Hyphenation Of Capillary Electrophoresis Research Articles

Speciation of selenium-containing small molecules in urine and cell lysate by CE-ICPMS with in-capillary enrichment

The quantitative speciation of selenium in biological systems is highly important for evaluating health status and elucidating transformations of Se species in physiological and pathological processes. Hyphenation of capillary electrophoresis with inductively coupled plasma mass spectrometry (CE-ICPMS) is promising for this purpose. However, the unfavorable or insufficient sensitivity for selenium analysis with CE-ICPMS seriously limits its practical applications in biological analysis, e.g., cell analysis. Therefore, it is crucial to improve the detection sensitivity for Se species. In this study, CE-ICPMS sensitivities for five selenium species (selenocystamine (SeA), methyl-2-acetamido-2-deoxy-1-seleno-β-d-galactopyranoside (SeSug 1), selenomethionine (SeMet), Se-Methylselenocysteine (MeSeCys) and selenocystine (SeCys)) were improved by in-capillary stacking via pH gradient between the zones of sample-leading buffer and the incorporation of isopropanol. The improvement on sensitivity of up to 9.9 folds was achieved in different biological samples, with LODs of 0.29–0.52 μg L−1. This approach was further applied for Se speciation in cell lysate, urine and culture medium. It showed that SeMet was more readily reduced in the medium and favorably accumulated by HepG2, HuH-7 and HCCLM3 cells with respect to SeSug 1 and MeSeCys. In cells, all the three Se species were largely transformed into other Se species. Furthermore, more than 70 % of SeMet reduced in medium was transformed into unknown Se species after 48-h interaction with cells.

Effective monitoring of Platinum-DNA adducts formation under simulated physiological conditions by CE-ICP-MS/MS

The leading Pt(II)-based anticancer drugs have been used for decades; however, chemotherapy with their application is burdened with severe side effects. The administration of compounds capable of DNA platination in the form of prodrugs has the potential to overcome the drawbacks associated with their use. Progress toward their clinical application depends on establishing proper methodologies that would allow assessing their ability to bind to DNA in the biological environment. Herein, we propose implementing the approach based on the hyphenation of capillary electrophoresis with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) for studying Pt-DNA adduct formation. The presented methodology opens the possibility to employ the multielement monitoring for studying the differences in the behavior of Pt(II) and Pt(IV) complexes and, interestingly, revealed the formation of various adducts with DNA and cytosol components for the latter one.

Applications of capillary electrophoresis for biopharmaceutical product characterization.

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is not surprising that CE-based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015-2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

Hyphenation of capillary electrophoresis with MC-ICP-MS – A novel tool for uranium age dating

Capillary electrophoresis (CE) was hyphenated to multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) to determine the model age of a highly enriched uranium (HEU) sample using the 234U/230Th radiochronometer. The use of hydroxymethylbutyric acid (HMBA) as the CE electrolyte was investigated, and a complexation stacking method was developed to increase the thorium signal obtained. The age of the material was determined by measuring the 230Th content of the HEU sample using isotope dilution in conjunction with the CE-MC-ICP-MS protocol. The CE-MC-ICP-MS protocol and a standard offline protocol using gravitational chromatography both gave results in accordance within uncertainties with the production date of the HEU sample (March 1965). Liquid waste production was only of a few microliters with the use of CE. The hyphenation of CE with MC-ICP-MS render the measurement of the age of the HEU material in less than one day possible. Obtaining results in a timely fashion is of particular importance for nuclear forensics studies.

A robust sheath-flow CE-MS interface for hyphenation with Orbitrap MS.

The hyphenation of capillary electrophoresis with high-resolution mass spectrometry, such as Orbitrap MS, is of broad interest for the unambiguous and exceptionally sensitive identification of compounds. However, the coupling of these techniques requires a robust ionization interface that does not influence the stability of the separation voltage while coping with oxidation of the emitter tip at large ionization voltages. Herein, we present the design of a sheath-flow CE-ESI-MS interface which combines a robust and easy to operate set-up with high-resolution Orbitrap MS detection. The sheath liquid interface is equipped with a gold coated electrospray emitter which increases the stability and overall lifetime of the system. For the characterization of the interface, the spray stability and durability were investigated in dependence of the sheath-flow rate, electrospray voltage, and additional gold coating. The optimized conditions were applied to a separation of angiotensin II and neurotensin resulting in LODs of 2.4 and 3.5ng/mL.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

Recent advances in capillary electrophoresis-mass spectrometry: Instrumentation, methodology and applications.

