Two-dimensional Capillary Electrophoresis Research Articles

Determination of thiamine and pyridoxine in food supplements by a green ultrasensitive two-dimensional capillary electrophoresis hyphenated with mass spectrometry

Determination of thiamine and pyridoxine in food supplements by a green ultrasensitive two-dimensional capillary electrophoresis hyphenated with mass spectrometry

Ultrasensitive determination of serotonin in human urine by a two dimensional capillary isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry

A two-dimensional capillary isotachophoresis-capillary zone electrophoresis method hyphenated with tandem mass spectrometry was developed and validated for ultrasensitive quantification of serotonin in real human urine samples. Under optimal conditions, the separation was achieved within 12 min (including on-line sample preparation) with the limit of detection of 34 pg mL−1 (due to a large volume sample injection, here 10 µL, and isotachophoretic preconcentration). This concentration limit represents the lowest value for serotonin in comparison to other previously published separation methods employing mass spectrometry detection and applied to urine matrices. Thanks to high orthogonality, on-line concentration and clean-up effects of this approach, other excellent validation parameters such as linearity (coefficient of determination > 0.99), inter-day and intra-day precision (relative standard deviations 3.5–12.2%), accuracy (relative errors within 99–109.4%), and recovery (96–102%) could be easily obtained too. To demonstrate applicability of the method, we monitored serotonin levels in various real samples (from a healthy volunteer and clinical ones). The determined levels, normalized on the creatinine concentrations, were in the range of 6.81–12.86 ng mmol−1 creatinine This advanced method is suggested for an effective, reliable, high sample throughput, and low cost routine clinical screening or targeted metabolomic studies of serotonin in urine samples.

Online top-down mass spectrometric identification of CE(SDS)-separated antibody fragments by two-dimensional capillary electrophoresis

Size heterogeneity analysis by capillary sieving electrophoresis utilizing sodium dodecyl sulfate (CE(SDS)) with optical detection is a major method applied for release and stability testing of monoclonal antibodies (mAbs) in biopharmaceutical applications. Identification of mAb-fragments and impurities observed with CE(SDS) is of outstanding importance for the assessment of critical quality attributes and development of the analytical control system. Mass spectrometric (MS) detection is a powerful tool for protein identification and characterization. Unfortunately, CE(SDS) is incompatible with online MS-hyphenation due to strong ionization suppression of SDS and other separation buffer components.Here, we present a comprehensive platform for full characterization of individual CE(SDS)-separated peaks by CE(SDS)-capillary zone electrophoresis-top-down-MS. The peak of interest is transferred from the first to the second dimension via an 8-port valve to remove MS-incompatible components. Full characterization of mAb byproducts is performed by intact mass determination and fragmentation by electron transfer dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation. This enables online determination of intact mass as well as sequence verification of individual CE(SDS)-separated peaks simultaneously. A more substantiated characterization of unknown CE(SDS) peaks by exact localization of modifications without prior digestion is facilitated. High sensitivity is demonstrated by successful mass and sequence verification of low abundant, unknown CE(SDS) peaks from two stressed mAb samples. Good fragmentation coverages are obtained by MS2, enabling unequivocal identification of these mAb-fragments. Also, the differentiation of reduced/non-reduced intra-protein disulfide bonds is demonstrated. In summary, a reliable and unambiguous online MS2 identification of unknown compounds of low-abundant individual CE(SDS) peaks is enabled.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300–800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL−1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02–1.17% and 5.25–7.88%, respectively), and recovery (ranging in the interval of 90.0–93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4–491.2 ng·mL−1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

On-line two-dimensional capillary electrophoresis with mass spectrometric detection using a fully electric isolated mechanical valve.

CE is becoming more and more important in many fields of bioanalytical chemistry. Besides optical detection, hyphenation to ESI-MS detection is increasingly applied for sensitive identification purposes. Unfortunately, many CE techniques and methods established in research and industry are not compatible to ESI-MS since essential components of the background electrolyte interfere in ES ionization. In order to identify unknown peaks in established CE methods, here, a heart-cut 2D-CE separation system is introduced using a fully isolated mechanical valve with an internal loop of only 20 nL. In this system, the sample is separated using potentially any non-ESI compatible method in the first separation dimension. Subsequently, the portion of interest is cut by the internal sample loop of the valve and reintroduced to the second dimension where the interfering compounds are removed, followed by ESI-MS detection. When comparing the separation efficiency of the system with the valve to a system using a continuous capillary only a slight increase in peak width is observed. Ultraviolet/visible detection is integrated in the first dimension for switching time determination, enabling reproducible cutting of peaks of interest. The feasibility of the system is successfully demonstrated by a 2D analysis of a BSA tryptic digest sample using a nonvolatile (phosphate based) background electrolyte in the first dimension.

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.

Nicked-sleeve interface for two-dimensional capillary electrophoresis

We report an improved interface for two-dimensional capillary electrophoresis. This interface is based on capillary tubing and a Plexiglas chip, both of which were milled using a micro-dicing saw. The interface was evaluated and compared to a traditional interface design for both pseudo one-dimensional and two-dimensional capillary electrophoresis. We observe less than 70% transfer efficiency for the traditional design and greater than 90% transfer efficiency with this new interface.

