In this work, a new purpose-made portable capillary electrophoresis instrument with a contactless conductivity detector was used for the determination of degradation products of nitrogen mustards in different water samples. The capillary was coated with poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate) to avoid analyte-wall interactions. The coating procedure was studied to obtain the best repeatability of the migration time of the analytes. Four different coating procedures were compared; flushing the capillary with the co-polymer at 100 psi for 2 min at 60°C provided the best RSD values (<4%). The analytical method was also optimized. The use of 20 mM of MES adjusted to pH 6.0 with His as running buffer allowed a good baseline separation of the three analytes in different water samples without matrix interferences. The method permitted the detection of the three degradation products down to 5 μM.

Two flat gold electrodes are placed vis-à-vis with an epoxy spacer layer that is etched out to give a ca. 100 μm deep electrochemically active trench. A water-insoluble oil phase, here the redox system N,N-diethyl-N’N’-didodecyl-phenylenediamine (DDPD) in 4-(3-phenylpropyl)-pyridine (PPP), is immobilised into the trench to allow anion transfer upon oxidation of DDPD(oil) to DDPD⁺(oil). In “mono-potentiostatic mode” quantitative transfer/expulsion of anions into the trench oil phase occurs. However, in “bi-potentiostatic mode” feedback currents dominated by rapid plate-to-plate diffusion normal to the electrode surfaces are observed. Comparison of “normal” diffusion and “lateral” diffusion shows that the rate of diffusion-migration charge transport across the oil film is anion hydrophobicity dependent.

A simple analysis of chlorophenols (2-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) and phenol was accomplished by coupling a pressurized capillary electrochromatography (pCEC) with amperometric detection. Efficient and reproducible separation of these compounds was achieved within 9 min on a capillary monolithic stationary phase bonded with octadecyl ligands and sulfonate groups, where the selectivity and the retention of analytes can be functionally controlled by optimizing experimental variables, including organic modifier content, mobile phase pH, ionic strength, working electrode potential, separation voltage, and supplementary pressure. A mixed-mode retention mechanism consisting of reverse-phase chromatographic partition, electrostatic repulsion and electrophoresis is considered to play roles in the separation. The use of acetonitrile-based media seems effectual in preventing the unfavorable irreversible adsorption on both wall and electrode, and offer higher sensitivity and less electrode fouling in amperometric detection of phenols. The limits of detection were in the range from 0.02 to 0.2 μg mL⁻¹ with a wide linear dynamic range of 5000-fold, while the peak area precision ranged from 3.2% to 7.5%. The feasibility of using this method in real analysis was evaluated by recovery estimates and comparative experiment on spiked tap water samples. Good recoveries of 80–110% were achieved. Additionally, a paired t-test was used to correlate the two methods.

It is difficult to detect biogenic amines in biological fluids because of their very low concentrations. In this paper, we reported an on-line sample preconcentration method in capillary electrophoresis-amperometric detection (CE-AD) based on a dynamic pH junction, and a concentration enhancement of approximately 100-fold was achieved compared with the classical CE-AD methods in the simultaneous analysis of six biogenic amines in urine (dopamine, epinephrine, norepinephrine, tyramine, tryptamine, and serotonin). The technique is proposed based on the sharp pH change generated at the boundary between an acidic sample and the basic background electrolyte (BGE) zone. Under optimised conditions, all analytes were successfully focused and well-separated within 20 min with high efficiency and sensitivity (LODs at S/N = 3 ranging from 5.34 to 68.3 nM). For the analysis of urine samples by this method, satisfactory recoveries were obtained without a complicated pretreatment step or derivatisation process. Therefore, it is self-evident that this approach for the analysis of real biological samples has great potential in the future.

In this paper, we present two new methodologies of improving the performance of microchip-based electrochemical detection in microfluidic devices. The first part describes the fabrication and characterization of epoxy-embedded gold microelectrode arrays that are evenly spaced and easily modified. Electrodepositions using a gold plating solution can be performed on the electrodes to result in a 3-dimenional pillar array that, when used with microchip-based flow injection analysis, leads to an 8-fold increase in signal (when compared to a single electrode), with the limit of detection (LOD) for catechol being 4 nM. For detecting analytically challenging molecules such as nitric oxide (NO), platinization of electrodes is commonly used to increase the sensitivity. It is shown here that microchip devices containing either the pillar arrays or more traditional glassy carbon electrodes can be modified with platinum black for NO detection. In the case of using glassy carbon electrodes for NO detection, integration of the resulting platinized electrode with microchip-based flow analysis resulted in a 10 times signal increase relative to use of a bare glassy carbon electrode. In addition, it is demonstrated that these electrodes can be coated with Nafion to impart selectivity towards NO over interfering species such as nitrite. The LOD for NO when using the platinum black/Nafion-coated glassy carbon electrode was 9 nM. These electrodes can also be embedded in a polystyrene substrate, with the applicability of these sensitive and selective electrodes being demonstrated by monitoring the ATP-mediated release of NO from endothelial cells immobilized in a microfluidic network without any adhesion factor.

A new and simple strategy based on nanostructured CaCO₃-PEI MPs for phenol sensing using PDMS/glass fluidic microchip is developed. This fluidic microsystem including integrated screen-printed-electrodes (SPE) modified with CaCO₃-PEI MPs and tyrosinase (Tyr) through cross-linking with glutaraldehyde, represents a low cost platform for phenol detection. The designed fluidic microsystem improves the sensitivity of the biosensor allowing the detection of very low concentrations of phenol (up to 10 nM). This device shows high repeatability and low detection limit, is easy to be fabricated, inexpensive, disposable and amenable to mass production.

Glycomics has become a rapidly emerging field and monitoring of protein glycosylation is needed to ensure quality and consistency during production processes of biologicals such as therapeutic antibodies or vaccines. Glycoanalysis via multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) represents a powerful technique featuring high resolution, high sensitivity as well as high-throughput performance. However, sample data retrieved from this method exhibit challenges for downstream computational analysis due to inter-sample migration time shifts as well as stretching and compression of electropherograms. Here, we present glyXalign, a freely available and easy-to-use software package to automatically correct for distortions in xCGE-LIF-based glyco-data. We demonstrate its ability to outperform conventional algorithms such as DTW and COW in terms of processing time and alignment accuracy for high-resolution datasets. Built upon a set of rapid algorithms the tool includes an intuitive graphical user interface and allows full control over all parameters. Additionally, it visualizes the alignment process and enables the user to re-adjust misaligned results. Software and documentation are available at http://www.glyxera.com.

AC electroosmotic (ACEO) flow above the gap between coplanar electrodes is mapped by the measurement of Stokes forces on an optically-trapped polystyrene colloidal particle. E²-dependent forces on the probe particle are selected by amplitude modulation (AM) of the ACEO electric field (E) and lock-in detection at twice the AM frequency. E²-dependent dielectrophoresis (DEP) of the probe is eliminated by driving the ACEO at the probe's DEP crossover frequency. The location-independent DEP crossover frequency is determined, in a separate experiment, as the limiting frequency of zero horizontal force as the probe is moved toward the midpoint between the electrodes. The ACEO velocity field, uncoupled from probe DEP effects, was mapped in the region 1–9 μm above a 28 μm gap between the electrodes. By use of variously-sized probes, each at its DEP crossover frequency, the frequency dependence of the ACEO flow was determined at a point 3 μm above the electrode gap and 4 μm from an electrode tip. At this location the ACEO flow was maximal at ∼117 kHz for a low-salt solution. This optical trapping method, by eliminating DEP forces on the probe, provides unambiguous mapping of the ACEO velocity field.

In this study we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on microfluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microliters of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multivalent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within five hours using 200 μl of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMAs) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalized with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity (MFI) of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilized. The cells with reduced expression of MHC-I molecules showed after cytotoxicity tests an NK susceptibility 3.5 times higher than normal cells.

A novel open-tubular capillary electrochromatography (OT-CEC) column coated with chitosan-graft-(β-cyclodextrin) (CDCS) was prepared using sol-gel technique. In the sol-gel approach, owing to the three-dimensional network of sol-gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating isomers were shown. The column efficiencies of 55 000∼163 000 plates/m for the isomeric xanthopterin and phenoxy acid herbicides using the sol-gel-derived CDCS columns were achieved. Good stabilities were demonstrated that the RSD values for the retention time of thiourea and isoxanthopterin were 1.3% and 1.4% (n = 5), 1.6% and 2.0% (n = 3), 2.9% and 3.1% (n = 3), respectively. The sol-gel-coated CDCS columns have shown improved separations of isomeric xanthopterin in comparison with CDCS-bonded capillary column.

Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, Mitotracker Red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilisation, denaturation and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analysed by MS, resulting into the identification of 10 proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or “stainomics”, can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.

The site selective electrodeposition of silver metal onto a conducting object such as carbon microtubes (CMTs) in an electrolytic solution could be achieved by means of bipolar electrochemistry (BPE). Two half-reactions are simultaneously carried out at both extremities of the CMT which act as a bipolar electrode. The thermodynamic threshold value of the process which consists in metal electro-reduction and concomitant water oxidation is directly related to the length of CMT. That is the reason why, when scaling down the methodology to microscale objects, electric fields in the range of tenths of kilovolts per meter are necessary. In that context, a capillary electrophoresis (CE) apparatus provides a convenient experimental platform to achieve in a straightforward manner such experimental conditions. We exemplify this methodology with the efficient and quick electro-reduction of Ag⁺ on CMTs from a low-concentration aqueous electrolytic solution during the migration across a fused capillary. CE allows applying safely a large enough electric field (typically ∼ 30 kV/m) for the successful modification of 15 to 20 μm-long substrates. The corresponding hybrid materials have been characterized by optical microscopy as well as scanning electronic microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX).

A SU-8/Pyrex single-channel microchip integrating three 100 μm Pt electrodes (ME-ED) for class-selective electrochemical index determination (CSEID) of phenolic acids and flavonoids in complex extracts of Tagetes lucida (Tl), Mentha piperita (Mp), Cymbopogon citratus (Cc), Calendula officinalis (Co) and Cynara scolymus (Cs) is proposed. Under strategic conditions controlled by a MES buffer (pH 5.0; 25mM) and accordingly to the antioxidant acid-base properties, the simultaneous measurement of total acids and flavonoids indexes was achieved in less than 100s with excellent analytical performance. The reliability of ME-ED approach was demonstrated towards the high agreement between the total phenolic content obtained using microchip approach with those obtained by the well-established HPLC-DAD; revealing both identical order regarding to the total phenolic content in the target samples. In addition, further comparison of ME-ED with the traditional Folin-Ciocalteu (FC) antioxidant capacity assay, showed that ME-ED approach could become a class-selective antioxidant capacity assay revealing that the sample antioxidant capacity was decreasing as Tl>Mp>Cs>Cc>Co according to their endogenous polyphenol content. These results suggested that the microchip approach is not only a reliable method for fast assessment of class-selective antioxidants constituting a very good alternative to the long analysis times and the using of toxic solvents required in HPLC but a novel truly antioxidant capacity assay. This excellent analytical performance is connected with the key-features of the “ready-to-use” system employed in this work such as portability, full integration of electrochemical detection, easy-operation and potential ME-ED disposability.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D) was successfully applied to the investigation of performate, peracetate, and perpropionate in aqueous medium. Ionic mobilities, diffusion coefficients, and hydrodynamic radii were obtained for the first time for these species. CE-C⁴D was also used to estimate the pK_a values of the peroxycarboxylic acids. Because the peroxycarboxylates (POCs) undergoes hydrolysis while migrating, a simple calibration curve cannot be used for quantitation. Thus, an indirect calibration approach was used. The new method was used to monitor the formation of peroxycarboxylic acids from hydrogen peroxide and the carboxylic acid as well as to the quantitation of peracetic acid in a commercial sample. The CE-C⁴D method compares favorably with the conventional titration method because of the possibility of speciation of the POC, the low sample consumption, and the low LOD (14, 8, and 24 μmol/L for performate, peracetate, and perpropionate, respectively). Although POCs are structural isomers of monoalkyl carbonates (MACs), they have greater hydrodynamic radii, which suggests that the positions of the oxygen atoms in the molecules have a direct impact in the charge density and consequently on the hydration atmosphere.

