Head-column Field-amplified Sample Stacking Research Articles

Highly Sensitive SDS Capillary Gel Electrophoresis with Sample Stacking Requiring Only Nanograms of Adeno-Associated Virus Capsid Proteins.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.

Imidazolium ionic liquids as dynamic and covalent modifiers of electrophoretic systems for determination of catecholamines

The subject of this study is comparison of imidazolium based dynamic and covalent coatings of the quartz capillary wall on the example of catecholamines determination. A way of synthesis of covalent coatings was proposed. For the first time different type of on-line sample preconcentration techniques (field-amplified sample stacking (FASS), head-column field amplified sample stacking (HC FASS), electrostacking, sweeping) were performed for catecholamines determination in N-alkylsubstituted imidazolium coated capillary.It was found that long chain imidazolium ionic liquids (C12MImCl and C16MImCl) in background electrolyte content create a dynamic coating of the quartz capillary walls and generate anode electroosmotic flow. Same is true for covalent coatings based on N-alkylsubstituted imidazolium. Both coatings prevent sorption of catecholamines on the internal surface of the quartz capillary. As a result efficiency and peak symmetry are increased.The maximum stacking efficiency factor (SEF) values for dynamic coatings were in range of 70–85, and limits of detection (LODs) were about 0.05 µg/ml under sweeping condition (micelle forming agent – C16MImCl) when the electric conductivity of sample matrix was higher than electric conductivity of BGE. In case of covalent coating, the maximum SEF values were higher than 1000, and LODs were about 1–2 ng/ml under sweeping condition (micelle forming agent – sodium dodecyl sulphate) in combination with electrostacking.

Fundamental aspects of field-amplified electrokinetic injection of cations for enantioselective capillary electrophoresis with sulfated cyclodextrins as selectors

Head-column field-amplified sample stacking of cations from a low conductivity sample followed by enantiomeric separation using negatively charged chiral selectors was studied experimentally and with computer simulation. Aspects investigated include the direct electrokinetic injection of the analytes into the background electrolyte, the use of a selector free buffer plug, the contribution of complexation within the buffer plug and the application of an additional water plug between sample and buffer plug. Attention was paid for changes of ionic strength which is known to have a significant impact on complexation and thus effective mobility. Racemic methadone was selected as a model compound, randomly substituted sulfated β-cyclodextrin as chiral selector and phosphate buffers (pH 6.3) for the background electrolyte and the buffer plug. Results confirm that the buffer plug is providing a spacer between cationic analytes and the negatively charged selector during electrokinetic injection. Simulation predicts the required length and composition of the plug for a given injection time to avoid an interference with the selector. A short water plug added between the low conductivity sample and a high conductivity buffer plug is demonstrated to provide best conditions to achieve high sensitivity in enantioselective drug assays with sulfated cyclodextrins as selectors.

Insights into head-column field-amplified sample stacking: Part II. Study of the behavior of the electrophoretic system after electrokinetic injection of cationic compounds across a short water plug

Part I on head-column field-amplified sample stacking comprised a detailed study of the electrokinetic injection of a weak base across a short water plug into a phosphate buffer at low pH. The water plug is converted into a low conductive acidic zone and cationic analytes become stacked at the interface between this and a newly formed phosphoric acid zone. The fundamentals of electrokinetic processes occurring thereafter were studied experimentally and with computer simulation and are presented as part II. The configuration analyzed represents a discontinuous buffer system. Computer simulation revealed that the phosphoric acid zone at the plug-buffer interface becomes converted into a migrating phosphate buffer plug which corresponds to the cationically migrating system zone of the phosphate buffer system. Its mobility is higher than that of the analytes such that they migrate behind the system zone in a phosphate buffer comparable to the applied background electrolyte. The temporal behaviour of the current and the conductivity across the water plug were monitored and found to reflect the changes in the low conductivity plug. Determination of the buffer flow in the capillary revealed increased pumping caused by the mismatch of electroosmosis within the low conductivity plug and the buffer. This effect becomes elevated with increasing water plug length. For plug lengths up to 1% of the total column length the flow quickly drops to the electroosmotic flow of the buffer and simulations with experimentally determined current and flow values predict negligible band dispersion and no loss of resolution for both low and large molecular mass components.

