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An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CEMS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CEMS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg·mL-1 and the limit of detection (LOD) was 0.05 µg·mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (< 0.5 % and 2.4 %, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100 % cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control.

Protein profiling of major bovine milk proteins (i.e., whey and casein proteins) is of great interest in food science and technology. This complex set of protein proteoforms may vary with breed, genetics, lactation stage, health, and nutritional status of the animal. Current routine methods for bovine milk protein profiling at the intact level are typically based on capillary electrophoresis-ultraviolet, which does not allow confirming unequivocally the identity of the separated proteins. As an alternative, in this study, we describe for the first time a novel and simple capillary electrophoresis-mass spectrometry method in positive electrospray ionization mode. Under the optimized conditions, capillary electrophoresis-mass spectrometry allowed the separation and identification at the intact level of major bovine milk whey and casein proteins in less than 15 min. Furthermore, high-resolution mass spectrometry confirmed its importance in the reliable characterization of bovine milk protein proteoforms, especially those with slight molecular mass differences, such as β-casein A1 and A2, which are relevant to unequivocally identify milk with specific β-casein compositions (e.g., A2A2 milk, which is widely known as A2 milk). This differentiation was not possible by matrix-assisted laser desorption/ionization mass spectrometry, which provided rapidly and easily a rich but less accurate fingerprint of bovine milk proteins due to the lower mass resolution.

In this study, on-line solid-phase extraction capillary liquid chromatography-mass spectrometry (SPE-CapLC-MS) and on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) were compared for the analysis of the opioid peptide biomarkers dynorphin A (1-7) (DynA), endomorphin 1 (End 1), and methionine-enkephalin (Met). First, a capillary liquid chromatography-mass spectrometry (CapLC-MS) method was established, which allowed limits of detection (LODs) of 0.5 μg/mL for Dyn A and Met, and 0.1 μg/mL for End 1. Then, a column switching setup operated by a 2-position/6-port micro-valve with a C18 enrichment column was assembled for SPE-CapLC-MS. Under optimized conditions, the LODs for the three peptides were lowered up to 1000-fold compared to CapLC-MS, until detecting 0.5 ng/mL concentrations. Repeatability (

Several strategies have been developed to decrease the concentration limits of detection (LODs) in capillary electrophoresis (CE). Nowadays, chromatographic-based preconcentration using a microcartridge integrated in the separation capillary for in-line solid-phase extraction capillary electrophoresis (SPE-CE) is one of the best alternatives for high throughput and reproducible sample clean-up and analyte preconcentration. This review covers different designs (geometrical configurations, with frits or fritless, capillary types, compatibility with commercial instrumentation, etc.) and materials (sorbents, supports, affinity ligands, etc.) applied for almost 30 years to prepare in-line SPE-CE microcartridges (i.e. analyte concentrators), with emphasis on the conventional unidirectional configuration in capillary format. Advantages, disadvantages and future perspectives are analyzed in detail to provide the reader a wide overview about the great potential of this technique to enhance sensitivity and address trace analysis.

A hybrid material (nano-metal organic framework@organic polymer, named as nano-MOF@polymer) was applied for the first time as sorbent for on-line solid-phase extraction capillary electrophoresis with ultraviolet detection (SPE-CE-UV). The resulting material was prepared building layer-by-layer a HKUST-1 (Hong Kong University of Science and Technology-1) nano-MOF onto the polymer surface, which allowed controlling the thickness and maximizing the active surface area. The sorbent was widely characterized at micro- and nano-scale to validate the synthesis and to establish the material properties. Then, fritless microcartridges (2 mm) were assembled by packing only a few micrograms of sorbent particles and investigated for preconcentration of fluoroquinolones (FQs) in several real samples (river water, human urine and whole cow milk). Under the optimized conditions, the sample (ca. 60 μL) was loaded in separation background electrolyte (BGE, 50 mM phosphate (pH 7)), and retained analytes were eluted using a small volume of 2% v/v formic acid in methanol (ca. 50 nL). The SPE-CE-UV method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability, reproducibility and reusability. The developed method showed a LOD decreasing until 1 ng L−1 when larger volumes of sample were loaded (ca. 180 μL), which was 500,000 times lower than by CE-UV. This undescribed sensitivity enhancement would arise from the homogenous and populated MOF nano-domains and the appropriate permeability of the hybrid material, which would promote high extraction efficiency and loading capacity. Furthermore, the sorbent showed appropriate selectivity regardless the analyzed complex environmental, biological or food matrix samples, achieving excellent detectability and recoveries (>90%).

En els últims anys ha augmentat el consum dels derivats sintètics de la catinona, un alcaloide que es troba de forma natural en les fulles del khat, ja que representen una alternativa més assequible i accessible respecte a altres drogues il·lícites més conegudes com les amfetamines. Després de ser consumits, aquests compostos es poden trobar en l'organisme ja sigui metabolitzats o en la seva forma pura a baixes concentracions. Per tant, solen ser necessaris mètodes altament sensibles per determinar aquestes substàncies en mostres biològiques com sang, cabell, saliva o, més comunament, orina. Les catinones presenten un centre quiral i, per tant, es poden trobar en dues formes enantiomèriques (R i S). Cada enantiòmer pot presentar un comportament farmacocinètic i farmacodinàmic diferent i, per tant, tenir diferents efectes en l'organisme. En conseqüència, la enantioseparació de les catinones sintètiques pot ser de gran utilitat. En aquest sentit, l'electroforesi capil·lar és una tècnica molt adequada per a aquest propòsit, ja que la enantioseparació es pot aconseguir simplement afegint un selector quiral a l'electròlit de separació. En los últimos años ha aumentado el consumo de derivados sintéticos de la catinona, un alcaloide que se encuentra de forma natural en las hojas del khat, puesto que representan una alternativa más asequible y accesible respecto a otras drogas ilícitas más conocidas como las anfetaminas. Después de ser consumidos, estos compuestos se pueden encontrar en el organismo ya sea metabolizados o en su forma pura a bajas concentraciones. Por lo tanto, suelen ser necesarios métodos altamente sensibles para determinar estas sustancias en muestras biológicas como sangre, cabello, saliva o, más comúnmente, orina. Las catinonas presentan un centro quiral y, por tanto, se pueden encontrar en dos formas enantioméricas (R y S). Cada enantiómero puede presentar un comportamiento farmacocinético y farmacodinámico diferente y, por consiguiente, tener diferentes efectos en el organismo. En consecuencia, la enantioseparación de las catinonas sintéticas puede ser de gran utilidad. En este sentido, la electroforesis capilar es una técnica muy adecuada para este propósito, puesto que la enantioseparación se puede lograr simplemente agregando un selector quiral al electrolito de separación In recent years, the consumption of synthetic derivatives of cathinone, an alkaloid naturally found in the leaves of the khat plant, has increased as they represent a more affordable and accessible alternative to other well-known illicit drugs such as amphetamines. After their consumption, these compounds can be found in the organism either metabolized or in their pure form at low concentrations. Therefore, highly sensitive methods are usually necessary to determine these substances in biological samples such as blood, hair, oral fluids, or, more commonly, urine. Cathinones present a chiral centre, so they can be found in two enantiomeric forms (R and S). Each enantiomer can present different pharmacokinetic and pharmacodynamic behaviour and have different effects on the organism. Consequently, the enantioseparation of synthetic cathinones can be very useful. In these sense, capillary electrophoresis represents an excellent technique for this purpose, since enantioseparation can be achieved simply by adding a chiral selector to the background electrolyte. However, despite of this and other well-known advantages of this analytical technique, one of its main limitations is its poor sensitivity

