Simultaneous determination of inorganic anions and cations in waters by capillary electrophoresis

A simple and rapid assay method for three stimulant drugs (amphetamine, methamphetamine, and dimethamphetamine) in human urine using solid-phase microextraction was developed. In solid-phase microextraction, the drugs were equilibrated between the adsorbent coated-fiber and aqueous sample matrix. After adsorption of the analytes, the fiber was directly transferred to the injector of a gas chromatograph, where the analytes were thermally desorbed and subsequently separated by the gas chromatograph and detected by mass spectrometer. The solid-phase microextraction method, which did not require solvents, was found to be a fast and simple analytical method. We optimized the solid-phase microextraction technique, for factors such as the NaCl salt effect (30%), pH effect (pH=12.4), equilibration time (30 min), desorption time (1 min) and coated-fiber type (100 microm poly(dimethylsiloxane)) and detected the stimulants in human urine, obtained from human subjects. The detection limits of each drug were below 1-10 ng/ml. The developed method can be applied to the abused drug test.     

Genetic typing by capillary electrophoresis with the allelic ladder as an absolute standard

We demonstrate a genetic typing method based on capillary electrophoresis/laser-induced fluorescence (CE-LIF). VNTR polymorphism in the human D1S80 locus was studied. A pooled allelic ladder, which contains the 27 most common human alleles, was used as the absolute standard. Extracted genomic DNA from an individual was amplified by polymerase chain reaction (PCR). Typing can be accomplished by co-injection of the PCR product and the D1S80 ladder and then running CE. Separation by a polymer solution of poly(ethylene oxide) in uncoated fused-silica capillaries allows high-resolution, repeated runs in the same capillary. Sensitive detection with minimal sample preparation is possible by using ethidium bromide as the intercalating dye. Statistical analysis of the data indicates a high level of confidence in matching the bands despite variations in the injection process or in the CE system. Future adaptation to a multiple-capillary array system should allow high-speed, high-throughput operation.     

Stereoselective determination of S-naproxen in tablets by capillary electrophoresis

A capillary electrophoresis (CE) method was developed for the stereoselective determination of the non-steroidal anti-inflammatory drug (NSAID), S-naproxen, in tablets. Several beta-cyclodextrin derivatives (CDs) were tested as chiral selectors, including sulfobutyl-beta-CD (SBCD), carboxymethyl-beta-CD (CMCD), dimethyl-beta-CD (DMCD) and trimethyl-beta-CD (TMCD), in a phosphoric acid/triethanolamine pH 3 buffer. Under these conditions, the analyte was mainly present in an uncharged form and therefore, the use a neutral CD (DMCD or TMCD) alone could not lead to enantiomeric separation. On the contrary, by addition of a charged CD (SBCD or CMCD) to the running buffer, giving the analyte enantiomers an adequate mobility, chiral resolution could be achieved, although the resolution values obtained in this case were not quite satisfactory (Rs < 1.5). Dual systems, based on the use of mixtures of charged and neutral CDs, were then investigated. The SBCD/TMCD system was found to be particularly well suited to the enantioseparation of naproxen and after optimisation of the concentrations of both CDs, a resolution value of 5.4 could be obtained. The method was validated for the determination of R-naproxen (enantiomeric impurity) in the 0.1-2% range, using the racemic mixture of the analyte. A second validation was performed in the 50-150% range for the quantitation of S-naproxen. In both cases, good results with respect to linearity, precision and accuracy were obtained.     

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin-piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated.     

Aptamers as ligands in affinity probe capillary electrophoresis

A DNA aptamer against IgE was labeled with fluorophore and used as a selective fluorescent tag for determining IgE by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). CE-LIF separations of samples containing fluorescently labeled aptamer and IgE were complete in less than 60 s and revealed two zones, one corresponding to free aptamer and the other to aptamer bound to IgE. The free aptamer peak decreased and bound aptamer peak increased in proportion to the amount of IgE in the sample so that IgE could be detected with a linear dynamic range of 10(5) and a detection limit of 46 pM. The assay was highly selective as aptamer was unaffected by the presence of IgG and IgE did not bind other DNA sequences. IgE was determined in serum samples with similar analytical figures of merit. Similar conditions using a thrombin aptamer allowed detection of thrombin.     

Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.     

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.     

