selected that enabled correct clas-sification of prostate cancer patients with 92% sensitivityand 96% specificity [45]. The same working group appliedthis technique for diagnosis of urothelial carcinoma. Apattern of 22 polypeptides was identified. A prominent

polypeptide from the diagnostic pattern for urothelialcarcinoma was identified as fibrinopeptide A – a knownbiomarker of ovarian cancer and gastric cancer. Themethod allowed correct identification of patients withurothelial carcinoma among those with other malignantand non-malignant genitourinary diseases with a sensi-tivity ranged from 86% to 100% [46].

Recently Rossing et al. [47] applied CE-MS as a fast andsensitive tool for identification of biomarkers and urin-ary polypeptide patterns that can be used to discriminatebetween diabetic patients with and without diabeticnephropathy, and furthermore to monitor treatmenteffects. The data also indicate that several potential bio-markers in addition to albumin can be defined using CE-MS and, if required, MS/MS sequences. Differences inurinary polypeptide patterns between normo- andmacroalbuminuric patients permitted the establishmentof a “diabetic renal damage” pattern consisting of 113polypeptides. Eleven of these polypeptides had beensequenced and identified. These 113 polypeptides wereutilized to monitor the treatment with candesartan overthree different dose levels. A simple pattern recognitionalgorithm was used that searched for polypeptideswithin a distinct time and mass window (Fig. 2). Wittke etal. [48] reported use of the same technology for biomar-ker discovery in patients with renal diseases. The authorsscreened urine samples of healthy individuals and ofpatients for differences in protein patterns. Twenty-sevenpolypeptides were additionally present, others wereabsent in the urine of patients. In a more comprehensiveapproach, human urine from healthy volunteers, dia-betes mellitus and septic patients, and patients withchronic renal diseases were investigated for ultimate bio-markers. Typical polypeptide patterns were related tothe respective state of health [43, 49].

2.2 Amino acids

The measurement of urinary amino acids is importantsince proper screening can help in the diagnosis ofinborn errors of amino acid metabolism such as phenyl-ketonuria, maple syrup urine disease, and histidinemia[50, 51]. Sogo et al. [52] described a (CE-ESI-MS/MS) methodto identify and quantify amino acids. A fused silica capil-lary was employed for separation under acidic pH condi-tions using 1 M formic acid as running buffer. A sheathliquid around the outside of a CE capillary was appliedby pumping 10 mL/min of 5 mM ammonium acetate in50% v/v methanol–water to the CE interface, to provide astable electrical connection between the tip of the capil-lary.

Annovazzi et al. [53] described a CE-LIF method forinvestigating chronic obstructive pulmonary disease andPseudoxanthoma elasticum; two disorders characterizedby an abnormal turnover of elastin. The method is based



on separation and identification of minute amounts ofurinary desmosines; amino acids that can be used aspotential markers of elastin alteration. The urine sam-ples were derivatized with FITC, and then injected. Theseparation was carried out using 20 mM sodium tetrabo-rate pH 9.0 containing 60 mM SDS and 15% v/v methanolas a background electrolyte. Figure 3 shows the electro-phoretic pattern obtained from a micropreparative run,performed on derivatized urine of patient P5. Peak 1 wasdesalted and collected for MS identification. The signalat m/z = 916.3 corresponds to FITC-desmosines and thatat m/z = 390.3 corresponds to a fragment obtained fromthe FITC molecule. The same technique was used also byLochman et al. [54] for determination of biogenous ami-nothiols – homocysteine, cysteine, glutathione, cystei-nylglycine, c-glutamylcysteine in plasma and urine. Theanalysis of aminothiol compounds in body fluids isimportant in diagnosing inherited cystinuria, homocys-tinuria, and other diseases. Samples were reduced withtris(carboxyethyl)phosphine and labeled with 5-(bromo-methyl)fluorescein. Capillary electrophoretic separa-tions were performed in 60 mM borate–15 mM SDS–2-amino-2-methyl-1-propanol, pH 10.0, with laser-inducedfluorescence detection (Fig. 4).

