Pressure-assisted Capillary Electrophoresis Research Articles

Migration time correction for dual pressure capillary electrophoresis in semi-targeted metabolomics study.

Migration time fluctuation strongly affects peak alignment and identification of unknown compounds, making migration time correction an essential step in capillary electrophoresis (CE)-based metabolomics. To obtain more reliable information, metabolites with different apparent mobilities are analyzed by tandem mass spectrometry. Applying a small pressure is a common practice for reducing the analysis time of anions in a positive mode CE, known as the pressure-assisted CE. However, applying pressure may reduce the separation efficiency and can be undesirable for cation analysis. A simple way to address this issue is to increase the pressure after a certain time, during the separation. We term this practice as dual pressure CE. However, changing the pressure during the CE separation complicates migration time correction. Previous migration time correction methods were established based on a consistent electroosmotic flow and a constant pressure-driven bulk-flow velocity. We proposed a new correction method to support the peak alignment when dual pressure CE is used. A Python-based script was developed to implement dual pressure CE migration time correction for semi-targeted metabolomics study performed by a multiple reaction monitoring-based method. This script can help select suitable endogenous metabolites as correction markers, perform migration time correction, and conduct peak alignment. A case study showed that migration time precision of 156 metabolites in 32 samples can be improved from 4.8 to 11.4%RSD (relative standard deviation) to less than 1.8%RSD.

Determination pk <sub>a</sub> Values of Six Phenolic Acids by Pressure-Assisted Capillary Electrophoresis and Simultaneous Determination of These Six Phenolic Acids in <i>Euphorbiae Lathyris L.</i> By Capillary Zone Electrophoresis with Heptapkis (2,6-di-o-Methyl)-β-Cyclodextrin as Modifier

Determination pk <sub>a</sub> Values of Six Phenolic Acids by Pressure-Assisted Capillary Electrophoresis and Simultaneous Determination of These Six Phenolic Acids in <i>Euphorbiae Lathyris L.</i> By Capillary Zone Electrophoresis with Heptapkis (2,6-di-o-Methyl)-β-Cyclodextrin as Modifier

Characterization of interaction between Bcl-2 oncogene promoter I-Motif DNA and flavonoids using electrospray ionization mass spectrometry and pressure-assisted capillary electrophoresis frontal analysis

B-cell lymphoma 2 (Bcl-2) is an antiapoptotic protein which is believed to be a triggering factor in developing human tumors. The Bcl-2 C-rich promoter element has been shown to form the i-motif (IM) via cytosine−cytosine (C–C+) base pair building blocks, which can be targeted through the binding of ligands associated with Bcl-2 expression modulation. In this work, we monitored IM development and thermodynamic stability within the Bcl-2 promoter via circular dichroism (CD) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The results demonstrated that at an acidic pH, as well as in a crowded molecular environment, the Bcl-2 promoter element predominantly exists in a stable intramolecular IM folded state. We further explored the potential of targeting of the Bcl-2 IM to increase chemotherapeutic efficacy. We first used a rapid ESI-MS screening assay to identify possible ligands, finding that three natural flavonoids (P1, P5 and P6) exhibited a clear affinity for IM binding at 1:1 stoichiometry. Relative to P6, P1 and P5 were expected to form the more stable complexes with the Bcl-2 IM in gas phase according to MS/MS data. We further used ESI-MS and pressure-assisted capillary electrophoresis frontal analysis (PACE-FA) to assess the binding constants for these flavonoids in gas and liquid phases, respectively, with the latter considering both specific and non-specific binding. We found P5 and P6 to specifically bind the Bcl-2 IM with binding constants of ~104 M−1. P1 binding was confirmed to be due to both specific and nonspecific interactions, and the specific binding constant (8.67 × 103 M−1) was found much less significant than the binding constant in gas phase. Taken all these observations into consideration, the specific binding of selected flavonoids to the Bcl-2 IM may prove to be a potential ligand for modulating Bcl-2 gene expression.

Pressure-assisted capillary electrophoresis frontal analysis for faster binding constant determination.

