Zone Electrophoresis Method Research Articles

Simultaneous detection of zearalenone, citrinin, and ochratoxin A in pepper by capillary zone electrophoresis

ABSTRACT In the present study, a simple and fast method for simultaneous detection of zearalenone, citrinin, and ochratoxin A utilising capillary zone electrophoresis with an ultraviolet detector was developed. The optimised approach was validated and applied using pepper samples. The proposed method yielded satisfactory linearity between the signal and the mycotoxin concentration in the range of 1.5–150 μg/kg for zearalenone, 4.5–150 μg/kg for citrinin, and 0.8–150 μg/kg for ochratoxin A. The limits of detection for these mycotoxins ranged from 0.3 to 1.5 μg/kg. The corresponding intra- and inter-day precisions were less than 3.5 % and 4.1 %, respectively. Moreover, the matrix effect was also assessed and the result was compared using the capillary zone electrophoresis and high-performance liquid chromatography methods. The developed approach could be used for simultaneous detection of zearalenone, citrinin, and ochratoxin A in pepper.

Determination of Commonly Used Excipients in Pharmaceutical Preparations by Microchip Electrophoresis with Conductivity Detection

In this study, a microchip electrophoresis method for simultaneous determination of three frequently used pharmaceutical excipients (methylparaben, propylparaben and erythrosine) was described. Anionic zone electrophoresis (ZE) separations were performed on the microchip with contact conductivity detection using a high pH buffer (9.8). Sufficient resolution of the analytes (≥ 2.6) was achieved by 1 mmol L−1 β-cyclodextrin added to the buffer. Adsorption of the analytes on the walls of a poly(methylmethacrylate) microchip was suppressed by adding 1 mmol L− 1 sulfate to the sample. The LOD values for the excipients ranged from 0.5 to 11.1 µmol L− 1. High repeatability of the migration times of the analytes in model (< 2.9% RSD) and real pharmaceutical (< 4.1% RSD) samples was reached on the microchip. Peak areas of the analytes in all analyzed samples were characterized by RSD values ranging from 0.4 to 5.7%. The ZE method was successfully applied to the analysis of pharmaceutical syrups after a simple pretreatment (centrifugation and dilution), and gave recoveries of the analytes in the range 86.1–96.6%. The results showed a good agreement with those obtained by CZE with UV detection (254 nm) at 95% confidence level.

Capillary Electrophoresis Method for the Assessment of Erythropoiesis-Stimulating Agents in Final Formulations.

Capillary electrophoresis (CE) comprises several separation modes that can be used to characterize proteins in terms of physico-chemical properties such as isoelectric point or molecular weight, or in terms of purity/heterogeneity for the presence of charge or size variants. In glycoproteins the heterogeneity occurring as a consequence of variable amounts of terminal sialic acid residues on glycan moieties can be detected by CE. As such, a capillary zone electrophoresis (CZE) method was found suitable for the detection of isoforms of several erythropoiesis-stimulating agents (Bietlot and Girard, J Chromatogr A 759:177-184, 1997; Boucher et al., J Pharm Biomed Anal 71:207-213, 2012). In particular, the method can be used to analyze finished products containing erythropoietin-α, erythropoietin-β, or darbepoetin-α regardless of the formulation and without the need for sample pretreatment. The major excipients encountered in the various formulations included polysorbate 80, polysorbate 20, or human serum albumin. The ability of the method to resolve isoforms of the active ingredient in finished product enables the comparison of the isoform profile with that of the corresponding drug substance, allowing the assessment of the structural integrity and content of the active ingredients in finished products.

Application of capillary electrophoresis with electrokinetic supercharging and sweeping for the on-line preconcentration of phenolic acids.

