New insights into the conversion of electropherograms to the effective electrophoretic mobility scale.

CE-MS is increasingly gaining momentum as an analytical tool in metabolomics, due to its ability to obtain information about the most polar elements in biological samples. This has been helped by improvements of robustness in peak identification by means of mobility-scale representations of the electropherograms (mobilograms). As a necessary step toward facilitating the use of CE-MS for untargeted metabolomics data, the authors previously developed and introduced ROMANCE, a software automating mobilogram generation for large untargeted datasets through a simple and self-contained user interface. Herein, we introduce a new version of ROMANCE including new features such as compatibility with other types of data (targeted MS data and 2D UV-Vis absorption-like electropherograms), and the much needed additional flexibility in the transformation parameters (including field ramping and the use of secondary markers), more measurement conditions (depending on detection and integration modes), and most importantly tackling the issue of quantitative peak conversion. First, we present a review of the current theoretical framework with regard to peak characterization, and we develop new formulas for multiple marker peak area corrections, for anticipating peak position precision, and for assessing peak shape distortion. Then, the new version of the software is presented and validated experimentally. We contrast the multiple marker mobility transformations with previous results, finding increased peak position precision, and finally we showcase an application to actual untargeted metabolomics data.

Quantitative CE analysis of punicalagin in Combretum aculeatum extracts traditionally used in Senegal for the treatment of tuberculosis.

Mycobacterium tuberculosis is the causative agent of tuberculosis, an infectious bacterial disease, which most commonly affects the lungs. In the search for novel active compounds or medicines against tuberculosis, an ethnopharmacological survey combined with a host-pathogen assay has recently highlighted the potency of an aqueous extract of Combretum aculeatum. C. aculeatum is used in traditional medicine and has demonstrated a significant in vitro antimycobacterial activity. Punicalagin, an ellagitannin, was isolated and found to be related to the biological activity of the extract. An analytical method for the evaluation of punicalagin in C. aculeatum was developed by capillary electrophoresis. After method optimization, the quantification of punicalagin was achieved for the evaluation of various plant extracts to determine the content of punicalagin related to the extraction modes and conditions, origin of the plant material, and harvesting period. The developed method demonstrated that the leaves presented the highest punicalagin content compared to the seeds and stems. A decoction of 30 min in boiling water was found to be the best extraction mode of C. aculeatum.

Evolution in the design of a low sheath-flow interface for CE-MS and application to biological samples.

Although several interfaces for CE-MS hyphenation are commercially available, the development of new versatile, simple and yet efficient and sensitive alternatives remains an important field of research. In a previous work, a simple low sheath-flow interface was developed from inexpensive parts. This interface features a design easy to build, maintain, and adapt to particular needs. The present work introduces an improved design of the previous interface. By reducing the diameter of the separation capillary and the emitter, a smaller Taylor cone is spontaneously formed, minimizing the zone dispersion while the analytes go through the interface and leading to less peak broadening associated to the ESI process. Numerical modeling allowed studying the mixing and diffusion processes taking place in the Taylor cone. The analytical performance of this new interface was tested with pharmaceutically relevant molecules and endogenous metabolites. The interface was eventually applied to the analysis of neural cell culture samples, allowing the identification of a panel of neurotransmission-related molecules. An excellent migration time repeatability was obtained (intra-day RSD <0.5% for most compounds, and <3.0% for inter-day precision). Most metabolites showed S/N ratios >10 with an injected volume of 6.7 nL of biological extract.

Determination of 16 antineoplastic drugs by capillary electrophoresis with UV detection: Applications in quality control.

Two capillary electrophoresis (CE) methods were developed for the analysis of 16 antineoplastic drugs contained in injectable pharmaceutical formulations. A capillary zone electrophoresis (CZE) method coupled to UV was developed with a background electrolyte (BGE) made of a 100 mM phosphate buffer at pH 2.5 containing 50% v/v of acetonitrile and dynamic coating of capillaries with Ceofix®. This method allowed the analysis of doxorubicin, epirubicin, idarubicin, daunorubicin, irinotecan, topotecan, vincristine, vindesine, vinblastine, and vinorelbine in less than 8 min. A micellar electrokinetic chromatography (MEKC) method coupled to UV was also developed for the determination of methotrexate, pemetrexed, etoposide, etoposide phosphate, fludarabine phosphate, and 5-fluorouracil. A run time of 16 min was obtained with a BGE made of 50 mM borate buffer at pH 9.2 with 80 mM of sodium dodecyl sulfate (SDS) and 20% v/v of acetonitrile. For both methods, the applied voltage was 30 kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in fused silica capillaries with an internal diameter of 50 µm and a total length of 64.5 cm. Both methods were validated and trueness values between 99.4 and 101.3% were obtained with repeatability and intermediate precision values of 0.5-1.8% for all drugs. These methods were found appropriate for controlling injectable pharmaceutical formulations containing antineoplastic drugs and successfully applied in quality control.

