Determination of Fexofenadine in Tablets by Capillary Electrophoresis in Free Solution and in Solution with Cyclodextrins as Analyte Carriers

ABSTRACT

Capillary electrophoresis (CE) methods for the determination of fexofenadine (FEX) in commercial pharmaceuticals were developed. It was demonstrated that FEX could be effectively analyzed in free solution cationic CE at low pH. Another analytical approach studied was based on cyclodextrin (CD) modified CE where highly charged CD derivatives served as analyte carriers. In this way, the separation range was spread to physiological pH region and a CE analysis of FEX, present actually in its zwitterionic form, could be accomplished. Several parameters affecting the separations were studied, including the type and concentration of carrier ion, counterion, analyte carrier, and pH of the buffer. The methods based on the free solution CE and CD-modified CE were compared each other, validated, and applied for the determination of FEX in tablets.     

Mechanistic study on analyte focusing by dynamic pH junction in capillary electrophoresis using computer simulation

Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.     

Anodic destruction of 4-chlorophenol solution

The electrochemical oxidation of 4-chlorophenol solutions was studied using a dimensional stable anode (DSA), made of pure titanium sheet mesh coated with Ti/TiO(2) and RuO(2) film. An electrochemical cell with one working electrode and two counter-electrodes was designed. A gas collecting system to collect the electrolysis gaseous products was also designed. The influence of current density (6.51-21.58 mA/cm(2)), pH (2.0-12.6) and initial 4-chlorophenol concentration (25-100 mg/l) on the destruction was investigated. Complete elimination was successfully achieved within 2 h for most investigated conditions. Highest rates of elimination were achieved at a pH of 12.6.A new approach to calculate the current efficiency (CE) of the cell was proposed. The volumes of the gases produced at the anode and at the cathode were the basis for the new CE calculations. It was observed that the worst CE was approximately 20% and the best CE was approximately 89%. The most efficient pH was at 12.6 and the most efficient current density was at 11.39 mA/cm(2).     

Gadolinium hydroxide coprecipitation system for the separation-preconcentration of some heavy metals

A simple and new procedure for the determination of trace amounts of lead(II), cobalt(II), manganese(II) and copper(II) is described, that combines atomic absorption spectrometry-gadolinium hydroxide coprecipitation. One milliliter of 1% gadolinium(III) solution was added to each sample; the pH was then adjusted to 11 in order to collect trace heavy metals on gadolinium(III) hydroxide. The precipitate was separated by centrifugation and dissolved in 1 mL of 1 mol L(-1) HNO3. The influences of analytical parameters including amount of gadolinium(III), sample volume, etc. were investigated on the recoveries of analyte ions. The effects of concomitant ions were also examined. The recoveries of the analyte ions were greater than 95. The detection limits for the analyte elements based on 3 sigma (n=20) were in the range of 0.52-12.0 microg L(-1). The method was applied to the determination of analytes in real samples and good results were obtained (relative standard deviations <10%, recoveries >95%).     

Distance-dependent metal-enhanced quantum dots fluorescence analysis in solution by capillary electrophoresis and its application to DNA detection

Here the distance dependence of metal-enhanced quantum dots (QDs) fluorescence in solution is studied systematically by capillary electrophoresis (CE). Complementary DNA oligonucleotides-modified CdSe/ZnS QDs and gold nanoparticles (Au NPs) were connected together in solution by the hybridization of complementary oligonucleotides, and a model system (QD-Au) for the study of metal-enhanced QDs fluorescence was constructed, in which the distance between the QDs and Au NPs was controlled by adjusting the base number of the oligonucleotide. In our CE experiments, the metal-enhanced fluorescence of the QDs solution was only observed when the distance between the QDs and Au NPs ranged from 6.8 to 18.7 nm, and the maximum enhancement by a factor of 2.3 was achieved at 11.9 nm. Furthermore, a minimum of 19.6 pg of target DNA was identified in CE based on its specific competition with the QD-DNA in the QD-Au system. This work provides an important reference for future study of metal-enhanced QDs fluorescence in solution and exhibits potential capability in nucleic acid hybridization analysis and high-sensitivity DNA detection.     

Selective focusing of catecholamines and weakly acidic compounds by capillary electrophoresis using a dynamic pH junction

A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(-8) M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known.     

