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Current COVID-19 antigen testing is primarily carried out by obtaining a specimen via nasopharyngeal swab and performing a rapid lateral flow immunoassay (LFIA) or related immunoassays. On average, a nasopharyngeal antigen-based LFIA for COVID-19 remains positive for approximately one week from symptom onset, and levels of infectivity and duration of the symptoms may depend primarily on carrying a high viral load enough to infect others. It has been proposed that patients with long-COVID, a syndrome in which patients continue to have complications of COVID with ongoing symptoms, may have occurring viral replication, despite testing negative via rapid COVID antigen testing.We therefore propose a modified antigen-based method that exposes hidden or masked antigenic sites of viral specimens, or lingering fragments of viral proteins, present in sputum using a home-based rapid immunoassay for COVID-19. Almost all protocols for testing were performed according to LFIA kit manufacturer’s instructions for the detection of SARS-CoV-2 nucleocapsid protein antigen, one of the most predominant proteins encoded by the SARS-CoV-2 virus. However, in challenging the manufacturer instructions, one or more digestive enzymes and a detergent were added to the collected biosamples (nasopharyngeal, oropharyngeal, saliva, buccal, gargle, and sputum); this modified procedure expose hidden or masked antigenic sites of the coronavirus or cross-reactant antigenic sites of related or non-related viruses, or some of the plethora of epitopes generated by the sample corresponding microbiota to accomplish an optimal binding to the commercial antibody used in the diagnostic test. The modified protocol can enhance detection sensitivity by making the resultant test band in sputum samples visible, that would otherwise not be seen, and consequently may generate a false negative result, in a nasopharyngeal sample from a patient with mild symptoms of COVID-19 and/or low viral load. Therefore, a need exists for an improved sample pre-treatment extraction procedure that allows optimization of sample preparation to attain a more accurate test result. Although the experiments described here were performed using commercial platforms, with antibodies directed to SARS-CoV-2 nucleocapsid antigen, this method may also be viable for the detection of any other pathogen in sputum by using antibodies directed to the key antigens present in the pathogen of interest. Furthermore, this modified method to expose the content of sputum can be used as a simple protocol to study the sputome, the proteome of sputum, and other omics (sputomics). In summary, this simple method is non-invasive, rapid, inexpensive, accurate, and may provide increased sensitivity and specificity in the detection of COVID-19 antigens for several weeks or even months.

Over the years, multiple biomarkers have been used to aid in disease screening, diagnosis, prognosis, and response to therapy. As of late, protein biomarkers are gaining strength in their role for early disease diagnosis and prognosis in part due to the advancements in identification and characterization of a distinct functional pool of proteins known as proteoforms. Proteoforms are defined as all of the different molecular forms of a protein derived from a single gene caused by genetic variations, alternative spliced RNA transcripts and post-translational modifications. Monitoring the structural changes of each proteoform of a particular protein is essential to elucidate the complex molecular mechanisms that guide the course of disease. Clinical proteomics therefore holds the potential to offer further insight into disease pathology, progression, and prevention. Nevertheless, more technologically advanced diagnostic methods are needed to improve the reliability and clinical applicability of proteomics in preventive medicine. In this manuscript, we review the use of immunoaffinity capillary electrophoresis (IACE) as an emerging powerful diagnostic tool to isolate, separate, detect and characterize proteoform biomarkers obtained from liquid biopsy. IACE is an affinity capture-separation technology capable of isolating, concentrating and analyzing a wide range of biomarkers present in biological fluids. Isolation and concentration of target analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (CE) coupled to one or more detectors. IACE has the potential to generate rapid results with significant accuracy, leading to reliability and reproducibility in diagnosing and monitoring disease. Additionally, IACE has the capability of monitoring the efficacy of therapeutic agents by quantifying companion and complementary protein biomarkers. With advancements in telemedicine and artificial intelligence, the implementation of proteoform biomarker detection and analysis may significantly improve our capacity to identify medical conditions early and intervene in ways that improve health outcomes for individuals and populations.

