Optimization of the micellar electrokinetic capillary chromatographic determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine using experimental design and radial basis function neural network

Orthogonal design has been applied to the optimization of separation and determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine by micellar electrokinetic capillary chromatography. Operational variables, such as the voltage, micelle concentration, buffer concentration and pH were optimized. Their different effects on peak resolution were studied by the experimental design method. Optimized separation conditions were obtained and successfully applied to the separation and determination of dauricine and daurisoline in real samples. The proposed method allows alkaloids in real samples to be determined within 15 min using a buffer system composed of 25 mM HAc, 25 mM NaAc and 2% polyoxyethylene sorbitan monolaurate (Tween-20) (pH 4.5). In addition, a radial basis function neural network with a “4-18-1” structure was developed based on the experimental results of orthogonal design and uniform design, and applied to the prediction of peak resolution of dauricine and daurisoline under the optimum separation conditions given by orthogonal design. The predicted results were in good agreement with the experimental values, indicating that radial basis function neural network may be a potential method for the selection of separation conditions in capillary electrophoresis.     

Determination of triazine compounds in ground water samples by micellar electrokinetic capillary chromatography

In this work, a capillary electrophoresis (CE) method for the determination of a group of eleven triazine compounds by micellar electrokinetic capillary chromatography (MEKC) with diode array detection was developed. The eleven herbicides studied were: desethylatrazin-2-hydroxy (DEA), simazine, prometon, atrazine, simetryn, ametryn, propazine, prometryn, trietazine, terbutylazine, and terbutryn The separation of these compounds was optimised as a function of buffer concentration and pH, concentration of sodium dodecyl sulphate (SDS) and voltage applied. To increase the selectivity of the separation and the resolution of the solutes, different organic solvents were tested as buffer additives, obtaining the best results when 1-propanol was used. The optimised buffer (24 mM of sodium borate, 18 mM of disodium hydrogen phosphate, 25 mM of SDS, pH 9.5, and 5% of 1-propanol) provides the best separation in terms of resolution and migration time. This method allowed the determination of these compounds at concentrations of 0.05 μg l⁻¹ in ground water samples pretreated using solid-phase extraction (SPE).     

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 μg mL⁻¹ SiNPs.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

Application of Artificial Neural Networks in Multifactor Optimization of Selectivity in Capillary Electrophoresis

ABSTRACT

A methodology based on the coupling of experimental design and artificial neural networks (ANNs) was proposed in the optimization of selectivity in capillary electrophoresis. The effect of the buffer composition, concentration, SDS concentration, ethanol percentage and the applied voltage on the separation of six choice solutes was examined by using orthogonal design. Feedforward-type neural networks with faster back propagation (BP) algorithm were applied to model the separation process, and then optimization of the experimental conditions was carried out in the modeled neural network with 5-7-1 structure, which had been confirmed to be able to provide the maximum performance. It was demonstrated that by combining ANN modeling with experimental design, the number of experiments necessary to search and find optimal separation conditions can be reduced significantly. Because of its general validity, the new proposed approach can also be applied in other separation conditions.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

Far infrared-assisted extraction followed by capillary electrophoresis for the determination of bioactive constituents in the leaves of Lycium barbarum Linn.

In this work, a method based on capillary electrophoresis with amperometric detection and far infrared-assisted extraction has been developed for the determination of rutin, gentisic acid, and quercetin in the leaves of Lycium barbarum Linn. The effects of detection potential, irradiation time, and the voltage applied on the infrared generator were investigated to acquire the optimum analysis conditions. The detection electrode was a 300-μm-diameter carbon disc electrode at a detection potential of +0.90 V. The three analytes could be well separated within 12 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) of 0.31, 0.48, and 0.78 μM for rutin, gentisic acid, and quercetin, respectively. The proposed method has been applied to determine the three bioactive constituents in real plant samples.     

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20 s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 μg/dL (1 μM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.     

Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

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.     

