Fast Capillary Electrophoresis Method Research Articles

Determination of sorbate and benzoate in beverage samples by capillary electrophoresis—Optimization of the method with inspection of ionic mobilities

The aim of this study was to develop a fast capillary electrophoresis method for the determination of benzoate and sorbate ions in commercial beverages. In the method development the pH and constituents of the background electrolyte were selected using the effective mobility versus pH curves. As the high resolution obtained experimentally for sorbate and benzoate in the studies presented in the literature is not in agreement with that expected from the ionic mobility values published, a procedure to determine these values was carried out. The salicylate ion was used as the internal standard. The background electrolyte was composed of 25 mmol L −1 tris(hydroxymethyl)aminomethane and 12.5 mmol L −1 2-hydroxyisobutyric acid, at pH 8.1. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 μm I.D.), with short-end injection configuration and direct UV detection at 200 nm for benzoate and salicylate and 254 nm for sorbate ions. The run time was only 28 s. A few figures of merit of the proposed method include: good linearity ( R 2 > 0.999), limit of detection of 0.9 and 0.3 mg L −1 for benzoate and sorbate, respectively, inter-day precision better than 2.7% ( n = 9) and recovery in the range 97.9–105%. Beverage samples were prepared by simple dilution with deionized water (1:11, v/v). Concentrations in the range of 197–401 mg L −1 for benzoate and 28–144 mg L −1 for sorbate were found in soft drinks and tea.

Development and validation of a method for quantitative determination of econazole nitrate in cream formulation by capillary zone electrophoresis

A simple, fast, inexpensive and reliable capillary zone electrophoresis (CZE) method for the determination of econazole nitrate in cream formulations has been developed and validated. Optimum conditions comprised a pH 2.5 phosphate buffer at 20 mmol L −1 concentration, +30 kV applied voltage in a 31.5 cm × 50 μm I.D. capillary. Direct UV detection at 200 nm led to an adequate sensitivity without interference from sample excipients. A single extraction step of the cream sample in hydrochloric acid was performed prior to injection. Imidazole (100 μg mL −1) was used as internal standard. Econazole nitrate migrates in approximately 1.2 min. The analytical curve presented a coefficient of correlation of 0.9995. Detection and quantitation limits were 1.85 and 5.62 μg mL −1, respectively. Excellent accuracy and precision were obtained. Recoveries varied from 98.1 to 102.5% and intra- and inter-day precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CZE method presented advantageous performance characteristics and it can be considered suitable for the quality control of econazole nitrate cream formulations.

Micellar Electrokinetic Capillary Chromatography of 1,4-Benzodiazepine Derivates and their Degradation Products

The aim of this work is to elaborate a fast and reliable capillary electrophoresis method, for the separation of 1,4-benzodiazepine (BZD) derivates and their degradation products in acidic medium and in UV light. Because BZDs are neutral, hydrophobic compounds, the most efficient method proved to be micellar elektrokinetic capillary chromatography (MECC). For the separation we used a sodium tetraborate buffer with tensioactive agent (sodium dodecyl sulphate - SDS) and an organic modifier (methanol). We also optimized the analytical conditions, and studied the effects of the SDS and methanol concentrations and buffer pH on the determination efficiency.

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.

Development of a fast capillary electrophoresis method for determination of creatinine in urine samples

The aim of this work was to develop a fast method using capillary electrophoresis for the determination of creatinine in human urine samples. The pH and constituents of the background electrolyte were selected by inspection of effective mobility of creatinine and candidate urine interferents versus pH curves. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated by the Peakmaster software and considered in the optimization of the background electrolyte, composed by 10 mmol L −1 tris(hydroxymethyl)aminomethane and 20 mmol L −1 2-hydroxyisobutyric acid (HIBA) at pH 3.93. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 μm I.D.), with short-end injection configuration and direct UV detection at 215 nm. The migration time of creatinine was only 22 s. A few figures of merit of the method are as follows: good linearity in the concentration interval of 5–70 mg L −1 ( R 2 > 0.99), limit of detection of 0.5 mg L −1, inter-day precision better than 2.7% ( n = 9) and recovery in the range 99.0–103.7% at three concentration levels (50, 100 and 150 mg L −1). Urine samples were prepared by deproteination with acetonitrile (1:3 sample:acetonitrile, v/v), centrifugation and dilution of a deproteinated aliquot with 12.5 mmol L −1 HIBA (1:4, v/v). Creatinine concentrations between 489 and 1063 mg L −1 were obtained in the urine of four healthy volunteers.