The present paper describes the results related to the optical and electrochemical characterization of thin carbon films fabricated by spin coating and pyrolysis of AZ P4330-RS photoresist. The goal of this paper is to provide comprehensive information allowing for the rational the selection of the conditions to fabricate optically-transparent carbon electrodes (OTCE) with specific electro-optical properties. According to our results, these electrodes could be appropriate choices as electrochemical transducers to monitor electrophoretic separations. At the core of this manuscript is the development and critical evaluation of a new optical model to calculate the thickness of the OTCE by variable angle spectroscopic ellipsometry (VASE). Such data was complemented with topography and roughness (obtained by AFM), electrochemical properties (obtained by cyclic voltammetry), electrical properties (obtained by electrochemical impedance spectroscopy), and structural composition (obtained by Raman spectroscopy). Although the described OTCE were used as substrates to investigate the effect of electrode potential on the real-time adsorption of proteins by ellipsometry, these results could enable the development of other biosensors that can be then integrated into various CE platforms.

This paper reports a method for rapid, simple, direct, and reproducible determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA). The platform described herein uses polyester-toner (PT) microchips incorporating capacitively coupled contactless conductivity detection (C⁴D) and electrophoresis separation of the analytes. The PT microchip presented 150-μm-wide and 12-μm-deep microchannels, with injection and separation lengths of 10- and 40-mm long, respectively. The best results were obtained with 320 kHz frequency, 4.5 V_pp excitation voltage, 80 mmol L⁻¹ CHES/Tris buffer at pH 8.8, injection in −1.0 kV for 7 s, and separation in −1.5 kV. RSD values related to the peak areas for glyphosate and AMPA were 1.5 and 3.3% and 10.1 and 8.6% for intra- and inter-chip assays, respectively. The detection limits were 45.1 and 70.5 μmol L⁻¹, respectively, without any attempt of preconcentration of the analytes. Finally, the method was applied to river water samples in which glyphosate and AMPA (1.0 mmol L⁻¹ each) were added. The recovery results were 87.4% and 83.7% for glyphosate and AMPA, respectively. The recovery percentages and LOD values obtained here were similar to others reported in the literature.

A single-strand conformation polymorphism (SSCP) analysis and targeted sequencing approach was used for the genetic characterization of some major pathogens from a cohort of 227 people with histories of gastrointestinal disorders. Genomic DNAs from faecal samples were subjected to PCR-amplification of regions in the glycoprotein (gp60) or triose phosphate isomerase (tpi) gene, or the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2). Cryptosporidium, Giardia and strongylid nematodes were detected in 94, 132 and 12 samples. Cryptosporidium hominis subgenotypes IbA10G2, IdA15G1, IgA17, IgA18 and IfA13G1 were identified in 74.6, 16.9, 5.6, 1.4 and 1.4% of 71 samples, respectively. For C. parvum, subgenotypes IIaA17G2R1 (47.6%) and IIaA18G3R1 (23.8%) were identified in 23 samples. Giardia duodenalis assemblage B (78%) was more common than assemblage A (22%). In addition, DNA of the nematodes Ancylostoma ceylanicum (n = 2), Ancylostoma duodenale (4), Necator americanus (5) and Haemonchus contortus (1) was specifically detected. This is the first report of A. ceylanicum in two persons in Australia and, we provide molecular evidence of H. contortus in a child. This SSCP-based approach should provide a useful diagnostic and analytical tool for a wide range of pathogens.

A new type of polymethacrylate based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely D,L-phenylalanine, D,L-tyrosine and D,L-tryptophan by capillary electrochromatography (CEC). The monolithic column was prepared by in-situ polymerization of butyl methacrylate (BMA), N-methacryloyl-L-histidine methyl ester (MAH) and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included N,N-dimethyl formamide (DMF) and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate-based monolith showed that MAH was incorporated into the polymeric structure via in-situ polymerization. Some experimental parameters including pH, concentration of the mobile phase and MAH concentration with regard to the chiral (CEC) separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that L-enantiomers of aromatic amino acids migrated before D-enantiomers. The reversal of enantiomer migration order for tryptophan was observed upon change of pH. Using the chiral monolithic column (100 μm i.d. and 375 o.d.), the best chiral separation was performed in 35/65% acetonitrile (ACN)/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. Scanning electron microscopy (SEM) images showed that the chiral monolithic column has a continuous polymeric skeleton and large through-pore structure.

iMole is a platform that automatically extracts images and captions from biomedical literature. Images are tagged with terms contained in figure captions by means of a sophisticate text mining tool. Moreover iMole allows the user to upload directly their own images within the database and manually tag images by curated dictionary. Using iMole the researchers can develop a proper biomedical image database, storing the images extracted form paper of interest, image found on the web repositories and their own experimental images. In order to show the functioning of the platform, we used iMole to build a Two-dimensional gel electrophoresis (2DE gels) database (DB). Briefly, tagged 2DE gel images were collected and stored in a searchable 2DE gel database, available to users through an interactive web-interface. Images were obtained by automatically parsing 16 608 proteomic publications, which yielded more than 16 500 images. The database can be further expanded by users with images of interest trough a manual uploading process. iMole is available with a preloaded set of 2DE gel data at http://imole.biodigitalvalley.com.

Application of Tris–Tricine gels for two-dimensional gel electrophoresis is hampered by formation of mixed CHAPS-SDS micelles resulting in typical swirling pattern in the low mass range which makes reliable quantitative and qualitative gel evaluation impossible. Modification of 2DE strip equilibration procedure prevented the direct interaction between both detergents during equilibration process, thus substantially improving gel separation.

The wall ζ-potential ζ_w, the potential at the shear plane of the electric double layer, depends on the properties of the BGE solution such as the valence and type of electrolyte, the pH and the ionic strength. Most of the methods estimate ζ_w from measurements of the EOF velocity magnitude u_eo, usually spatially averaged over the entire capillary. In these initial studies, evanescent-wave particle velocimetry was used to measure u_eo in steady EOF for a variety of monovalent aqueous solutions to evaluate the effect of small amounts of divalent cations, as well as the pH and ionic strength of BGE solutions. In brief, the magnitude of the EOF velocity of NaCl-NaOH and borate buffer-NaOH solutions was estimated from the measured velocities of radius a = 104 nm fluorescent polystyrene particles in 33 μm fused-silica microchannels. The particle ζ-potentials were measured separately using laser-Doppler microelectrophoresis; ζ_w was then determined from u_eo. The results suggest that evanescent-wave particle velocimetry can be used to estimate ζ_w for a variety of BGE solutions, and that it can be used in the future to estimate local wall ζ-potential, and hence spatial variations in ζ_w.

A non-linear dipolophoretic analysis is applied to analytically explain the counter-intuitive experimental results of Gangwal et al. (2008) that an uncharged micro/nano size dielectric Janus particle is attracted to the wall of a micro-channel when exposed to an AC uniform electric field in the direction parallel to the no-slip boundaries. We employ the so-called ‘weak’ field assumption and consider a metallodielectric Janus colloid comprising of two semi-spheres of distinct dielectric properties subject to an oscillating uniform electric field with moderate frequency (below the Maxwell-Wagner limit). The Debye scale (ratio of electric double layer (EDL) thickness to particle size) is considered unrestricted. Under the low-Reynolds number hypothesis, Faxén's theorem and the Green's function (Stokeslet) method of singularities, including appropriate images with respect to the no-slip boundary, are applied under the remote-field approximation to determine the dynamics and trajectory of a small colloid moving near a wall. When assuming maximum dielectric contrasts between hemispheres and relatively low Debye scale (compared to particle radius), a rather simple relation for the equilibrium position of the colloid (i.e. tilt angle and distance from the wall) is obtained and found to be in qualitative good agreement with the experimental observations of Gangwal et al. (2008) and the predictions of Kilic and Bazant (2011).

A high aspect ratio three-dimensional electrokinetic nanoprobe is used to trap polystyrene particles (200 nm), gold nanoshells (120 nm), and gold nanoparticles (mean diameter 35 nm) at low voltages (less than 1 V_rms). The nanoprobe is fabricated using room-temperature self-assembly methods, without the need for nanoresolution lithography. The nanoprobe (150 nm to 500 nm in diameter, 2 μm to 150 μm in length) is mounted on the end of a glass micropipette, enabling user-specified positioning. The nanoprobe is one electrode within a point-and-plate configuration, with an indium tin oxide coverslip serving as the planar electrode. The three-dimensional structure of the nanoprobe enhances dielectrophoretic capture; further, electrohydrodynamic flow enhances trapping, increasing the effective trapping region. Numerical simulations show low heating (1 K), even in biological media of moderate conductivity (1 S/m).

Nanoparticles (NPs) can be used as pseudostationary phases (PSPs) in electrokinetic chromatography (EKC), which is similar to the use of micelle additives as applied in micellar electrokinetic chromatography (MEKC). To date, the use of NPs to enhance enantiomeric separation by EKC with β-CD or its derivative as chiral selector has been reported only in two papers. However, to the best of our knowledge, there has been no prior effort to use NPs for achieving enantioseparation with polysaccharides as chiral selector. This paper describes for the first time the use of carbon nanoparticles (CNPs) as PSPs to modify chiral separation system employing dextrin as chiral selector for the enantioseparations of several basic drugs in capillary EKC. Three different types of CNPs, including carbogenic nanoparticles (NPs), carboxylated single-walled carbon nanotubes (c-SWCNTs) and carboxylated multi-walled carbon nanotubes (c-MWCNTs), were used as running buffer additives, respectively. The potential of the PSPs and the effects of dextrin concentration, buffer pH and buffer concentration on the enantioseparations were evaluated. Four pairs of tested enantiomers were successfully resolved in less than 15 min with the resolution values in the range of 1.41–4.52 under optimized conditions. Compared to the buffer without NPs, the introduction of NPs into the buffer enhanced the separation of the enantiomers.

Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has been difficult due to the distinct physicochemical properties of the peptidyl and glycan moieties, the variable and dynamic nature of the glycosylation process, their heterogeneous nature, and the low relative abundance of each glycoform. In this study, we explore a new pipeline developed for large-scale empirical identification of N-linked glycoproteins of tomato fruit as part of our ongoing efforts to characterize the tomato secretome. The workflow presented involves a combination of lectin affinity, tryptic digestion, ion-pairing HILIC and precursor ion-driven data dependent MS/MS analysis with a script to facilitate the identification and characterization of occupied N-linked glycosylation sites. A total of 212 glycoproteins were identified in this study, in which 26 glycopeptides from 24 glycoproteins were successfully characterized in just one HILIC fraction. Further precursor ion discovery (PID)-based MS/MS and deglycosylation followed by high accuracy and resolution MS analysis were used to confirm the glycosylation sites and determine site occupancy rates. The workflow reported is robust and capable of producing large amounts of empirical data involving N-linked glycosylation sites and their associated glycoforms.

The test used in clinics as prostate cancer biomarker, based on the concentration of the glycoprotein prostate-specific antigen (PSA) in serum, leads to an elevated number of false positives. In the search for new prostate cancer biomarkers, analysis of the proportions of different groups of glycoforms of PSA is promising. Peaks of PSA, called isoforms and containing one or several glycoforms of the glycoprotein, can be separated by CE. For those samples in which PSA concentration is very low, a very sensitive detection technique, such as LIF would be required. However, CE separation of fluorescently labeled isoforms of glycoproteins is challenging. In this work three different methods of fluorescent derivatization of PSA were assayed with the aim of finding conditions allowing labeling of the glycoprotein compatible with CE resolution of its isoforms. NanoOrange, as non-covalent label, 5-(iodoacetamide) fluorescein and BODIPY® FL C₁-IA, as covalent tags of thiol groups, and Chromeo™ P503 as covalent tag of amino groups were tried. Only the derivatization with the P503 fluorogenic dye led to the resolution by CE-LIF of several isoforms of labeled PSA. Adapting this derivatization method to be performed on-column lead to a reduction in labeling time from 4 hours to 45 seconds. Automation of the whole analysis permitted to carry out fluorescent labeling and CE separation of PSA isoforms in less than 12 min.

Heavily O-glycosylated membrane-tethered or secreted proteins often escape identification by gel-based proteomics due to weak staining and due to low identification rates in tandem mass spectrometry. The present protocol refers to a chemical in-gel de-O-glycosylation of proteins based on repeated oxidation/elimination of glycans leaving the protein backbone intact at the gel position of the native glycoprotein. On restaining prior to spot picking the deglycosylated proteins are detectable at increased staining intensities when applying fluorescent dyes or silver stains. Evidence is provided that de-O-glycosylation of proteins in gels is efficient and does not introduce structural artefacts into the protein backbones. In-gel trypsin digestion of deglycosylated proteins, like human glycophorin A, revealed strongly enhanced sequence coverage in LC-ESI tandem mass spectrometry. The protocol is applicable in 1D and 2D gel settings within one working day.

