Electrophoresis 2014, 00, 1–17 1 Virginia Rodr´ ıguez Robledo 1 William Franklin Smyth 2 1 Faculty of Pharmacy, Department of Analytical Chemistry and Food Technology, University of Castilla—La Mancha (UCLM), Albacete, Spain 2 School of Pharmacy and Pharmaceutical Sciences, University of Ulster, Coleraine, Northern Ireland, UK Received November 14, 2013 Revised January 24, 2014 Accepted January 24, 2014 Review Review of the CE-MS platform as a powerful alternative to conventional couplings in bio-omics and target-based applications Many recent papers and reviews have confirmed the powerful coupling between CE and MS due to efficient and selective separation in combination with selective detection al- lowing detailed characterization of many biomolecules. It is known that CE-MS is an increasingly used and sought after technique for analysis in different fields such as en- vironmental science, food analysis, biotechnology, pharmaceutical analysis, biomedical science, forensic science, toxicology, and genetic analysis. CE-MS is particularly used in bio-omic applications (proteomic, metabolomic, and also genomic applications) for the determination of biomarkers, disease diagnosis, and therapeutic treatment monitoring. Biomarker qualification, clinical proteomics, and its implementation in routine clinical analysis have certain limitations associated with reproducibility and analytical robustness. However, CE-MS has been successfully used in numerous clinical applications in recent years when compared with other platforms. The main advantage lies in the availability of large comparable datasets that were all obtained by using the same procedure for sam- ple preparation, analysis, and subsequent data evaluation. The scope of this review is to discuss the performance of CE-MS as a platform for bio-omics analysis and target-based applications focusing on quadrupole (Q), IT, and TOF analyzers, and the types of bioap- plications that apply to the particular analyzers. Papers and reviews that were published in 2012 and 2013 with relevant CE-MS applications are considered in this review. Keywords: Biofluids / CE-MS / Genomics / Metabolomics / MS analyzer / Proteomics / Target- based applications DOI 10.1002/elps.201300561 1 Introduction 1.1 Hybrid/symbiotic techniques Hybrid or symbiotic analytical techniques are described as couplings between, at least, two instrumental techniques with an appropriate interface in order to obtain the advan- tages of the constituent techniques. These coupling technolo- Correspondence: Dr. Virginia Rodr´ ıguez Robledo, Faculty of Phar- macy, Department of Analytical Chemistry and Food Technology, University of Castilla—La Mancha (UCLM), Albacete 02071, Spain E-mail: [email protected]Abbreviations: 6AM, 6-acetylmorphine; AD, Alzheimer’s dis- ease; BIA, bacterial inhibition assay; CKD, chronic kidney disease; COD, codeine; CSF, cerebrospinal fluid; ECA, en- zymatic colorimetry assay; EDDP, 2-ethylidene-1,5-dimethyl- 3,3-diphenylpyrrolidine; EPO, erythropoietin; FAN, fangchi- noline; FTLD, frontotemporal lobe dementia; HCOD, hy- drocodeine; LBD, Lewy body disease; MCI, mild cogni- tive impairment; MRB, moving reaction boundary; nLC, nanoflow LC; Q, quadrupole; QC, quality control; QqQ, triple quadrupole; RF, radio frequency; SIN, sinomenine; SPME, solid-phase microextraction; TET, tetrandrine; UHPLC, ultra- HPLC gies result in novel solutions to analytical problems [1] when compared to the instrumental techniques on their own. The most popular commercial hybrid techniques are based on the coupling of separation techniques (chromatography or elec- trophoresis) with sensitive and/or selective detection (spec- troscopy and MS) [2]. Historically, GC was coupled with MS in the 1960s and this proved a robust and long-lasting hybrid technique. Due to advanced technologies in MS, recent years have seen the coupling of LC and MS, and CE and MS, which provides many possible analytical strategies. The main goals being the introduction extra selectivity and high peak capac- ity, ultimately performing the most comprehensive analy- ses [3]. Papers comparing the advantages and disadvantages of LC-MS and CE-MS have been extensively reviewed re- cently [4–6]. Currently, RPLC-MS is the core technology for peptide, protein, and metabolite identification; and it certainly will remain so in the foreseeable future [7–12]. The extensive development of the last generation of mass spectrometers coupled to highly efficient LC systems has brought better res- olution, sensitivity, and reproducibility in a relatively short time-frame [13]. Colour Online: See the article online to view Figs. 1, 2, 4, and 5 in colour. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Transcript
Electrophoresis 2014, 00, 1–17 1
Virginia Rodrıguez Robledo1
William Franklin Smyth2
1Faculty of Pharmacy,Department of AnalyticalChemistry and FoodTechnology, University ofCastilla—La Mancha (UCLM),Albacete, Spain
2School of Pharmacy andPharmaceutical Sciences,University of Ulster, Coleraine,Northern Ireland, UK
Received November 14, 2013Revised January 24, 2014Accepted January 24, 2014
Review
Review of the CE-MS platform as a powerfulalternative to conventional couplings inbio-omics and target-based applications
Many recent papers and reviews have confirmed the powerful coupling between CE andMS due to efficient and selective separation in combination with selective detection al-lowing detailed characterization of many biomolecules. It is known that CE-MS is anincreasingly used and sought after technique for analysis in different fields such as en-vironmental science, food analysis, biotechnology, pharmaceutical analysis, biomedicalscience, forensic science, toxicology, and genetic analysis. CE-MS is particularly used inbio-omic applications (proteomic, metabolomic, and also genomic applications) for thedetermination of biomarkers, disease diagnosis, and therapeutic treatment monitoring.Biomarker qualification, clinical proteomics, and its implementation in routine clinicalanalysis have certain limitations associated with reproducibility and analytical robustness.However, CE-MS has been successfully used in numerous clinical applications in recentyears when compared with other platforms. The main advantage lies in the availability oflarge comparable datasets that were all obtained by using the same procedure for sam-ple preparation, analysis, and subsequent data evaluation. The scope of this review is todiscuss the performance of CE-MS as a platform for bio-omics analysis and target-basedapplications focusing on quadrupole (Q), IT, and TOF analyzers, and the types of bioap-plications that apply to the particular analyzers. Papers and reviews that were publishedin 2012 and 2013 with relevant CE-MS applications are considered in this review.
Keywords:
Biofluids / CE-MS / Genomics / Metabolomics / MS analyzer / Proteomics / Target-based applications DOI 10.1002/elps.201300561
1 Introduction
1.1 Hybrid/symbiotic techniques
Hybrid or symbiotic analytical techniques are described ascouplings between, at least, two instrumental techniqueswith an appropriate interface in order to obtain the advan-tages of the constituent techniques. These coupling technolo-
Correspondence: Dr. Virginia Rodrıguez Robledo, Faculty of Phar-macy, Department of Analytical Chemistry and Food Technology,University of Castilla—La Mancha (UCLM), Albacete 02071, SpainE-mail: [email protected]
gies result in novel solutions to analytical problems [1] whencompared to the instrumental techniques on their own. Themost popular commercial hybrid techniques are based on thecoupling of separation techniques (chromatography or elec-trophoresis) with sensitive and/or selective detection (spec-troscopy and MS) [2]. Historically, GC was coupled with MSin the 1960s and this proved a robust and long-lasting hybridtechnique.
Due to advanced technologies in MS, recent years haveseen the coupling of LC and MS, and CE and MS, whichprovides many possible analytical strategies. The main goalsbeing the introduction extra selectivity and high peak capac-ity, ultimately performing the most comprehensive analy-ses [3]. Papers comparing the advantages and disadvantagesof LC-MS and CE-MS have been extensively reviewed re-cently [4–6]. Currently, RPLC-MS is the core technology forpeptide, protein, and metabolite identification; and it certainlywill remain so in the foreseeable future [7–12]. The extensivedevelopment of the last generation of mass spectrometerscoupled to highly efficient LC systems has brought better res-olution, sensitivity, and reproducibility in a relatively shorttime-frame [13].
Colour Online: See the article online to view Figs. 1, 2, 4, and 5 in colour.