The development of a data system for a combination of liquid chromatography or capillary electrophoresis with an ion trap storage/reflectron time-of-flight mass detector

A data system based upon a 200 MHz transient recorder interface card in a Pentium PC computer is demonstrated for on-line analysis of microbore high-performance liquid chromatography (HPLC), capillary HPLC and capillary electrophoresis (CE) separations using a fast and sensitive ion-trap storage/reflectron time-of-flight mass spectrometric detector (IT-reTOFMS). Under the control of a user-written program, the system is capable of conducting the data acquisition and storage for a minimum of 30 min, at rates exceeding 10 Hz, of individual mass spectra containing 16,000 data points having 10 nsec resolution. The capability is mainly attributed to the use of a data reduction scheme in which only mass intensities higher than a preset threshold are saved as indexed flight-time/intensity pairs. This produces a typical reduction ratio of 30:1 in data set size, yielding faster storage with smaller file size, and permits the complete set of mass spectra to be held in the computer's memory. In addition, the data system is capable of displaying, for real-time evaluation of the analysis, each individual mass spectrum and the total-ion chromatogram. Further, the selected-ion chromatograms of given masses and a 3-dimensional topographic map describing a separation process can be rapidly generated from the collected data for the unambiguous and high fidelity identification of target analytes in a complex mixture.     

Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations

The properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3-5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 10(2) L/mol, as that found for beta-cyclodextrin (beta-CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 x 10(2) L/mol, is one order of magnitude higher than that of enantiomers separated with beta-CD. Analytically convenient mobility differences up to 1-2 x 10(9) m2V-1s-1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250,000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin-analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.     

A computer-controlled variable light attenuator for protection and autoranging of a laser-induced fluorescence detector for capillary zone electrophoresis

Photodetectors have a limited range over which they can measure light intensities for any particular setting. The intensity of light reaching the detector can be kept within this range by using a liquid crystal variable light attenuator controlled by a computer that continuously checks the amount of light reaching the detector and adjusts the attenuation to an appropriate level. Using such a system we have constructed an intensified charge-coupled device (ICCD) camera-based detector with a dynamic range of over six orders of magnitude which is never exposed to damaging or saturating light levels.     

Optimized determination of carbohydrate-deficient transferrin isoforms in serum by capillary zone electrophoresis

Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI > 5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 microL) and 500 mM NaHCO3 (12 microL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (20 microm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were separated in human serum. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-transferrin, and 0.4% of trisialo-transferrin, expressed as percentages of the terasialo-transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo-transferrin) were < 0.1%. Intra-day and day-to-day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo- and trisialo-transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo- and trisialo-transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).     

Validated capillary electrophoresis method for the determination of atropine and scopolamine derivatives in pharmaceutical formulations

The simultaneous determination of atropine and scopolamine derivatives, which have similar structures, was investigated by using capillary zone electrophoresis. The effects of buffer pH, buffer concentration and hydroxypropyl-beta-cyclodextrin concentration on migration time and resolution of the investigated compounds were systematically studied. The selected electrophoretic buffer consisted of a 80 mM sodium citrate pH 2.5, containing 2.5 mM hydroxypropyl-beta-cyclodextrin as the complexing agent. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 1 and 2% for the intermediate precision of migration times and peak area ratios, respectively. The linearity of the method was assessed between 50 and 150% of the theoretical content (coefficient of correlation greater than 0.99). The proposed method was found to be suitable and accurate for the determination of these basic drugs in pharmaceutical preparations.     

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.     

Stable cationic capillary coating with successive multiple ionic polymer layers for capillary electrophoresis

A coated capillary modified with a cationic polymer was developed by using a novel coating procedure, successive multiple ionic-polymer (SMIL) coating. The SMIL coating was achieved by first attaching the cationic polymer to the capillary inner wall, and then the anionic polymer to the cationic polymer layer, and finally the cationic polymer to the anionic polymer layer. The stability of Polybrene (PB)-modified capillary made by SMIL coating was remarkably improved in comparison with a conventional PB-modified capillary. It endured during 600 replicate analyses and also showed strong stability against 1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating was all below 1%, and good reproducibilities were obtained. The PB-modified capillary made by SMIL coating was applied to the basic protein analyses. It gave good performances for the protein analyses even when the pH of the electrolyte was near the isoelectric point (pI) of the protein. In addition, 0.1 M NaOH rinse prior to the sample injection allowed the reproducible analysis of a highly adsorptive sample such as plasma because the adsorbed sample could be flushed out of the capillary. Besides protein analyses, an efficient analysis of the cationic drugs by capillary electrophoresis/mass spectrometry (CE/MS) was also possible.     

