Fast determination of ethambutol in pharmaceutical formulations using capillary electrophoresis with capacitively coupled contactless conductivity detection

A method for the determination of ethambutol (EMB), a first‐line drug against tuberculosis, based on CE with capacitively coupled contactless conductivity detection is proposed. The separation of EMB and its main product of degradation were achieved in less than 3 min with a resolution of 2.0 using a BGE composed of 50 mmol/L histidine and 30 mmol/L MES, pH 6.30. By raising the pH to 8.03, the analysis time was reduced to 1.0 min, but with a significant loss of resolution (0.7). Using the best separation conditions, linearity of 0.9976 (R², five data points), sensitivity of 1.26×10^?4 V min μmol^?1 L, and LOD and quantification of 23.5 and 78.3 μmol/L, respectively, were obtained. Recoveries at four levels of concentration ranged from 95 to 102% and the concentration range studied ranged from 100 to 500 μmol/L. The results obtained for the determination of EMB in pharmaceutical formulations were compared with those obtained by using CE with photometric detection.     

A review of the recent achievements in capacitively coupled contactless conductivity detection

Capacitively coupled contactless conductivity detection (C⁴D) in the axial electrode configuration was introduced in 1998 as a quantification method for capillary electrophoresis. Its universality allows the detection of small inorganic ions as well as organic and biochemical species. Due to its robustness, minimal maintenance demands and low cost the popularity of this detector has been steadily growing. Applications have recently also been extended to other analytical methods such as ion chromatography, high-performance liquid chromatography and flow-injection analysis. C⁴D has also found use for detection on electrophoresis based lab-on-chip devices. Theoretical aspects of C⁴D in both the capillary and microchip electrophoresis format have been comprehensively investigated. Commercial devices are now available and the method can be considered a mature detection technique. In this article, the achievements in C⁴D for the time period between September 2004 and August 2007 are reviewed.     

Membraneless vaporization unit for direct analysis of solid sample

A new apparatus, called ‘membraneless vaporization’ (MBL-VP) unit was designed and developed for direct analysis of solid samples. Solid analyte was converted into a gaseous form which then reacts with an indicator reagent. Change in absorbance was used to quantitate the analyte. Stirring with a magnetic bar was employed to facilitate the evaporation of the gas. Unlike the pervaporation technique, hydrophobic membrane was not required for this MBL-VP technique.Application of the membraneless technique for direct determination of calcium carbonate in calcium supplements, has shown to be very precise (R.S.D. = 2.5% for 0.16 mmol CO₃²⁻), with detection limit of 0.5 mg CaCO₃. Results by this method agreed well with flame atomic absorption spectrometric method. Sample throughput was 20 samples h⁻¹.     

New membraneless vaporization unit coupled with flow systems for analysis of ethanol

This work presents the development of a new design for a membraneless vaporization (MBL-VP) unit, called dual chamber MBL-VP for measurement of volatile compounds. With this unit, exact volumes of sample and reagent are introduced into their respective cone-shaped chambers from the base of the cones. Diffusion of volatile analyte then takes place. After an appropriate time interval, the acceptor solution is withdrawn from the chamber into the detector flow-cell, while the sample solution is withdrawn to waste. Unlike the previous MBL-VP design, problems with overflow of solutions are eliminated by precise control of the input volume to be less than the volume of the chamber. The developed flow system with the dual chamber MBL-VP unit was applied to the determination of the ethanol content of various liquid samples, using the oxidation reaction between potassium dichromate and the diffused ethanol. In addition, in order to accelerate the gas diffusion process, the donor chamber was aerated. As the result, relatively short analysis time of 144 s was achieved for ethanol content in the range of 5–50% (v/v). The proposed method was successfully validated against a gas chromatographic method for 17 alcoholic samples. Percentage recovery was in the range of 96–109%.     