The N-glycosylation in pea and lentil seedling amine oxidases (PSAO and LSAO, respectively) was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of PSAO by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and “trimmed” N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF mass spectrometry. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a reversed phase using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, for example in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow pump (LVSEP) and field-amplified sample injection (FASI), was investigated in capillary electrophoresis (CE) to achieve highly sensitive oligosaccharide analysis. In CE with LVSEP-FASI, analytes injected throughout the capillary were concentrated on the basis of LVSEP, followed by an electrokinetic introduction of concentrated analytes from the inlet vial by the FASI mechanism. After switching the inlet vial solution from the sample to running buffer, the concentrated analytes were then separated by capillary zone electrophoresis (CZE). In the present LVSEP-FASI-CZE, pressure was applied to the capillary inlet until the inlet vial solution was exchanged. The applied pressure generated a counter-flow against the EOF. It kept the stacked sample zone within the capillary, minimizing loss of concentrated analytes. Fluorescein was first analyzed by LVSEP-FASI-CZE to optimize preconcentration condition. Up to 110,000-fold sensitivity increase was obtained with 200 μL of sample, compared to normal CZE with sample injection of 0.3 psi for 3 s (ca, 1.7 nL). From the results, the pressure application improved the efficiency of the FASI-mode concentration significantly at total concentration time longer than 10 min. In the analysis of maltoheptaose, a 10,000-fold sensitivity increase was achieved, which is the highest concentration efficiency ever reported in CE of oligosaccharides. The relative standard deviations of the detection time and peak height were 2.4% and 11%, respectively. In the analysis of glucose oligomer, up to 8,600-fold sensitivity increases were achieved without reducing the separation performance of conventional CZE.

A capillary electrophoresis based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease (COPD) and lung cancer patients by means of two haptoglobin specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled and profiled by capillary electrophoresis. Disease associated changes of core and antennary fucosylation were identified by targeted exoglycosidase digestions and their levels were compared in the different patient groups. Terms such as of core- and arm-fucosylation degree, as well as branching-degree were introduced for easier characterization of the changes and statistical analysis was used to examine which structures are responsible for the observed differences. Increased level of α1–6 fucosylated tri-antennary glycans was found in all disease groups compared to the control. Elevated amounts of core- and arm fucosylation on tetra-antennary glycans were detected in the lung cancer group compared to the COPD group. A larger scale study is necessary to confirm and validate these preliminary findings in the glycosylation changes of haptoglobin, so could then be used as biomarkers in the diagnosis of malignant and inflammatory lung diseases.

Multiple hydroxy-, keto-, di-, and tri-carboxylic acids are among the cellular metabolites of central carbon metabolism (CCM). Sensitive and reliable analysis of these carboxylates is important for many biological and cell engineering studies. In this work, we examined 3-nitrophenylhydrazine as a derivatizing reagent and optimized the reaction conditions for the measurement of ten CCM related carboxylic compounds, including glycolate, lactate, malate, fumarate, succinate, citrate, isocitrate, pyruvate, oxaloacetate, and α-ketoglutarate as their 3-nitrophenylhydrazones using LC/MS with electrospray ionization. With the derivatization protocol which we have developed, and using negative-ion multiple reaction monitoring on a triple-quadrupole instrument, all of the carboxylates showed good linearity within a dynamic range of ca. 200 to more than 2000. The on-column limits of detection and quantitation were from high femtomoles to low picomoles. The analytical accuracies for eight of the ten analytes were determined to be between 89.5 to 114.8% (CV≤7.4%, n = 6). Using a quadrupole time-of-flight instrument, the isotopic distribution patterns of these carboxylates, extracted from a ¹³C-labeled mouse heart, were successfully determined by UPLC/MS with full-mass detection, indicating the possible utility of this analytical method for metabolic flux analysis. In summary, this work demonstrates an efficient chemical derivatization LC/MS method for metabolomic analysis of these key CCM intermediates in a biological matrix.

This study developed capillary electrophoresis (CE) and ultra-high-pressure liquid chromatography (UHPLC) methods coupled with UV detectors to characterize the metabolomic profiles of different rhubarb species. The optimal CE conditions used a back ground electrolyte (BGE) with 15 mM sodium tetraborate, 15 mM sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholate, and 30% acetonitrile (ACN) (v/v) at pH 8.3. The optimal UHPLC conditions used a mobile phase composed of 0.05% phosphate buffer and ACN with gradient elution. The gradient profile increased linearly from 10% ACN to 21% ACN within the first 25 minutes, then increased to 33% ACN for the next 10 minutes. It took another 5 minutes to reach the 65% ACN, then for the next 5 minutes it stayed unchanged. Sixteen samples of Rheum officinale and Rheum tanguticum collected from various locations were analyzed by CE and UHPLC methods. The metabolite profiles of CE were aligned and baseline corrected before chemometric analysis. Metabolomic signatures of rhubarb species from CE and UHPLC were clustered using principle component analysis and distance-based redundancy analysis; the clusters were not only able to discriminate different species but also different cultivation regions. Similarity measurements were performed by calculating the correlation coefficient of each sample with the authentic samples. Hybrid rhizome was clearly identified through similarity measurement of UHPLC metabolite profile and later confirmed by gene sequencing. The present study demonstrated that CE and UHPLC are efficient and effective tools to identify and authenticate herbs even coupled with simple detectors.

DNA analysis has widespread applicability in biology, medicine, biotechnology and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5’-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whether the mechanism involves specific interactions between the phosphates and the DNA strands or is a result of other properties of the separation medium. Phosphate generally has been avoided in DNA separations by capillary gel electrophoresis because its high ionic strength exacerbates Joule heating. Our results suggest that phosphate compounds should be examined for separation of DNA based on sequence.

This article reports a simple methodology using dispersive liquid–liquid microextraction (DLLME) combined with CZE. It has been applied for the simultaneous determination of phenolic compounds like : caffeic, gallic, vanillic, syringic, cinnamic, p-coumaric acids and oleuropein, apigenin, luteolin, 3-hydroxytyrosol and tyrosol, in virgin olive oil (VOO). The optimized extraction conditions for 20 g of VOO were: extractant solvent: 400 μL boric acid 30 mM at pH 9.5; dispersive solvent: 300 μL carbon tetrachloride; vortex: 8 min; centrifugation: 3 min. The composition of the BGE was optimized resulting in the selection of a solution made of 30 mM boric acid at pH 9.5. As a strategy for on-line preconcentration a stacking step was applied, injecting a plug of water before sample injection. The short extraction time, centrifugation and electrophoretic steps allow the selective determination of phenolic compounds in VOO with satisfactory LODs (0.004–0.251 mg Kg⁻¹), recoveries (89.4–101.0%) and RSD (less than 7.44% in peak area and less than 0.69% in migration time), compatible with the concentration levels present in the samples.

Biologically active peptides derived from complex bovine milk protein hydrolysates are of particular interest in food science and nutrition because they have been shown to play different physiological roles, providing benefits in human health. In this study we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) for separation and identification of bioactive peptides in three hypoallergenic infant milk formulas (IF). An appropriate sample clean-up using a citrate buffer with dithiothreitol and urea followed by solid-phase extraction (SPE) with C18 and StrataX cartridges allowed detection of a large number of low-molecular-mass bioactive peptides. This preliminary identification was solely based on the measured experimental monoisotopic molecular mass values (M_exp). Later, we evaluated the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m_e versus q/M^α, α = 1/2 for the classical polymer model) to describe their migration behaviour. The assistance of migration prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of 24, 30 and 38 bioactive peptides was confirmed in each of the three IFs. A significant number of these peptides were reported as inhibitors of angiotensin converting enzyme (ACE), however, the presence of sequences with other biological activities such as antihypertensive, antithrombotic, hypocholesterolemic, immunomodulation, cytotoxicity, antioxidant, antimicrobial, antigenic or opioid was also confirmed.

We developed the dual-micropillar-based microfluidic platform to direct embryonic stem (ES) cell fate. 4 × 4 dual-micropillar-based microfluidic platform consisted of 16 circular-shaped outer micropillars and 8 saddle-shaped inner micropillars in which single ES cells were cultured. We hypothesized that dual-micropillar arrays would play an important role in controlling the shear stress and cell docking. Circular-shaped outer micropillars minimized the shear stress, whereas saddle-shaped inner micropillars allowed for docking of individual ES cells. We observed the effect of saddle-shaped inner micropillars on cell docking in response to hydrodynamic resistance. We also demonstrated that ES cells cultured for 6 days within the dual-micropillar-based microfluidic platform differentiated into neural-like cells. Therefore, this dual-micropillar-based microfluidic platform could be a potentially powerful method for screening of lineage commitments of single ES cells.

A novel sulfoalkylbetaine-based zwitterionic organic-silica hybrid monolith was synthesized by using 3-dimethyl- (3-(N-methacrylamido) propyl) ammonium propane sulfonate (DMMPPS, neutral sulfoalkyl-betaine monomer). The added amount of zwitterionic monomer was significantly increased when DMMPPS was used instead of the conventionally used acidic sulfoalkyl-betaine monomer, i.e., the N, N-dimethyl-N- methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE), and this led to a significantly improved hydrophilicity of the monolith. The DMMPPS-based organic-silica hybrid monolith exhibited good mechanical stability and excellent separation performance. About ∼20 μm plate height (corresponding to column efficiency of ∼50 000 plates/m) was obtained for nucleoside at the linear velocity of 1mm/s. The proposed monolithic column was successfully applied to separate purines/pyrimidines, nucleotides, and tryptic digest of bovine hemoglobin (BHb) in a nano-HILIC mode, and the results demonstrated that such monolith has the potential for separation of a variety of hydrophilic substances.

Adult tapeworms of the genus Echinococcus (family Taeniidae) occur in the small intestines of carnivorous definitive hosts and are transmitted to particular intermediate mammalian hosts, in which they develop as fluid-filled larvae (cysts) in internal organs (usually lung and liver), causing the disease echinococcosis. Echinococcus species are of major medical importance and also cause losses to the meat and livestock industries, mainly due to the condemnation of infected offal. Decisions regarding the treatment and control of echinococcosis rely on the accurate identification of species and population variants (strains). Conventional, phenetic methods for specific identification have some significant limitations. Despite advances in the development of molecular tools, there has been limited application of mutation scanning methods to species of Echinococcus. Here, we briefly review key genetic markers used for the identification of Echinococcus species and techniques for the analysis of genetic variation within and among populations, and the diagnosis of echinococcosis. We also discuss the benefits of utilizing mutation scanning approaches to elucidate the population genetics and epidemiology of Echinococcus species. These benefits are likely to become more evident following the complete characterisation of the genomes of E. granulosus and E. multilocularis.

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open-tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallydimethylammonium chloride (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet-layer-like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused-silica capillary via electrostatic assembly.

Several new fast staining protocols for the visualization of proteins separated by SDS-PAGE utilizing Coomassie Blue staining have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS-PAGE of serially diluted protein samples.

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor is a key xenobiotic sensor that regulates the expression of drug-metabolizing enzymes and many other genes. Radiolabeled ³²P-DNA-probes had been used in studies of PXR-DNA interactions. There is an increasing need for non-radioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the non-radioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.

The ischemic cascade starts when atherosclerotic plaques decrease the supply of oxygen and substrates to cells and finalizes with myocardial infarction. These states have been here studied at metabolite level by optimization of a metabolomics profiling approach based on high-accuracy mass spectrometry. For this purpose, serum samples from patients diagnosed with coronary artery disease (CAD) and affected by stable angina or myocardial infarction (acute myocardial infarction or non-ST elevation myocardial infarction) were analyzed by LC–QTOF/MS after deproteinization to compare the profile of serum metabolites. The data set, composed by tentative molecular features detected in mass spectrometry analyses, was filtered with statistical algorithms to remove entities resulting in redundant information. Tentative molecules were identified finding mainly lipids as statistically significant metabolites in the discrimination study due to their change in concentration. Lipids such as bile acid derivatives, phospholipids and triglycerides were identified as relevant compounds for discrimination of individuals who suffered acute or non-ST elevation myocardial infarction from those suffering stable angina. The results achieved by this research could support the capability of metabolomics to go inside the study of artery diseases, and in addition to other omics disciplines could help to detect the occurrence of these disorders at initial stages or even to prognosticate their appearance.

