Rapid Capillary Zone Electrophoresis Research Articles

Application of capillary zone electrophoresis to determine second-generation H1 antihistaminic drugs, loratadine and rupatadine

The second generation of H1 antihistamines from the piperidine group are often used for treating allergic diseases due to their action on histaminic receptors, the primary mediator of allergy. Moreover, the antihistamines have anti-inflammatory action, mediated through platelet-activating factor blocking activity. A simple and rapid capillary zone electrophoresis method was developed and validated for the determination of loratadine (LOR) and rupatadine (RUP) in tablets. The analyses were carried out using a fused silica capillary of 50.2 cm (40 cm effective length), 75 µm i.d. The background electrolyte was composed of boric acid 35 mmol/L, pH 2.5. Voltage of 20 kV, hydrodynamic injection of 3447.3 Pa for 3s, temperature at 25 ºC, and UV detection at 205 nm were applied. Electrophoretic separation was achieved at 1.8 and 2.8 min for RUP and LOR, respectively. The method was linear for both drugs in a range of 50.0 to 400.0 μg/mL (r>0.99). The limits of detection and quantification were 46.37 and 140.52 μg/mL, for LOR and 29.60 and 89.69 μg/mL for RUP respectively. The precision was less than 5.0 % for both drugs. The average recovery was approximately 100 %. The proposed novel method can significantly contribute to the rapid detection of counterfeit products and in quality control of drug products containing antihistamines.

Validated Capillary Zone Electrophoretic Determination of Avanafil and Dapoxetine Hydrochloride in their Pure form and Pharmaceutical Preparation

Aims: In this study, a simple, green, and rapid capillary zone electrophoresis (CZE) method coupled with a diode array detector (DAD) was applied for the analysis of avanafil (AVA) and dapoxetine hydrochloride (DAP) as a binary mixture using vardenafil (VAR) as an internal standard (IS) in pure form and pharmaceutical formulation.
 Methodology: The separation was done using fused silica capillary (58.5 cm total length, 50 cm effective length, and 50 μm internal diameter) and the running background electrolyte (BGE) was 100 mM acetate buffer at pH 3.6. During the separation process, the applied voltage was 30 KV, while the temperature was 25 °C. The sample injection was applied at a pressure of 50 mbar for 10 s, and detection was carried out at 210 nm for DAP and 248 nm for AVA and VAR.
 Results: Analysis of the tested drugs and the internal standard was carried out in less than 6.5 min, where the migration times were 4.29, 4.90, and 6.02 min for IS, DAP and AVA respectively. The proposed method showed linearity in the concentration range 5-80 and 5-70 μg/mL with correlation coefficients 0.9996 and 0.9999 for AVA and DAP respectively. The limit of detection (LOD) was 0.523 and 0.531 for AVA and DAP respectively, while the limit of quantification (LOQ) was 1.585 and 1.608 in respective order. The Peak purity and identity in the proposed method were validated by DAD.
 Conclusion: The proposed CZE method was validated according to ICH guidelines and applied successfully for the estimation of AVA and DAP in their combined pharmaceutical preparation.

Use of Rapid Capillary Zone Electrophoresis to Determine Amino Acids Indicators of Herring Ripening during Salting.

Currently, herring fillets are salted with acetic acid to activate muscle proteases. This causes a change in the composition of free amino acids, compared to salting of whole fish with viscera proteases. Therefore, old indicators of the ripening dynamics of salted fish based on amino acids are not current. Determination of free amino acids can be performed by many methods, but most are labor intensive and expensive. Therefore, a capillary electrophoresis method without derivatization (CZE) was used to determine the actual ripening rates of salted herring fillets. A group of hydrophobic and basic amino acids were determined in trichloroacetic acid (TCA) extracts of meat and brine to develop 16 indicators. Statistical regression analysis of the indicators (R2adj, RMSE, cluster analysis) followed by principal component analysis (PCA) correlation analysis of the indicators vs sensory evaluation parameters of texture and TPA-hardness of salted fillet meat allowed the choice of the most precise indicators. The best indicator in meat was Phe/Tyr-height, which value increased during salting. A more precise indicator of ripening was His/Tyr-height in brine, which value decreased during salting. Sensory evaluation parameters of salted herring texture correlated strongly with TPA-hardness and traditional indicators such as non-protein nitrogen and protein hydrolysis product fraction content. However, the most precise indicators were those obtained from amino acids determined by the CZE method. Results obtained in this study may be suitable for fast monitoring of the salted herring ripening process in industry.

