Quantification of buserelin in a pharmaceutical product by multiple-injection CZE

An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 microg/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 +/- 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).     

Fiber optic Z-cell for CZE

Fiber optic Z-cell for CZE was designed, constructed, tested and compared with on-column detection. Ten times higher sensitivity for Z-cell in comparison with on-column detection was achieved as expected from optical pathlength ratio. Linear dynamic range was > 4 orders of magnitude for both cells.     

CZE of human alpha-1-acid glycoprotein for qualitative and quantitative comparison of samples from different pathological conditions

Alpha-1-acid glycoprotein (AGP) presents different forms, which may arise from differences in the amino acid sequence and/or in the glycosidic part of the protein. Changes in forms of AGP have been described in literature as a possible tumor marker. While most previous works have approached the study of glycopeptides and/or glycans obtained after fragmentation of the protein, in this work, a CZE method is developed to separate up to eleven peaks of intact forms of AGP. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Electropherograms of AGP samples purified from sera of cancer patients and healthy donors are qualitatively and quantitatively compared. Percentages of correct assignment of AGP peaks close to 100% are achieved by using either the migration time of each peak relative to that of the EOF marker or the effective electrophoretic mobility of the peaks. The computer program permits to select, among different hypotheses for peak allotment, that one providing the highest accuracy of assignment. In this way, some peaks with different charge-to-mass ratio and a different distribution of area percentage of AGP forms are observed when comparing samples from sick and healthy individuals. Thus, a method that permits to compare AGP forms existing in sera of individuals with different pathophysiological situations has been developed. A potential for using AGP forms analyzed by CZE as a disease marker and for using this technique for screening purposes is envisaged.     

Large-volume sample stacking for the analysis of seven beta-lactam antibiotics in milk samples of different origins by CZE

A method for the simultaneous determination of eight beta-lactam antibiotics (nafcillin, cloxacillin, oxacillin, dicloxacillin, ampicillin, amoxicillin, and penicillin G) in fortified milk samples of different origins has been proposed by using CZE with diode-array detection (CZE-DAD). Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using 175 mM Tris buffer with 20% ethanol at pH 8.0. Methylparaben has been used as an internal standard (IS). Taking into account the lack of sensitivity of the UV-Vis detection, a solvent extraction/SPE method was applied for off-line preconcentration and sample cleanup, and also an on-line preconcentration methodology, such as large-volume sample stacking (LVSS) with polarity switching, was developed, providing LODs ranging from 2 to 10 microg/L. The method permits the quantification of these residues below the levels established in milk by the EU Regulation. Satisfactory recoveries ranging from 86 to 93% were also obtained in milk samples of different origins.     

Optimization of CZE for analysis of phytochemical bioactive compounds

Advantages of CZE such as high efficiency, low cost, short analysis time, and easy implementation result in its wide applications for analysis of phytochemical bioactive compounds (e.g. flavonoids, alkaloids, terpenoids, phenolic acid, saponins, anthraquinones and coumarins). However, several aspects, including sample preparation, separation, and detection have significant effects on CZE analysis. Therefore, optimization of these procedures is necessary for development of the method. In this review, sample preparation such as extraction method and preconcentration, separation factors including buffer type, concentration and pH, additives, voltage and temperature, as well as detection, e.g. direct and indirect UV detection, LIF and MS were discussed for optimization of CZE analysis on phytochemical bioactive compounds. The optimized strategies were also reviewed.     

CZE Separation of New Drugs for Treatment of Leukemia

Imatinib, bosutinib, dasatinib, pazopanib, erlotinib, canertinib and vatalanib are new developed anticancer drugs, especially for treatment of leukemia. In this article, a fast and high throughput capillary zone electrophoresis method has been developed and validated for analysis of these new drugs in pharmaceutical formulas. The method can be easily utilized for determination of all the drugs in one run what is advantageous for the quality control in pharmaceutical industry because there is no need for changing and optimization of separation conditions when changing the analyte. The separation was performed using an uncoated fused silica capillary with 100 mmol L⁻¹ sodium phosphate buffer pH 2.75, voltage of 25 kV, hydrodynamic injection time of 5 s by 50 mbar, and detection at 214 nm. Under these conditions, the analysis took about 8 min. The validation of all the drugs resulted in recoveries in the range of 84–100 %. The method showed to be precise for all the drugs with RSDs of migration times lower than 0.9 % (interday precision). A very good linearity in the validated range (5–100 μg mL⁻¹) and the limits of detection (LODs) in the range of 0.5–2.0 (μg mL⁻¹) were achieved. Finally, we proved that the method is robust by the Youden’s test. Therefore, our method can be successfully applied for analysis of the real pharmaceutical samples.

