Capillary Zone Electrophoresis Conditions Research Articles

Stability-Indicating Quantification of Ciprofloxacin in the Presence of Its Main Photo-Degradation Product by CZE and UPLC: A Comparative Study

Active pharmaceutical ingredients (APIs) can be assured to be safe and effective with the help of stability-indicating procedures. An accurate comparison comprising the utilization of capillary zone electrophoresis (CZE) and ultra-performance liquid chromatography (UPLC) for the sensitive and accurate measurement of ciprofloxacin (CPF) in the presence of its major photo-degradation product was conducted. The CZE and UPLC working conditions were optimized to obtain the best pattern of separation for CPF and its photo-degradant. The linearity range of the cited techniques was confirmed to be 0.5 to 50 µg/mL. A thorough validation scheme according to the ICH-Q2B criteria was performed, including linearity, accuracy, precision, robustness, detection, and quantification limits. Selective quantification of CPF in the presence of up to 90% of its main photo-degradant was carried out using the proposed methods. For the analysis of CPF in tablet and intravenous (I.V.) solution forms, the CZE and UPLC procedures were applied. The suggested methods can be applied to keep an eye on the safety and efficacy of CPF in either bulk or dosage forms.

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 the degree of deacetylation of chitosan by capillary zone electrophoresis

In this study we have developed a capillary zone electrophoresis (CZE) method for the determination of the degree of deacetylation (DDA) of chitosan. The electrophoretic mobilities of chitosans were measured by using the optimized CZE conditions. An internal standard, hexammine cobalt(III) chloride, was used to improve the precision of the electrophoretic mobility measurement. We have constructed a linear calibration curve between the CZE measured mobilities and the 1H NMR determined DDAs ranging from 55.3% to 96.2%. Based on the established linear calibration equation and the CZE measured mobilities, we were able to obtain not only the average DDA values but also the DDA distribution profiles of the chitosan samples. Without prior sample treatment or purification, we have successfully employed the newly developed CZE method to characterize the reacetylation derivatives of chitosans and to detect the DDA variability in different lots of chitosan products.

Analysis of tartrazine aluminum lake and sunset yellow aluminum lake in foods by capillary zone electrophoresis

A novel analytical method for tartrazine aluminum lake and sunset yellow aluminum lake using capillary zone electrophoresis (CZE) was studied. The pigments contained in the color lakes were successfully separated from the aluminum matrix in the pre-treatment process, which included the following steps: dissolve the color lakes in 0.1 mol/L H2SO4, adjust the pH of the solution to 5.0, then mix it with the solution of EDTA x 2Na and heat it in a water bath, then use polyamide powder as the stationary phase of solid phase extraction to separate the pigments from the solution, and finally elute the pigments with 0.1 mol/L NaOH. The CZE conditions systematically optimized for tartrazine aluminum lake were: 48.50 cm of a fused silica capillary with 40.00 cm effective length and 50 microm i. d., the temperature controlled at 20.0 degrees C, 29.0 kV applied, HPO4(2-)-PO4(3-) (0.015 mol/L, pH 11.45) solution as running buffer, detection at 263 nm. The conditions for sunset yellow aluminum lake were: the same capillary and temperature, 25.0 kV applied, HPO4(2-)-PO4(3-) (0.025 mol/L, pH 11.45) solution as running buffer, detection at 240 nm. The limits of detection were 0.26 mg/L and 0.27 mg/L, and the linear ranges were 0.53-1.3 x 10(2) mg/L and 0.54-1.4 x 10(2) mg/L for tartrazine aluminum lake and sunset yellow aluminum lake, respectively. The RSDs were 4.3% and 5.7% (run to run, n = 6), 5.6% and 6.0% (day to day, n = 6) for tartrazine aluminum lake and sunset yellow aluminum lake, respectively. Further developments for this method could make it a routinely used method analyzing color lakes in foods.