Ionic liquids based on imidazole for online concentration of catecholamines in capillary electrophoresis

Potential possibilities of long-chain ionic liquids based on imidazole (1-dodecyl-3-methylimidazolium chloride and 1-cetyl-3-methylimidazolium chloride) for online sample concentration techniques (field-amplified sample stacking, head-column field-amplified sample stacking, and sweeping) of catecholamines were studied in both capillary zone electrophoresis and micellar electrokinetic chromatography. The use of a high-conductivity sample matrix in sweeping was found to significantly increase the separation efficiency of analyte up to 2×106 theoretical plates per meter and remarkably reduce limits of detection for catecholamines up to 50ng/mL. This approach was shown to be suitable for the determination of trace amounts of catecholamines in biological fluids.

Insights into head-column field-amplified sample stacking: Part I. Detailed study of electrokinetic injection of a weak base across a short water plug

The fundamentals of electrokinetic injection of the weak base methadone across a short water plug into a phosphate buffer at low pH were studied experimentally and with computer simulation. The current during electrokinetic injection, the formation of the analyte zone, changes occurring within and around the water plug and mass transport of all compounds in the electric field were investigated. The impact of water plug length, plug injection velocity, and composition of sample, plug and background electrolyte are discussed. Experimental data revealed that properties of sample, water plug and stacking boundary are significantly and rapidly altered during electrokinetic injection. Simulation provided insight into these changes, including the nature of the migrating boundaries and the stacking of methadone at the interface to a newly formed phosphoric acid zone. The data confirm the role of the water plug to prevent contamination of the sample by components of the background electrolyte and suggest that mixing caused by electrohydrodynamic instabilities increases the water plug conductivity. The sample conductivity must be controlled by addition of an acid to prevent generation of reversed flow which removes the water plug and to create a buffering environment. Results revealed that a large increase in background electrolyte concentration is not accompanied with a significant increase in stacking.

Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins.

Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

On-line concentration of biogenic amines by capillary electrophoresis using synthesized covalent coating based on imidazolium ionic liquid

Covalent coatings of internal quartz capillary walls based on the ionic liquids were synthesized within this research. The synthesis of the covalent coating included a step of silylation followed by the functionalization with imidazole and 1-bromobutane. The resulting coating created anodic electroosmotic flow (EOF) at pH = 2. The range of pH at which coatings do not change their properties was 2.0-5.5. The possibilities of different ways of on-line concentration ( large volume sample stacking, head-column field-amplified sample stacking, sweeping, sweeping in combination with electrostacking) of catecholamines were examined. Stacking efficiency factors (SEF) were 105-135 and the detection limits were decreased to 11-17 ng/ml for analytes under the large volume sample stacking. Under the sweeping conditions, sodium dodecyl sulfate (micelle forming agent) was sorbed on the modified walls, provided a negative charging surface and acted as a strong cation exchanger. This fact led to the additional reserves in the separation selectivity of catecholamines. Under the electrostacking, the values of SEF were above 1000 and the detection limits were reduced to 1-2 ng/ml. An approbation of established patterns in the analysis of urine samples was carried out. Keywords: covalent coating, ioniс liquids, biogenic amines, on-line concentration (Russian) DOI: http://dx.doi.org/10.15826/analitika.2017.21.1.006 E.A. Kolobova 1,2 , L.A. Kartsova 1 , E.A. Bessonova 1 , A.V. Kravchenko 1 1 Saint-Petersburg State University, Institute of Chemistry, 26 Universitetskii prospect, St. Petersburg, Petergof 198504, Russian Federation 2 The Nikiforov Russian Center of Emergency and Radiation Medicine, 54 Optikov st., St. Petersburg, 199034, Russian Federation

Simultaneous determination of pharmaceutical and personal care products in wastewater by capillary electrophoresis with head-column field-amplified sample stacking

This article reports a sensitive and reliable solid-phase extraction coupling capillary zone electrophoresis with head-column field-amplified sample stacking method for trace determination of nineteen pharmaceutical and personal care products in wastewater.