Validation and application of capillary electrophoresis for the analysis of lidocaine in a skin tape stripping study

A fast and simple capillary zone electrophoresis method was developed and validated for the determination of lidocaine in skin using tape samples. Separation was performed in a 350 mm (265 mm to window) x 50 microm i.d. fused silica capillary using a background electrolyte of phosphoric acid-Tris pH 2.5. The extraction of lidocaine from tape samples was achieved using methanol, which was diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and lidocaine were 2.9 and 3.2 min, respectively. The limit of quantification for lidocaine was 50 microg, with signal to noise ratio greater than 10. The calibration curve was linear from 50 to 1000 microg with r(2) greater than 0.99. The CV for both within- and between-assay imprecision and the percentage of inaccuracy for the quality control samples including lower and upper limits of quantitation were <or=2% and <or=14%, respectively. The absolute recovery of lidocaine was >97%. The accuracy and selectivity of this method allowed the measurement of lidocaine in tape samples obtained from a skin tape stripping study of local anesthetics in healthy subjects.

Chiral separation of ephedrine alkaloids from Ephedra sinica extract and its medicinal preparation using cyclodextrin-modified capillary zone electrophoresis

A fast capillary zone electrophoresis (CZE) method using dimethyl-β-cyclodextrin (DM-β-CD) as modifier with tetrabutylammonium chloride (TBAC) as addition has been developed for the chiral separation of (±)-ephedrine ((±)-EP), (±)-pseudoephedrine ((±)-PP), (±)-N-methylephedrine ((±)-MP), and (±)-norephedrine ((±)-NP). The electrophoretic separation was performed using a 35-cm × 50 μm I.D. (30-cm effective length) fused silica capillary. Samples were introduced under electromigrated injection at 5 kV for 4 s, and the running voltage was 15 kV at the injector end of the capillary. Within 19 min, eight ephedrine compounds were separated and detected at 210 nm. The method was successively applied to the determination of the ephedrine enantiomers in the Chinese herbal extract from Ephedra sinica and its medicinal preparation (Xiaoerqing feiwan). Parameters affecting the resolution between (+) and (-)-enantiomers, such as pH, cyclodextrin type and concentration, organic modifier, and tetraalkylammonium reagents, were reported.

Development and validation of a capillary electrophoresis method for the determination of sulfate in indinavir sulfate raw material

In this work, a simple and fast capillary electrophoresis method using indirect UV detection (230 nm) for the quantitative determination of sulfate in the indinavir sulfate raw material was described. A running electrolyte consisting of 10 mmol L-1 ammonium molybdate containing 0.15 mmol L-1 cetyltrimethylammonium bromide (pH 7.5) was used. Samples were injected hydrodynamically by applying 5000 Pa pressure during 3 s. The response was linear from 10.1 µg mL-1 to 79.8 mg µL-1. Limits of detection and quantification were 0.34 and 1.13 mg µL-1, respectively. The intra-day precision expressed as relative standard deviation was 2.8% for 10 independent sample preparations. Recoveries varying from 96.9 to 102.4% at three concentration levels were obtained. The methodology was successfully applied for real samples. The concentration of sulfate in indinavir sulfate raw material varied from 13.3 to 13.5% (m/m calculated as anhydrous base).

Fast method to determine furosine in breakfast cereals by capillary zone electrophoresis

Furosine is a well-know marker of the extent of the thermal treatment applied to foodstuffs. A fast, reproducible and accurate capillary zone electrophoresis (CZE) method to analyse furosine focused on complex matrices, such as cocoa-added breakfast cereals, is presented. A representative group of 17 commercial cocoa-added breakfast cereals were determined by the reference method (HPLC) and by CZE. In addition, eight ready-to-eat breakfast cereals were tested to point out the application of the procedure to a simpler matrix. Data from both methodologies are compared by F-test and unpaired Student's t-test, showing no significant differences among them and an adequate correlation between methods was found (r 2=0.9852; p=0.000). The CZE method provides a valuable analytical tool for determination of furosine in cocoa-added breakfast cereals and is suitable for routine analysis where presence of highly retained material could limit the resolution and shelf-life of the analytical columns applied for HPLC. The analysis is carried out in 3 min. The stability of the migration time and the good correlation between HPLC–CZE results support the effectiveness of the method.

Simple and Rapid Method for the Differentiation of Lens culinaris Medik. from False Lentil Species

The practice of using both common vetch (Vicia sativa L.) and single-flower vetch (Vicia articulata Hornem.) seeds as food or feed is encouraged by the very high resemblance of their seeds with those of small-seeded lentil cultivars. Among the Vicieae, antinutritional and toxic factors are particularly important, because many species, containing high levels of these compounds, are not safe. A simple and fast capillary electrophoresis (CE) method was proposed for the differentiation of lentil cultivars from false lentil species (i.e., single-flour vetch and common vetch). Proteins were extracted from defatted milled seeds with an alcoholic/saline solution. Extracts were separated in an uncoated fused-silica capillary with iminodiacetic acid (IDA) isolectric buffer containing 0.05% hydroxypropylmethylcellulose (HPMC) and 20% acetonitrile. The presented method is useful also for the detection of contamination of whole or split seeds of lentil by vetch species. With respect to alternative techniques, such as DNA-based markers or thin-layer chromatography (TLC), CE has the advantages of being less expensive, faster, and fully automated.

