Capillary Electrophoretic Separation Research Articles

Sulfonic Functionalized Polydopamine Coatings with pH-Independent Surface Charge for Optimizing Capillary Electrophoretic Separations.

Enhancing the pH-independence and controlling the magnitude of electroosmotic flow (EOF) are critical for highly efficient and reproducible capillary electrophoresis (CE) separations. Herein, we present a novel capillary modification method utilizing sulfonated periodate-induced polydopamine (SPD) coating to achieve pH-independent and highly reproducible cathodic EOF in CE. The SPD-coated capillaries were obtained through post-sulfonation treatment of periodate-induced PDA (PDA-SP) coatings adhered on the capillary inner surface. The successful immobilization of the SPD coating and the substantial grafting of sulfonic acid groups were confirmed by a series of characterization techniques. The excellent capability of PDA-SP@capillary in masking silanol groups and maintaining a highly robust EOF mobility was verified. Additionally, the parameters of sulfonation affecting the EOF mobilities were thoroughly examined. The obtained optimum SPD-coated column offered the anticipated highly pH-independent and high-strength cathodic EOF, which is essential for enhancing the CE separation performance and improving analysis efficiency. Consequently, the developed SPD-coated capillaries enabled successful high-efficiency separation of aromatic acids and nucleosides and rapid cyclodextrin-based chiral analysis of racemic drugs. Moreover, the SPD-coated columns exhibited a long lifetime and demonstrated good intra-day, inter-day, and column-to-column repeatability.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

(Keynote) Electrochemical and Mass Spectrometry Measurements of Nanometer Vesicles

We originally developed a tool to measure the chemical messenger contents of single nanometer vesicles starting with capillary electrophoretic separation, lysing and electrochemical detection. We called this flow vesicle impact cytometry (FVIEC). The method used a microfluidic device with sheath flow and electrode for detection. The next generation approaches led to static electrochemical measurements and mass spectrometry imaging of vesicular content. First, micro-/nano-electrochemical approaches have been developed to carry out quantitative measurements of intravesicular content and real-time monitoring of vesicle release dynamics. We have used stochastic vesicle impact electrochemical cytometry (VIEC) and intracellular VIEC (IVIEC) to provide a highly effective way to quantify the electroactive contents inside vesicles. This has allowed direct comparison of the quantity of molecules released by exocytosis to those in the vesicles. Partial release is observed across all cell types examined to date by the electrochemical methods and appears to be regulated.More recently, we have used mass spectrometry imaging with NanoSIMS as a complimentary approach to image the neurotransmitter dopamine across the internal volume of these incredibly small vesicular structures. We have developed standards to carry out quantitative NanoSIMS and subsequently used it to quantify the chemical contents across the nanometer structure of vesicles. We then used NanoSIMS imaging of dopamine inside and drug into vesicles to quantify the fraction of messenger and demonstrate drug entry during exocytosis providing definitive evidence of partial release and a means to introduce drug into cells.

Chitosan derivatives as dynamic coatings for transferrin glycoform separation in capillary electrophoresis

Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Nonaqueous Capillary Electrophoretic Separation of Analogs of (24R)-1,24-Dihydroxyvitamin D3 Derivative as Predicted by Quantum Chemical Calculations.

Nonaqueous capillary electrophoretic (NACE) separation was obtained of analogs of (24R)-1,24-dihydroxyvitamin D3 derivative (calcipotriol) as predicted by quantum chemical calculations supported by the density functional theory (DFT). Among the key electronic properties investigated, absolute values of the dipole polarizability and energy gap between HOMO and LUMO molecular orbitals of the analog molecules differ significantly for particular analogs, and there is a direct relationship with their electrophoretic migration time. These differences and relationships suggest that the structurally related analogs should be separable in the electrostatic field. Indeed, the robust, sensitive, and rapid NACE method was first developed for the identification and determination of the anticancer analog of calcipotriol (coded PRI-2205) and its process-related impurities (coded PRI-2201, PRI-2203, and PRI-2204) in organic and aqueous biological solutions. The direct relation between the calculated electronic properties of the analogs and the experimental electrophoretic migration time could be a promising prospect for theoretically predicting the electrophoretic separations.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX-MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

Fluorescently Labeling Amino Acids in a Deep Eutectic Solvent.

The increased use of deep eutectic solvents (DESs) in recent years has been significant and provides new approaches to sample collection and preparation. At the same time, the use of these new solvents to prepare samples can present challenges for subsequent analyses. Common analytical approaches, such as fluorescent labeling, may not be compatible with the solvents. In this work, we explore how effective three traditional fluorescent labels can be at derivatizing amino acids in the most common DESs, formed from choline chloride and ethylene glycol. We demonstrate that the unique solvent characteristics of the DESs still allow for two of the fluorophores, fluorescein isothiocyanate and 5-carboxyfluorescein succinimidyl ester, to effectively label amino acids. Initial optimizations of the reaction conditions demonstrate that we can effectively label both d- and l-amino acids, in solution with concentrations of amino acids down to 4 μM. Capillary electrophoretic separations following this preparation can detect as little as 50 nM. This is possible without removal of any DES from the sample matrix. These results represent the first complete fluorescent labeling reaction in a DES and subsequent capillary electrophoretic separation of the analytes.