A targeted approach has been applied to quantitative analysis of eicosanoids derived from omega-6 fatty acids in serum from individuals diagnosed with coronary artery disease. The target metabolites were series-2 prostaglandins (PG), thromboxane B2, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs). The method was based on SPE–LC­–MS/MS in selected reaction monitoring mode (SRM) for highly selective and sensitive determination of the target eicosanoids. The combination of SPE and LC–MS/MS involved the benefits from both direct analysis of serum without a step for protein precipitation and fully automation of the analysis. The method allowed comparison of omega-6 derived eicosanoids in serum from patients diagnosed with coronary artery disease and from control individuals. The effect of treatment with aspirin on the profile of the target compounds was evaluated through its incidence on the different pathways. Finally, the serum levels of the target metabolites in patients diagnosed with coronary artery disease were also statistically examined according to the severity of the coronary lesion stratified as stable angina, non-ST-elevation acute coronary syndrome (NSTE-ACS) and acute myocardial infarction (AMI).

Multiple myeloma (MM) and chronic lymphocytic leukaemia (CLL) are closely related B-cell non-Hodgkin's lymphomas. MM, a plasma cell malignancy, is the second most common haematopoietic cancer in Western countries, with the median survival time of 3–4 years. CLL, a lymphocyte B malignancy, is the most common leukaemia in Western countries. About 25–30% of all CLL patients do not survive the period of 5 years following diagnosis. Both malignancies are complicated, not fully understood and incurable with the current standard treatment. Biologically, MM and CLL may be preceded by associated precursor conditions, that is monoclonal gammopathy of undetermined significance for MM and its cellular counterpart and monoclonal B-cell lymphocytosis for CLL. Similarities and differences in the biology of these malignancies prompted us to evaluate their metabolomics in stages requiring chemotherapy. Fingerprinting of serum metabolites by the use of LC-MS has never been applied in studies on MM and CLL patients. Obtained results revealed metabolites common for both malignancies (e.g. fatty acids, acylcarnitines, sphingolipids, phospholipids, phenylalanylphenylalanine, and isoprene) as well as those which render them different (e.g. lysophosphatidylcholines, monoacylglycerols, aminocaproic acid, phenylacetylglutamine).

In this study, an ultra fast liquid chromatography/ion trap-time of flight mass spectrometry (UFLC/MS-IT-TOF) -based serum lipidomics method was employed to characterize the serum lipid profile of patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma (HCC). After data collection and processing, 96 lipids including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins, triacylglycerides and cholesterol esters were identified and used for subsequent data analysis. Partial least squares-discriminant analysis (PLS-DA) revealed that patients with liver diseases had distinctly different serum lipid profile from that of healthy controls; while cirrhosis and HCC patients had a similar serum lipid profile, but different from that of hepatitis patients. The ANOVA analysis found 75 out of 96 identified lipids to be abnormally regulated, among which most of these lipids were down-regulated in cirrhosis and HCC patients compared with those of healthy controls and hepatitis patients, while hepatitis patients induced several lipids down-regulated and others up-regulated compared with those of healthy controls, indicating the aberrant lipid metabolism in patients with liver diseases. This work demonstrated the utility of UFLC/MS-IT-TOF-based serum lipidomics as a powerful tool to investigate the lipid metabolism of liver diseases.

A capillary zone electrophoresis method with two different MS detection conditions (MRM and Full Scan) was developed to determine qualitative and quantitative changes in the metabolic profile of avocado fruits (Persea americana). LODs in MRM approach were found between 20.1 and 203.0 ppb for abscisic acid and perseitol, respectively, whilst in Full Scan varied within the range 0.22–1.90 ppm for the same metabolites. The RSDs for reproducibility test did not exceed 11.45%. The two MS approaches were used to quantify 10 metabolites (phenolic acids, flavonoids, a carbohydrate, an organic acid, a vitamin and a phytohormone) in 18 samples of avocado at different ripening states, and the achieved results were compared. Perseitol, quinic, chlorogenic, trans-cinnamic, pantothenic and abscisic acids, as well as epicatechin and catechin decreased during the ripening process, whereas ferulic and p-coumaric acids showed the opposite trend. Moreover, some other unknown compounds whose concentration changed largely during ripening were also studied by MS/MS and Q-TOF MS to get a tentative identification.

Alzheimer's disease (AD) is a neurodegenerative multifactorial disease whose cause is still unknown. The majority of AD biochemical markers currently available have been developed as an extension of targeted physiological studies on the basis of the “amyloid hypothesis”. The potential of Metabolomics for the discovery of novel biomarkers and elucidation of new biochemical pathways modified in the progression of AD is highlighted in this review work. A variety of non-targeted metabolomic approaches for the discrimination between healthy subjects and AD patients are described. Moreover, the feasibility of Metabolomics to predict progression to AD in individuals with mild cognitive impairment (MCI) is also presented.

Lactoferrin is app. 80 kDa iron binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of lactoferrin (LF) from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimized steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 μg) was linked. Lactoferrin was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase (HRP) reducing 3,3′,5,5′-Tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng.ml⁻¹. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 μg.ml⁻¹. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-lactoferrin-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R² higher than 0.95, indicating a very good agreement of the results obtained.

A three-dimensional, finite element model was developed to optimize the kinetics and mass transfer characteristics of low concentration, 18 base pair (bp) single stranded DNA (ssDNA) targets in bulk media solution, to 18 bp complimentary oligonucleotide probes immobilized on electrochemical detection electrodes positioned along the length of a microfluidic channel. Conditions considered in the model were fluid flow rate, diffusion time, DNA melting temperature, number of matching base-pairs, and temperature of the fluid in the channel. System optimization was based on maximizing the uniformity and surface concentration of the specifically-bound hybridized DNA, minimizing waste volume generation, and minimizing the hybridization time. With the coupled simulation method used, the total experiment time was reduced from 150 minutes to 60 minutes and the simulated results were consistent with experimental results found in the literature. A stopped flow procedure was investigated as a means to improve hybridization. This procedure can improve uniformity and capture efficiency, and reduce waste, but also decreases overall signal intensity relative to continuous flow operation Finally, the use of temperature in reducing mismatched hybridization and improving duplex stability was also successfully modeled and simulated.

The determination of fatty acids by non-aqueous capillary zone electrophoresis with capacitively coupled contactless conductivity detection was investigated. A new deoxycholate based background electrolyte, which had previously been found to give significantly improved baseline stability in the determination of lipophilic organic ammonium ions, was found to be similarly beneficial for the determination of the anions. The use of a poly(vinyl alcohol) coated capillary was required for suppression of the electroosmotic flow and to obtain well reproducible results. The complete separation of twelve fatty acids could be achieved with 10 mM sodium deoxycholate in methanol within 6 minutes under optimized conditions. The poly(vinyl alcohol)-coated capillary demonstrated outstanding stability over 300 runs with no sign of depletion of the poly(vinyl alcohol) layer. Method validation showed a good linearity range from 0.75 to 25 μM with correlation coefficients between 0.9949 to 0.9979. The limit of detection was determined as 0.5 μM for all fatty acids. The developed approach was successfully demonstrated for the separation of free fatty acids in commercial and home-made edible oil.

Microchannel plate (MCP), a high-porosity glass membrane used as an electron multiplier in analytical/scientific instruments for the detection of energetic photons and charged particles is demonstrated here as a highly effective bipolar electrode for electrokinetic focusing of anions. Assembled between a pair of microfluidic channels filled with an electrolyte buffer and subjected to a sufficient bias potential, MCP supports faradaic reactions, owing to its semiconducting characteristics. Thousands of microcapillary tubes fused together define MCP and act in unison such that each microcapillary serves as a tiny bipolar electrode surrounding an infinitesimal element of bulk electrolyte with a large surface-area-to-volume ratio and hence performs highly effective as compared to a planar electrode inlaid into a microchannel. This performance has been validated here where concentration enrichment of a fluorescent tracer has been demonstrated at a remarkable rate of up to 175-fold/s exceeding those reported for planar bipolar electrodes. We attribute such high performance to the rapid onset of ion-depletion zone and subsequent steep field gradient, signifying the high-porosity structure of MCP as an effective bipolar electrode.

The proteome of Goji berries (Lycium barbarum) has been investigated in depth by using the combinatorial peptide ligand libraries (CPLL) technology. Since, in general, when plant samples are solubilized under native conditions, a limited number of identifications ensues, we have tried here solubilization under both native and denaturing conditions. In the latter case, since up to 3% SDS is used, the amount of surfactant is diluted to 0.1% or else proteins are precipitated with acetone/methanol, followed by CPLL capture. Under these conditions, a total amount of 350 proteins has been univocally identified, which represents by far the deepest proteomic investigation of Goji berries.

Isomerization and enantiomerization of Asp in the tetrapeptide Ala-Phe-Asp-GlyOH was studied at pH 10 and 80°C as well as 25°C. CE-MS allowed the distinction between α-Asp and β-Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfey's reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order Asp > Ala > Phe. The CE assay was validated with respect to linearity, LOQ, LOD and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β-Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.

In this paper, a rapid thermal cycling procedure is combined with a direct amplification from a paper punch, permitting a high-speed amplification of a 7-locus multiplex that requires no extraction step. When coupled with a short 1.8 cm microfluidic electrophoresis system, the entire procedure from paper punch to genotype can be completed in under 25 minutes. The paper describes selection and optimization of enzyme, direct amplification conditions, the reproducibility of the procedure and concordance with standard forensic genotyping methods. The procedure utilizes a small high-speed thermal cycler and microfluidic device along with a small laptop and is highly portable. Overall, this technique should provide a useful and reliable procedure for rapid determination of identity of individuals retained at checkpoints as well as a quick method for preliminary identification of individuals at remote locations following mass disasters.

The performance of high-resolution capillary zone electrophoresis (CZE) for determination of carbohydrate-deficient transferrin (CDT) in human serum based on internal and external quality data gathered over a ten-year period is reported. The assay comprises mixing of serum with a Fe(III) ion-containing solution prior to analysis of the iron saturated mixture in a dynamically double coated capillary using a commercial buffer at alkaline pH. CDT values obtained with a human serum of a healthy individual and commercial quality control sera are shown to vary less than 10%. Values of a control from a specific lot were found to slowly decrease as function of time (less than 10% per year). Furthermore, due to unknown reasons, gradual changes in the monitored pattern around pentasialo-Tf were detected which limit the use of commercial control sera of the same lot to less than two years. Analysis of external quality control sera revealed correct classification of the samples over the entire ten-year period. Data obtained compare well with those of HPLC and CZE assays of other laboratories. The data gathered over a ten-year period demonstrate the robustness of the high-resolution CZE assay. This is the first account of a CZE-based CDT assay with complete internal and external quality assessment over an extended time period.

Tumor microenvironment plays very important roles in the carcinogenesis. A variety of stromal cells in the microenvironment have been modified to support the unique needs of the malignant state. This study was to discover stromal differentially expressed proteins (DEPs) that were involved in colon carcinoma carcinogenesis. Laser capture microdissection (LCM) was captured and isolated the stromal cells from colon adenocarcinoma (CAC) and non-neoplastic colon mucosa (NNCM) tissues, respectively. Seventy DEPs were identified between the pooled LCM-enriched CAC and NNCM stroma samples by iTRAQ-based quantitative proteomics. Gene Ontology (GO) relationship analysis revealed that DEPs were hierarchically grouped into 10 clusters, and were involved in multiple biological functions that were altered during carcinogenesis, including extracellular matrix organization, cytoskeleton, transport, metabolism, inflammatory response, protein polymerization, and cell motility. Pathway network analysis revealed 6 networks and 56 network eligible proteins with Ingenuity pathway analysis. Four significant networks functioned in digestive system development and its function, inflammatory disease, and developmental disorder. Eight DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ) were validated by Western blotting, and four DEPs (DCN, FN1, PKM2, and HSP90B1) were validated by immunohistochemical analysis. It is the first report of stromal DEPs between CAC and NNCM tissues. It will be helpful to recognize the roles of stromas in the colon carcinoma microenvironment, and improve the understanding of carcinogenesis in colon carcinoma. The present data suggest that DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ might be the potential targets for colon cancer prevention and therapy.

