Capillary Electrophoretic Analysis Research Articles

Fractionation and High Performance Capillary Electrophoretic Analysis of Phospholipids

Phosphatidylcholine of high purity (PC, content 92.80%) was prepared from market soybean power phospholipids (PC content 14.05%) by using solvent extraction and column chromatography. As the main objective, micellar electrokinetic capillary chromatography (MECC) was established for the separation and analysis of phospholipids. MECC conditions such as surfactant and its concentration, pH of running buffer solution, organic modifier and its volume content, concentration of buffer solution, temperature etc were optimized to provide good separation and larger peak area of phospholipids. The optimum MECC conditions were as follows: running buffer system of 35 mmol/L sodium deoxycholate (SDC)-1 mmol/L borax buffer solution/n-propanol (57 : 43, v/v) with pH 8.30, column temperature of 44 degrees C, applied voltage of 25 kV and ultraviolet (UV) detection at 200 nm. Addition of standards was used to identify the components of phospholipids. External standard method was used to determine PC content. As the results shown, five components of phospholipids could be effectively separated under the optimum MECC conditions. Correlation coefficient within 0.1 - 1 g/L PC concentration reached 0.999 0. The average recovery of PC was 98.0%. The intra-day relative standard deviation (RSD) and inter-day RSD of peak area of PC were 1.36% and 3.27%, respectively. The qualitative result of PC obtained by MECC was consistent with that determined by thin layer chromatography and infrared analysis, respectively. So MECC can be used as an effective tool for the separation, analysis and quality control of phospholipids.

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome™). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis–Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K m = 167 ± 23 μM, V max = 3418 ± 234 pmol/min/mg for S-VER, and K m = 168 ± 35 μM, V max = 2502 ± 275 pmol/min/mg for R-VER.

Capillary electrophoretic analysis of the antibiotic vancomycin in innovative microparticles and in commercial formulations

A new fast capillary electrophoretic method has been developed for the analysis of the glycopeptide antibiotic vancomycin in formulations. An electrophoretic run is completed within 3.0 min; fused silica capillaries (100 μm i.d., 8.5 cm effective length and 48.5 cm total length) and a background electrolyte consisting of 12.5 mM, pH 2.5 phosphate buffer are used. The applied voltage is −20.0 kV; samples are injected by pressure (30 mbar × 3 s) at the anodic end of the capillary. The method was successfully applied to innovative controlled release microparticles consisting of a coated albumin core containing vancomycin. A simple procedure has been developed to obtain complete vancomycin extraction from microparticles using a 5% (w/v) sodium dodecyl sulphate aqueous solution. The method has been validated in terms of linearity, precision and accuracy. Good linearity was found in the 0.25–5.00 μg/mL range. Satisfactory precision was obtained, with relative standard deviation values always lower than 3.9%; accuracy was satisfactory, with recovery values between 97.8 and 102.2%. The method is also suitable for vancomycin determination in commercial capsules.

Rapid Determination of Diterpenoids in Andrographis paniculata by Microemulsion Electrokinetic Capillary Chromatography with Short-End Injection

A rapid, simple, and reliable method has been developed for routine capillary electrophoretic analysis of two diterpenoids, andrographolide and dehydroandrographolide, in Andrographis paniculata. For this system, using ethyl acetate oil-based microemulsion electrokinetic chromatography (MEEKC) and the short-end injection technique, analysis was complete in less than 2.5 min. In method validation the relative standard deviations of migration time and peak area of the two constituents were, respectively, 0.54% and 1.70% for andrographolide and 0.45% and 2.11% for dehydroandrographolide. Regression equations revealed linear relationships (correlation coefficients 0.9994 for andrographolide and 0.9993 for dehydroandrographolide) between peak area and concentration. The effects of buffer pH, borate concentration, SDS concentration, co-surfactant type and concentration, injection time, and running potential were systematically investigated. The method can be successfully implemented in routine quality-control testing.

