Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures.

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .

Simultaneous determination of flavonoids and anthraquinones in honey by using SPE-CE-LIF.

Based on advantages of capillary electrophoresis (CE), a new solid-phase extraction (SPE) coupled with CE has been developed for preconcentration, enrichment and determination of anthraquinones and flavonoids (rutin, emodin, quercetin, 1,8-dihydroxyanthraquinone) in honey. The environmental-friendly chitin activated after an easy processing is selected as the adsorbent to enrich analytes. Then, chitin was filled into the filter as the solid phase. To improve the extraction effect, some key parameters of extraction were optimized. Under the optional extraction conditions, the chitin showed excellent adsorption capacity and selectivity over rutin, emodin, quercetin, and 1,8-dihydroxyanthraquinone, with enrichment factors reaching 5 folds. The CE coupled with fluorescence detection was used for the detection. Results prove the method is simple, fast, and highly sensitive, with the limit of detection (LOD) is 3.00-200.0 ng/mL; the recovery is 90.0-107.0%, and relative standard deviation of (RSD) is 1.8-8.3%.

Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries.

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.

Chip-Based Capillary Zone Electrophoresis Mass Spectrometry for Rapid Resolution and Quantitation of Critical Quality Attributes in Protein Biotherapeutics.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

Evaluation of a nanoflow interface based on the triple-tube coaxial sheath-flow sprayer for capillary electrophoresis-mass spectrometry coupling in metabolomics.

The performance of an original CE-MS interface that allows the in-axis positioning of the electrospray with respect to the MS inlet was evaluated. The variations in the geometrical alignment of this configuration in the absence of a nebulizing gas afforded a significant reduction in the sheath-liquid flow rate from 3 µL/min to as low as 300 nL/min. The sheath liquid and BGE were respectively composed of H2O-iPrOHCH3COOH 50:50:1 (v/v/v) and 10% acetic acid (pH 2.2). A significant gain in sensitivity was obtained, and it was correlated to the effective mobility of the analytes. Compounds with low mobility values showed a greater sensitivity gain. Special attention was paid to the detection of proteinogenic amino acids. Linear response functions were obtained from 15 ng/mL to 500 ng/mL. The limits of quantification, as low as 34.3 ng/mL, were improved by a factor of up to six compared to the conventional configuration. The in-axis setup was ultimately applied to the absolute quantification of four important amino acids, alanine, tyrosine, methionine and valine, in standard reference material (NIST plasma). The accuracies ranged from 78 to 113%, thus demonstrating the potential of this configuration for metabolomics.

Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis.

Kinetic reactions of the transphosphorylation with creatine kinase (CK) were individually investigated between creatine (Cr) and creatine phosphate (CrP) by pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The transphosphorylations are reversible between Cr and CrP, and reverse reactions inevitably accompany in general batch analyses. In pCE/DFA, the kinetic reaction proceeds in a separation capillary and the product is continuously resolved from the substrate zone. Therefore, the formation rate is kept constant at the substrate zone without the reverse reaction, and the product is detected as a plateau signal. This study demonstrates the direct and individual analyses of both the forward and the backward kinetic reactions with CK by pCE/DFA. A plateau signal was detected in the pCE/DFA with ADP or ATP as one of the products on either the forward or the backward reactions. The Michaelis-Menten constants of Km,ATP (from Cr to CrP) and Km,ADP (from CrP to Cr) were successfully determined through the plateau signal. Determined values of Km,ATP and Km,ADP by pCE/DFA were smaller than the ones obtained by the pre-capillary batch analyses. The results agree with the fact that the reverse reaction is excluded in the analysis of the kinetic reactions. The proposed pCE/DFA is useful on individual analyses of both forward and backward kinetic reactions without any interference from the reverse reaction.

Two capillary approach for a multifunctional nanoflow sheath liquid interface for capillary electrophoresis-mass spectrometry.

