Chapter 15 - Capillary electrophoresis

Two capillary electrophoresis (CE) methods were developed for the analysis of 16 antineoplastic drugs contained in injectable pharmaceutical formulations. A capillary zone electrophoresis (CZE) method coupled to UV was developed with a background electrolyte (BGE) made of a 100 mM phosphate buffer at pH 2.5 containing 50% v/v of acetonitrile and dynamic coating of capillaries with Ceofix®. This method allowed the analysis of doxorubicin, epirubicin, idarubicin, daunorubicin, irinotecan, topotecan, vincristine, vindesine, vinblastine, and vinorelbine in less than 8 min. A micellar electrokinetic chromatography (MEKC) method coupled to UV was also developed for the determination of methotrexate, pemetrexed, etoposide, etoposide phosphate, fludarabine phosphate, and 5-fluorouracil. A run time of 16min was obtained with a BGE made of 50mM borate buffer at pH 9.2 with 80mM of sodium dodecyl sulfate (SDS) and 20% v/v of acetonitrile. For both methods, the applied voltage was 30kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in fused silica capillaries with an internal diameter of 50μm and a total length of 64.5cm. Both methods were validated and trueness values between 99.4 and 101.3% were obtained with repeatability and intermediate precision values of 0.5-1.8% for all drugs. These methods were found appropriate for controlling injectable pharmaceutical formulations containing antineoplastic drugs and successfully applied in quality control.

This review gives a survey of selectivity modes in capillary electrophoresis separations in pharmaceutical analysis and bioanalysis. Despite the high efficiencies of these separation techniques, good selectivity is required to allow quantitation or identification of a particular analyte. Selectivity in capillary electrophoresis is defined and described for different separation mechanisms, which are divided into two major areas: (i) capillary zone electrophoresis and (ii) electrokinetic chromatography. The first area describes aqueous (with or without organic modifiers) and nonaqueous modes. The second area discusses all capillary electrophoretic separation modes in which interaction with a (pseudo)stationary phase results in a change in migration rate of the analytes. These can be divided in micellar electrokinetic chromatography and capillary electrochromatography. The latter category can range from fully packed capillaries, via open-tubular coated capillaries to the addition of microparticles with multiple or single binding sites. Furthermore, an attempt is made to differentiate between methods in which molecular recognition plays a predominant role and methods in which the selectivity depends on overall differences in physicochemical properties between the analytes. The calculation of the resolution for the different separation modes and the requirements for qualitative and quantitative analysis are discussed. It is anticipated that selectivity tuning is easier in separation modes in which molecular recognition plays a role. However, sufficient attention needs to be paid to the efficiency of the system in that it not only affects resolution but also detectability of the analyte of interest.

Chapter 10 Separation of nucleic acids and oligonucleotides and sequencing of DNA Fragments by capillary electrophoresis

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis

Nine xanthones from a Chinese traditional medicine, Securidaca inappendiculata Hassk, were separated by different capillary electrophoresis (CE) modes, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC), and capillary electrochromatography (CEC). The comparison of separation selectivity and efficiency for xanthones by using these CE methods indicated that different CE modes greatly varied in the separation selectivity and efficiency for xanthones; their advantages and disadvantages are discussed. Moreover, compared with traditional chromatographic methods, e.g., high performance liquid chromatography (HPLC), CE shows higher separation ability and analytical speed for these xanthones.

