High Performance Anion Exchange and Hydrophilic Interaction Liquid Chromatography Approaches for Comprehensive Mass Spectrometry-Based Characterization of the N-Glycome of a Recombinant Human Erythropoietin.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

Preparation and characterisation of anion-exchange latex-coated silica monoliths for capillary electrochromatography.

Silica monoliths coated with functionalised latex particles have been prepared for use in monolithic ion-exchange capillary electrochromatography (IE-CEC) for the separation of inorganic anions. The ion-exchange monoliths were prepared using 70 nm quaternary ammonium, anion-exchange latex particles, which were bound electrostatically to a monolithic silica skeleton synthesised in a fused silica capillary. The resulting stationary phases were characterised in terms of their chromatographic performance and capacity. The capacity of a 50 microm diameter 25 cm latex-coated silica monolith was found to be 0.342 nanoequivalents and 80,000 theoretical plates per column were typically achieved for weakly retained anions, with lower efficiency being observed for analytes exhibiting strong ion-exchange interaction with the stationary phase. The electroosmotic flow (EOF) was reversed after the latex-coating was applied (-25.96 m2 V(-1) s(-1), relative standard deviation (RSD) 2.8%) and resulted in anions being separated in the co-EOF mode. Ion-exchange interactions between the analytes and the stationary phase were manipulated by varying the ion-exchange selectivity coefficient and the concentration of a competing ion (phosphate or perchlorate) present in the electrolyte. Large concentrations of competing ion (greater than 1M phosphate or 200 mM perchlorate) were required to completely suppress ion-exchange interactions, which highlighted the significant retention effects that could be achieved using monolithic columns compared to open tubular columns, without the problems associated with particle-packed columns. The latex-coated silica monoliths were easily produced in bulk quantities and performed reproducibly in acidic electrolytes. The high permeability and beneficial phase ratio makes these columns ideal for micro-LC and preconcentration applications.

Development of a polar-embedded stationary phase with unique properties.

This paper describes a new polar-embedded stationary phase that contains an internal sulfonamide functional group coupled with an ether linkage. The synthesis involves functionalization of spherical silica particles with ligands prepared in a multi-step synthesis. The resulting material contains 16.5% carbon, corresponding to a ligand coverage of 2.4mumol/m(2). Chromatographic evaluations indicates that the new stationary phase exhibits lower polarity than any other polar-embedded packings investigated, with additional features such as low silanol activity, excellent compatibility with 100% aqueous mobile phases, higher shape selectivity for polycyclic aromatic hydrocarbons, and strong affinity to nitro-containing compounds.

On-line preconcentration of organic anions in capillary electrophoresis by solid-phase extraction using latex-coated monolithic stationary phases.

Quaternary ammonium functionalised polymeric latex particles were coated onto the wall of a fused-silica capillary or onto a methacrylate monolithic bed synthesised inside the capillary in order to create ion-exchange stationary phases of varying ion-exchange capacity. These capillaries were coupled in-line to a separation capillary and used for the solid-phase extraction (SPE), preconcentration and subsequent separation of organic anions by capillary electrophoresis. A transient isotachophoretic gradient was used for the elution of bound analytes from the SPE phase using two modes of separation. The first comprised a low capacity SPE column combined with a fluoride/octanesulfonate discontinuous electrolyte system in which peak compression occurred at the isotachophoretic gradient front. The compressed anions were separated electrophoretically after elution from the SPE preconcentration phase and resolution was achieved by altering the pH of the electrolyte in which the separation was performed. In the second approach, a latex-coated monolithic SPE preconcentration stationary phase was used in combination with a fluoride/perchlorate electrolyte system, which allowed capillary electrochromatographic separation to occur behind the isotachophoretic gradient front. This method permitted the removal of weakly bound anions from the SPE phase, thereby establishing the possibility of sample clean-up. The effect of the nature of the strong electrolyte forming the isotachophoretic gradient on the separation and also on the preconcentration step was investigated. Capillary electrochromatography of inorganic and organic species performed on the latex-coated monolithic methacrylate column highlighted the presence of mixed-mode interactions resulting from the incomplete coverage of latex particles onto the monolithic surface. Analyte preconcentration prior to separation resulted in compression of the analyte zone by a factor of 300. Improvement in the limit of detection of up to 10400 times could be achieved when performing the preconcentration step and the presented methods had limits of detection (S/N=3) ranging between 1.5 and 12 nM for the organic anions studied.

Latex-coated polymeric monolithic ion-exchange stationary phases. 1. Anion-exchange capillary electrochromatography and in-line sample preconcentration in capillary electrophoresis.