ROMANCE: A new software tool to improve data robustness and feature identification in CE-MS metabolomics.

The use of capillary electrophoresis coupled to mass spectrometry (CE-MS) in metabolomics remains an oddity compared to the widely adopted use of liquid chromatography. This technique is traditionally regarded as lacking the reproducibility to adequately identify metabolites by their migration times. The major reason is the variability of the velocity of the background electrolyte, mainly coming from shifts in the magnitude of the electroosmotic flow and from the suction caused by electrospray interfaces. The use of the effective electrophoretic mobility is one solution to overcome this issue as it is a characteristic feature of each compound. To date, such an approach has not been applied to metabolomics due to the complexity and size of CE-MS data obtained in such studies. In this paper, ROMANCE (RObust Metabolomic Analysis with Normalized CE) is introduced as a new software for CE-MS-based metabolomics. It allows the automated conversion of batches of CE-MS files with minimal user intervention. ROMANCE converts the x-axis of each MS file from the time into the effective mobility scale and the resulting files are already pseudo-aligned, present normalized peak areas and improved reproducibility, and can eventually follow existing metabolomic workflows. The software was developed in Scala, so it is multi-platform and computationally-efficient. It is available for download under a CC license. In this work, the versatility of ROMANCE was demonstrated by using data obtained in the same and in different laboratories, as well as its application to the analysis of human plasma samples.

Development of a New Extraction Device Based on Parallel-Electromembrane Extraction.

A new device for parallel-electromembrane extraction (Pa-EME) was developed to enable simultaneous and high-throughput extraction of ionic and ionizable compounds from biofluids. The new system is composed of a reusable conductive well-plate used as an acceptor compartment and a filtration well-plate used as a donor compartment. A design of experiments was implemented to optimize the main experimental parameters (agitation, voltage, and time) with standard solutions in formic acid 50 mM. The stirring rate was found the primary influent parameter. The Pa-EME device showed excellent extraction yields from 84% to 101% with RSD lower than 7.5% on model compounds. Optimized parameters were then applied to plasma samples and process efficiencies from 59% to 62% and RSD of less than 8.0% were obtained. The whole extraction process took less than 20 min to prepare 8 samples simultaneously, greatly enhancing the sample preparation throughput (<3 min per sample).

Sample preparation for polar metabolites in bioanalysis.

Sample preparation is a primary step of any bioanalytical workflow, especially in metabolomics analysis where maximum information has to be obtained without spoiling the analytical instrument. Because of their biological implication, highly polar metabolites, such as amino acids, nucleobases, and catecholamines seem to attract growing interest in the field of comprehensive metabolomics analysis although their extraction from the matrix remains a real challenge. In this paper, we discuss about the actual practice and issues of hydrophilic metabolites' extraction, including new solutions and perspectives to improve their phase transfer from a complex biological sample to a clean extract prior to analysis.

Dynamic-Electromembrane Extraction: A Technical Development for the Extraction of Neuropeptides.

In this work, a dynamic-electromembrane extraction (d-EME) device was developed for the extraction of neuropeptides. On the basis of a thin polypropylene hollow fiber (50 µm of wall-thickness and 280 µm i.d.), this setup allowed for a continual renewal of the acceptor compartment. Because of the reduced size of the device, high preconcentration factors were obtained (up to 50-fold). The extraction remained constant regardless of the extraction time (from 15 to 45 min); accordingly, this new setup minimized the effect of electrolysis on extraction performance while enabling high extraction yield (up to 72%) for most lipophilic neuropeptides.

Evaluation of a new low sheath-flow interface for CE-MS.

The current trend for increasing technical complexity in the field of CE-ESI-MS interfaces has incited for more accessible alternatives. In this work, a simple low sheath-flow ESI interface operating in the submicroliter nanospray regime without nebulizing gas assistance was evaluated. The use of sheath liquid enabled improving the ionization of the analytes, while the absence of nebulizing gas minimized sample dilution and loss of efficiency. After a rapid qualification, the effect of main operational parameters such as sheath liquid composition and flow rate, working distance and ESI potential was studied. Simulation of the mixing processes inside the Taylor cone proved its size to be of utmost importance in band broadening processes. As a proof of concept, the interface was eventually applied to a set of representative basic drugs analyzed by CE-TOF/MS. Limits of detection reached the 25-100 ppb range with suitable robustness and repeatability results. This design has demonstrated good performance while being simple and accessible to the user.

