Simple Capillary Zone Electrophoresis Research Articles

Simple, fast and reliable CE method for simultaneous determination of ciprofloxacin and ofloxacin in human urine

A simple, fast, and accurate capillary zone electrophoresis method has been developed for the determination of ciprofloxacin and ofloxacin. This method uses liquid–liquid extraction. Therefore, it is characterized by a very simple procedure of sample preparation but at the same time satisfactory precision and accuracy. The extraction process of the same urine sample was repeated three times. The extraction protocol was performed each time for 15 min with 1 mL of dichloromethane and chloroform mixture in a 3:1 volume ratio. A 0.1 mol/L phosphate-borate buffer (pH 8.40) was selected as the background electrolyte. UV detection was performed at 288 nm. The separation was carried out at a voltage of 16 kV, at a temperature of 25 °C. Experimentally evaluated LOQ values for ciprofloxacin and ofloxacin were 0.2 nmol/mL urine and 0.05 nmol/mL urine, respectively. For both analytes the calibration curves exhibited linearity over the entire tested concentration range of 1–6 nmol/mL urine. The precision of the method did not exceed 15%, and the recovery was in the range of 85–115%. The developed and validated procedure was applied to analyze human urine for the content of ciprofloxacin and ofloxacin.

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.

Green, Cost-Effective Simultaneous Assay of Chloramphenicol, Methylparaben, and Propylparaben in Eye-Drops by Capillary Zone Electrophoresis.

A green, cost-effective, and simple capillary zone electrophoresis (CZE) method was developed and validated for simultaneous determination of chloramphenicol, methylparaben, and propylparaben in eye-drops. With sodium tetraborate as background electrolyte (BGE), the apparent mobilities of chloramphenicol, methylparaben, and propylparaben increased and analysis time reduced when pH of BGE increased from 8.5 to 10.0 and concentration of BGE decreased from 40 mM to 15 mM, but complete separation of chloramphenicol from other matrix components was achieved only with sodium tetraborate concentration at 30 mM or higher and at pH = 9.3 or lower. The most suitable electrophoretic conditions for the intended application were a 30 mM sodium tetraborate solution, pH 9.3 as BGE, working voltage set at 25 kV, and UV detection at 280 nm at the cathodic extremity of the capillary. The final method was validated and proved to be reliable for assay of chloramphenicol, methylparaben, and propylparaben in eye-drops.

Field amplified sample stacking and in-capillary derivatization for forensic analysis of morphine and morphine-6-glucuronide in human urine by capillary electrophoresis

A rapid, sensitive, selective and simple capillary zone electrophoresis (CZE) method, by integration of field amplified sample stacking (FASS) and in-capillary derivatization (in-CapD) coupled with fluorescence (FL) detection (CZE-FASS-in-CapD-FL), has been developed and validated for simultaneous determination of morphine and its metabolite, morphine-6-glucuronide (morphine-6-G)in human urine. The method was based on in-line derivatization by introducing the sample to running background electrolytes (BGE) of disodium tetraborate decahydrate and potassium ferricyanide, whereas analytes were oxidized into highly fluorescent dimer products. Various parameters influencing the sensitivity and efficiency of the proposed method were investigated, and the optimal conditions of the BGE were as follow: 60 mM disodium tetraborate decahydrate (pH 10.5), 0.25 mM potassium ferricyanide and separation voltage of 9 kV. For FASS, morphine and morphine-6-G were electrokinetically injected for 14 s at 16 kV into the capillary that was pre-injected with water plug for 4 s. The fluorescence signals were then detected at excitation and emission wavelengths of 340 and 450 nm, respectively. Analysis was performed at ambient temperature (22±1 °C). Validation of the method showed good linearity over morphine and morphine-6-G concentrations ranging from 2 to 2000 ng/mL and 2.5 to 2000 ng/mL, respectively. The mean recoveries were ranged from 97.72 to 87.26% and 97.14 to 85.43% for morphine and morphine-6-G, respectively. The validated CZE-FASS-in-CapD-FL method was effectively applied as quantitative forensic analysis of morphine and morphine-6-G in clinical urine samples.

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.

Determination of homocysteine thiolactone in human urine by capillary zone electrophoresis and single drop microextraction

A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 μA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50–200 nM, with R2 0.9995. The intra- and inter-day precision and recovery were 4.0–14.5% (average 8.7% and 9.3%) and 92.7–115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples.