     

CZE Separation of New Drugs for Treatment of Leukemia

Imatinib, bosutinib, dasatinib, pazopanib, erlotinib, canertinib and vatalanib are new developed anticancer drugs, especially for treatment of leukemia. In this article, a fast and high throughput capillary zone electrophoresis method has been developed and validated for analysis of these new drugs in pharmaceutical formulas. The method can be easily utilized for determination of all the drugs in one run what is advantageous for the quality control in pharmaceutical industry because there is no need for changing and optimization of separation conditions when changing the analyte. The separation was performed using an uncoated fused silica capillary with 100 mmol L^?1 sodium phosphate buffer pH 2.75, voltage of 25 kV, hydrodynamic injection time of 5 s by 50 mbar, and detection at 214 nm. Under these conditions, the analysis took about 8 min. The validation of all the drugs resulted in recoveries in the range of 84–100 %. The method showed to be precise for all the drugs with RSDs of migration times lower than 0.9 % (interday precision). A very good linearity in the validated range (5–100 μg mL^?1) and the limits of detection (LODs) in the range of 0.5–2.0 (μg mL^?1) were achieved. Finally, we proved that the method is robust by the Youden’s test. Therefore, our method can be successfully applied for analysis of the real pharmaceutical samples.     

Coordination modes and different hapticities for fullerene organometallic complexes

The different coordination modes in fullerene organometallic complexes are reviewed. The main modes are η2 and η5, but there are some interesting studies about the other four, all of them are revised in order to show which is the state of art of this kind of compounds with the respect of the hapticity.     

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.     

CZE and ESI-MS of Borate-Sugar Complexes

Capillary zone electrophoresis (CZE) with electrospray ionization (ESI) mass spectrometry (MS) was used to study borate (B^?1) and sugar (L) complexes (L _x B^?1). Boric acid was adjusted to pH 10 with ammonium hydroxide to create an ESI-MS compatible CZE background electrolyte. We show for the first time that the electrophoretic peaks for each injected sugar contained both the substrates (i.e., sugar and/or multimers) and products (i.e., L _x B^?1). The effects of sheath liquid, temperature, and borate concentration were studied. The molecular mass information obtained from the ESI-MS provided new evidence on the mechanisms of borate-sugar complexation. Direct infusion ESI-MS and CZE-ESI-MS experiments strongly suggest that the formation of L _x B^?1 was from the direct reaction of a sugar or sugar multimer (L _x ) and B^?1. Larger L _x B^?1, where x > 2 were observed. Separation in the CZE dimension allows for the simultaneous analysis of a sugar mixture and simplified the ESI-MS analysis of sugars of the same molecular mass. The increase in sugar electrophoretic mobility caused by the increase in borate concentration was discussed in terms of the formation of L _x B^?1 complexes. In addition, the separation of five nucleosides by CZE using a borate electrolyte and detection using ESI-MS is demonstrated.     

CZE for the speciation of arsenic in aqueous soil extracts

We developed two separation methods using CZE with UV detection for the determination of the most common inorganic and methylated arsenic species and some phenylarsenic compounds. Based on the separation method for anions using hydrodynamic sample injection the detection limits were 0.52, 0.25, 0.27, 0.12, 0.37, 0.6, 0.6, 1.2 and 1.0 mg As/L for phenylarsine oxide (PAO), p-aminophenylarsonic acid (p-APAA), o-aminophenylarsonic (o-APAA), phenylarsonic acid (PAA), 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenite or arsenious acid (As(III)) and arsenate (As(V)), respectively. These detection limits were improved by large-volume sample stacking with polarity switching to 32, 28, 14, 42, 22, 27, 26 and 27 microg As/L for p-APAA, o-APAA, PAA, roxarsone, MMA, DMA, As(III) and As(V), respectively. We have applied both methods to the analysis of the arsenic species distribution in aqueous soil extracts. The identification of the arsenic species was validated by means of both standard addition and comparison with standard UV spectra. The comparison of the arsenic species concentrations in the extracts determined by CZE with the total arsenic concentrations measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) indicated that CZE is suited for the speciation of arsenic in environmental samples with a high arsenic content. The extraction yield of phenylarsenic compounds from soil was derived from the arsenic concentrations of the aqueous soil extracts and the total arsenic content of the soil determined by ICP-AES after microwave digestion. We found that 6-32% of the total amount of arsenic in the soil was extractable by a one-step extraction with water in dependence on the type of arsenic species.     

Principles of classifying diagrams for different types of biazeotropic ternary mixtures

A new equation for two-phase systems in integer units is obtained by transformations of the Serafimov equation. The total number of types of phase portraits for biazeotropic ternary mixtures is determined on the basis of this equation.     

Measurement of dynamic properties of polymeric glasses for different modes of deformation

Methods for measuring various storage moduli and damping factors of glassy polymers in the frequency range 10^?2 to 10^?7 Hz are critically reviewed. At frequencies between about 10^?2 and 200 Hz, components of the complex tensile modulus E¹ and complex Poisson's ratio ν¹ have been determined at 21 °C for polymethyl methacrylate (PMMA) and rigid polyvinyl chloride (PVC) using a non-resonance technique with bidirectional strain gauges. From these measurments, components of the complex shear modulus G¹ and bulk modulus K¹ have been evaluated and shown to vary with frequency in a manner consistent with data obtained by other methods. Molecular motions responsible for the broad secondary relaxation regions in PMMA and PVC are thus able to couple with both shear and dilatational stress fields.     