On-line separation and quantification of virus antigens of different serotypes in multivalent vaccines by capillary zone electrophoresis: A case study for quality control of foot-and-mouth disease virus vaccines

Accurate quantification of effective antigens of different serotypes is crucial for quality control of multivalent vaccines but challenging. A simple and rapid capillary zone electrophoresis (CZE) method was developed for on-line separation and quantification of foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent FMDV vaccines. The FMDV peak identity in CZE was demonstrated by the study of FMDV dissociation combined with high performance size exclusion chromatography (HPSEC) analysis. After optimizing CZE conditions including UV detecting wavelength, injection volume, and separation voltage, both serotype A and O FMDV showed good reproducibility (RSD <5%) and linear responses (R2=0.999) between the peak area and FMDV content in the concentration range of 15-400 μg/mL. The two serotypes of FMDV with similar size had different migration time in CZE according to their different zeta potential, which allows them to be separated and quantified, with accuracy of <10% relative error. CZE was then successfully applied for antigen quantification of commercial O monovalent and A/O bivalent FMDV vaccines. Compared with HPSEC, CZE was not only able to quantify each serotype of FMDV, but also free from interference of nucleic acids impurities. In summary, the CZE can be a simple, rapid, and reliable tool for quality control of monovalent and bivalent FMDV vaccines. The CZE method can also be further extended to the quality control of other multivalent virus and virus like particle vaccines.

Rapid Capillary Zone Electrophoresis of Recombinant Erythropoietin by the Use of Dynamic Double Layer Coating

The determination of erythropoietin (EPO) isoforms by using capillary zone electrophoresis (CZE) has been a key study in the quality control of erythropoietin products. However, the current European Pharmacopeia method has been reported to provide poor reproducibility, tedious preconditioning, long separation times exceeding 60 min, and the employment of toxic reagents. In this study, a novel rapid CZE method, in which a double layer dynamic coating technique was developed for the determination of erythropoietin isoforms by the minimization of protein adsorption to the inner capillary walls and to accelerate the throughout of the protocol. The results obtained by the measured procedure were demonstrated to meet the European Pharmacopeia requirements. Real samples from different vendors were also analyzed. This rapid CZE method was shown to be suitable for the quality control (QC) erythropoietin with better reproducibility and higher sample throughput.

Rapid separation and quantification of self-assembled nanoparticles from a liquid food system by capillary zone electrophoresis

Food processing generates a large amount of self-assembled colloidal nanoparticles (NPs), which have defined structures and directly interact with macrophages. Their promising potential as a new source of functional NPs and a key to elucidate food-body interactions prompt the importance of the method development. This study attempts to apply capillary zone electrophoresis (CZE) in studying self-assembled nanoparticles in a real liquid food system of freshwater clam (Corbicula fluminea) soup, a popular delicacy in East Asia with proven hepatoprotective effects. The soup sample was satisfactorily separated into one nanoparticle fraction and multiple molecular fractions within five minutes by the single and rapid CZE analysis, which was of high repeatability (peak area RSD < 4%, migration time RSD < 1%) and accurate quantification with a linear area-number relationship in the range of (7.5–110) × 1011 particles/mL (R2 > 0.99). Therefore, CZE analysis can be an efficient tool for the investigation of self-assembled nanoparticles in real liquid food systems.

"Development of a rapid capillary zone electrophoresis method to quantify Levofloxacin and Meloxicam from transdermal therapeutic systems "

"Development of a rapid capillary zone electrophoresis method to quantify Levofloxacin and Meloxicam from transdermal therapeutic systems "

Simultaneous separation and rapid determination of spironolactone and its metabolite canrenone in different pharmaceutical formulations and urinary matrices by capillary zone electrophoresis.