Extracellular vesicles (EVs) are membrane enclosed vesicles (

[spa] La presente tesis doctoral se centra en el desarrollo de nuevos métodos de análisis para la identificación y separación de glicopéptidos y glicanos utilizando técnicas microseparativas acopladas a la espectrometría de masas (cromatografía de líquidos capilar y electroforesis capilar, CapLC-MS y CE-MS). Aunque el análisis de los glicopéptidos es una estrategia habitualmente utilizada para la caracterización de productor farmacéuticos, continúa siendo difícil la detección de glicoformas poco abundantes que podrían ser esenciales para diferenciar proteínas endógenas de variantes recombinantes. De este modo, en esta tesis se han desarrollado dos métodos para la purificación selectiva de glicopéptidos, obtenidos de digestos de glicoproteínas, utilizando la eritropoetina humana recombinante (rhEPO) como glicoproteína modelo. Por otro lado, en muchas patologías se han descrito alteraciones en los glicanos de ciertas glicoproteínas en presencia de procesos inflamatorios importantes y muchos tipos de cáncer. Además, dado que ciertas estructuras de glicano así como tipos de enlace han estado relacionados con enfermedades específicas, la caracterización exhaustiva de los tipos de enlace de los isómeros de los glicanos ha suscitado gran interés biomédico en los últimos años. En este sentido, se han establecido en esta tesis doctoral diferentes metodologías para la caracterización de los isómeros de los glicanos de la alfa-1-glicoproteína ácida (hAGP), la cual ha generado interés biomédico dada la alteración de sus glicanos en ciertos procesos inflamatorios y cáncer. Primero, la digestión con diversas exoglicosidasas (sialidasa, fucosidasa y galactosidasa) en combinación con el análisis de los glicanos per cromatografía de líquidos capilar de interacción hidrofílica zwitteriónica acoplada a la espectrometría de masas (CapZIC-HILIC-MS) y la estrategia GRIL utilizando [12C6]/[13C6]- anilina, permitió asignar los enlaces de los ácidos siálicos, así como de las fucosas de los isómeros de los glicanos de la hAGP. Por otro lado, se desarrolló un método para asignar los isómeros de los glicanos per espectrometría de masas en tándem de alta resolución. La posibilidad de separar los isómeros antes de la detección, juntamente con la obtención de la masa exacta de los iones fragmentos, permitió el establecimiento de un método de caracterización de los glicanos fiable por CapZIC-HILIC-MS/MS. Además, la doctoranda realizó una estancia en la Freie Universität Berlin (Berlin, Alemania), bajo la supervisión del Dr. Kevin Pagel, para estudiar la separación de los isómeros de glicanos per espectrometría de movilidad iónica acoplada a la espectrometría de masas (IM-MS). Finalmente, el método por CapZIC-HILIC-MS en combinación con la estrategia GRIL utilizando [12C6]/[13C6]-anilina se aplicó en esta tesis para el análisis de muestras patológicas, con el fin de evaluar posibles alteraciones en los isómeros de los glicanos de dos glicoproteínas de fase aguda, l’hAGP y la transferrina de ratón (mTf), en presencia de ciertos procesos inflamatorios y cáncer. Además, se emplearon herramientas quimiométricas con el objetivo de identificar glicanos como candidatos biomarcadores de estas patologías., [eng] The present thesis is focused on the development of novel analytical methods for the identification and separation of glycopeptides and glycans by microseparative techniques coupled to mass spectrometry. Although the analysis of glycopeptides is a commonly used strategy for glycan characterization of biopharmaceuticals, the detection of low abundant glycopeptides that could be essential to differentiate endogenous and recombinant variants of certain proteins continues to be a challenge. In this thesis, methods to selectively enrich glycopeptides from glycoprotein digests were developed using recombinant human erythropoietin (rhEPO) as model glycoprotein. On the other hand, alterations in glycan structures have been described in presence of important inflammatory processes and many types of cancer. As certain glycan isomers or linkage-types have been related to specific pathologies, the in depth characterization of glycan isomer linkage-types has aroused great interest in last years. In this regard, different methodologies were established in this thesis to characterize human alpha-1-acid glycoprotein (hAGP) glycan isomers, whose glycosylation has been described to be altered in several diseases. Exoglycosidase digestions, tandem mass spectrometry (MS/MS) as well as ion-mobility mass spectrometry (IM-MS) were used with this purpose. Finally, the established method for glycan isomer separation in combination with the GRIL strategy with [12C6]/[13C6]aniline was used in this thesis to evaluate possible glycan isomer modifications in two acute-phase proteins, hAGP and mouse transferrin (mTf), caused by certain inflammatory diseases and cancer. Moreover, multivariate data tools, such as PCA and PLS-DA, were also employed in these studies to identify glycan-based biomarker candidates.

This work provides the pKa at the biorelevant temperature of 37 °C for a set of compounds proposed as internal standards for the internal standard capillary electrophoresis (IS-CE) method. This is a high throughput method that allows the determination of the acidity constants of compounds in a short time, avoiding the exact measurement of the pH of the buffers used. pH electrode calibration at 37 °C can be avoided too. In order to anchor the pKa values obtained through the IS-CE method in the pH scale, the acidity constant at 37 °C of some of the standards has been determined also by the reference potentiometric method. In general, a decrease in the pKa value is observed when changing the temperature from 25 to 37 °C, and the magnitude of the change depends on the nature of the compounds. Once the pKa values at 37 °C of the internal standards have been established, the method is applied to the determination of the acidity constants of seven polyprotic (5 diprotic and 2 triprotic) drugs. The obtained mobility-pH profiles show well-defined curves, and the fits provide precise pKa values. Due to the lack of reference data at 37 °C only the pKa values of labetalol can be compared to values from the literature, and a very good agreement is observed.

In this study, polymeric monoliths with gold nanoparticles (AuNP@monolith) were investigated as microcartridges for the analysis of protein biomarkers by on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS). “Plug-and-play” microcartridges (7 mm) were prepared from a glycidyl methacrylate (GMA)-based monolithic capillary column (5 cm x 250 µm i.d.), which was modified with ammonia and subsequently functionalized with gold nanoparticles (AuNPs). The performance of these novel microcartridges was evaluated with human transthyretin (TTR), which is a protein related to different types of familial amyloidotic polyneuropathies (FAP). Protein retention depended on the isoelectric point of the protein (TTR pI~5.4) and elution was achieved with a basic phosphate solution. Under the optimized conditions, limits of detection (LODs) for TTR by AuNP@monolith-SPE-CE-MS were 50 times lower than by CE-MS (5 vs 250 mg•L−1, with an ion trap (IT) mass spectrometer). The sensitivity enhancement was similar compared to SPE-CE-MS using immunoaffinity (IA) microcartridges with intact antibodies against TTR. Linearity, repeatability in migration times and peak areas, reusability, reproducibility and application to serum samples were also evaluated. Fil: Pont, Laura. Universidad de Barcelona; España Fil: Marin, Gemma. Universidad de Barcelona; España Fil: Vergara Barberán, María. Universidad de Valencia; España Fil: Gagliardi, Leonardo Gabriel. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Laboratorio de Investigación y Desarrollo de Métodos Analíticos. - Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Laboratorio de Investigación y Desarrollo de Métodos Analíticos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Sanz Nebot, Victoria. Universidad de Barcelona; España Fil: Herrero Martinez, José Manuel. Universidad de Valencia; España Fil: Benavente, Fernando. Universidad de Barcelona; España

This study evaluates zwitterionic-hydrophilic interaction capillary liquid chromatography (capZIC-HILIC) and capillary electrophoresis (CE) with ultraviolet (UV) and mass spectrometry (MS) detection for the direct, label free and multiplex analysis of microribonucleic acids (miRNAs). CapZIC-HILIC-UV and CE-UV methods were first optimized, resulting in similar separations for a mixture of three miRNAs (hsa-iso-miR-16-5p, hsa-let-7g-5p, and hsa-miR-21-5p) but with reversal of elution/migration orders and small differences in repeatability, linearity, limit of detection (LOD) and separation efficiency. The established UV methods were transferred and validated in these terms with mass spectrometry (MS) detection, which allowed identifying the miRNAs and characterizing their post-transcriptional modifications. LOD by capZIC-HILIC-MS was 1 mu M of miRNA, around 5 times lower than by CE-MS due to the analyte dilution with the sheathflow CE-MS interface and to the slightly increased abundance of alkali metals adducts in the CE-MS mass spectra. In addition, the suction effect promoted by the nebulizer gas in CE-MS negatively affected the already compromised separations. In contrast, CE-MS showed superior repeatabilities with spiked serum samples, as well as reduced costs, extended capillary column durabilities and shorter conditioning times. The comparison of the different methods allows disclosing the current advantages and disadvantages of capZIC-HILIC and CE for the analysis of miRNA biomarkers.

