Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.     

Quantitative analysis of non-steroidal anti-inflammatory drugs by capillary zone electrophoresis

The potential utility of capillary zone electrophoresis (CZE) for the separation and quantitative determination of some non-steroidal anti-inflammatory drugs (NSAIDs) was investigated. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the nature and concentration of the anionic and cationic components of the separation buffer. A buffer consisting of 75 mM glycine adjusted to pH 9.1 with triethanolamine was found to provide a very efficient and stable electrophoretic system for the CZE analysis of NSAIDs, giving RSD values of about 0.1 and 0.5% for the within-day reproducibility of migration times and peak areas, respectively at a concentration of 25 micrograms ml-1 (n = 5). Response was linear from 2-100 micrograms ml-1 for both sulindac and tiaprofenic acid, for which the LOQ values were 2.8 and 1.9 micrograms ml-1, respectively, using UV detection at 280 nm. Accuracy for each drug was 102-103%.     

Principles and practice of peptide analysis with capillary zone electrophoresis

Capillary electrophoresis provides a new analytical tool that complements reversed-phase high-performance liquid chromatography separations of peptides. The principles of this technique and their application to developing peptide analyses are described. Detailed recipes for useful buffers and for preparation of the instrument are provided. A sequence of generally useful experiments is suggested. Basic troubleshooting guidelines are provided.     

Application of capillary zone electrophoresis in cephalosporin analysis

Cephalosporins have structures and antibiotic activity similar to those of penicillins which represent a class of compounds with closely related structures. Most of the cephalosporins contain aromatic groups and show distinctive UV spectra. Separating the different types of cephalosporins is a challenging task for HPLC. but the resolving power of capillary zone electrophoresis (CZE) makes this separation fast and simple. The present study reports the application of CZE for cephalosporin analysis and the separation of cephalosporins from plasma. Both field strength and temperature were shown to influence the plate number. The influence of injection time on the peak height was studied. Furthermore, the influence of pH value on the separation of cephalosporins by CZE was investigated. The low sample amount required and the relatively short analysis time are the main advantages of this method.     

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.     

Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking

The present paper describes the methodological optimisation and validation of a capillary zone electrophoresis method for the determination of morphine, cocaine and 3,4-methylenedioxymethamphetamine (MDMA) in hair, with injection based on field-amplified sample stacking. Diode array UV absorption detection was used to improve analytical selectivity and identification power. Analytical conditions: running buffer 100 mM potassium phosphate adjusted to pH 2.5 with phosphoric acid, applied potential 10 kV, temperature 20 degrees C, injection by electromigration at 10 kV for 10 s, detection by UV absorption at the fixed wavelength of 200 nm or by recording the full spectrum between 190 and 400 nm. Injection conditions: the dried hair extracts were reconstituted with a low-conductivity solvent (0.1 mM formic acid), the injection end of the capillary was dipped in water for 5 s without applying pressure (external rinse step), then a plug of 0.1 mM phosphoric acid was loaded by applying 0.5 psi for 10 s and, finally, the sample was injected electrokinetically at 10 kV for 10 s. Under the described conditions, the limit of detection was 2 ng/ml for MDMA, 8 ng/ml for cocaine and 6 ng/ml for morphine (with a signal-to-noise ratio of 5). The lowest concentration suitable for recording interpretable spectra was about 10-20-times the limit of detection of each analyte. The intraday and day-to-day reproducibility of migration times (n = 6), with internal standardisation, was characterised by R.S.D. values < or = 0.6%; peak area R.S.D.s were better than 10% in intraday and than 15% in day-to-day experiments. Analytical linearity was good with R2 better than 0.9990 for all the analytes.     

Application of capillary zone electrophoresis for analysis of thyreostatics

Capillary zone electrophoresis was optimized for the separation of thiouracil, methylthiouracil and propylthiouracil. Methylthiouracil could be determined in various types of urine (human, bovine, horse), either without any pretreatment or in ethyl acetate extracts, within 15 min. For identification, the simultaneous detection at three UV wavelengths (216, 245 and 278 nm) was advantageously used while for quantification the wavelength of the absorbance maximum at 278 nm was preferred. Under optimized conditions a linear response of the detector in the concentration range 0.1-100 ppm was obtained. On analysis of untreated urine, a detection limit of 0.5 ppm was found; for urine extracts the detection limit was 0.1 ppm. Univocal peak identification, based on absorption at three wavelength, was only possible above 2 ppm. Relative standard deviation for standard solutions of methylthiouracil, diluted in the background electrolyte, was 1%; for methylthiouracil in extracts dissolved in the background electrolyte it was 4.5%, and for methylthiouracil in untreated urine, 12.7%.     