Monitoring of threo‐methylphenidate enantiomers in oral fluid by capillary electrophoresis with head‐column field‐amplified sample injection

Threo-methylphenidate is a chiral psychostimulant drug widely prescribed to treat attention-deficit hyperactivity disorder in children and adolescents. An enantioselective CE-based assay with head-column field-amplified sample stacking for analysis of threo-methylphenidate enantiomers in liquid/liquid extracts of oral fluid is described. Analytes are electrokinetically injected across a short water plug placed at the capillary inlet and become stacked at the interface between plug and buffer. Enantiomeric separation occurs within a few minutes in a pH 3.0 phosphate/triethanolamine buffer containing 20 mg/mL (2-hydroxypropyl)-β-CD as chiral selector. The assay with six point multilevel internal calibration provides a linear response for each enantiomer in the 10-200 ng/mL concentration range, is simple, inexpensive, and reproducible, and has an LOQ of 5 ng/mL. It was applied to oral fluid patient samples that were collected up to 12 h after intake of an immediate release tablet and two different extended release formulations with racemic methylphenidate. Drug profiles could thereby be assessed in a stereoselective way. Almost no levorotary threo-methylphenidate enantiomer was detected after intake of the two extended release formulations, whereas this enantiomer was detected during the first 2.5 h after intake of the immediate release preparation. The noninvasive collection of oral fluid is an attractive alternative to plasma for the monitoring of methylphenidate exposure in the pediatric community.

SPE of 5-lipoxygenase metabolites and the effect of head-column field-amplified sample stacking in MEKC

The SPE of leukotrienes and eicosatetraenoic acids using anion exchange materials was compared to the classical extraction with C18 columns. A silica-based strong anion exchanger, a polymer-based weak anion exchanger, and a polymer-based mixed-mode strong anion exchanger were studied. All anion exchange materials displayed a higher recovery of the analytes with values between 70 and 90% when extracting standard solutions and analyzing by HPLC. The effect was less pronounced for the analysis of the compounds in incubations of polymorphonuclear leukocytes. Using MEKC with head-column field-amplified sample stacking for analyte quantification, much lower values of the peak areas were observed compared to the determination of the recovery of the analytes by HPLC. Using MEKC analysis, the highest values were found for the polymer-based weak anion exchange material, while values below 10% were found for the polymer-based mixed mode strong anion exchanger. This could be attributed to the presence of electrolytes in the eluates that compromised the stacking efficiency. The extent of residual electrolytes depended on the SPE protocol, resulting in large differences of the amount of analyte determined by MEKC when applying head-column field-amplified sample stacking for online analyte concentration.

Structural Characterization of Pyridylaminated Oligosaccharides Derived from Neutral Glycosphingolipids by High-Sensitivity Capillary Electrophoresis–Mass Spectrometry

High-sensitivity capillary electrophoresis-electrospray ionization quadrupole ion trap time-of-flight mass spectrometry (CE-ESI-QIT-TOF MS) was developed to structurally characterize four kinds of pyridylaminated (PA) oligosaccharides, i.e., lactose (Lac)-PA, globotriose (Gb3)-PA, globotetraose (Gb4)-PA, and IV(3) αGalNAc-Gb4 (Forssman antigen)-PA, derived from neutral glycosphingolipids. The CE-MS system included the head-column field-amplified sample stacking (HC-FASS) method for effective sample injection into a capillary column in CE, a sheathless interface between CE and a mass spectrometer, and MS and tandem MS (MS(2)) measurements with narrow mass range repeated high-speed switching. The total sensitivity of the developed CE-MS system was about 20,000 times higher than that of the conventional CE-MS system consisting of pressure injection, a sheath-flow interface, and a wide mass range measurement. The MS and MS(2) spectra of the four PA-oligosaccharides at a concentration of 25 amol/μL in mixtures (each 250 amol/10 μL in a tube) clearly showed protonated molecular ions ([M + H](+)) and the fragment ions responsible for the sequential elimination of saccharides. The developed CE-MS system is a powerful method for the structural characterization of glycosphingolipids extracted from very small amounts of biological materials and could be extended to the structural characterization of oligosaccharides derived from glycoproteins.