A fast determination of acidity constants (pKa) of very insoluble drugs has become a necessity in drug discovery process because it often produces molecules that are highly lipophilic and sparingly soluble in water. In this work the high throughput internal standard capillary electrophoresis (IS-CE) method has been adapted to the determination of pKa of water insoluble compounds by measurement in methanol/aqueous buffer mixtures. For this purpose, the reference pKa values for a set of 46 acid-base compounds of varied structure (internal standards) have been established in methanol-water mixtures at several solvent composition levels (with a maximum of 40% methanol). The IS-CE method has been successfully applied to seven test drugs of different chemical nature with intrinsic solubilities lower than 10-6 M. pKa values have been determined at different methanol/aqueous buffer compositions and afterwards Yasuda-Shedlovsky extrapolation method has been applied to obtain the aqueous pKa. The obtained results have successfully been compared to literature ones obtained by other methods. It is concluded that the IS-CE method allows the determination of aqueous pKa values using low proportions of methanol, becoming then more accurate in the extrapolation procedure than other reference methods.

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: −5.4 and −5.6 Kcal/mol for HSA and −4.3 and −3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins., AcknowledgementsThe generous gift of the serum sample by the cancer patientdonor is highly appreciated. We would also like to acknowledgethe financial support from the Spanish Ministry of Economy andCompetitiveness (MINECO, projects CTQ2013-43236-R, BIO2010-16922, and BIO2015-66356-R) and from the Generalitat deCatalunya, Spain (grant 2014 SGR 229). N. F.-G. and M. G.acknowledge the Ph.D. JAE-pre grant and the JAE-doc grant, respec-tively, from CSIC co-financed by the European Social Fund. D.N-C.acknowledges the contract in the frame of the Youth GuaranteeImplementation Plans financed by the European Social Fund (ESF)and the Youth Employment Initiative (YEI). J.E. G.-L. acknowledgessupport by Award Number R01GM089759 from the National Insti-tute of General Medical Sciences (NIH, U.S.A.).

This study presents for the first time an in-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method for the enantiodetermination of drugs of abuse in urine samples. The enantioseparation of R,S-3,4-methylenedioxypyrovalerone (R,S-MDPV) was achieved with a 10 mM ammonium acetate BGE (pH 7) that contained 0.5% (m/v) of sulphated-α-CD as chiral selector. At these pH conditions, this CD was negatively charged, which prevented its entrance into the mass spectrometer since it migrates in the opposite direction. To improve sensitivity, an in-line SPE-CE-MS method using high pressure for sample introduction (i.e. 20 min at 3 bars) was developed. Furthermore, the conditioning procedure and the first part of the electrophoretic separation were performed by switching off the nebulizer gas and the ionization source voltage to avoid non-volatile contaminant arrival into the mass spectrometer. The developed methodology was validated by analyzing urine samples, which required a very simple liquid-liquid extraction (LLE) sample pretreatment. Linearity ranged from 30 to 250 ng mL-1, limit of detection (LOD) was 10 ng mL-1, relative standard deviation (RSD) values were below 10.5% in terms of intra-day and inter-day precision and the relative error values were below 9% for peak areas accuracy.

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the background electrolyte necessary for an efficient separation and sensitive MS detection. Here, we present SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS) to overcome these drawbacks while keeping the design simple. The nvSPE-CE-MS system is operated with a single CE instrument and two capillaries for independent and orthogonal SPE preconcentration and CE separation, which are interfaced through an external and electrically isolated valve with a 20 nL sample loop. The instrumental setup is proved for the analysis of opioid and amyloid beta peptide biomarkers in standards and plasma samples. NvSPE-CE-MS allowed decreasing the limits of detection (LODs) 200 times with regard to CE-MS. Compared to unidirectional SPE-CE-MS, peak efficiencies were better and repeatabilities similar, but total analysis times longer and LODs for standards slightly higher due to the heart-cut operation and the limited volume of the valve loop. This small difference on the LODs for standards was compensated for plasma samples by the improved tolerance of nvSPE-CE-MS to complex sample matrices. In view of these results, the presented setup can be regarded as a promising versatile alternative to avoid complicated matrix samples entering the separation capillary in SPE-CE-MS.

The use of organic acids (e.g. acetic acid and gluconic acid) as additives during protease production is regarded as one of the simplest alternatives to increase enzyme stability and activity in many industrial processes. However, no methods have been described for the determination of organic acids in proteases and their contents have not been established yet. In this work, a novel, rapid and simple method for the determination of organic acids in proteolytic enzymes by capillary electrophoresis (CE) with indirect ultraviolet (UV) detection has been developed. Under the optimized conditions, the method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and intra-day and inter-day repeatability. Later, a sample pretreatment based on a hydroalcoholic microextraction was carefully optimized to obtain good recovery and repeatability and determine acetic and gluconic acids in a commercial protease sample. The complete procedure was validated using the standard-addition calibration method, finding matrix effects on the studied compounds. Finally, acetic acid and gluconic acid were quantified at 80 mg/Kg (0.0080% (m/m)) and 69 mg/Kg (0.0069% (m/m)) in the protease sample, respectively.

[spa] En las últimas décadas, el análisis de compuestos biomarcadores en muestras biológicas se ha convertido en una herramienta esencial para el diagnóstico, seguimiento y pronóstico de diversas enfermedades. Las principales dificultades en muchos casos son la baja concentración de los biomarcadores, la complejidad de la matriz de la muestra y la limitada disponibilidad de muestra. En esta tesis doctoral, se presentan nuevas estrategias basadas en electroforesis capilar- espectrometría de masas (CE-MS) para la separación, detección, caracterización y cuantificación de péptidos, proteínas y microARNs (miRNAs) biomarcadores en fluidos biológicos. La CE-MS es una técnica excelente para la separación de biomoléculas cargadas y su identificación inequívoca. En esta tesis, se han desarrollado estrategias novedosas para la predicción y optimización de las separaciones de mezclas complejas de péptidos en CE. Para evaluar estas estrategias, se han estudiado los péptidos beta amiloides. Una limitación importante de la CE es su baja sensibilidad en términos de concentración para la mayoría de analitos a causa del pequeño volumen de inyección de muestra. La extracción en fase sólida en línea con la electroforesis capilar (SPE-CE) es una excelente estrategia para disminuir los límites de detección. En esta tesis se han investigado diferentes metodologías de SPE-CE-MS unidireccional empleando sorbentes selectivos, como los sorbentes de inmunoafinidad, afinidad a aptámero, afinidad a metal inmovilizado y el carburo de silicio, para el análisis de proteínas intactas (transtiretina y α-sinucleína), digestos de proteínas y miRNAs en fluidos biológicos. También se ha investigado el potencial de la SPE-CE-MS con una nanoválvula (nvSPE-CE-MS) para el análisis de péptidos en fluidos biológicos. Se ha desarrollado un método de nvSPE-CE-MS empleando un sorbente C18 para el análisis de péptidos opioides y péptidos beta amiloides y se han comparado las ventajas e inconvenientes de la nvSPE-CE-MS respecto a la SPE-CE-MS unidireccional. Finalmente, se han investigado nuevas estrategias para el análisis rápido y eficiente de digestos de proteínas. La tripsina es la enzima proteolítica más comúnmente utilizada y la digestión se realiza habitualmente en disolución, requiriéndose largos tiempos de digestión. Con el objetivo de disminuir el volumen de muestra, su manipulación y el tiempo total de análisis, en esta tesis se ha desarrollado una metodología de análisis de proteínas empleando microrreactores empaquetados con partículas con tripsina inmovilizada en línea con la CE-MS (IMER-CE-MS)., [eng] In recent years, an increased emphasis has been placed on the analysis of biomarker compounds in biological samples for the diagnosis, follow-up and prognosis of numerous diseases. The leading difficulties in many of these analyses are the low concentration of the biomarkers, their structural complexity, the sample matrix effects and the limited availability of sample. In this doctoral thesis, we present novel strategies based on capillary electrophoresis- mass spectrometry (CE-MS) for the separation, detection, characterization and quantification of peptide, protein and microRNA (miRNA) biomarkers in biological fluids. MS is a powerful technique for the unequivocal identification of biomolecules due to its potential with regard to the detailed structural characterization of unknown compounds. However, due to the complexity of most biological fluids, the hyphenation of high-performance separation techniques is essential prior to MS analysis. In this regard, CE is a microscale technique that is very suitable for the separation of charged biomolecules. Nevertheless, electrophoretic separation methods must be properly optimized to achieve rapid, efficient, sensitive and high-resolution separations. In this thesis, we have investigated novel strategies to predict and optimize the separation of complex mixtures of peptides in CE. To assess these strategies, amyloid beta peptides, which are biomarkers of Alzheimer’s disease, were studied. A major limitation of CE is the relatively poor concentration sensitivity for most analytes due to the small sample volume that can be injected in the separation capillary. On-line solid- phase extraction capillary electrophoresis (SPE-CE) is a powerful approach for sample clean-up and reduction of the limits of detection. In SPE-CE, analytes from a large volume of sample are retained on a sorbent contained in a microcartridge. In unidirectional SPE-CE, the most typical configuration, the microcartridge is mounted in series to the separation capillary, inserted near the capillary inlet. After sample loading, the microcartridge is washed to remove non-selectively retained molecules. Then, the retained analytes are desorbed in a small volume of eluent, resulting in sample clean-up and concentration enhancement before electrophoretic separation and detection. In the present thesis, selective sorbents, such as immunoaffinity, aptamer affinity, immobilized metal affinity and silicon carbide sorbents have been explored in unidirectional SPE-CE-MS for the analysis of intact proteins, such as transthyretin, which is a biomarker of familial amyloidotic polyneuropathy type I, and α-synuclein, which is related to Parkinson’s disease, protein digests and miRNAs, which are related to cancer, in biological fluids. Unidirectional SPE-CE-MS is straightforward to implement. However, this simple setup has some inherent limitations. On the one hand, the sample volumes introduced using pressure depend on the dimensions of the separation capillary. On the other hand, sample loading is conducted in the same direction as the subsequent separation. Therefore, some of the matrix components could be irreversibly adsorbed in the inner wall of the separation capillary. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the BGE necessary for an efficient separation or sensitive MS detection. In order to overcome these drawbacks, some new configurations where the sample is introduced in an orthogonal direction to the separation have been proposed, requiring the use of valves. We have investigated SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS). A nvSPE-CE-MS method with a C18 sorbent for the analysis of opioid peptides and amyloid beta peptide fragments has been developed and the advantages and disadvantages compared to the unidirectional SPE-CE- MS have been discussed. New strategies for high-throughput bottom-up analysis of proteins have also been investigated. Tryptic digestion has been traditionally conducted in solution and requires long digestion times. In contrast, immobilized enzymes allow decreasing the sample volume and the total digestion times, minimize the sample handling, improve the digestion yields, as well as to stabilize the enzyme, avoid its autoproteolysis and simplify its recovery making it reusable. In this thesis, we have investigated on-line immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (IMER-CE-MS) using microreactors packed with immobilized trypsin particles.