2.3 Purine and pyrimidines

Purines and pyrimidines represent an important class ofmetabolites, which serve as vital precursors for biosyn-thesis of DNA and RNA nucleotides in a cell. The analysisof purine and pyrimidine levels in biological fluids plays

an important role in the diagnosis of a variety of meta-bolic disorders. Modified purines and pyrimidinesexcreted in urine have been also used as potential bio-markers for early detection of cancer. Adam et al. [55] andFriedecky et al. [56] developed CE methods for diagnosisof inherited purine and pyrimidine enzyme deficiencies.Characteristic abnormalities were detected in electro-pherograms of urine samples from patients with purineand pyrimidine enzyme deficiencies. Electrophoreticseparations were achieved at pH 1.8 using 200 mM phos-phate buffer. The authors observed that the addition oflithium and aspartinium as co-ions had no influence onseparation efficiencies and resolution. In anotherapproach the electrophoretic separations were carriedout in an uncoated fused silica capillary using 45 mMborate, 55 mM N-tris(hydroxymethyl)methylglycine,10 mM tartrate, 1 mM cetyltrimethylammonium bro-mide, and 0.44% tetrabutylammonium hydroxide–2-amino-2-methyl-1,3-propanediol (pH 8.6) as BGE. Cationicsurfactant was added to give reproducible reversed EOF[55].

Britz-Mckibbin et al. [57] reported on the analysis ofpurine metabolites with multiplexed capillary electro-phoresis and UV detection with online preconcentrationby a dynamic pH junction. The optimization of purinefocusing was assessed by systematically altering the sam-ple matrix properties, such as the buffer co-ion, pH, andionic strength using a 96-capillary array format. Samplestacking was enhanced by low ionic strength samplematrix, to amplify the local analyte velocity relative toBGE, thereby focusing a large injection volume to


Figure 2. Typical polypeptides in normo-, micro-, and macroalbuminuric patients. Polypeptides with frequencies of occurrencegreater than 70% in each individual group are displayed with their mass over the migration time. A total of 231 polypeptides wereclassified as typical for normoalbuminuric patients without diabetic retinopathy (A), 266 for normoalbuminuric patients with dia-betic retinopathy (B) and 256 for microalbuminuric patients with retinopathy (C). While these patterns appear quite similar, someof the 140 polypeptides classified as typical for macroalbuminuria (D) show a different migration time behaviour and are of highermolecular mass. Reprinted from reference [47], with permission.


enhance the sensitivity. The ionic strength of samplematrix relative to BGE also plays an important role inpurine focusing and resolution. The optimum conditionsfor purine analysis were 20 mM phosphate buffer pH 6.0.

2.4 Nucleosides, nucleotides, and DNA adducts

Modified nucleosides are formed post-transcriptionallyin RNA. During RNA turnover, free modified nucleosidesare formed which circulate in the blood stream and areexcreted in urine. Their levels are increased in a numberof malignant diseases, and they can be used in clinicalchemistry as tumour markers. A pattern recognitionmethod has been used to reveal more information on thedifferences between healthy individuals and cancerpatients. La et al. [58] described a CE method in MEKCmode for profiling of 15 nucleosides in urine from 12female thyroid cancer patients and 12 healthy females.The nucleosides from urine samples were first isolatedwith phenylboronate gel and separated on uncoated col-

umns using 25 mM borate–42.5 mM phosphate (pH 6.7)containing 200 mM SDS as a running buffer. RecentlyLiebich et al. [59] reported the same mode for identifica-tion of nucleosides using MALDI-TOF for clinical studiesof children with leukemia and on women with breastcancer. Candidate markers are pseudouridine, 1-methyl-guanosine, N2-methylguanosine, 3-methyluridine, and 1-methyl-inosine.