On-column labeling for capillary electrophoretic analysis of individual mitochondria directly sampled from tissue cross sections

This technical note reports on a new procedure to on-column-label organelles sampled from a tissue cross section into a fused silica capillary. These organelles are then analyzed by capillary electrophoresis with postcolumn laser-induced fluorescence detection. In this procedure, the fluorescent label does not come in contact with the tissue, which facilitates visualization of the sampled tissue cross section. In addition, on-column labeling allows for better control of the reaction time and fluorescent label concentrations. As a proof-of-principle, we show results of mitochondria from rat gastrocnemius muscle cross sections that were on-column-labeled with 10-N-nonyl acridine orange (NAO), a mitochondrion-specific probe, and compare them with results for NAO in-tissue labeling of the same tissue. The new organelle labeling procedure reported here may easily be extended to the analysis of individual organelles in other biological samples and may become a valuable tool in studies investigating the role of mitochondria in muscle aging and exercise physiology.

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.

Capillary electrophoretic analysis of fragment length polymorphism in ribosomal markers of Cryptosporidium from humans

Cryptosporidium oocyst DNA samples (n=80) from humans with cryptosporidiosis in Australia and the UK were characterized genetically and categorized by capillary electrophoretic (CE) analysis of part of the small subunit gene (pSSU; approximately 300bp) and second internal transcribed spacer (pITS-2; approximately 230bp) of nuclear ribosomal DNA. The amplicons were heat denatured and subjected to capillary electrophoresis in LPA matrix (Amersham) in a MegaBACEtrade mark 1000 system (Amersham). The chromatograms captured were stored electronically and then analysed using MegaBACEtrade mark Fragment Profiler software. Using reference DNA control samples representing Cryptosporidium hominis and Cryptosporidium parvum, particular peaks in the profiles were defined for their specific identification and differentiation. The two species could be readily differentiated based on their profile in the pSSU, the peak differences being associated with a nucleotide difference of <1.7%. While no variation was detectable in the pSSU profiles within each species, significant intraspecific variability in the positions of peaks in the pITS-2 chromatograms was displayed. For the 80 samples subjected to CE analysis of the pITS-2, four different genetic variants (genotypes) were detected within C. hominis and seven within C. parvum. Based on CE analysis of either pSSU and pITS-2 amplicons, it was readily possible to detect both species in 'mixed samples'. This CE method is time- and cost-effective, and may find applicability as a tool for the high throughput analysis of oocyst DNA samples for epidemiological surveys and for the monitoring of cryptosporidiosis outbreaks.

The potential of inductively coupled plasma‐mass spectrometric detection for capillary electrophoretic analysis of pesticides

AbstractSee original http://dx.doi.org/10.1002/elps.200410098

Evaluation of Sieving Polymers for Fast, Reproducible Electrophoretic Analysis of Short Tandem Repeats (STR) in Capillaries

Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.

Capillary electrophoretic analysis of advanced glycation endproducts formed from the reaction of reducing sugars with the amino group of glucosamine

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.

The potential of inductively coupled plasma‐mass spectrometric detection for capillary electrophoretic analysis of pesticides

In this work, the potential of inductively coupled plasma-mass spectrometry (ICP-MS) coupled to capillary electrophoresis (CE) to determine organophosphorus pesticides (OPPs) is demonstrated. Element specific detection of (31)P with ICP-MS is performed for the detection of OPPs. Three common OPPs, including glyphosate, glufosinate, and aminomethylphosphonic acid (AMPA), were analyzed by CE-ICP-MS to demonstrate its applicability for the analysis of OPPs. The advantages of using ICP-MS with respect to other common detectors, such as flame photometric detection (FPD), for CE analysis of OPPs are shown. Additionally, different CE separation conditions were studied to achieve complete baseline separation of the pesticide compounds in short migration times. Two CE buffer systems were evaluated for the separation of OPPs using ICP-MS detection. A buffer solution containing 40 mmol.L(-1) ammonium acetate at pH 9.0 and an applied voltage of +20 kV were finally selected leading to a separation time of 10.0 min. Both migration time and area relative standard deviations (%RSD) were evaluated and their respective values were in the intervals of 1.1-3.3% and 2.7-5.3%. Detection limits obtained with the CE-ICP-MS system were in the range of 0.11-0.19 mg.L(-1) (as compound) yielding an enhancement of 130- to 230-fold with respect to FPD. The proposed methodology was finally applied for the determination of the OPPs mentioned above in natural river water samples.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

Capillary electrophoretic and mass spectrometric analysis of a polydisperse fluorosurfactant

A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.

