Microextraction techniques combined with capillary electrophoresis in bioanalysis

Over the past two decades, many environmentally sustainable sample-preparation techniques have been proposed, with the objective of reducing the use of toxic organic solvents or substituting these with environmentally friendly alternatives. Microextraction techniques (MEs), in which only a small amount of organic solvent is used, have several advantages, including reduced sample volume, analysis time, and operating costs. Thus, MEs are well adapted in bioanalysis, in which sample preparation is mandatory because of the complexity of a sample that is available in small quantities (mL or even μL only). Capillary electrophoresis (CE) is a powerful and efficient separation technique in which no organic solvents are required for analysis. Combination of CE with MEs is regarded as a very attractive environmentally sustainable analytical tool, and numerous applications have been reported over the last few decades for bioanalysis of low-molecular-weight compounds or for peptide analysis. In this paper we review the use of MEs combined with CE in bioanalysis. The review is divided into two sections: liquid and solid-based MEs. A brief practical and theoretical description of each ME is given, and the techniques are illustrated by relevant application

Rapid determination of p K a values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK a) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C4D) was used as an additional detection mode. The C4D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK a determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50mM. pK a values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK a values were in accordance with the literature. Figure a UV (1) and C4D (2) detectors placed on-line on the CE capillary. b Curve of effective mobility as a function of pH for histidin

Drug-protein binding: a critical review of analytical tools

The extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed. This review focuses on the most important approaches used to characterize drug-protein binding. A description of the principle of each method with its inherent strengths and weaknesses is outlined. The binding affinity ranges, information accessibility, material consumption, and throughput are compared for each method. Finally, a discussion is included to help users choose the most suitable approach from among the wealth of methods presented. Figure Range of binding constants (log Ka) assessable by the main separative and non-separative analytical tools used to characterize drug-protein interactions. ED: equilibrium dialysis, UF: ultrafiltration, PAMPA: parallel artificial membrane permeability assay, HPAC/ZE: high-performance affinity chromatography/zonal elution approach, HPAC/FA: high-performance affinity chromatography/frontal analysis approach, ACE: affinity capillary electrophoresis (mobility shift assay), CE/FA: capillary electrophoresis/frontal analysis, Spectro.: spectroscopic assays, ITC: isothermal titration calorimetry, comp.: competition studies, titration: titration studies, DSC: differential scanning calorimetry, SPR: surface plasmon resonance-based assay

Analyse de composés pharmaceutiques par électrophorèse capillaire couplée à des techniques de détection alternatives

La présente thèse présente le couplage de l'électrophorèse capillaire à différents systèmes de détection pour l'analyse de composés pharmaceutiques dans les fluides biologiques. Trois techniques de détection alternatives à la détection UV/Vis conventionnelle ont été couplées afin d'augmenter la sensibilité, la sélectivité et/ou d'étendre le domaine d'applications. Il s'agit de la spectrométrie de masse, de la fluorescence induite par laser et de l'électrochimie. La première section est constituée d'une introduction qui reporte les notions théoriques et bibliographiques essentielles concernant les différents couplages étudiés. La deuxième partie traite de l'ensemble des travaux effectués, ayant fait ou non l'objet de publications, et offre un résumé des résultats obtenus. La troisième section regroupe les différents articles rédigés en langue anglaise et publiés. Finalement, la dernière section comprend les conclusions et perpectives, ainsi que les annexes

Capillary Electrophoresis-Ultraviolet-Mass Spectrometry (CE-UV-MS) for the Simultaneous Determination and Quantification of Insulin Formulations

This chapter describes a CE-UV-MS method for the identification and quantification of insulin in pharmaceutical formulations in a single run. The CE conditions are optimized to avoid the adsorption of the protein onto the capillary wall. Particular attention is paid regarding the choice of the internal standard. A strategy based on multiple injections is applied to correct both ionization and injection variabilities. The methodoligy is validated according to internal guidelines and the obtained accuracy profile demonstrates the ability of the CE-UV-MS method to quantify insulin in pharmaceutical formulations within a 15% acceptance range. This strategy can be implemented in the field of quality control, as well as in the detection of counterfeits

Non-aqueous capillary electrophoresis for the analysis of acidic compounds using negative electrospray ionization mass spectrometry