Determination of the retention factor of ionized compounds in microemulsion electrokinetic chromatography requires two mobility measurements at the same pH: one in the presence of the microemulsion and another in plain buffer. However, it has been observed that in some cases subtracting one mobility from another determined in a different medium leads to negative retention factors, which makes no sense from a chemical point of view. This indicates that there is some error in the process which has a direct impact when retention factors are used for further applications. Here, we evaluate how the components of the microemulsion confer different properties to the buffer medium, particularly varying the viscosity parameter (which is inversely related to mobility). Whereas sodium dodecyl sulfate, the surfactant used in the microemulsion, has little effect on the medium viscosity (only an increase of 5%-6%), the presence of 1-butanol, used as a stabilizer, increases it by around 30%. Meanwhile, heptane, which is used as an oil, provokes a slight decrease. Consequently, the mobilities obtained in the microemulsion system are shifted to higher values (less negative mobilities) compared to mobilities obtained in the aqueous buffer, and so one cannot be directly subtracted from the other. Since the microemulsion-buffer medium cannot be directly reproduced, we propose a correction that takes into account the variation of viscosities. This is determined from the electrophoretic mobility of the benzoate ion. As this ion does not interact with the microemulsion, the ratio of its mobilities (measured in plain buffer and microemulsion) is equivalent to the ratio of viscosities, and can be used as the correction factor for other measurements. Thus, mobilities in buffer and microemulsion media are placed on the same scale, overcoming the errors in retention factor determination.

In this paper we extend the use of the quality criterion t' to optimize separations in capillary electrophoresis (CE). The theoretical parameter t' takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T', for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization. Here, we demonstrate the applicability of T' using as a variable the electrophoretic mobility (m(e)) for the optimization of pH in the separation of a mixture of amyloid beta (A beta) peptide fragments. In addition, it is shown the versatility of T' using other variables related to m(e), which do not require experimental measurements. This is the case with ionizable compounds as the A beta peptide fragments, whose charge-to-mass ratios can be calculated if accurate pK(a) values are available in the literature. The excellent performance of T' for A beta peptide fragments is further validated optimizing the pH for the separation of mixtures of harmala alkaloids (HAlks) and quinolone antibiotics.

The analysis and detection of targeted peptides has received due attention in many fields of proteomics research, such as discovery of biomarkers. In this regard, capillary electrophoresis-mass spectrometry (CE-MS) is widely used, however, direct analysis of low-abundance peptides such as histidine-containing peptides (His-peptides) in complex matrixes by CE-MS remains an analytical challenge. In the present study, an immobilised metal affinity solid-phase extraction containing Ni(II) coupled on-line to capillary electrophoresis-mass spectrometry (IMA-SPE-CE-MS) method has been developed to selectively enrich His-peptides from protein tryptic digests and enhance sensitivity. The method was optimised with α-casein and validated with other standard proteins (β-casein and κ-casein). Later, it was applied to an Escherichia Coli (E. Coli) whole cell lysate. IMA-SPE-CE-MS was very selective and allowed an enrichment factor up to 100-fold. The on-line enrichment and separation method coupled to MS detection is straightforward and advantageous over off-line pretreatment methods in terms of simplicity, cost-effectiveness and throughput.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2019, Tutor: Fernando Benavente, MicroRNAs (miRNA) are a class of small endogenous RNA molecules of about 19-23 nucleotides that act as regulators of genes. Aberrant expression of these oligonucleotides has been associated with a plethora of diseases ranging from nervous system diseases, heart diseases, inherited diseases and different types of cancer. The increasing association between miRNA malfunction and diseases coupled with the advances in detection and characterization of these kinds of analytes have turned miRNAs into an interesting topic of study, especially within the molecular biology field. Due to their stability towards pH, temperature and other factors, applications of microRNAs in forensic studies have also been studied. In this work we have optimized a capillary electrophoresis with ultraviolet detection method (CE-UV) that allows separating a mixture of synthetic miRNAs with similar nucleotide sequence and length. Compatibility with on-line mass spectrometry detection was also considered during the optimization. The method was validated in terms of repeatability of migration times and peak heights, linearity and limits of detection (LOD). For characterization of miRNAs matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used, where mass accuracy and other quality parameters were evaluated and compared between different modes. Better LOD than the CE-UV method and good mass accuracy were achieved using one of the miRNAs as an internal standard for the calibration, though loss of repeatability of peak area and height was observed, making this method unsuitable for quantitative analysis. Sequencing of microRNAs with the tandem mass spectrometry (MS/MS) mode was discarded due to instrument limitations

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9–11% and 6–11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L−1 and 10–50 mg L−1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.

Los factores más importantes a tener en cuenta cuando se desarrollan nuevas técnicas y métodos de separación son la simplificación, rapidez y coste de los análisis, la reducción de la cantidad de reactivos y muestra necesarias, la mejora de la selectividad, eficacia y resolución de las separaciones, la disminución de los límites de detección y la reproducibilidad de los resultados. En este contexto han ido madurando en los últimos tiempos las técnicas microseparativas, implementándose en microchips o dispositivos microfluídicos o en tubos y columnas capilares [1-6]. Estas técnicas de separación en microescala tienen un gran potencial, porque permiten cumplir prácticamente con todos los requisitos anteriormente indicados. Sin embargo, a menudo los límites de detección (LODs) en unidades de concentración no son lo suficientemente bajos debido a los pequeños volúmenes de muestra necesarios para los análisis con estas técnicas [7-11]. Esto, unido a la siempre cuestionada reproducibilidad, han sido los principales impedimentos para se apliquen ampliamente a muestras complejas reales.