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point.

The hyphenation of capillary electrophoresis and mass spectrometry (CE/MS) remains a minor technique compared with liquid chromatography/mass spectrometry (LC/MS), which represents nowadays the standard instrumentation, regardless of its introduction thirty years ago. However, from a theoretical point of view, CE coupling should be quite favorable especially with electrospray ionization mass spectrometry (ESI-MS). At the time, the sensitivity provided by CE/MS was often limited, due to hyphenation requirements, which at some point appeared to disqualify CE/MS from benefiting from the performance gain driving the evolution of MS instruments. However, this context has been significantly modified in a matter of a few years. The development of innovative CE/MS interfacing systems has enabled an important improvement regarding sensitivity and reinforced robustness in order to provide an instrumentation accessible to the largest scientific community. Because of the unique selectivity delivered by the electrophoretic separation, CE/MS has proved to be particularly relevant for the analysis of biological molecules. The conjunction of these aspects is motivating the interest in CE/MS analysis and shows that CE/MS is mature enough to enrich the toolbox of analytical techniques for the analysis of complex biological samples. Here we discuss the characteristics of the major types of high-sensitivity CE/ESI-MS instrumentation and emphasize the late evolution and future positioning of CE/MS analysis for the characterization of biological molecules like peptides and proteins, through some pertinent applications.

Revealing cooperative binding of polycationic cyclodextrins with DNA oligomers by capillary electrophoresis coupled to mass spectrometry

Gene delivery is critical for the development of nucleic acid-based therapies against a range of severe diseases. The conception of non-viral (semi)synthetic vectors with low cytotoxicity and virus-like efficiency is gathering a lot of efforts, but it represents a fantastic challenge still far from accomplishment. Carbohydrate-based scaffolds offer interesting features towards this end, such as easy availability, relatively cheap cost, tuning properties and a good biocompatibility. The lack of analytical methods providing quantitative and qualitative data on their binding properties with oligonucleotides (DNA/RNA), with a minimal time and sample consumption, represents a limitation for these channels. Here, we attempted to fill the gap by hyphenation of capillary electrophoresis with mass spectrometry (CE-MS). This coupling strategy allows discriminating free and complexed DNA oligomers with cationic cyclodextrins (CDs), determining the stoichiometry where the highest observed is always DNAn: n/3(CD), and unambiguously assigning the partners through m/z detection. Very reliable data were obtained with migration time within 5.5 (standard deviation < 0.5%) and 25 min (standard deviation < 1.1%) for UV and MS detection, respectively. Furthermore, varying the nitrogen/phosphorus ratio (N/P), key parameters relating to the thermodynamics e.g. the micro and macroscopic dissociation constants Kd and KD, respectively (both in low μM range) and the Gibbs free energy ΔG (−16.3 to −26.9 kJ mol−1), and also the cooperativity as Hill number (nH between 0.98 and 15.75) of the supramolecular process can be delineated, providing a unique tool for the high throughput screening and selection of efficient gene delivery carriers.

The fate of differently functionalized gold nanorods in human serum: A response from capillary electrophoresis–inductively coupled plasma mass spectrometry

A highly selective and sensitive method was developed to characterize intrinsic intramolecular interactions between potential theranostic agents, gold nanorods (AuNRs), and plasma proteins. The method is based on a hyphenation of capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS), which enables monitoring the speciation changes of AuNRs under physiologically compatible conditions. To improve the separation resolution between the intact nanorods and different gold–protein conjugates, the CE system was optimized by varying the type and concentration of background electrolyte, applied voltage, and sample loading. Optimization allowed also for acquiring the acceptable figures of merit such as migration time and peak area precision of 4.7–8.2% and 5.1–6.3%, respectively, detection limits in the range of 5.5–5.7μgL−1 Au, and recoveries on the order of 91–99%. With the developed method the metal-specific profiles were recorded for differently functionalized AuNPs in combinations with individual serum proteins and in human serum. In case of carboxy-modified AuNPs, proteinization in real-serum environment occurs without albumin participation, apo-transferrin dominating the protein corona under equilibrium conditions. On the contrary, the AuNRs with surface amino-groups first form the albumin conjugate but albumin in this “soft” corona becomes slowly replaced by other, less abundant proteins, exhibiting a higher affinity toward the aminated surface.