Capillary electrophoretic analysis of dimethylsulfoniopropionate in sugarcane and marine algal extracts

A simple and high-resolution analytical method for the determination of dimethylsulfoniopropionate (DMSP) in sugarcane and marine algae is described. Effective extraction of DMSP from plant samples was also investigated using organic solvents, 5% perchloric acid or deionised water. To increase the sensitivity, DMSP in the extracts was first converted to a phenacyl ester, and the reaction mixture was applied directly to capillary electrophoresis without any pretreatment. Water extraction followed by esterification in a pH 4 reaction buffer was found most suitable for the measurement of alkaline-labile DMSP. This method was applied to the determination of DMSP levels in marine algae samples collected from the seashore of Nagasaki, Japan. An increase in DMSP content in Ulva pertusa in the winter period was observed.

Automated analysis of individual particles using a commercial capillary electrophoresis system

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.

General experimental aspects for capillary electrophoretic analysis of gliadins in Triticum species

Capillary electrophoresis (CE) of gliadins from Triticum species in acidic or isoelectric buffers is currently used. The performance of five buffers are compared here. The highest resolving power and the shortest time of analysis resulted associated to the isoelectric buffers. The slight different performance of the iminodiacetic acid (IDA) and aspartic acid (Asp) buffer systems should be taken into consideration before to start CE analyses. IDA buffer should be preferred when screening large germplasm collections or when a detailed characterisation of ω-gliadins is required. Asp buffer proved to be most satisfactory for cultivar discrimination or varietal identification.

Rapid and direct analysis of γ-hydroxybutyric acid in urine by capillary electrophoresis–electrospray ionization ion-trap mass spectrometry

The present work was aimed at the development of a capillary electrophoretic analysis of γ-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silica capillary: 100 cm × 50 μm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 °C, nebulizer pressure: 10 psi; sheath liquid solution: methanol–water (90:10) containing 0.1% ammonia delivered at 3 μl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/ z for GHB and 115 m/ z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 μg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 μg/ml in water and 20 μg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.

Determination of bromide and potassium in saline groundwaters by capillary electrophoresis without prior dilution

Two capillary electrophoretic analysis methods are presented; one optimized for the analysis of Br− and the other for determination of K+ in groundwaters of high conductivity. The water sampling was performed from the planned final disposal facility area for spent nuclear fuel at Olkiluoto in the municipality of Eurajoki, Finland. Bromide was analysed in an acidic electrolyte solution containing 5 mM formic acid and 42 mM NaCl (pH 3.5) and using direct UV detection (200 nm). Sample stacking was needed for the preconcentration. Potassium was analysed at pH 4.5 using imidazole-18-crown-6 ether solution. The accuracies of the Br− and K+ methods were tested using laboratory-made reference sample mixtures with high salt concentration. In the Br− analyses, the limits of detection and determination were 0.1 and 1 mg L−1, respectively. The developed CE analysis for K+ in saline water was repeatable (RSD% 14.5–18.0) and the detection and the determination limits were 0.5 and 2.0 mg L−1, respectively. The interlaboratory results showed that CE measurements of Br− and K+ were compatible with those made with traditional solvent chemistry techniques.

In-capillary derivatization approach applied to the analysis of insulin by capillary electrophoresis with laser-induced fluorescence detection

An in-capillary derivatization procedure of insulin for its subsequent capillary electrophoretic analysis (with laser-induced fluorescence detection) was developed. The in-capillary derivatization performed using the 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) in a borate buffer pH 8.9, was achieved by successive introduction of plugs of sample and AQC reagent followed by application of a voltage (30 kV). Derivatization reaction results from the differential transport velocities that permit the distinct zones to penetrate each other under the applied field. Reagent/sample molar ratio ( R m) and plug lengths ratio were shown to have an influence on the efficiency of the derivatization reaction. A single peak could be obtained with a high reagent/sample molar ratio ( R m ≥ 68). The tagged derivative peak intensity and efficiency were improved when reagent solution time injection was at least twice higher than that of insulin sample. The validation of the method showed a good linearity between the corrected area of the derivative peak and insulin concentrations. The relative standard deviations of the migration times and the corrected areas obtained for the tagged derivative were 2.3 and 4.6%, respectively. An efficient derivatization and separation of a mixture of insulin and two glycated forms of insulin was obtained using the technique.

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis.

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.