A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of pre-cast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected.

Nucleophosmin (NPM) is a novel prognostic biomarker for Ewing's sarcoma. To evaluate the prognostic utility of NPM, we conducted an interactomic approach to characterize the NPM protein complex in Ewing's sarcoma cells. A gene suppression assay revealed that NPM promoted cell proliferation and the invasive properties of Ewing's sarcoma cells. FLAG-tag-based affinity purification coupled with liquid chromatography-tandem mass spectrometry identified 106 proteins in the NPM protein complex. The functional classification suggested that the NPM complex participates in critical biological events, including ribosome biogenesis, regulation of transcription and translation, and protein folding, that are mediated by these proteins. In addition to JAK1, a candidate prognostic biomarker for Ewing's sarcoma, the NPM complex, includes 11 proteins known as prognostic biomarkers for other malignancies. Meta-analysis of gene expression profiles of 32 patients with Ewing's sarcoma revealed that 6 of 106 were significantly and independently associated with survival period. These observations suggest a functional role as well as prognostic value of these NPM complex proteins in Ewing's sarcoma. Further, our study suggests the potential applications of interactomics in conjunction with meta-analysis for biomarker discovery.

Methylarginines (MAs) are potent vasoconstrictors that have been reported to be present at elevated concentrations in the blood of patients suffering from cardiovascular disease (CVD). To determine the diagnostic potential of MAs for CVD, a method capable of rapidly quantifying their endogenous concentrations from serum samples is required. To that end, a heat-assisted extraction method was developed. Serum was first rapidly heated, causing it to congeal into a gel, and then subjected to solid-liquid extraction. The extraction solution was then derivatized with a fluorogenic dye and analyzed by CE-LIF to permit quantitation of the MAs. This heat-assisted extraction procedure allowed a no-net-dilution extraction of the analytes to be performed into a solvent compatible with the subsequent CE analysis. This enabled direct detection of low abundance analytes, such as MAs, without the need for a preconcentration step. This sample preparation method was compared with a commonly used solid-phase extraction method for MA analysis. Endogenous MA concentrations determined by both the heating and SPE methods were found to be in good agreement with each other and with values previously reported in the literature.

Post-translational modifications are significant reactions that occur to proteins. One of these modifications is a non-enzymatic reaction between the oxo-group(s) of sugars and amino-group(s) of protein – glycation. This reaction plays an important role in the chronic complications of diabetes mellitus, or in the aging process of organisms, that is, it has an important role in the pathophysiology and “normal” physiology of animals. In the work presented here, we studied the glycation of albumins (HSA and BSA). Methodologically, we used nano-LC coupled to a QTOF mass spectrometer. In vitro-modified proteins were cleaved by trypsin and the arising peptides were separated on a C18 nano column with a trap-column. Peptides and their modifications were analysed with a high-resolution QTOF mass spectrometer with a mass determination precision of better than 5 ppm. Non-enzymatic in vitro reaction products between albumin and ribose were identified. Besides well-known carboxymethyl lysine, new modifications were determined – creating mass shifts of 78 and 218. The origin of the first modification is discussed and its possible structure is presented. In addition, a mass shift of 132 belonging to a Schiff base was also identified. The location of all the modifications within the structure of the proteins was determined and their reactivity to various oxo-compounds was also examined.

A CE protocol was developed to separate reduced glutathione and its four novel analogues UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), UPF17 (Tyr(Me)-α-Glu-Cys-Gly), UPF50 (β-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), and UPF51 (β-Ala-His-Tyr(Me)-α-Glu-Cys-Gly), and their homo- and heterodimers by varying the ionic strength and/or pH of different BGEs. For the determination of dissociation constants (pK_a) of the above-mentioned peptides the CE method was used. Effective electrophoretic mobilities of analytes were measured in the pH range 5.50–10.00 using optimized BGE with an ionic strength of 50 mM at 25°C. pK_a values were calculated by fitting the experimental points to a suitable model with correlation coefficients higher than 0.99. The pK_a values for imidazolyl, amino and thiol moieties of the analyzed peptides were in the range 5.94–6.29, 8.81–9.10, and 7.86–8.13, respectively.

This article describes the development of a reliable CZE-ESI-MS method to simultaneously separate and quantitate three specific metabolites (3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), and picolinic acid (PA)) of the kynurenine pathway (KP) of tryptophan catabolism. Using a covalently bonded sulfonated capillary, the parameters such as pH, type of background electrolyte, type of organic solvent, nebulizer pressure as well as both negative and positive ESI-MS modes were optimized to achieve the best Rs and S/N of three KP metabolites. The developed CZE-ESI-MS assay provided high resolution of PA/QA, high specificity, a total analysis time of 10 min with satisfactory intraday and interday repeatability of migration time and peak areas. Under optimized CZE-ESI-MS conditions, the calibration curves over a concentration range of 19–300 μM for 3-HAA and QA, and 75–300 μM for PA were simultaneously generated. The method was successfully applied for the first time to profile the concentrations of initial substrate, 3-HAA, and its eventual products, PA and QA, formed in the complex multienzyme system. As the ratio of two enzymes, 3-hydroxyanthranilate 3,4-dioxygenase (HAO) and α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) decreases, the concentration of QA approaches essentially zero indicating that all ACMS formed by the action of HAO is consumed by ACMSD rather than its spontaneous decay to QA.

A simple data reconstruction technique in CE-UV-ESI-MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE-MS interfaces. We present here a method to transform the traditional time-based electropherogram into the corresponding temperature-correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer-filled capillary. The temperature-correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature-correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE-UV separations to be correlated with the corresponding peak obtained by MS detection in CE-MS even for differences in capillary dimensions and thermostatic control.

Melatonin (MT) presence in higher plants was recently discovered and the knowledge of its function in vivo is limited. Several studies have recently shown the occurrence of MT and related compounds in grapes and wines. The analysis of MT in plants and foods represents a highly challenging task due to its wide concentration range, the difficulty in the selection of the extraction solvents because of its amphipathic nature, and the fact that it reacts quickly with other matrix components. Thus, sample processing factors; preparation/cleanup procedures; and chromatographic/detection parameters, such as HILIC and reverse phase (C₈ and C₁₈) chromatographic modes, ESI, and atmospheric pressure chemical ionization (APCI) in both negative and positive modes were evaluated. Taken together, we have demonstrated that optimal conditions were quite different for each of the matrices under study. A sonication-mediated extraction step was necessary for grape skin (100% v/v methanol) and plant tissues (50% v/v methanol), while wine and must required a SPE preconcentration step. HILIC−(+) APCI ionization was better for MT standards, while C₈−(+) APCI was the best choice for grape skin and C₁₈−(+ESI) was suitable for wine. On the other hand, C₈−(+)ESI was the most appropriate for vegetal tissues of Arabidopsis thaliana. Proposed methods were validated and the LODs were in the low picogram levels range. The optimized approaches were applied to the determination of MT and its isomer in different vegetal/food samples; levels found within the range: 4.9–440 ng/g.

This paper describes a novel real-time impedance chip for the detection of squalene-water emulsion phase separation. Each impedance chip contains eight pairs of indium tin oxide microelectrode arrays for detecting eight samples, and six chips can be connected with the switch relay to measure 48 samples in the system simultaneously. The proposed impedance chip has the advantages of needing only a small sample volume (0.5 mL), and provides parallel, continuous, and real-time detection. The effects of the surfactant concentration on the stability of a squalene/water emulsion were studied by means of a visual inspection, a conductance probe, and by impedance chip. Three different concentrations of Tween 20 surfactant (9, 17, and 29 wt%) were employed for the examinations. The results indicated that the phase separation rate was faster in the lower surfactant concentration. However, the emulsion of 29 wt% Tween 20 was fairly stable for more than 2 days since there were no signal changes according to the three detection methods. The reaction time (TR) for completing the measured phase separation process differed for each of the three methods (measuring aqueous phase height, conductance, and impedance, respectively). For the 9 wt% Tween 20, the reaction times were 24 h, 20 min, and 5 min in the tests using visual inspection, conductance probe, and impedance chip, respectively. For the 17 wt% Tween 20, the TR was also shorter when using the impedance chip method compared to the other two methods. Therefore the proposed impedance chip has a quick reaction response and provides an alternative and effective method to detect emulsion stability.

Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme-to-substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE-MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.

The influence of various cations as metal ions (barium, calcium, copper, magnesia, manganese, and nickel), pharmaceuticals (ephedrine, ethambutol, pilocarpine, and pirenzepine), arginine, and guanidine has been tested on BSA, β-lactoglobulin, and ovalbumin. Influences on proteins regarding changes in size, charge, or mass were of particular interest. ACE proved to be a suitable method to investigate these effects. ACE is able to observe slight but significant changes on proteins due to the excellent precision of the measurements. Therefore, some unexpected behaviors of protein–ligand interactions were found. The protein charge becomes more negative under metal ion influence and some pharmaceutical cations. Probably metal ions bound to the proteins form additional complexes with anions from the surrounding solution. Furthermore, already bound cations could be displaced at the protein surface. Both effects would change the overall charge of the ligand–protein complexes. In all studied cases, multiple-binding stoichiometries have been observed.

PTMs enable cells to adapt to internal and external stimuli in the milliseconds to seconds time regime. Protein phosphorylation is probably the most important of these modifications as it affects protein structure and interactions, critically influencing the life cycle of a cell. In the last 15 years, new insights into phosphorylation have been provided by highly sensitive MS-based approaches combined with specific phosphopeptide enrichment strategies. Although so far research has mainly focused on the discovery and characterization of O-phosphorylation, this review also briefly outlines the current knowledge about N-phosphorylation depicting its ubiquitous relevance. Further, common pitfalls in sample preparation, LC-MS analysis, and subsequent data analysis are discussed as well as issues regarding quality and comparability of studies on protein phosphorylation.

Electromigration methods including CE and ITP are attractive for incorporation in microfluidic devices because they are relatively easily adaptable to miniaturization. After its popularity in the 1970s, ITP has made a comeback in microfluidic format (μ-ITP, micro-ITP) driven by the advantages of the steady-state boundary, the self-focusing effect, and the ability to aid in preconcentrating analytes in the sample while removing matrix components. In this review, we provide an overview of the developments in the area of μ-ITP in a context of the historic developments with a focus on recent developments in experimental and computational ITP and discuss possible future trends. The chip-ITP areas and topics discussed in this review and the corresponding sections include: PC simulations and modeling, analytical μ-ITP, preconcentration ITP, transient ITP, peak mode ITP, gradient elution ITP, and free-flow ITP, while the conclusions provide a critical summary and outlook. The review also contains experimental conditions for μ-ITP applications to real-world samples from over 50 original journal publications.

Metabolomics represents the global assessment of metabolites in a biological sample and reports the closest information to the phenotype of the biological system under study. Mammalian cell metabolomics has emerged as a promising tool with potential applications in many biotechnology and research areas. Metabolomics workflow includes experimental design, sampling, sample processing, metabolite analysis, and data processing. Given their influence on metabolite content and biological interpretation of data, a good experimental design and the appropriate choice of a sample processing method are prerequisites for success in any metabolomic study. The use of mammalian cells in the metabolomics field involves harder sample processing methods, including metabolism quenching and metabolite extraction, as compared to the use of body fluids, although such critical issues are frequently overlooked. This review aims to overview the common experimental procedures used in mammalian cell metabolomics based on mass spectrometry, by placing special emphasis on discussing sample preparation approaches, although other aspects, such as cell metabolomics applications, culture systems, cellular models, analytical platforms, and data analysis, are also briefly covered. This review intends to be a helpful tool to assist researchers in addressing decisions when planning a metabolomics study involving the use of mammalian cells.

Asparagine deamidation is a common nonenzymatic post-translational modification comprising the conversion of asparaginyl residues to aspartyl and isoaspartyl residues, respectively. As a result an additional negative charge is introduced that can affect the tertiary structure as well as the biological activity of a protein. Since deamidation reduces the protein's pI value, differentially deamidated forms of a protein can be separated in 2D gels. We have analyzed a dataset of 430 protein spots from 2D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a Coomassie-stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin-2, V-type proton ATPase subunit B, and protein disulfide-isomerase A3 were also found in 2D gels of proteins extracted from rat hippocampus. All identified deamidation sites contained a glycine residue on the carboxyl side of the asparaginyl residue. Strikingly, a second glycine residue at the +3 position was found in the majority of the deamidated peptides. We propose that the NGxG motif confers exceptional susceptibility to in vivo asparagine deamidation.