In this paper is described the implementation of the frequency-dependent line model (FD-Line) in a real-time digital power system simulator. The main goal with such development is to describe a general procedure to incorporate new realistic models of power system components in modern real-time simulators based on the Electromagnetic Transients Program (EMTP). In this procedure are described, firstly, the steps to obtain the time domain solution of the differential equations that models the electromagnetic behavior in multi-phase transmission lines with frequency dependent parameters. After, the algorithmic solution of the FD-Line model is implemented in Simulink environment, through an S-function programmed in C language, for running off-line simulations of electromagnetic transients. This implementation allows the free assembling of the FD Line model with any element of the Power System Blockset library and also, it can be used to build any network topology. The main advantage of having a power network built in Simulink is that can be executed in real-time by means of the commercial eMEGAsim simulator. Finally, several simulation cases are presented to validate the accuracy and the real-time performance of the FD-Line model. En este artículo se describe la implementación del modelo de línea dependiente de la frecuencia FD-Line en un simulador digital en tiempo-real de sistemas eléctricos de potencia. El objetivo principal con este desarrollo es describir un procedimiento general para incorporar nuevos modelos realistas de componentes de sistemas eléctricos de potencia en los modernos simuladores de tiempo real basados en el programa de transitorios electromagnéticos (o EMTP, por sus siglas en inglés). En dicho procedimiento se describen, primeramente, los pasos para obtener la solución en el dominio del tiempo de las ecuaciones diferenciales que modelan el comportamiento electromagnético de una línea de transmisión polifásica con parámetros dependientes de la frecuencia. Posteriormente, se implementa la solución algorítmica del modelo de línea en Simulink, por medio de una función S en lenguaje C, para ejecutar simulaciones fuera de línea de transitorios electromagnéticos. Esta implementación permite ensamblar libremente el modelo FD-Line con cualquier elemento de la librería de sistemas de potencia en Simulink y también, se puede construir cualquier topología de red eléctrica. La principal ventaja de tener una red eléctrica construida en Simulink es que puede ser ejecutada en tiempo real por medio del simulador comercial eMEGAsim. Finalmente, se presentan varios casos de simulación de sistemas de potencia para probar la precisión y el desempeño en tiempo-real del modelo FD-Line.

In the last few years, there has been a greater appreciation by the scientific community of how separation science has contributed to the advancement of biomedical research. Despite past contributions in facilitating several biomedical breakthroughs, separation sciences still urgently need the development of improved methods for the separation and detection of biological and chemical substances. In particular, the challenging task of quantifying small molecules and biomolecules, found in low abundance in complex matrices (e.g., serum), is a particular area in need of new high-efficiency techniques. The tandem or on-line coupling of highly selective antibody capture agents with the high-resolving power of CE is being recognized as a powerful analytical tool for the enrichment and quantification of ultra-low abundance analytes in complex matrices. This development will have a significant impact on the identification and characterization of many putative biomarkers and on biomedical research in general. Immunoaffinity CE (IACE) technology is rapidly emerging as the most promising method for the analysis of low-abundance biomarkers; its power comes from a three-step procedure: (i) bioselective adsorption and (ii) subsequent recovery of compounds from an immobilized affinity ligand followed by (iii) separation of the enriched compounds. This technology is highly suited to automation and can be engineered to as a multiplex instrument capable of routinely performing hundreds of assays per day. Furthermore, a significant enhancement in sensitivity can be achieved for the purified and enriched affinity targeted analytes. Thus, a compound that exists in a complex biological matrix at a concentration far below its LOD is easily brought to well within its range of quantification. The present review summarizes several applications of IACE, as well as a chronological description of the improvements made in the fabrication of the analyte concentrator-microreactor device leading to the development of a multidimensional biomarker analyzer.

Over the years, analytical chemistry and immunology have contributed significantly to the field of clinical diagnosis by introducing quantitative techniques that can detect crucial and distinct chemical, biochemical and cellular biomarkers present in biosamples. Currently, quantitative two-dimensional hybrid immuno-analytical separation technologies are emerging as powerful tools for the sequential isolation, separation and detection of protein panels, including those with subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. One such technique to perform this challenging task is immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies and/or other affinity ligands as highly selective capture agents with the superior resolving power of capillary electrophoresis. Since affinity ligands can be polyreactive, i.e., binding and capturing more than one molecule, they may generate false positive results when tested under mono-dimensional procedures; one such application is enzyme-linked immunosorbent assay (ELISA). IACE, on the other hand, is a two-dimensional technique that captures (isolation and enrichment), releases, separates and detects (quantification, identification and characterization) a single or a panel of analytes from a sample, when coupled to one or more detectors simultaneously, without the presence of false positive or false negative data. This disruptive technique, capable of preconcentrate on-line results in enhanced sensitivity even in the analysis of complex matrices, may change the traditional system of testing biomarkers to obtain more accurate diagnosis of diseases, ideally before symptoms of a specific disease manifest. In this manuscript, we will present examples of the determination of biomarkers by IACE and the design of a miniaturized multi-dimensional IACE apparatus capable of improved sensitivity, specificity and throughput, with the potential of being used as a point-of-care instrument and holding promise for precision medicine and P4 medicine.