Non-aqueous capillary electrophoresis (NACE) is an attractive CE mode, in which water solvent of the background electrolyte (BGE) is replaced by organic solvent or by a mixture of organic solvents. This substitution alters several parameters, such as the pKa, permittivity, viscosity, zeta potential, and conductivity, resulting in a modification of CE separation performance (i.e., selectivity and/or efficiency). In addition, the use of NACE is particularly well adapted to ESI-MS due to the high volatility of solvents and the low currents that are generated. Organic solvents reduce the number of side electrochemical reactions at the ESI tip, thereby allowing the stabilization of the ESI current and a decrease in background noise. All these features make NACE an interesting alternative to the aqueous capillary zone electrophoresis (CZE) mode, especially in combination with mass spectrometry (MS) detection. The aim of this work was to evaluate the use of NACE coupled to negative ESI-MS for the analysis of acidic compounds with two available CE-MS interfaces (sheath liquid and sheathless). First, NACE was compared to aqueous CZE for the analysis of several pharmaceutical acidic compounds (non-steroidal anti-inflammatory drugs, NSAIDs). Then, the separation performance and the sensitivity achieved by both interfaces were evaluated, as were the impact of the BGE and the sample composition. Finally, analyses of glucuronides in urine samples subjected to a minimal sample pre-treatment (“dilute-and-shoot”) were performed by NACE-ESI-MS, and the matrix effect was evaluated. A 20- to 100-fold improvement in sensitivity was achieved using the NACE mode in combination with the sheathless interface and no matrix effect was observed regardless of the interfaces

New supported liquid membrane for electromembrane extraction of polar basic endogenous metabolites

tExtraction of polar endogenous compounds remains an important issue in bioanalysis although differenttechniques have been evaluated. Among them, electromembrane extraction (EME) is a relevant approachbut supported liquid membranes (SLMs) dedicated to polar molecules are still lacking. In this study 22organic solvents were evaluated as SLMs on a set of 45 polar basic metabolites (log P from −5.7 to1.5) from various biochemical families. To investigate a large variety of organic solvents, a parallel elec-tromembrane extraction device was used and a constant current approach was applied to circumventthe heterogeneous conductivities of the different SLMs. Among the tested organic solvents, 2-nitrophenylpentyl ether (NPPE) appeared the most efficient SLM with the extraction of a large variety of polar cationicmetabolites, high extraction yields, and low extraction variabilities. The applied current and the compo-sition of the acceptor and donor solutions were also evaluated and 300 A per well and acetic acid 1%(v/v), both as acceptor and donor compartments, were the most efficient conditions. The new SLM and theoptimized experimental parameters were successfully applied to the extraction of precipitated plasmasamples. Although the extraction recovery decreased for most compounds in the biological matrix, pro-cess efficiency (PE) up to 90% and low extraction variability (RSD between 2 and 18%) were obtainedfor several very polar compounds such as choline or acetylcholine, emphasizing the potential of EME forpolar compounds

Capillary electrophoresis–electrospray ionization-mass spectrometry interfaces: Fundamental concepts and technical developments

Capillary electrophoresis (CE) hyphenated to electrospray ionization (ESI) mass spectrometry (MS) is a powerful tool for analyzing a wide variety of analytes in different matrices. The major issue with CE–ESIMS lies in finding a suitable and versatile interface to ensure the best CE and ESI operations. Thus, the development and improvement of CE–ESI-MS interfaces have been the subjects of much research. The first part of the present review focuses on the fundamental aspects of the three steps of the ESI process, i.e., spray formation, droplet evolution, and the production of gas-phase ions. In the second part of the review, the electrochemical reactions involved in the ESI and CE processes and their influences on the sensitivity and performance are discussed in detail. Then, the existing interfaces are divided into two major classes according to their operating flow rate (electrospray vs. nanospray regime). The particular characteristics of these two regimes are discussed by considering their practical impacts on ionization and the MS response. Finally, the current CE–ESI-MS interfaces are summarized, including their major advantages, drawbacks, and fields of application

Microextraction techniques combined with capillary electrophoresis in bioanalysis

Over the past two decades, many environmentally sustainable sample-preparation techniques have been proposed, with the objective of reducing the use of toxic organic solvents or substituting these with environmentally friendly alternatives. Microextraction techniques (MEs), in which only a small amount of organic solvent is used, have several advantages, including reduced sample volume, analysis time, and operating costs. Thus, MEs are well adapted in bioanalysis, in which sample preparation is mandatory because of the complexity of a sample that is available in small quantities (mL or even μL only). Capillary electrophoresis (CE) is a powerful and efficient separation technique in which no organic solvents are required for analysis. Combination of CE with MEs is regarded as a very attractive environmentally sustainable analytical tool, and numerous applications have been reported over the last few decades for bioanalysis of low-molecularweight compounds or for peptide analysis. In this paper we review the use of MEs combined with CE in bioanalysis. The review is divided into two sections: liquid and solid-based MEs. A brief practical and theoretical description of each ME is given, and the techniques are illustrated by relevant applications