Phenolic acids are natural antioxidants. Many studies have confirmed that these compounds can reduce the risk of developing chronic diseases such as diabetes, cardiovascular diseases, and certain cancers. In this work, we developed a rapid and efficient capillary electrophoresis method with an on-line preconcentration technique that could be used to simultaneously analyze 10 commonly found phenolic acids in plants. Briefly, phosphate buffer solution (pH 2) was filled into an uncoated fused silica capillary as the leading electrolyte, and then samples which were prepared in borate buffer (as the terminating ion) were loaded by electrokinetic injection (-10 kV, 900 s). After sample injection, both ends of the capillary were switched to the vial containing phosphate buffer with sodium dodecyl sulfate. The separation was then performed in micellar electrokinetic chromatography (MEKC) mode at -20 kV. During the method validation, the correlation coefficient of the regression curve was measured as greater than 0.997 and the relative standard deviation and relative error were lower than 9.63 % and 4.7 %, respectively. The limits of detection (LODs, S/N = 3) of these 10 analytes ranged from 0.01 to 2.5 ng/mL. Compared with the conventional capillary zone electrophoresis (CZE) method, the sensitivity for the analytes could be increased up to 25,000-fold. The method that we developed here was applied successfully to the detection of phenolic acids in fruit juices.

Determination of Amino Acids in Plasma and Nutritional Supplements by Capillary Electrophoresis with Copper(II) Coordination

A capillary zone electrophoresis (CZE) method was developed using interactions between amino acids and copper(II) without derivatization. The optimized separation buffer included 10% isopropanol, 50 mmol L−1 CuSO4, 50 mmol L−1 acetic acid, and 50 mmol L−1 sodium acetate at pH = 3.98 at a detection wavelength of 233 nm. This method allowed baseline separation and determination of ten amino acids in less than 21 min. Quality parameters such as linearity (r > 0.99), precision (relative standard deviation <2.51%), limits of detection (0.05 µg mL−1), and recovery (82.25% to 117.6%) were measured. The separation of amino acids in high salt solutions was explored. The method was used to directly determine amino acids in human plasma and nutritional supplements.

A rapid, low-cost quantitative diagnostic method for hepatitis C virus infection using capillary zone electrophoresis

Hepatitis C virus (HCV)-RNA amplification is a costly procedure in terms of time and reagents. Consequently, the search for more a cost-effective specific HCV diagnostic method is of great interest. Capillary zone electrophoresis (CZE) methods that detect HCV in serum, plasma, whole blood, and ascites without the need for sample pretreatment are not currently available. Here, a CZE method was developed that detects a larger specific peak in serum and other body fluids of HCV-infected patients than that found in healthy or hepatitis B virus (HBV)-infected individuals. The nature of the HCV peak was investigated using biochemical treatments, including RNase, DNase, and chymotrypsin enzymes. Electroeluted HCV peak was applied to transmission electron microscopy; electron micrographs showed that the HCV peak was attributed to virus-like particles with diameter and morphological properties similar to non-enveloped HCV nucleocapsids. The determination of CZE-HCV and HCV-RNA levels using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in 258 subjects revealed that these two tests were highly correlated (r = 0.92, p < 0.0001). One important issue of HCV testing is the storage conditions of serum to obtain reliable results. Serum samples at -20 °C showed the best preservation of the HCV peak up to one year. In conclusion, we detected HCV using CZE in a microliters volume from different body fluids. Besides the stability of samples in maintaining their peak height, the HCV-CZE test is rapid (<15 min) and a well-suited and low-cost technique. Thus, a major improvement in the quantitative diagnosis of HCV infection was established.

RAPID AND SIMPLE QUANTITATIVE DETERMINATION OF ATRACTYLENOLIDE I AND ATRACTYLENOLIDE III IN ATRACTYLODES MACROCEPHALA AND ITS DIFFERENT PROCESSED PRODUCTS BY CAPILLARY ZONE ELECTROPHORESIS

Atractylenolide I (AO-I) and atractylenolide III (AO-III) are the major sesquiterpenes considered to be bioactive from Atractylodes macrocephala (RAM) and its different processed products, popularly used for the treatment of the various digestive diseases and tumors. A rapid and simple capillary zone electrophoresis (CZE) method to quantitatively determine the AO-I and AO-III is described. Using an optimized method, AO-I and AO-III were separated in less than 10 min with A 50 mM borate buffer and a separated voltage 20 kV at 25°C. The CZE method was validated for linearity, sensitivity, accuracy, and precision and then used to determine the content of AO-I and AO-III. This method was successfully applied to determine AO-I and AO-III in real sample RAM and its different processed products.

