Simple Capillary Electrophoresis Method Research Articles

Front Cover: Capillary electrophoresis method for simultaneous analysis of caffeine, vanillin and ethyl vanillin in beverages

This cover picture describes a fast and simple capillary electrophoresis method for the simultaneous analysis of caffeine, vanillin, and ethyl vanillin in beverages. The optimal separation conditions were 25 mM phosphate buffer at pH 8.5, containing 10% acetonitrile and detected at 200 nm. Employing the optimum conditions, the three compounds were separated in less than 3 min. The procedure was validated with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), (RSD%) accuracy (recovery) and precision (repeatability). Calibration curves with (r2 > 0.9986) in the concentration range of 5-80 μg/mL were obtained for the three analytes. The LODs and LOQs in the range of 2.9-5.6 and 5.7-7.8 μg/mL were obtained, respectively. Recoveries of analytes in range from 85 to 108%, (n = 3) were attained. The validated method was successfully utilized for the analysis of the analytes in various beverage samples.

Capillary electrophoresis method for simultaneous analysis of caffeine, vanillin and ethyl vanillin in beverages

AbstractFast and simple capillary electrophoresis method was proposed for the simultaneous determination of caffeine, vanillin, and ethyl vanillin in beverages. The optimal separation conditions were 25 mM phosphate buffer at pH 8.5, containing 10% (v/v) acetonitrile and UV‐absorption detection at the wavelength of 200 nm. Employing the optimum conditions, the three compounds were separated in less than 3 min. The procedure was validated with respect to linearity, limits of detection, limits of quantitation, (RSD%) accuracy (recovery), and precision (repeatability). Calibration curves with (r2 > 0.9986) in the concentration range of 5–80 μg/mL were obtained for the three analytes. The limits of detection and quantitation in the range of 2.6‐4.3 and 4.1‐5.2 μg/mL were obtained, respectively. Recoveries of analytes in range from 92.1 to 104.4%, (n = 3) were attained. The validated method was successfully utilized for the analysis of the analytes in beverage samples.

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein.

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

A rapid and simple capillary electrophoresis method for indirect determination of the biocide 2,2-dibromo-3-nitrilo-propionamide (DBNPA) in cooling waters.

A capillary zone electrophoresis (CE) method was developed for the determination of the biocide 2,2-dibromo-3-nitrilo-propionamide (DBNPA) in water used in cooling systems. The biocide is indirectly determined by CE measurement of the concentration of bromide ions produced by the reaction between the DBNPA and bisulfite. The relationship between the bromide peak areas and the DBNPA concentrations showed a good linearity and a coefficient of determination (R²) of 0.9997 in the evaluated concentration range of 0-75 μmol L⁻¹. The detection and quantification limits for DBNPA were 0.23 and 0.75 μmol L⁻¹, respectively. The proposed CE method was successfully applied for the analysis of samples of tap water and cooling water spiked with DBNPA. The intra-day and inter-day (intermediary) precisions were lower than 2.8 and 6.2%, respectively. The DBNPA concentrations measured by the CE method were compared to the values obtained by a spectrophotometric method and were found to agree well.

Development of a capillary electrophoresis method for the simultaneous determination of cephalosporins

A rapid and simple capillary electrophoresis method has been developed for the simultaneous determination of six extensively used cephalosporin antibiotics (cefaclor, cefadroxil, cefalexin, cefuroxim, ceftazidim, ceftriaxon). The determination of cephalosporins was performed at a pH 6.8, using a 25 mM phospate - 25 mM borate mixed buffer, + 25 kV voltage at a temperature of 25 ?C. We achieved a baseline separation in approximately 10 minutes. The separation resolution was increased by addition of an anionic surfactant, 50 mM sodium dodecyl sulfate, to the buffer solution. The proposed separation was evaluated on the basis of detection and quantification limits, effective electrophoretic mobility and relative standard deviation for migration times and peak areas.

Capillary electrophoresis analysis of isoniazid using luminol‐periodate potassium chemiluminescence system

A rapid and simple capillary electrophoresis method coupled with chemiluminescent (CL) detection was proposed for analysis of isoniazid (ISO) based on the enhancement effect of ISO to CL emission of luminol-periodate potassium reaction. Under the optimal conditions, ISO can be assayed in the range of 7.0 × 10(-7) to 3.0 × 10(-5) g mL(-1) (R(2) = 0.9990) with a limit of detection of 3.0 × 10(-7) g mL(-1) (signal-to-noise ratio of 3). The whole analysis process can be completed within 2.5 min with a theoretical plate number of 6258. The relative standard deviations of the signal intensity and the migration time were 3.1 and 1.4% for a standard sample at 1.0 × 10(-5) g mL(-1) (n = 5), respectively. The presented novel strategy was successfully applied to the determination of ISO in commercial pharmaceutical preparations and spiked human serum samples. Copyright © 2010 John Wiley & Sons, Ltd.