Separation and determination of magnolol and honokiol inMagnolia officinalis bark by capillary zone electrophoresis

The effects of pH on separation parameters such as migration mobility, resolution, sensitivity, column efficiency and peak shape were emphatically studied. Better separation of magnolol and honokiol using capillary zone electrophoresis was achieved by optimizing pH in the range 5.0–11.7. The influences of applied voltage and temperature were also investigated. We adopted a better sample extraction procedure by which higher contents of honokiol and magnolol with sample compositions unchanged were obtained. The analysis was performed with direct UV detection using a 10 mM borate-10 mM phosphate buffer at pH of 11.6. The method was successfully applied to the simultaneous determination of magnolol and honokiol inMagnolia officinalis bark within 9 min.     

Determination of diphenhydramine by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection

Tris(2,2′-bipyridyl)ruthenium(II) electrochemiluminescence detection in a capillary electrophoresis separation system was used for the determination of diphenhydramine. In this study, platinum disk electrode (300 μm in diameter) was used as a working electrode and the influence of applied potential and buffer conditions were investigated. Under optimal conditions: 1.2 V applied potential, pH 8.50, 15 kV separation voltage and 10 mmol l⁻¹ running buffer, the calibration curve of diphenhydramine was linear over the range of 4×10⁻⁸ to 1×10⁻⁵ mol l⁻¹. This technique gave satisfactory precision, and relative standard deviations of migration times and chemiluminescence peak intensities were less than 1 and 6%, respectively. The technique was applied to animal studies for determination of diphenhydramine extracted from rabbit plasma and urine samples, and the extraction efficiency were between 92 and 98.5%.     

Separation and determination of homovanillic acid and vanillylmandelic acid by capillary electrophoresis with electrochemical detection

A method of separation and determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA) was developed based on capillary zone electrophoresis/amperometric detection with high sensitivity, good resolution and selectivity. In order to achieve complete separation and good response, several factors including pH, buffer concentration, separation voltage, detection potential and the length of separation capillary, were studied in detail. The method has been used to determine both HVA and VMA in human urine. Uric acid (UA) in human urine did not interference with their determination. The limit of detection of the method was 1.3×10⁻⁶ mol/l (1.4 fmol) for HVA and 7.9×10⁻⁷ mol/l (0.87 fmol) for VMA at a signal-to-noise ratio of 3.     

Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-beta-cyclodextrin as modifier

A fast capillary zone electrophoresis (CZE) method, using dimethyl-β-cyclodextrin (DM-β-CD) as modifier, has been developed for the determination of three flavonoids (quercetin (QU), kaempferol (KA) and isorhamnetin (IS)) in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon tablet). Optimum separation was achieved with 20 mM borate buffer at pH 10.0 containing 5 mg ml⁻¹ of DM-β-CD. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992 and 0.9996) between the peak area of each compound (QU, KA and IS) and its concentration. The relative standard deviations of migration times and peak areas were <1.53 and 4.14%, respectively. The effects of several CE parameters on the resolution were studied systematically. The contents of three flavonoids in H. rhamnoides were successfully determined with 4.5 min, with satisfactory repeatability and recovery. It was also tested that the possibilities of using this method for the determination of flavonoids in Chinese medicinal preparation.     

Optimization and Parameter Study for Chiral Separation by Capillary Electrophoresis

Orthogonal design and uniform design were used for the optimization of separation of enantiomers using 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) as a chiral selector by capillary zone electrophoresis. The concentration of DM-β-CD, buffer pH, running voltage, and capillary temperature were selected as variable parameters, their different effects on peak resolution were studied by the design methods. It was concluded that orthogonal design offers a rapid and efficient means for testing the importance of individual parameters and for determining the optimum operating conditions. However, for a large number of both factors and levels, uniform design is more efficient. The effect of addition of methanol and citric acid buffer on the separation of enantiomers was also examined.     