A sulfonated methacrylate monolithic polymer has been synthesized inside fused-silica capillaries of diameters 50-533-microm i.d. and coated with 65-nm-diameter fully functionalized quaternary ammonium latex particles (AS18, Dionex Corp.) to form an anion-exchange stationary phase. This stationary phase was used for ion-exchange capillary electrochromatography of inorganic anions in a 75-microm-i.d. capillary with Tris/perchlorate electrolyte and direct UV detection at 195 nm. Seven inorganic anions (bromide, nitrate, iodide, iodate, bromate, thiocyanate, chromate) could be separated over a period of 90 s, and the elution order indicated that both ion exchange and electrophoresis contributed to the separation mechanism. Separation efficiencies of up to 1.66 x 10(5) plates m(-1) were achieved, and the monoliths were stable under pressures of up to 62 MPa. Another latex-coated monolith in a 250-microm-i.d. capillary was used for in-line preconcentration by coupling it to a separation capillary in which the EOF had been reversed using a coating of either a cationic polymer or cationic latex particles. Several capillary volumes of sample were loaded onto the preconcentration monolith, and the analytes (inorganic anions) were then eluted from the monolith with a transient isotachophoretic gradient before being separated by electrophoresis in the separation capillary. Linear calibration curves were obtained for aqueous mixtures of bromide, nitrite, nitrate, and iodide. Recoveries of all analytes except iodide were reduced significantly when the sample matrix contained high levels of chloride. The preconcentration method was applied to the determination of iodide in open ocean water and provided a limit of detection of 75 pM (9.5 ng/L) calculated at a signal-to-noise ratio of 3. The relative standard deviation for migration time and peak area for iodide were 1.1 and 2.7%, respectively (n = 6). Iodide was eluted as an efficient peak, yielding a separation efficiency of 5.13 x 10(7) plates m(-1). This focusing was reproducible for repeated analyses of seawater.

Latex-coated polymeric monolithic ion-exchange stationary phases. 2. Micro-ion chromatography.

Latex-coated monolithic polymeric stationary phases are used for micro-ion chromatography (mu-IC) of inorganic anions. Monolithic columns were prepared by the in situ polymerization of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid within fused-silica capillaries of varying internal diameters. Introduction of ion-exchange sites was achieved by coating the anionic polymeric monolith with either Dionex AS10 or Dionex AS18 quaternary ammonium functionalized latex particles to give total ion-exchange capacities in the range 9-24 nequiv for a 30-cm column. The resultant mu-IC columns were used for the separation of anionic analytes using chloride or acetate as the eluent-competing ion and direct UV spectrophotometric detection at 195 nm or using hydroxide as the eluent-competing ion and suppressed or nonsuppressed contactless conductivity detection. Separation efficiencies of 13,000 plates/m were observed (for iodate), and separation efficiency was maintained for large increases in flow rate (up to 42 microL/min, corresponding to a linear flow velocity of 18.5 mm/s), enabling highly reproducible, rapid separations to be achieved (seven analyte anions in less than 2 min). Use of a hollow fiber micromembrane suppressor enabled effective suppression of hydroxide eluents over the range 0.5-5.0 mM, thereby permitting suppressed conductivity detection to be performed. However, the relatively large size of the suppressor resulted in reduced separation efficiencies (e.g., 5400 plates/m for iodate). Detection limits obtained with suppressed conductivity detection were in the range 0.4-1.2 microM.

Recent developments in electrolytic devices for ion chromatography.

Recent developments in new electrolytic devices that utilize the electrolysis of water and charge-selective electromigration of ions through ion-exchange media have significantly changed the routine operation of ion chromatographic methods. Examples of these new electrolytic devices include on-line eluent generators that produce high-purity electrolyte eluents using deionized water as the carrier stream, continuously regenerated trap columns that remove ionic contaminants in the eluents, and continuously regenerated suppressors that reduce eluent background conductance prior to conductivity detection. The combined use of these electrolytic devices has made it possible to perform various ion chromatographic separations using only deionized water as the mobile phase. This paper reviews the operation principles of these electrolytic devices and their applications in the ion chromatographic determination of anionic and cationic analytes.

Analysis of amino acid-carbohydrate mixtures by anion exchange chromatography and integrated pulsed amperometric detection.

A review is presented of recent developments in the area of analysis of amino acid-carbohydrate mixtures. Based on its broad selectivity, the AminoPac PA10 column exhibits a remarkable capability to perform simultaneous separations of amino acids and carbohydrates. This ability is further enhanced by the equal sensitivity for carbohydrates and amino acids exhibited by the "amino acid" integrated pulsed electrochemical detection (IPAD) waveforms. Equimolar levels of carbohydrates and amino acids are separated either by optimized elution gradients alone or by a combination of modified gradients and a bi-modal IPAD waveform. Samples containing large amounts of carbohydrates may be analyzed after suitable sample preparation. Both a manual off-line method and a fully automatic on-line method are discussed. In addition, we will review the application of these methods to various types of samples, including cell culture media, glycoprotein hydrolysates, beverages, condiments and soil extracts.