Oxidative damage of DNA has attracted a great deal ofinterest in relation to mutagenesis, carcinogenesis, andaging. One of the major products of oxidative degrada-tion of nucleic acid is 8-hydroxy-29-deoxyguanosine(8OHdG), which has been, therefore, proposed as a usefulbiomarker. Recent reports on analysis of urinary 8OHdGin healthy individuals have found that 8OHdG concen-tration varies from 1 to 50 nM. Enhanced levels of urin-ary 8OHdG above these values have been found in vari-ous groups of patients, in particular cancer patients, andthose undergoing chemotherapy or radiation therapy[60, 61]. Yao et al. [62] developed a CE method with end-


Figure 3. Typical electrophoretic pattern obtained from a micropreparative run performed on derivatized urine of patient P5 on acapillary of 57 cm length and 100 mm ID. Peak 1 was that desalted and collected for MS identification. The peak indicated by anarrow is the excess of FITC. Inset: mass spectrum of material collected under peak 1. The signal at m/z = 916.3 corresponds toFITC-desmosines and that at m/z = 390.3 corresponds to a fragment obtained from the FITC molecule. Conditions: Uncoatedfused silica capillary (57 cm650 lm ID. 50 cm to the detector maintained at 258C; 30 kV; 20 mM sodium tetraborate pH 9.0 con-taining 60 mM SDS and 15% v/v methanol as BGE. Hydrodynamic injection at 3.5 kPa for 10 s. Reprinted from reference [53],with permission.


column amperometric detection for analysis of urinary8OHdG. A single-step solid-phase extraction procedurewas optimized and used for extracting 8OHdG fromhuman urine. To improve the sensitivity of this method,a new focusing technique based on a dynamic pH junc-tion was used. Sample stacking was achieved when ana-lytes possess different velocities in the sample and theBGE zones caused by pH difference in two segments ofelectrolyte in the capillary. A 30 mM phosphate buffer,pH 6.5 was used as sample matrix whereas 30 mM boratebuffer pH 9.12 was used as running buffer. In borate buf-fer solutions, the anionic mobility of 8-OHdG is signifi-cantly enhanced, which is probably due to the formationof a complex of boric acid with a sugar moiety of 8-OHdG.Direct analysis of 8OHdG in untreated urine with UVdetection has been reported [63, 64]. 80 mM 2-(cyclo-hexylamino) ethanesulfonic acid (CHES), 9 mM LiOH(pH 8.6) buffer system containing 0.1 mM CTAB was used

as the separation electrolyte [64] instead of borate buffer[63]. The introduction of CTAB led to inversion and ratelowering of EOF in a way that EOF has the same directionas anionic nucleosides/nucleotides migrating towardsthe anode. Li+ has been chosen as the counter-ion toCHES because of its low mobility as compared to Na+ orK+. The lower mobility of the buffer ions allows the bufferconcentration to be increased without influencing thereleased Joule heat, leading to a peak broadening.Enhanced buffer capacity is probably responsible for asuppression of fluctuations of the electric current, ascompared with the analysis of much more diluted aque-ous model samples.

3 Biomarkers in blood

3.1 Peptides

Only a limited number of applications of the detection ofsmall peptides in human body fluids have beendescribed. An automated sheathless CE-MS platform wasdeveloped for discovery of biomarkers in human serum[65]. The method is based on analysis of low molecularweight peptides in human serum. The method includedtransient isotachophoresis for in-line preconcentrationby injecting a plug of ammonium stacking solution afterinjecting the sample. A 3-methacryloylaminopropyl tri-methylammonium chloride coated capillary was usedfor CE separation using 60 mM acetic acid in 20% metha-nol (pH 3.2) as a running buffer. The buffer compositionwas chosen to support stable electrospray and to mini-mize signal loss due to ion suppression.

3.2 Proteins

Evidently, blood (serum or plasma) is a very rich sourceof information. Candidate proteomic biomarker discov-ery from human plasma holds both incredible clinicalpotential as well as significant challenges. The dynamicrange of proteins within plasma is known to exceed 1010,and many potential biomarkers are probably present atlower protein abundances.

Kolch et al. [42] has described a CE coupled to ESI-TOF-MS method in positive mode for analysing plasma sam-ples without any sample pre-treatment. A polybrene-coated fused silica capillary was used to avoid hydropho-bic interaction with the capillary wall and to obtain astrong reversed electroosmotic flow. This separationmethod provides two advantages: first, the proteins arecompletely desalted, because the small cations aremigrating in the other direction and never reach thedetector. Secondly, modified proteins like glycosylatedforms are separated from non-modified forms because ofthe lower positive charge due to non-charge-bearinggroups or negatively charged modifications.


