Determination of thermodynamic pKa values of benzimidazole and benzimidazole derivatives by capillary electrophoresis

Thermodynamic pK_a values for benzimidazole and several substituted benzimidazoles were determined by CE. Electrophoretic mobilities of benzimidazoles were determined by CE at different pH levels and ionic strengths. The dependence of electrophoretic mobilities on pH was used to obtain pK_a values at different ionic strengths. Extrapolations to zero ionic strength were used to determine the thermodynamic pK_a values. Using this method the thermodynamic pK_a values of 15 benzimidazoles were determined and found to range from 4.48 to 7.38. Results from the CE measurements were compared with spectrophotometric measurements which were evaluated at wavelengths where the highest absorbance difference for varying pH was recorded. The two analytical techniques were in good agreement.     

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.     

Determination of terbinafine in pharmaceuticals and dialyzates by capillary electrophoresis

A capillary electrophoresis method has been developed for the separation and determination of terbinafine (TER) in various pharmaceutically relevant matrices. Capillary zone electrophoresis (CZE) separation and UV absorbance photometric detection were carried out in a 160 mm capillary tube with a 300 μm i.d., hydrodynamically (membrane) closed. The influences of pH, carrier cation and counterion on migration parameters of TER were studied and the following conditions were selected: a 20 mmol l⁻¹ glycine running buffer adjusted to pH 2.7 with acetic acid, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC) as an electro-osmotic flow (EOF) suppressor, a 250 μA driving current, and 20 °C. The optimized separation conditions were convenient for the determination of TER in commercial tablets and spray and in dialyzates. Here, the dialysis was used to investigate in vitro permeation of TER through the skin from the gel. The samples of dialyzates were examined with and without simple extraction procedure and the results were compared. A permeation profile of the drug present in the gel of given composition was obtained analyzing pretreated samples. The proposed electrophoretic method was successfully validated. It was suitable for the simple, sensitive, rapid and highly reproducible assay of TER. CZE analysis was completed within 5.5 min. The detection limit of TER was 1.73 μmol l⁻¹ at a 224 nm detection wavelength. The intra- and inter-laboratory precisions over the concentration range 6.0-60.0 μmol l⁻¹ were between 0.32-0.69% and 1.04-1.44% including R.S.D. of migration times and peak areas, respectively. The mean absolute recoveries of drugs from samples were found to be 98.34 (tablets) and 99.47% (spray). It is suggested that there are potentialities to determine TER present in unpretreated complex samples, as CZE in a hydrodynamically closed separation system may be easily on-line combinable with purification and preconcentration CE modes (e.g., isotachophoresis, ITP).     

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis

Acid dissociation constants (pK_a) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pK_a values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pK_a values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pK_a values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pK_a values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pK_a values of such analogues as possessing carboxylic acid moiety.     

Determination of organic acid drug counter-ions by capillary electrophoresis

Summary A capillary electrophoresis method is described for the novel application of quantifying levels of the simple organic acid counter-ions of a variety of basic drugs. These counter-ions are organic acids such as succinic and maleic. The method uses indirect UV detection and an electroosmotic flow modifier. Acceptable precision and detector linearity were obtained using internal standards. Method validation was completed and acceptable data was generated. The method is now in routine use for this type of testing.     

Critical evaluation of buffering solutions for pKa determination by capillary electrophoresis

The performance of the most common and also some other less common CE buffers has been tested for the pKa determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH+/BisTris, TrisH+/Tris, CHES/CHES-, and CAPS/CAPS- can be used with all type of analytes, others like ammonium/ammonia, butylammonium/butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES-, HEPES/HEPES-, and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.     