Detection of C-reactive protein based on magnetic nanoparticles and capillary zone electrophoresis with laser-induced fluorescence detection

A simple and fast method based on magnetic nanoparticles (MNPs) and capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection was developed for the detection of C-reactive protein (CRP). To optimize the CZE conditions, several factors including buffer compositions, buffer ionic strength, buffer pH, applied voltage and capillary temperature have been examined. The optimal separation buffer selected was a 30mM sodium phosphate (PB) buffer, pH 8.0. The optimal CE applied voltage and temperature selected were 20kV and 35°C, respectively. The CZE profile of the MNP-1°Ab-CRP-2°Ab/FITC bioconjugates showed good reproducibility. One major peak was observed for the MNP bioconjugates. The quantitative analysis also showed good results. The coefficient of variation (CV%) for the major peak area was 8.7%, and the CV% for the major peak migration time was 2.5%. The linear range for CRP analysis was 10–150μg/mL, and the concentration limit of detection (LOD) was 9.2μg/mL. Non-specific interactions between bovine serum albumin (BSA) and the system can be prevented by including 10% (v/v) of human plasma in the binding buffers. The CE/LIF method might be helpful for analyzing high concentrations of CRP in a patient's plasma after an acute-phase inflammation. This new method demonstrated the possibility of using MNPs and CE/LIF for the detection of proteins, and provided information for the establishment of appropriate CE conditions.

Capillary Electrophoretic Fingerprint Investigation of Gel Pen Inks

In this study, two capillary electrophoresis modes, capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were employed for the examination of gel ink fingerprints. The optimized CZE conditions were as follows: running buffer, 0.2M boric acid and 0.05M sodium tetraborate; pH, 6.47; detection, 214 nm; separation voltage, 20 kV MEKC conditions were as follows: 20 mM sodium tetraborate-20 mM sodium dodecyl sulphate; detection, 214 nm; separation voltage, 20 kV. The CZE and MEKC fingerprints of 18 el pen inks from Beijing markets were analyzed and the inks were classified according to the fingerprints of the two CE modes.

A study on the alkaline hydrolysis of isatin-β-thiosemicarbazone by capillary electrophoresis with enhanced sample loadability

An analytical potential of capillary zone electrophoresis (CZE) with enhanced sample loadability (a 200nL injection volume) in determination of alkaline hydrolysis products of isatin-β-thiosemicarbazone (IBT), a compound with important biological activity, has been studied. The CZE separation conditions for a complete resolution of transformation products, i.e. 2-aminophenylglyoxalate, 2-(2-aminophenyl)-2-semicarbazonoethane, anthranilate and E-Z geometric isomers of 2-(2-aminophenyl)-2-thiosemicarbazonoethane, have been optimized. CZE separations with UV detection at 240nm were performed using glycine running buffer at high pH (9.2) and containing an uncharged β-cyclodextrin as a complexing agent. High sensitivity (with detection limits ranging from 0.1 to 1.2μM), good repeatability (RSD of migration times less than 0.4% and 0.4–3.4% RSD of peak areas) and linearity over two orders of magnitude were achieved for the compounds studied. The employed CZE method, characterized by simple sample handling (only dilution step needed) and total analysis time of less than 15min, has been applied successfully to time monitoring of the transformation of IBT in alkaline media. Under optimized CZE conditions, the effect of pH of reaction media, implemented by different concentration of NaOH (0.1–100mM), on the course of the alkaline hydrolysis of IBT was studied in this respect, as well.

Determination of puerarin in pharmaceutical and biological samples by capillary zone electrophoresis with UV detection

A rapid and efficient capillary zone electrophoresis (CZE) method with ultraviolet detection has been successfully developed for the determination of puerarin in Xinkeshu capsules and biological samples. The optimal CZE conditions were determined to be 70mM H3BO3–Na2B4O7, pH 9.2, injection time 5s, applied voltage 25kV, working temperature 25°C and detection wavelength 254nm. Under these conditions, a linear range from 17.3 to 138μg/mL with the correlation coefficient of 0.9998 and limit of detection of 34.6ng/mL (S/N=3) for puerarin were obtained. The relative standard deviation of the migration time and peak area of puerarin were 0.23–0.91, 0.43–1.62, 0.10–0.64 and 0.64–1.32% for intra-day and inter-day analyses, respectively. Satisfactory results were obtained for recovery and repeatability. The proposed method was successfully applied to analyze the puerarin content in Xinkeshu capsules and some biological samples with good results. It is suitable for applications in pharmaceutical industries for quality control and in clinical laboratories for therapeutic drug monitoring purposes.