The caffeine content of foods and beverages varies considerably, interfering with our ability to obtain valid interpretations in many human studies with regard to the mechanism of action(s) of caffeine and/or its metabolites. The rate of metabolism of caffeine and other xanthine drugs also varies greatly from one individual to another. Therefore, it is extremely important to develop accurate, reliable analytical methods to quantify caffeine and its metabolites in simple and complex matrixes. A simple method is described for the separation and characterization of caffeine and its major metabolites employing capillary electrophoresis (CE) coupled to ultraviolet-absorption and mass spectrometry (MS) detection. After optimization of the electrophoresis separation conditions, a reliable separation of caffeine and 11 of its major metabolites was achieved in 50 mM ammonium carbonate buffer, pH 11.0. The volatile aqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by MS. The CE method achieved baseline resolution for all 12 compounds in less than 30 min. The CE-MS method is suitable for use as a routine procedure for the rapid separation and characterization of caffeine and its metabolites. The usefulness of this method was demonstrated by the extraction, separation, and identification of caffeine and its 11 metabolites from normal urine samples. The urine specimens were first acidified to obtain optimum binding efficiency to the sorbents of the off-line, solid-phase extraction procedure employed here, and an acidified eluent solvent was employed for the desorption step to maximize the recovery of the bound analytes.

Macro-hybrid penalized finite element approximations are studied for steady filtration problems with seawater intrusion. On the basis of nonoverlapping domain decompositions with vertical interfaces, sections of coastal aquifers are decomposed into subsystems with simpler geometries and small scales, interconnected via transmission conditions of pressure and flux continuity. Corresponding local penalized formulations are derived from the global penalized variational formulation of the two-free boundary flow problem, with continuity transmission conditions modelled variationally in a dual sense. Then, macro-hybrid finite element approximations are derived for the system, defined on independent subdomain grids. Parallel relaxation penalty-duality algorithms are proposed from fixed-point problem characterizations. Numerical experiments exemplify the macro-hybrid penalized theory, showing a good agreement with previous primal conforming penalized finite element approximations (Comput. Methods Appl. Mech. Engng. 2000; 190:609–624). Copyright © 2005 John Wiley & Sons, Ltd.

Parallel proximal-point algorithms for mixed finite element models of flow in the subsurface are presented. The applied methodology corresponds to operator splitting and non-overlapping domain decomposition methods, combined with resolvent or proximation characterizations and proximal-point algorithms. In this manner, macro-hybrid mixed finite element models are systematically derived, in general being globally non-conforming on the basis of non-matching grids. Also, iterative algorithms of the Uzawa penalty–duality type are produced, which are well-conditioned and robust. These parallel iterative algorithms generalize optimization and augmented Lagrangian procedures, and can be interpreted as implicit and semi-implicit numerical time marching schemes. Copyright © 2004 John Wiley & Sons, Ltd.