CE hyphenated to ESI-MS (CE-ESI-MS) is a well-established technique to analyze charged analytes in complex samples. Although various interfaces for CE-MS coupling are commercially available, the development of alternatives which combine sensitivity, simplicity, and robustness remains a topic of research. In this work, a nanoflow sheath liquid CE-MS interface with two movable capillaries inside a glass emitter is described. The setup enables a separation mode and a conditioning mode to guide the separation capillary effluent either into the electrospray or to the waste, respectively. This enables to exclude parts of the analysis from MS detection and unwanted matrix components reaching the mass spectrometer, comparable to divert valves in LC-MS coupling. Also, this function improves the overall robustness of the system by reduction of particles blocking the emitter. Preconditioning with electrospray interfering substances and even the application of coating materials for every analysis is enabled, even while the separation capillary is built into the interface with running electrospray. The functionality is demonstrated by analyses of heavy matrix bioreactor samples. Overall, this innovation offers a more convenient installation of the interface, improved handling with an extended lifetime of the emitter tips and additional functions compared to previous approaches, while keeping the higher sensitivity of nanoflow CE-MS-coupling.

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve.

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.

Separation and determination of alkyl sulfate surfactants in wastewater by capillary electrophoresis coupled with contactless conductivity detection after preconcentration by simultaneous adsorption using alumina beads.

The aim of the present study is to determine four anionic alkyl sulfate (AS) surfactants with different alkyl chains, namely, C8, C10, C12, and C14, in wastewater by CE with capacitively coupled contactless conductivity detection (CE-C4 D). The conditions effective for the separation of the four AS surfactants were systematically optimized and found to be in a Tris-His (50 mM/20 mM) BGE solution at a pH of 8.95, using a separation voltage of +15 kV, hydrodynamic injection by siphoning using a 20 cm injection height and an injection time of 20 s. The LODs for C8, C10, C12, and C14 were 2.58, 2.30, 2.08, and 3.16 mg/L, respectively. The conditions used to achieve the simultaneous adsorption and preconcentration of the AS surfactants using Al2 O3 beads were pH of 3 and 0.1 mM NaCl. The adsorption efficiencies were found to be 45.6, 50.8, 81.7, and 99.9%, while the desorption efficiencies reached 66.1, 70.4, 83.9, and 100.0% for C8, C10, C12, and C14, respectively. The concentrations of the AS surfactants in wastewater samples were quantified by CE-C4 D after preconcentration by simultaneous adsorption using Al2 O3 beads. The results obtained from the proposed method were consistent with those obtained by HPLC-MS/MS, with a deviation of less than 15%. Our results indicate that the CE-C4 D performed after preconcentration by an adsorption technique using Al2 O3 beads is a new, inexpensive, and suitable method for quantifying AS surfactants in wastewater samples.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Selective Enrichment of Low-abundance Compounds in a Mixture by Capillary Electrophoresis.

Recently, we developed a new approach for the selective enrichment of low-abundance compounds in biological samples by capillary electrophoresis. As a model test, the low-abundance compound lysozyme was successfully fractionated from a mixture containing high-abundance compound BSA (1:4500) using a custom-made apparatus. The feasibility of this approach for real complex biological samples was verified by rat serum, wherein three low-abundance proteins with high charge/mass ratios were detected.

A simple method for the analysis of extracellular vesicles enriched for exosomes from human serum by capillary electrophoresis with ultraviolet diode array detection.