Separation of 5-Lipoxygenase Metabolites Using Cyclodextrin-Modified Microemulsion Electrokinetic Chromatography and Head Column Field-Amplified Sample Stacking

A cyclodextrin-modified microemulsion electrokinetic chromatography method employing head column field-amplified sample stacking was developed for the analysis of arachidonic acid metabolites of the lipoxygenase pathways. The influence of the concentration of boric acid, the surfactant sodium dodecyl sulfate, the co-surfactant 1-butanol and the oil phase octane as well as the pH of the background electrolyte, the separation voltage and the separation temperature was studied. The optimized microemulsion consisting of 20 mM boric acid buffer, pH 9.0, 3.0 % (m/v) sodium dodecyl sulfate, 0.5 % (v/v) octane, 5.0 % (v/v) 1-butanol and 15 mM α-cyclodextrin enabled the separation of 20-hydroxy-leukotriene B4, leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid, 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid as well as the internal standard prostaglandin B1 in <10 min employing a separation voltage of 17.5 kV at a temperature of 23 °C. A matrix peak from solid-phase extraction sample workup co-migrated with 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid affecting peak integration. The addition of 5 % (v/v) 2-propanol to the microemulsion resulted in the separation of this eicosatetraenoic acid and the matrix components at the expense of analysis time and peak resolution between the diastereomers 6-trans-leukotriene B4 and 6-trans-12-epi-leukotriene B4. In summary, the MEEKC method appeared to be especially suitable for the more polar arachidonic acid metabolites.

Chemometrics-guided development of a cyclodextrin-modified micellar electrokinetic chromatography method with head-column field amplified sample stacking for the analysis of 5-lipoxygenase metabolites

A new sensitive method using α-cyclodextrin-modified micellar electrokinetic chromatography has been developed to separate and quantify arachidonic acid metabolites of the lipoxygenase pathways in human polymorphonuclear leukocytes, i.e. leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, and 15(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid. The electrophoresis system was optimized with regard to the pH, boric acid, SDS and α-cyclodextrin concentration as well as separation voltage and temperature using a three level resolution IV fractional factorial design and a five level circumscribed central composite design. The resulting optimized conditions included 80mM sodium borate buffer, pH 10.07, containing 16.6mM sodium dodecyl sulfate, and 15mM α-cyclodextrin, using a separation voltage of 12.5kV at 23°C. Sensitivity was enhanced employing head-column field amplified sample stacking which resulted in limits of quantification between 30 and 50ng/mL and limits of detection between 10 and 17ng/mL after solid phase extraction of the lipoxygenase products. The method was validated according to the recommendations of the International Conference on Harmonization and applied to the determination of the lipoxygenase metabolites in polymorphonuclear leukocytes upon stimulation with Ca2+-ionophore A23187 and arachidonic acid. Robustness was confirmed using a three level resolution IV fractional factorial design. The novel method is suitable for the analysis of various arachidonic acid metabolites produced by cells and may be used for evaluation of lipoxygenase inhibitors.

Gas dispersion concentration of trace inorganic contaminants from fuel gas and analysis using head-column field-amplified sample stacking capillary electrophoresis

The presence of inorganic elements in fuel gas generally accelerates the corrosion and depletion of materials used in the fuel gas industry, and even leads to serious accidents. For identification of existing trace inorganic contaminants in fuel gas in a portable way, a highly efficient gas-liquid sampling collection system based on gas dispersion concentration is introduced in this work. Using the constructed dual path gas-liquid collection setup, inorganic cations and anions were simultaneously collected from real liquefied petroleum gas (LPG) and analyzed by capillary electrophoresis (CE) with indirect UV absorbance detection. The head-column field-amplified sample stacking technique was applied to improve the detection limits to 2-25 ng mL(-1). The developed collection and analytical methods have successfully determined existing inorganic contaminants in a real LPG sample in the range of 4.59-138.69 μg m(-3). The recoveries of cations and anions with spiked LPG samples were between 83.98 and 105.63%, and the relative standard deviations (RSDs) were less than 7.19%.