A simple solid-phase microextraction device was fabricated for use in on-line immunoaffinity capillary electrophoresis (CE). The device, designed in the form of a four-part cross-shaped or cruciform configuration, includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes) permitting the confinement of beads or suitable microstructures. The surface of the beads in the analyte concentrator carried a molecular recognition adsorbing chemical or affinity ligand material. The improved cruciform configuration of the analyte concentrator-microreactor device, designed for use in on-line immunoaffinity CE, enables it to specifically trap, enrich, and elute an analyte from any biological fluid or tissue sample extract without any sample pretreatment except filtration, centrifugation, and/or dilution allowing the separation and characterization of target analyte(s) with improved speed, sensitivity, and lower cost than existing techniques. As a model system, Fab' fragments derived from a purified immunoglobulin G (IgG) antibody were covalently bound to controlled-porosity glass and used as constituents of the analyte-microreactor device. The high-specificity polyclonal antibodies employed in these experiments were individually raised against the acidic nonsteroidal anti-inflammatory drugs ibuprofen and naproxen, and the neuropeptides angiotensin II, and neurotensin. These compounds, which were present in simple and complex matrices were captured by and eluted from the analyte concentrator-microreactor using a 50 mM sodium tetraborate buffer solution, pH 9.0, followed by a 100 nL plug of 300 mM glycine buffer, pH 3.4. Two analyte concentrators were tested independently: one containing Fab' fragments derived from antibodies raised against ibuprofen and naproxen; the other containing Fab' fragments derived from antibodies raised against angiotensin II and neurotensin. Each resulting electropherogram demonstrated the presence of two eluted materials in less than 20 min. Immunoaffinity CE performed in a cruciform structure was simpler and faster than previously reported in the literature using on-line microextraction devices designed in a linear format. The new concentration-separation system operated consistently for many runs, maintaining reproducible migration times and peak areas for every analyte studied.

A simple method for the separation and characterization of a group of nine basic compounds, comprising seven tricyclic antidepressant and two bronchodilator drugs, by nonaqueous capillary electrophoresis (NACE) employing ultraviolet and mass spectrometry detection is described. After optimization of the electrophoresis separation conditions, including the compositions of the electrolyte and the organic solvent, a reliable separation of all nine basic analytes was achieved in 80 mM ammonium formate dissolved in a methanol-acetonitrite (80:20 v/v) mixture, having an apparent pH of 8.7. The volatile nonaqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by mass spectrometry. When results were compared with reversed-phase gradient and isocratic high-performance liquid chromatography (HPLC) methods, the NACE method provided greater efficiency, achieving baseline resolution for all nine basic compounds in less than 30 min. The NACE method is suitable for use as a routine procedure for the rapid separation and characterization of basic compounds and is a viable alternative to HPLC for the separation of a wide range of pharmaceutical drugs.

Numerical estimation of Lyapunov exponents in non-linear dynamical systems results in a very high computational cost. This is due to the large-scale computational cost of several Runge–Kutta problems that need to be calculated. In this work we introduce a parallel implementation based on MPI (Message Passing Interface) for the calculation of the Lyapunov exponents for a multidimensional dynamical system, considering a weakly coupled algorithm. Since we work on an academic high-latency cluster interconnected with a gigabit switch, the design has to be oriented to reduce the number of messages required. With the design introduced in this work, the computing time is drastically reduced, and the obtained performance leads to close to optimal speed-up ratios. The implemented parallelisation allows us to carry out many experiments for the calculation of several Lyapunov exponents with a low-cost cluster. The numerical experiments showed a high scalability, which we showed with up to 68 cores.

A new prototype of contactless conductivity detector, smaller and easier to operate than the former version, is described. For a fused-silica capillary with 142-microm wall thickness and voltages up to 25 kV, it can be placed at the low- or high-voltage end of the column. This feature allowed implementation of an apparatus with sample introduction at the grounded end of the column. The input signal is an important parameter for determining the signal-to-noise ratio (S/N) of the detection system. An optimization procedure of its amplitude and frequency is proposed. Although the SIN must be determined by introduction of actual samples, the operating conditions can be optimized merely by changing the signal parameters and by using a mathematical procedure. Thus, an easy and fast optimization routine can be carried out. Mathematical and instrumental backgrounds are discussed, and experimental support of the technique's effectiveness is presented.

Advances in instrumentation and methodologies are urgently needed to achieve, rapid, simultaneous and sensitive determination of multiple substances found at a wide range of concentrations in biological fluids, tissues and cells. The application of immunoaffinity capillary electrophoresis in life sciences is already having an impact on the quantification of many biomarkers for diagnosis and monitoring the prognosis of diseases. This review explains how immunoaffinity capillary electrophoresis, the combination of highly selective antibody capture agents with the high resolving power of capillary electrophoresis, can provide highly specific assays leading to the selective isolation, concentration, separation and quantification of analytes of interest in complex biological matrices. In addition to a discussion of the technology, some applications of clinical and pharmaceutical relevance will be presented.