Figure 4. Electropherogram of urine from (a) healthy person;(b) patient with cystinuria type I/I on penicillamine treatment.Actual concentration of aminothiols: (a) Hcy, 1.1 mM/M crea-tinine; Cys, 30.3 mM/M creatinine; creatinine, 1.7 M; (b) Hcy,4.7 mM/M creatinine; Cys, 157.2 mM/M creatinine; PA,12.1 mM/M creatinine; creatinine, 2.1 M. BGE: 60 mMborate–15 mM sodium dodecyl sulfate–2-amino-2-methyl-1-propanol buffer, pH 10.0. Peaks: Hcy, homocysteine; Cys,cysteine; GSH, glutathione; PA, penicillamine; IS, internalstandard (mercaptoacetic acid). Reprinted from reference[54], with permission.


A capillary electrophoretic enzyme immunoassay withelectrochemical detection was developed for determina-tion of tumour marker cancer antigen 125 (CA125), anovarian cancer associated antigen. The antigen is locatedon the surface of ovarian tumour cells, with restrictedexpression in normal adult tissues. In sera of patientswith carcinoma, the CA125 antigen is not exclusive toovarian carcinoma, but is shown to be elevated in a largenumber of different cancers. In this method, after thenoncompetitive enzyme immune reaction, the freeenzyme (horseradish peroxidase)-labeled anti-CA125 anti-body (Ab*) and the bound enzyme-labeled complex (Ag–Ab*) were separated in a polyacrylamide-coated capillarywith borate buffer pH 7.4 as a running buffer [66].

Carbohydrate-deficient transferrin (CDT) is considereda reliable marker of alcohol abuse. CDT is not a singlemolecule but a group of isoforms: asialo-, monosialo-, anddisialotransferrins. These three isoforms lack one chaincompared with the two N-glycan chains of the predomi-nant tetrasialotransferrin. Deglycosylation/desialylationof transferrin has been related to immoderate ethanolconsumption compared with abstinence or moderateconsumption [67–69]. Sialylated transferrin isoforms inserum were analyzed by CE to differentiate moderatefrom heavy alcohol consumption. The separation capil-lary was coated by “dynamic double coating” to obtainstable EOF and to minimize protein adsorption to thecapillary wall. Double coating was obtained by injectingmalic acid buffer (pH 4.8) to create a polycation layer fol-lowed by the separation buffer, Tris-borate (pH 8.5), to pro-duce a polyanion double layer, then just before sampleinjection a 10 g/L solution of sodium dodecyl sulfate wasinjected. The double layer is eliminated after each electro-phoretic run and regenerated before the next. Detectionof asialotransferrin in sera of alcohol abusers offers thehighest discrimination between excessive and moderatedrinking, as the asialylated isoform was missing in 95% ofmoderate drinkers but present in 92% of alcohol misusers[67, 70]. In another study Ramdani et al. [71] investigatedtransferrin glycosylation in patients with cancer, inwhich aberrant glycosylation is also induced.

3.3 Uric acid

Uric acid is an important clinical biomarker for a host ofmedical disorders. High levels of uric acid in blood(hyperuricemia) are linked with gout, renal failure, leu-kemia, and lymphoma, as well as other pathological con-ditions and have also been encountered in cases of alco-hol and drug overdose. CE has been used with electroche-mical amperometric detection for the determination ofuric acid in serum [72]. Co-migration of anions with theelectroosmotic flow is accomplished with reversed polar-ity and the buffer additive CTAB in a MES buffer system,giving rise to rapid and sensitive analyses. Optimal buffer

conditions (pH 7.0), detection potential (0.80 V vs. Ag/AgCl), and electrokinetic injection are employed to allowfor maximal resolution and signal intensity.

Another CE method for the analysis of uric acid, urea,creatinine, and creatine in plasma and urine has beendeveloped [73]. These non-protein nitrogen compoundsserve as biomarker for renal function. Baseline separa-tion was achieved in 25 mM phosphate buffer (pH 3.45).The buffer concentration affected the thickness of theelectric double layer on the capillary inner wall, therebyaffecting the separation. Separation was improved withincreasing concentration of buffer. On the other hand,the migration time was delayed and the electric currentwas elevated, which results in peak broadening. Decreas-ing the buffer concentration led to co-elution of creatineand urea.