Validated capillary zone electrophoretic method for simultaneous analysis of benazepril in combination with amlodipine besylate and hydrochlorothiazide

In this work, a novel, simple, and quick capillary zone electrophoresis (CZE) method was proposed for simultaneous analysis of benazepril (BEN) with other co-administrated antihypertensive drugs, amlodipine besylate (AML) and hydrochlorothiazide (HCT), using a diode array detector (DAD). A fused silica capillary (78.5 cm total length, 70 cm effective length, and 75 μm id) was used in separation using a 40 mM phosphate buffer pH 7.5 as a running background electrolyte (BGE) under a positive potential of 30 KV, at a stable temperature of 25 °C for capillary during separation. Hydrodynamic injections were performed for 12 s at 50 mbar, and detection was performed at 210 nm for AML and BEN, at 225 nm for HCT, and at 232 nm for xipamide (XIP) added as an internal standard (IS). Separation of the three analyzed drugs and the IS was performed in less than 8 min. Migration times were 4.06, 5.23, 6.69, and 7.3 min for AML, HCT, BEN, and XIP, respectively. The findings proved that the proposed method was linear in the range of 10–80 μg/mL for all drugs with correlation coefficients &gt;0.9994. The limit of detection (LOD) values of AML, HCT, and BEN were 1.004, 1.224, and 0.896 μg/mL, respectively, whereas the limit of quantification (LOQ) values were 3.124, 3.727, and 2.749 μg/mL for the cited drugs, respectively. Peak identity and purity were confirmed by DAD. The developed CZE method was applied for the analysis of the three antihypertensive drugs successfully in their combined pharmaceutical tablets, and it can be used for the quality control of single-pill combination (SPC) samples of these drugs in short time.

Identification and characterization of infectious bursal disease virus subviral particles by capillary zone electrophoresis: potential application for vaccine production and quality control

The infectious bursal disease (IBD) causes immunosuppression in chicken of all ages and high mortality in young chicken, posing serious threat to poultry industry worldwide. One promising strategy for preventing this highly contagious disease is using recombinant subunit vaccine, employing VP2 subviral particles (SVP) as epitomic antigen. Analytical techniques of viral-like particles such as SDS-PAGE, western blot, or high-performance size-exclusion chromatography have been widely applied, but mostly unsatisfactory. In the present study, a simple, fast and cost-effective capillary zone electrophoresis (CZE) method with UV-detection was developed to analyze purified IBDV-SVP (expressed by Escherichia coli system) using commercial monoclonal antibody (mAb) against VP2. To find satisfying CZE conditions, injection mode, separation voltage, and separation buffer were explored. Through the modified CZE, mAb and SVP could be well separated and shown distinct peaks in the electropherogram. Furthermore, to determine the stoichiometry, the area of the mAb peak versus SVP/mAb binding ratio was plotted and indicated that 2 or 3 receptor molecules were bound per SVP. The purity and integrity of SVP and the interactions between SVP and mAb could be analyzed by the developed simple CZE-UV method in less than half hour. This CZE-UV method proved to be a valuable and useful tool in detection, characterization, and quantification of IBDV-SVP and the mAb, offering potential applications of in-process quality control of vaccine production, surveillance of serum antibody produced against IBDV infection, or vaccine immunization.

Stability Indicating Assay of Florfenicol using Capillary Zone Electrophoresis in Veterinary Formulation and in Milk

A simple and fast capillary zone electrophoresis method with direct ultraviolet detection has been developed for the quantitative analysis of veterinary drug florfenicol. Migration time of 1.22 minute was achieved using 1 mM borate buffer of pH 9.3 as background electrolyte. Limit of detection and limit of quantification for this method are 3.34 μg and 19.5 μg, respectively. Stability indicating assay of florfenicol is studied by exposing the drug to various stress conditions such as acidic, basic, oxidative, thermal, and ultraviolet for inducing degradation. This method was also employed for the analysis of florfenicol in its formulation and its residue in commercially available milk.

Sensitive determination of aldehyde metabolites in exhaled breath condensate using capillary electrophoresis with laser-induced fluorescence detection.