Behavior of peptides in capillary electrophoresis: effect of peptide charge, mass and structure

In the last decade the large potential of capillary electrophoresis as a technique for separation and characterization of peptides has been demonstrated extensively. In this field, a large number of chemical structures has to be taken into consideration, for which very often no data or even standards are available. As a result, there has been a strong desire to relate electrophoretic behavior to molecular properties and structure of the compounds. The activities in that direction, in the area of capillary zone electrophoresis, are critically reviewed. Special attention is paid to peptide charge, mass, hydrophobicity and structure, and their influence on the selectivity of the separation. Also, some complexation phenomena are discussed.     

Evaluation of capillary zone electrophoresis and micellar electrokinetic capillary chromatography with direct injection of plasma for the determination of cefotaxime and its metabolite

Quantitative aspects of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were investigated for the determination of cefotaxime (C) and its deacetyl metabolite (DA) in human plasma in a concentration range of therapeutic interest. For CZE, plasma samples spiked with C and DA were injected after deproteinization with acetonitrile, and analytes were separated in a fused silica capillary using a borate buffer at pH 9.2 as electrolyte; no suitable internal standard was found. For MECC, plasma samples spiked with C, DA, and theobromine as internal standard were directly injected after dilution with water and analyzed using a phosphate buffer, pH 8.00, containing 165 mM SDS as separation electrolyte and a fused silica capillary. Both methods gave satisfactory interday precision with respect to migration times (RSD < 1%) and gave linear responses over the concentration ranges investigated (5-100 mg L-1 C and 5-20 mg L-1 DA). For CZE, intraday RSD (n = 4 graphs) between the slopes of the calibration graphs was acceptable (5.7%) for C. The corresponding figures for interday precision (n = 4 days) were fair (16.1%) in comparison to those obtained with MECC, for which the RSD was 1.49% when theobromine was used as internal standard. A satisfactory interday precision between slopes was also obtained with MECC even without the use of an internal standard (RSD = 4.38%), which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 2 mg L-1 (CZE) and 1 mg L-1 in plasma (MECC) for C and DA. MECC was shown to be superior with regard to simplicity, rapidity, precision, and sensitivity.     

Direct determination of ephedrine and norephedrine in human urine by capillary zone electrophoresis

This paper describes a simple and fast method for the simultaneous determination of ephedrine and norephedrine in urine by capillary zone electrophoresis. Separation and determination of these stimulants in human urine was performed in 50 mM phosphate buffer at pH 9.5, modifying the electroosmotic flow with acetonitrile. The method allows direct determination of the stimulants in urine in concentrations lower than 20.0 micrograms/ml, and has a limit of determination of 2.6 +/- 0.2 micrograms/ml for ephedrine and 2.3 +/- 0.2 micrograms/ml for norephedrine in urine and may be applied for doping control.     

Simultaneous determination of dextromethorphan and its metabolites in human plasma by capillary electrophoresis

A sensitive capillary electrophoretic method was developed and validated for the simultaneous determination of dextromethorphan and its metabolites, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan, in human plasma. After cleavage of conjugates by enzymatic hydrolysis with beta-glucuronidase, dextromethorphan and its metabolites were extracted from 1.5 ml of plasma by a liquid liquid extraction procedure using heptane-ethylacetate (50:50, v/v) and re-extracted to aqueous phase. The compounds were separated within 8 min on a fused silica capillary, 75 microm internal diameter using sodium borate (pH 9.4; 50 mM) as running buffer, and measured by UV-detection at 195 nm using a detection cell with a path length of 1.2 mm. The method was accurate and precise. Linear relationships were observed between the peak response and the concentration in the range of 1-400 ng ml(-1) plasma with correlation coefficients above 0.998. The limit of detection was 0.5-1 ng ml(-1) plasma for all compounds. The method was used for determination of plasma levels of dextromethorphan and its metabolites after transdermal and oral administration of dextromethorphan.     

Rapid determination of volatile bases in fish by using an ammonia ion-selective electrode

A simple and rapid method using an ammonia ion-selective electrode (ISE) to measure volatile bases in fish is proposed. Accuracy and precision were determined with 5, 10, 20, and 30 ppm NH3 standard solutions. Ammonia values obtained with the method correlate strongly with total volatile basic nitrogen (r2 = 0.88). Recoveries of added ammonia to homogenized fish samples ranged from 83.7 to 96.0%. Responses of the probe to trimethylamine (TMA), calculated as NH3 (mg/100 mL), ranged from 74.9 to 91.7%. These findings indicate that the probe measured TMA as well as ammonia. Storage trials on 8 fish species illustrated that the results obtained with the ISE method reflected nitrogen concentrations based on total volatile base (TVB) analysis. This procedure may be used in lieu of the traditional TVB method for on-site rapid screening of fish.