2 V. R. Robledo and W. F. Smyth Electrophoresis 2014, 00, 1–17
1.2 Conventional advantages of CE-MS
The use of CE, as an alternative separation technique to LCwith interfacing to a mass spectrometer, has increased sig-nificantly in the recent years. CE has emerged as a highlyefficient and versatile separation technique [14], given that itcan be used in a number of separation modes such as CZE,CEC, MEKC, and capillary IEF. In particular, CZE is a veryattractive technique with its separation mechanism based oncharge-to-size ratio differences of compounds migrating inan electric field. Apart from highly efficient, versatile, andselective separations, CE separations are fast, relatively in-expensive (simple fused-silica capillaries vs. LC columns),robust [15–17], and require only small amounts of sampleand reagents [18, 19].
1.3 Current advantages of CE-MS for bio-omic
(clinical) analysis
It is believed that CE-MS is now considered as a particularlyattractive technique as it combines high-resolution separa-tions with high detection selectivity and sensitivity. Apartfrom the well-known conventional advantages in analyticalseparations, this technique can, in addition, facilitate thecomparison of complex samples in a reproducible mannerbecause of consistent migration profiles and the provisionof complementary information on the composition of a bio-logical sample [20]. For these reasons, the use of CE-MS forbiomolecule analysis, with the emphasis on proteomic de-terminations, has increased significantly in the last 5 yearsmostly in the development of new analytical methods for bothlarge and small molecules [21].
The present review will partially focus on the main CE-MS applications of biomedical, pharmaceutical [22], toxico-logical, and forensic science called bio-omic applications (pro-teomic, metabolomic, and genomic applications). Determina-tions in the bio-omics applications field can have problemswith their implementation and biomarker qualification inclinical proteomic analysis, defining this term as applicationsof proteome analysis aiming at improving the current clinicalsituation. In this sense, the success of clinical proteome anal-ysis should be assessed based on the clinical impact followingimplementation of findings [23]. Although there have beenseveral technological advancements in MS recently [24, 25],with a large number of proteomic biomarkers described, mostof the methods were not subsequently validated and certainlyhave had no impact in clinical decision making as yet [26,27].However, CE-MS has been successfully used in numerousstudies in biomarker discovery in biofluids for several dis-eases [28, 29], verification, and also in clinical applications inthe last few years, as reviewed in [23, 30]. A major advantageof the approach is the availability of large comparable datasetsthat were all obtained by using the same procedure for sam-ple preparation, analysis, and subsequent data evaluation.Many of the reviews’ authors were not aware of the availabil-ity of similar datasets from any other MS-based platform inuse [31]. CE-MS is currently the profiling method with the
shortest route from the analytical laboratory to clinical prac-tice since it uses the same platform for discovery, validation,and clinical implementation missing out the costly and time-consuming process of changing technology with the risk ofloosing biomarkers en route [13].
1.4 Instrumental considerations
In this review, the applicability of CE-MS platforms for rou-tine target-based and bio-omics applications in general andparticularly in clinical proteomic analysis will be describedand discussed from an instrumental point of view, bearing inmind the different types of analyzers used.
CE can be hyphenated with a variety of mass spectrom-eters for detection, especially considering the high efficiencyof the CE peaks. So the main features of an MS detector usedfor coupling with CE should be speed and sensitivity, par-ticularly when determinations of compounds at traces levelsin biofluids samples are considered. Compatible detectorsused for CE are the quadrupole (Q), triple quadrupole (QqQ),Fourier transform-ion cyclone resonance technique, TOF,and IT [14]. Different MS analyzers are used depending onthe type of information that is required. For example, identifi-cation of new compounds requires an MS analyzer to be usedin structure elucidation or for additional selectivity in orderto gain sensitivity by reducing the background noise [32–34].In this sense, IT and TOF detectors are the most desirableanalyzers [35] for detection and identification, respectively, ofbiomolecules, focusing on clinical applications for biomark-ers discovery, disease diagnosis, and therapeutic treatmentmonitoring [36–42]. Q and QqQ analyzers are predominatelyused for their high sensitivity for targeted compounds with arelatively low sample volume [43].
As an example of a current approach, Simo et al. [44]have recently described two different strategies that can befollowed for peptide analysis by CE-MS based on the analyzerused: an IT target-based approach or a proteomics strategy[45, 46] including the peptidomics approach [39] using theTOF detector.
1.5 Reviews from 2012 to 2013
In this review, the focus will be on CE-MS papers publishedin 2012 and 2013 with specific attention paid to the type ofanalyzers used and the applications in the bio-omics field. Sothe text is subdivided according to the analyzer mostly usedin different bioapplications such as TOF, IT, and Q or QqQ.
Table 1 shows a summary of review papers covering theliterature published in 2012 and 2013. In this period, severalreviews have been published describing different applicationsof CE-MS to analyze profiles of compounds with potentialinterest in protein or peptide analysis also called proteomics[13, 18, 20, 23, 31].
A few review papers have been published in themetabolomics field [47–50]. For example, a particular
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review provided an update on the most recent developmentsin CE-MS for polar and charged metabolites in biologicalsamples covering the scientific literature from July 2010 toJune 2012 [48]. Yet again, CE-MS is considered as valuableinstrumental analysis platform for metabolic profiling andfingerprinting.
De novo natural product identification, as well as the au-thentication, distribution, and quantification of constituentsin biogenic raw materials, natural medicines, and biologi-cal materials obtained from model organisms, animals, andhumans, has also been reviewed [49]. Over the past 5 years(2008–2013), a mini-review [50] discussed the latest and im-portant advances of CE-MS-based metabolomics in the sup-port of drug discovery and development stages from screen-ing drug targets, evaluating drug efficacy to tracking drugtoxicity and adverse effects.
Another review has focused on recent approaches and ap-plications of CE-MS relevant to pharmaceutical and biotech-nological applications [21] showing actual developments andfuture prospects.
Because the use of CE-MS for biomolecule analysis hasincreased significantly in the last 5 years, the applicationsfor complex samples have been also reviewed from 2007 to2011 [22]. In this review, the applications have been catego-rized according to the types of analytes studied, including theanalysis for proteins and peptides [51], carbohydrates [52],and small biomolecules, developing new methods for largemolecules, while analyses of smaller molecules are involvedwith more complex tissues and other matrices.
Finally, various techniques are employed for transgenecharacterization including analytical techniques such as GC(GC-MS) and CE-MS. Transgenics are characterized at phe-notypic and molecular levels for understanding the locationof transgene insertion site, ploidy level, copy number, inte-grated vector sequences, protein expression, and stability ofthe transgene. Evaluation of transgene expression involvesthe application of integrated phenomics, transcriptomics,proteomics, and metabolomics approaches [53].
2 Mass spectrometric analyzers in CE-MS
As explained previously, CE is a valuable tool for the analysisof complex biological samples because it provides high reso-lution and low sample consumption [39, 54, 55]. CE-MS en-ables direct analyte identification. As a hyphenated technique,CE-MS is well suited to protein, peptide, and metabolomicanalysis [56–62]. The major advantages of CE-MS particularlyare its high resolution, high separation efficiency, selectivity,sensitivity, speed of analysis, structural information [63–66],and that almost any charged species can be infused into themass spectrometer. By far, the favorite ionization source inCE-MS is electrospray that is characterized by high ionizationefficiency and the soft nature of the ionization process. Thus,CE-MS using the ESI interface for the on-line coupling hasgiven rise to a powerful hyphenated technique able to com-bine the advantages of both CE and MS instruments [32, 67].
Many papers and reviews have been published comparingdifferent ionization sources for biological applications usingthe CE-MS coupling. However, this review will focus on thedifferent types of analyzers. For this reason, it is crucial toknow the instrumental features for the main analyzers usedfor biological samples. Currently, the most commonly usedmass analyzers in metabolomics and even proteomics appli-cations are the quadrupole (Q) in the SIM mode or the QqQconfiguration using the MS/MS mode, IT, and TOF.