Much attention has recently been directed to the development and application of on-line sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes. Properly bound to a solid support, an “affinity ligand” can selectively adsorb a “target analyte” found in a simple or complex mixture at a wide range of concentrations. As a result, the isolated analyte is enriched and highly purified. When this affinity technique, allowing noncovalent chemical interactions and biochemical reactions to occur, is coupled on-line to high-resolution capillary electrophoresis and mass spectrometry, a powerful tool of chemical and biological information is created. This paper describes the concept of biological recognition and affinity interaction on-line with high-resolution separation, the fabrication of an “analyte concentrator-microreactor”, optimization conditions of adsorption and desorption, the coupling to mass spectrometry, and various applications of clinical and pharmaceutical interest.

Capillary electrochromatography (CEC) is an emerging high-resolution separation technique that combines features of both capillary electrophoresis (CE) and micro-high-performance liquid chromatography (HPLC). CEC is capable of resolving neutral and ionic species with very high separation efficiencies because of its high selectivity and versatile properties. The difficulties in preparing reliable CEC columns and using CEC with regular CE instruments for routine analysis has limited its application. Nevertheless, many recent improvements of the technique, optimization of method development, and the potential use in pharmaceutical analysis and bioanalysis has made CEC a technique that is rapidly gaining acceptance. In this report, we have developed a CEC method for the rapid separation of a contraceptive drug substance, norgestimate, and its related degradation compounds. Total analysis time is reduced by more than 50% when compared with the current in-house HPLC method. The relative standard deviations for both the retention time and peak areas are less than 2%. Effects of other experimental parameters on the separation characteristics of norgestimate using CEC, such as effects of solvent composition, solvent strength, and buffer concentration, are discussed.

The use of capillary electrophoresis (CE) for clinically relevant assays is attractive since it often presents many advantages over contemporary methods. The small-diameter tubing that holds the separation medium has led to the development of multicapillary instruments, and simultaneous sample analysis. Furthermore, CE is compatible with a wide range of detectors, including UV-Vis, fluorescence, laser-induced fluorescence, electrochemistry, mass spectrometry, radiometric, and more recently nuclear magnetic resonance, and laser-induced circular dichroism systems. Selection of an appropriate detector can yield highly specific analyte detection with good mass sensitivity. Another attractive feature of CE is the low consumption of sample and reagents. However, it is paradoxical that this advantage also leads to severe limitation, namely poor concentration sensitivity. Often high analyte concentrations are required in order to have injection of sufficient material for detection. In this regard, a series of devices that are broadly termed 'analyte concentrators' have been developed for analyte preconcentration on-line with the CE capillary. These devices have been used primarily for non-specific analyte preconcentration using packing material of the C18 type. Alternatively, the use of very specific antibody-containing cartridges and enzyme-immobilized microreactors have been demonstrated. In the current report, we review the likely impact of the technology of capillary electrophoresis and the role of the CE analyte concentrator-microreactor on the analysis of biomolecules, present on complex matrices, in a clinical laboratory. Specific examples of the direct analysis of physiologically-derived fluids and microdialysates are presented, and a personal view of the future of CE in the clinical environment is given.

The techniques of capillary electrophoresis (CE) are often the methods of choice for the analysis of complex mixtures of chemically similar molecules. Their applicability to studies across many diverse disciplines is quite remarkable. However, poor concentration limits of detection of CE methods often preclude their use for the analysis of dilute analyte mixture. This limitation has been addressed by the development of analyte concentrator and membrane preconcentration cartridges. These devices are used to insert an appropriate adsorptive phase into the inlet of the CE capillary, to enable the analysis of samples amounts that are in vast excess of the total capillary volume. An ability to perform on-line microreactions is a further advantage afforded by these devices. In this report, we present all attempted approaches at on-line preconcentration and microreaction with analyte separation by CE. We discuss the potential merits of each technique and describe our views of the future developments of analyte concentrator and membrane preconcentration technologies.

A method to perform on-line sample preconcentration of serum immunoglobulin E by affinity capture is described. Purified anti-IgE antibodies were covalently bound to an analyte concentrator-reaction chamber or cartridge. The immunoglobulins (IgE) were bound to and eluted from the cartridge by the optimum dissociating buffer system, and the eluent(s) were then subjected to capillary electrophoresis. The first design used was a 5 mm solid-phase cartridge fabricated by assembling a bundle of multiple microcapillaries in which a monoclonal antibody directed against IgE was covalently bound to the surface of every microcapillary. The whole assembly was connected, through sleeve connectors, to the capillary column for affinity capillary electrophoresis. The second design used consisted of an analyte concentrator-reaction chamber that was fabricated from a solid rod of glass. Several small diameter passages or through holes containing a similar surface area was tested for the same experiments and perfor...