Adding external pressure during the process of capillary electrophoresis usually add to the band broadening, especially if the pressure induced flow is significant. The resolution is normally negatively affected in pressure-assisted capillary electrophoresis (PACE). Frontal analysis (FA), however, can potentially benefit from using an external pressure while avoiding the drawbacks in other modes of CE. In this work, possible impact from the external pressure was simulated by COMSOL Multiphysics®. Under a typical CE-FA set-up, it was found that the detected concentrations of analyte will not be significantly affected by an external pressure less than 5psi. Besides, the measured ligand concentration in PACE-FA was also not affected by common variables (molecular diffusion coefficient (10-8 to 10-11 m2 /s), capillary length etc). To provide an experimental proof, PACE-FA is used to study the binding interactions between hydroxypropyl β-cyclodextrin (HP-β-CD) and small ligand molecules. Taking the HP-β-CD /benzoate pair as an example, the binding constants determined by CE-FA (18.3±0.8 M-1 ) and PACE-FA (16.5±0.5M-1 ) are found to be similar. Based on the experimental results, it is concluded that PACE-FA can reduce the time of binding analysis while maintaining the accuracy of the measurements.

Detection and quantification of chitosan aggregates by pressure-assisted capillary zone electrophoresis

An approach to the detection and determination of chitosan aggregates in acetic acid solutions is proposed using pressure-assisted capillary zone electrophoresis. Processes of chitosan aggregation are studied depending on the composition of dispersion medium and storage time. The presence of several species of positively charged chitosan aggregates is revealed for the first time. Particle sizes in the range 20–2500 nm are determined by scanning electron microscopy and static and dynamic light scattering. The dependence of the shape of electropherograms on particle size distribution obtained under the same conditions is found. A trend to changing electrophoretic mobility depending on the size of the aggregate is observed, which enables the approximate evaluation of the polydispersity of chitosan solutions. Chitosan is used for the effective dynamic modification of capillaries, which does not require the introduction of a modifier into the background electrolyte.

Rapid determination of branched chain amino acids in human blood plasma by pressure-assisted capillary electrophoresis with contactless conductivity detection.

A CE method with contactless conductivity detection has been developed for the clinical determination of the branched chain amino acids (BCAAs) valine, isoleucine and leucine in human blood plasma. The CE separation was performed in an optimised BGE with composition of 3.2 M acetic acid in 20% v/v methanol, pH 2.0. The achieved separation time was 125 s when using a capillary with an effective length of 14.7 cm, electric field intensity of 0.96 kV/cm and simultaneous application of a hydrodynamic pressure of 50 mbar. The separation efficiency in blood plasma equalled 461 000 theoretical plates/m for valine and isoleucine, and 455 000 theoretical plates/m for leucine; the detection limits are equal to 0.4 μM for all three amino acids. The RSD values for repeatability of the migration time equalled 0.1% for measurements during a single day and 0.3% for measurements on different days; the RSD values for repeatability of the peak areas equalled 2.3-2.6% for measurements during a single day and 2.7-4.6% for measurements on different days. It followed from the performed tests that the plasmatic levels of BCAAs attain a maximum 60 min after intravenous application of an infusion of BCAAs.

New peak broadening parameter for the characterization of separation capability in capillary electrophoresis.

The influence of separation conditions on peak broadening is usually estimated by the number of theoretical plates. Using the data available in literature and experimental data, it is shown that in pressure-assisted capillary electrophoresis the plate number is not directly related to the separation capability of conditions used. The experiments at different electrolyte flow velocities demonstrate that a higher plate number (the best separation efficiency) can be obtained with a lower peak resolution. Since ions are separated by electrophoresis due to the difference in electrophoretic mobilities, the peak width in terms of electrophoretic mobility is suggested as a new peak broadening parameter describing the separation capability of the conditions used. The parameter can be calculated using the tailing factor and the temporal peak width at 5% of the peak height. A simple equation for the resolution calculation is derived using the parameter. The advantage of the peak width in terms of mobility over other parameters is shown. The new parameter is recommended to be used not only in pressure-assisted capillary electrophoresis but also in general capillary electrophoresis when in a number of runs the virtual separative migration distance and separation capability of the conditions used change widely.

Characterization of interactions between methoxatin disodium salt and human serum albumin by pressure-assisted capillary electrophoresis/frontal analysis and circular dichroism spectroscopy.

Pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA) and circular dichroism spectroscopy were utilized to investigate the interactions between methoxatin disodium salt (PQQ-2Na) and human serum albumin (HSA). With the PACE/FA method, sodium phosphate buffer solution (67 mm, pH 7.4) was used as the background electrolyte. Hydrodynamic injection at 50 mbar for 50 s and external pressure of 50 mbar were applied. The binding constant and the number of primary binding sites to HSA were obtained under fixed concentration of PQQ-2Na (100 µm) and increasing HSA concentration (0~475 µm). The thermodynamic parameters characterized the main acting forces of hydrophobic and electrostatic interactions. The displacement experiments using phenylbutazone and flurbiprofen as ligand markers suggested that the binding site was the Sudlow site I of the HSA molecule. Circular dichroism spectroscopy was further employed to evaluate the conformation changes of HSA under the interaction of PQQ-2Na. This work provides comprehensive information for understanding the interactions between PQQ-2Na and HSA.

Poly(glycidyl methacrylate)/silver nanocomposite microspheres as a radioiodine scavenger: Electrophoretic characterisation of carboxyl- and amine-modified particles

Silver nanoparticles possess potent antibacterial properties and have extremely high affinities to radioiodine. For several applications, it is essential to anchor the nanoparticles to microparticles or solid surfaces to make them insoluble while retaining their unique properties. This current work is related to the design of anionic and cationic macroporous polymer microspheres based on poly(glycidyl methacrylate) (PGMA) obtained using a multistep swelling polymerisation. According to scanning electron microscopy, the microspheres were monodisperse in size and 4.2μm in diameter. The presence of the carboxyl and amino groups in the PGMA-COOH and PGMA-NH2 microspheres was confirmed by FT-IR spectroscopy. Capillary electrophoresis (CE) and pressure-assisted capillary electrophoresis (PACE) were used to study the electrophoretic behaviour of both types of microparticles. The electrophoretic mobility of the microparticles was changed into ζ potential using Smoluchowski modelling. Finally, silver-containing microspheres were prepared by reducing silver nitrate in the presence of the microspheres, and they proved effective for scavenging radioiodide ions from a model medium.

Determination of pKavalues of a triptolide derivative and its impurities by pressure-assisted capillary electrophoresis

A pressure-assisted capillary zone electrophoresis method was developed to determine the pKa values of a triptolide derivative (LLDT-246) and its impurities. The method was performed in an uncoated fused-silica capillary under the electric voltage of 18kV and 50mbar of external pressure applied simultaneously. A series of running electrolyte buffers were used with pH ranging between 2.2 and 10.0 with the constant ionic strength of 0.05M. The values of pKa of LLDT-246 and two impurities were calculated based on the pH dependence of effective mobilities (μeff). The pKa value of LLDT-246 was in good agreement with that of determined by potentiometric titration method.

Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures

Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

Fast separations by capillary electrophoresis hyphenated to electrospray ionization time-of-flight mass spectrometry as a tool for arsenic speciation analysis

Fast capillary electrophoresis (CE) hyphenated to time-of-flight mass spectrometry (TOF-MS) of four organoarsenic species (glycerol oxoarsenosugar, sulfate oxoarsenosugar, arsenobetaine, arsenocholine) are presented using short length CE capillaries under high electric field strengths of up to 1.3 kV cm(-1) with small inner diameter (ID). The separation of arsenosugars by CE is demonstrated for the first time. An aqueous formic acid solution was employed as the background electrolyte (BGE) for the separation. Various acid concentrations were evaluated for their influence on migration times, separation efficiency as well as with regard to controlling the charge of the arsenic species. A 0.1 M formic acid/ammonium formate buffer (pH 2.8) proved to be suitable for the separation of the four species. A non-aqueous BGE was tested as an alternative buffer system for fast speciation analysis. Separation of arsenobetaine and arsenocholine could even be achieved within 10 s by pressure-assisted CE. Application of the optimized method for the analysis of extracts of a seagrass and a Wakame algae sample as well as the brown algae homogenate reference material IAEA-140/TM revealed a clear signal for the glycerol arsenosugar.