In this paper, an on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-Sp) and hsa-let-7g-Sp (let-7g-Sp). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the micro cartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol.L-1, and the limit of detection (LOD) was around 10 nmol.L-1 (50 times lower than by CE-MS). In order to analyze human serum samples, an offline sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-Sp and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

Filiació URV: SI Nowadays, so-called drugs of abuse (DOAs) are widely used and pose an undeniable problem at a social level. These compounds have incalculable consequences for society, such as the cost of medical treatment, higher incidence of criminality and economic repercussions. Innovative, modern separation and determination techniques are therefore required for analysis of DOAs. In this work, we review methods based on capillary electrophoresis applied to the determination of illicit drugs and new psychoactive substances in different kinds of matrix, published between 2007 and 2015. We critically discuss the main strengths and weaknesses of all of these approaches by means of relevant applications in clinical practice, forensics, pharmacokinetics, metabolic monitoring and environmental analyses.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2018, Tutors: Martí Rosés Pascual, Elisabet Fuguet Jordà, The aim of this work is to characterize a tetradecytrimethylammonium bromide (surfactant), heptane (oil) and butan-1-ol (co-surfactant) microemulsion by microemulsion electrokinetic chromatography (MEEKC) through the solvation parameter model. This model is especially useful to describe the distribution of neutral solutes between two phases (the aqueous phase and the microemulsion). To do that, 69 neutral compounds with representative enough properties were analysed. The solvation parameter model is based on the linear free energy relationships (LFERs), which can be written as: log k = c + eE + sS + aA + bB + vV (3) where k is the MEEKC retention factor and E, S, A, B and V are the Abraham solute descriptors. The coefficients of the system (e, s, a, b and v) can be obtained by multiple linear regression and provide the properties of the studied microemulsion system. Once the coefficients of the system are determined, the studied system can be compared with other systems with known coefficients. In this work, the TTAB MEEKC system was correlated with octanol-water partition and SDS MEEKC systems. The coefficients are similar enough, thus the TTAB MEEKC system can be used to emulate the octanol-water partition. The octanol-water partition emulation is especially useful as it is a measure of the lipophilicity of compounds, which plays an important role in drug discovery and development

Previous studies have shown that a microemulsion electrokinetic chromatography (MEEKC) system can estimate the logarithm of the octanol-water partition coefficient (log Po/w) of neutral solutes. In the present work, the applicability of the method to partially and fully ionized acids has been evaluated. Naproxen, a monoprotic acid, has been used as test solute. The retention factor (k) of this compound has been measured in MEEKC at several values of pH and the retention factor-pH profile has been established. As log Po/w correlates with log kMEEKC for neutral compounds, this correlation has been used to estimate the logarithm of the octanol-water partition coefficient of the neutral (log Po/w(HA)), and the fully ionized (log P o/w(A-)) forms of naproxen. Then, the logarithm of the octanol-water distribution coefficient (log Do/w) of the partially ionized form of the acid has been estimated. The comparison of the estimated values with the ones obtained experimentally using the classical procedures, such as the shake-flask method, shows differences under 0.4 log Do/w units either if the acid is partially ionized or in its neutral form in the most part of the pH range. However, the method overestimates the log Do/w of the highly (>99.5 %) or fully ionized form of naproxen.

In the present study a pressure-assisted MEEKC method with reversed-polarity using a conventional CE instrument with UV detection and uncoated fused silica capillaries is validated as a high-throughput methodology for the lipophilicity determination of the neutral species of acidic compounds (pKa > 3.5). After the calibration of the system with four standard compounds of known log Po/w, mass distribution ratios (log kMEEKC) of new molecules can be directly converted into log Po/w values by means of a simple linear equation (log Po/w=a·log kMEEKC+b). The method was internally and externally validated for a log Po/w range between -1.54 and 4.75, with higher accuracies than conventional liquid chromatographic methods.

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra. Twenty-two compounds were tentatively identified as antihypertensive bovine casein fragments through a mass search in a database of bioactive peptides. The identity of 17 antihypertensive peptides was reliably confirmed by capillary electrophoresis-tandem mass spectrometry. The proposed analytical approach demonstrated the potential to obtain a characteristic and activity-related fingerprint for quality control and authentication of the antihypertensive nutraceutical.

[spa] Esta tesis doctoral se ha centrado en el desarrollo de metodologías analíticas adecuadas que permitan la separación, detección, caracterización e identificación de proteínas y metabolitos como biomarcadores de diferentes enfermedades o procesos patológicos. Concretamente, se ha centrado en el análisis de biomarcadores proteicos tales como la transtiretina (TTR), una proteína oligomérica con una gran tendencia a formar agregados amiloides y relacionada con la polineuropatía amiloidótica familiar tipo I (FAP-I), y el antígeno carcinoembrionario humano (CEA), una glicoproteína biomarcadora del cáncer de colon y de la metástasis de hígado (derivada del cáncer de colon). También se ha hecho especial énfasis en el estudio de biomarcadores metabólicos relacionados con la progresión de la enfermedad del Huntington, una enfermedad neurodegenerativa hereditaria caracterizada también por la formación de agregados amiloides. La dificultad de las metodologías empleadas para el análisis de biomarcadores proteicos y metabólicos reside principalmente en el tratamiento de muestra que se tiene que llevar a cabo para aislar y preconcentrar los compuestos de interés presentes en muestras biológicas complejas (tales como suero y plasma) y en la utilización de técnicas de separación y detección adecuadas que permitan la correcta caracterización de dichos biomarcadores. Por este motivo, se han empleado diferentes estrategias para el análisis dirigido (targeted analysis) y no dirigido (untargeted analysis, global profiling) de biomarcadores proteicos y metabólicos, así como para el análisis de proteínas mediante estrategias de top-down y bottom-up, cada una con sus ventajas e inconvenientes. También se han evaluado diferentes técnicas de separación de alta resolución miniaturizadas como la electroforesis capilar (CE), la cromatografía de líquidos capilar (CapLC) y la movilidad iónica (IM), todas ellas acopladas a la espectrometría de masas (CE-MS, CapLC- MS y IM-MS, respectivamente). En resumen, esta tesis doctoral presenta una visión global de las estrategias analíticas más utilizadas actualmente para el estudio de biomarcadores en el campo de la investigación proteómica y metabolómica, con el objetivo de poder diagnosticar, predecir un debut temprano y proponer nuevas dianas terapéuticas a diferentes enfermedades y procesos patológicos., [eng] The present thesis is focused on the separation, detection and characterization of proteins, glycoproteins and metabolites in biological fluids that may be able to act as disease biomarkers. Specifically, it addresses the analysis of amyloidotic neurodegenerative and cancer biomarkers from different proteomics and metabolomics perspectives, depending on the analytical and the sample preparation strategies employed: (i) targeted or untargeted strategies and (ii) top-down or bottom-up strategies (only in the case of proteomics analysis). Furthermore, different microscale separation techniques, such as capillary electrophoresis (CE), capillary liquid chromatography (CapLC) and ion mobility spectrometry (IMS), all of them hyphenated to mass spectrometry (CE-MS, CapLC-MS and IM-MS, respectively) are evaluated for the analysis of different proteins and metabolites of interest. Transthyretin (TTR) is a homotetrameric protein known to misfold and aggregate causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 of the monomeric sequence (Met30). In this thesis, we describe different off-line and on-line immunoprecipitation (IP) procedures under denaturing and non-denaturing conditions for the isolation of TTR from healthy controls and FAP-I human serum samples prior analysis by CE- MS, CapLC-MS and IM-MS. The obtained results permit us to propose complementary and reliable methods to screen, diagnose, and follow-up patients with suspected TTR amyloidosis, as well as to gain a novel insight into the structural changes on TTR proteoforms or oligomers to understand the mechanism underlying FAP-I. Huntington’s disease (HD) is also subject of study of this thesis. HD is an inherited neurodegenerative disorder caused by an expansion of the cytosine-adenine-guanine (CAG) repeat in the exon 1 of the huntingtin gene (HTT), which encodes a stretch of glutamines in the huntingtin protein that make it prone to form amyloid fibrils. However, the mechanisms involving aggregation onset or progression are not fully understand yet. Therefore, studies targeting metabolism as an alternative to the huntingtin protein are regarded as necessary. The present thesis describes the optimization of a plasma pretreatment for optimized recovery of low molecular mass compounds prior to on-line solid-phase extraction capillary electrophoresis mass spectrometry with a C18 chromatographic sorbent (C18-SPE-CE-MS), a novel sensitive method for on-line sample clean-up and concentration enhancement in metabolomics studies. Moreover, it presents the capacity of the C18-SPE-CE-MS methodology to analyze low molecular mass compounds in plasma samples from wild-type (wt) and HD mice in combination with advanced chemometric data analysis tools, such as multivariate curve resolution alternating least squares (MCR-ALS), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), for the identification of potential metabolite biomarker candidates involved in HD progression, which could provide a window of opportunity for prediction of disease onset, evaluation of HD early progression and response to treatment. Colorectal cancer (CRC) and liver metastatic disease are also studied in this thesis. Specifically, human carcinoembryonic antigen (CEA) is a highly N-glycosylated protein (60% m/m of glycans and 28 N-linked glycosylation sites) found in normal human colonic epithelial cells, as well as in tumor forming CRC cell lines. Despite the demonstrated usefulness of CEA as a biomarker for monitoring CRC and liver metastatic disease patients’ therapy, improvement in the selectivity of CEA detection remains a challenge for clinical tumor diagnosis. This thesis describes a multienzyme sheathless capillary electrophoresis tandem mass spectrometry (CE- MS/MS) strategy for the N-glycosylation analysis of CEA samples purified from human colon carcinoma and human liver metastases. The obtained results permit us to propose novel potential N-glycopeptide markers, which could provide important biologic information on how N-glycosylation may influence CEA structure in CRC and liver metastatic disease.