Capillary electrochromatography (CEC) has been realised as a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) allowing the separation of analytes based on their differing adsorption and migration behaviour. There is a considerable interest in developing new methodologies for CEC, which uses chromatographic beads or monoliths as stationary phases or alternatively employs open tubular (OT) capillaries relying on surface modifications. Besides the bulk flat flow profile of the mobile phase in CEC columns allowing an enhanced resolution, due to the influence of the applied electrical field there are other possibilities to provide a higher resolution with better detection. As an additional newly means for peak sharpening and sample enrichment isotachophoresis and a “sweeping mechanism” could be envisaged, which both take advantage of the inhomogeneous electric field pattern in the capillary by choosing appropriate leading and terminating background electrolytes (BGEs). Transient isotachophoresis would be another technique to provide an increased plate height in the separations, using an optimized sample matrix, e.g. containing a relatively high acetonitrile content, with the same leading and terminating BGEs. In this thesis, a new method to generate a “sharpening zone”, which is similar to the concept of transient isotachophoresis, was established using an aqueous sample matrix containing an organic solvent. Target analytes within this zone could be concentrated whilst they migrated through the capillary. A novel technique was developed that used the sharpening zone for sweeping and concentrating several unresolved analyte peaks allowing an enhanced peak resolution. As the aim was to concentrate only some part of the analyte zone containing compounds of interest, the position and width of the sharpening zone needed to be well controlled. Various techniques to tune the width and position of the sharpening zone were thus developed, involving modifying the organic modifier content in the BGE, applying pressure additionally to the electroosmotic flow (EOF) and modifying the inner surface of the capillaries. This work also critically evaluated some related existing knowledge and theoretical approaches in CEC including the concept of tuneable system eigenpeaks (noncomigrating or ghost peaks caused by waves of an electric field in the capillary). Although peak broadening caused by eigenpeaks (which often suppresses resolution in CEC) has been well described for aqueous or pure non-aqueous BGEs, a systematic study of eigenmobility in aqueous/acetonitrile BGEs had not been reported. The experiments related to the eigenmobility led to a new approach to tune the eigenpeak position in chromatograms whereby a selective sample peak sharpening mechanism was elaborated using the sharpening zone. The novel approach was demonstrated for the separation of various peptides in the CE mode and for the resolution of incompletely resolved chiral amino acids in CEC. These investigations furthermore led to the development of an appropriate stationary phase, which was produced using techniques for an in-column synthesis of a molecular imprinted polymer (MIP). Well prepared MIPs are capable of specifically enhancing the retention of target analytes, herein focusing on enantio-selective isolation of chiral molecules. Since such a MIP was rather rigid, which often caused capillary clogging when used as a conventional monolithic stationary phase, this MIP was incorporated into porous layer open tubular (PLOT) capillaries. Numerous experimental difficulties were overcome with respect to the vinylisation of long fused-silica capillaries and for the polymerisation of the MIP within using thermal- and photo-polymerisations. A method for the characterization of such MIP-PLOT capillaries was elaborated involving the Langmuir adsorption theory. The new columns were evaluated using chromatography, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and CEC analysis. All experiments were designed according to the principles of Green Chemistry. These concepts involved the use of small scale analysis, simulation studies reducing the number of performed experiments, systematic studies with low amounts of solvents and materials, and optimisation of synthesis methods to reduce time, energy and reagent consumption. The theories, methods and results presented in this thesis will, in the future, allow further targeted developments of novel analytical tools for a green, sensitive, selective and improved resolution analysis of chemical and biological compounds including chiral compounds, peptides and proteins.

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

The operating parameters that affect the performance of analyte focusing by micelle collapse (AFMC) to neutral analytes (i.e., dialkyl phthalates) in normal migration micellar electrokinetic chromatography (NM-MEKC) are examined. NM-MEKC is characterized by an electroosmotic flow greater than the electrophoretic velocity of the micellar pseudostationary phase, and was performed using sodium dodecyl sulfate (SDS) with neutral to high pH electrolytes in fused silica capillaries. AFMC is a recently introduced on-line sample preconcentration technique in capillary electrophoresis that can provide hundreds fold improvement in detection sensitivity. The mechanism of AFMC is based on the analyte transport, release, and accumulation by the moving surfactant micelles [e.g., SDS at concentrations closer to the critical micelle concentration (CMC)] in the sample that dilutes below its CMC into a liquid phase zone. The sample is prepared in a matrix that contains SDS micelles and high mobility anions, where the conductivity of the sample matrix is higher compared to that of the separation solution. The sample injection length, sample and separation solution conductivity ratio, and surfactant micelle concentration in the sample were found to affect the AFMC performance, as well as the effective separation length in NM-MEKC. The use of a different electrolyte salt in the sample and separation solution also affected AFMC NM-MEKC results. In particular, sodium chloride in the sample matrix can induce a micelle-mediated neutral analyte isotachophoretic concentration that is detrimental to the technique.

Various modes of capillary electrophoresis offer numerous possibilities for biopharmaceutical analysis. Chapter 2 studied capillary zone electrophoresis (CZE) for characterization of glycoprotein erythropoietin by its isoform distribution. The CZE method for identification of recombinant human erythropoietin described in the European Pharmacopoeia has shown poor reproducibility. In this study, it was found that the root cause of the irreproducibility was due to inadequate capillary conditioning. The method was optimized to make it more robust and suitable for quality control laboratory for analysis of epoetin alfa. Further, a procedure for removal of polysorbate 80 from formulated epoetin alfa was developed, allowing the material to be analyzed using the modified CZE method. Chapter 3 describes a micellar electrokinetic chromatography (MEKC) method for the determination of residual cell culture media components including folic acid, hypoxanthine, mycophenolic acid, nicotinic acid, riboflavin, and xanthine. Acceptable limits of detection and quantitation were obtained. The MEKC method was compared to the corresponding CZE method using the same running buffer containing no sodium dodecyl sulfate (SDS). The effect of SDS concentration on separation, the pH of the running buffer, and the detection wavelength were studied and optimal MEKC conditions were established. Chapter 4 describes a capillary SDS gel electrophoresis method for purity and impurity analysis of monoclonal antibodies. The study investigated the effect of sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide, and reduction conditions with 2-mercaptoethanol. It was observed that the sample buffer at slight acidic conditions (pH 5.5-6.5) greatly decreased thermally induced fragmentation of non-reduced CNTO3157. As such, a citrate-phosphate buffer at pH 6.5 was used for sample preparation to replace the commonly used Beckman sample buffer (pH 9.0). Chapter 5 details an investigation of the effect of sample injection length on efficiency and detection sensitivity enhancement in MEKC using the sweeping technique under conditions of suppressed electroosmotic flow. The relationship between injection length and retention factor was derived and experimentally confirmed. A method for predicting the optimal injection length for a given analyte was developed.