Diethylenetriaminepentaacetic anhydride (DTPA) conjugated to IgG (DTPA-IgG) and labeled with 111 In is useful for detecting focal sites of infection and inflammation (R.H. Rubin, A.J. Fischman, R.J. Callahan, B. Khaw, F. Keech, M. Ahmad, R. Wilkinson and H.W. Strauss, 111 In-labeled nonspecific immunoglobulin scanning in the detection of focal infection, N. Engl. J. Med., 321 (1989) 935–940). MACROSCINT ® contains DTPA-IgG formulated as a lyophile from a citrate buffer containing maltose. Exposure of both reconstituted and lyophilized MACROSCINT ® to intense light resulted in degradation primarily via formation of precipitating aggregates. However, lyophilized and reconstituted MACROSCINT ® responded differently to thermal stress. Reconstituted MACROSCINT ® subjected to thermal stress (65°C) also degraded through formation of precipitating aggregates. In contrast, exposure of lyophilized MACROSCINT ® to thermal stress (65°C) resulted primarily in an increase in the molecular size of the MACROSCINT ® DTPA-IgG monomer. This increase in molecular size was a function of both the moisture content in the vial and the amount of time for which the sample was stressed, but was not a function of the conjugation with DTPA. Monosaccharide analysis of the samples demonstrated that this increase in molecular size corresponded to an increase in the amount of glucose covalently attached to the IgG. These data suggest that the increase in molecular size as a function of thermal stress is due to the covalent attachment of maltose, which is a glucose disaccharide present in the lyophile as an excipient, to the IgG. This degradation pathway was only observed in the lyophile.

Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a collinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10−10M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10−12M) of this compound in biological samples should be possible. Fourth, narrow -bore capillaries (5–10 μm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.

A method to measure glutamate in brain dialysates was developed. Nanomolar concentration of glutamate labeled with naphthal-ene-2,3-dicarboxaldehyde (NDA) was measured by using capillary electrophoresis and laser-induced fluorescence detection. The limit of mass detection was in the zeptomole concentration (10−21 M) range. The technique was linear between 10−5 M to 10−8 M. It was possible to reduce the migration time of brain dialysate glutamate to 60 seconds when capillaries of 15 μm inside diameter were used. The potential of capillary electrophoresis coupled to laser-induced fluorescence detection for in vivo monitoring of neurotransmitters is discussed.

The link between brain and reproduction is the release of the decapeptide luteinizing hormone-releasing hormone (LHRH) from the brain median eminence (ME), a release modulated by other neuropeptides such as β-endorphine (BEND) and neuropeptide Y (NPY) impinging on the LHRH neuron. Analysis of brain perfusates is the metnod of choice to determine in vivo release from discrete brain areas. For this, push-pull cannula (PPC) and microdialysis sampling are the most prominent technologies. However, sample volume is a limiting issue when attempting to measure by radioimmunoassays (RIA) in vivo release of multiple neuropeptides from these sources, especially when neuropeptide concentration is low and the whole sample (100 μl PPC perfusate/10 min) has to be dedicated to measure only one neuropeptide. We developed a capillary electrophoresis (CE)-based assay for the simultaneous determination of LHRH, BEND and NPY in PPC samples from the ewe ME. This assay uses CE for the separation of these neuropeptides ...

Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long-term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. However, this can be challenging, in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below. This review highlights the need for state-of-the art methodologies, with high-sensitivity and high-throughput capabilities, for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology has failed to successfully bring this advancement to the patient's bedside. We present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases; in particular, we review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two-dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point-of-care biomarker analyzer in translational medicine.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.

Humanized anti-Tac monoclonal antibody has been characterized by both SDS-PAGE and isoelectric focusing using traditional polyacrylamide methods. In addition to these techniques, capillary electrophoresis has been adapted to isoelectric focusing in a liquid phase. The humanized antibody has a pl in the range of 8.2–9.0, a region in which precast polyacrylamide or agarose gels are not stable in the 7–10 pH range. Experiments have been conducted to generate gradients in the 3–10 pH range and these gradients have successfully separated differently charged subspecies of humanized anti-TAC. Because of the simplicity in generating a variety of gradients, the power of resolution in different modes, its high efficiency, and the minimum sample volume requirements, capillary electrophoresis may become the method of choice for the routine characterization of therapeutic monoclonal antibodies and their stability profiles.