CarboPac PA20: a new monosaccharide separator column with electrochemical detection with disposable gold electrodes.

This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3x150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 microm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.

Use of coupled open-tubular capillaries for in-line ion-exchange preconcentration of anions by capillary electrochromatography with elution by a transient isotachophoretic gradient.

Open-tubular capillaries have been joined together for use in on-column ion-exchange preconcentration of anions by capillary electrochromatography (CEC) with elution by a transient isotachophoretic gradient. This involved the coupling of a preconcentration capillary and a separation capillary using a PTFE sleeve. Such coupling allowed precise lengths of differently coated capillaries to be joined in-line to form a single multi-mode column. The different segments could be tailored to optimize a separation by either altering the length of each segment to precisely manipulate the amount of stationary phase present or by changing the internal diameter of each segment to alter the phase ratio in the chromatographic column without affecting the path length for UV detection. In this work, a segmented in-line capillary was used in conjunction with a fluoride-octanesulfonate discontinuous electrolyte system to increase the number of anions that could be preconcentrated and separated. Quaternary ammonium functionalised latex particles were used for creating the preconcentration segment and the separation segment was coated with poly(diallyldimethylammonium chloride). This allowed the detection of trace anions in drinking water and in situ sampling of river water for the analysis of trace inorganic anions. The repeatability of producing the quaternary ammonium functionalized latex-coated segments was assessed and the effect of segmentation on peak efficiency was investigated.

Environmental applications of a cryptand adjustable-capacity anion-exchange separator.

A 2.2.2 cryptand-based anion exchanger was recently introduced as a commercial product. This new technology relies on a covalently bonded 2.2.2 cryptand, which allows one to selectively control the capacity of the column simply by the choice of eluent. This provides the analyst with more flexibility over conventional anion exchangers to suit the needs of the sample matrix. Since that time, a 2.2.1 version has also been developed and studied. In this paper we will compare the two types of columns and choose the best one for analyzing several environmental samples.

Use of disposable gold working electrodes for cation chromatography-integrated pulsed amperometric detection of sulfur-containing amino acids.

We have prepared disposable thin-film gold working electrodes on polymeric substrates. Our microfabrication process allows for inexpensive and reproducible mass production of such electrodes. We utilize this new type of electrode in flow-through electrochemical cells to replace the conventional non-disposable gold working electrodes for integrated pulsed amperometric detection (IPAD) of compounds separated by high-performance cation-exchange chromatography. Using two S-containing amino acids (homocysteine and cysteine) as test compounds, we have modified a previously reported waveform for optimum performance with disposable gold electrodes. With the help of the same two test substances we have characterized the analytical performance of disposable gold electrodes under the new conditions. Compared to non-disposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration and reproducibility. When used with a new IPAD waveform, the disposable electrodes functioned reproducibly for 3 days. At the end of the specified usage period of 3 days, the disposable electrodes are simply replaced. Reconditioning by polishing is thus no longer required.

Development and characterization of microfabricated disposable gold working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection.

We have developed a new type of microfabricated thin-film electrode on polymeric substrates. The microfabrication process allows for inexpensive and reproducible mass production of disposable working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection (IPAD). These microfabricated electrodes are disposable and have been optimized for use in flow-through low-dead-volume electrochemical cells. The analytical performance of microfabricated gold electrodes was characterized with the help of the IPAD method for amino acid detection under alkaline conditions required for anion-exchange separations. When used with a new optimized six-potential IPAD waveform, the electrodes functioned properly for weeks. Compared to nondisposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration, and reproducibility. Disposable electrodes make it possible to avoid polishing and reconditioning, which are required with nondisposable electrodes.

Simulation and optimization of retention in ion chromatography using virtual column 2 software.

A new software package, Virtual Column 2, is described for the simulation and optimization of the separation of inorganic anions by ion chromatography (IC). The software uses a limited amount of experimental retention data acquired according to a correct experimental design to predict retention times for analytes over a designated search area of eluent compositions. The experimental retention data are used to solve a new retention model, called the linear solvent strength model, empirical approach (LSSM-EA), which then enables prediction of retention times for all eluent compositions in the search area. The theoretical development of LSSM-EA and the processes used for solving the equations are discussed. Virtual Column 2 can be used for eluents containing one or two competing ions, and the software contains retention databases for up to 33 analytes on the Dionex AS9A-HC, AS4A-SC, and AS14A analytical columns with carbonate-bicarbonate eluents and the Dionex AS10, AS15, and AS16 analytical columns with hydroxide eluents (results for the AS10 and AS15 columns are not discussed in the present study). Virtual Column 2 has been evaluated extensively and is shown to give predicted retention times that in most cases agree with experimentally determined data to within 5%. The software has uses in practical IC method development, education and training in IC, and refinement of existing IC methodology. A free version of this program is available by download at www.virtualcolumn.com.