The aim of this study was to develop a novel, sensitive, precise, simple, and rapid capillary zone electrophoresis method for the quality control of spironolactone in three different formulation types and a rapid simultaneous determination of the content of spironolactone and canrenone in urine samples using fluocinonide as an internal standard. After optimization of separation conditions, the electrolyte solution was the pH 5.5, 20 mM phosphate buffer containing 4.5 g/L sulfated-β-cyclodextrin, 15 kV of electric filed across the capillary applied at 25°C. A diode array detector was used, and the detection wavelength was 260 nm. Under optimum conditions, good linearity was achieved with correlation coefficients from 0.9976 to 0.9997. Detection limits were 0.56 and 0.20 μg/mL, and the quantitation limits were 1.87 and 0.67 μg/mL, respectively. Excellent accuracy and precision were obtained. Recoveries of the analytes varied from 100.8 to 103.1%. The results indicated that baseline separation of analytes was obtained and this method was suitable for quantitative determination of spironolactone in pharmaceutical preparations and rapid simultaneous determination of the content of spironolactone and its major metabolite canrenone in urine samples.

Rapid Determination of Coumarins in Plants by Capillary Electrophoresis

The goal of this study was to optimize the parameters for capillary electrophoresis to separate and determine aesculin, aesculetin, umbelliferone, and dihydrocoumarin in plant materials. A simple and rapid capillary zone electrophoresis method is reported for this separation that required less than nine minutes. A bare fused silica capillary was employed using 20 mM borax in 5% methanol at pH 10.1 as the background electrolyte with an applied voltage of 30 kV at 27°C. Good linearity and reproducibility were obtained with high correlation coefficients. The analytes were determined by molecular absorption at 194 and 206 nm. Coumarins were determined in Aesculus hippocastanum L., Cichorium intybus L., Melilotus officinalis L., and Juniperus communis L. “Pendula.” The concentrations of aesculin and aesculetin were 3.07 and 6.31 mg g−1 in Aesculus hippocastanum. In Cichorium intybus, the aesculetin concentration was 2.42 mg g−1. The dihydrocoumarin concentration was 0.54 mg g−1 in Melilotus officinalis, and the concentration of umbelliferone was 0.58 mg g−1 in Juniperus communis “Pendula.”

Determination of Shikonin and Rosmarinic Acid in Echium vulgare L. and Echium russicum J.F. Gmel. by Capillary Electrophoresis

A simple and rapid capillary zone electrophoresis (CZE) procedure for determination of shikonin and rosmarinic acid in roots of Echium vulgare L. and Echium russicum J.F. Gmel. is described. The separation was achieved in less than 4 min. The analysis was performed in fused silica capillary (i.d. 75 µm, 48.5 cm total length) using 50 mM borate, at pH 9.5 as the run buffer with applied voltage of 30 kV and constant temperature (27 °C). The method ensures good linearity and repeatability with high correlation coefficients (0.994 and 0.998 for shikonin and rosmarinic acid, respectively). The compounds were detected by UV absorption at 218 (shikonin) and 202 nm (rosmarinic acid). The limits of detection (LOD) for shikonin and rosmarinic acid were 0.603 ppm and 0.270 ppm, respectively. The method was used for estimation of both components in Echium plants. We found out that the content of shikonin and rosmarinic acid were 4.4–4.8 mg/kg air-dry material and 20.0–54.2 mg/kg air-dry material, respectively, in the Echium species analyzed.

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.