The combination of flow injection with electrophoresis using capillaries and chips

The technique of combined flow injection CE (FI-CE) integrates the essential favorable merits of FI and CE. It utilizes the various excellent on-line sample pretreatments and preconcentration (such as cloud point extraction, SPE, ion-exchange, dynamic pH junction and head-column field-amplified sample stacking technique) of FI, which has the advantages of high speed, accuracy, precision and avoiding manual handling of sample and reagents. Therefore, the coupling of FI-CE is an attractive technique; it can significantly expand the application of CE and has achieved many publications since its first appearance. The basic principles, instrumental developments and applications of FI-CE system from 2006 to 2008 are reviewed.

Online Concentration by Head-column Field-amplified with Large-volume Sample Stacking Using Flow Injection-Capillary Electrophoresis for the Analysis of Four Active Components in Cold Medicines

A simple, effective, and sensitive online concentration method for the detection of dextromethorphan hydrobromide (Dex), chlorphenamine hydrogen maleate (Chl), pseudoephedrine hydrochloride (Pse) and paracetamol (Par) based on flow injection-capillary electrophoresis (FI-CE) analysis with head-column field-amplified sample stacking and large-volume sample stacking was developed. The background electrolyte (BGE) was a solution composed of 55 mM borate-15% (v/v) acetonitrile (ACN) (pH 9.3). The sample was injected electrokinetically between plugs of water. Under the optimum conditions, about a 30-fold improvement in the concentration sensitivity relative to normal CE methods was achieved, giving low limits of detection (LOD) of 1.94 x 10(-5), 0.64 x 10(-5), 1.16 x 10(-5) and 2.84 x 10(-5) mg/mL for Dex, Chl, Pse and Par, respectively. The repeatability (defined as RSD) was 1.01, 1.91, 0.89 and 0.92% with the peak-area evaluation and 1.94, 3.98, 2.66 and 3.27% with the peak-height evaluation for Dex, Chl, Pse and Par, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing Dex, Chl, Pse and Par.

Electrophoretic determination of albumin in urine using on-line concentration techniques

To improve the sensitivity of the UV-detection for the determination of trace amounts of albumin by capillary zone electrophoresis (CZE), five on-line preconcentration techniques, including field-amplified sample stacking (FASS), head-column field-amplified sample stacking (HC-FASS), stacking with a polymer solution, dynamic pH junction and large volume sample stacking (LVSS ) with reversed polarity, were compared. Sensitivity enhancement factor and reproducibility were two factors that were used to assess the suitability of each method. To minimize protein adsorption on the capillary wall, capillaries were covalently modified with anionic polymer, poly(sulfopropylmethacrylate) coating. All used methods have good reproducibility. The maximum sensitivity enhancement factor (about 67-fold in terms of peak heights) was achieved with LVSS technique. The concentration limit of detection (LOD) (S/N = 3) for the human serum albumin obtained with the optimized LVSS approach was 15 μg/ml with UV-detection. The method was further evaluated for the analysis of urine samples with gel-filtration-based sample-desalting procedure.

Head-column field-amplified sample stacking in a capillary electrophoresis-flow injection system

A simple, effective, and continuous online concentration method for the sensitive detection of alkaloids applying CE-flow injection analysis with head-column field-amplified sample stacking was developed. A series of samples was continuously introduced into the capillary by electrokinetic means without interrupting the high voltage. A short water plug was introduced by the EOF at the capillary inlet end prior to sample introduction. Under optimum conditions, 15-fold improvement in concentration sensitivity was achieved, giving an LOD of about 0.67 and 0.73 microg/mL for ephedrine (E) and pseudoephedrine (PE), respectively. The separation could be achieved within 4 min and sample throughput rate could reach up to 7/h. The repeatability (defined as RSD) was 3.62, 1.51% with peak area evaluation and 1.30, 2.58% with peak height evaluation for E and PE, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing E and PE, and the recoveries were 92.3-102.4%.

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60–360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of α1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower p I values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher p I values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.