A major concern in treating premature infants with birth-associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip-based immunoaffinity CE device has been applied to determine the concentrations of inflammation-associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r² values in the range of 0.903–0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

Proteome analysis using capillary electrophoresis coupled to mass spectrometry (CE-MS) has been used in a number of clinical applications in the past years. The main focus of CE-MS-based studies has been on the investigation of urine, due to the stability of the urinary proteome, ease of collection, and also the low molecular weight range of the urinary proteome, mostly peptides below 30 kDa. The reproducibility of this approach has enabled analysis of over 20 000 samples in a comparable way, giving enormous statistical power to any additional study involving this methodological setup. In this article, we review the major technological issues associated with the application of CE-MS in the routine investigation of the urinary proteome for clinical applications. We pinpoint recent developments that may have a chance to improve on the currently used approach, and highlight obstacles that need to be solved. In the second part of the article, we review the recent clinical applications, aiming to highlight relevant issues, and possible future routine applications in clinical diagnosis. In the end, we provide a short outlook, and indicate future developments to be expected, as well as problems that need to be solved to enable routine application of CE-MS in a clinical setting.

Chemical characterization and monitoring of fermentation broths and cell culture media provide significant information on the changes occurring within these complex and dynamic systems. Analytical methods based on CE in capillaries and microchips are attractive for integration in instrumental tools to obtain this critical data, improving the understanding and control of bioprocesses. In this review, the use of CE for chemical characterization and monitoring fermentations is discussed, organized by analyte class, including organic acids, pharmaceuticals, proteins, sugars, amino acids, and metabolites published between 1992 and October 2012. A section is dedicated to the roles CE plays throughout the wine making process, where applications range from characterization and increase in fundamental understanding of the fermentation to forensic applications, verifying the authenticity of the wine.

Human rhinoviruses (HRVs) are valuable tools in the investigation of early viral infection steps due to their far reaching (although still incomplete) characterization. During endocytosis, native virions first loose one of the four capsid proteins (VP4); corresponding particles sediment at 135S and were termed subviral A particles. Subsequently, the viral RNA genome leaves the viral shell giving rise to empty capsids. In continuation of our previous work with HRV serotype 2 (HRV2) intermediate subviral particles, in which we were able to discriminate by CE even between two intermediates (AI and AII) of virus uncoating, we further concentrated on the characterization of AI particles with the electrophoretic mobility of around –17.2 × 10⁻⁹ m²/Vs at 20°C. In the course of our present work we related these particles to virions as previously described at the subviral A stage of uncoating (and as such sedimenting at 135S) by determination of their protein and RNA content—in comparison to native virions AI particles did not include VP4, however, still 93% of their initial RNA content. Binding of an mAb specific for subviral particles demonstrated antigenic rearrangements on the capsid surface at the AI stage. Furthermore, we investigated possible factors stabilizing intermediates of virus uncoating. We could exclude the influence of the previously suspected so-called contaminant of virus preparation on HRV2 subviral particle formation. Instead, we regarded other factors being part of the virus preparation system and found a dependence of AI particle formation on the presence of divalent cations.

Quantitative and qualitative analysis of amino acids in biofluids offers relevant information in diagnosis of diseases, evaluation of nutritional state, and in elucidating metabolic influences on physiology. A simple, rapid, and robust procedure in terms of sample treatment, separation, and quantitation based on CE-LIF has been optimized for use in human plasma samples. Time required for derivatization was 15 min and analysis time was 35 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was the labeling agent used for obtaining fluorescent derivatives. Electrophoretic conditions were: 175 mM borate buffer at pH 10.25 prepared with 12.5 mM β-cyclodextrin. The voltage applied was +21 kV. Fourteen amino acids could be quantified: l-proline, l-phenylalanine, l-leucine, l-isoleucine, l-ornithine, d-ornithine, l-glutamine, l-alanine, l-threonine, glycine, l-serine, d-serine, taurine and l-glutamate. With this chiral CE-LIF method, l- and d-amino acids are adequately separated. The method was validated for a representative group of amino acids in human plasma: l-proline, l-isoleucine, l-ornithine, l-glutamine, l-alanine l-threonine, glycine, l-serine, d-serine, and glutamate. The method has been successfully applied to human plasma from patients with bipolar disorder, all of whom were taking lithium as a mood stabilizer. Eleven amino acids were quantified in plasma from nine patients, aged 24–55 years. The results were in accordance to published values for the bipolar patients. The method is useful particularly in studies where plasma amino acid levels can be used as biomarkers for diagnosis of diseases, evaluating the disease progression, and monitoring response to drug therapy.

Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated immobilized metal affinity chromatography (IMAC) system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

Molecular profiling techniques have provided extensive sets of molecular features characterizing clinical phenotypes, but further extrapolation to mechanistic molecular models of disease pathophysiology faces major challenges. Here, we describe a computational procedure for delineating molecular disease models utilizing omics profiles, and exemplify the methodology on aspects of the cardiorenal syndrome describing the clinical association of declining kidney function and increased cardiovascular event rates. Individual molecular features as well as selected molecular processes were identified as linking cardiovascular and renal pathology as a combination of cross-organ mediators and common pathophysiology. The molecular characterization of the disease presents as a set of molecular processes together with their interactions, composing a molecular disease model of the cardiorenal syndrome. Integrating omics profiles describing aspects of cardiovascular disease and respective profiles for advanced chronic kidney disease on molecular interaction networks, computation of disease term-specific subgraphs, and complemented by subgraph segmentation allowed delineation of disease term-specific molecular models, at their intersection providing contributors to cardiorenal pathology. Building such molecular disease models allows in a generic way to integrate multi-omics sources for generating comprehensive sets of molecular processes, on such basis providing rationale for biomarker panel selection for further characterizing clinical phenotypes.

Chondroitin sulfate (CS)/dermatan sulfate (DS) are often found in nature as hybrid glycosaminoglycan chains in various proteoglycans. In the recent years, several MS methods were developed for the determination of over-, regular-, and undersulfated CS/DS chains. In the present work, the released hybrid CS/DS isolated and purified from mouse brain were digested with chondroitin AC lyase. The depolymerized chains were separated by gel filtration chromatography. Collected tetrasaccharides were analyzed by fully automated (NanoMate robot) chip-based nanoESI high capacity ion trap multistage MS (MS²–MS⁴) recently introduced in glycosaminoglycan research by our laboratory. The obtained data were confirmed by high resolution MS screening and MS/MS performed on QTOF instrument. NanoMate-high capacity ion trap MS and QTOF MS screening revealed the presence in the mixture of oversulfated tetrasaccharides bearing three and four sulfate groups as well as traces of regularly and undersulfated hexamers. Additionally, several saturated species as either tetramers or hexamers exhibiting different sulfate content were discovered in the analyzed fraction. This diversity of the sulfation status indicates that the mouse brain might contain several types of proteoglycans. The molecular ions corresponding to trisulfated-[4,5Δ-GlcA-GalNAc-IdoA-GalNAc] were subjected to multistage fragmentation by CID. Sequence analysis data allowed for the postulation of two rare structural motifs: [4,5Δ-GlcA-GalNAc(4S)-IdoA(2S,3S)-GalNAc] and [4,5Δ-GlcA-GalNAc-IdoA(2S,3S)-GalNAc(4S)], previously not reported in neural tissue.

Unlike the nanoparticles with surface plasmon resonance, the optical response of polystyrene microparticles (PSMPs) is insensitive to the chemical components of the surrounding medium under the wavelength-dependent differential interference contrast microscopy. This fact is exploited for the measurement of the refractive index of cytoplasm in this study. PSMPs of 400 nm in diameter were loaded into the cell to contact cytoplasm seamlessly, and the refractive index information of cytoplasm could be extracted by differential interference contrast microscopy operated at 420 nm illumination wavelength through the contrast analysis of PSMPs images.

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)-specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin–biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio-rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished.

A detailed study on the effect of the buffer concentration and the magnitude of the superimposed hydrodynamic flow on separation performance in CZE with contactless conductivity detection was carried out with capillaries of 10, 25, and 50 μm internal diameter. It was confirmed that capillaries of narrow internal diameters require higher buffer concentrations for best sensitivities. For all diameters it was found that electrodispersion was the most pronounced band-broadening factor for relatively long residence times. For shorter times, Joule heating related band broadening appears to be the most significant factor, which means that best separation efficiencies are obtained with the narrowest capillaries. As detection limits are as good for capillaries of 10 μm internal diameters as for the other diameters when using contactless conductivity detection, these narrow capillaries are, therefore, generally of benefit when employing this detection technique. Hydrodyamic flow was found to have only a very limited effect on band broadening; an effect was only noticeable for the 50 μm capillary and relatively high flow rates.

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.

An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining and isolated from tissue sections using LCM for FASP and proteomic analysis. Using this method, we isolated a small population of CD90⁺ stem-like cells from glioblastoma multiforme tissue sections and identified 674 high-confidence (false discovery rate < 0.01) proteins from 32 nL of CD90⁺ cells by LC-MS/MS using an Orbitrap Elite mass spectrometer. We further quantified the relative abundance of proteins identified from equal volumes of LCM-captured CD90⁺ and CD90⁻ cells, where 109 differentially expressed proteins were identified. The major group of these differentially expressed proteins was relevant to cell adhesion and cellular movement. This ILFAPT method has demonstrated the ability to provide in-depth proteome analysis of a very small specific cell population within tissues. It can be broadly applied to the study of target cell populations within clinical specimens.

Fabry condition, a lysosomal storage disease (LSD) is characterized by the absence or reduction of the α-galactosidase A activity. Recently, a new diagnostic method for detection of α-galactosidase activity from dried blood spots (DBS) using a chemical substrate and quantification of reaction mixture was developed. To improve this method in the terms of automation, reproducibility, sensitivity, and data reliability, we introduce here an innovative analytical approach based on chip-nanoESI MS. The α-galactosidase assay products derived from DBS of 11 healthy donors and 11 Fabry disease patients were analyzed by NanoMate robot coupled to a high-capacity ion trap MS. Confirmation and structural analysis of the reaction products was achieved by CID and electron transfer dissociation (ETD) MS/MS. The cleavage of a substrate GLA-S generated a product, GLA-P, which was quantified related to an internal standard GLA-IS. Comparative patient versus control analysis indicated a 13-fold reduction in GLA-P/GLA-IS ratio in the case of the patients. Moreover, our method provided direct data on the enzyme, from which it was for the first time possible to discriminate between the patients lacking the enzyme and those presenting a less active one. GLA-IS and GLA-P were confirmed by CID/ETD, which applied together, increased considerably the sequence coverage and provided complementary information for unambiguous product identification. The present chip-nanoESI CID and ETD MSⁿ strategy introduced here for first time in LSD diagnosis, provided a maximum confidence in assay product identification, a high sensitivity, speed of analysis, and result reproducibility.

In this study, we determined serum levels of metallothioneins (MTs) and zinc in children with solid tumours (neuroblastoma, Hodgkin lymphoma, medulloblastoma, osteosarcoma, Ewing sarcoma and nephroblastoma) by differential pulse voltammetry Brdicka reaction and ELISA. Zn(II) level in patients sera was 40% compared to controls, contrariwise, MT level was 4.2 × higher in patients. No significant differences among single diagnoses were found both for Zn(II) and MT. When determined Zn(II)/MT ratio, in controls its value was 24.6, but it was 2.6 in patients. After Western-blotting with anti-MT and anti-Zn chicken antibodies, variable intensities of the bands within the samples were observed. The brightness curve obtained for each sample both for MT- and Zn blots was further analysed to produce a list of band positions together with some complementary information related to the intensity of the observed bands by the optimised algorithm. We constructed from those profiles decision trees that enable to distinguish different groups of tumours. The blood samples were heat-treated, in which we supposed mainly MT, but samples contained other thermostable Zn-containing proteins that were helpful for identification of embryonal tumours with 88% accuracy and for identification of sarcomas with 78% accuracy. In MT blots the accuracies were 53 and 45%, respectively. Simultaneous analysis of MT and Zn blots did not increased accuracy of identification neither in embryonal tumours (80%) nor in sarcomas. Those results are promising not only from diagnostic point of view but particularly in the area of studying of individual MT isoforms and their aggregates in malignant tumours.