Characterization of a Porous Nano-electrospray Capillary Emitter at Ultra-low Flow Rates

Biopharmaceuticals, especially therapeutic monoclonal antibodies, have emerged as a very promising new generation of protein-based drugs. However, their comprehensive analysis continues to pose new challenges for the bioanalytical field. Hyphenation of capillary electrophoresis with electrospray ionization (CE-MS) is a promising technique to address these challenges. One of the main advantages of CE-MS is the ability to produce stable electrospray at ultra-low flow rates (5-20 nl/min range). In this short communication we report on the characterization of a porous nano-electrospray capillary emitter focusing on the effects of ultra-low flow rate on ionization efficiency, ion suppression and detection sensitivity. Ion suppression effect of a poorly-ionizable sugar in the presence of an easily-ionized peptide was reduced by almost 2-fold. Intact therapeutic antibody infusion analysis demonstrated that MS detection sensitivity increased by an order of magnitude with the decrease of flow rate from 250 nL/min to 20 nL/min using the nano-electrospray capillary emitter.

CZE-CZE ESI-MS Coupling with a Fully Isolated Mechanical Valve.

The hyphenation of capillary electrophoresis and electrospray ionization-mass spectrometry is a powerful tool for peptide and protein analysis. It provides high separation power in combination with sensitive and selective detection and the possibility of analyte identification. Unfortunately, many proven capillary electrophoresis methods are not compatible with electrospray-mass spectrometry since several compounds of best separating background electrolytes are interfering in the electrospray ionization. Here, we describe a two-dimensional capillary electrophoresis system using the second dimension as a cleanup stage in order to remove interfering compounds to enable electrospray-mass spectrometry coupling.

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, β-blockers) emphasized high sensitivity and efficiency, while repeatability, expressed as relative standard deviation of corrected heights and areas, was suitable for quantitative purposes (<5%).

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Metal complexes stability constant determination by hyphenation of capillary electrophoresis with inductively coupled plasma mass spectrometry: The case of 1:1 metal-to-ligand stoichiometry

Nuclear energy development has raised new issues like radionuclides biogeochemistry. The modelling of their biochemical properties involves the accurate determination of thermodynamical data, like stability constants. This can be achieved by using hyphenated capillary electrophoresis (CE)–ICPMS and the method was applied successfully on 1:1 lanthanum–oxalate and uranyl–oxalate complexes. Several significant steps are discussed: choice of analytical conditions, electrophoretic mobility calculation, mathematical treatment of experimental data by using linear regressions, ligand concentration and ionic strength corrections. The following values were obtained with a good precision for lanthanum–oxalate and uranyl–oxalate complexes: log( K°(LaOxa +)) = 6.10 ± 0.10 and log( K°(UO 2Oxa)) = 6.40 ± 0.30, respectively, at infinite dilution. These values are consistent with the literature data, showing CE–ICPMS potential for metal complexes stability constants determination.

Utilization of capillary electrophoresis/mass spectrometry (CE/MSn) for the study of anthocyanin dyes

Hyphenation of capillary electrophoresis with electrospray ionization mass spectrometry was utilized for the monitoring of anthocyanins in wine and wine musts. CE/MS was performed in two electrolytes: 1) an acidic one (chloroacetate-ammonium, pH 2) and 2) a basic one with high selectivity towards derivatives containing vicinal hydroxy groups (borate-ammonium, pH 9). The setup of MS was optimized and the fragmentation of common anthocyanins was studied in detail. Attention was also focused on the fragmentation of anthocyanidin skeleton. The anthocyanidins substituted with hydroxy groups fragment via a cascade of neutral losses of water and carbon monoxide. Fragmentation of anthocyanidins containing a methoxy group on their B-ring starts with the cleavage of methane and/or methyl radical. The optimized method was utilized for the monitoring of changes in anthocyanin profile in red wines as well as the process of release of anthocyanins to wine must.