Stability of metallothionein isoforms by capillary zone electrophoresis

Advanced glyco-oxidation end products (AGEs) generate oxygen free radicals that potentiate the development of atherosclerosis. Thus, AGEs may potentiate the aggregation of human platelets through oxidative stress. AGE-bovine serum albumin (BSA) and AGE-poly-L-lysine were evaluated for aggregation of human platelets. Superoxide in platelet-rich plasma (PRP) was measured using lucigenin-derived chemiluminescence. The platelet aggregation induced by ADP or U46619 was potentiated by preincubation with AGE-BSA, by 40% and by 59%, P < .05, respectively, vs BSA. Aggregation was increased by AGEs in a dose-dependent manner. The production of superoxide was significantly greater in PRP incubated with AGE-BSA vs BSA. The other Maillard reaction products, such as Amadori-, pentosidine-, and carboxymethyl lysine (CML)-BSA had no effect. Superoxide dismutase or indomethacin abolished the enhancing effect of AGEs on the platelet aggregation. AGEs potentiate platelet aggregation possibly with superoxide anions and prostanoids. AGE-induced potentiation of platelet aggregation may be involved in the development of atherosclerosis.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

Commercially pure titanium (CPT) substrate was subjected to porcelain firing and bond strengths under three-point bending mode (span length: 15 mm; crosshead speed: 0.5 mm/min) were evaluated. Experimental variables included surface treatments of CPT and porcelain firing schedules. Variables for the surface treatments were (1) sandblasting, (2) mono- and triple-layered nitridation, and (3) mono-layered chrome-doped nitridation. Variables for the porcelain firing schedule included (4) bonding agent application, (5) bonding agent plus gold bonding agent application, and (6) Procera porcelain application. All together eleven sample groups were prepared with different combination of aforementioned experimental variables. Statistically all of them exhibited no significant differences. Hence, we employed two further criteria; (I) the minimum bond strength should exceed the maximum porcelain strength per se, and (II) the CPT substrate should not be heated close to the beta-transus temperature. After applying these criteria, it was concluded that mono-layered nitridation and mono-layered application of chrome-doped nitridation on both (with and without) sandblasted and non-sandblasted surfaces were the most promising conditions for a successful Titanium-Porcelain System.     

Analysis of carbohydrate deficient transferrin by capillary zone electrophoresis

We report a capillary zone electrophoresis method to separate the various sialylated isoforms of transferrin. The separation is carried out under nondenaturing conditions and at basic pH. Under these conditions, transferrin exhibits two major and three minor peaks. Plasma samples from a population consuming varying amounts of alcohol at different intervals were studied. A cut-off value of 3% carbohydrate deficient transferrin (CDT: disialo, monosialo, and asialo transferrin), results in a clinical sensitivity of 88% in a population consuming at least 70 g/day alcohol for a minimum of two weeks. The sensitivity dropped significantly in a population consuming less than 70 g/day. This confirms previous reports of CDT as a specific marker for significant and chronic use of alcohol. Capillary electrophoresis offers an alternative method with respect to analysis time and throughput in the clinical laboratory.     

Peak broadening in capillary zone electrophoresis

A review on peak broadening in capillary zone electrophoresis in free solutions is given which covers a selection of the literature published on this topic over the period mainly between 1992 and the beginning of 1997 (consisting of 71 publications). The contributions to peak dispersion from extracolumn effects (e.g. due to the finite length of the injection zone, or the aperture of the detector), from longitudinal diffusion, Joule heating, electromigration dispersion (concentration overload), a different path length of the solute ions, wall adsorption, laminar flow and the (longitudinally) homogeneous or nonhomogeneous electroosmotic flow are described. The latter may also occur when a longitudinally nonhomogeneous radial electric field is applied. Peak dispersion is depicted either by the plate-height model, or the concentration of the solute as a function of space and time is calculated either analytically or numerically by solving the equation of continuity with appropriate initial and boundary conditions and possibly completed by equations governing further quantities.     