This paper reports the use of novel phenyl-hexyl core-shell particles packed into fused silica capillaries in nano-LC. Capillary columns of different id of 25, 50, 75, 100, and 150 μm were packed employing the slurry packing method. The columns were used for the separation of a model mixture containing five aromatic hydrocarbons. Benzene, toluene, ethylbenzene, n-propylbenzene, and n-butylbenzene were separated utilizing an isocratic elution mode. Mixtures of water/ACN at different ratio were studied to find optimal experimental conditions for baseline separation of all sample components. As expected with this novel stationary phase, an RP chromatographic mechanism was observed. A mixture of water/ACN, 30:70, v/v allowed the complete resolution of the studied analytes. Efficiency increased by decreasing the capillary id recording the highest number of plates per meter with capillaries of 25 μm id. The decrease of the column id also resulted in a flatter dependence of the plate numbers on the linear flow rate of the mobile phase allowing the increase of the flow rate of the mobile phase without significant decrease of efficiency.

A highly sensitive capillary electrophoretic profiling of nicotine (NIC) and nornicotine (NNIC) was developed and applied to mushrooms with amperometric detection (AD). Effects of the experimental factors including detection potential, separation parameters, and sample pretreatment conditions were investigated. Under the optimal conditions, the electrophoretic analysis of NIC and NNIC was achieved within 8 min on a pencil carbon disc working electrode at 0.95 V, which was lower than those reported previously. Good calibration curves were obtained in 0.01–2.0 μg/mL and 0.02–3.0 μg/mL with the LOD of 2 ng/mL and 5 ng/mL for NIC and NNIC, respectively. The feasibility of the resultant method was verified. Average recoveries of different fortified levels ranged in 80.7–86.0% and 94.0–98.6% for NNIC and NIC were gained, respectively. Applied to a range of mushrooms (Boletus edulis and Lentinus edodes), the NIC contained naturally was successfully found in the level of 19.71–79.20 μg/kg. The results obtained with CE-AD method were acceptable and close to that of HPLC-MS.

Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.

Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100–500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100–500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100–200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR.

This review, compiled for celebrating the decennial of the series of issues of Electrophoresis on bioanalysis, does not cover much heri (yesterday in Latin), since this was extensively described by my previous review Electrophoresis 2004, 25, 2111–2127, nor, for that matter cras (tomorrow in Latin) since making predictions on future scenarios is decidedly a nonscientific endeavor, but is concentrated onto hodie (today in Latin). Recent progress in the following fields is covered: SDS electrophoresis; IEF and the unraveling of the chemistry and composition of soluble carrier ampholytes; 2D maps; CE with microchip technology and some coatings of unique performance; chemistry and function of combinatorial peptide ligand libraries. This latter technique is becoming fundamental in the detection and identification of low-abundance proteins in proteome analysis.

The one-carbon cycle is composed of four major biologically important molecules: methionine (l-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (l-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of l-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle.

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10⁴–1.5 × 10⁶ Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.

Hydrophobically modified quaternized celluloses (HMQCs), containing the quaternary ammonium groups and hexadecyl groups, were homogeneously synthesized as novel dynamic coatings for CE. Compared with quaternized cellulose coating, HMQC coating is able to generate stronger reversed EOF (ca. 10% increase) and has better efficiency in suppressing protein adsorption. The effects of the polymer concentration, the degree of hydrophobic substitution, and the buffer pH on the EOF mobility as well as on the separation of basic proteins were investigated in detail. It was shown that the use of HMQC coating could obviously improve the separation performance of basic proteins within a broad pH range. Five basic proteins (lysozyme, ribonuclease A, cytochrome c, bovine pancreatic trypsin inhibitor, and chymotrypsinogen) could be completely separated at pH 8.0. The successful performance of HMQC was further demonstrated by the analyses of lysozyme in tear and urine samples.

Off-line SPE and CE coupled with electrochemical detection have been used for the determination of bisphenol A (BPA), bisphenol F, 4-ethylphenol, and bisphenol A diglycidyl ether in bottled drinking water. The use of boron-doped diamond electrode as an electrochemical detector in amperometric mode that provides a favorable analytical performance for detecting these endocrine-disrupting compounds, such as lower noise levels, higher peak resolution with enhanced sensitivity, and improved resistance against electrode passivation. The oxidative electrochemical detection of the endocrine-disrupting compounds was accomplished by boron-doped diamond electrode poised at +1.4 V versus Ag/AgCl without electrode pretreatment. An off-line SPE procedure (Bond Elut® C18 SPE cartridge) was utilized to extract and preconcentrate the compounds prior to separation and detection. The minimum concentration detectable for all four compounds ranged from 0.01 to 0.06 μM, having S/N equal to three. After exposing the plastic bottle water container under sunlight for 7 days, the estimated concentration of BPA in the bottled drinking water was estimated to be 0.03 μM. This proposed approach has great potential for rapid and effective determination of BPA content present in water packaging of plastic bottles that have been exposed to sunlight for an extended period of time.

A simple procedure for preparing inexpensive paper-based three-electrode electrochemical cells is described here. They consist of small circular pads of hydrophilic paper defined by hydrophobic barriers printed on paper with wax-based ink. The back face of these pads is insulated by thermally laminating a polyethylene layer and working, reference and counter electrodes are drawn on paper by using commercial pencil leads. At last, a controlled volume of sample containing a supporting electrolyte was laid to soak in paper channels. Their performance was evaluated by assaying these devices as both simple cells suitable for recording voltammograms on static samples and low-cost detectors for flowing systems. Voltammetric tests, conducted by using potassium hexacyanoferrate(II) as model prototype, were also exploited for identifying the brand and softness of graphite sticks enabling paper to be marked with lines displaying the best conductivity. By taking advantage of the satisfactory information thus gained, pencil drawn electrodes were tested as amperometric detectors for the separation of ascorbic acid and sunset yellow, which were chosen as prototype electroactive analytes because they are frequently present concomitantly in several food matrices, such as soft drinks and fruit juices. This separation was performed by planar thin layer chromatography conducted on microfluidic paper-based devices prepared by patterning on filter paper two longitudinal hydrophobic barriers, once again printed with wax-based ink. Factors affecting both separation and electrochemical detection were examined and optimised, with best performance achieved by using a 20 mM acetate running buffer (pH 4.5) and by applying a detection potential of 0.9 V. Under these optimum conditions, the target analytes could be separated and detected within 6 min. The recorded peaks were well separated and characterized by good repeatability and fairly good sensitivity, thus proving that this approach is indeed suitable for rapidly assembling inexpensive and reliable electrochemical detectors for flow analysis systems.

The prevalent use of silver nanoparticles (AgNPs) in commercial goods has brought forth an urgent need for environmental salvation. With the global river systems being contaminated by AgNPs, fast and efficient detection systems are needed to trace the presence of AgNPs in common water to prevent detrimental effects to the public health. In this work, the detection of AgNPs via electrochemical oxidation has been achieved on a “Lab-on-chip” platform. This platform provides a fast, convenient, and portable detection system for the detection of AgNPs in common water.

A magnet-assisted alignment device was designed and fabricated for the amperometric detection of CE. It mainly consisted of a magnet-containing electrode holder, a capillary-based microdisc detection electrode, a detection cell, and a micrometer adjuster. To demonstrate the feasibility and performance of the alignment device, it was used in combination with a carbon nanotube/polypropylene (CNT/PP) composite electrode for the determination of p-phenylenediamine, m-aminophenol, and m-dihydroxybenzene in commercial hair dye by CE. The CNT-based electrode was fabricated by packing a melt mixture of CNTs and PP in a piece of fused silica capillary under heat, offering significantly lower operating potentials, substantially enhanced signal-to-noise characteristics, and high resistance to surface fouling. Because magnetic force was employed to move the detection electrode, the alignment system was significantly simplified. It is characterized by simple design and fabrication, high alignment reproducibility, reduced alignment time, and low cost. Both the alignment device and the CNT/PP composite electrode should find a wide range of applications in microchip CE, flowing injection analysis, and other microfluidic analysis systems.

A fused-silica capillary with a common outer diameter, 360 μm, but containing seven internal channels, each 28 μm in diameter (a multichannel capillary), has been tested on electrophoretic separations of mixtures of dopamine, adrenaline, and noradrenaline, using a contactless conductivity and UV photometric detection. It has been demonstrated that the sensitivity of the detection of these neurotransmitters in multichannel capillary, in comparison with those obtained for a standard singlechannel capillary with similar cross-sectional area, is comparable to that for the contactless conductivity and is about 50% higher for the UV photometry. The sensitivity is increased without loss of the separation efficiency, in contrast to UV detection with bubble cell. Further possibilities of using a multichannel capillary are demonstrated on separations of mixtures of inorganic cations (K⁺, Ba²⁺, Na⁺, Mg²⁺, and Li⁺) and mixtures of glucose and ribose. The main advantage of multi-channel capillary in comparison with a singlechannel capillary with the same cross-sectional area becomes apparent in separations in background electrolytes of high conductivity.

A highly sensitive capillary isotachophoresis method with LIF detection for microbial analysis was developed. This allowed the reliable analysis of Escherichia coli bacteria with a LOD of 14 cells in a sample volume of 100 μL, or 1.35 × 10² cell/mL, which is 47 times lower than reported by CE-LIF and 148 times lower than CE-UV with on-line concentration. A leading electrolyte of 50 mM Tris-HCl was used while the cells were diluted in 5 mM Tris HEPES as the terminator. To facilitate detection, cells were stained with the universal nucleic acid fluorophore SYTO 9. Continuous electrokinetic injection of the cells from the terminator under field amplified conditions concentrated cells into a single peak at the leader/terminator boundary allowing quantitation by measurement of peak height. The method was applied to water collected from two local streams, with only filtration through a 5-μm syringe filter to remove large particulate matter followed by a ten times dilution in terminator, with total analysis time approximately 40 min. The detected cell numbers in the water samples by the isotachophoresis method were 3.70 × 10⁵ cell/mL and 2.62 × 10⁴ cell/mL, which were slightly higher than the 9.50 × 10⁴ cell/mL and 1.96 × 10⁴ cell/mL obtained by conventional microbiological plate counting.

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS-PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA-F8 (anti-MHC slow/I), SC-71 (anti-MHC 2a and 2x), 10F5 (anti-MHC 2b), and BF-35 (anti-MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS-PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS-PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.

The analysis of drugs of abuse in microfluidic devices has the potential to provide solutions to today's on-site analysis challenges. The use of such devices has not been limited to miniaturising conventional analytical methods used routinely in forensic laboratories; new and interesting approaches have been implemented in microfluidics and benefit from the ability to control minute amounts of liquids in the small channels. The microfluidic platforms developed so far have been used successfully to carry out single or multiple analytical processes and offer a great opportunity for new technologies for on-site drug testing.

Microemulsion electrokinetic chromatography (MEEKC) is a CE separation technique, which utilizes buffered microemulsions as the separation media. In the past two decades, MEEKC has blossomed into a powerful separation technique for the analysis of a wide range of compounds. Pseudostationary phase composition is so critical to successful resolution in EKC, and several variables could be optimized including surfactant/co-surfactant/oil type and concentration, buffer content, and pH value. Additionally, MEEKC coupled with online sample preconcentration approaches could significantly improve the detection sensitivity. This review comprehensively describes the development of MEEKC from the period 1991 to 2012. Areas covered include basic theory, microemulsion composition, improving resolution and enhancing sensitivity methods, detection techniques, and applications of MEEKC.

The review gives an update about the methodological and instrumental developments in micellar electrokinetic capillary chromatography as a type of CE analytical technique. Here, the last two years development of the technique are particularly presented. Recent approaches to improve sensitivity are discussed. Newly introduced concentration techniques and experimental methods for verification of the different mechanisms and processes of micellar electrokinetic chromatography analysis are highlighted. A theoretical model to explain changes in separation and electrophoretic mobility order of fully charged analytes are demonstrated. Modern approaches for improving compatibility of micellar electrokinetic capillary chromatography to mass spectrometry are also reported.