After ions are generated in MS, they are separated accord-ing to their masses. There are many factors to be taken intoaccount when choosing the MS analyzer because they cansignificantly influence its qualitative and quantitative perfor-mance [68, 69]. These factors are (i) transmission of ions tothe detector defined as the ratio between the number of ionsreaching the detector and the number of ions produced in thesource, and this is also largely dependent on the ion guideand focusing lenses; (ii) the resolution as the smallest incre-ment of mass that can be distinguished by the analyzer; and(iii) scan rate (or acquisition rate), which is the time takenfor the analyzer to scan a given mass range in atomic massunits. The high-mass limit is also an important factor as it de-termines the highest value of the mass-to-charge ratio (m/z)that can be measured (and is expressed in Daltons or atomicmass units).
2.1 Quadrupole (Q) analyzer
The quadrupole analyzer is one of the most commonly usedmass analyzers being more compact, less expensive, andmore robust than most other types of mass spectrometers. Itconsists of four parallel cylindrical or hyperbolic rods, equallyspaced around a central axis, that serve as electrodes. Oppos-ing sets of rods, which are connected electrically, have both adirect current (DC) and an alternating current (or radio fre-quency (RF)) applied to them, with one pair being attached tothe positive side of a variable DC source and the other pair tothe negative terminal [68]. The pair of positive rods forms ahigh-mass filter for positive ions traveling toward the detectorwhile the pair of negative rods forms a low-mass filter. Thecombination of high- and low-mass filters creates an area ofmutual stability, where ions of certain m/z ratios transit tothe center of the Q, in a spiral/corkscrew-like trajectory, enroute to the detector [70, 71].
It should be noted that the Q is not dependent on thekinetic energy of the ions when they leave the source. Theonly requirements are that the time for crossing the analyzershould be short compared to the time necessary to switchfrom one mass to the other, and that the ions remain longenough between the rods for few oscillations of the alternat-ing potential to occur [72]. The resolution of a quadrupoleis approximately 1 Da over the entire mass range, thus itis generally considered a low-resolution instrument. Thismost relevant handicap for quadrupole analyzers is due tothe fact that high resolutions are an indispensable require-ment for identification of new compounds. Thus, detailed
structural information on molecules would be obtained byCID [73] that couples three quadrupoles in series to createa triple quadrupole (QqQ). Using this tandem mass spec-trometer, the first (Q1) and third (Q3) quadrupoles can beused for scanning (or using the SIM mode) while the centralquadrupole (RF-only quadrupole, Q2) serves as a collisioncell. The resulting fragment ions are then analyzed by thethird quadrupole [72]. An important consideration when us-ing a triple quadrupole is its low-mass cutoff. The low-massproduct ions from a high-mass precursor ion may be lostbelow approximately one-third of the mass of the precursormass [70].
2.1.1 Applications using the triple quadrupole (QqQ)
The high sensitivity of the QqQ, compared to TOF or IT an-alyzers, is of value in carrying out complex analyses in phar-maceutical and biological matrices. CZE-ESI-QqQ can alsobe applied for (i) the investigation of weak associated speciesof the drugs and (ii) monitoring of predicted (targeted) com-pounds derived from the original drugs, for example drug im-purities or metabolites (when standards of targets are miss-ing). Maraakova et al. [74] have illustrated the potential ofCE-ESI-MS/MS using the QqQ configuration. This work pro-posed the simultaneous determination and identification ofa multidrug mixture, namely pheniramine, phenylephrine,and paracetamol. They were determined and identified withhigh reliability. Successful validation and application of theproposed method suggested its routine use in advanced drugcontrol and biomedical research.
Other recently published papers have used the QqQ con-figuration in order to obtain the advantages that the CE-MSplatform provides such as very low sample volume and highsensitivity. Thus, Ji-Seon et al. described a CE-MS methodfor newborn screening of phenylketonuria, a representativeamino acid metabolic disease in a dried blood spot. CE-MSwas equipped with an ionophore membrane packed sheath-less ESI interface that was developed by their own researchgroup [43]. Under the optimized conditions, a rapid determi-nation was developed, obtaining low LODs and CVs lowerthan 4% for migration time and area ratio. Finally, the ana-lytical method was applied to real clinical samples of Koreanneonates and the results were compared with those of theconventional methods for phenylketonuria diagnosis such asthe screening methods, bacterial inhibition assay (BIA) andenzymatic colorimetry assay (ECA), in addition to HPLC-MS.The results showed good agreement with HPLC-MS, and thepotential of the method as a reference method for the use ofECA and BIA as routine analytical methods for clinical ap-plications was illustrated. While other methods (ECA, BIA,and HPLC-MS) will continue to play a dominant role in thisfield, CE may evolve as an effective alternative due to its differ-ent separation mechanism, speed, high separation efficiency,and extremely low sample consumption.
The same research team developed a CE-MS method us-ing a similar membrane sheathless ESI interface in order to
acquire high sensitivity for underivatized amino acids [75]. InFig. 1, the images of the blunt-tapered emitter tip end includ-ing the Taylor cone and electrospray plume are illustrated.
Others papers using QqQs for macromolecules with highm/z ratios such as methacrylic monomers [76] have been alsopublished.
2.1.2 Applications using the SIM mode
Using the Q analyzer, a single m/z can be monitored at max-imum collection efficiency and maximum sensitivity if oper-ated in the SIM mode. In this mode, the mass analyzer isprogramed to allow only ions of specific m/z values to passthrough the detector, preventing all other ions from reach-ing the detector [73]. Apart from the increase in sensitivityobtained from the SIM mode, this may also add to the selec-tivity of the Q when two or more ions of different m/z co-elutebecause the SIM mode can employ different m/z channels toshow individual peaks for these ions at their respective reten-tion/migration times [71]. By scanning through multiple SIMevents, it is also possible to measure numerous compounds ina single run [57]. Thus, a study carried out by Bhowmik andJung [77] established a simultaneous quantitative determi-nation of nine endogenous nucleotides in rat plasma usingMEKC/ESI-MS. The samples were analyzed using CE andthe SIM mode with positive ionization with a silica capillarycolumn in a reversed polarity mode. Analyses of nucleotidesare important and often critical to the fields of clinical, bio-chemical, environmental, and pharmaceutical research be-cause nucleotides are the most fundamental constituents ofliving cells and are involved in cell metabolism. The monitor-ing of metabolic products using such analytical technologiesin both healthy and diseased cellular metabolism situationshas been useful for assessing the toxicities and understandingthe mechanisms of certain drug therapies.
Because of the sensitivity and selectivity that SIM pro-vides, the quadrupole in this mode is ideally suited to thequantitative analysis of metabolites [78]. As an example,Yu [79] published a paper that explored the relationshipbetween urinary metabolites and the clinical chemother-apy response in breast cancer by CE-MS coupled with on-line concentration. In this study, it was found that mostof the amino acids and organic acids dramatically modifiedbetween the patients and healthy volunteers. After receiv-ing chemotherapy, chemotherapy-sensitive patients showed a30% change in metabolite levels compared to healthy people,with chemotherapy-insensitive patients showing only a 9%change in metabolite levels compared to healthy people show-ing recurrence. It can be translated that oxaloacetate, pyruvatesuccinate, malate, and �-ketoglutarate decreased, but lactate,�-hydroxybutyrate, cis-aconitate, isocitrate, citrate, proline,valine, cysteine, leucine and isoleucine, aspartate, glutamate,methionine, histidine, phenylalanine, arginine, tryptophan,and cystine increased in breast cancer patients comparedwith the normal controls. Among them, eight metabolites,pyruvate, malate, �-ketoglutarate, �-hydroxybutyrate, lactate,
6 V. R. Robledo and W. F. Smyth Electrophoresis 2014, 00, 1–17
Figure 1. The images of theblunt-tapered emitter tip end(A), Taylor cone (the asteriskpoint; B), and the electrosprayplume under light (C) operat-ing at 2.4 kV ESI voltage. CEconditions: capillary, uncoatedfused-silica (95 cm separationcapillary and 4 cm emitter tip× 50 m id); electrolyte, 5 mMNH4Ac (pH 10.8); separationvoltage, 25 kV. Reproducedfrom [73] with permission.
citrate, oxaloacetate, and cysteine, were the most importantvariables for the classification. The extent of energy insuffi-ciency for chemotherapy-insensitive patients was greater thanthat for chemotherapy-sensitive patients. Therefore, urinarymetabolic products may be new potential predictive markersfor therapy efficacy.