Influencing of the migration time in CZE

For selective influencing of the ion mobility in CZE several electrolyte additives were used. With acetone as organic solvent the migration times of inorganic cations are changed. The separation of Cl^– and I^– is optimized by adding -cyclodextrin to the electrolyte. By adding 18-crown-6 it is possible to slow down the mobility of K⁺. In this way the disturbing effect of matrix ions can be prevented.     

CZE Determination of Flavonoids in Halenia elliptica

A capillary zone electrophoretic method with UV detection at 254 nm has been developed and validated for quantitative analysis of three flavonoids, luteolin?7?O?β?D-glycoside, genkwanin, and luteolin, in Halenia elliptica. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. The effects on migration of buffer pH, electrolyte concentration, and organic modifier were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 9.00 containing10% (v/v) acetonitrile. Regression equations revealed a good linear relationship between the peak area of each compound and its concentration. All correlation coefficients were 0.9999. The relative standard deviations of migration time and peak areas were, respectively, <1.58 and 5.02%(intra-day) and <1.69% and 5.36% (inter-day). The amounts of the three compounds in the different parts of H. elliptica (stems, leaves, and flowers) and in the extracts of the flowers obtained with different solvents (100% methanol and 50% and 75% methanol in water) were successfully determined with satisfactory repeatability and recovery.     

Separation of multiphosphorylated peptide isomers by CZE

A separation of mono, doubly and triply phosphorylated isomers was developed with CZE with an aqueous electrolyte containing 3.9 mol/L formic acid and 30% v/v trifluoroethanol. Thus a mixture of ten phosphopeptides corresponding to the human tau sequence 226-240 was separated within 70 min. Although peptides with different phosphorylation degrees, i.e. 0-3 phosphate groups, were well separated, some of the phosphopeptide isomers containing one or two phosphate groups were only partially separated. The electrolyte system is compatible with both MALDI- and ESI-MS, allowing a direct coupling, and thus could have some interesting applications in proteomics.     

Fast Screening for Tobacco-Specific N-nitrosamines by CZE Using Dynamically Coated Capillaries

In this paper, we propose a rapid capillary zone electrophoretic method with dynamically coated capillaries for separation of and screening for six tobacco-specific nitrosamines, a group of strong carcinogens found only in tobacco products. Dynamic coating of the capillary surface with a polycation coating (CElixir Reagent A) followed by a polyanion coating (CElixir Reagent B; pH 2.5) was accomplished by use of a commercially available reagent kit. With this method, six tobacco-specific nitrosamines: N??-nitrosonornicotine, N??-nitrosoanatabine, N??-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitroamino)-4-(3-pyridyl)-1-butanol were simultaneously separated and determined within 3 min, with a satisfactory detection limit of 1.0 ??g mL^?1 for all the tobacco-specific nitrosamines at S/N = 3, and a linear relationship between UV absorbance and analyte concentration (R ² > 0.98). The repeatability of the method ranged from 0.24 to 0.73% (RSD, n = 5) for migration time and 0.25?C1.54% (RSD, n = 5) for peak area, demonstrating that this capillary electrophoretic method is better than conventional capillary zone electrophoresis.     

A Simple Stability Indicating CZE Method for the Analysis of Octreotide Acetate

A simple stability indicating capillary zone electrophoretic method was developed and validated for the analysis of octreotide acetate (OCT-Ac). The best separation was achieved by bare fused silica capillaries (50 μm i.d.; 65.5 cm total and 57.0 cm effective length), phosphate buffer (pH = 3.25; 50 mM), at 32.5 °C. The samples were injected using 50 mbar for 5 s and subjected to the applied voltage of 27.5 kV for separation. The detection was carried out using a PAD at a wavelength of 195 nm. For improving the repeatability of the method, l-histidine was applied as an internal standard. According to the validation results, the method was linear in the concentration range of 3.30–400 μg mL^?1 (correlation coefficient of 0.9996) with a limit of detection of 1.08 μg mL^?1 and a limit of quantification of 3.30 μg mL^?1; accuracy of the method was between 100.4 ± 0.2 and 101.1 ± 0.2 %; intra-assay precision was 0.5–2.6 % and intermediate precision was 1.3–3.2 %. The proposed method was successfully applied for the quantification of OCT-Ac in both a pharmaceutical formulation and force-degraded samples and for the detection and separation of degradation products; besides, the obtained results were used for the evaluation of the degradation kinetics of OCT-Ac under different stress conditions. So, it is concluded that the developed method could be employed as a simple, accurate and precise stability-indicating method in quality control laboratories to assess the quantity and stability of OCT-Ac pharmaceutical products.     

An observation of unusual temperature effects for enantioselective CZE employing highly sulfated-beta-cyclodextrin

In this paper data are presented suggesting that for enantiomers of basic and neutral analytes, the concentrations of highly sulfated CDs typically used are above the optimum since lower concentrations result in unacceptable migration times. This situation arises because of the high binding affinities often obtained with sulphated CDs. In a system being over the optimum CD concentration increasing the temperature of the capillary offers a capability to improve selectivity and reduce migration times.