Determination of bisphenol A and naphthols in river water samples by capillary zone electrophoresis after cloud point extraction

As a first attempt, cloud point extraction (CPE) was developed to preconcentrate bisphenol A (BPA), α-naphthol and β-naphthol prior to performing capillary zone electrophoresis (CZE) analysis. The parameters influencing the CPE efficiency, such as Triton X-114 concentrations, pH value, extraction time and temperature were systematically evaluated.After diluting with acetonitrile, the surfactant-rich phase of CPE can be injected directly into the CE instrument. The CZE baseline separation was achieved with running buffer (pH 9.5) composed of 50mM sodium tetraborate in 30% (v/v) methanol, and an applied voltage of 25kV. Under the optimized CPE and CZE conditions, an preconcentration factor of 50 times could be obtained and the limit of quantification for the three analytes were found to be 1.67μgL−1, 0.80μgL−1 and 0.67μgL−1 for BPA, α-naphthol and β-naphthol, respectively. The proposed methods have shown to be a green, rapid and effective approach for determination of three analytes present in river water samples.

Improved capillary electrophoresis determination of carbohydrate-deficient transferrin including on-line immunosubtraction

The instrumental analysis of carbohydrate-deficient transferrin (CDT), a recognized marker of chronic alcohol abuse, is most commonly carried out by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Between these two techniques, CZE shows higher efficiency and productivity, but is often reported to be inferior to HPLC in terms of selectivity, because of a less specific ultraviolet detection wavelength than HPLC. On these grounds, the present work was aimed at the development of an improved CZE method for CDT determination, including an on-line immunosubtraction step specifically aimed at enhancing the analytical specificity of CZE determination. The analytical conditions were as follows: uncoated fused silica capillary, 30 microm x 60 cm (L = 50 cm to detector); running buffer, 100 mmol/L borate and 6 mmol/L DAB (1,4-diaminobutane), pH 8.3; voltage, 30 kV; temperature, 25 degrees C; detection, 200 nm. Under the described CZE conditions, a baseline separation between all the CDT related peaks was achieved with good analytical performances in terms of both precision and accuracy. In order to achieve unequivocal recognition of the CDT peaks, an in-capillary immunosubtraction step was included by loading a plug of anti-human transferrin antibody solution after the sample plug. This analytical approach was applied successfully to recognize CDT peaks in the presence of potential interferences.

Capillary Zone Electrophoresis method for determination of (+)-S clopidogrel carboxylic acid metabolite in human plasma and urine designed for biopharmaceutic studies

Fast and reproducible Capillary Zone Electrophoresis (CZE) method for the quantification of (+)-S clopidogrel carboxylic acid metabolite in human fluids was elaborated for the first time. Optimal buffer and CZE conditions were established to obtain the complete separation of clopidogrel, its metabolite and piroxicam (internal standard), during one analytical run. Finally, resolution of the analytes was obtained in an uncoated silica capillary filled with a phosphate buffer of pH 2.5. The analytes were isolated from plasma and urine samples using solid phase extraction (SPE). Validation of the CZE method was carried out. The calibration curve of clopidogrel was linear in the range of 0.5–10.0 mg/L in plasma and urine, whereas for (+)-S carboxylic acid metabolite linearity was confirmed in the range of 0.25–20.0 mg/L in plasma and 0.25–10.0 mg/L in urine. Intra- and inter-day precision and accuracy were repeatable. LOD and LOQ were also estimated. SPE recovery of the analytes from plasma and urine was comparable and greater than 80%. The validated method was successfully applied in pharmacokinetic investigations of (+)-S carboxylic acid metabolite of clopidogrel following the oral administration of clopidogrel to patients prior to percutaneous coronary intervention.