Rapid, Simple and Ultrasensitive Analysis of Paraquat in Drinking Water by Field-Amplified Sample Injection Combined with Pressure-Assisted Capillary Zone Electrophoresis

Rapid, Simple and Ultrasensitive Analysis of Paraquat in Drinking Water by Field-Amplified Sample Injection Combined with Pressure-Assisted Capillary Zone Electrophoresis

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-D: -glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.

Pressure-assisted capillary electrophoresis for cation separations using a sequential injection analysis manifold and contactless conductivity detection

Pressure assisted capillary electrophoresis in capillaries with internal diameters of 10μm was found possible without significant penalty in terms of separation efficiency and sensitivity when using contactless conductivity detection. A sequential injection analysis manifold consisting of a syringe pump and valves was used to impose a hydrodynamic flow in the separation of some inorganic as well as organic cations. It is demonstrated that the approach may be used to optimize analysis time by superimposing a hydrodynamic flow parallel to the electrokinetic motion. It is also possible to improve the separation by using the forced flow to maintain the analytes in the capillary, and thus the separation field, for longer times. The use of the syringe pump allows flexible and precise control of the pressure, so that it is possible to impose pressure steps during the separation. The use of this was demonstrated for the speeding up of late peaks, or forcing repeated passage of the sample plug through the capillary in order to increase separation.

Ultra-fast adenosine 5′-triphosphate, adenosine 5′-diphosphate and adenosine 5′-monophosphate detection by pressure-assisted capillary electrophoresis UV detection

Herein, we report a new CE method to measure adenine nucleotides adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate in red blood cells. For this purpose, 20 mmol/L sodium acetate buffer at pH 3.80 was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 25 kV and an overimposed pressure of 0.2 psi from inlet to outlet. A rapid separation of these analytes in less than 1.5 min was obtained with a good reproducibility for intra- and inter-assay (CV<4 and 8%, respectively) and an excellent analytical recovery (from 98.3 to 99%). The applicability of our method was proved by measuring adenine nucleotides in red blood cells.

Analysis of nucleotides by pressure-assisted capillary electrophoresis–mass spectrometry using silanol mask technique

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis–electrospray ionization mass spectrometry (PACE–MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide–adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations ( n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.

Determination of dissociation constants of folic acid, methotrexate, and other photolabile pteridines by pressure-assisted capillary electrophoresis

Pressure-assisted CE (PACE) was applied to determine the previously inaccessible complete set of pK values for folic acid and eight related multiprotic compounds. PACE allowed the determination of all acidity macroconstants at low (<or=0.1 mM) concentration without interferences of selfassociation or photodegradation throughout the pH range. The accuracy of the constants was verified by NMR-pH, UV-pH, and potentiometric titrations and the data could be converted into physiological ionic strength. It was shown that even three overlapping pK values can be determined by CE with good precision (<0.06) and accuracy if an appropriately low sample throughput is used. Experimental aspects of PACE for the quantitation of acid-base properties are analyzed. The site-specific basicity data obtained for folic acid and methotrexate (MTX) reveal that apparently slight constitutional differences between folic acid and MTX carry highly different proton-binding propensities at analogous moieties, especially at the pteridine N(1) locus, providing straightforward explanation for the distinctive binding to dihydrofolate reductase at the molecular level.

Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis

We have developed a method based on capillary electrophoresis (CE) coupled to mass spectrometry (MS) that is a powerful tool for metabolome analysis. In this paper, a simple method for the simultaneous analysis of anionic metabolites such as sugar phosphates, organic acids, nucleotides and CoA compounds based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS) is described. The feature of this method is that CE polarity, where the inlet of the capillary is at the anode and the outlet at the cathode, is inverted from conventional CE analysis for anions. Moreover, an ordinary fused silica capillary was chosen instead of a specific cationic polymer-coated capillary (SMILE (+) capillary). A robust and inexpensive analytical method was established from the above-mentioned CE polarity and noncoated fused silica capillary. A trimethylamine acetate electrolyte, pH 10.0, provides a high resolution between isomers. Electrolyte flow using an air pump after electrophoresis enables the comprehensive and simultaneous analyses of sugar phosphates, organic acids, nucleotides and a CoA compound that to date have been impossible to analyze. Our method can be used as a de facto standard for metabolome analysis.

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry.

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.