Characterization of molecularly imprinted and nonimprinted polymer submicron particles specifically tailored for removal of trace 17β‐estradiol in water treatment

AbstractThis study investigated the potential use of molecularly imprinted polymer (MIP) submicron particles for the selective removal of trace 17β‐estradiol (E2) in water treatment. Methacrylate‐based MIP submicron particles were synthesized, in a one‐step suspension polymerization procedure, using ethylene glycol dimethacrylate (EGDMA) as the cross‐linker. After template removal, the particles could be used as a smart material for specific binding of E2. The submicron size of MIP particles facilitated uniform dispersion in water for up to 17 days. These particles were meritorious in mass transfer behavior, allowing phase partitioning of E2 molecules in water during a short treatment time. After 1‐mL water samples of different E2 concentrations were treated with 20 mg of MIP particles for 2 min, recovery percentages as high as 97% ± 3% were achieved. The specific binding capacity of these MIP particles was determined to be 15 mg E2/g. Nonimprinted polymer nanoparticles were also evaluated for nonspecific binding of E2, using 0.5 mg in 1 mL of water, to attain 64% ± 3% efficiency in 3 min towards general water treatment. A simple capillary electrophoresis method was successfully developed for the characterization of MIP and NIP particles. Apparently the less negative the electrophoretic mobility, the higher binding efficiency and faster binding kinetics the particles would exhibit with E2 due to less hindered Brownian diffusion. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Determination of gentisin, isogentisin, and amarogentin in Gentiana lutea L. by capillary electrophoresis

A novel, fast, and simple capillary electrophoresis method has been developed for the analysis of gentisin, isogentisin, and amarogentin in roots of Gentiana lutea (yellow gentian), an herb traditionally used as gastric stimulant. Gentisin and isogentisin are xanthones showing potent inhibition of monoamine oxidase type A and B, amarogentin represents one of the bitter principles of Gentiana, responsible for its gastric-roborant effects. Optimal CE-separation conditions comprise a 100 mM sodium tetraborate buffer of pH 9.3, containing 10 mM beta-cyclodextrin as additive; optimum temperature and applied voltage were found to be 30 degrees C and 25 kV, respectively. Direct diode array detection at 260 nm (gentisin, isogentisin) and 242 nm (amarogentin) was performed, and the required analysis time was only 11 min. The developed method was validated for linearity, sensitivity, precision, and accuracy, and utilized to assay several commercially available G. lutea samples. Quantitative data obtained with the developed CE method are compared with HPLC results, and the advantages of each approach are discussed.

Separation of the positional isomers of phthalic acids in hydroorganic solvents using capillary electrophoresis

The positional isomers of phthalic acids (ortho-, meta-, and para-) and benzoic acid could be completely separated by capillary electrophoresis (CE). A simple CE method employing direct detection in mixed methanol/water buffers is presented. The effect of the electrolyte buffer system, including pH, buffer concentration, and organic solvent on the electrophoretic mobility of the analytes, is investigated. The electroosmotic flow is reversed using cationic surfactant and cetyltrimethylammonium bromide so that anions are separated under the co-EOF mode. The resolution of the analytes and selectivity could be improved by the adjustment of the methanol content. Ion association with the surfactant in methanol/water buffer is discussed. The validity of the method in terms of sensitivity, reproducibility, and linearity is also reported.

A simple capillary electrophoresis method for the rapid separation and determination of intact low molecular weight and unfractionated heparins

A simple, selective and accurate capillary electrophoresis (CE) method has been developed for the rapid separation and identification of various low molecular weight heparins (LMWHs) and unfractionated heparin. Separation and operational parameters were investigated using dalteparin sodium as the test LMWH. The developed method used a 70 cm fused silica capillary (50 μm i.d.) with a detection window 8.5 cm from the distal end. Phosphate electrolyte (pH 3.5; 50 mM), an applied voltage of −30 kV, UV detection at 230 nm and sample injection at 20 mbar for 5 s were used. The method performance was assessed in terms of linearity, selectivity, intra- and inter-day precision and accuracy. The method was successfully applied to the European Pharmacopeia LMWH standard, dalteparin sodium, enoxaparin sodium and heparin sodium with a significant reduction in the run time and increased resolution compared with previously reported CE methods. Different CE separation profiles were obtained for various LMWHs and unfractionated heparin showing significant structural diversity. The current methodology was sensitive enough to reveal minor constituent differences between two different batches of enoxaparin sodium. This CE method also clearly showed chemical changes that occurred to LMWHs under different stress conditions. The sensitivity, selectivity and simplicity of the developed method allow its application in research or manufacturing for the identification, stability analysis, characterization and monitoring of batch-to-batch consistency of different low molecular weight and unfractionated heparins.