Determination of target compounds in cefoperazone sodium and tazobactam sodium for injection by capillary electrophoresis

A capillary zone electrophoresis (CZE) method was developed for the simultaneous determination of cefoperazone sodium and tazobactam sodium in the injectable powder of cefoperazone sodium and tazobactam sodium with hydrochlorothiazide as the internal standard. The operation was carried out on a quartz capillary (75 cm x 75 microm i. d. , 63 cm effective length). The electrophoretic conditions were as follows: 40 mmol/L borax solution as the back ground electrolyte (BGE), 12. 0 kV applied voltage, 220 nm as the detection wavelength; the sample solution was injected by hydraulic pressure for 10 s at the height of 10 cm. The cefoperazone and tazobactam showed good linear relationship in the ranges of 0.25-3.96 g/L and 0.062-0.99 g/L with the correlation coefficients of 0.999 5 and 0.999 6, respectively. The relative standard deviations of relative peak areas were less than 3%. The preparation was stable in 208 min. The recovery results met the methodology requirements. The method is simple, rapid, reproducible, and suitable to control the quality of cefoperazone sodium and tazobactam sodium injectable powder.

Separation and Sensitive Detection of α -Lactalbumin and β -Lactoglobulin by Capillary Zone Electrophoresis

α-lactalbumin and β-lactoglobulin are main impurities in the lactose products extracted from whey,which may cause allergy.Therefore,the separation and detection of race α-lactalbumin and β-lactoglobulin are of great importance to food and pharmaceutical industry.In this work,a simple,fast,sensitive and reproducible capillary zone electrophoresis(CZE) method has been developed for this purpose.After simple sample preparation procedure,α-lactalbumin and β-lactoglobulin can be baseline separated within 2 minutes under following conditions:25 mM phosphate buffer at pH 7.0,+ 30 kV applied voltage,25 ℃ capillary temperature,205 nm UV detection,and sample injection at 50 mbar for 10 s。The limit of detection(LOD) at 3 times of S/N is 3.0 and 12 mg/L for α-lactalbumin and β-lactoglobulin,respectively.The repeatability(RSD,n=6) is below 1% for migration time and below 6% for peak area.These data qualified the method to the analysis of real lactose samples.The proposed method also shows a great potential in other fields of food and pharmaceutical industry.

Characterization of in Vitro Modified Human Very Low-Density Lipoprotein Particles and Phospholipids by Capillary Electrophoresis

A simple capillary zone electrophoresis (CZE) method was used to characterize human very low-density lipoprotein (VLDL) particles for four healthy donors. One major peak was observed for native, in vitro oxidized and glycated VLDL particles. The effective mobilities and peak areas of the capillary electrophoresis (CE) profiles showed good reproducibility and precision. The mobility of the oxidized VLDL peak was higher than that of the native VLDL. The mobility of the glycated VLDL peak was similar to that of the native VLDL. Phospholipids isolated from VLDL particles were analyzed by our recently developed micellar electrokinetic chromatography (MEKC) with a high-salt stacking method. At absorbance 200 nm, the native VLDL phospholipids showed a major peak and a minor peak for each donor. For oxidized VLDL phospholipids, the area of the major peak reduced for three donors, possibly due to phospholipid decomposition. For glycated VLDL phospholipids, the peak mobilities were more positive than native VLDL phospholipids for two donors, possibly due to phospholipid-linked advanced glycation end products (AGEs). Very interestingly, at absorbance 234 nm, the major peak of oxidized VLDL phospholipids was resolved as two peaks for each donor, possibly due to conjugated dienes formed upon oxidation.