Evaluation of adsorption preconcentration/capillary zone electrophoresis/nanoelectrospray mass spectrometry for peptide and glycoprotein analyses

The use of an on-line adsorption preconcentrator coupled with capillary zone electrophoresis/nanoelectrospray mass spectrometry (PC/CZE/nESMS) is described for the analysis of peptides and protein digests. The investigation was focused on the production of disposable preconcentrators made of large particle size (40 microns irregular packing), thereby eliminating the use of a retaining frit without loss of performance. These preconcentration devices were made of commercially available components which can be easily interfaced to current CZE/nESMS systems. Practical issues such as the composition of the stationary phase, the elution volume and sample breakthrough and carry-over were evaluated in order to optimize the analytical performance of this technique. Under optimized elution conditions, the PC/CZE/nESMS technique provided separation efficiencies in excess of 100,000 theoretical plates for a sample loading of 8 microliters. Sample carry-over was minimized by proper reconditioning of the preconcentrator prior to the CZE separation. Alternatively, the sample carry-over resulting from small elution volumes could be used advantageously to provide multiple analyses from a single injection of sample. The application of this technique is demonstrated for the analysis of proteolytic peptides from a Bauhinia purpurea lectin at a concentration level of 30 nM. Further structural information was obtained using on-line tandem mass spectrometry to elucidate the structure of N-linked glycans and the amino acid sequences of the glycopeptides.     

Determination of denatured serum proteins in the casein fraction of heat-treated milk by capillary zone electrophoresis

The amount of heat-denatured serum proteins in heat-treated milk could be estimated by analyzing the casein fraction, obtained by isoelectric precipitation at pH 4.6, by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at pH.3. Optimization of the sample and running buffer minimized adsorption of serum proteins, especially that of bovine serum albumin (BSA). This afforded a detection limit down to ca. 5-65 micrograms/mL of the three main serum proteins in milk. The detector response (UV at 214 nm) was linear in the range of 0.05-0.35 and 0.05-0.85 mg/mL for alpha-lactalbumin and beta-lactoglobulin, respectively. BSA showed a slightly less linear behavior, due to residual adsorption to the capillary wall. The recovery of serum proteins was in the range of 89-107%. The method was evaluated by analyzing Dutch commercial milks and cheese milk, which had received increasing heat loads. The addition of milk powder to pasteurized milk could be detected by this method as well as the serum protein to casein ratio in various products.     

Purity testing of recombinant proteins by capillary electrophoresis

Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties. The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E. coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells. The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies. MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins.     

Simultaneous determination of S-adenosylmethionine and S-adenosylhomocysteine by capillary zone electrophoresis

A reproducible, rapid procedure for the simultaneous quantitative separation of S-adenosylmethionine and S-adenosylhomocysteine by capillary zone electrophoresis has been developed. Separations were performed by using an uncoated capillary of 60 cm effective length and 50 microm ID, 40 mM sodium phosphate buffer, pH 2.50, as background electrolyte solution, and 30 kV. On-line detection was carried out at 254 nm. Under the conditions selected we resolved a standard solution containing S-adenosylmethionine and S-adenosylhomocysteine in a run time shorter than 8 min. A mass detection limit in the range of 10 fmol was achieved. Adenosyl-L-methionine, S[methyl-3H] has also been assayed under the same experimental conditions. Other related compounds did not show interference, including those derived from the hydrolysis of S-adenosylmethionine. The present method allows simultaneous determination of these compounds, which play an important role in many microbiological and enzymatic research studies.     

Urine protein analysis by capillary electrophoresis

Increased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost-effective than agarose electrophoresis.     

Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy

A previous study showed a portion of HIV-1 plasma virus was lysed by the addition of exogenous human AB+ seronegative complement. The current study was performed to determine whether infectious plasma virus was inactivated by complement. Incubation of plasma virus with AB+-seronegative serum resulted in substantial decreases in infectious titers, demonstrating that infectious plasma virus is susceptible to complement-mediated inactivation. Although complement also induced some lysis of plasma virus samples, virus was neutralized to a significantly higher degree, suggesting neutralization did not occur solely by lysis. Additionally, C5-deficient complement substantially neutralized virus, indicating coating of virus by early complement components was an important mechanism of neutralization. A portion of some freshly isolated plasma virus samples bound to complement receptor 2 in the absence of exogenous complement, indicating that early complement components bound virus in vivo. Furthermore, plasma virus samples that had less C3 deposited on their surface in vivo had higher infectious titers than samples with a larger fraction with surface C3. These findings suggest that complement can neutralize HIV-1 plasma virus in vivo by coating with complement proteins. This is the first study to provide evidence that coating by complement leads to functional inactivation of a virus in vivo.     

High-performance capillary electrophoresis of cereal proteins

Cereal grains are widely used of human foods and animal feed throughout the world. Cereals provide dietary protein, which also often has a functional role, as wheat gluten does in bread. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. This review describes the development of charge- and size-based HPCE methods for analysis of cereal grain proteins, and the use of these methods for cultivar identification, classification, and prediction of quality. HPCE is versatile, rapid, easily automated, readily quantified, and provides high-resolution separations. Clearly, HPCE is a valuable addition to other methods of cereal protein analysis and should, in time, be applicable to all protein classes from all cereals.