CZE Determination of Calixarenes and Related Derivatives Using Acetonitrile as a Modifier

The applications of third-generation host compound calixarenes have been restricted due to their high price and shortage of supply. To obtain high pure calixarenes with high yield, synthesis strategy and analytical method investigations are necessary. The present work describes capillary zone electrophoresis (CZE) for the separation and determination of calixarene derivatives under the optimized CZE conditions within 20 min. The CZE method was evaluated with linear ranges, detection limits, recoveries and repeatability. The results indicated that the method was accurate and reproducible. As expected, it could be utilized to separate and evaluate calixarene derivatives during their synthesis and purification.

Enantiomeric separation of metolachlor and its metabolites using LC–MS and CZE

The stereoisomers of metolachlor and its two polar metabolites [ethane sulfonic acid (ESA) and oxanilic acid (OXA)] were separated using liquid chromatography–mass spectrometry (LC–MS) and capillary zone electrophoresis (CZE), respectively. The separation of metolachlor enantiomers was achieved using a LC–MS equipped with a chiral stationary phase based on cellulose tris(3,5-dimethylphenyl carbamate) and an atmospheric pressure chemical ionization source operated under positive ion mode. The enantiomers of ESA and OXA were separated using CZE with gamma-cyclodextrin (γ-CD) as chiral selector. Various CZE conditions were investigated to achieve the best resolution of the ESA and OXA enantiomers. The optimum background CZE electrolyte was found to consist of borate buffer (pH = 9) containing 20% methanol (v/v) and 2.5% γ-CD (w/v). Maximum resolution of ESA and OXA enantiomers was achieved using a capillary temperature of 15 °C and applied voltage of 30 kV. The applicability of the LC–MS and CZE methods was demonstrated successfully on the enantiomeric analysis of metolachlor and its metabolites in samples from a soil and water degradation study that was set up to probe the stereoselectivity of metolachlor biodegradation. These techniques allow the enantiomeric ratios of the target analytes to be followed over time during the degradation process and thus will prove useful in determining the role of chirality in pesticide degradation and metabolite formation.

Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis.

A systematic investigation has been made into the mechanisms of the capillary zone electrophoresis (CZE) separation of 12 common nucleotides (mono-, di- and triphosphorylated) and polydeoxythymidylic acid oligonucleotides (pd(T)5-18) using electrophoretic mobility values calculated from migration time data. Relationships between electrophoretic mobility and the physicochemical characteristics of the analytes (charge, dissociation constants, charge-to-mass ratio) and the background electrolyte conditions (buffer strength, percentage organic modifier and buffer pH) were characterised. Nucleotide migration was dominated by the negatively charged phosphate groups. Additionally, there were important contributions to migration behaviour from the ionised amide groups of the nucleobases guanine and uracil at higher buffer pH values or with the presence of methanol in the electrolyte. Calculated electrophoretic mobility values for the nucleotides showed a substantially improved (5-fold) inter-run repeatability compared with migration time data. These studies show the value of representing nucleotide migration data as electrophoretic mobility in CZE for obtaining a more thorough analysis of separation mechanisms and to compensate for variation in migration time data caused by small changes in electrosmotic flow. Oligonucleotides pd(T)5-11 could be adequately resolved from their nearest neighbour, but the limit of single-base separation was pd(T)10 from pd(T)11 under the conditions used. It was calculated that a difference in charge-to-mass ratio of 2.64 x 10(-5) was required for resolution under the CZE conditions used.

OPERATING PARAMETER EFFECTS IN CAPILLARY ZONE ELECTROPHORESIS (CZE): ANALYSIS OF PENTAMIDINE, HYDROXYSTILBAMIDINE, PROPAMIDINE, DAPI, AND STILBAMIDINE IN BODY FLUIDS

A study was undertaken of the effects of varying those parameters that most affect analyses by capillary zone electrophoresis (CZE), electrolyte buffer, concentration of electrolyte, pH, voltage, temperature, and diameter of capillary, in respect to the following aromatic diamidines of therapeutic value: pentamidine, hydroxystilbamidine, propamidine, 6-amidino-2-(4-amidinophenyl) indole dilactate (DAPI), and stilbamidine. The data obtained permit the design of specific analytic methodology for each of these chemicals under differing conditions. As a demonstration, an analytical method is proposed in which all these chemicals are analyzed, simultaneously, in serum and urine under the following CZE conditions: citrate buffer 25 mM, pH = 3.75, T = 30°C, and V = 14 kV. The detection limits for the method for serum and urine are (μg/mL): pentamidine, 0.3; hydroxystilbamidine, 0.4; propamidine, 0.2; DAPI, 0.3; and stilbamidine, 0.3. Oasis HLB cartridges were used for prior treatment of both serum and urine samples.