4 Biomarkers in cerebrospinal fluid

Cerebrospinal fluid (CSF) is a clear liquid produced in theventricles of the brain that surrounds and protects thecentral nervous system from trauma. CSF directly con-tacts the extracellular space of the brain, making it aunique medium to detect biochemical changes in thecentral nervous system. Recent evidence suggests thatCSF is also involved in clearance of toxic molecules frominterstitial space of the brain and changes in CSF physiol-ogy may be associated with aging and the developmentof neurodegenerative disease [74]. Wittke et al. [44] pre-sented the analysis of cerebrospinal fluid for identifica-tion of low molecular weight proteins and peptides,which can be used as biomarker for schizophrenia andAlzheimer's disease. The analysis is accomplished usingbare fused silica with running buffer consisting of 20%acetonitrile and 0.25 M formic acid. Also, Ullsten et al.[75] showed that CSF can be analyzed using CE-MS. Capil-laries coated with PolyE-323 enabled fast analyses for pro-teins up to 66 kDa, in a protein tryptic digest and mix-tures of various neuron peptides, within a few minutes,with high resolution and efficiency. A chip-based capil-lary electrophoresis system has been designed for mea-suring the concentrations of inflammatory cytokines incerebrospinal fluid of patients with head trauma. Isola-tion of reactive cytokines was achieved by immune affi-nity capture using a panel of six immobilized antibodies,directly attached to the injection port of the chip. Separa-tion of isolated cytokines was achieved by electrophor-esis in 2 min with quantification of resolved peaks beingachieved by on-line laser-induced fluorescence and inte-gration of each peak area [76].

5 Biomarkers in tissue and tissue extracts

Many challenges remain for studies of tissue proteomics,including issues related to complexity, dynamic range,



sensitivity, and quantification of biomarkers associatedwith various diseases and disease states. Identification ofdifferentially expressed proteins that are characteristicof a clearly defined tumour stage will pave the way fordefining the molecular and biochemical pathways bywhich normal cells progress to cancerous states. Thegreatest expectations for targeted proteomics researchusing enriched malignant cells from high quality speci-mens reside in the identification of diagnostic, prognos-tic, and predictive biological markers in the clinical set-ting, as well as the discovery and validation of new pro-tein targets in the biopharmaceutical industry. One ofthe main problems with analysis of tissue samples is theheterogeneous nature of the sample. Many different celltypes are typically present in tissue biopsies, and in caseof diseased tissue, a small number of abnormal cells maylie within or adjacent to unaffected areas.

Kawata et al. [77] improved a diagnostic method forchronic hepatic disorder by analysing metallothionein(MT) isoforms and trace metals (copper and zinc) in theliver of patients with hepatocellular carcinoma by CEand inductively coupled plasma-mass spectrometry,respectively. The authors found that the determinationof both liver MT isoforms and metal concentration, andsubsequent MDA analysis using three parameters, MT-1/total MT ratio, Cu, and Zn concentrations, are highlyeffective in discriminating the juxta-tumour and tumourportion of human liver with the development of hepato-cellular carcinoma.

Glycosaminoglycans (GAGs) have documented implica-tions for the growth and progression of malignanttumours. The composition of GAGs in disaccharidesderived from human gastrointestinal carcinomas arecharacterized by increased concentrations of GAGs,which have quite different disaccharide compositionwhich, in turn, is associated with a marked increase ofnon-sulfated (Ddi-nonS) and 6-sulfated (Ddi-mono6S) D-disaccharides. An HPCE method has been developed todetermine the composition and the disaccharide pat-terns of hyaluronan, chondroitin sulfate, and dermatansulfate in malignant tissues. Separation and analysiswere carried out on an uncoated fused-silica capillary at258C with 15 mM orthophosphate pH 3.0 as operatingbuffer using reversed polarity. At this low pH, the electro-phoretic mobility of sulfated 2x-disaccharides is muchlarger than the EOF, which, with reversed polarity, isdirected against the migration of carbohydrates [78].