On-line pH modification of carbonate eluents using an electrolytic potassium hydroxide generator for ion chromatography.

Classical gradient elution, based on the application of a gradient pump used for mixing two or more prepared eluent components in pre-determined concentrations, was replaced by a chromatography system equipped with an isocratic pump and an electrolytic KOH generator. The isocratic pump delivered a constant concentration eluent composed of pure hydrogencarbonate solution. Carbonate ions, the main component of carbonate/hydrogencarbonate-based eluents, were formed by titration of hydrogencarbonate with KOH formed on-line in the electrolytic KOH generator. By changing the concentration of electrolytically-generated KOH, the eluent composition could be changed from pure hydrogencarbonate to a carbonate/hydrogencarbonate buffer, and finally to a carbonate/hydroxide-based eluent. The described system was tested to achieve pH-based changes of retention behavior of phosphate under constant inflow eluent composition conditions.

Determination of anions at trace levels in power plant water samples by ion chromatography with electrolytic eluent generation and suppression.

An ion chromatographic method was developed for the determination of nine inorganic and organic acid anions at sub- to low-microg/l levels in power plant water samples. In this method, samples were injected using a large-volume direct injection technique, the analyte anions were separated on a hydroxide-selective anion-exchange column using high-purity hydroxide eluents generated by an on-line electrolytic eluent generator and detected using the suppressed conductivity detection method. The method performance was evaluated by analyzing synthetic water samples containing additives encountered in the power plant water samples and four water samples from a fossil fuel power plant. The relative standard deviations of retention times of analyte ions separated on the hydroxide-selective anion-exchange column were less than 0.4%. The recoveries of analyte ions spiked into the synthetic water samples at concentrations of 0.13-1.0 microg/l were in the range of 70-120%. The method detection limits for analyte ions in deionized water were 0.0099, 0.0056, 0.019, 0.057, 0.0084, 0.023, 0.067, 0.037, and 0.079 microg/l for fluoride, acetate, formate, chloride, nitrite, sulfate, bromide, nitrate, and phosphate, respectively.

Adjustable-capacity anion-exchange separator.

A cryptand-based anion exchanger has been developed in which the capacity and to a lesser degree, selectivity are adjustable simply by the choice of the mobile phase. Although much work has been done in the past using cryptand-based anion exchangers, these stationary phases were based on adsorbed cryptands rather than covalently bound cryptands. These phases suffered from the usual problems associated with adsorbed systems. A novel styrene-based cryptand has been synthesized which can be covalently attached to a solid support. A brief review of cryptands and binding constants as well as comparisons of adsorbed phases versus covalently bound phases will be discussed. Some of the unique chromatographic properties of this prototype column will be illustrated as well.

On-column ion-exchange preconcentration of inorganic anions in open tubular capillary electrochromatography with elution using transient-isotachophoretic gradients. 3. Implementation and method development.

A solid-phase extraction method based on an ion-exchange retention mechanism has been used for in-line preconcentration of inorganic anions prior to their separation by capillary electrophoresis (CE). A single capillary containing a preconcentration and a separation zone has been used in a commercial CE instrument without instrumental modification. Analyte anions were retained on a preconcentration zone comprising an adsorbed layer of cationic latex particles, while separation was achieved in a separation zone comprising fused silica modified by adsorption of a cationic polymer. Elution of the adsorbed analytes was achieved using an eluotropic gradient formed by a transient isotachophoretic boundary between a fluoride electrolyte and a naphthalenedisulfonate electrolyte. Optimization of the electrolyte concentrations, sample injection times, and back-flushing times allowed the successful separation of sub-ppb levels of inorganic anions using a 100-min injection at 2 bar pressure, introducing over 40 capillary volumes of sample. A method based on a 10-min injection allowed a 100-fold increase in sensitivity over conventional hydrodynamic injection for Br-, I-, NO3-, CrO4(2-), and MoO4(2-) with a total analysis time of 25 min. Detection limits were dependent on the injection time but were in the range 2.2-11.6 ppb for a 10-min injection time. This approach was used to determine NO3- in Antarctic ice cores where the analysis could be performed using a sample volume 100 times less than that used for ion chromatography.