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis.

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.

Capillary zone electrophoresis analysis of gliadin proteins from Chinese and Yugoslav winter wheat cultivars

Gliadin proteins extracted from fifteen Chinese and Yugoslav winter wheat cultivars were fractionated using a new separation technique – Capillary Zone Electrophoresis (CZE). Different CZE conditions were defined to optimize resolution and reproducibility of gliadin separations. Excellent resolution and high reproducibility of gliadin CZE patterns were obtained by using 47 cm length, 50 μm i.d. capillaries at 15 kV and 30° C in sodium borate buffer system with acetonitrile (ACN) and sodium dodecyl sulfate. By using these CZE conditions, gliadin proteins from each cultivar were easily separated into more than 35 components. This resolution is generally superior to that of one- and two-dimensional electrophoresis and RH-HPLC. Analysis of reproducibility of gliadin CZE patterns from Chinese cultivar ‘Lumai 6’ showed that the average relative standard deviation (RSD) for peak migration times and heights was 0.21% and 4.06%, respectively. Gliadin electrophoregrams of all cultivars studied showed clear qualitative and quantitative differences, including presence or absence of some major peak, migration times and heights of peaks. Specifically, some closely related cultivars that were not differentiable by A-PAGE, were readily differentiated by CZE. In addition, winter wheat cultivars from China and Yugoslavia showed greater differences in gliadin compositions revealed by CZE.

Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides.

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

The Statistical Evaluation of Migration Parameters of Flavonoids in Capillary Electrophoresis with Reference to Structural Descriptors

Under each of 38 buffer conditions, six migration parameters of 14 flavonoids were obtained in capillary zone electrophoresis (CZE) and nine parameters in micellar electrokinetic capillary chromatography (MEKC). A total of 183 structural descriptors for each flavonoid were calculated to represent the molecular structures in a quantitative way. The relationship between the structural descriptors and the migration parameters of flavonoids in CZE or MEKC were evaluated by cluster and factor analyses. Under all CZE conditions the apparent solute mobility (μa) was always the first variable to associate with all molecule-level structural descriptors and the electrotopological state indices (Si) of skeletal carbons, whereas the effective mobility (μe) was closer to Si of oxygens on skeletons. On the other hand, in MEKC retention factor or its logarithm did not depended more on the structural descriptors than other migration parameters. This indicated that the parameter did not represent the properties of the flavonoids better than other parameters. Thus, the results confirmed that hydrophobic interaction was not the only underlying force that influenced the migration behaviour of the flavonoids in MEKC.

Capillary electrophoretic analyses of β-trace protein and other low molecular weight proteins in cerebrospinal fluid from patients with central nervous system diseases

Ordinary capillary-zone electrophoresis (CZE), as well as CZE in a sodium dodecylsulfate-containing polymer solution (SDS-CZE) and capillary isoelectrofocusing (CIEF), was applied to the analysis of low molecular weight proteins in cerebrospinal fluid (CSF) from patients with various neuropsychiatric disorders. Under the CZE conditions employed, a peaks of β-trace protein (βTP), which is the most abundant low MW protein in CSF, was clearly detected on the electropherograms of all the samples examined, and the CSF βTP level could be tentatively determined using allylamine added at a constant concentration as the internal standard. The results revealed that βTP in CSF was non-specifically increased in organic diseases in the central nervous system (CNS), especially in ones giving severe physical damage to the brain tissues. On the other hand. SDS-CZE allowed us to determine simultaneously the CSF minor low MW proteins other than βTP, such as β 2-microglobulin, ψ-trace protein, myelin basic protein, etc., while the CIEF electropherograms suggested that βTP were separated into several fractions with the different PI values. These capillary electrophoresis systems seem to be powerful as aids in the biochemical examinations of βTP and other low molecular weight proteins in CSF from patients with CNS diseases.