A quantitative spectrophotometrc assay for the determination of proline and hydroxyproline is described. The assay is based on the derivatization of the imino acids with the fluorogenic chromophore fluorescamine and the separation of the derivatized analytes by capillary electrophoresis. Fluorescamine reacts efficiently with secondary amino acids to form nonfluorescent aminoenone type chromophores which are easily detected at the low UV region. The assay is simple, rapid, and offers a significant improvement in detection sensitivity when compared with standard colorimotric methods for determining these imino acids. Derivatization with fluorescamine also enhances the degree of resolution of the two imino acids, enabling baseline separation of the derivatized analytes.

A procedure to improve on-column fluorescence detection for capillary zone electrophoresis is reported. A fluorescence detector was built using an epillumination fluorescence microscope and an argon-ion air-cooled laser. The 488-nm line was isolated with a band pass filter to eliminate the ultraviolet line. A dichroic mirror which reflected wavelengths under 510 nm and a 0.75 numerical aperture (NA), 0.3 mm working distance objective of the microscope condensed the laser beam on a fused-silica capillary and a cross-shaped fluorescence spot was observed. The emitted light was collected with the same objective, filtered with a 520-nm high pass filter, a spatial filter and a notch filter, and focused on a photodetector. The photodetector was either a gallium-arsenide or a multialkali photomultiplier tube. The signal generated was fed to a current-to-voltage converter and registered on a strip chart recorder. The detector was tested with fluorescein-derivatized amino acids. For the fluorescein thiocarbamyl (FTC)-amino acids the limit of concentration detection varied according to the amino acid. Taking FTC-arginine as the best example, the limit of concentration detection (LOCD) was 3.75·10−2M. Assuming that 1 nl was injeted, the limit of mass detection (LOMD) was 3.75·10−3 amol or about 2250 molecules. This represents an improvement of about three orders of magnitude with respect to previous laser induced fluorescence on-column detection. The potential and advantages when using an epillumination fluorescence microscope with laser induced fluorescence as a detection system for capillary electrophoresis are discussed.

Several methods are available for determining the components present in a typical pharmaceutical dosage form. Although some of these methods are simple and have received wide pharmaceutical industry acceptance over many years, several limitations still need to be overcome. Capillary electrophoresis was examined as an alternative method for the determination of a recombinant cytokine in a pharmaceutical dosage form. The results show that capillary electrophoresis can separate the recombinant cytokine from its dosage form matrix adequately, rapidly and with minimum sample volume requirements. Owing to its high efficiency, high resolution and simplicity, capillary electrophoresis appears to be very applicable to pharmaceutical samples. For reliable quantification of the resulting data, however, it is essential to optimize the experimental conditions, such as separation buffer and capillary column temperature. The advantages and potential of capillary electrophoresis for the separation of active ingredients and excipients in pharmaceutical formulations are discussed.

Two methods were developed using capillary electrophoresis to concentrate urinary constituents in order to collect adequate amounts of separated analytes. the first method utilizes multiple capillaries arranged in bundles and coupled to a single capillary through a glass connector. the second method consists of the use of an analyte concentrator containing an antibody covalently bound to a solid support material. Both methods allow loading of increased amounts of samples into the capillary, and the continuous collection of purified material in nanogram-microgram quantities. the samples collected by the two methods were further analyzed by mass spectrometry.

A preparation of a highly purified monoclonal antibody was subjected to capillary electrophoretic determination. Through the use of multiple parallel capillaries it was possible to increase both the detector response and sample load in direct proportion to the number of capillaries. While the amount of antibody solution assessed by ultraviolet detection was in the range of tenths of μg ml −1 , values in the range of tenths of ng ml −1 were obtained using fluorescence detection. The results are discussed in terms of their practical values for biological samples, targeted for human consumption as pharmaceutical drugs.