Effective separation and simultaneous detection of gatifloxacin, aminomethylbenzoic acid, cefazolin and cefminox in human urine by capillary zone electrophoresis

Gatifloxacin (GAFX), P-aminomethylbezoic acid (PAMBA), cefazolin (CFZL) and cefminox (CMNX) can be combined and administered perorally or intravenously, and are excreted mainly into the urine. A novel capillary zone electrophoresis (CZE) method was developed, for the first time, for an effective separation and simultaneous determination of the said four drugs in human urine. The electrophoresis conditions were investigated and optimized. A 25 mM borate buffer (pH 9.2) was used and the peak area was determined with UV detection. Under optimal conditions, the four drugs can be separated effectively. Good linearity was achieved with the correlation coefficients (r2) of > 0.999. The limit of detection (LOD) for PAMBA, GAFX, CFZL and CMNX was 5.8, 3.9, 7.8 and 15.0 μg ml−1, respectively. Their average recovery in human urine was 93.2, 98.3, 108 and 92.7%, respectively, with the RSDs ranging from 1.0% to 3.5%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for the selective and simultaneous analysis of the test drugs in human urine at clinically relevant concentrations.

Simultaneous Determination of Strychnine, Brucine, Strychnine N-Oxide and Brucine N-Oxide in Crude and Fermented Nux Vomica by CZE with Ephedrine Hydrochloride as an Internal Standard

A rapid capillary zone electrophoresis (CZE) method with ephedrine hydrochloride as an internal standard was developed and validated for separating and quantifying the alkaloids strychnine (1), brucine (2), strychnine N-oxide (3), and brucine N-oxide (4) in the seed of Strychnos nux-vomica L. (crude nux vomica) and its fermented products by fungus Trametes cinnabarina (Jacq.: Fr.) Fr. (fermented nux vomica). The contents of alkaloids (1 and 2) in crude nux vomica were higher than those in fermented drugs, whereas alkaloids (3 and 4) were only detected in fermented ones. The method developed is sensitive, specific and accurate, as well as effective and easy, which can be used to assess the qualities of crude and fermented nux vomica.

Determination of doripenem and related substances in medicinal product using capillary electrophoresis

Doripenem, the latest carbapenem antibiotic licensed in the United States (15 October 2007) and the European Union (25 July 2008), has been implemented into therapeutic use along with imipenem, meropenem and ertapenem. The described method of zone electrophoresis in a low pH buffer for the separation of doripenem from its impurities has been successfully performed using field-amplified sample stacking (FASS), followed by UV absorption detection at 214 nm. The best results were obtained with phosphate buffer (100 mM) pH 2.9 containing 10% (v/v) of methanol, as the background electrolyte. Uncoated fused-silica capillary (60/52 cm; 75 μm id) with normal polarity, and voltage values of 25 kV, was used throughout the investigation. The optimised method of doripenem determination was validated in terms of linearity, accuracy and precision, and provides a detection limit of 3.0 μg/mL of doripenem. The repeatability, expressed by relative standard deviation (RSD) of the migration time, for doripenem and its degradation products varied from 1.37 to 2.51%, whereas the corrected peak areas were about 0.91-9.87%. Satisfactory separation was achieved within 20 min of electrophoresis; moreover, all carbapenems (imipenem, meropenem, ertapenem and doripenem) were well separated from each other during this time. The evaluated CZE method was applied in the analysis of a medicinal product containing doripenem Doribax(®) powder for solution for infusion.

ASSAY AND STABILITY-INDICATING CAPILLARY ZONE ELECTROPHORETIC METHOD FOR THE DETERMINATION OF MODAFINIL IN BULK AND ITS PHARMACEUTICAL PREPARATIONS

A simple, sensitive, and cost effective capillary zone electrophoresis (CZE) method for the determination of the novel wake promoting agent, modafinil in pharmaceutical formulations has been developed and validated. The CZE separation was performed using 50 μm i.d × 56 cm fused silica capillary under the following conditions: capillary temperature, 25°C; applied voltage, 25 kV; 20 mM H3PO4 − 1 M tris running buffer (pH 9.0); detection wavelength, 225 nm. Phenobarbital was used as the internal standard. The method was validated and showed not only good precision and accuracy but also good robustness. The calibration was linear from 5 to 250 µg mL−1. The accuracy values ranged from 101.6 to 105.3%. The good accuracy values obtained indicate the potential of this method for the determination of the analyte in pharmaceutical formulations. The LOD and LOQ were 1.2 and 3.5 μg mL−1, respectively. The method has been successfully applied to the determination modafinil in pharmaceutical tablet formulations. Excipients present in the tablets and degraded products from different stress conditions did not interfere in the assay.