Determination of critical micelle concentration values by capillary electrophoresis

An improved, simple capillary electrophoresis method for the determination of critical micelle concentration values in an easy way by plotting the corrected electric current values versus the surfactant concentrations at a given field was described. The critical micelle concentrations of sodium dodecyl sulfate in different solutions were obtained, and the values were in good agreement with those found in the literature.

Quantitative Analysis of β-Blockers inPharmaceutical Preparations by Capillary Electrophoresis

A simple capillary electrophoresis method was developed for the analysis of four β-blockers (atenolol, metoprolol, pindolol, and propranolol) in pharmaceutical preparations. The method was validated regarding accuracy, precision, linearity, and detection/quantification limits, and the obtained values were in accordance to those reported in the literature. The method was applied to the determination of the drugs in commercial tablet preparations and proved to be fast and reliable for the quantitative analysis of the β-blockers.

Enantiomeric separation of N′-nitrosonornicotine by capillary electrophoresis

N′-Nitrosonornicotine (NNN) is the most abundant tobacco-specific nitrosamine (TSNA) found in tobacco and tobacco smoke, and is formed by the nitrosation of nicotine, amongst other possible mechanisms. Tumor induction is believed to occur by way of 2′-hydroxylation of NNN, and others have shown that ( S)-NNN undergoes significantly more 2′-hydroxylation than does ( R)-NNN. As such, there is a need to be able to separate and quantify the two enantiomeric forms of this important analyte. In the current work, we present a simple capillary electrophoresis method capable of achieving the desired separation. By employing uncoated, fused-silica capillaries with a citric acid buffer (pH 2.8) containing hydroxypropyl-β-cyclodextrin (HP-β-CD), the ( R)- and ( S)-forms of NNN are resolved in about 4 min. The identities of the two enantiomers were confirmed by comparison to reaction products from the nitrosation of an ( S)-(−)-nicotine sample. Furthermore, this method was applied to a crude tobacco extract to establish its potential for resolving NNN enantiomers from a complex mixture containing more dominant tobacco alkaloids.

Rapid Simultaneous Determination of Three Methylxanthines in Human Plasma by Capillary Electrophoresis

A simple capillary electrophoresis method is described for simultaneous determination of three methylxanthines, theophylline, dyphylline, and caffeine in human plasma. Plasma proteins are precipitated by acetonitrile and the supernatant was performed in Tris buffer (20 mM; pH 9) with sodium dodecyl sulfate (SDS) (150 mM) as an anionic surfactant. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and SDS. Application of the proposed method to the determination of theophylline and dyphylline in human plasma proved to be feasible. The practicability of the proposed method is demonstrated on a healthy volunteer.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

The Determination of Plasma Nitrite and Nitrate by Capillary Ion Analysis

Quantitative studies of the release of NO in vivo are difficult due to its short half-life. Consequently, most investigators have assayed nitrite and/or nitrate because these are relatively stable markers of NO production. Although various methods have been developed, all are limited to some extent by the degree of sample handling required. To overcome this we have developed a simple capillary electrophoresis method which requires very little sample handling and can measure both nitrite and nitrate in a single short run time. The method is based on the principles of capillary ion analysis and offers high sensitivity, good precision, and high throughput. The limit of detection is approximately 200 nM for nitrite and 300 nM for nitrate. Regression analyses for both anions routinely give values of greater than 0.99. Basal values obtained with this method are in good agreement with literature values.

Analysis of isoflavones by capillary electrophoresis

A simple capillary electrophoresis method is described for the assay of several isoflavones and coumestrol isolated from plant extracts. The method has good reproducibility; it compares well to HPLC, and it can be performed in less than 10 min.

Measurement of vitamin C by capillary electrophoresis in biological fluids and fruit beverages using a stereoisomer as an internal standard

Ascorbic acid (or vitamin C) is an important component of many biological systems and various physiological roles have been described for it. A rapid and simple capillary electrophoresis method for ascorbic acid measurements in biological fluids as well as in beverages was developed. A stereoisomer of ascorbic acid, isoascorbic acid, not normally found in nature, was used as the internal standard for this assay. The analysis was performed in a 30 cm × 75 μm I.D. fused-silica capillary with 100 m M tricine buffer, pH 8.8, and measured by UV absorbance at 254 nm. The method was sensitive to 1.6 μ/ml and linear to 480.1 μg/ml. Within-run R.S.D. was 3.2% (93.5 ± 3.0 μ/ml, mean ± S.D., n = 18) and run-to-run R.S.D. was 3.3% (35.6 ± 1.2 μ/ml, mean ± S.D., n = 10) and 1.9% (149.4 ± 2.8 μ/ml, mean ± S.D., n = 10). Average spiked recovery from human plasma samples was 98.0%. The technique has been demonstrated to be suitable for assay of vitamin C in biological samples and some fruit juices.