2D Capillary Electrophoresis Hyphenated with Spectral Detection for the Determination of Quinine in Human Urine

The possibilities of a column coupling two-dimensional capillary electrophoresis (2D CE) combined with fiber-based diode array detection (DAD) for the direct, highly reliable and ultrasensitive quantitative determination of quinine in real multicomponent ionic matrices (human urine) are demonstrated in this work. The capillary isotachophoresis (CITP) stage provided an on-line sample pretreatment (elimination of interfering matrix constituents, preseparation and preconcentration of the analyte) before the capillary zone electrophoresis (CZE) separation. Due to the large volume (30 µL) sample injection and CITP sample preconcentration, a simple absorbance photometric detection was sufficient for obtaining very low concentration limits of detection (∼8.6 ng/mL). The combination of the different separation mechanisms (CITP and CZE) resulted in enhanced separation selectivity. This enabled us to obtain a pure analyte zone in the directly injected real samples suitable for qualitative and quantitative evaluation. The spectral DAD allowed (i) characterization of the purity (i.e., spectral homogeneity) of the analyte zone; and (ii) preliminary indication of structurally related compounds (i.e., potential biodegradation products of quinine), via characteristic spectra recorded in intervals of 200-800 nm. The CITP-CZE-DAD method was characterized by favorable performance parameters that are suitable for its routine biomedical use. One of the primary benefits of the CITP-CZE-DAD method is the possibility of performing direct injections of real biological samples while avoiding external sample preparation procedures and, therefore, enhancing the reliability and applicability of analyses and the potential for method automatization and miniaturization.

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.

Sweeping with Electrokinetic Injection and Analyte Focusing by Micelle Collapse in Two-Dimensional Separation via Integration of Micellar Electrokinetic Chromatography with Capillary Zone Electrophoresis

A novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri and mouse blood samples. First, a new sweeping with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhance detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely high resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug lengths for flavonoids under 15 kV for 60 min were experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8% (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3 × 10(4) plates/m. This method has been successfully applied to determine flavonoids in Herba Leonuri and postdosing mouse blood samples. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples.

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.

Two‐dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser‐induced fluorescence detection

CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2-D separation method for proteins. In this method, two capillaries are joined through a buffer-filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first-dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second-dimension separation. A fraction was transferred to the second-dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.

The multi-concentration and two-dimensional capillary electrophoresis method for the analysis of drugs in urine samples

A novel method has been developed by integration of multi-concentration and two-dimensional (2D) capillary electrophoresis (CE) for simultaneous enhancement of detection sensitivity and separation power in complex samples. Capillary zone electrophoresis (CZE) was used as the first dimension separation according to mobilities, from which the effluent fractions were further analyzed by micellar electrokinetic capillary chromatography (MEKC) acting as the second dimension. Cation-selective exhaustive injection (CSEI) preconcentration method was used to introduce more analytes into the capillary. Furthermore, pH junction and sweeping dual concentration strategies were employed to avoid sample zone diffusion at the interface. The resulting electrophoregram was quite different from that of either CZE or MEKC separation. Up to (0.5–1.2) × 104 fold improvements in sensitivity were obtained relative to the conventional electrokinetic injection method. The proposed method was successfully applied to the determination of drugs in human urine.

Multi-concentration and two-dimensional capillary electrophoresis method for the analysis of drugs in urine samples

A novel method has been developed by integration of multi-concentration and two-dimensional (2D) capillary electrophoresis (CE) for simultaneous enhancement of detection sensitivity and separation power in complex samples. Capillary zone electrophoresis (CZE) was used as the first dimension separation according to mobilities, from which the effluent fractions were further analyzed by micellar electrokinetic capillary chromatography (MEKC) acting as the second dimension. Cation-selective exhaustive injection (CSEI) preconcentration method was used to introduce more analytes into the capillary. Furthermore, pH junction and sweeping dual concentration strategies were employed to avoid sample zone diffusion at the interface. The resulting electrophoregram was quite different from that of either CZE or MEKC separation. Up to (0.5-1.2)×104 fold improvements in sensitivity were obtained relative to conventional electrokinetic injection method. The proposed method was successfully applied to the determination of drugs in human urine.

Automated Enzyme-Based Diagonal Capillary Electrophoresis: Application to Phosphopeptide Characterization

Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have different migration times in the second capillary and will generate spots that fall off the diagonal in the electropherogram. We demonstrate the system with immobilized alkaline phosphatase to monitor the phosphorylation status of a mixture of peptides. This enzyme-based diagonal capillary electrophoresis assay appears to be generalizable; any post-translational modification can be detected as long as an immobilized enzyme is available that reacts with the modification under electrophoretic conditions.

Improvement in protein separation in Barretts esophagus samples using two-dimensional capillary electrophoresis analysis in presence of cyclodextrins as buffer additives

In two-dimensional capillary electrophoresis (2DCE) components are separated based on their size and hydrophobicity. A preliminary run separates analytes in the first capillary based on size (CSE). Following that, fractions are electrokinetically transferred across an interface into a second capillary, where components are further resolved according to hydrophobicity. In order to succeed in this analysis, two orthogonal methods should be selected for the different modes. The transfers from the first to the second capillary must be efficient in order to reduce tailing effects and lost of resolution. We report a new method to improve the resolution with our 2DCE instrumentation using CD doped buffers. When methyl beta cyclodextrin (mβCD) is added to the 2DCE interface buffer a stacking effect is described in the transfers from the first to the second dimension. In addition to that, changes in retention times are observed when proteins form complex with CD's helping in the separation. Protein fingerprints were obtained from BE homogenates using this method in presence of methyl beta cyclodextrin (mβCD). Within-day and between-day precision has been studied in order to establish the reproducibility of the methodology proposed.

Reaction of fluorogenic reagents with proteins: III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503

The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were determined by continually infusing Chromeo P503-labeled α-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concentration than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis analysis of Chromeo P503-labeled α-lactalbumin produced concentration detection limits (3σ) of 12 pM and mass detection limits of 0.7 zmol, but with modest theoretical plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis analysis of a homogenate prepared from a Barrett's esophagus cell line; the separation quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.

Miniaturized two‐dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.