A novel capillary electrophoresis with laser-induced fluorescence detection method has been developed for the analysis of aldehyde metabolism biomarkers for oxidative stress in exhaled breath condensate (EBC), and fluorescein 5-thiosemicarbazide was used as a derivatization reagent. In a simple capillary zone electrophoresis mode, ten low molecular weight aldehydes (LMWAs) could be well separated within 30min. The reaction efficiency was doubled by increasing sample solution pH and magnetic stirring, and the LODs of this method reached 0.16-3.4nM (S/N = 3). Acceptable recoveries (82.1-115%) were obtained for EBC samples, and the RSD data were within 7.9%. This developed method has been applied for the analyses of EBC samples and evaluation of the correlation between smoking and the contents of aldehyde metabolites in EBC. Due to no need of buffer additives and sample preconcentration, this proposed method may provide an appealing alternative for the trace analyses of LMWAs in noninvasive biofluids. Graphical abstract ᅟ.

A simple, low-cost and robust capillary zone electrophoresismethod with capacitively coupled contactless conductivity detection for the routine determination of four selected penicillins in money-constrained laboratories.

A simple and robust capillary zone electrophoresismethod was developed and validated for the determination of amoxicillin and clavulanate, ampicillin, phenoxymethyl penicillin (Pen V) as well as flucloxacillin. Capacitively coupled contactless conductivity detection was employed as detectionmode thatmakes CE a simple and economic tool formoney-constrained laboratories. The developedmethod is straightforward and user-friendly. It offers good sensitivity and sufficient selectivity for the routine assay of the selected penicillins. The repeatabilities were <1.9% RSD for relative peak areas and <1% RSD formigration times for all the analytes. Themethod showed good linearity (R2 >0.995) within the 80-120% range of the target concentration (0.5mg/mL) for each antibiotic. The accuracy of themethod, evaluated by standard fortification at three levels, was good for all the analytes. An extended robustness study was performed by varying ±10% of the optimum value of TRIS concentration, l-histidine concentration and temperature in a full factorial design at two levels. This was to evaluate larger than usual variability of factors on the assay value, in order to better cover potential global variance in lab conditions and equipment. Finally, themethod was applied for the determination of percent (%) content of all antibiotics in available formulations.

Enantioselective determination of aspartate and glutamate in biological samples by ultrasonic-assisted derivatization coupled with capillary electrophoresis and linked to Alzheimer’s disease progression

A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0–20.0 μg mL−1l-Glu and 0–2.0 μg mL−1d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85–0.96 μg mL−1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer’s disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = −0.158) between plasma l-Asp concentration and AD severity.

Differentiation of modern and ancient varieties of common wheat by quantitative capillary electrophoretic profile of phenolic acids

Phenolic compounds have received great attention among the health promoting phytochemicals in common wheat (Triticum aestivum L.), mainly because of their strong antioxidant properties. In the present study a simple Capillary Zone Electrophoresis (CZE) method with UV detection was optimized and validated for the quantitation of six of the most important phenolic acids in whole grain i.e., sinapic, ferulic, syringic, p-coumaric, vanillic and p-hydroxybenzoic acid. The separation was achieved in a running buffer composed of sodium phosphate solution (50 mM) in water/methanol 80:20 (v/v) at pH 6.0 and using a fused-silica capillary at the temperature of 30 °C under application of 27 kV. By means of diode array detector, and made possible by the favorable characteristic UV spectra, the quantitation of the solutes was carried out at 200, 220 and 300 nm, in the complex matrices represented by the soluble and bound fractions of wheat flours. The validation parameters of the method i.e., linearity, sensitivity, precision, accuracy and robustness were in line with those obtained by consolidated separation techniques applied for the same purposes (e.g., HPLC-UV), with a significant advantage in term of analysis time (less than 12 min). Ten varieties of soft wheat (five modern Italian and five old Italian genotypes) were analysed and the data were subjected to Principal Components Analysis (PCA). Interestingly, significant differences of the quantitative phenolic acids profile were observed between the modern and the ancient genotypes, with the latter showing higher amount of the main represented phenolic acids.

The use of experimental design for the development of a capillary zone electrophoresis method for the quantitation of captopril.