A field amplified sample injection-capillary zone electrophoresis (FASI-CZE) method for the analysis of benzophenone (BP) UV-filters in environmental water samples was developed, allowing the separation of all compounds in less than 8 minutes. A 9- to 25-fold sensitive enhancement was obtained with FASI-CZE, achieving limits of detection down to 21-59 µg/L for most of the analyzed BPs, with acceptable run-to-run and day-to-day precisions (relative standard deviations lower than 17%). In order to remove water sample salinity and to enhance FASI sensitivity, an off-line solid-phase extraction (SPE) procedure using a Strata X polymeric reversed-phase sorbent was proposed, obtaining recoveries up to 72-90% for most of benzophenones. With the combination of off-line SPE and FASI-CZE, limits of detection in the range 0.06-0.6 µg/L in a river water matrix, representing a 2400- to 6500-fold enhancement, were obtained. Method performance was evaluated by quantifying a blank river water sample spiked at 1 µg/L. For a 95% confidence level, no statistical differences were observed between found concentrations and spiked concentrations (probability at the confidence level, p value, of 0.60), showing that the proposed off-line SPE-FASI-CZE method is suitable for the analysis of benzophenone UV-filters in environmental water samples at low µg/L levels. The method was successfully applied to the analysis of BPs in river water samples collected before and after industrialized and urban areas, and in some drinking water samples.

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest., This work was supported by the Spanish Ministerio de Economía y Competitividad (Grant Nos. BIO 2010–16922, BIO2015-66356-R and 2015-66356-R, CTQ 2013-43236-R and CDTI Grant No. IDI20130186) and by the Generalitat de Catalunya, Spain (Grant No. 2014 SGR 229). N. F.-G. acknowledges the Ph.D. JAE-pre grant from CSIC co-financed by the European Social Fund. The collection of prebiopsy urines was made possible by grant funding from the Prostate Cancer Foundation (Young Investigator Award, A. P.) and Movember (GAP1 award), along with support from the HRB-funded Dublin Centre for Clinical Research Network, which assisted with patient recruitment, follow-up, and sample collection

In this paper we describe a strategy to estimate by CE the acidity constants (pKa) of complex polyprotic peptides from their building peptide fragments. CE has been used for the determination of the pKas of five short polyprotic peptides that cover all the sequence of amyloid beta (Aβ) peptides 1-40 and 1-42 (Aβ fragments 1-15, 10-20, 20-29, 25-35 and 33-42). First, the electrophoretic mobility (me) was measured as a function of pH of the background electrolyte (BGE) in the pH range 2-12 (bare fused silica capillary, I=25mM and T=25ºC). Second, the mes were fitted to equations modelling the ionisable behaviour of the different fragments as a function of pH to determine their pKas. The accuracy of the pKas was demonstrated predicting the electrophoretic behaviour of the studied fragments using the classical semiempirical relationships between me and peptide charge-to-mass ratio (me vs. q/Mr1/2, classical polymer model, q=charge and Mr=relative molecular mass). Separation selectivity in a mixture of the fragments as a function of pH was evaluated, taking into account the influence of the EOF at each pH value, and a method for the simple and rapid simulation of the electropherograms at the optimum separation pH was described. Finally, the pKas of the fragments were used to estimate the pKas of the Aβ peptides 1-40 and 1-42 (tC and D 3.1, E 4.6 and Y 10.8 for acidic amino acids and tN-D 8.6, H 6.0, K 10.6 and R 12.5 for basic amino acids), which were used to predict their behaviour and simulate their electropherograms with excellent results. However, as expected due to the very small differences on q/Mr1/2 values, separation resolution of their mixtures was poor over the whole pH range. The use of poly(vinyl alcohol) (PVA) coated capillaries allowed reducing the electroosmotic flow (EOF) and a slight improvement of resolution.

In the present work, several MEEKC systems are studied to assess their suitability for lipophilicity determination of acidic, neutral, and basic compounds. Thus, several microemulsion compositions over a wide range of pH values (from 2.0 to 12.0), containing heptane, 1-butanol and different types and amounts of surfactant (SDS or sodium cholate: from 1.3 to 3.3%) are characterized using Abraham's solvation model. The addition of acetonitrile (up to 10%) is also studied, since it increases the resolution of the technique for the most lipophilic compounds. The system coefficients obtained are very similar to those of the 1-octanol/water, used as the reference lipophilicity index, allowing simple and linear correlations between the 1-octanol/water partition values (log Po/w ) and MEEKC mass distribution ratios (log kMEEKC ). Variations in the microemulsion composition (aqueous buffer, surfactant, concentration of ACN) did not significantly affect the similarity of the MEEKC systems to log Po/w partition.

Filiació URV: SI A suitable method has been developed and validated for simultaneously determining cocaine and its major metabolite, benzoylecgonine, 6-acetylmorphine, codeine, morphine and the methadone enantiomers in human hair samples by the in-line coupling between SPE and CyD-assisted CE with a previous sample pretreatment procedure based on pressurized liquid extraction. Optimal separation was achieved on a fused silica-capillary of 50 μm i.d. and 80 cm total length using 11 mM α-CyD in an aqueous solution of 80 mM sodium phosphate at pH 2.5 as the separation medium and an applied voltage of 30 kV. The SPE-CE device consisted of a short length of capillary packed with Oasis HLB sorbent, which was inserted near to the inlet end of the CE capillary. Several parameters affecting the in-line preconcentration were evaluated. The LOQs reached for hair samples were in the range of 0.3–2.5 ng/mg with satisfactory analytical precision in both intraday and day-to-day experiments (RSDs

Toxicity has been emulated in tadpole species through chromatographic systems. The parameter studied to evaluate the non-specific toxicity of a compound is the narcosis concentration (Cnar), which is defined as the concentration needed for the immobilization of the organism. Because experimental investigation with animals is lengthy, costly, technically difficult, and ethically questionable, there is a great interest in developing surrogate physicochemical systems able to emulate biological systems to obtain the same information in a faster, more economic, and easier manner. In order to see which chromatographic systems would be able to emulate tadpole narcosis, both, tadpole narcosis data and data in several chromatographic and electrophoretic systems, were fitted to a linear solvation energy relationship (LSER) model. Thus, by comparison of the models it was possible to see which of the chromatographic systems were more similar to the biological one. The physicochemical systems that best emulate tadpole narcosis were an HPLC system based on an immobilized artificial membrane (IAM) column, and two micellar electrokinetic chromatography (MEKC) systems based on sodium taurocholate (STC) and a mixture of sodium dodecylsulphate (SDS) and Brij 35 as surfactants. A system based on a RP18 HPLC column also was selected for comparison because it is a common column in most analytical laboratories. To establish the models, a set of compounds with known Cnar values were analyzed in the chromatographic, and electrophoretic selected systems and, then, the retention factor (k) was correlated to the concentration of narcosis. Statistics showed that the system based on STC micelles was the best to emulate toxicity in tadpoles. The robustness and predictive ability of the developed models were validated.