Analysis of flavonoids by CE using capacitively coupled contactless conductivity detection

A CE method employing capacitively coupled contactless conductivity (C(4)D) compared to indirect UV-detection was developed for the analysis of phytochemically relevant flavonoids, such as 6-hydroxyflavone, biochanin A, hesperetin and naringenin. To ensure fast separation at highest selectivity, sensitivity and peak symmetry, the pH value and the concentration of the running BGE had to be optimized regarding both co- and counter-EOF mode. Optimum conditions were found to be 1.0 and 5.0 mM chromate BGE (pH 9.50) in the counter- and co-EOF mode, respectively. Validation of the established CE-C(4)D method pointed out to be approximately seven times more sensitive compared to indirect UV-detection applying the same conditions. The lower LOD defined at an S/N of 3:1 was found between 0.12 and 0.21 microg/mL for the analytes of interest using C(4)D and between 0.77 and 1.20 microg/mL using indirect UV-detection. Compared to an earlier published CE method employing direct UV-detection, C(4)D was found to be approximately two times more sensitive. Due to the lower baseline noise, C(4)D showed an excellent regression coefficient >0.99 compared to 0.93 when using indirect UV detection calibrating within a concentration range between 1 and 10 microg/mL. The influence of the sugar moiety on the conductivity of a flavonoid was studied upon the analysis of the aglycon hesperetin and the rutinosid hesperidin. The sugar moiety in hesperedin shows a higher conductivity compared to hesperetin. Finally, the optimized established CE-C(4)D method was applied to the determination and quantification of naringenin in Sinupret.     

Determination of heavy metal ions by microchip capillary electrophoresis coupled with contactless conductivity detection

An integrated detection circuitry based on a lock-in amplifier was designed for contactless conductivity determination of heavy metals. Combined with a simple-structure electrophoresis microchip, the detection system is successfully utilized for the separation and determination of various heavy metals. The influences of the running buffer and detection conditions on the response of the detector have been investigated. Six millimole 2-morpholinoethanesulfonic acid + histidine were selected as buffer for its stable baseline and high sensitivity. The best signals were recorded with a frequency of 38 kHz and 20 V(pp). The results showed that Mn(2+), Cd(2+), Co(2+), and Cu(2+) can be successfully separated and detected within 100 s by our system. The detection limits for five heavy metals (Mn(2+), Pb(2+), Cd(2+), Co(2+), and Cu(2+)) were determined to range from about 0.7 to 5.4 μM. This microchip system performs a crucial step toward the realization of a simple, inexpensive, and portable analytical device for metal analysis.     

Rapid determination of NSAIDs by capillary and microchip electrophoresis with capacitively coupled contactless conductivity detection in wastewater

A simple, rapid method using CE and microchip electrophoresis with C⁴D has been developed for the separation of four nonsteroidal anti-inflammatory drugs (NSAIDs) in the environmental sample. The investigated compounds were ibuprofen (IB), ketoprofen (KET), acetylsalicylic acid (ASA), and diclofenac sodium (DIC). In the present study, we applied for the first time microchip electrophoresis with C⁴D detection to the separation and detection of ASA, IB, DIC, and KET in the wastewater matrix. Under optimum conditions, the four NSAIDs compounds could be well separated in less than 1 min in a BGE composed of 20 mM His/15 mM Tris, pH 8.6, 2 mM hydroxypropyl-beta-cyclodextrin, and 10% methanol (v/v) at a separation voltage of 1000–1200 V. The proposed method showed excellent repeatability, good sensitivity (LODs ranging between 0.156 and 0.6 mg/L), low cost, high sample throughputs, portable instrumentation for mobile deployment, and extremely lower reagent and sample consumption. The developed method was applied to the analysis of pharmaceuticals in wastewater samples with satisfactory recoveries ranging from 62.5% to 118%.     

Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection

Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE‐C⁴D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm ) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non‐UV‐absorbing L‐valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28°C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused‐silica capillary (75 μm, i.d. × 52 cm). The CE‐C⁴D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5–250 μg/mL for the studied analytes. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Capacitively coupled contactless conductivity detection with dual top–bottom cell configuration for microchip electrophoresis

An optimized capacitively coupled contactless conductivity detector for microchip electophoresis is presented. The detector consists of a pair of top–bottom excitation electrodes and a pair of pickup electrodes disposed onto a very thin plastic microfluidic chip. The detection cell formed by the electrodes is completely encased and shielded in a metal housing. These approaches allow for the enhancement of signal coupling and extraction from the detection cell that result in an improved signal‐to‐noise‐ratio and detection sensitivity. The improved detector performance is illustrated by the electrophoretic separation of six cations (NH, K⁺, Ca²⁺, Na⁺, Mg²⁺, Li⁺) with a detection limit of approximately 0.3 μM and the analysis of the anions (Br^?, Cl^?, NO, NO, SO, F^?) with a detection limit of about 0.15 μM. These LODs are significantly improved compared with previous reports using the conventional top–top electrode geometry. The developed system was applied to the analysis of ions in bottled drinking water samples.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Optimization of a capillary zone electrophoresis–contactless conductivity detection method for the determination of nisin

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative–nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance‐to‐digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 μg/mL to 9.307 ± 0.437 μg/g.     

Carbon nanotube-enhanced separation of DNA fragments by a portable capillary electrophoresis system with contactless conductivity detection

It was demonstrated that separation of DNA fragments by a CE-contactless conductivity detection system (CE-CCD) could be enhanced with multiple-wall carbon nanotubes (MWCNs) as buffer additive. For HaeIII digest of PhiX174 DNA, optimized MWCN concentration was obtained when the MWCN was above its threshold concentration, at which MWCN could form a network in the buffer as pseudostationary phase to provide additional interaction sites. In the case of larger DNA, MWCN near or below its threshold concentration was enough to provide great improvement of the resolution, which was shown by the separation of the 2-Log DNA ladder. Furthermore, the buffer containing MWCN could provide a more stable baseline in the CE-CCD system, owing to less fluctuation of its conductivity. Compared with CE-UV, CE-CCD with MWCN could provide lower LODs as well as better resolution.     

Capillary electrophoresis with capacitively coupled contactless conductivity detection method development and validation for the determination of azithromycin,clarithromycin, and clindamycin

A capillary electrophoresis with capacitively coupled contactless conductivity detection based method for the assay of azithromycin, clarithromycin and clindamycin was optimized and validated in this study. A buffer solution of 20 mM 2‐(N‐morpholino) ethane sulfonic acid, 40 mM l ‐histidine and 0.6 mM cetyltrimethylammonium bromide (pH 6.39) was used for the electrophoresis. An uncoated, bare‐fused silica capillary (total length 60 cm, effective length 32 cm, 75 μm id) was used at 25°C. The sample was injected hydrodynamically at 0.5 psi for 5 s. The electrophoresis was conducted at 30 kV in reverse polarity for 6 min with 3 and 2 min of in‐between sodium hydroxide (0.1 M) and background electrolyte rinsing, respectively. Ammonium acetate was used as internal standard. This simple and robust method showed reasonable limit of detection and limit of quantitation for azithromycin (0.0125/0.03 mg/mL), clarithromycin (0.017/0.03 mg/mL), and clindamycin (0.038/0.06 mg/mL), with good selectivity, precision both intraday (relative standard deviation ≤ 1.0%) and interday (relative standard deviation < 3.7%), linearity (R ² > 0.999) and recovery (99 – 101.7%). The method was successfully applied for the determination of azithromycin, clarithromycin and clindamycin in formulations.     

Determination of carbethopendecinium bromide in eye drops by capillary electrophoresis with capacitively coupled contactless conductivity detection