The Effect of Molecular Mass of Hydroxyethyl Cellulose on the Performance of Capillary Electrophoretic Separation of Proteins

Capillary electrophoresis (CE) is a versatile analytical separation method in the field of biochemistry. Although it has been proved that the relative molecular mass (Mr) of the polymers determines the threshold concentration of the entangled polymer solution, which will affect the separation performance of DNA molecules, there is still no report on the effect of Mr on the separation performance of proteins. Herein, we have thoroughly performed the CE of proteins ranged from 14.3 kDa to 116 kDa in a mixed hydroxyethyl cellulose (HEC) solution. The mixed solution was obtained with various Mr including 90,000, 250,000, 720,000, and 1,300,000. Then, we found that the mixed polymer provided a high resolution for small protein molecules while increasing the efficiency of large ones. Results demonstrated that the migration time decreased if HEC (1,300,000) was mixed with the lower Mr one, and the mixed solution (1,300,000/250,000) offered the highest resolution. The resolution was negatively correlated with the electric field strength. Finally, we have employed the optimal electrophoretic conditions to separate proteins in human tears, and it showed that lysozyme, lipocalin, and lactoferrin from human tears were successfully resolved in the mixed HEC. Such work indicates that CE has the potential to be developed as a tool for the diagnosis of xerophthalmia, meibomian gland dysfunction, or other eye diseases.

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations.

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40×10-9 m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.

Comparative Determination of Melittin by Capillary Electrophoretic Methods

Bee venom from honey bees (Apis Mellifera L.) is known to have many pharmacological and biological properties. Melittin, a peptide consisting of 26 amino acids, is known as the main component of bee venom. The study aims to develop a rapid capillary electrophoresis method for separating and quantifying melittin in honeybee venom. Since melittin is a basic peptide, it will adhere to the capillary wall during separation. Two different methods were developed in this study for the capillary electrophoretic separation of melittin. As a first approach, a low pH buffer system was used. For the second approach, the capillary column was coated with a positively charged polymer (PEI). With both methods developed, the migration of melittin in the capillary was achieved by preventing wall adsorption. Melittin migrated in 6 min when the low-pH buffer system was applied, whereas its migration time is longer than 10 min in the PEI-coated capillary column. Thus, a low-pH buffer system was preferred for the analysis of the actual bee-venom sample. 100 mmol L-1 phosphoric acid/sodium dihydrogen phosphate system at pH 1.55 was chosen as separation buffer. As a conclusion, a fast and reliable method was developed for the determination of melittin in honeybee venom. The method was applied to an Anatolian bee venom sample to highlight the melittin amount. The melittin amount was found as 24.5 ± 3.4 g 100 g-1 in the bee venom sample.

Covalent cationic copolymer coatings allowing tunable electroosmotic flow for optimization of capillary electrophoretic separations

Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30–40) × 10−9 m2V−1s−1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0–60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2–10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Quantifying 35 transcripts in a single tube: model-based calibration of the GeXP multiplex RT-PCR assay

BackgroundQuantitative analysis of differential gene expression is of central importance in molecular life sciences. The Gene eXpression Profiling technology (GeXP) relies on multiplex RT-PCR and subsequent capillary electrophoretic separation of the amplification products and allows to quantify the transcripts of at least 35 genes with a single reaction and one dye.ResultsWe provide a kinetic model of primer binding and PCR product formation as the rational basis for taking and evaluating calibration curves. The calibration procedure and the model predictions were validated with the help of a purposefully designed data processing workflow supported by easy-to-use Perl scripts for calibration, data evaluation, and quality control. We further demonstrate the robustness and linearity of quantification of individual transcripts at variable relative abundance of other co-amplified transcripts in a complex mixture of RNAs isolated from differentiating Physarum polycephalum plasmodial cells.ConclusionsWe conclude that GeXP analysis is a robust, sensitive, and useful method when the transcripts of tens to few hundred genes are to be precisely quantified in a high number of samples.

Simultaneous determination of four phenolic acids in traditional Chinese medicine by capillary electrophoresis-chemiluminescence.

