Separation of the high-ceiling diuretic Torasemide and its metabolites by capillary zone electrophoresis with diode-array detection

A capillary zone electrophoretic method was developed for the separation of the high-ceiling loop diuretic Torasemide and three of its metabolites (M1, M3 and M5) using an experimental design approach. Two different experimental designs were employed to optimize the developed method: (i) a fractional factorial design examining six factors at two levels (2(6-2)) and (ii) a central composite design examining two factors at two levels (2(2)+2x2+p). The factors studied were: pH, buffer concentration, proportion of boric acid in the mixed boric acid:potassium dihydrogen phosphate background electrolyte, temperature, applied voltage and percentage of organic modifier. Resolution between peaks was established as response. Separation of the four studied compounds was achieved in less than 8 min, using an electrolyte of 20 mM boric acid:potassium dihydrogen phosphate (75:25 v/v) with 15% MeOH adjusted to pH 9.7 with KOH, at a potential of 28 kV. Detection wavelength and temperature were 206 nm and 35 degrees C, respectively.     

三角形法和四面体法优化选择毛细管区带电泳背景电解质

建立了两种高效、快速的毛细管区带电泳背景电解质(BGE)的优化方法三角形优化法和四面体优化法。以色谱指纹图谱指数F和色谱指纹图谱相对指数Fr作为评价毛细管电泳分析系统的目标函数,以雪莲药材水提取液为样品,考察一定浓度的硼砂、硼酸、磷酸氢二钠和磷酸二氢钠溶液按三角形优化法和四面体优化法构成背景电解质时对样品的分离情况,通过添加有机改性剂和调节pH进行再优化。用三角形法优化出以50 mmol/L硼砂-含3%乙腈的150 mmol/L磷酸二氢钠(体积比为1∶1)作为BGE时分离效果最佳,用四面体法优化出以50 mmol/L硼砂-150 mmol/L磷酸二氢钠-200 mmol/L硼酸(体积比为1∶1∶2,用0.1 mol/L氢氧化钠调pH 8.55)作为BGE时分离效果最佳,分别获得28个和25个电泳峰。所建立的方法操作简捷,适用于中药材水提取液或醇提取液的毛细管区带电泳BGE的选择。     

Stability-indicating method for determination of oxyphenonium bromide and its degradation product by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for determination of oxyphenonium bromide (OX) and its degradation product. The method was based on the HPLC separation of OX from its degradation product, using a cyanopropyl column at ambient temperature with mobile phase of acetonitrile-25 mM potassium dihydrogen phosphate, pH 3.4 (50 + 50, v/v). UV detection at 222 nm was used for quantitation based on peak area. The method was applied to the determination of OX and its degradation product in tablets. The proposed method was also used to investigate the kinetics of the acidic and alkaline degradation of OX at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The pH-rate profile of the degradation of OX in Britton-Robinson buffer solutions within the pH range 2-12 was studied.     

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Binding of phosphate with a simple hexaaza polyammonium macrocycle

A mixture of dihydrogen phosphate and phosphoric acid has been crystallized with a hexaprotonated 26-membered polyammonium macrocycle, 1,4,7,14,17,20-hexaazacyclohexacosane, as the counterion. The complex crystallizes in the monoclinic space group P2(1)/c with unit cell parameters of a = 10.006(2) A, b = 12.525(1) A, c = 19.210(2) A, beta = 102.91(1) degrees, and V = 2346.6(5) A3. The hexaprotonated macrocycle is located on a crystallographic center of inversion and is surrounded by eight phosphate anions. Six of the phosphates are dihydrogen phosphates (H2PO4-), and the other two are neutral phosphoric acid molecules. Intricate hydrogen-bonding networks, involving the anionic and neutral phosphates and the protonated macrocycle, dominate the crystal lattice. Potentiometric studies using NaCl as the supporting electrolyte indicate high formation constants for the triprotonated macrocycle, H3L3+, with PO4(3-) at pH approximately 9.5 (log K = 4.55(4)), for the tetraprotonated macrocycle, H4L4+, with monohydrogen phosphate, HPO4(2-), at pH approximately 8.0 (log K = 6.01(3)), and for ditopic complexes with H5L5+ and H6L6+ and dihydrogen phosphate, H2PO4-, at pH approximately 4.0 (log K = 6.16(6)) and pH approximately 2.5 (log K = 6.44(5)), respectively. The ditopic behavior in the simple polyazamacrocycle receptor is a somewhat unusual occurrence, as is the finding of phosphoric acid species in the crystal structure.     