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

[cat] Aquesta Tesi es titula Determinació de constants d’acidesa de substàncies d’interès biològic per electroforesi capil·lar. La Dissertació es focalitza llavors en l’estudi de la determinació de la constant d’acidesa de fàrmacs utilitzant aquesta tècnica d’electroseparació, l’electroforesi capil·lar. La constant d’acidesa o pKa és una propietat fisicoquímica útil en el descobriment i desenvolupament de fàrmacs, on el coneixement de l’estat de ionització d’un grup funcional és sovint essencial per a comprendre les propietats farmacocinètiques i farmacodinàmiques de nous fàrmacs potencials. Gràcies a l’ús d’un patró intern (IS), es va desenvolupar una nova metodologia per a la determinació d’aquestes constants per electroforesi capil·lar (CE), el mètode IS-CE. No obstant això, aquest mètode només es va desenvolupar per a substàncies simples monopròtiques. Així doncs, l’objectiu principal d’aquesta Tesi és establir el mètode IS-CE com a mètode d’alt rendiment per a la determinació de constants d’acidesa de compostos bioactius, així com també dissenyar un prototip específic per aquest mètode per tal d’implementar-lo per a l’anàlisi rutinari de fàrmacs potencials en laboratoris d’anàlisis fisicoquímiques. Aquesta fita ha estat perseguida al llarg d’aquesta Dissertació, cobrint durant el seu desenvolupament cada necessitat que presentava el mètode. Per començar, en aquesta Tesi s’estableix el pKa d’un llistat de patrons interns àcids i bàsics de diferent estructura que cobreixi tot l’interval de pH útil en electroforesi capil·lar i s’amplia l’aplicabilitat del mètode a compostos amb diverses constants d’acidesa. Posteriorment s’optimitzen els paràmetres instrumentals per tal de convertir l’IS-CE en un mètode high throughput IS-CE. Després s’avalua la capacitat del mètode per a la determinació de compostos de baixa solubilitat en aigua i s’amplia per a la determinació de constants d’acidesa de compostos de molt baixa solubilitat utilitzant mescles metanol-aigua. Arribat aquest punt es dissenya una instrumentació específica adaptable al mètode IS-CE, tot simplificant i agilitzant el procés per a la determinació del pKa. En conjunt, aquest treball d’investigació científica ha permès el desenvolupament consolidat d’un mètode ràpid, robust, precís i acurat per a la determinació de constants d’acidesa de compostos bioactius. Així mateix, també ha permès dissenyar un nou prototip per a la implementació d’aquest mètode a l’anàlisi rutinari de fàrmacs potencials en laboratoris de mesures fisicoquímiques i de drug discovery and development., [eng] The Dissertation is then focused on the study of the acidity constant determination of drugs using this electroseparation technique, capillary electrophoresis. The acidity constant or pKa is a useful physicochemical property in drug discovery and development, where the knowledge of the ionization state of a functional group is often essential to understand the pharmacodynamic and pharmacokinetic properties of new potential drugs. In the past, a new method to determine acidity constants by capillary electrophoresis (CE) was developed by the use of an internal standard (IS), the IS-CE method. However, this method was only developed for simple monoprotic compounds. Thus, the main objective of this Thesis is to establish the IS-CE method as a high throughput method for the determination of acidity constants of bioactive compounds as well as to design a specific prototype for this method in order to implement it for the routine analysis of potential drugs in physicochemical analysis laboratories. This goal has been pursued throughout this Dissertation in order to meet the need during the development of this method. First of all, this Thesis establishes the pKa of a list of acidic and basic internal standards with different structures that covers the entire pH-range useful in capillary electrophoresis, and expands the applicability of this method to compounds with several acidity constants. Afterwards, instrumental parameters and experimental conditions are optimized in order to transform the IS-CE method into a high throughput method. Subsequently, the method is evaluated for sparingly soluble compounds in water and extended for the determination of acidity constants of highly insoluble compounds using methanol-water mixtures. At this point a specific instrumentation is designed and adapted to the IS-CE method, simplifying and accelerating the pKa determination process. Overall, the conducted scientific research has allowed the development of a consolidated fast, robust, precise and accurate method for the determination of acidity constants of bioactive compounds. Likewise, a new prototype has been designed for the implementation of this method to the analysis of potential drugs in routine laboratory of physicochemical measurements and drug discovery and development.

In this work, the electrophoretic behavior of hydrophobic fullerenes [buckminsterfullerene (C-60), C-70, and N-methyl-fulleropyrrolidine (C-60-pyrr)] and water-soluble fullerenes [fullerol (C-60(OH)(24)); polyhydroxy small gap fullerene, hydrated (C-120(OH)(30)); C-60 pyrrolidine tris acid (C-60-pyrr tris acid); and (1,2-methanofullerene C-60)-61-carboxylic acid (C60CHCOOH)] in micellar electrokinetic capillary chromatography (MECC) was evaluated. The aggregation behavior of the water-soluble compounds in MECC at different buffer and sodium dodecyl sulfate (SDS) concentrations and pH values of the background electrolyte (BGE) was studied by monitoring the changes observed in the electrophoretic pattern of the peaks. Broad and distorted peaks that can be attributed to fullerene aggregation were obtained in MECC which became narrower and more symmetric by working at low buffer and SDS concentrations (below the critical micelle concentration, capillary zone electrophoresis (CZE) conditions). For the characterization of the suspected aggregates formed (size and shape), asymmetrical flow field-flow fractionation (AF4) and transmission electron microscopy (TEM) were used. The results showed that the increase in the buffer concentration promoted the aggregation of the particles, while the presence of SDS micelles revealed multiple peaks corresponding to particles of different aggregation degrees. Furthermore, MECC has been applied for the first time for the analysis of C-60 in two different cosmetic products (i.e., anti-aging serum and facial mask).

[eng] Fullerenes or Buckyballs are a group of carbon nanoparticles, classified as engineered nanoparticles. The advent of fullerene nanoparticles in commercial, industrial and biomedical applications raises concern about their potential ecological and human health risks. Fullerenes have not been regulated, although the European Commission (European Commission, 2002), the European Parliament (European Parliament, 2008) and the US Environmental Protection Agency (EPA, 2015) have prioritised legislation on nanomaterials handling and disposal. Given the current lack of studies regarding their presence, fate and behaviour in consumer products and environmental matrices and their associated human and environmental risks it is becoming increasingly important to be able to characterise and quantitate fullerene nanoparticles, especially derivatives, in a wide range of matrix types. To fill these knowledge gaps, one of the objectives of this thesis was the development of analytical methodologies for the determination of pristine and surface modified fullerenes by ultra-high performance liquid chromatography coupled to MS (UHPLC-MS) and by nonaqueous capillary electrophoresis (NACE) and micellar electrokinetic capillary chromatography (MECC) with UV-Vis detection in different environmental matrices (water and sediment samples) and cosmetic products, respectively. In addition, in this thesis the size and shape characterisation of surface modified fullerenes in aqueous solutions of different pH and ionic strength values was studied by combining several techniques (CE, AF4-MALS and TEM). Regarding the analysis of fullerenes, the use of a sub-2 µm C18 column, toluene-methanol as a mobile phase and of APPI ionisation source allowed to develop a UHPLC method coupled to MS/(MS) for the analysis of five pristine (C60-C84) and three surface modified fullerenes (PCBM, PCBB and C60-pyrr) in less than 4.5 min showing high sensitivity and selectivity. Furthermore, the employment of H-SIM mode for pristine fullerenes (mass resolving power >12,500 FWHM), and SRM mode for fullerene derivatives, allowed achieving MLODs lower than most of those previously reported in the literature. For the extraction of the water samples, liquid-liquid extraction (LLE) with toluene and the addition of salt is proposed obtaining recoveries higher than 83 %. For sediments we propose the use of pressurised liquid extraction (PLE) performed at a high extraction temperature (150 °C) using one extraction cycle of 10 min achieving good recoveries (70-92 %). The developed methodology allowed us to report for the first time the presence of C60-pyrr, PCBM and PCBB in sediments (2.0 - 8.5 ng Kg-1 levels) and of PCBM and PCBB in pond water samples (0.1- 5.1 pg L-1 levels). Two CE methods, a non-aqueous (NACE) and a micellar (MECC) method have been developed. LOQs at mg L-1 levels were obtained with both methods making possible their application for the analysis of cosmetic products. Both methods (NACE and MECC) were applied for the quantitation of C60 in cosmetic products and the results are comparable to those obtained by LC-MS (in an anti-aging serum). With respect to the characterisation of fullerene aggregates, the use of AF4-MALS demonstrated that fullerene aqueous solutions contain particles of different aggregation degree in agreement with TEM micrographs. The size determination of the studied compounds by AF4 at different salt concentrations demonstrated that the enhanced aggregation of fullerenes with the increase in the ionic strength could explain the broad, multiple and distorted peaks obtained in MECC which were more obvious at high buffer concentration. The hydrodynamic radii of polyhydroxy-fullerenes increased more than 5 times and those of the carboxy-fullerene derivatives up to 180 nm (3rd peak) (for C60-pyrr tris acid) for 0.1M NaCl. The propensity of fullerenes to aggregate in both aqueous solutions and organic solvent mixtures justify the electrophoretic peak profiles observed in MECC (i.e., broad peaks at high electrolyte concentration) and their higher retention in C18 columns using toluene-acetonitrile compared to toluene-methanol mobile phases (due to the formation of bigger aggregates), respectively., [spa] Los fulerenos son cada vez más utilizados en medicina (administración de fármacos), en procesos ambientales (remediación) y en la industria (células solares) debido a sus propiedades estructurales y electrónicas únicas. En este contexto, es importante aumentar el conocimiento actual con respecto a las características, el comportamiento, el destino y la toxicidad de los fulerenos, una nueva clase de contaminantes emergentes orgánicos. Por lo tanto, es cada vez es más importante ser capaz de caracterizar y cuantificar las nanopartículas de fulerenos en una amplia gama de tipos de matriz y para este fin, se necesita el desarrollo de métodos analíticos para su análisis cuantitativo y cualitativo. Además de la determinación de las concentraciones de fulerenos en matrices complejas, se requiere su caracterización en términos de grado de agregación, distribución del tamaño y morfología de la superficie en soluciones acuosas de diferentes características (fuerza iónica y pH) con el fin de proporcionar herramientas para establecer su riesgo. En esta tesis se han establecido metodologías para el análisis de ocho fulerenos en muestras ambientales (agua y sedimentos) por cromatografía líquida acoplada a espectrometría de masas y se ha detectado por la primera vez la presencia de tres derivados de fulerenos. Además, se ha desarollado metodología para el análisis de fulerenos hidrofobicos y solubles en agua mediante la electroforesis capilar en medio no-acuoso (NACE) y la cromatografía capilar electrocinética micelar (MECC) y los metodos se han aplicado para la determinación de C60 en productos cosmeticos. Por último, se ha estudiado el comportamiento de agregación de los fulerenos en soluciones acuosas de diferentes características (pHs, fuerzas iónicas) mediante diferentes técnicas (electroforesis capilar (CE), asymetrical flow-field flow fractionation (AF4), microscopia electrónica de transmisión (TEM)). En este contexto se han determinado los tamaños de los agregados formados en las condiciones evaluadas, y la morfología de las partículas.