A NEW CAPILLARY ZONE ELECTROPHORESIS METHOD FOR THE SCREENING OF CONGENITAL DISORDERS OF GLYCOSYLATION (CDG)

Background: The Congenital Disorders of Glycosylation (CDG) are an expanding group of metabolic diseases with a broad clinical presentation. We sought to validate a new Capillary Zone Electrophoresis (CZE) method (Sebia CAPILLARYS™ CDT) to screen for CDG. Methods: We analyzed 119 serum samples from children of varying ages and of both sexes to establish a reference range of transferrin glycoforms including CDT (Carbohydrate Deficient Transferrin). We then studied serums from 8 known CDG patients and compared the CZE results to the isoelectric focusing (IEF) profiles. We also analyzed serums after extraction from spotted Guthrie cards. Results: The mean (SD) percentage of transferrin glycoforms is 18.5 (4.4), 78.5 (4.2), 2.5 (1.3) and 0.6 (0.3) for penta-, tetra-, trisialotransferrin and CDT, respectively. There is no statistically significant difference between the different age groups analyzed (0-5, 6-11, 12-15, 16-18, and &gt; 18 years) or between sexes. We observed a good correlation between the CZE and IEF profiles with both fresh serum and serum extracted from Guthrie cards. Conclusions: The Sebia CAPILLARYS™ CDT system is a simple and reliable method to screen for CDG in pediatric and adult patients with an unexplained clinical syndrome, particularly when the nervous system is involved.

Simultaneous Determination of Atenolol and Chlorthalidone in Pharmaceutical Preparations by Capillary-Zone Electrophoresis

A capillary zone electrophoresis (CZE) method for the simultaneous determination of the β-blocker drugs atenolol and chlorthalidone in pharmaceutical formulations has been developed. The CZE separation was performed under the following conditions: capillary temperature, 25°C; applied voltage, 25 kV; 20 mM H3PO4–NaOH running buffer (pH 9.0); and detection wavelength, 198 nm. Phenobarbital was used as internal standard. The method was validated and showed not only good precision and accuracy but also good robustness. The method has been successfully applied to the simultaneous determination of both atenolol and chlorthalidone in pharmaceutical tablets.

Online concentration of aristolochic acid I and II in Chinese medicine preparations by micellar electrokinetic chromatography

In this study, an online concentration method in micellar electrokinetic chromatography (MEKC) applying field-enhanced sample injection (FESI) mode was developed for the detection of aristolochic acids (AAs) in Chinese medicine preparations. AA-I and AA-II were baseline separated with high separation efficiency, and 100-fold enhancement of the detection sensitivity was achieved compared with those obtained from normal capillary zone electrophoresis (CZE) or simple MEKC method. The proposed method was successfully applied for the determination of AAs in Chinese medicine preparations.

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.

Determination of Four Anthraquinones in Xanthophytum Attopvensis Pierre by Capillary Zone Electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for the simultaneous determination of four anthraquinones in Xanthophytum Attopvensis Pierre. The effects of several factors such as the pH, concentration of the running buffer and the separation voltage were investigated to find the optimum conditions. The optimum conditions were an electrolyte containing 15 mmol L−1 borate, at pH 9.64 and 17.5 kV applied voltage. The four analytes were completely separated within 5 min. Regression equations gave linear relationships between the peak area of each analyte and the concentration. The recoveries ranged from 96.5% to 106.2%. The method was successfully used in the analysis of extracts from Xanthophytum Attopvensis Pierre with a relatively simple extraction procedure, and the assay results were satisfactory.

Separation of Peptides and Proteins by Capillary Electrochromatography

Capillary electrochromatography (CEC) is a separation method that has gained increasing attention in recent years. It combines the advantages of high performance liquid chromatography (HPLC) selectivity with the high efficiency of electrophoresis. Due to its promise of high peak capacities, CEC appears to be promising for resolution of complex mixtures, such as those generated during protein and peptide mapping. However, direct transfer of HPLC and capillary zone electrophoresis (CZE) methods to CEC is not trivial due to the unique requirements for CEC in terms of stationary phase and operating conditions. In this paper, we discuss the various approaches undertaken for the CEC separation of peptides and proteins.