Extracellular vesicles (EVs) are membrane enclosed vesicles (<1 µm), such as exosomes (30-150 nm), involved in cell communication, which have important biological implications. In this study, EV preparations were enriched for exosomes from human serum by polyethylene glycol (PEG) precipitation. Different variables of the PEG precipitation method (i.e. concentration of PEG, filtration and centrifugation of the resuspended pellets) were evaluated by measuring the size of the isolated particles by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). In addition, a novel capillary electrophoresis-ultraviolet diode array (CE-UV-DAD) method was developed to obtain characteristic multiwavelength electrophoretic profiles of the EV preparations. Using EV preparations precipitated with 10% m/v of PEG, a background electrolyte (BGE) of 0.1 M Tris and 0.25 M boric acid at pH 7.9 with 0.5% m/v of hydroxypropyl cellulose (HPC) allowed reducing the adsorption of the EVs to the inner wall of the fused silica separation capillary. Sodium dodecyl sulfate (SDS) at 0.1% m/v was also necessary to enhance dispersibility, while homogenizing the charge of the particles to improve the size-dependent separation induced by HPC. Under these optimized conditions, a characteristic electrophoretic multiwavelength profile of the EV preparation and a standard of exosomes was obtained, and separation showed excellent reproducibility and appropriate analysis times. The obtained electrophoretic fingerprints are a simple, effective and complementary tool for the quality control of EV preparations.

An electrophoretic ion analyzer for on-site autonomous water monitoring.

An on-site ion analyzer based on capillary electrophoresis with pressure-driven flow through injection and capacitively coupled contactless conductivity detection has been developed for field monitoring of cations and anions in environmental waters. Automated time-pressure based hydrodynamic injection provides stable pL-nL scale injection (RSD = 1.96%, n = 30). A mixture of 400 mM Bis-Tris, 400 mM MOPS and 2 mM 18-crown-6 is used as the background electrolyte to provide repeatable separations. A proprietary hydrophilic coated 25 µm id capillary is used to suppress the electroosmotic flow. Separations of anions (Cl-, NO3-, NO2-, SO42-, F- and PO43-) and cations (NH4+, K+, Na+, Ca2+ and Mg2+) are achieved by switching the polarity of the high voltage power supply in two individual runs. Signal fluctuations caused by the temperature or viscosity changes in on-site monitoring are corrected by on-line introduction of internal standards. RSDs of the migration time and the corrected peak height over ~35 h and 350 analysis cycles are <4.06%. The LODs of inorganic ions are in the range of 2.1 µM (K+) to 6.8 µM (PO43-).  The feasibility for on-site water monitoring with this system has been validated by a standard Ion chromatography method with comparable results obtained.

Advantages and Pitfalls of Capillary Electrophoresis of Pharmaceutical Compounds and Their Enantiomers in Complex Samples: Comparison of Hydrodynamically Opened and Closed Systems.

Several research disciplines require fast, reliable and highly automated determination of pharmaceutically active compounds and their enantiomers in complex biological matrices. To address some of the challenges of Capillary Electrophoresis (CE), such as low concentration sensitivity and performance degradation linked to the adsorption and interference of matrix components, CE in a hydrodynamically closed system was evaluated using the model compounds Pindolol and Propranolol. Some established validation parameters such as repeatability of injection efficiency, resolution and sensitivity were used to assess its performance, and it was found to be broadly identical to that of hydrodynamically opened systems. While some reduction in separation efficiency was observed, this was mainly due to dispersion caused by injection and it had no impact on the ability to resolve enantiomers of model compounds even when spiked into complex biological matrix such as blood serum. An approximately 18- to 23-fold increase in concentration sensitivity due to the employment of wide bore capillaries was observed. This brings the sensitivity of CE to a level similar to that of liquid chromatography techniques. In addition to this benefit and unlike in hydrodynamically opened systems, suppression of electroosmotic flow, which is essential for hydrodynamically closed systems practically eliminates the matrix effects that are linked to protein adsorption.

Low-cost and versatile analytical tool with purpose-made capillary electrophoresis coupled to contactless conductivity detection: Application to antibiotics quality control in Vietnam.