Capillary zone electrophoresis for simultaneous determination of seven nonsteroidal anti-inflammatory drugs in pharmaceuticals

A simple capillary zone electrophoresis (CZE) method has been developed for analyzing seven nonsteroidal anti-inflammatory drugs (NSAIDs)—sulindac (SU), ketoprofen (KE), indomethacin (IN), piroxicam (PI), nimesulide (NI), ibuprofen (IB), and naproxen (NA). The separation was run using borate buffer (60 mmol L^–1, pH 8.5) containing 13% (v/v) methanol at 20 kV, and detected at 200 nm. Several conditions were studied, including concentration and pH of borate buffer, methanol percentage, and separation voltage. In method validation, the calibration plots were linear over the range 40.0–500.0 mol L^–1. In intra-day and inter-day analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 5%. The limits of detection were 10 mol L^–1 for SU, IN, PI, and 20 mol L^–1 for KE, NI, IB, NA (S/N = 3, sampling 6 s by pressure). All recoveries were greater than 95%. This method was applied to the quality control of six NSAIDs in pharmaceuticals using NI as internal standard (IS). The assay results were within the labeled amount required by USP 25.     

An evaluation of the use of capillary electrophoresis to monitor trace drug residues following the manufacture of pharmaceuticals

Summary The novel use of capillary electrophoresis to the important and developing area of monitoring possible drug residues on pharmaceutical manufacturing equipment is reported. The CE method is applicable to a wide range of basic drugs with sensitivity as low as 25ng/ml (equivalent to 8×10^–8 M). This sensitivity is equivalent to that obtainable for HPLC for the drugs tested and is obtained by employing a combination of a wider bore capillary with low wavelength UV detection. Preliminary evaluation of the method performance shows acceptable precision, linearity, sensitivity and accuracy. Features of the method compared to HPLC include simplicity, ease of method transfer, reductions in analysis set-up time, and reduced costs of solvents and columns.     

Determination of the ionization constants of weak electrolytes by capillary zone electrophoresis

Capillary zone electrophoresis in untreated fused silica capillaries has proved suitable for the determination of the ionization constants of weak electrolytes. Several fundamental equations relating the electrophoretic mobilities of ionized solutes to hydronium ion concentrations in the running electrolyte have been verified experimentally. The observed dependence of the electrophoretic mobilities of weak bases and ampholytes on the pH of the electrolyte showed good agreement with predicted behavior. The pK_a values calculated from electrophoretic mobility data obtained by capillary zone electrophoresis were reasonably close to those reported in the literature.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8 % and <1.2 %, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8% and <1.2%, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

High-performance capillary electrophoresis using open tubes and gels

Summary This paper overviews several aspects of high performance capillary electrophoresis (HPCE), a promising new method of analytical and micropreparative separation of biochemically important samples. The basic migration equations of electrophoresis are first presented and the benefit of high fields for rapid analysis and high performance emphasized. Since power is generated with high voltages, Joule heating results and this heat must be dissipated. The use of capillary columns is shown to be important in efficient heat removal and in minimizing the temperature differences within the column. The various factors influencing band broadening are next described, and it is shown how plate counts close to 10⁶ can be achieved. Various results from our laboratory on open tube and gel columns are then presented to illustrate the potential of this method. Chiral resolution of dansylated amino acids using a chiral metal chelate micelle in open tube HPCE is shown. With the gel columns, the baseline separation of a 2-chain variant from methionine growth hormone (met-hGH) under non-denaturing conditions at fields close to 1000 V/cm is presented. Finally, the micropreparative purification of a 20-mer oligonucleotide using the gel column is described.     

On-line solution stability determination of pharmaceuticals by capillary electrophoresis

Summary Good agreement between the impurity levels in a batch of a related impurity of ranitidine were obtained by CE and HPLC. A solution of the impurity was positioned on the CE autosampler and analysed sequentially. The extent of degradation was monitored by loss of main peak and the formation of two principal degradation products. It was found that after 9.25 hours only 2% area/area of the original impurity remained. Buffering of the sample solution to pH 7 was shown to minimise this degradation.Unattended in-situ stability testing of an solution of the impurity in water was performed by CE.     