Effects of experimental parameters on the signal intensity of capillary electrophoresis electrospray ionization mass spectrometry in protein analysis

In recent years, the hyphenation of capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS) has been widely used for the analysis of biological molecules with high efficiency and high accuracy. However, the signal intensity of CE-ESI-MS is restricted by various parameters. This paper reports the effects on the CE-ESI-MS signal of the pH and the electrolyte concentration, the formic acid concentration of the sheath liquid, and the sheath liquid composition, using several proteins as samples. The study was performed systematically by experimental and theoretical analyses. The maximum signal intensity of three proteins (cytochrome c, insulin, bovine serum albumin) was attained with a pH 4.40 buffer containing 75 mM formic acid and 75 mM ammonium acetate. Investigation of the influence of the formic acid concentration in the sheath liquid (over the range of 0%–1%) on the ESI-MS signal of brovine serum albumin showed that the feasible amount of the formic acid in sheath liquid could improve the signal intensity of the sample ions. However, considerable band broadening was observed that should be attributed mainly to column overloading, band spreading at the interface, and scanning data acquisition.

Recent advances in capillary electrophoresis/electrospray-mass spectrometry.

In this review, the progress in hyphenation of capillary electrophoresis (CE) with electrospray ionization-mass spectrometry (ESI-MS) since the article of Banks (Banks, J. F., Electrophoresis 1997, 18, 2255-2266) is reported. In all capillary-based electromigration techniques, such as capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focussing (CIEF), micellar electrokinetic chromatography (MEKC), affinity capillary electrophoresis (ACE), as well as in the hybrid techniques capillary electrochromatography (CEC), and pressurized capillary electrochromatography (pCEC) progress has been made in experimental setups, and for many groups of analytes, such as peptides, proteins, nucleotides, saccharides, drugs and their metabolites, CE/ESI-MS has been successfully applied. Electromigration is further miniaturized. New preconcentration methods allow the investigation of compounds, which are not sensitively detected with ESI-MS. Coordination ion spray (CIS) MS is another method for sensitivity enhancement by on-line formation of charged coordination compounds.

Recent advances in capillary electrophoresis/electrospray-mass spectrometry

In this review, the progress in hyphenation of capillary electrophoresis (CE) with electrospray ionization-mass spectrometry (ESI-MS) since the article of Banks (Banks, J. F., Electrophoresis 1997, 18, 2255–2266) is reported. In all capillary-based electromigration techniques, such as capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focussing (CIEF), micellar electrokinetic chromatography (MEKC), affinity capillary electrophoresis (ACE), as well as in the hybrid techniques capillary electrochromatography (CEC), and pressurized capillary electrochromatography (pCEC) progress has been made in experimental setups, and for many groups of analytes, such as peptides, proteins, nucleotides, saccharides, drugs and their metabolites, CE/ESI-MS has been successfully applied. Electromigration is further miniaturized. New preconcentration methods allow the investigation of compounds, which are not sensitively detected with ESI-MS. Coordination ion spray (CIS) MS is another method for sensitivity enhancement by on-line formation of charged coordination compounds.

Iodine speciation in biological samples by capillary electrophoresis-inductively coupled plasma mass spectrometry.

A hyphenation of capillary electrophoresis (CE) to inductively coupled plasma mass spectrometry (ICP-MS) was employed for the speciation of iodine. The separation method used a buffer sandwich of phosphate (pH 2.3), NaOH, sodium dodecyl sulfate (SDS) and borate buffer (pH 8.3) for stacking, aiming at sufficient separation of iodide, iodate, thyroxine (T4) and triiodothyronine (T3). These four iodine species were separated within 15 min and subsequently detected during a pressure-driven detection step (baseline-separated) at 19.5, 29.1, 36.6 and 42.2 s. The detection limits were determined at 0.08 microg I/L (iodide), 0.3 microg I/L (iodate), 3.5 microg I/L (thyroxine) and 2.5 microg I/L (triiodothyronine). This method was applied on iodine speciation in human serum ("healthy" and after thyroid gland operation) and urine. The serum from the healthy person contained iodide (13 microg I/L), T4 (61 microg I/L) and T3 (7.5 microg I/L), whereas the serum from the thyroid-operated person lacked T3. As no "free" I-hormones are known in serum, the role of the thyroid hormone binding globulin (TBG) was investigated. We found that spiked T4 or T3 immediately bound to TBG. Investigations on human urine showed only a peak for iodide.