2.2 IT analyzer
The classical IT analyzer is best described as a closed or 3Dversion of the quadrupole. It consists of a doughnut-shapedcentral ring electrode that is flanked by two convex-shapedend caps with entry and exit orifices in their center [68,69,71].Ions are only allowed into the trap at certain intervals (i.e. a se-ries of pulsed positive to negative voltages repel or attract ionsto the entrance of the end cap aperture) in order to maximizetheir transmission to the detector and minimize space-chargeeffects, which may lead to a reduction in the performance ofthe analyzer [80]. Once inside the trap, ions are subjected tooscillating electric fields that are applied by an RF voltage tothe ring electrode and are trapped via helium (He) gas, rotat-ing in complex trajectories according to their m/z ratios for aspecific length of time. As ions are stored together, they in-teract by electrostatic repulsion resulting in the expansion oftheir trajectories, often leading to ion losses. To control suchinteractions, the interior of the trap is kept under low pressureby He gas. The gas–ion collisions reduce the kinetic energyand trajectory expansion of the ions and force them towardthe center of the IT. In this region of the trap, ions of certainm/z ratios rotate together in stable orbits. The stability of ionmotion is dependent on the m/z ratio of the ion, the size of theIT, the oscillating frequency of the RF, and the amplitudes ofthe applied DC and RF voltages. However, the normal massselective instability mode of IT operation utilizes no DC volt-age on the ring electrode [69,71,80]. In addition to scanning,the IT can also operate in a mode similar to that of SIM. Inthis mode, a specific ion is accumulated in the trap by ejectingall other ions during the accumulation period, which is fol-lowed by the ejection of the selected ion. This technique can
improve the S/N of the measurement [69]. An example, us-ing such an SIM mode, has been developed by Botello et al.[81] for the determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine(HCOD), and 6-acetylmorphine (6AM) in urine. The mainproblem in the analysis of biological samples is that, in thesecomplex matrices, interfering endogenous compounds arepresent at higher concentrations than the target analytes. Sothe objective of this work was to establish a method usingonline SPE CE-MS (SIM mode) for simultaneous determina-tion of these drugs in a complex matrix such as urine. Thedrugs in the study were detected as protonated molecules[M+H]+ for all the compounds (EDDP: 278 m/z, COD:300 m/z, HCOD: 302 m/z, and 6AM: 328 m/z). The advan-tages of the method are the high efficiency of CE combinedwith the selectivity and sensitivity of MS together with thecomplete automation of the process and significant precon-centration. In addition, the hyphenation with MS also allowsaccurate identification of the target compounds in this kindof complex sample.
2.2.1 Applications using MSn
The main advantage of the IT is the ability to conduct theMS measurements without the need of an additional massanalyzer such as the extra Q in QqQ and Q-TOF instrumentsor a collision cell as He gas is already present in the IT.Using the normal scan operation, the kinetic energy of theions is not enough to induce fragmentation. Therefore, thekinetic energy of selected ions is adjusted to cause CID withHe. Fragment ions can then be sequentially ejected as in thenormal scan operation [80]. Unlike in QqQ and Q-TOF instru-ments, this fragmentation process can be repeated n times,providing MSn spectra and, hence, more detailed structuralinformation.
The application of IT-MS in structure elucidation,fragmentation behavior, and fragmentation pathways ofcompounds is well established. Moreover, several reportsshow that IT-MS can also be very useful for quantitative
Figure 2. Peptide (A) and pro-tein (C) identification sensitiv-ity comparison of solid-phasemicroextraction (SPME), mul-tistep elution, transient ITP(tITP) CE-MS/MS (blue), di-rect injection CE-MS/MS pre-viously described (green), andnanoflow LC (nLC-MS/MS,red) of Pyrococcus furiosus(Pfu) tryptic digests of 16 pro-teins run in duplicate. Venncomparisons of identified pep-tides (B) and proteins (D) fromcombined duplicate runs of 5and 100 ng of Pfu digests us-ing SPME-tITP-CE, direct in-jection CE-MS/MS, and nLC.Reproduced from [84] withpermission.
analysis [82–84]. As a representative work [85], a nonaque-ous CE-IT-MS method with a nanospray ionization interfacefor the identification and quantification of tetrandrine (TET),fangchinoline (FAN), and sinomenine (SIN) in a positive ionmode can be cited. In this paper, MSn (n = 2, 3) can be eas-ily used for compound identification with much lower noiselevel for quantification. The fragmentation pattern of a targetanalyte is useful in performing MSn for either qualitative orquantitative analysis. Therefore, it is important to investigatethe fragmentation pathways of the compounds of interest. Nocomprehensive ESI-MS fragmentation study of TET, FAN,and SIN and their simultaneous determination has been re-ported by CE-MS. In their paper, Chen et al. [85] describeda new method of structure elucidation based on MSn. Directinfusion was used to collect the MS1 profile and then the MS2
and MS3 data of selected ions (TET, FAN, and SIN) in the pos-itive ion mode. The major fragment ions of the analytes wereconfirmed and main fragmentation pathways of fragmentions were studied. The CE-MS method was validated for lin-earity, sensitivity, accuracy, and precision and then used todetermine the content of analytes in real samples of radix andrhizomes (Stephaniae tetrandrae and Menispermum dauricum,respectively).
Another example is the study by Wang et al. [86], whichemployed a solid-phase microextraction (SPME), multistep
elution, transient ITP (tITP) CE-MS/MS procedure. In orderto improve sensitivity over a previously reported proteomicapplication of conventional nanoflow LC (nLC)-MS/MS re-sults, a high-sensitivity porous ESI using a 30 �m id poroussprayer capillary for the proteomic analysis of a moderatelycomplex protein mixture was employed. In addition to maxi-mizing separation efficiency, electrokinetic methods for elu-tion and separation after loading the sample by applicationof pressure was used. Evaluation of sensitivity of the sheath-less SPME-tITP-CE-MS/MS platform for analysis of a mod-erately complex proteomic mixture was developed. Figure 2illustrates a sensitivity comparison of the different processessuch as SPME-CE-MS/MS, direct injection CE-MS/MS, andnLC-MS/MS.
2.2.2 Applications in the analysis of amino acids,
peptides, and proteins
Currently there are an increasing number of applications us-ing CE-IT-MS that focuses on amino acids, peptides, and pro-teins [87–93]. In order to illustrate these applications, theserepresentative papers are now discussed [87–89].
Chen et al. [87] optimized parameters in CE-ESI-IT basedon moving reaction boundary (MRB) so as to improve its
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sensitivity, specificity, and stability for determination of 19standard amino acids. Early detection is the most effectiveway to reduce mortality for some types of cancers such asgastric cancer, because it is usually asymptomatic at earlystage. Up to now, most GCs were diagnosed when they arealready metastatic, so there is an urgent need of sensitiveand reliable tumor markers for its early diagnosis. In thisway, metabonomic, which is the comprehensive profiling ofmetabolic changes, may shed light on this need. This studyestablished feasible and useful methodology via MRB-CE-MSin searching for potential tumor markers of gastric cancerin patients’ urine samples. For that, the optimized methodwas also validated and finally applied to urine samples fromgastric cancer patients and control subjects. Several aminoacids were recognized as potential tumor markers, whichdeserve further investigation and successfully distinguishedgastric cancer patients from controls, as well as early-stagepatients from advanced-stage patients.
Similarly, another type of biological sample, which needshigh sensitivity and specificity, is whole saliva. Rossetti et al.[88] described a study for quantitative analysis of thymosin�4 in saliva using CE-IT-MS with multiple ion monitoring.This scan modality, by reducing the baseline noise and in-terferences, increases the sensitivity and specificity necessaryfor biological matrices. Thymosin �4 (T�4) is a peptide, mostabundant in humans and other mammals, present in almostany tissue and in extracellular media (except for erythrocytes),and in several body fluids. A small quantity of this peptidewas found in extracellular media. Recently, many authorshave focused their attention on this peptide for its amazingmultiple functions and involvement in diseases. T�4 playsan important role as G-actin sequestering peptide, havingmultiple amazing functions as wound healing, stimulationof angiogenesis, and suppression of inflammation. The roleof T�4 in tumor development could suggest the activationof fetal programs in the tumor cells. Some proteins, com-monly expressed in fetal development but not in adults, maybe highly synthesized during neoplastic transformation andT�4 peptide has been found among them. The authors opti-mized CE-MS separation and isolated the four most intensemulticharged ions present in the MS spectra of the peptide.This can unambiguously identify in a short time thymosin �4in saliva after a very fast and reduced sample pretreatmentprocedure.