Determination of acidity constants,ionic mobilities,and hydrodynamic radii of carborane-based inhibitors of carbonic anhydrases by capillary electrophoresis

Capillary electrophoresis (CE) has been applied for determination of the thermodynamic acidity constants (pK_a) of the sulfamidoalkyl and sulfonamidoalkyl groups, the actual and limiting ionic mobilities and hydrodynamic radii of important compounds, eight carborane-based inhibitors of carbonic anhydrases, which are potential new anticancer drugs. Two types of carboranes were investigated, (i) icosahedral cobalt bis(dicarbollide)(1-) ion with sulfamidoalkyl moieties, and (ii) 7,8-nido-dicarbaundecaborate with sulfonamidoalkyl side chains. First, the mixed acidity constants, pK_a^mix, of the sulfamidoalkyl and sulfonamidoalkyl groups of the above carboranes and their actual ionic mobilities were determined by nonlinear regression analysis of the pH dependences of their effective electrophoretic mobility measured by capillary electrophoresis in the pH range 8.00−12.25, at constant ionic strength (25 mM), and constant temperature (25°C). Second, the pK_a^mix were recalculated to the thermodynamic pK_as using the Debye–Hückel theory. The sulfamidoalkyl and sulfonamidoalkyl groups were found to be very weakly acidic with the pK_as in the range 10.78−11.45 depending on the type of carborane cluster and on the position and length of the alkyl chain on the carborane scaffold. These pK_as were in a good agreement with the pK_as (10.67−11.27) obtained by new program AnglerFish (freeware at https://echmet.natur.cuni.cz ), which provides thermodynamic pK_as and limiting ionic mobilities directly from the raw CE data. The absolute values of the limiting ionic mobilities of univalent and divalent carborane anions were in the range 18.3−27.8 TU (Tiselius unit, 1 × 10⁻⁹ m²/Vs), and 36.4−45.9 TU, respectively. The Stokes hydrodynamic radii of univalent and divalent carborane anions varied in the range 0.34−0.52 and 0.42−0.52 nm, respectively.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Determination of synthetic pharmaceuticals in phytotherapeutics by capillary zone electrophoresis with contactless conductivity detection (CZE-CD)

In this work, a method for simultaneous determination of amfepramone, fenproporex, sibutramine and fluoxetine was developed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection (C⁴D) using a homemade capillary electrophoretic system. The optimized conditions for the separation of the pharmaceuticals by CZE were as follows: 50 mmol L^− 1 phosphate buffer (pH 5.0) in 50/50 (v/v) mixture of water/acetonitrile as the working electrolyte, 15 kV separation voltage, 25 °C separation temperature, hydrodynamic injection by gravity using 20 cm injection height and 60 s injection time. The detection by C⁴D was carried out by using a homemade detector, which employs a sinusoidal wave generator operating at 600 kHz frequency and 2 V_pp wave amplitude. The optimized and validated CZE-C⁴D method was applied for the determination of the studied pharmaceuticals as adulterants in phytotherapeutic formulations commercialized in Brazil for slimming purposes.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

An overview of method validation and system suitability aspects in capillary electrophoresis

Summary Although capillary electrophoresis (CE) methods have been included in some regulatory submissions, the formal requirements for validation of such methods are not well-defined or universally understood. This paper provides a general overview of the aspects of method performance which need to be considered during the validation of CE methods particularly emphasising areas of similarity with, and differences between, HPLC and CE. The individual aspects of method performance are reviewed and guidance given, with illustrative literature examples where possible, on how to carry out suitable validation studies. Performance criteria are also provided, wherever possible. In this way, a suggested validation approach for CE methods is presented, reflecting the similarity with that generally adopted for HPLC methods, but accommodating those aspects which are different for, or peculiar to, CE.     

Analysis of dissolution test sample solutions by high speed capillary electrophoresis

Summary The quantitative use of high speed capillary electrophoresis (HSCE) is examined by applying high voltages across short capillaries. Acceptable performance in terms of injection precision and migration times were achieved within 1–2 minute analysis times. HSCE was used for the novel CE application of dissolution test sample solution analysis. The results generated by HSCE compared well with those generated using validated on-line UV absorbance measurements. It is concluded that HSCE is a viable alternative and supplement to standard analytical methods employed in dissolution test analysis.     

Determination of pKa values of anthraquinone compounds by capillary electrophoresis

In this paper, the dissociation constants (pKa1, pKa2) of five anthraquinones were determined from the relation between the effective mobility at different pH values and the buffer pH value, which was derived from the basic electrophoresis theory and the dissociation equilibrium of a binary acid. In addition, the changes of pKa values of the five compounds were also investigated when organic modifiers were added to the buffer system.