Simultaneous Separation and Determination of Benzoic Acid Compounds in the Plant Medicine by High Performance Capillary Electrophoresis

A simple and inexpensive high performance capillary electrophoresis (HPCE) was applied to separate five benzoic acid compounds simultaneously. The investigation was carried out by micellar electrokinetic capillary chromatography (MECC). To avoid a time‐consuming and tedious procedure, orthogonal experimental design OA₉ (3⁴) for separation experiments was applied to find the optimal conditions in terms of the resolution and analytical time. The best conditions for separation were obtained using a 20 mM borax and 30 mM sodium dodecyl sulfate (SDS) buffer (pH 9.8) containing 2 mM β‐CD and 4% methanol (v/v). Online UV detection was performed at 250 nm. A voltage of 16 kV was applied and the temperature was controlled at 25 °C. Injection was performed for 5 s. The method was validated for the quantification of benzoic acid, salicylic acid and ortho‐aminobenzoic acid in Radix Isatidis, a traditional plant medicine with removal of endotoxin. The separation and determination were satisfactory and quick.     

Enantiomeric analysis of methotrexate in pharmaceuticals by cyclodextrin-modified capillary electrophoresis

A capillary electrophoresis for the chiral separation of racemic methotrexate (rac-MTX) was developed and validated. The two enantiomers were separated by using fused-silica capillary and a running buffer containing phosphate and hydroxypropyl-β-cyclodextrin (HP-β-CD). Several parameters were studied, including concentration and pH of phosphate buffer, separation voltage, and type and concentration of CD. The quantitative ranges were 12.5-200.0 μM for each enantiomer. The intra- and inter-day relative standard deviations (R.S.D.) and relative errors (R.E.) (n=5) were all <5%. The detection limits were found to be about 4 μM (S/N=3, injection 5 s) at 280 nm. All recoveries were greater than 93%. This method was applied to the assay of l-MTX in pharmaceuticals.     

Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers. The three analytes could be perfectly analyzed by using the buffer of extreme pH. The effects of several important factors were investigated to find optimum conditions. A carbon-disk electrode was used as working electrode. The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate (pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V (versus saturated calomel reference electrode, SCE) as detection potential. Under the optimum conditions, o-, m- and p-nitroaniline were separated successfully and good linearity, reproducibility and recovery results were obtained. The detection limit for m-nitroaniline was as low as at 9.06 × 10⁻⁹ mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8% for migration time and 1.1% for peak areas. The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.     

Simultaneous determination of two active ingredients in Flos daturae by capillary electrophoresis with electrochemiluminescence detection

The coupling of Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the simultaneous determination of the two major active ingredients (atropine and scopolamine) in Flos daturae. Parameters related to the separation and detection were discussed and optimized. It was proved that 20 mM phosphate buffer at pH 8.48 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.2 V. Under the optimized conditions: ECL detection at 1.2 V, 20 mM phosphate buffer at pH 8.48, 5 mM Ru(bpy)₃²⁺ and 50 mM phosphate buffer at pH 7.48 in the detection reservoir, detection limits of 5 × 10⁻⁸ mol/l for atropine and 1 × 10⁻⁶ mol/l for scopolamine were obtained. Relative standard derivations of the ECL intensity and the migration time were 5.16 and 0.71% for atropine and 5.07 and 1.22% for scopolamine, respectively. Developed method was successfully applied to determine the amounts of both alkaloids in Flos daturae. A baseline separation for atropine and scopolamine was achieved within 11 min.     

Fast enantiomeric separation of propranolol by affinity capillary electrophoresis using human serum albumin as chiral selector: application to quality control of pharmaceuticals

In the last years, capillary electrophoresis (CE) has gained considerable interest in pharmaceutical laboratories for controlling the chiral purity of drugs. This paper describes a simple and fast method for resolution of propranolol enantiomers by affinity capillary electrophoresis (ACE) using human serum albumin (HSA) as chiral selector. The effect of several experimental variables such as HSA concentration, temperature, chiral selector plug length and addition of organic modifiers, on the separation is evaluated. Complete enantioresolution of R- and S-propranolol was achieved in less than 5 min when the capillary was completely filled with 100 μM HSA solution and the electrophoresis was carried out with 67 mM phosphate buffer (pH 7.4) at 20 kV and 35 °C. Peaks were assigned to each propranolol enantiomer according to their relative affinities to HSA. The proposed method was applied to the analysis of pharmaceutical preparations containing propranolol. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed method make it suitable for quality control of the enantiomeric composition of propranolol in pharmaceuticals.