A capillary zone electrophoresis (CZE) method for the quantitation of captopril (CPT) using UV detection was developed. Influence of electrolyte concentration and system variables on electrophoretic separation was evaluated and a central composite design (CCD) was used to optimize the method. Variables investigated were pH, molarity, applied voltage and capillary length. The influence of sodium metabisulphite on the stability of test solutions was also investigated. The use of sodium metabisulphite prevented degradation of CPT over 24 hours. A fused uncoated silica capillary of 67.5cm total and 57.5 cm effective length was used for analysis. The applied voltage and capillary length affected the migration time of CPT significantly. A 20 mM phosphate buffer adjusted to pH 7.0 was used as running buffer and an applied voltage of 23.90 kV was suitable to effect a separation. The optimized electrophoretic conditions produced sharp, well-resolved peaks for CPT and sodium metabisulphite. Linear regression analysis of the response for CPT standards revealed the method was linear (R2 = 0.9995) over the range 5-70 μg/mL. The limits of quantitation and detection were 5 and 1.5 μg/mL. A simple, rapid and reliable CZE method has been developed and successfully applied to the analysis of commercially available CPT products.

Enantiomeric separation of five acidic drugs via capillary electrophoresis using streptomycin as chiral selector.

A simple capillary zone electrophoresis (CZE) method was developed to achieve the rapid enantiomeric separation of a set of acidic drugs using streptomycin as chiral selector. The enantiomers of 5 chiral phenyl-containing acidic pharmaceutical compounds were separated excellently by CE using an uncoated silica capillary. Several experimental parameters such as the concentration of streptomycin, buffer concentration and pH, running voltage, and capillary temperature were all investigated systematically in order to optimize the chiral separation. All analytes were got baseline separation within 10min. And the results showed that streptomycin can be used as a chiral selector to the enantioseparation of five acidic drugs by CZE method.

Capillary zone electrophorsis for the analysis of naturally occurring 2-hydroxycitric acids and their lactones.

A simple capillary zone electrophoresis method with direct ultraviolet detection has been developed for the analysis of naturally occurring diastereomeric 2-hydroxycitric acid lactones. Using 50mM sodium phosphate buffer of pH 7, a baseline resolution Rs > 3.0 was observed for all organic acids selected for the present study. This method was employed for the quantitative determination of title acids present in the plant sources namely Garcinia cambogia fruit rinds and Hibiscus sabdariffa calyx. Conversion of 2-hydroxycitric acids to their lactones on heating the above plant sources is deliberated. The Hydrolysis of hydroxycitric acid lactones in aqueous solution is reported for the first time.

Analysis of Aliphatic Organic Acids in Commercial Fruit Juices by Capillary Electrophoresis with Indirect UV Detection: Application to Differentiation of Fruit Juices

A simple, cheap, and reliable capillary zone electrophoresis (CZE) method with indirect UV detection to determine the main organic acids in several fruit juices has been developed in this work. The parameters affecting CZE separation including the chromophore reagent (2,6-pyridinedicarboxylic acid, PDC) concentration and pH in background electrolyte (BGE), temperature, and applied voltage were studied. The analytical parameters of the method as linearity, precision, and detection and quantification limits were also investigated. The proposed method was applied to the evaluation of organic acid contents of commercial fruit juices from apple, grape, mandarin, orange, and pineapple and compared with the levels allowed by European legislation. A chemometric technique such as linear discriminant analysis (LDA) was also applied to differentiate fruit juices based on CZE data. This simple and reliable developed procedure allows a rapid control of adulteration of high-value commercial fruit juices, which constitutes an important tool for authenticity texting in food industries and regulatory agencies.

Simultaneous separation and determination of four uncaria alkaloids by capillary electrophoresis using dual cyclodextrin system

The purpose of this study was to develop a simple, quick and precise capillary zone electrophoresis method (CZE) for the separation and determination of uncaria alkaloids using dual cyclodextrins as additives for the separation. The four analytes were baseline separated within 15min at the applied voltage of 15kV with a running buffer (pH 5.7) consisting of 40.0mM phosphate buffer, 161.7mM 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and 2.21mM mono-(6-ethylenediamine-6-deoxy)-β-cyclodextrin (ED-β-CD). Under the optimum conditions, a good linearity was achieved with correlation coefficients from 0.9989 to 0.9992. The detection limits and the quantitation limits ranged from 0.63 to 0.98μg/mL and from 2.08 to 3.28μg/mL, respectively. Excellent accuracy and precision were obtained. Recoveries of the analytes varied from 97.1 to 103.2%. This method was suitable for the quantitative determination of these alkaloids in the stem with hook of Uncaria rhynchophylla and the formulations of Uncaria rhynchophylla.

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.