Chlorogenic, ferulic, vanillic, and caffeic acids are phenolic acids found in natural drugs. They possess the biological activities of scavenging free radicals and inhibiting thrombus formation. Phenolic acids can inhibit the oxidation of low-density lipoprotein, as well as have anti-inflammatory effects. This paper reports for the first time a capillary electrophoresis–chemiluminescence (CE–CL) method for the simultaneous determination of the four phenolic acids found in traditional and proprietary Chinese medicine, including Lycium chinense Miller, Shuanghuanglian oral liquid, and Taraxacum mongolicum granules. Capillary electrophoretic separation was performed on a self-assembled CE–CL device with an uncoated fused-silica capillary (66 cm effective length, 50 μm i.d.), and the background electrolyte was composed of 3.0 × 10−5 M Ag(iii) (pH = 12.01), 3.0 mM luminol (pH = 9.20), and 10 mM sodium tetraborate solution. The injection time was 12 s (under gravity) and the separation voltage was 22 kV. The combination of solid-phase extraction (SPE) and CE–CL improves the sensitivity. Under optimal conditions, calibration graphs displayed a linear range between 0.625 and 20.0, 1.000 and 30.0, 0.150 and 1.50, and 0.045 and 1.00 μg mL−1 for chlorogenic, ferulic, vanillic, and caffeic acid, respectively. The detection limit ranged from 0.014 to 0.300 μg mL−1. The practicality of using the proposed method to determine the four target analytes in traditional Chinese medicine was also validated, in which recoveries ranged from 90.9% to 119.8%. Taken together, these results indicate that the developed method is sensitive and reliable. Furthermore, the method was successfully applied to real traditional Chinese medicine samples.

Research progress of electrically-driven force based online rapid separation and enrichment techniques

Sample preparation is a critical step in complex sample analysis, which enables analyte isolation and preconcentration from a complex matrix. Therefore, sample preparation is an effective approach to enhance the sensitivity, selectivity, and accuracy of the analytical method. However, the transfer of target analytes from a random state in the original sample matrix to a highly ordered pre-analysis state involves an entropy reduction process that cannot occur spontaneously. Therefore, sample preparation is always a time-consuming, labor-intensive, and error-prone process. Introducing additional energy or reducing the entropy of the system can enhance the separation and enrichment effects as well as accelerate sample preparation. The introduction of an electric field into an online sample preparation system can not only introduce additional energy into the system, but also drive the directional migration of the sample among the separation, enrichment, and detection processes, ensuring that the entropy reduction progresses smoothly. These advantages of electrically-driven force based online separation and enrichment techniques make them effective for accelerating sample preparation. Typically, there are four acceleration strategies in electrically-driven force based online separation and enrichment techniques: (1) the additional energy of the electric field is added into the system to accelerate mass transfer and energy exchange; (2) electrically-driven flows, including electroosmotic flow and electrophoretic flow, are applied to drive the directional migration of the sample among the separation, enrichment, and detection processes, ensuring that sample preparation and analysis are executed smoothly; (3) the online integration technique is applied to enhance the automaticity of the entire sample preparation and analysis processes, and reduce errors from manual operation; (4) device miniaturization or size reduction methods such as microextraction are applied to enhance the sample preparation efficiency and reduce the time consumed. This review summarizes the progress in electrically-driven force based online rapid separation and enrichment techniques in the last ten years. In this specific research area, more than one hundred research papers are published each year, and can be classified into three types based on the electrically-driven force based online rapid separation and enrichment techniques considered: capillary, microchip, and membrane extraction. Among these, over 50% of the studies focused on electrically-driven capillary force based online rapid separation and enrichment techniques. By applying a high-voltage electric field at the two ends of a capillary, charged species in the capillary can migrate along the direction of the medium. This makes the electrically-driven capillary technique not only fast, highly efficient, and low-cost, but also effective for rapid sample preparation. Typically, two modes of electrically-driven capillary force based online rapid separation and enrichment techniques are employed: online capillary electrophoretic separation and enrichment, and online microextraction-capillary electrophoresis. Device miniaturization from the capillary to microchip through microelectronic mechanical systems and microfluidics enables small-amount sample preparation and analysis, and is also advantageous due to being rapid and efficient, as well as low energy- and sample-consuming. The specific easy-integration trait of microchip devices enables the online integration of multi-step sample preparation and analysis. On the other hand, the controllable electrically-driven force can be used for both, directional flow transfer between different functional units in the microchip as well as for dynamic control of the electrically fluid pump and valve. Online microchip electrophoretic separation and enrichment, and online microextraction-microchip electrophoresis, are two common modes of microchip electrically-driven force based online rapid separation and enrichment techniques. In electrically-driven membrane extraction online separation and enrichment techniques, a supporting liquid membrane is used to eliminate the matrix interference, enabling large-scale real sample application. These techniques have received increasing attention in the research area of electrically-driven force based online separation and enrichment. Overall, rapid separation and enrichment techniques are highly desired in complex sample analysis, and electrically-driven force based online approaches offer significant application potential, especially in food, the environment, and medicine.