Sodium cysteamine phosphate is a prodrug derivative ofcysteamine that can be used in cystinosis treatment. Cysti-nosis is a rare autosomal recessive storage disease, resultingfrom over 32 different mutations in the gene CTNS that,encodes an integral lysosomal membrane protein. Cysteine,which is quickly oxidized to cystine, is hydrolyzed in thelysosome. In normal individuals, both cysteine and cystinecan be transported through the lysosomal membrane to thecytoplasm, where cystine is reconverted to cysteine via glu-tathione. The lack of functional cystinosin leads to the ac-cumulation of cystine, which crystallizes and causes severalinjuries to the cells. Zatkovskis Carvalho et al. [89] evaluatedthe possibility to assess sodium cysteamine phosphate by
Figure 3. Schematic representation of the setup used for MSand CE-MS experiments. 1, make-up liquid reservoir; 2, tee-connection for make-up liquid pressurization; 3, make-up liquidpressure line; 4, make-up liquid transfer line; 5, CE BGE/samplereservoir; 6, polyoxymethylene block for CE pressurization andhigh-voltage power supplies connection to the CE anode; 7, CEpressure line; 8, separation capillary; 9, platinum CE electrode(anode); 10, electric circuit of the CE-ESI setup, composed by twohigh-voltage power supply units and a 54 M� resistor; 11, �tee(P-775 Upchurch) used to merge CE BGE with make-up liquid;12, stainless steel needle used as CE electrode (cathode) and ESIemitter, simultaneously. Reproduced from [87] with permission.
CE by means of the quantification of its oxidation product,cystamine, which is a more suitable substance to be used asprimary standard than sodium cysteamine phosphate. A well-established and precise determination of sodium cysteaminephosphate by an iodimetric technique is in general used, al-though considerably more care and time is required from theanalyst. CE-MS was employed to aid in the investigation in or-der to get a more precise assay compared with the titrimetricassay and for this purpose a noncommercial, simple, and in-expensive ESI source was constructed. In Fig. 3, a schematicrepresentation of the setup used for MS and CE-MS experi-ments is shown. The ESI interface used in this study, togetherwith the liquid chromatography model (LCQ) using a ion trap(IT) MS, was relatively simple and inexpensive for the identifi-cation of the oxidation products of cysteamine and cystamine.
2.2.3 Instrumental improvements to increasing
sensitivity
Hyphenation of CE with MS has shown to be highly suit-able for the analysis of hydrophilic compounds in biologicalsamples and also it has gained popularity in forensic toxi-cology. But the sensitivity of mentioned technique may belimited due to several reasons such as very small sample
Figure 4. Schematic of the on-line laser ablation sample transfercollection system for CE/ESI. Reproduced from [94] with permis-sion.
injection volumes and the use of the sheath-flow interface.Several papers have described instrumental improvementsin order to improve the sensitivity such as the pressurizedliquid junction nanoflow interface [93] and the online fritlessSPE procedure [94].
Cocaine has become one of the most frequently abuseddrugs in many countries and the second most abused sub-stance in Europe (according to the European Monitoring Cen-tre for Drugs and Drug Addiction). Taking into account theshort half-life of cocaine in biological fluids, the assessmentof the metabolites is of utmost importance for monitoringcocaine abuse in toxicological analysis. Hezinova et al. [94]developed a new method for the simultaneous separationof cocaine and four metabolites in urine via a pressurizednanoliquid junction interface by CE-ESI-MS. The detectionof the studied compounds was performed using an IT massspectrometer in the positive ionization mode. Several analyt-ical methods (immunoassays, GC-MS, and HPLC-MS) havebeen previously published for their determination but cur-rently CE-MS is one of the most attractive methodologies forforensic laboratories, because it combines the advantageousfeatures of CE with the selectivity and sensitivity of MS. In thispaper, two previously evaluated pressurized liquid junctiondevices have been used for the hyphenation of CE systemwith the MS detector with the aim of improving the sen-sitivity. In addition, to enhance sensitivity, a field-amplifiedsample injection was evaluated in terms of injection timeand sample solvent composition. The LODs achieved withthe field-amplified sample injection method ranged from 1.5to 10 ng/mL for cocaine, cocaethylene, benzoylecgonine, nor-cocaine, and ecgonine methyl ester.
For analysis of low analyte concentrations, sample pre-concentration is often needed. When larger sample volumesshould be concentrated, the sensitivity of CE can be enhancedby SPE to trap and desorb analytes prior to CE analysis. In thework published by Tak et al. [95], online frit-free SPE has beenstudied for the preconcentration of test compounds prior toanalysis by CE-MS. The mixed-mode sorbent Oasis HLB wasselected for the trapping of compounds of different polaritysuch as EDDP, dihydrocodeine, and codeine. Among differ-ent modalities of SPE with respect to CE (off-line, on-line,at-line), in-line approach employs an SPE sorbent positionedinside the CE capillary, either as an inserted segment or asa coating or filling. With the sorbent as an integrated part of
the analytical system, no separate control unit is required forin-line SPE-CE and modification of the CE instrumentation isnot needed. In this paper, it is concluded that the frit-free in-line preconcentration construct proved to be highly useful forimproving the sensitivity of CE obtaining very low detectionlimits (pg/mL levels), because an injected sample volume of30 times more than the capillary volume was used.
Another relevant method on instrumental improvementhas been described by Sung-Gun and Kermit [96] for directanalysis of biological samples in their native environment us-ing ambient laser ablation sampling. Several ambient ioniza-tion techniques have been developed recently to avoid prob-lems associated with an ion source under vacuum. For exam-ple, laser desorption and ablation methods include electro-spray laser desorption ionization, MALD-ESI, laser ablationESI, and laser electrospray MS techniques, which all use alaser to remove materials from the sample, which then in-teracts with charged electrospray droplets to form ions. Laserablation can also be used for ambient sample loading prior toseparation and ionization for MS. These authors have there-fore coupled ambient laser ablation sample transfer to a CEseparation of the transferred analyte (see Fig. 4). ESI-MS wasused to detect the separated components. This system wastested using mixtures of peptide and protein standards. Cou-pling ambient IR laser ablation on-line sampling to CE-ESI-MS was required in the development of a novel collection andelectrokinetic transfer system. Finally, it can concluded thatthe interface is generally applicable and has potential utilityfor MS imaging as well as the loading of microfluidic devicesfrom untreated ambient samples.
2.3 TOF analyzer
The principle of TOF analysis is based on the fact that ionswith the same initial kinetic energy travel at different veloci-ties, which are proportional to their m/z ratio [67].
Basically, ions from the source are accelerated to thesame kinetic energy by a pulsed electric field at the entranceof the flight tube (1–2 m in the simplest analyzer). Ionsthen transit the flight tube en route to the detector. Lighter,multiple-charged ions reach the detector before heavier,single-charged ions. The next set of ions is then pulsed onlyafter all ions have reached the detector. Due to high trans-mission efficiency, the TOF analyzer achieves a very highsensitivity. Variations in ion flight times, resulting from dif-ferent spatial and kinetic energy distributions, can lead toa significant reduction in mass resolution and accuracy. Tocompensate for these variations, modern TOF instrumentsare equipped with a series of ring electrodes (with increasingpotential) at the end of the flight tube, called reflectrons. Bydoing so, the flight distance toward the detector is doubled,which leads to a significant improvement in mass resolutionand accuracy. In fact, TOF is generally considered to be ofhigh resolution and one of the fastest mass analyzers makingit ideal for highly efficient analytical separation methodolo-gies (e.g. GC × GC, ultra-HPLC (UHPLC), and CE) [97]. Mass
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resolution and accuracy are therefore essential to the unam-biguous determination of empirical formulae (from elemen-tal composition) for the identification of unknowns [98, 99].Compounds with the same nominal mass, but different em-pirical formulae, can only be distinguished with high reso-lution and not unit mass resolution as with Q and IT massspectrometers. [97]. Due to its high sensitivity, high massresolution, and accuracy, TOF has a theoretically unlimitedmass range and an extremely high scan rate and is ideallysuited to metabolomics [100–102] and proteomics [103] appli-cations. The next sections (2.3.1 and 2.3.2) therefore discussthe most significant applications in the bio-omics (proteomicsand metabolomics) field using CE-MS when TOF is used asthe analyzer.