A new MEKC method with large-volume sample stacking and polarity switching was developed for on-line preconcentration and detection of sulfonylurea herbicide (SUH) residues in cereals, including nicosulfuron (NS), thifensulfuon (methyl) (TFM), tribenuron-methly (TBM), sulfometuron-methyl (SMM), pyrazosulfuron-ethyl (PSE), and chlorimuron-ethyl (CME). In order to achieve a high resolution and enrichment factor, several parameters were optimized, such as the pH of the running buffer, the concentration of the BGE and the SDS, the separate voltage, the sample size, the pH, and the electrolyte concentration of the sample. The optimal running buffer was composed of 30 mM borate and 80 mM SDS at pH 7.0. The borate concentration in the sample was 30 mM and the pH value of the sample was the same as that of the running buffer. The concentrating voltage and the separating voltage were –15 kV and 15 kV, respectively. The sample size was 1.455 kPa × 780 s (33.11 cm). Under the optimum conditions, for NS, TFM, TBM, SMM, PSE, and CME, the enrichment factors were 613, 642, 835, 570, 709, and 599; the LODs were 0.29–0.50 ng/g, 0.22–0.36 ng/g, 0.60–0.89 ng/g, 0.39–0.72 ng/g, 0.28–0.56 ng/g, and 0.31–0.57 ng/g; the LOQs of six SUHs were all 5 ng/g; the average recoveries of the spiked sample were 86.68–92.99%, 80.73–93.65%, 81.49–94.40%, 82.97–95.1%, 82.96–98.84%, and 80.41–92.94%, respectively.

A new trimethylamine amination polychloromethyl styrene nanolatex (TMAPL) and TMAPL coated capillary column (ccc-TMAPL) were successfully prepared. The TMAPL coating was characterized with reversed steady EOF values of ca. ‒16.8 × 10⁻⁵ cm² V⁻¹ s⁻¹. It was applied to establish open-tubular (OT) CEC and field-amplified sample stacking (FASS) OT-CEC methods for the determination of bromate in tap water. Compared to OT-CEC, the LOD with FASS-OT-CEC was improved from 80 to 8 ng/mL. The developed FASS-OT-CEC method was practically used for the analysis of bromate in tap water samples with recoveries ranging from 93.6 to 103.5%.

Sweeping, an on-line sample concentration technique in CE, is the picking and accumulation of analytes by the pseudostationary phase or complexing additive. In the presence of an electric field, the analytes concentrated at the additive front that initially penetrated the sample zone. Here, we describe the sweeping of cationic alprenolol enantiomers using sulfated β-CD and organic solvent. The separation solution contained the anionic additive while ACN was in the sample solution. With fused silica capillaries, positive polarity, and solutions buffered at pH 3, the direction of the enantiomers' effective electrophoretic mobility was the same as the electrophoretic mobility (or electrophoretic mobility without additive). When the amount of ACN in the sample was increased (i.e. 60%), the interaction between the analytes and additive became negligible. This caused the sweeping boundary to shift from the electrophoretically moving β-CD front to the zone between the sample and separation solution. The equation that described the narrowing of injected sample zone was derived. The performance of sweeping with 60% ACN in the sample was then studied under different operating conditions (e.g. type of injection, injection time, and CD concentration). The low interaction between enantiomers and additive gave only moderate increases in sensitivity (approximately tenfold), but was improved when field enhancement was used during electrokinetic injection. With a conductivity difference (separation/sample solution) of 70 and a short injection time of 30 s at 20 kV, peak improvements of >100-fold was easily achieved.

The content of α-hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl-malic, dl-tartaric and dl-isocitric acids, and citric acid. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α-hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.

In this study, the dissolution of polysaccharides into an ionic liquid was investigated and applied as a coating onto the capillary walls of a fused-silica capillary in open-tubular CEC. The coating was evaluated by examining the chiral separation of two analytes (thiopental, sotalol) with three cellulose derivatives (cellulose acetate, cellulose acetate phthalate, and cellulose acetate butyrate). Baseline separation of thiopental enantiomers was achieved by use of each polysaccharide coating (Rs: 7.0, 8.1, 7.1), while sotalol provided partial resolution (Rs: 0.7, 1.0, 0.9). In addition, reproducibility of the cellulose-coated capillaries was evaluated by estimating the run-to-run and capillary-to-capillary RSD values of the EOF. Both stability and reproducibility were very good with RSD values of less than 7%.

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open-tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle-coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d,l-tryptophan (dl-Trp) was performed in monolayer BSA-modified capillary and PS/BSA nanoparticle-coated columns. The results indicated that the nanoparticle-modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl-Trp (within 3 min) was achieved with the PS/BSA-immobilized column using an electroosmotic pump-assisted CEC. Enantiomer separations of other compounds like dl-tyrosine and warfarin were also achieved with the column. Besides, run-to-run and column-to-column repeatabilities of the PS/BSA-coated column in the chiral separation were systematically introduced.

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

A novel pH-responsive coating technique was developed and applied to CE successfully in this paper. The coating was formed by bonding mixed opposite charge poly(acrylic acid) and poly(2-vinylpyridine) randomly onto the inner wall of a silica capillary. The coating processes were first characterized by ellipsometry and atomic force microscopy at macroscale and microscale, respectively. Measurements of EOF were implemented to confirm the coating. Direction and velocity of EOF became controllable from negative to positive, showing a perfect sigmoidal curve as the coating net charges alternated by the pH of BGE. The control of the EOF makes it possible to analyze different kinds of small molecules, peptides, and proteins successfully in the same capillary. Results showed that the stability and reproducibility for separations of fluoroquinolone standards were satisfactory for more than a hundred separations. A series of basic and acidic protein standards were separated with admirable efficiency and minimal adsorption using both polarities. The separation of tryptic BSA digest showed that the prepared capillary has immense potential in analyzing a single sample with both acidic and basic separations, which achieved the expectation in proteomics study by CE-MS.

The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up- and down-regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.

Traditionally, CE with SDS (CE-SDS) places many restrictions on sample composition. Requirements include low salt content, known initial sample concentration, and a narrow window of final sample concentration. As these restrictions require buffer exchange for many sample types, sample preparation is often tedious and yields poor sample recoveries. To improve capacity and streamline sample preparation, an automated robotic platform was developed using the PhyNexus Micro-Extractor Automated Instrument (MEA) for both the reduced and nonreduced CE-SDS assays. This automated sample preparation normalizes sample concentration, removes salts and other contaminants, and adds the required CE-SDS reagents, essentially eliminating manual steps during sample preparation. Fc-fusion proteins and monoclonal antibodies were used in this work to demonstrate benefits of this approach when compared to the manual method. With optimized conditions, this application has demonstrated decreased analyst “hands on” time and reduced total assay time. Sample recovery greater than 90% can be achieved, regardless of initial composition and concentration of analyte.

To extend the application of molecularly imprinted polymers, the dual-templates molecularly imprinted monolithic columns were developed in a capillary format. Two templates serotonin and histamine were simultaneously imprinted using two different functional monomers such as methacrylic acid (MAA) and methylenesuccinic acid (MSA) in a mixture of ethylene glycol dimethacrylate (EDMA) as a cross-linker and AIBN as polymerization initiator dissolved in DMF as porogen. The resulting molecular imprinted polymers (MIPs) were characterized based on their performance in the CEC separation of two imprinted templates. The optimization parameters such as pH, ACN composition, and concentration of the eluent were varied to achieve best resolution and efficiency for CEC separation of templates with each MIP column. It was found that the MIP monolith column fabricated using MSA offered better resolution and separation efficiency compared to column fabricated with MAA. This work utilized the dual-templates imprinting approach successfully and broadens the scope of multi-templates imprinting capabilities in capillary format in CEC application.

This is the first time when protein-imprinted polymers are prepared with “pending templates.” The polymers were synthesized in the presence of a real sample (chicken egg white), rather than any known commercial proteins. Compared with a simultaneously synthesized nonimprinted control polymer, the polymers show higher adsorption capacity for abundant components (as “pending templates”) in the original sample. Chromatography experiments indicated that the columns made of the imprinted polymers could retain abundant species (imprinted) and separate them from those not imprinted. Thus, the sample could be split into dimidiate subfractions with reduced complexities. “Pending template imprinting” suggests a new way to investigate molecular imprinting, especially to dissect, simplify, and analyze complicated samples through a series of polymers just imprinted by the samples per se.

A new design for a compact portable lab-on-a-chip instrument based on MCE and dual capacitively coupled contactless conductivity detection (dC⁴D) is described. The instrument is battery powered with total dimension of 14 × 25 × 8 cm³ (w × l × h), and weighs 1.2 kg. The device consists of a front electrophoresis compartment which has the chip holder and the chip, the associated high-voltage electrodes for electrophoresis injection and separation and the detector. The detection cell is integrated into the device housing with an exchangeable plug-and-play cartridge format. The design of the dC⁴D cell has been optimized for maximum performance. The cartridge includes the top–bottom excitation and pick up electrodes incorporated into the cell and connected to push-pull self-latching pins that are insulated with plastic. The metal frame of the cartridge is grounded completely to eliminate electronic interferences. The cartridge is designed to clamp a thin fluidic chip at the detection point. The cartridges are replaceable whereby different cartridges have different detection electrode configurations to employ according to the sensitivity or resolution needed in the specific analytical application. The second compartment consists of all the electronics, data acquisition card, high-voltage modules of up to ±5 kV both polarity, and batteries for 10 h of operation. The improved detector performance is illustrated by the electrophoresis analysis of six cations (NH₄⁺, K⁺, Ca²⁺, Na⁺, Mg²⁺, Li⁺) with a detection limit of approximately 5 μM and the analysis of the anions (Br⁻, Cl⁻, NO₂⁻, NO₃⁻, SO₄²⁻, F⁻) with a detection limit of about 3 μM. Analytical capabilities of the instrument for food and medical applications were evaluated by simultaneous detection of organic and inorganic acids in fruit juice and inorganic cations and anions in rabbit blood samples and human urine samples are also demonstrated.

This work demonstrates the use of thin film heaters to enhance electrothermal pumping in microfluidic systems. Thin film heating electrothermal pumping is more efficient than Joule heating alone. Numerical simulations of an asymmetric electrode array are performed to demonstrate the advantages of incorporating thin film heaters. This specific simulation shows that thin film heater electrothermal pumping provides approximately two and one-half times more volumetric flow than Joule heating alone for the same input power to both systems. In addition, external heating allows for electrothermal pumping to be applicable to low conductivity media.

Dielectrophoresis is a versatile tool for the sorting, immobilization, and characterization of cells in microfluidic systems. The performance of dielectrophoretic systems strongly relies on the configuration of microelectrodes, which produce a nonuniform electric field. However, once fabricated, the microelectrodes cannot be reconfigured to change the characteristics of the system. Here, we show that the reorientation of the microfluidic channel with respect to the microelectrodes can be readily utilized to alter the characteristics of the system. This enables us to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, change the deflection pattern of nonviable cells along the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. We demonstrate that the reorientation of the microfluidic channel is an effective tool to create versatile dielectrophoretic platforms using the same microelectrode design.

We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

This paper presents a method for fast and simultaneous determination of diclofenac (DCF) and its common counter-ions (potassium, sodium, and diethylammonium) using CE with capacitively coupled contactless conductivity detection (CE-C⁴D). On the basis of a single electropherogram (about 50 s), the proposed method allows the determination of the stoichiometry, absolute quantification and evaluation of the degradation degree of the active pharmaceutical ingredient (DCF). A linear working range from 100 to 500 μmol/L was obtained for all analytes in an equimolar TRIS/TAPS (10 mmol/L) solution as the background electrolyte as well as adequate LOD (7, 6, 7, and 10 μmol/L for K⁺, Na⁺, diethylammonium, and DCF, respectively). The proposed method was applied to the analysis of pharmaceutical formulations (tablets and spray form) with similar results to those achieved by HPLC (DCF) or flame photometry (K and Na) at a 95% confidence level.

Determination of pregabalin in urine samples was carried out by nonaqueous CE with TOF-MS via ESI, with a mixture of 10 mM ammonium formate and 0.05% acetic acid in methanol. By using TOF-MS, accurate mass information was obtained, thus causing a great improvement in qualitative ability. In order to avoid ionic suppression, urine samples dilution 1:10 was used. This was the only treatment to urine samples before the injection. Despite this dilution, the detection limit was as low as 0.03 μg/mL for pregabalin. The method was validated with respect to accuracy, precision, and linearity, LOD, and LOQ. This method was applied to the analysis of urine samples from seven different cancer patients undergoing treatment with pregabalin. The developed method may find wide application for the routine determination of pregabalin in biological samples in order to establish a more efficient and safe dosage.