The use of selective adsorbents in capillary electrophoresis-mass spectrometry for analyte preconcentration and microreactions: a powerful three-dimensional tool for multiple chemical and biological applications.

Much attention has recently been directed to the development and application of online 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.

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry.

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE-MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.

Monitoring the purity of a synthetic peptide by capillary electrophoresis: utilization of an on-line preconcentration method for improved separation and detection sensitivity.

To date, there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics utilizing peptides. The success of peptide pharmaceuticals has, however, been accompanied by some failures, both prior to entry and in the clinic. Progress has been made in various areas to improve the effectiveness of the final drug product. One major advance has been in the area of peptide synthesis and control of the purity of the peptide of interest. Recent advances in analytical instrumentation, including advances in capillary electrophoresis, have had a great impact on the ability to separate and detect low quantities of impurities and degradation products during the synthesis of a peptide drug. In this work, affinity capillary electrophoresis (ACE) was developed for the identification and characterization of a chemically synthesized peptide fragment of a snake toxin called fasciculin. The affinity capillary electrophoresis technology utilized in this study employed two powerful techniques coupled on-line for the direct and rapid determination of analytes in simple and complex matrices. The first technique, aimed for the nonselective extraction and concentration of one or more analytes of interest, utilizes a solid-phase analyte concentrator device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated target analyte(s), after elution from an analyte concentrator device. The on-line preconcentration step has proved valuable in terms of improving separation conditions as well as enhancing detection sensitivity values for the peptide fragment with a sensitivity increase ranging from 100- to 10,000-fold. Different types of analyte concentrator devices and a few binding-desorption conditions were tested. Bare and internal-wall-coated fused-silica capillaries were used. Comparative performance with HPLC in terms of selectivity and sensitivity is also discussed.

A comparative study of fish species identification by gel isoelectrofocusing two-dimensional gel electrophoresis, and capillary zone electrophoresis.

A comparative study of fish species identification was accomplished using three different electrophoretic techniques. Sarcoplasmic proteins were extracted from three related fish species and subjected to gel isoelectrofocusing (IEF), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and capillary zone electrophoresis (CZE). The fish species--Genypterus chilensis, Genypterus blacodes, and Genypterus maculatus--were from the Ophidiidae family. The three electrophoretic techniques provided suitable fish species identification. Nevertheless, CZE demonstrated several advantages over the other two conventional techniques. Some of the benefits include the use of small amounts of reagents; short separation times, permitting fast comparative analysis; data reproducibility; and ease with which the technique is performed.

Affinity capillary electrophoresis: important application areas and some recent developments.

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting ('plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen-antibody, hapten-antibody, lectin-sugar, drug-protein, and enzyme-substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.

New approaches in clinical chemistry: on-line analyte concentration and microreaction capillary electrophoresis for the determination of drugs, metabolic intermediates, and biopolymers in biological fluids.

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.

Enhancement of concentration limits of detection in CE and CE-MS: a review of on-line sample extraction, cleanup, analyte preconcentration, and microreactor technology.

The techniques of CE and on-line capillary electrophoresis-mass spectrometry (CE-MS) have been widely used for the analysis of many chemically diverse molecules. These methods of analysis allow analyte separations in aqueous solutions that are complementary to classical techniques such as HPLC and HPLC-MS. However, the one major limitation of CE is the fact that the best performance is normally obtained in analyzing small sample volumes (typically < 50 nL for a 50-micron-i.d. capillary). Ultimately, this leads to a relatively poor concentration limit of detection (CLOD) for CE and CE-MS when compared to that of HPLC or HPLC-MS. Recently, the analyte concentrator and membrane preconcentration cartridge have been described for use on-line with the CE capillary. Common to many of the approaches that led to the development of these devices is the incorporation of a suitable stationary phase at the inlet of the CE capillary. This relatively simple modification permits the introduction of much larger sample volumes (> 100 microL) into the CE capillary, and lowers both the CE and CE-MS CLOD. Detection of analytes present in complex mixtures at concentrations of < 200 fg/mL is reported to be possible when using such techniques in conjunction with CE and CE-MS. Additionally, the analyte concentrator has been developed to analyze microreactions on-line with the CE capillary. Recently, analyte derivatization and enzymatic protein digestion on-line with CE separations were reported. The purpose of this manuscript is to discuss and critically review these techniques and all described attempts at improving the CLOD of CE and CE-MS techniques using an adsorptive mechanism at the inlet of the CE capillary.

Degradation of lyophilized and reconstituted MACROSCINT (DTPA-IgG): precipitation vs. glucosylation.

Diethylenetriaminepentaacetic anhydride (DTPA) conjugated to IgG (DTPA-IgG) and labeled with 111In 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, 111In-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 degrees C) also degraded through formation of precipitating aggregates. In contrast, exposure of lyophilized MACROSCINT to thermal stress (65 degrees 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.