Capillary zone electrophoresis (CZE) and high performance liquid chromatography (HPLC) were applied to the authentication of fruit products based on the compositional profiles of polyphenols. Various sample treatments were used to maximize the overall recovery of polyphenols or specific fractions, such as phenolic acids or anthocyanins. The resulting CZE and HPLC data were treated with Principal Component Analysis (PCA) showing that samples were mainly clustered according to the fruit of origin, with cranberry- and grape-based products clearly separated in groups. A possible adulterated cranberry extract was analyzed more deeply by high resolution mass spectrometry (HRMS) in order to identify the presence of A-type proanthocyanidins, which are characteristic and more abundant in cranberry-based products. In accordance with PCA interpretation, HRMS results indicated that the suspicious sample was not a cranberry-based product, allowing us to validate and demonstrate the suitability of both CZE- and HPLC-proposed methods for the characterization of fruit-based products.

La determinació de biomarcadors peptídics en fluids biològics resulta de gran interès pel diagnòstic, la monitorització i el pronòstic de nombrosos desordres. En aquesta tesi doctoral s’han explorat diverses metodologies d’extracció en fase sòlida acoblada en línia amb l’electroforesi capil•lar amb detecció per espectrometria de masses (SPE-CE-MS) per a l’anàlisi de biomarcadors peptídics, que es troben a baixa concentració en matrius complexes. S’han estudiat principalment pèptids opiacis que estan relacionats amb desordres neurològics i síndromes de dolor crònic. S’han avaluat diverses fases estacionàries comercials per a l’anàlisi de pèptids opiacis per SPE-CE emprant preconcentradors amb frits i la fase estacionària C18 és la que ha proporcionat millors resultats per a l’anàlisi de mostres de plasma humà. La saturació del preconcentrador s’ha previngut mitjançant un tractament de mostra previ que consta de dues etapes, una precipitació amb acetonitril seguida d’una filtració per centrifugació. Com que l’etapa de filtració és crítica per tal d’obtenir les màximes recuperacions dels anàlits i eliminar la matriu de la mostra, s’han provat filtres de diversos talls moleculars i diverses condicions de filtració. L’acoblament de la SPE-CE a un espectròmetre de masses amb analitzador de temps de vol (TOF-MS) mitjançant una interfase amb líquid auxiliar convencional ha permès disminuir els LOD per a la majoria dels pèptids opiacis en mostres de plasma humà. Per tal de poder detectar concentracions inferiors, s’ha demostrat que la combinació de la SPE-CE amb la tècnica de separació electroforètica isotacoforesi transitòria (tITP) és una bona alternativa. També, s’ha comparat el comportament de preconcentradors amb i sense frits per a l’anàlisi de pèptids opiacis per C18-SPE-CE-UV i C18-SPE-CE-TOF-MS. S’ha avaluat un prototip recentment desenvolupat d’una interfase nanoelectroesprai (nanoESI) sense líquid auxiliar basada en una punta porosa per a l’anàlisi de pèptids opiacis per CE-MS i també, s’ha establert una metodologia de C18-SPE-CE-TOF-MS emprant un innovador preconcentrador sense frits compatible amb les dimensions específiques del capil•lar de separació requerit per a la interfase nanoESI sense líquid auxiliar. Una altra possibilitat per a millorar la detecció de pèptids és utilitzar fases estacionàries amb una selectivitat més elevada que el C18, com les fases estacionàries d’immunoafinitat. S’han explorat dos procediments de preparació d’aquest tipus de fases estacionàries: la immobilització d’anticossos intactes oxidats (immunoglobulina G, IgG) a través dels carbohidrats a partícules de sílice activades amb grups hidrazida i la immobilització de fragments d’IgG (fragment d’unió a l’antigen, Fab’) a partícules de sílice activades amb grups succinimidil. Seguint els procediments establerts, s’ha preparat una fase estacionària amb IgG i una altra amb Fab’ contra les endomorfines 1 i 2 (End1 i End2). S’han optimitzat i avaluat metodologies d’IA-SPE-CE-TOF-MS per a l’anàlisi de mescles de patrons d’End1 i d’End2 i s’han analitzat mostres de plasma humà. També, s’ha explorat l’aplicabilitat d’una fase estacionària d’immunoafinitat amb IgG per a l’anàlisi de biomolècules d’elevat pes molecular per IA-SPE-CE-TOF-MS emprant la glicoproteïna transferrina com a model., The determination of peptide biomarkers in biological fluids has become crucial for the diagnosis, monitoring and prognosis of numerous disorders. This doctoral thesis explores several on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) methodologies for the analysis of peptide biomarkers that are found at low concentration in complex matrices, such as opioid peptides. Several commercial sorbents have been compared for the analysis of opioid peptides by SPE-CE using microcartridges with frits and the C18 sorbent has provided the best results for the analysis of human plasma samples. The saturation of the on-line SPE microcartridge has been prevented by a double-step sample clean-up pretreatment that consists of precipitation with acetonitrile and centrifugal filtration. The coupling of SPE-CE with a time-of-flight mass spectrometer (TOF-MS) by a conventional sheathflow interface has provided better LODs. In order to detect lower concentrations, the combination of SPE-CE with the electrophoretic preconcentration technique transient isotachophoresis (tITP) has demonstrated to be a good alternative. The performances of frit and fritless microcartridges have been compared for the analysis of opioid peptides by C18-SPE-CE-UV and C18-SPE-CE-TOF-MS. A recently developed sheathless nanoelectroespray (nanoESI) interface based on a porous tip has also been evaluated for the analysis of opioid peptides by CE-TOF-MS and a C18-SPE-CE-TOF-MS using a novel fritless microcartridge. Finally, two different procedures for the preparation of immunoaffinity sorbents (IA) have been explored: the immobilization of intact antibodies (immunoglobulin G, IgG) or IgG fragments (antigen binding fragment, Fab’) to silica particles. Two IA sorbents against Endomorphins 1 and 2 have been prepared following the established procedures. IA-SPE-CE-TOF-MS methodologies have been optimized and evaluated for the analysis of standards solutions of End1 and End2 and human plasma samples. The suitability of an immunoaffinity sorbent with IgG has also been explored for the analysis of large biomolecules by IA-SPE-CE-TOF-MS using the glycoprotein transferrin as a model.