Static coatings for the analysis of intact monoclonal antibody drugs by capillary zone electrophoresis.

This work reports on the investigation of the optimal conditions associated with good isoform resolution, minimized adsorption, and acceptable repeatability for the analysis of intact monoclonal antibodies. To this end, various static capillary coatings were evaluated, and major trends were observed. For a positively charged coating, a low EOF mobility value was the main criterion for satisfactory resolution. For neutral coatings, adsorption was the main concern. If the coating does not fully cover the surface silanols, electrostatic interactions tend to decrease protein recovery through irreversible adsorption. The influence of the BGE pH was also investigated, as the resolution of monoclonal antibody isoforms is driven by their charge difference. The nature and concentration of the BGE were proven to be important, as an ampholytic compound such as ε-aminocaproic acid was found to decrease adsorption compared with ammonium acetate. With the optimal conditions determined during method development, a complete investigation according to four criteria, i.e. migration time, separation efficiency, EOF mobility, and protein recovery, did not present evidence of any adsorption. Finally, the repeatability and intermediate precision were assessed, and low variability was demonstrated with an RSD less than 1.3% for the main peak migration time.

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 applications.

Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling.

The hyphenation of capillary electrophoresis (CE) with mass spectrometry (MS) is a powerful method to obtain high efficient, sensitive, and selective analyses. The successful coupling with electrospray ionization (ESI) source requires closed electric circuits for both the CE separation and the ESI processes. A wide range of interfaces has been proposed to satisfy this requirement. Among them, the new high sensitivity porous sprayer based on a porous tip achieves the electric connection by inserting the capillary outlet made of a porous material into an ESI needle filled with a conductive liquid and independently grounded. This device is compatible with the minute flow rates exhibited in CE and therefore makes possible the use of a nano-electrospray behavior. In this work, this interface was evaluated for hyphenating a CE with a single quadrupole MS instrument for low molecular weight analytes. Investigations aimed at highlighting the most influent parameters thanks to a design of experiments, reaching the best performance in terms of sensitivity and stability. MS signal intensities of various pharmaceutical compounds (e.g. amphetamines, ß-blockers) emphasized high sensitivity and efficiency, while repeatability, expressed as relative standard deviation of corrected heights and areas, was suitable for quantitative purposes (<5%).

Characterization of drug-protein interactions by capillary electrophoresis hyphenated to mass spectrometry.

The demand for analytical techniques to evaluate and measure drug-plasma protein interactions continues to increase. The binding of drugs to plasma proteins is an important parameter to determine during the drug development process because it impacts both pharmacokinetics and pharmacodynamics. Among the numerous methods that have been proposed to perform such studies, CE in frontal analysis mode (CE/FA) is attractive because it consumes a relatively low amount of samples, is fast, and enables analyses under near-physiological conditions. Most CE/FA applications have been performed with UV detection and often lack sensitivity. In this study, CE was hyphenated to MS to enhance the sensitivity of the method and to evaluate strong drug-plasma protein interactions. To adapt the previously developed CE/FA-UV method to CE/FA-MS, different parameters were considered, such as the buffer composition, the rinsing step, and the ESI and MS parameters. The most critical aspect involved obtaining stable MS signals. Good results were achieved due to careful optimization of the ESI and MS parameters, among which the sheath liquid composition appeared to be the most significant. Interactions between six drugs and α(1) -acid glycoprotein and three drugs and BSA, including basic, neutral, and acidic drugs, were measured with the optimized CE/FA-MS method. The obtained affinity constants ranged from 1·10(-4) M(-1) to 2·10(-5) M(-1) and were in good agreement with the results that were obtained by CE/FA-UV and equilibrium dialysis.

New insights in pharmaceutical analysis.

The research unit of pharmaceutical analytical chemistry (PAC) has been active in the field of separation sciences for many years. Liquid chromatography (LC) and its latest improvements such as ultra-high performance chromatography (UHPLC) and supercritical fluid chromatography (SFC) are deeply and thoroughly studied, from a fundamental viewpoint to its various application capabilities. Electro-driven separations such as capillary electrophoresis (CE) are also a major field of interest, especially for macromolecules, and low cost. All these techniques are investigated with various detection modes including mass spectrometry (MS) for various applications where high sensitivity and selectivity is needed. Extracting the relevant information from the overwhelming amount of data generated by modern analytical platforms has become an important issue for knowledge discovery in various research fields. The appropriate treatment of such data is therefore of crucial importance to provide valuable information. Numerous works in our research group have demonstrated the usefulness of statistical and mathematical methodologies to improve quality of the results. Therefore, well-established chemometric approaches (e.g. design of experiments, multivariate data analysis, etc.) are implemented to optimize the analytical process from method development to data analysis.