Separation of anaesthetic ketamine and its derivates in PAMAPTAC coated capillaries with tuneable counter-current electroosmotic flow

Capillary electrophoretic separation of ketamine, norketamine, hydroxynorketamine, and dehydronorketamine was performed in the counter-current regime under the influence of oppositely-directed electroosmotic flow. For this purpose, the fused silica capillaries were covalently coated with the poly(acrylamide-co-3-acrylamidopropyl trimethylammonium chloride) copolymer (PAMAPTAC). The content of the cationic monomer APTAC in the polymerization mixture varied in the range 0–6 mol. % and the generated electroosmotic flow increased continuously in the 0–20 · 10−9 m2V−1s−1 interval. Importantly, it resulted in improved electrophoretic resolution of ketamine/norketamine, which increased from 0.8 for neutral PAM coating (i.e. 0% PAMAPTAC) to 3.0 for 6% PAMAPTAC. The determination of ketamine and its derivates in rat serum was performed in a 4% PAMAPTAC capillary with an inner diameter of 25 μm. The separation was performed in a 500 mM aqueous solution of acetic acid (pH 2.3). The clinical sample was deproteinized by the addition of acetonitrile to the serum and a large volume of the treated sample was injected directly into the capillary. The achieved limit of detection ranged from 2.2 ng/mL for dehydronorketamine to 4.1 ng/mL for hydroxynorketamine; the intra-day repeatability was 1.0–1.5% for the migration time and 2.8–3.3% for the peak area. The developed methodology was employed for time monitoring of ketamines in rat serum after intra venous administration of low doses of anaesthetic at a level of 2 μg per g of body weight.

Non-hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single-stranded DNA.

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label-free, and economical non-hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single-stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single-base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84-mer, in which guanine to adenine single-base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.

Solid phase extraction and capillary electrophoretic separation of racemic catecholamines by using magnetic particles coated with a copolymer prepared from poly(3,4-dihydroxyphenylalanine) and polyethyleneimine.

A method is presented for chiral separation of catecholamines present in bovine and mice blood. It combines magnetic solid phase extraction (MSPE) and chiral capillary electrophoresis (ch-CE). A copolymer consisting of poly(3,4-dihydroxyphenylalanine) and polyethyleneimine was coated onto magnetic particles (MPs) by co-deposition using the CuSO4/H2O2 system as a polymerization initiator. The coated MPs are spherical and the average diameter is about 168 ± 4nm. The thickness of the coating is approximately 19nm. The functional MPs are used as sorbents in MSPE to simultaneously extract the catecholamines epinephrine, norepinephrine and isoprenaline. Under the optimum conditions, the extraction efficiencies for those catecholamines are in the range from 92.3 to 98.3%, with relative standard deviations (RSDs) of <5.3%. The extraction can be performed within 4min. The extracts were then submitted to ch-CE. A method for field-enhanced sample injection (FESI) was used to enhance the detection sensitivities of the enantiomers. The limits of detection for catecholamine enantiomers range from 400 to 600pgmL-1. In comparison with the FESI-ch-CE method, the sensitivity enhancement factors of the MSPE/ch-CE method for catecholamines are about 10-fold. The method was applied to the determination of trace levels of catecholamine enantiomers in (spiked) bovine and mice blood. The recoveries ranged from 88.2 to 93.8%, with RSDs of <5.5%. The whole detection procedure takes less than 30min. Graphical abstract Schematic representation of the separation and detection of catecholamine enantiomers in blood by combination of polyDOPA/PEI-magnetic particles-based solid phase extraction and chiral-capillary electrophoresis.

A Review on Nanotechnology: Analytical Techniques Use and Applications

The combination of nanotechnology with molecular biology, information technology and instrumentation, is opening the door to a new industrial age. The aim of this review article is to summarize the current knowledge of nanotechnology in synthesizing, identifying and characterization of nanomaterials using analytical techniques. Physical and chemical approach synthesis of nanomaterials befalls challenges in the development of analytical techniques used to characterize them. The major techniques include: Transmission Electron Microscopy, Scanning Electron Microscopy, Atomic Force Microscopy, Dynamic Light Scattering, X ray Photoelectron Spectroscopy, X-ray Diffraction, Single Phase Inductively Coupled Plasma Mass Spectroscopy, X ray Fluorescence Spectroscopy, Auger Electron Spectroscopy, X ray Absorption Fine Structure, Capillary Electrophoretic Separations, Magnetic nano particles coupled HPLC and Dynamic light scattering. Nanomaterials have been characterized for the extensive potential applications in optics, electronics, magnetics, and catalysts; chemical sensing, biomedicine, micro reactor, and they have been applied in food, biological, environmental and pharmaceuticals uses. In spite of the extended use of nano particles in diverse consumer products, there is a great concern over the unexpected impact or effects on humans due to exposure.