Determination of saikosaponins a, c, and d in Bupleurum Chinese DC from different areas by capillary zone electrophoresis

A fast capillary zone electrophoresis (CZE) method was developed for the determination and separation of saikosaponins a, c, and d in Chinese herbal extracts of Bupleurum Chinese DC from different areas. Detection at 214 nm with a system containing sodium borate buffer and mono-3-phenylcarbamoyl-beta-CD was found to be the most suitable approach for this analysis. Saikosaponins a, c, and d could be easily determined within 8 min. The effect of the concentration of mono-3-phenylcarbamoyl-beta-CD, the concentration of the running buffer and buffer pH value on the migration behavior of the saikosaponins is discussed.

Capillary zone electrophoresis as a tool for the quality control of goldenseal extracts

The root extracts of goldenseal (Hydrastis canadensis L.) are popular phytomedicines for the treatment of gastrointestinal disorders and upper respiratory tract infections. Here we describe a simple and fast capillary zone electrophoresis (CZE) method with ultraviolet detection at 225 nm for the quantification of the major goldenseal constituents, berberine and hydrastine, in herbal remedies containing goldenseal root extracts. Tritoqualine, an antihistaminic drug with a hydrastine-like phthalidisoquinoline structure, was applied as an internal standard. The running buffer was a 1:5 mixture of 500 mM ammonium acetate (adjusted to pH 3.4 with acetic acid) and methanol. Our newly developed CZE method was validated regarding limit of detection (LOD), limit of quantification, linearity, accuracy and precision. For both berberine and hydrastine, the LOD was 1.0 microg/mL and the linearity was obtained between 2.5 and 500 microg/mL. Using our newly developed method, both the alkaloids could be analysed in herbal remedies containing goldenseal root extracts within 8 min.

Oxalate distribution in soils under rhubarb (Rheum rhaponticum)

Oxalate in soils may enhance phosphate availability, promote mineral dissolution, and increase the mobility of aluminium and heavy metal cations by complexation. Rhubarb (Rheum rhaponticum L.) has very high content of oxalate in leaves and petioles, and therefore the topsoil under rhubarb might have elevated contents of oxalate. Soil samples were collected at depths of 0–2.5 and 2.5–5 cm from 10 cm sections along 100 cm transects from rhubarb plants at four locations in Denmark, and from seven layers in a soil profile to 80 cm depth at one location. Oxalate was extracted from the soil with 0.2 M phosphate at pH 2 by reciprocal shaking for 24 h and then determined by a new fast capillary zone electrophoresis method with 300 mM KH2PO4 and 0.30 mM TTAB electrolyte adjusted to pH 7, developed and tested to analyse high-ionic-strength soil extracts. Rhubarb increases the oxalate content in soil under the leaves slightly. The average content of oxalate in the upper 0–5 cm soil was 444 µmol/kg at the Kaldred site, and 111–333 µmol/kg at the three other locations. In the soil profile, the content of oxalate decreased from 500 µmol/kg in 0–5 cm depth to 110 µmol/kg at 75–80 cm depth. No significant seasonal changes in oxalate contents were observed, while an annual variation of 100 µmol/kg could be observed at 0–2.5 cm depth. During plant decay in autumn, a slight increase in oxalate content was observed at 30 cm soil depth. In conclusion, the role of oxalate in weathering and metal transport appears to be limited in soils under rhubarb. Oxalate might stimulate microbiological growth and phosphate mobilisation in the rhizosphere, but concentrations observed are too low to impose any toxic effects to organisms.

Very fast and direct capillary zone electrophoresis method for the determination of creatinine and creatine in human urine

A capillary zone electrophoresis (CZE) method was investigated for the determination of creatinine and creatine in human urine by using a fused-silica capillary (75μm ID × 30cm total length, 10cm effective length). The separation was performed using an hydrodynamic injection time of 3s (0.5psi), a voltage of −15kV and a 60mM phosphoric buffer solution at pH 2.2 as electrolyte separation. Under these conditions, the analysis takes less than 2.7min. A linear response over the 3.0–120mgL−1 concentration range was investigated for compounds. A dilution of the sample (water:urine) was the only step necessary before the electrophoresis analysis. Detection limits of 0.7 and 1.3mgL−1 for creatinine and creatine (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine creatinine and creatine in different human urine samples.

Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 μm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.

Analysis of impurities in crude and highly-purified terephthalic acid by capillary electrophoresis

In this work, a simple and fast capillary electrophoresis method for the simultaneous analysis of 4-carboxybenzaldehyde (4-CBA), p-toluic acid (pTOL) and benzoic acid (BZ) in industrial batches of crude (CTA) and highly-purified (PTA) terephthalic acid was developed. The electrophoretic conditions comprise: 20 mmol L-1 tetraborate buffer at pH 9, hydrodynamic injection (5 s/17 kPa), applied voltage of +30 kV and direct UV detection at 200 nm. The concentrations of 4-CBA and pTOL in five batches of CTA and four batches of PTA as determined by the proposed CE methodology were in agreement with the results from gas chromatography and polarography methods in current use in Tereftalatos Mexicanos S.A. (TEMEX). Levels of BZ were determined only by the CE methodology and ranged from 60 - 300 ppm in CTA and 5 - 7 ppm in PTA. Several impurities deriving from the incomplete oxidation of p-xylene were investigated. So far the positive identification of 4-hydroxymethylbenzoic acid was accomplished by comparison with pure standards.

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cm × 75-μm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5 mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.

Capillary electrophoretic method for determination of protease inhibitor indinavir sulfate used in human immunodeficiency virus therapy

A simple, fast and reliable capillary electrophoresis (CE) method for determination of indinavir sulfate, a potent protease inhibitor used in human immunodeficiency virus (HIV) therapy, in commercial and simulated capsule formulations is described. The analysis was performed in a 75 μm i.d. uncoated fused-silica capillary with 27 cm length (effective length of 19.4 cm) using a 20 mmol l −1 phosphate buffer at pH 2.52. Samples were injected hydrodynamically by applying 0.5 psi pressure during 2 s. The applied voltage was 28 kV. Direct UV detection at 214 nm led to an adequate sensitivity without interference from sample excipients and known impurities. For quantitative purposes, diazepam was used as internal standard. Under optimized conditions, the migration times for indinavir sulfate and diazepam were 1.06 and 1.66 min, respectively. Analytical curve of peak area ratios versus concentration in the range of 20.0–100.0 μg/ml gave a coefficient of correlation of 0.9992, establishing the method linearity. The limits of detection and quantitation were 4.61 and 14.0 μg/ml, respectively. The within-day precision expressed as relative standard deviation was <1.5% for 10 consecutive sample injections. An average recovery of 100.81±0.56% at three concentration levels was obtained. Based on the performance characteristics, the proposed methodology was found suitable for the determination of indinavir sulfate in capsule formulations, presenting additional advantages inherent to the CE technology, such as low consumption of reagents and column endurance.

Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-β-cyclodextrin as modifier

A fast capillary zone electrophoresis (CZE) method, using dimethyl-β-cyclodextrin (DM-β-CD) as modifier, has been developed for the determination of three flavonoids (quercetin (QU), kaempferol (KA) and isorhamnetin (IS)) in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon tablet). Optimum separation was achieved with 20 mM borate buffer at pH 10.0 containing 5 mg ml −1 of DM-β-CD. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992 and 0.9996) between the peak area of each compound (QU, KA and IS) and its concentration. The relative standard deviations of migration times and peak areas were <1.53 and 4.14%, respectively. The effects of several CE parameters on the resolution were studied systematically. The contents of three flavonoids in H. rhamnoides were successfully determined with 4.5 min, with satisfactory repeatability and recovery. It was also tested that the possibilities of using this method for the determination of flavonoids in Chinese medicinal preparation.

92 The BPSU study of biliary atresia — outcome after 8 years

Hereditary tyrosinemia type I is a serious metabolic disorder leading to liver failure. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is a relatively new drug which is used to prevent the accumulation of toxic metabolites in patients with hereditary tyrosinemia type I. In the present study, we have developed a new, simple, fast, and cost-effective capillary electrophoresis method for the quantitative monitoring of this drug in serum samples. Micellar electrochromatographic separation of NTBC was performed using 20 mmol/L phosphate and 40 mmol/L sodium dodecylsulfate (SDS) at pH 12 as running electrolyte. Separation of NTBC was achieved in around 4 min. Reproducibilities of migration times and corrected peak areas of NTBC (as R.S.D.%) were found as 0.73 and 1.99, respectively. The detection limit was 3.17 and the quantification limit was 10.6 μmol/L for NTBC using UV detection at 278 nm. The utility of the method was demonstrated by the detection of NTBC in serum samples from patients with hereditary tyrosinemia type I using this drug.