Background: The specific forms of pro–B-type natriuretic peptide (proBNP) that occur in human blood are not yet clear. We demonstrated the presence of several proBNP forms in human plasma with a new affinity chromatography method that can be used in combination with nano–liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC–ESI–MS/MS). Methods: For affinity chromatography, we coupled Fab′ fragments of polyclonal sheep antibodies specific for N-terminal proBNP (NT-proBNP) epitope 1–21 to silica beads. We connected a column (10 mm × 0.8 mm inner diameter) packed with these beads to a trypsin reactor and used a preconcentrator in combination with a fritless nanospray column to perform MS analyses of proBNP forms in preextracted and non-preextracted samples of plasma from patients with severe heart failure (HF). We used Western blotting in deglycosylation experiments to confirm the shifts in proBNP and NT-proBNP masses. Results: Tandem MS experiments demonstrated the presence of both NT-proBNP and circulating proBNP in preextracted samples of plasma from patients with severe HF, and Western blotting analyses revealed 2 bands of approximately 23 kDa and 13 kDa that shifted after deglycosylation to positions that corresponded to the locations of recombinant proBNP and synthetic NT-proBNP. Conclusions: We obtained clear evidence for circulating proBNP in patients with severe HF and provided the first demonstration of O-glycosylation of NT-proBNP. The higher molecular masses for NT-proBNP and proBNP observed in the Western blotting analyses than those expected from calculations can be explained by O-glycosylation of these peptides in vivo.

A quantitative ultraviolet detection method for the determination of urinary metabolites is described using capillary zone electrophoresis. The determination of these metabolites is simple, fast. reproducible and utilizes very small amounts of sample. This method is linear between 1.0 × 10−4 and 1.0 × 10−2 M for creatinine and between 1.0 × 10−1 and 1.0 M for urea. The ultraviolet method shows detection limits for creatinine in the picogram (femtomol) range, and for urea in the nanogram (picomol) range.

A fluorescence detector for capillary electrophoresis was built using an epillumination fluorescence microscope equipped with a 50-W mercury lamp and a photodiode. The performance of the detector was tested with riboflavin and fluorescamine-derivatized amino acids and amphetamine. We compared fluorite and conventional glass objectives and found that, by using a 25-μm I.D. capillary and a fluorite objective, an improvement in fluorescence detection by a factor of 56 can be achieved. A detection level of 500 amol for riboflavin was reached (using on-column detection). This sensitivity was at least comparable to that reported for other capillary electrophoresis systems using on-column non-coherent light fluorescence detectors. The potential and advantages of using a fluorescence microscope for capillary electrophoresis applications are discussed.

The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C18 derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100 μL). The captured analytes were eluted in a small volume of an appropriate solution (20–50 nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25 mM of formic acid in MeCN):H2O and a sheath liquid of 20 mM of acetic acid in 50:50 (v/v) methanol:H2O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.

Several CE methodologies have been described for the analysis of rHuEPO in concentrated solutions, but the inherently limited concentration sensitivity of CE precludes the detection of EPO at the levels found in biological fluids. In this work, we have investigated an on-line immunoaffinity solid-phase extraction capillary electrophoresis (IA-CE) methodology for the selective preconcentration of EPO in diluted solutions. The preliminary results obtained using a custom-made immunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutaraldehyde-glass beads show the potential of this novel approach. The summarized findings are discussed in detail as a starting point for our ongoing investigations.

The separation of the glycoforms of erythropoietin (EPO) by capillary electrophoresis (CE) was recently published as a monograph by the European Pharmacopoeia (European Pharmacopoeia 4 2002, 1316, 1123-1128). Although the experimental CE conditions employed a background electrolyte containing additives suitable for on-line UV-absorption detection, they were not appropriate for on-line mass spectrometry (MS) detection. In this work, an attempt was made to investigate experimental conditions employing volatile electrolyte systems to achieve the separation and characterization of EPO glycoforms using CE and ESI-MS methodologies. The influence of several operating conditions, such as the coating of the internal walls of the capillary as well as the composition, concentration, and the pH of the separation buffer were investigated. The results demonstrated that when the internal walls of the capillaries were permanently coated with Polybrene and a buffer electrolyte containing 400 mM of HAc-NH4Ac (acetic acid-ammonium acetate), pH 4.75, was used, a significantly reproducible separation was achieved for EPO glycoforms. Intact EPO was characterized by two mass spectrometry techniques: electrospray ionization (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS). The data demonstrated that MALDI-TOF-MS provided a good approximation to an average molecular mass of the EPO molecule. However, it was still necessary to carry out further separation of the intact EPO glycoforms in order to obtain molecular mass information when ESI-MS was used.