Estrogens, including the natural hormones estroneand estradiol, are thought to be involved in tumourinduction. Catechol estrogen quinones derived from 4-hydroxyestrone and 4-hydroxyestradiol react with DNAand form depurinating N7Gua and N3Ade adducts thatmight be responsible for tumour initiation [79]. Mar-kushin et al. [80] analysed the extracts of breast tissuefrom two women by CE interfaced with absorption and

luminescence spectroscopy. A bare fused-silica capillarywas used with 0.5% monomeric non-ionic surfactant in0.25 mM Tris–phosphate (pH 3.5) as a running buffer. n-Alkyl polyoxyethylene ethers (C16E8) were absorbed ontothe polar or hydrophilic silica/water interface to form aconstant-thickness layer for reducing EOF. It acts also assieving matrix for separation of DNA fragments [81]. Thelimit of detection with field-amplified sample stackingand absorbance detection is about 3610–8 M. Theauthors found that the level of 4-hydroxyestrone–N3Adeadduct in breast tissue extract from a woman with breastcarcinoma is larger by a factor of about 30 than that inthe tissue sample from a woman without breast cancer.

6 Other body fluids

Other body fluids such as saliva have also been subjectedto the search of biomarkers and have potential asmatrices for clinical diagnosis. A CE method with electro-chemical detector was developed for quantification ofuric acid in saliva samples in the presence of co-existingsubstances. An uncoated fused silica capillary was usedwith borax solution (pH 7.8) as running buffer. Themethod was found to be an alternative and convenientmethod for rapid diagnosis of gout in the clinic [82].

Another example is human seminal fluid. Prostate-spe-cific antigen (PSA) is a single chain glycoprotein that isused as a biomarker for prostate-related diseases. PSA hasone known posttranslational modification, a sialylateddiantennary N-linked oligosaccharide attached to theasparagine residue N45. Donohue et al. [83] developed arapid CE method to separate PSA isoforms purified fromhuman seminal fluid. High-resolution separation wasachieved by utilizing 1,4-diaminopropane (DAP) inborate buffer. DAP additive enhances the separation ofglycoproteins by balancing the velocities of EOF and elec-trophoretic migration of the analytes. In addition, borateion complexes to both fucose and N-acetyl-D-galactosa-mines’ two adjacent cis hydroxyl groups.

7 Concluding remarks

Capillary electrophoresis can serve as a highly efficientanalytical technique in biomarker identification, due toits advantages of requiring small sample volumes, highsensitivity, and great resolution. Although the CE tech-nique offers many advantages, it has not been generallyaccepted as a routine tool in the clinical laboratory. How-ever, robust CE or CE-MS analysis is applicable to smallmolecules, metabolites, amino acids, peptides, and pro-teins in inexpensive capillaries. Furthermore, compat-ibility is found with all kinds of buffers, providing fastseparation. Table 1 provides an overview of developed CEmethodology coupled to mass spectrometry for the anal-ysis of biomarkers.



This work was supported by the Austrian Genome Program (Gen-AU), Vienna, Austria and by the special research program “CellProliferation and Cell Death in Tumors” (SFB021, Austrian ScienceFund), FWF, Vienna, Austria.

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Table 1. Biomarkers analysed by CE-MS

Analyte Origin Disease Reference

Peptides Urine Prostate cancer [42]Peptides Urine Prostate cancer [43–45]Peptides Urine Urothelial carcinoma [46]Peptides Urine Diabetic nephropathy [47]Peptides Urine Renal disease [48]Peptides Urine Diabetes mellitus [49]Amino acids Urine Inborn errors of amino acids metabolism [50–52]Amino acids Urine Obstructive pulmonary disease [53]Amino acids Urine Cystinuria [54]Purines, pyrimidines Urine Enzyme deficiencies [55, 56]Nucleosides Urine Thyroid cancer [58]Nucleosides Urine Leukemia [59]DNA Urine Cancer patents after chemo- or radiotherapy [60, 61]Antigens Human serum Ovarian carcinoma [66]Transferrins Human serum Alcohol abuse [67–69]Uric acid Human serum Hyperuricemia [72]Peptides Cerebrospinal fluid Schizophrenia, Alzheimer [44]Metallothionein Tissue Hepatocellular carcinoma [77]Glycosaminoglycans Tissue Malignant tumours [80–82]PSA Seminal fluid Prostate cancer [83]


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Recent advances in capillary electrophoresis and capillary electrochromatography of pollutants

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