In this study, the development of our purpose-made capacitively coupled contactless conductivity detection (C4 D) for CE is reported. These systems have been employed as a simple, versatile, and cost-effective analytical tool. CE-C4 D devices, whose principle is based on the control of the ion movements under an electrical field, can be constructed even with a modest financial budget and limited infrastructure. A featured application was developed for quality control of antimicrobial drugs using CE-C4 D, with most recent work on determination of aminoglycoside and glycopeptide antibiotics being communicated. For aminoglycosides, the development of CE-C4 D methods was adapted to two categories. The first one includes drugs (liquid or powder form) for intravenous injection, containing either amikacin, streptomycin, kanamycin A, or kanamycin B. The second one covers drugs for eye drops (liquid or ointment form), containing either neomycin, tobramycin, or polymyxin. The CE-C4 D method development was also made for determination of some popular glycopeptide antibiotics in Vietnam, including vancomycin and teicoplanin. The best detection limit achieved using the developed CE-C4 D methods was 0.5 mg/L. Good agreement between results from CE-C4 D and the confirmation method (HPLC- Photometric Diode Array ) was achieved, with their result deviations less than 8% and 13% for aminoglycoside and glycopeptide antibiotics, respectively.

Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer.

OBJECTIVE: To evaluate the effect of a new type of automatic glycated hemoglobin analyzer on the separation of abnormal hemoglobin. METHODS: Samples diagnosed as hemoglobin variants by capillary electrophoresis and gene testing were selected, and HbA1c analyzer was used for separation and detection. RESULTS: A total of 13 hemoglobin variants in 40 samples could be separated from the normal peaks. CONCLUSIONS: The variant mode of hemoglobin HbA1c can identify a variety of hemoglobin variants, and the type of variants can be preliminarily determined according to the retention time and characteristic peak shape of the variants.

Dynamic pH barrage junction focusing of amino acids, peptides, and digested monoclonal antibodies in capillary electrophoresis-mass spectrometry.

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE-MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.

Enhanced light-emitting diode induced fluorescence detection system with capillary electrophoresis.

An enhanced fluorescence detection system of capillary electrophoresis (CE) was equipped with a concave silver mirror, by which the detection sensitivity of light-emitting diode induced fluorescence (LEDIF) can be increased greatly. The silver concave mirror and the cathode window in photomultiplier tube (PMT) were accurately set face to face at the same axis. When the two labeled tumor markers exactly moved to the center of detection window, the emission from analytes are excitated by LED source. Currently, the analytes may be regarded as a luminescent source point. When the source point exactly moves to the focus of the concave mirror, the emission of the labeled sample was collected effectively, enhanced by convergence and reflected by the concave mirror. Then it was sensitively detected by the PMT. The optical mechanism of enhancing detection sensitivity was explored. A simple comparative test on sensitivity was carried out, which aimed to compare sensitivity of the new detection system with concave mirror to that without concave mirror but the other conditions were kept the same. Two tumor markers labeled with FITC were selected for the test, using the simple LEDIF detect system. The results (LOD, 150 nM for L-Leu and L-Val) showed that the detection sensitivity matched with concave mirror reached more 16 times than the detection method without concave mirror.

Low Flow Voltage Free Interface for Capillary Electrophoresis and Mass Spectrometry Driven by Vibrating Sharp-Edge Spray Ionization.

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH. The versatility and ease of use of this ionization source is demonstrated using ß-blockers, peptides, and proteins. The cationic ß-blocker pindolol was injected electrokinetically, and detected at concentrations ranging from 10 nM to 5 µM, with an estimated detection limit of 2 nM. The vibrating sharp-edge spray ionization functions with flow rates from 70 to 200 nL/min and did not perturb the capillary electrophoresis separation electroosmotic flow as evidenced by the observation that most migration times differed less than 7% (n = 3) across a lab-built system interfaced to mass spectrometry and a commercial system that utilizes absorbance detection. For cationic beta-blockers the theoretical plates achieved in the capillary electrophoresis-mass spectrometry setup were 80%-95% of that observed with a commercial capillary electrophoresis-UV absorbance detection system.

Analysis of glycopeptide biomarkers by on-line TiO2 solid-phase extraction capillary electrophoresis-mass spectrometry.

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L-1 and 10-50 mg L-1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.