Cyclic chronopotentiometry as a detection tool for flowing solution systems

Cyclic chronopotentiometry provides a very simple detection method, which may be particularly useful in capillary electrophoresis (CE) and microseparation systems. It has been shown that for disk microelectrodes it is possible to define safe reduction and oxidation currents that would never lead to the formation of H2 or O2 gas bubbles, even if they are applied for an indefinitely long time period. During end-column CE detection, currents passing through the working microelectrode can be completely controlled by the external electronic circuit and they are not affected by the separation current. Consequently, problems created by the offset potential in CE can be completely eliminated. The detection can be accomplished through a variety of different mechanisms; however, generation of the electrode response as a result of analyte adsorption seems to be most common. The method is applicable to many analytes, which do not have to be electroactive. The analytical signal is obtained by monitoring the change in the average electrode potential (calculated for either a cathodic or an anodic half-cycle) caused by an analyte interacting with the electrode. The analytical signal is proportional to the analyte concentration, within a concentration range extending over approximately 2 orders of magnitude.     

CS-Cu酶膜的酶固定化工艺及其特性

以壳聚糖(CS)膜为载体,通过PPO与CS-Cu膜上的Cu2+鳌合,制备固定化PPO的CS-Cu酶膜.响应面法研究了酶膜在固定化过程中的Cu2+螯合量(QCu2+)、PPO浓度(CE)、pH三因素对固定化PPO的活力的影响,分别测定了溶液PPO和酶膜上固定化PPO的酶活性,并对CS-Cu-PPO膜的特性作了初步讨论.结果表明,PPO固定化的最优条件是:温度40℃,pH 7.67,QCu2+ 30.07mg/mL,CE2.12 mg/mL;酶膜中固定化PPO的米氏常数及其相对活力都比溶液PPO的大,半衰期约30 d,酶膜重复使用8次后的PPO活力降至79％.研究结果为开发特定功能的EMR提供了重要的基础.     

Capillary electrophoresis coupled on-line with ultraviolet resonance Raman spectroscopy

Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (lambda(ex): 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10-125 microg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm.     

On-line focusing of flavin derivatives using Dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection

Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.     

Chromium(III) porphyrin as a selective ionophore in a salicylate-selective membrane electrode

The construction, potentiometric response properties, and applications of a novel ion-selective electrode with high selectivity toward salicylate are described. Chromium(III) tetraphenylporphyrin chloride was used as ion carrier into plasticized PVC membrane. This ionophore is capable of serving as both a positively charged and neutral carrier, depending on the pH of the sample solution. The influence of several variables was investigated to optimize the potentiometric response and selectivity of the electrode. The resulting electrode demonstrates a near-Nernstian response over a wide range of salicylate concentration (10(-6)-10(-1) M). This electrode has a fast response time and micromolar detection limit and could be used over a wide pH range (3-9). The proposed electrode showed very high selectivity for salicylate over a number of common inorganic and organic anions. The specific interaction of salicylate with the central metal of porphyrin is described based on UV-visible absorption spectra. The electrode was successfully applied to the determination of salicylate in pharmaceutical preparations and clinical samples.     

Counterionic Detection by ICP-AES for Determination of Inorganic Anions in Water Elution Ion Chromatography Using a Zwitterionic Stationary Phase

The analytical methods for the determination of inorganic anions by water elution ion chromatography using a zwitterionic column (C14SB-coated column) have been explored, where pure water was used as the mobile phase. In the present ion chromatography, the complicated peaks derived from various "ion pairs" appeared on the chromatogram when the sample solution contained several kinds of cations and anions. The chromatograms with the complicated peaks were simplified by using a preconditioning cation-exchange column. In the preconditioning column, various kinds of countercations of the analyte anions were converted to a particular kind of common cation, and thus all the analyte anions were separated as the common cation form. The more effective separation was achieved by converting to a divalent cation (Mg(2+)) form than a monovalent cation (Na(+)) form, because the anions paired with the divalent cation provided longer elution times than those paired with the monovalent cation. All the analyte anions separated as the common cation form were simply determined from their calibration curves drawn from conductivity detection. In addition, the counterionic determination of the anions was also attempted by using ICP-AES. The analyte anions were determined by measuring their countercations with ICP-AES, where the calibration curve of the countercation measured by ICP-AES could be used.     