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

Application of capillary zone electrophoresis–inductively coupled plasma mass spectrometry and capillary isoelectric focusing–inductively coupled plasma mass spectrometry for selenium speciation

12 CE in impurity profiling of drugs

ABSTRACTFlavonoids are compounds which provide many pharmacological activities. In this paper, the literature with regard to the determination of flavonoids using capillary electrophoresis (CE) during 2008–2017 has been reviewed. The aim of the review is to overview the modes and specific determination conditions of CE for flavonoids. The advantages and disadvantages of CE in the determination of flavonoids are analyzed. The applications of the modes of CE for determination of flavonoids are introduced. Capillary zone electrophoresis, micelle electrokinetic capillary chromatography, microemulsion electrokinetic chromatography, and capillary electrochromatography are considered. Specific determination conditions including the pH, buffer, additive, capillary, voltage, temperature, injection, analysis time, detection, and online concentration methods are summarized. Flavonoids were generally separated in alkaline conditions at pH 8–10 in borate buffer. The advantages of CE are the short analysis time and low solvent consumption compared with the most popular high-performance liquid chromatography protocols. CE can be combined with a variety of detectors including ultraviolet spectrophotometry, mass spectrometry, chemiluminescence, electrochemical, potential gradient, dual opposite carbon fiber microdisk electrodes, and laser-induced fluorescence. In CE, short-light path length results in relative low sensitivity when combined with the most widely used ultraviolet spectrophotometry detector. The combination with more sensitive detectors and online concentration methods such as gradient elution, large volume sample stacking, sweeping, and electrokinetic supercharging alleviates this problem and improves the sensitivity. Capillary electrophoresis is an efficient, green, and valuable tool for the determination of flavonoids.

Capillary electrophoresis (CE) is an instrumental technique that employs narrow‐bore (10–100 µm ID, internal diameter) capillaries under the influence of a high‐gradient electric field to perform high‐efficiency separations. Different working modes of CE have been described. Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) have become the most popular modes of CE in environmental applications. Other kinds of electrokinetic chromatography (EKC) and other modes of CE like capillary electrochromatography (CEC) have also been used.CE techniques have been considered as a good alternative for analyzing pollutants because of their high performance and versatility related to selectivity and range of application. In addition, CE is a powerful tool in the field of chiral separations and it is considered one of the so‐calledcleananalytical techniques. Although the main drawback of CE techniques in environmental analysis is their insufficient sensitivity for detecting trace‐level pollutants, some procedures have been developed in order to overcome this problem.

Biopolymer such as DNA is important in living cells since its genetic information provides many building blocks for cell components. Also, DNA is employed for diagnosis of genetic disease, DNA sequencing, forensic investigation, and post-human genome project. Conventional methods such as slab gel electrophoresis (SGE) and ion chromatography (IC) for DNA analysis suffered long separation time and low resolution. However, recently developed capillary gel electrophoresis (CGE) showed high speed and high separation efficiency for especially DNA separation. Capillary electrophoresis (CE) without sieving medium does not have enough separation power for DNA sample with different lengths since each DNA has very similar charge-to-mass ratio, which deteriorates mobility differences for various sizes of DNA under electric field. Therefore, development of the sieving medium is the key for efficient DNA analysis. In early era for the capillary format, agarose or crosslinked polyacrylamide had been used at the expense of bubble formation at high electric field, gel instability, and no room for gel replacement. Later, water-soluble polymers such as cellulose derivatives including methyl cellulose (MC), hydroxyethyl cellulose (HEC), and hydroxypropylmethyl cellulose (HPMC) had been employed for relatively short DNA fragment analysis. These polymers exhibited strong stability under high electric field and ease of replacement. Liner type polymers such as linear polyacrylamide (LPA), polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) also provided similar effects on DNA separation. However, either cellulose or linear type polymers had relatively high solution viscosity for high resolution of DNA samples, which made it difficult for automation and fast analysis. In high throughput analysis for DNA sequencing, single nucleotide polymorphism (SNP), and polymerase chain reaction (PCR) products, effective but low viscosity sieving medium is essential for high sieving ability and easy replacement. Recently, mannitol added poly(N-isopropylacrylamide) (PNIPAM) or gold nanoparticle (GNP) as the sieving medium showed the potential for this purpose. In GNP case, the production of GNP took long time and the cost was high for preparation. In this paper, we have employed relatively cheap silica or α-alumina nanoparticle as the sieving medium. These nanoparticles were successfully applied for DNA analysis with the size range of 500 bp to 5000 bp. Figure 1 shows the electropherograms of 500 bp step ladder by capillary electrophoresis with different sieving matrices. Since the direction of the electosmtic flow (EOF) caused by surface silanol groups on fused silica capillary is toward negative electrode (opposite compared to migration of DNA) and the mangnitude of EOF is usually larger than that of electrophoretic mobility of DNA, the capillary surface needs to be covered in order to reduce EOF. The capillary surface in this experiment was coated either permanently (Figure 1(a) and (b)) or dynamically (Figure 1(c) to (e)). Figure 1(a) exhibited the significance of sieving