Development and validation of a rapid capillary zone electrophoresis method for determining charge variants of mAb

This work aimed to develop a rapid capillary zone electrophoresis (CZE) method to provide abundant purity and identity information of monoclonal antibodies. The CZE running buffer system was optimized to be 20mM acetate–acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene oxide (PEO) and 2mM triethylenetetramine (TETA), which was further tested with advantages on the peak resolution improvements. The conditioning period was scheduled to 1min for both 0.1M HCl and CZE running buffer to reduce total separation time. Additionally, the applied voltage and effective separation length were optimized at 30kV and 20cm separately. Compared with the method reported by Yan [1], this newly developed method showed a higher resolution in separating the two unknown basic peaks by testing monoclonal antibody sample (mAb1). The further validation results showed that for all five of charge isoform peaks of test mAb1, repeatability, intraday and interday precision had a RSD less than 0.58% for migration time and less than 3.18% for corrected area percent. The correlation coefficients of more than 0.98 for all peaks also demonstrated the good linearity for the method. In addition to the application of distinguishing intact antibody from C-terminal Lys variants, the method also has advantage in separating the Fab, Fc and intact antibody-relevant substances quickly, which facilitated the rough evaluation of papain induced digestion.

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R² = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).

Capillary electrophoretic analysis of whole blood samples for hemoglobin‐based oxygen carriers without the use of immunoprecipitation

Hemoglobin-based oxygen carriers (HBOCs) are blood substitutes, synthesized by polymerizing hemoglobin, which are being developed and investigated as alternatives to blood for medical purposes. However, due to their ability to increase the oxygen carrying capacity when taken by healthy individuals, HBOCs have been used as a doping agent among endurance athletes and are included in the World Anti-Doping Agency's Prohibited List. To maintain the fairness of competitions and continue the battle against doping, it is essential to be able to detect HBOCs if present in an athlete's blood. To achieve this goal, it is necessary to differentiate HBOCs from the native hemoglobin and to do so in a cost and time effective manner. We have developed a rapid capillary zone electrophoresis (CZE), UV absorbance, method capable of detecting HBOCs, in whole blood samples, at levels below those considered necessary to provide a performance enhancement. Our approach to the analysis for HBOCs utilizes the whole blood sample, not just the plasma, and does not require the use of immunoprecipitants to ensure accurate analysis. By lysing the red blood cells and using centrifugal filtration, followed by our CZE separation, we are able to effectively distinguish between native hemoglobin and HBOCs. Through this method, we have been able to reliably detect concentrations of HBOCs at the equivalent of 5.5 g/L, the equivalent to a 3.5% increase in blood hemoglobin concentration for an athlete.

Development and Validation of a Simple and Rapid Capillary Zone Electrophoresis Method for Quantification of Heparin in a Pharmaceutical Product and Stability Studies

A simple and rapid capillary zone electrophoresis method has been developed and validated for determination of heparin in a pharmaceutical product and evaluation of heparin stability under different stress conditions. The best separation was achieved by a bare fused silica capillary (50 μm i.d.; 50 cm total and 41.5 cm effective length), phosphate buffer (pH=3.50, 72 mM) at 35°C, hydrodynamic injection at 50 mbar for 40 seconds and applied voltage of -30 kV. Heparin and force degradation products were detected by a photo diode array detector at 200 nm and 257 nm, respectively. The proposed method was validated in terms of linearity, accuracy, precision, limit of quantification (LOQ) and limit of detection (LOD). For evaluation of heparin stability, heparin solutions were subjected to thermal (90 ± 1°C), acidic (pH=2.00, 70 ± 1°C) and basic (pH=12.00, 70 ± 1°C) stress conditions, also sun light exposure (in the lab). The results indicated that, the method was linear in the range of 0.312 to 15.0 mg/ml with LOD of 0.078 mg/ ml and LOQ of 0.312 mg/ml, accurate (between 97.27% and 101.0%) and precise (intra-day precision of 0.28 to 1.8 and inter-day precision of 0.78 to 3.2%). The migration time of heparin under optimized conditions, was 2.39 ± 0.03 minutes and force degradation products were separated within 7 minutes. Heparin content in the pharmaceutical product quantified by the method was 99.58 ± 0.70% of the label claim. Meanwhile the method could show the changes in the heparin electropherogram, also formation of degradation products under different stress conditions. Therefore the proposed method could be applied as a fast and suitable technique for determination of heparin in pharmaceutical dosage forms and stability studies.