Antiseptic agent carbethopendecinium bromide (septonex) was determined by capillary electrophoresis with capacitively coupled contactless conductivity detection. Optimal separation of this quaternary ammonium ion was achieved in BGE of pH 7.0 containing 30 mM 2-(N-morpholino)ethanesulfonic acid, 12.5 mg/mL of 2-hydroxypropyl-β-cyclodextrin and 20% v/v of acetonitrile. The separation was performed at 25°C in an uncoated fused silica capillary (50 μm id; total length, 60.5 cm; effective length, 50 cm) at 30 kV. Samples were injected hydrodynamically at 50 mbar for 6 s. For quantitative analysis, L-arginine (500 μg/mL) was used as internal standard. The calibration curve was rectilinear for 25-400 μg/mL of septonex (y=0.0113x-0.0063; r(2)=0.9992). The LOD was 7 μg/mL of septonex (at S/N=3). The run-to-run repeatability (n=6) was characterized by the RSDs of 0.18% for the migration time and 1.96% for the analyte/internal standard peak area ratio. Accuracy tested by recovery experiments at three concentration levels gave recoveries of 100.27-104.22% with RSD ≤2.19%. The method was successfully applied to the assay of carbethopendecinium bromide in eye drops. Quaternary ammonium ions having structure and size close to that of carbethopendecinium may not be resolved from the analyte.     

Identification of inorganic ions in post‐blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.     

Determination of PCR products by CE with contactless conductivity detection

The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2‐(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of –15 kV were employed and separations could be completed within 20–50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified.     

Study of the determination of inorganic arsenic species by CE with capacitively coupled contactless conductivity detection

The determination of arsenic(III) and arsenic(V), as inorganic arsenite and arsenate, was investigated by CE with capacitively coupled contactless conductivity detection (CE-C(4)D). It was found necessary to determine the two inorganic arsenic species separately employing two different electrolyte systems. Electrolyte solutions consisting of 50 mM CAPS/2 mM L-arginine (Arg) (pH 9.0) and of 45 mM acetic acid (pH 3.2) were used for arsenic(III) and arsenic(V) determinations, respectively. Detection limits of 0.29 and 0.15 microM were achieved for As(III) and As(V), respectively by using large-volume injection to maximize the sensitivity. The analysis of contaminated well water samples from Vietnam is demonstrated.     

A thin-layer contactless conductivity cell for detection in flowing liquids

The design is described of a thin-layer contactless conductivity detector suitable for liquid chromatography and flow-injection analysis. Its principal analytical parameters have been determined using a potassium chloride solution: the linear dynamic range extends from 7.5 × 10⁻⁶ to 1.5 × 10⁻² S m⁻¹, corresponding to the KCl concentration range from 0.5 to 1000 μM, the limit of detection equals 3.5 × 10⁻⁶ S m⁻¹ (0.2 μM KCl), the detection repeatability, expressed in terms of the relative standard deviation, amounts to 1.13% and the detection volume is 0.6 μL. The detector was applied to detection of ionic compounds, benzoic, lactic and octanesulfonic acids, and sodium capronate, after their separation by liquid chromatography in a Biospher PSI 100C 18 columns using a 60% aqueous acetonitrile mobile phase. The frequency characteristics of the detector are reasonably theoretically described on the basis of a simple model which is commonly used in the field of contactless impedance detectors.     

A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Determination of artificial sweeteners by capillary electrophoresis with contactless conductivity detection optimized by hydrodynamic pumping

The common sweeteners aspartame, cyclamate, saccharin and acesulfame K were determined by capillary electrophoresis with contactless conductivity detection. In order to obtain the best compromise between separation efficiency and analysis time hydrodynamic pumping was imposed during the electrophoresis run employing a sequential injection manifold based on a syringe pump. Band broadening was avoided by using capillaries of a narrow 10 μm internal diameter. The analyses were carried out in an aqueous running buffer consisting of 150 mM 2-(cyclohexylamino)ethanesulfonic acid and 400 mM tris(hydroxymethyl)aminomethane at pH 9.1 in order to render all analytes in the fully deprotonated anionic form. The use of surface modification to eliminate or reverse the electroosmotic flow was not necessary due to the superimposed bulk flow. The use of hydrodynamic pumping allowed easy optimization, either for fast separations (80 s) or low detection limits (6.5 μmol L⁻¹, 5.0 μmol L⁻¹, 4.0 μmol L⁻¹ and 3.8 μmol L⁻¹ for aspartame, cyclamate, saccharin and acesulfame K respectively, at a separation time of 190 s). The conditions for fast separations not only led to higher limits of detection but also to a narrower dynamic range. However, the settings can be changed readily between separations if needed. The four compounds were determined successfully in food samples.