In the same way when other analyzers are hyphenatedto CE, when TOF is coupled to CE in the sheath-flow mode,the ESI sprayer needs a sheath liquid to establish an electricalcontact between the ends of the capillary and maintain stableelectrospray. However, the main disadvantage of sheath-flowconfigurations is their low sensitivity because of dilution ofthe analyte by the sheath liquid [104]. So, sometimes for bio-logical applications, the sensitivity of the CE-TOF-MS methodcan be inadequate. Many authors therefore focus their stud-ies on using MS/MS in conjunction with on-line sample pre-concentration techniques and/or novel low-flow/sheathlessinterfaces for CE-MS to improve sensitivity [48]. This lim-itation and its improvements will be also discussed inthe following section (2.3.3) in addition to non-omicsapplications.
2.3.1 Proteomics applications
A current and relevant paper published by Simo et al. [44]demonstrated for peptide analysis the potential of the CE-MS technique using two different strategies, first one being atarget-based approach and the second one based on method-ology for proteomics applications, including the peptidomicsapproach [105–107]. In target-based analysis, the analyte (orgroup of analytes) is known and the method is developed todetermine target compounds from complex biological matri-ces using CE-ESI-IT MS/MS. Proteomic analysis is especiallycomplex since the proteome is variable in time and becauseof the heterogeneity of analytes (physicochemical propertiesand protein abundance). Taking into account the features ofthis analysis in addition to the need of powerful separationprocedures in order to allow their efficient identification, asensitive and selective MS analyzer is required. In such stud-ies, the use of CE on-line coupled to a TOF mass analyzer(CE-ESI-TOF-MS) for “a shotgun-like” proteomic study ofpeptides is proposed as adequate instrumentation.
In reviewing the papers based on the analysis of peptidesby CE-MS including applications in biological and biochem-ical research, clinical diagnosis, drug discovery, and phar-maceutical analysis, it is found that most of them focus onproteomics strategies and not on target-based analysis. Thereis an absence of standard studies on individual peptides and
proteins. The discussion now focuses on proteomics analy-sis for kidney diseases [108–110] and quality control (QC) ofintact proteins [111–114].
The number of patients with chronic kidney disease(CKD) is increasing steadily due to extended life expectancyof the population and the increased incidence of both dia-betes and obesity (around 25% of all cases), and etiologicallythere are others varieties such as hypertension, immunolog-ical or genetic disorders (hereditary kidney diseases [108]),infections, and drugs. For example, in order to quantify thenumber of sufferers just for diabetic people, it is believedthat by 2025, the number could reach 380 million of peopleworldwide. With such a potentially high number of patients,an early and unambiguous detection of patients at risk forCKD and staging of CKD are of high interest and becauseimproving the management and treatment of CKD is an im-portant healthcare requirement. Moreover, often the detec-tion of CKD at an advanced stage can lead to end-stage renaldisease characterized by the complete loss of kidney function.Progression from CKD to end-stage renal disease exposespatients to burdensome renal replacement therapy (e.g. dial-ysis or renal transplantation) and cardiovascular complica-tions. Therefore, noninvasive detection of diseases, based onurinary proteomics, is becoming an increasingly importantarea of research, especially in the area of CKD. Thus, Molinet al. [109] compared the performance of CE-MS with of thefact MALDI-MS in detecting CKD, based on a cohort of 137urine samples (62 cases and 75 controls) for the comparisonof detected biomarkers. The main conclusion obtained fromthis study was that the results demonstrated superior perfor-mance of the CE-MS approach in terms of peptide resolutionand obtained disease prediction accuracy rates. It is becauseof the fact MALDI-MS without a separation step before MSis a less-powerful technique than CE-MS. However, the dataalso demonstrated the ability of the MALDI-MS approach toseparate CKD patients from controls, at slightly reduced accu-racy, but expected reduced cost and time. The fact that severalCE-MS biomarkers can be detected by MALDI-MS supportsthe fact that the latter may well be used for initial screen-ing of a population at risk, identify those patients who likelyhave CKD, followed by reassessment of the controls by CE-MS. Another study conducted by Albalat et al. described theCE-MS approach for the clinical analysis of urine samplesaiming at proteomic profiling patients with CKD [110]. In aclinical setting, the technique has to be reproducible, robust,rapidly reconditioned for the subsequent sample, inexpen-sive, and should have good quality data for detected biomark-ers. In addition, it has proven itself to be capable of provid-ing sufficient sensitivity to allow clinical diagnosis and alsoprovides sufficient diagnostic information for the physician.In this study, an adequate reproducibility and good qualitydata on amino acid sequences of the detected biomarkers inaddition to provide sufficient sensitivity to allow clinical di-agnosis, using CE-MS (ESI-TOF) as analytical platform, havebeen proposed. In addition, urine has been used because it isan excellent sample source in the proteomic study of diseasesand at the moment has escalated with the aim of identifying
biomarkers that could be used for diagnosis or to predict theoutcome of renal pathologies.
A paper based on QC of proteins has been published byTaichrib et al. [111] for the characterization of erythropoietin(EPO). The glycoprotein EPO is an erythropoiesis-stimulatinghormone, mainly produced in the adult kidney. EPO has adistinct natural heterogeneity arising from its glycosylationthat shows strong composition variations. This heterogeneityincreases the complexity of the analysis of EPO considerably,but may also be used to distinguish different preparations.The characterization and determination of EPO is a mainissue, as variations in the number and type of the numer-ous isoforms caused by the uniqueness of each cell used forthe production are inevitable (even between batches). But theanalysis of EPO is of great interest regarding both QC of thepharmaceutical products and abuse detection. Apart from theverification of the production consistency, the characteriza-tion of the EPO glycoform distribution gains importance asthe patents on the innovator biopharmaceuticals have expiredor will expire soon, giving way to follow-on biologics, that isbiosimilars. Therefore, the paper presented a method for thedifferentiation of various EPO preparations by CE-MS andthe subsequent application of multivariate statistics. Usingmultivariate statistical methods based on CE-TOF MS exper-iments on intact glycoforms, such as principal componentanalysis and cluster analysis, discrimination of various EPOpreparations with differences in the origins and the produc-tion cell line but also batch-to-batch variations was described.The differentiation can be performed on the basis of a fewselected isoforms out of a pool of 100 or more various gly-coforms present in each preparation. EPO preparations fromcommercial suppliers and preproduction preparations wereanalyzed, and the relative peak areas of selected glycoformswere used to distinguish the preparations by grouping usingthe statistical evaluation.
A study developed by Klein et al. [112] evaluated the suit-ability of CE-TOF-MS to measure the consistency of differentlots of the probiotic formulation Pro-Symbioflor, which is abacterial lysate of heat-inactivated Escherichia coli and Ente-rococcus faecalis. Probiotics are defined as micro-organismsthat, when obtained in sufficient active quantities in thegut, lead to positive effects on health; most of the provenhealth effects probiotics elicit are provided in the gastroin-testinal tract. Probiotic bacteria have a wide range of appli-cations both in veterinary and human therapeutics. Inacti-vated probiotics are complex samples and QC should mea-sure as many molecular features as possible. In this paper,it confirmed that CE/MS is an appropriate QC tool to ana-lyze complex biological products such as inactivated probioticformulations and allows determination of the similarity be-tween lots. That conclusion was backed up by several CE-MSdeterminations developed in this work. Over 5000 peptideswere detected in five different lots of the bacterial lysate andin a sample of culture medium. Sequence analysis peptidesof both E. coli and E. faecalis and peptides originating fromthe culture medium were identified. Sequence analysis alsoidentified the presence of soybean, yeast, and casein pro-
tein fragments that are part of the formulation of the culturemedium.