Development and validation of a simple and rapid capillary zone electrophoresis method for determination of nnrti nevirapine in pharmaceutical formulations

A simple and fast capillary zone electrophoresis (CZE) method has been developed and validated for quantification of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, in pharmaceuticals. The analysis was optimized using 10 mmol L-1 sodium phosphate buffer pH 2.5, +25 kV applied voltage, hydrodynamic injection 0.5 psi for 5 s and direct UV detection at 200 µm. Diazepam (50.0 µg mL-1) was used as internal standard. Under these conditions, nevirapine was analyzed in approximately less than 2.5 min. The analytical curve presented a coefficient of correlation of 0.9994. Limits of detection and quantification were 1.4 µg mL-1 and 4.3 µg mL-1, respectively. Intra- and inter-day precision expressed as relative standard deviations were 1.4% and 1.3%, respectively and the mean recovery was 100.81%. The active pharmaceutical ingredient was subjected to hydrolysis (acid, basic and neutral) and oxidative stress conditions. No interference of degradation products and tablet excipients were observed. This method showed to be rapid, simple, precise, accurate and economical for determination of nevirapine in pharmaceuticals and it is suitable for routine quality control analysis since CE offers benefits in terms of quicker method development and significantly reduced operating costs.

Capillary zone electrophoresis as a potential technique for the simultaneous determination of sulfadoxine and pyrimethamine in tablet formulations

A novel, simple and rapid capillary zone electrophoresis method with UV detection has been developed for the simultaneous determination of pyrimethamine and sulfadoxine in tablet formulations. The compounds are separated in 6 min in a fused silica capillary, 30 cm long (20 cm to detector) × 50 μm using a 100 mM phosphate buffer pH 7.2 as background electrolyte, a 330 V cm −1 electric field and a detection wavelength of 214 nm. Analysis of different tablet formulations has shown a good agreement with the liquid chromatography method described in the United States Pharmacopoeia. Main advantages of the CZE method are the rapid set-up of instrumentation and capillary equilibration, short analysis time and low running cost.

Simultaneous determination of iridoid glycosides and flavanoids in Lamionphlomis rotate and its herbal preparation by a simple and rapid capillary zone electrophoresis method

Iridoid glycosides and flavanoids are two main effective components of Lamiophlomis rotata (Benth.) kudo. However, there is no method for simultaneous analysis of iridoid glycosides and flavanoids in L. rotata and its pharmaceutical preparations. A simple and rapid capillary zone electrophoresis (CZE) method was developed and validated for simultaneous determination of two iridoid glycosides (8-O-acetylshanzhiside methylester and 8-deoxyshanzhiside) and three flavanoids (apigenin, quercetin and luteolin) in L. rotata. Operational variables, such as the voltage, buffer concentration and pH were optimized, the final optimum separation condition was 10 mM sodium tetraborate-20 mM NaH(2) PO(4) (pH 8.5)-15% (v/v) methanol, 238 nm UV detection, 18 kV applied voltage. The linearity and the recovery of the proposed method were very satisfactory (correlation coefficients were 0.9994-0.9998 and the recoveries were 94.5-108.8% for the analytes) and the method allowed analytes in real samples to be determined within 9 min. The proposed CZE method can be used for quality control of iridoid glycosides and flavanoids in L. rotata and its herbal preparation.

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.

A rapid quantitative determination of phenolic acids in Brassica oleracea by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis method to quantitatively determine the phenolic acid contents in brassica vegetables is described. Phenolic compounds were extracted from broccoli, broccolini, Brussels sprouts, cabbage and cauliflower and the main hydroxycinnamic acids (sinapic, ferulic, p-coumaric and caffeic acids) were isolated by solid phase extraction with C18 cartridges. Using an optimised method, the four analytes were separated in less than 7min in a 50μm×60cm capillary with a 15mM borate buffer (pH=9.13) and a separation voltage of 30kV at 30°C. A linear relationship was observed for the method (r=0.9997–0.9999) with detection limits ranging from 1.1 to 2.3mg/kg of vegetables for the analytes. This method demonstrated good reproducibility with coefficients of variation of less than 5% for peak area and less than 1% for migration time (n=7). The method was successfully applied to quantitatively determine the phenolic acid contents in a range of brassica vegetables and the results were in good agreement when compared to those from high performance liquid chromatography analysis.