Oligomerized phospholipid bilayers as semipermanent coatings in capillary electrophoresis

Double-chained surfactants form semipermanent coatings that prevent protein adsorption in capillary electrophoresis (CE). To make such coatings more permanent, vesicles of the unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine were prepared and subjected to free-radical-initiated polymerization, both inside the capillary and in free solution. The latter generated oligomers of 2-5 units based on ESI-TOF MS, and formed the more stable coating in CE. Rinsing the capillary with a solution of the ex situ oligomerized DOPC suppressed EOF (0.8 x 10(-)(8) m(2)/V.s) for more than 20 h, whereas in situ oligomerized electroosmotic flow (EOF) suppressed the EOF for only 10 h. Mixtures of anionic and cationic proteins were separated under neutral pH and low ionic strength buffer with efficiencies of 480,000-930,000 plates/m and recoveries of 75-99%.     

One-step concentration of analytes based on dynamic change in pH in capillary zone electrophoresis

A novel strategy for one-step concentration of analytes during capillary electrophoresis (CE) is presented. A short platinum wire was inserted into the 75-microm-i.d. separation capillary. When a high voltage was applied for CE separation, a sharp pH gradient along the capillary was created dynamically by the electrolysis of water in the running buffer. Concentration of a large volume of injected analytes was accomplished by the change in analyte charge due to the dynamic pH gradient. Depending on the polarity of the applied potential and the direction of electroosmotic flow, either anions or cations can be concentrated. Several hundredfold concentration factors were achieved. Fluorescence imaging by a CCD camera was used to monitor 10 cm of the capillary near the platinum wire during the concentration process. The observations are consistent with a sweeping mechanism.     

Degradation kinetics of neostigmine in solution

The degradation kinetics of neostigmine were studied in aqueous solutions with varied pH from 1.5 to 9.9 under accelerated storage conditions. The stability of neostigmine in solutions containing propylene glycol or polyethylene glycol 400 was also investigated. The reaction order of neostigmine in these aqueous and solvent systems followed pseudo-first-order degradation kinetics. The degradation rates of neostigmine under various buffer concentrations within the investigated pH range were obtained. They indicated that the degradation was independent of the species of buffering agent. Maximum stability of neostigmine was determined at pH 5.0 buffer species conditions. The activation energy could be estimated from the Arrhenius plot as 15.72 kcal/mole. The half-life of 883.7 days was estimated at room temperature in 0.1 M, pH 4.9 acetate buffer solution (mu = 0.5). Ultraviolet (UV) irradiation at 254 nm of the neostigmine solutions in pH 4.9 acetate buffer showed an accelerated degradation in comparison with light-protected samples. Incorporation of propylene glycol into the neostigmine solution at pH 4.9 enhanced the stability; however, an adverse effect on the stability of neostigmine was noted when a polyethylene glycol 400 solvent system was used.     

The application of atomic absorption spectrometry for the determination of residual active pharmaceutical ingredients in cleaning validation samples

The objective of this work was the development and validation of atomic absorption spectrometric (AAS) methods for the determination of residual active pharmaceutical ingredients (API) in rinse samples for cleaning validation. AAS as an indirect method for the determination of API in rinse samples can be applied when it is in the form of salt with metal ions or when the metal ion is a part of the API's structure. The electrothermal AAS methods (aqueous and ethanol medium) for the determination of magnesium in esomeprazole magnesium and the flame AAS method for the determination of lithium in lithium carbonate in rinse samples were developed. Various combinations of solvents were tested and a combination of 1% aqueous or ethanol solution of nitric acid for esomeprazole magnesium and 0.1% aqueous solution of nitric acid for lithium carbonate were found to be the most suitable. The atomization conditions in the graphite furnace and in the flame were carefully studied to avoid losses of analyte and to achieve suitable sensitivity. The cleaning verification methods were validated with respect to accuracy, precision, linearity, limit of detection, and quantification. In all the cases, the limits of detection were at the microgram level. The methods were successfully applied for the determination of esomeprazole magnesium and lithium carbonate in rinse samples from cleaning procedures.     

Second-order calibration of excitation-emission matrix fluorescence spectra for the determination of N-phenylanthranilic acid derivatives

A spectrofluorimetric method has been developed for the quantitative determination of mefenamic, flufenamic, and meclofenamic acids in urine samples. The method is based on second-order data multivariate calibration (unfolded partial least squares (unfolded-PLS), multi-way PLS (N-PLS), parallel factor analysis (PARAFAC), self-weighted alternating trilinear decomposition (SWATLD), and bilinear least squares (BLLS)). The analytes were extracted from the urine samples in chloroform prior to the determination. The chloroform extraction was optimized for each analyte, studying the agitation time and the extraction pH, and the optimum values were 10 minutes and pH 3.5, respectively. The concentration ranges in chloroform solution of each of the analytes, used to construct the calibration matrix, were selected in the ranges from 0.15 to 0.8 microg mL-1 for flufenamic and meclofenamic acids and from 0.25 to 3.0 microg mL-1 for mefenamic acid. The combination of chloroform extraction and second-order calibration methods, using the excitation-emission matrices (EEMs) of the three analytes as analytical signals, allowed their simultaneous determination in human urine samples, in the range of approximately 80 mg L-1 to 250 mg L-1, with satisfactory results for all the assayed methods. Improved results over unfolded-PLS and N-PLS were found with PARAFAC, SWATLD, and BLLS, methods that exploit the second-order advantage.     