2.3.2 Metabolomics applications
Considering the two different strategies used in the method-ology for -omics applications and target-based approaches[44, 46], most of the papers for the determination of metabo-lites in biological samples, using CE-ESI-TOF, focus onmetabolomics strategies and a few papers are based on target-compounds analysis. Regarding the latter, a representativepaper described by Naz et al. [115] evaluated the perfor-mance and validation for carnitine, choline, ornithine, ala-nine, acetylcarnitine, betaine, and citrulline, covering the en-tire electropherogram of pool of rat serum. This work [115]focuses on the metabolome (quantification of some metabo-lites) to understand the control and regulation of complexmechanisms in the human body, because some metaboliteschange in response to specific diseases. Knowledge of theseconcentration changes of targeted metabolites may be usefulto detect the onset of disease in patients before the observa-tion of symptoms. In order to develop a noninvasive, reliable,rapid and simple screening method to detect biomarkers, ametabolomics fingerprinting approach was developed. An ul-trafiltration method was used for sample pretreatment andapplied to rat serum samples using CE-TOF-MS. The vali-dated method was applied to ventilator-induced lung injurysamples for the first time, and results obtained were in agree-ment with literature values.
Many papers, however, have been published on the de-termination of nontargeted compounds using CE-TOF-MS inbiological samples. Samples from animal models (mice andrats) [116–118] or using human samples of patients [119–126]have been proposed.
As an example, a relevant paper by Knolhoff et al.[118] combines small-volume metabolomic and transcrip-tomic platforms to determinate and characterize the chem-ical heterogeneity of the brain. For that, CE-MS usingTOF as analyzer and whole-genome gene expression arrays,aided by integrative pathway analysis, were utilized to sur-vey metabolomic/transcriptomic hippocampal neurochem-istry using the mouse brain (left caudal hippocampus) asbiological sample. This combination allowed the highlight-ing of pathways common to both platforms, thus producing asmall but validated list of metabolic pathways on which to fo-cus future studies. Compounds of particular interest includecholine (link to memory and neurogenesis), as well as acetyl-choline (relationship to stress, learning, and memory) and thedecrease in abundance of multiple amino acids. There may bea cumulative developmental link, which is suggested by priorstudies highlighting a significant decrease in neurogenesisobserved in mast cell deficient mice.
Representative examples, when human samples of pa-tients or control subjects were used, focus on Alzheimer’s dis-ease (AD) and neurodegenerative dementia disease [119,122]
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and others on the effect or benefits of dietary [127] and somediseases (foodomics) are described [121, 123].
Neurodegenerative dementia is one of the most increas-ingly common age-related disorders, which included AD,frontotemporal lobe dementia (FTLD), and Lewy body disease(LBD). Among various causes of dementia, AD is the mostprevalent (approximately 50% of all dementia patients), fol-lowed by LBD, FTLD, and other various mixed subtypes [121].Concretely for AD, a worldwide prevalence of over 30 millionpeople has been estimated, and its incidence is expected toincrease dramatically with an increasing elderly population.Thus, it is of extreme importance to solve the most impor-tant AD questions raised, that is origin, causes, treatment,prevention, and early and accurate diagnosis. Most of thebiomarkers studied so far are protein molecules. For exam-ple, AD has a beta-amyloid peptide (A�) and tau protein, inFTLD a protein named TDP-43 is recently identified, and forLBD �-synuclein and ubiquitin have been identified [119].These proteins cause neurodegeneration, leading to cogni-tive impairment, and are therefore thought to be therapeutictargets; however, the key aspects are still controversial.
A paper published by Ibanez et al. [119] has shown anontargeted metabolomic approach based on CE-MS to ex-amine metabolic differences in cerebrospinal fluid (CSF)samples from subjects with different cognitive status relatedto AD progression. To do this, CSF samples from 85 sub-jects were obtained from patients with (i) subjective cogni-tive impairment (SCI, i.e. control group), (ii) mild cognitiveimpairment (MCI) that remained stable after a follow-up pe-riod of 2 years, (iii) MCI that progressed to AD within a2-year time after the initial MCI diagnostic, and (iv) diag-nosed AD. Using multivariate statistical analysis based onCE-MS metabolomics for a total of 73 CSF samples, possibledisease progression biomarkers such as choline, dimethy-larginine, arginine, valine, proline, serine, histidine, creatine,carnitine, and suberylglycine were identified. So the results ofthis work suggest that CE-MS metabolomics of CSF samplescan be a useful tool to predict AD progression using humansamples.
In the same way, another paper by Tsuruoka et al. [122]showed a metabolomic analysis of serum and saliva obtainedfrom ten patients with neurodegenerative dementias (includ-ing AD, FTLD, and LBD), and from nine patients who wereage-matched healthy controls. The results obtained using CE-TOF-MS showed that six metabolites in serum (�-alanine,creatinine, hydroxyproline, glutamine, iso-citrate, and cyti-dine) and two in saliva (arginine and tyrosine) were signifi-cantly different between dementias and controls. In addition,the experiment confirmed that using multivariate analysis,serum was a more efficient biological fluid for diagnosis com-pared to saliva. A total of 45 metabolites in total were identifiedas candidate markers that could discriminate at least one pairof diagnostic groups from the healthy control group.
Metabolite analysis is especially useful for identifyingpathways modified in a given biological system after cer-tain pathology, perturbation, or treatment. Metabolomics hasbeen predominantly applied to discover biomarkers related to
prognosis, diagnosis, and therapeutic monitoring of diseases(mostly cancer). This discipline is particularly complex sincelow-molecular-weight metabolites represent a diverse rangeof chemicals in a wide dynamic range of concentrations inbiological samples. At present, numerous natural dietary con-stituents, for example polyphenols, are now under scrutinydue to their promising anticancer properties. The emergingfoodomics field and its omics tools are expected to play acrucial role in the investigation of the interactions betweennutrients or bioactive food compounds and genes.
In this regard, Ibanez et al. [121] presented an analyticalmultiplatform to carry out a broad metabolomic study on theantiproliferative effect of dietary polyphenols from rosemaryon human colon cancer cells. The multiplatform consisted ofCE-TOF and UHPLC-TOF using different modes as RF andHILIC, all them combined to achieve a global metabolomicexamination of the effect of dietary rich in polyphenols fromrosemary on HT29 colon cancer cells. Hydrophilic interac-tion liquid chromatography (HILIC) possesses a combina-tion of hydrophilic interaction, ion exchange, and RP reten-tion that result in enhanced retention of polar analytes. Sothis modality of LC is becoming a complementary analyticalmode to the more common RP/LC also in metabolomics, dueto the ability of HILIC to separate more hydrophilic metabo-lites. In this work, a nontargeted metabolomic approach wasused and metabolites showing significant different expres-sion after the polyphenols treatment were identified in coloncancer cells. Some changes and alterations were detected inpolyphenol-treated cells such as an enhanced reduced glu-tathione/oxidized glutathione ratio and polyamines contentwith important implications in cancer proliferation. Amongthe 65 tentatively identified metabolites founded to be sig-nificantly different (p � 0.05) after the polyphenol treatmentof colon cancer cells, 51 were determined to be significantlyupregulated and 14 downregulated. Additionally, due to largeamount of data generated in metabolomics studies in this pa-per, data were processed using statistical analysis. Main dataprocessing workflow steps include noise reduction, spectrumdeconvolution, electropherogram/chromatogram alignment,and peak integration.