Single-run separation of closely related cationic and anionic compounds by CE-ESI-MS: application to the simultaneous analysis of melamine and its analogs in milk.

In recent years, two adulteration incidents concerning the addition of melamine, a nitrogen-rich industrial small polar compound, to pet food and infant formula products have occurred in China. These issues prompted laboratories to develop methods for the analysis of melamine and related compounds in a wide variety of food products and ingredients. In this context, a CE-ESI-MS method was developed to simultaneously analyze melamine and its related products (ammeline, ammelide and cyanuric acid) that possess close physico-chemical properties. This method allows the simultaneous analysis of both cations and anions in a single run, using CE to divide the run into two time segments in normal polarity mode. For this purpose, ESI polarity was switched once during the run, increasing sensitivity and data quality. The method was applied to spiked powdered milk and melamine-contaminated powdered milk, with two sample preparation procedures.

Use of organic solvent to prevent protein adsorption in CE-MS experiments.

Protein adsorption onto capillary wall often hampers CE separations, particularly in the CZE mode. Electrostatic interactions are not the only factors affecting adsorption, as hydrophobic interactions and/or protein conformational changes are also involved in the adsorption phenomenon. Numerous methods can be used to reduce or avoid adsorption, such as (i) addition of low molecular weight molecules in the BGE, (ii) use of surfactants, or (iii) capillary coatings. However, most of these methods are not MS-compatible. In this study, we evaluated the addition of organic solvent as an alternative MS-compatible method to decrease protein adsorption. The effect of the solvent addition was emphasized using classical methods for estimating reversible and irreversible adsorption. In many cases, organic solvents were effective at decreasing adsorption. However, the influence of the organic solvent on protein adsorption should be evaluated case-by-case in CE method development.

Analysis of hemoglobin-based oxygen carriers by CE-UV/Vis and CE-ESI-TOF/MS.

Blood doping involves the use of products that enhance the uptake, transport, or delivery of oxygen to the blood. One approach uses artificial oxygen carriers, known as hemoglobin-based oxygen carriers (HBOCs). This study describes an analytical strategy based on CE for detecting intact HBOCs in plasma samples collected for doping control. On-capillary detection was performed by UV/Vis at 415 nm, which offered detection selectivity for hemoproteins (such as hemoglobin and HBOCs). On-line ESI-MS detection with a TOF analyzer was further used to provide accurate masses on CE peaks and to confirm the presence of HBOCs. An immunodepletion sample preparation step was mandatory prior to analysis, in order to remove most abundant proteins that interfered with CE separation and altered the ESI process. This analytical method was successfully applied to plasma samples enriched with Oxyglobin, a commercially available HBOC used for veterinary purposes. Detection limits of 0.20 and 0.45 g/dL were achieved in plasma for CE-UV/Vis at 415 nm and CE-ESI-TOF/MS, respectively.

High-throughput log P determination by MEEKC coupled with UV and MS detections.

A high-throughput screening method using MEEKC was developed for the determination of 1-octanol-water partition coefficients (log P(oct)). Two approaches were carried out to decrease determination times to about 20 min per compound: (i) a dynamically coated capillary was used to increase the EOF at low pH, allowing the measurement of log P(oct) of acidic compounds and (ii) a short-end injection was performed to reduce the capillary effective length. The analytical conditions were optimized to determine the lipophilicity of neutral, basic, and acidic compounds with log P(oct) ranging from 0 to 5. The developed method was first applied to a well-balanced set of 35 reference compounds, and second to a set of 21 acidic and 29 basic pharmaceutical compounds. Finally, determinations were achieved with MS detection, allowing a 20-fold throughput increase thanks to sample pooling. An atmospheric pressure photoionization source was selected to advantageously replace ESI as it was less affected by the non-volatile BGE additives used in MEEKC.

CE-ESI-TOF/MS for human growth hormone analysis.

CE is a powerful analytical tool used to separate intact biomolecules such as proteins. The coupling of CE with TOF/MS produces a very promising method that can be used to detect and identify proteins in different matrices. This paper describes an efficient, rapid, and simple CE-ESI-TOF/MS procedure for the analysis of endogenous human growth hormone and recombinant human growth hormone without sample preparation. Operational factors were optimized using an experimental design, and the method was successfully applied to distinguish human growth hormone and recombinant human growth hormone in unknown samples.

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.