Complexation Study of the Anticancer Agent EO-9 with 2-hydroxypropyl-β-Cyclodextrin

For the development of a bladder instillation of the indoloquinone agent EO-9, use of the complexing agent 2-hydroxypropyl-β-cyclodextrin (HPβCD) was considered. Therefore, a complexation study of EO-9 with HPβCD was performed. Complexation was studied in aqueous solution and in solid freeze-dried products. A phase solubility study, UV-visible spectroscopy (UV/VIS), and analysis of the effect of HPβD on the stability of EO-9 were performed. With the phase solubility study, a complexation constant (K1:1) of 32.9, a complexation efficiency (CE) of 0.0457, and a utility number (UCD) of 38.3 were calculated. These K1:1 and CE values indicate a weak complex, but the UCD shows that HPβCD can be very useful as solubilizer in the desired formulation. Furthermore, a positive effect of HPβCD on the chemical stability of EO-9 in solution was seen. Subsequently, complexation in the freeze-dried products was studied more thoroughly using Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses. HPβCD was found to be an excellent pharmaceutical complexing agent for application in formulations for EO-9 bladder instillations. Reconstitution before use of the developed freeze-dried products can be simply accomplished with water for injection.     

Electromigration injection from a small loop in capillary electrophoresis

Capillary electrophoresis (CE) is now clearly the separation technique of the decade. One aspect that is still in need of improvement is concentration sensitivity, especially when CE is used in conjunction with on-column UV-visible absorptiometry, by far the most common practice. Electromigration injection (EI) is among the most prominent techniques in CE that serves to improve the attainable limits of detection; it is also the most convenient and simplest of all injection modes. Unfortunately, EI is affected both from sample to sample (the amount of an analyte introduced depends on sample conductance) and from analyte to analyte within a sample (the introduction is strongly biased on analyte electrical mobility). Previously we have shown the utility of small loops affixed at the tip of a capillary (Anal. Chem. 1995, 67, 3853-3860; 1996, 68, 1164-1168). The present paper shows that there are remarkable advantages to be gained from forming a film of the sample solution on a wire loop and using the loop itself as the high-voltage electrode to perform electromigration from a very small sample volume. The sample constituents can be essentially exhaustively electromigrated from this volume in less than 1 min, and the mobility induced bias is dramatically lowered. The observed experimental behavior agrees with theoretical models.     

Application of solid-phase microextraction in the determination of diazepam binding to human serum albumin

In this paper, protein-drug interactions were studied by solid-phase microextraction (SPME) using diazepam binding to human serum albumin as a model system. Since drug compounds are normally polar and nonvolatile by nature, direct SPME is used in this work. The SPME extraction is an equilibrium process among the concentrations of the analyte partitioned onto the SPME fiber, free and bound drug in the solution. A calibration curve was first constructed by employing the amount of the analytes partitioned on the fiber versus the free analyte concentration in the solution in the absence of protein. In method I, the extraction was performed in the protein solution with known diazepam concentration. In method II, diazepam was first loaded onto the fiber by extracting in solution with known diazepam concentration. This fiber was subsequently transferred into the protein solution for desorption. The amount of the analyte left on the fiber was analyzed after the system reached equilibrium. The free drug concentration was then obtained from the calibration curve for both methods. The Scatchard plot was finally employed to obtain the number of binding sites and the equilibrium binding constants. Since only a very small amount of the protein solution is required (150 microL for each extraction), method II is very useful for circumstances where the protein amount is very limited. The direct measurement method proposed in this paper does not need a GC response factor, which significantly decreases the experimental error. The only measurement needed is the area count change (ratio) of the fiber injections before and after the protein was introduced into the solution. The difference between the direct measurement method for method I and method II is discussed. The result illustrated that the SPME direct measurement method provided both theoretical accuracy and simplicity in such applications.