A global foodomics strategy using whole-transcriptomemicroarray together with an MS-based nontargeted analyticalapproach (via CE-TOF MS and UHPLC-TOF-MS) have beenemployed to carry out transcriptomics and metabolomicsanalyses, respectively. This study has been developed byValdes et al. [123] who applied to study the antiprolifer-ative effect of dietary polyphenols from rosemary on twohuman leukemia lines, one showing a drug-sensitive phe-notype (K562), and another exhibiting a drug-resistant phe-notype (K562/R). Due to massive data obtained using theseanalytical platforms, a functional enrichment analysis wascarried out using ingenuity pathway analysis software asa previous step for a reliable interpretation. Integration ofdata obtained from transcriptomics and metabolomics plat-forms was attempted by overlaying datasets on canonical (de-fined) metabolic pathways using ingenuity pathway analy-sis software. Metabolomics analysis suggested that rosemary
polyphenols affected differently the intracellular levels ofsome metabolites in two leukemia cell sublines by meansof the identification of various differentially expressed genesmodulated by rosemary polyphenols. However, direct asso-ciations between the changes observed at transcriptome andmetabolome level could be established only in few cases.
2.3.3 Instrumental improvements
A review [46], published in 2011, paid special attention toemerging technological developments mainly related to theuse of new interfaces for CE-MS, enabling new possibili-ties for metabolomics studies. In this section of the review,instrumental improvements will be discussed for CE-ESI-TOF-MS. It is well known that one of the major points ofweakness of the ESI source comes from the possibility thatthe ionization process is affected by the nature and concen-tration of the compounds entering the ion source. The in-terfacing of the CE separation compartment with the ESI-MS section has been achieved by different means, namelysheath-flow, sheathless, and liquid-junction interfaces, theformer being, until recently, the only one commerciallyavailable.
Several papers have described instrumental improve-ments using ESI-TOF in order to gain sensitivity such asthe sheathless interface [128], a new cationic coating [129],and the use of negative mode [130].
To improve sensitivity, various sheathless CE-MS inter-faces have been developed. For example, the outer surface ofthe capillary has been coated with conductive materials suchas gold, silver, copper, or graphite, which allows the elec-trical contact to be established. However, these coatings areoften damaged by electrical discharge and this reduces thelife time of the electrical contact. In another approach, a plat-inum wire was inserted into the capillary to establish a stableelectric contact, but this caused bubble formation in the cap-illary. A paper published by Hirayama et al. [128] proposed asheathless CE-ESI-MS method for cationic metabolome anal-ysis. Under optimized conditions, 53 cationic metabolites,including amino acids and their derivatives, amines, nucleicacids, and small peptides, were successfully separated andselectively detected with TOF-MS. Detection limits for stud-ied compounds were between 0.004 and 0.8 �mol/L and in-creased more than fivefold for 21 (40%) of the compoundsdetected when a conventional sheath-flow CE-MS was used.
CE-MS is well suited for protein or peptide analysis. How-ever, it may suffer from analyte adsorption onto the silicasurface of the capillary, especially in case of basic or hy-drophobic intact proteins. As adsorption problems can leadto analyte loss, EOF instabilities, or even complete failureof analysis, many approaches have been addressed in or-der to overcome these drawbacks by means of modificationof the capillary surface with convenient coating materials.Pattky and Huhn [129] focused on statically adsorbed coat-ings due to the simple and fast coating procedures, theirrecoating possibilities, and MS compatibility. This study com-
Sheath-flow CE-MS has shown some limitations in termsof sensitivity, mainly due to very small sample injection vol-umes [130] or the use of the sheath-flow interface. Otherlimitations of sensitivity occur when MS detection in thenegative ion mode is used [46]. As example, Grundmannand Matysik [130] have currently published an experimentalapproach to conducting fast CE-MS measurements of verysmall samples (nanoliters) based on the concept of capillarybatch injection (Fig. 5II). In this paper, an automated, small-footprint injection device for CE-MS has been built and de-tails of design and specifications of the injection device areshown (see Fig. 5I). This device is capable of running truemultisample measurement series by using minimal sam-ple volumes and delivering an injection efficiency of up to100%.
Sensitivity obtained in the negative ion mode is usuallynot sufficient to provide a useful signal, and therefore MS de-tection in the positive ion mode using nonvolatile BGE [131]can be used. When CE is hyphenated to MS through asheath-liquid interface, the dilution (1:20–1:30) exerted bythe sheath-flow and the liquid sheath effect (BGE inorganicanions migrate toward the injection end) may further re-duce the ion suppression phenomena. Notwithstanding theabove-mentioned considerations, in real practice, the choiceof buffer electrolytes in CE-MS is traditionally limited to thosewith high volatility, such as ammonium acetate and formate.In the forensic field, only a few papers have been presentedon the use of nonvolatile buffers by CE-ESI-MS analysis ofdrugs of abuse in urine or plasma extracts without sacri-ficing analytical sensitivity. A study developed by Gottardoet al. [131] compared different electrolyte systems for the op-timization of the CE-ESI-MS analysis of a mixture of selectedforensic drugs, in terms of separation efficiency and detectionsensitivity.
3 Conclusions
In this review, CE-MS is regarded as a most importantplatform for use in bio-omic applications in proteomics,metabolomics, and genomics and also for target-based com-pounds in clinical analysis. There are, however, certain limi-tations associated with reproducibility and analytical robust-ness in clinical proteomics and its implementation in routineclinical analysis.
The major advantages of CE-MS are its particularly highresolution, high separation efficiency, sensitivity, speed of
14 V. R. Robledo and W. F. Smyth Electrophoresis 2014, 00, 1–17
Figure 5. (I) Injection cell (down) and injection capillary position-ing unit (up). Further items are: 13, stepper motor with leadscrew;14, linear guideway rail and block; 15, stirrer motor; 16, injectioncell holder with sample vials. (II) Schematic overview of the over-all experimental setup employed in this study, consisting of: a, mi-croliter syringe with injection capillary; b, sample; c, injection cellwith CE high-voltage source; d, separation capillary; e, electro-spray interface; f, TOF mass spectrometer. Reproduced from [130]with permission.
analysis, low sample consumption, and structural informa-tion that can be provided. The most popular ionization sourcein CE-MS is found to be electrospray, which is characterizedby high ionization efficiency and the soft nature of the ioniza-tion process. Many papers and reviews have been publishedcomparing different ionization sources for biological appli-cations using the CE-MS coupling. However, this review hasparticularly focused on the different types of MS analyzersused in hyphenation with CE, their instrumental features,and which kinds of bioapplications in the period 2012–2013are most suitable for the particular analyzer whether it be thequadrupole in the SIM mode or using the QqQ configuration(MS/MS), the IT using the MSn mode, or the TOF. The highsensitivity of the QqQ, compared to TOF or IT analyzers, is
of value in carrying out complex analyses in pharmaceuticaland biological matrices. CZE-ESI-QqQ can also be applied for(i) the investigation of weak associated species of the drugsand (ii) monitoring of predicted (targeted) compounds de-rived from the original drugs, for example drug impurities ormetabolites when standards of targets are missing. The SIMmode can add to the selectivity of the Q when two or moreions of different m/z values co-elute. The main advantage ofIT is the ability to conduct the MS measurements without theneed of an additional mass analyzer as with QqQ and Q-TOFinstruments or a collision cell as He gas is already presentin IT. The application of IT-MS in fragmentation behavior,fragmentation pathways, and structure elucidation of com-pounds is well established. Moreover, several reports showthat IT-MS can also be very useful for quantitative analysis.Currently, there are an increasing number of applications us-ing CE-IT-MS for the analysis of amino acids, peptides, andproteins. The sensitivity of IT can be limited due to relativelysmall sample injection volume and the use of the sheath-flow interface. Several papers have described instrumentalimprovements in order to improve the sensitivity such asthe pressurized liquid junction nanoflow interface and theon-line fritless SPE procedure.
Due to TOF’s high sensitivity, high mass resolution, andaccuracy, it has a theoretically unlimited mass range with anextremely high scan rate and is ideally suited to studies inmetabolomics and proteomics. This review gives examplesof studies using samples of patients or control subjects andfocuses on AD and neurodegenerative dementia disease. Oth-ers are involved with dietary studies and foodomics. Instru-mental improvements with ESI-TOF in order to gain sensi-tivity can be achieved with the sheathless interface, a cationiccoating, and the use of the negative mode.
The author V. R. R. would like to thank Professor Juan JoseBerzas Nevado for education on analytical chemistry field.
The authors have declared no conflict of interest.
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