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.     

Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography

A method of micellar electrokinetic capillary electrophoresis for determining the six flavonoids in Scutellariae Radix (i.e., baicalin, baicalein, wogonin 7-O-glucuronide, wogonin, oroxylin A 7-O-glucuronide, and oroxylin A) has been developed. The buffer solution (pH 7.24) composed of 20 mM sodium dodecyl sulfate (SDS), 20 mM sodium dihydrogen phosphate, and 25 mM sodium borate was found to be the most suitable electrolyte for the separation. The contents of the six flavonoids in crude Scutellariae Radix could easily be determined within about 15 min. On-column UV (254 nm) monitoring allowed the quantitative determination of baicalin. The effects of pH, surfactant concentration, and applied voltage on the migration behavior of the solutes were studied.     

New validated liquid chromatographic and chemometrics-assisted UV spectroscopic methods for the determination of two multicomponent cough mixtures in syrup

Multivariate spectrophotometric calibration and liquid chromatographic (LC) methods were applied to the determination of 2 multicomponent mixtures containing diprophylline, guaiphenesin, methylparaben, and propylparaben (Mixture 1), or clobutinol, orciprenaline, saccharin sodium, and sodium benzoate (Mixture 2). For the multivariate spectrophotometric calibration methods, principal component regression (PCR) and partial least-squares regression (PLS-1), a calibration set of the mixtures consisting of the components of each mixture was prepared in 0.1 M HCl. Analytical figures of merit such as sensitivity, selectivity, limit of quantitation, and limit of detection were determined for both PLS-1 and PCR. The LC separation was achieved on a reversed-phase C18 analytical column by using isocratic elution with 20 mM potassium dihydrogen phosphate, pH 3.3-acetonitrile (55 + 45, v/v) as the mobile phase and UV detection at 260 and 220 nm for Mixture 1 and Mixture 2, respectively. The proposed methods were validated and successfully applied to the analysis of pharmaceutical formulations and laboratory-prepared mixtures containing the 2 multicomponent combinations.     

Simultaneous assay of olanzapine and fluoxetine in tablets by column high-performance liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance liquid chromatographic (LC) and high-performance thin-layer chromatographic (TLC) methods for the simultaneous estimation of olanzapine and fluoxetine in pure powder and tablet formulations. The LC separation was achieved on a Lichrospher 100 RP-180, C18 column (250 mm, 4.0 mm id, 5 microm) using 0.05 M potassium dihydrogen phosphate buffer (pH 5.6 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1 mL/min and ambient temperature. The TLC separation was achieved on aluminum sheets coated with silica gel 60F254 using methanol-toluene (40 + 20, v/v) as the mobile phase. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 10-70 and 40-280 microg/mL with mean recovery of 99.54 +/- 0.89 and 99.73 +/- 0.58% for olanzapine and fluoxetine, respectively, by the LC method. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 100-800 and 400-3200 ng/spot with mean recovery of 101.53 +/- 0.06 and 101.45 +/- 0.35% for olanzapine and fluoxetine, respectively, by the TLC method with densitometry. These methods are simple, precise, and sensitive, and they are applicable for simultaneous determination of olanzapine and fluoxetine in tablet formulations.     

毛细管区带电泳法同时测定饮料中16种食品添加剂

建立了毛细管区带电泳法测定饮料中酸性红92、专利蓝V、荧光素二钠、酸性红1、靛蓝胭脂红、亮黑、丽春红6R、日落黄、苋菜红、柠檬黄等10种人工合成色素和苯甲酸、山梨酸、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯等6种防腐剂的分析方法。考察了缓冲溶液种类、浓度、pH值及运行电压、温度等对分离的影响,确定最佳电泳条件为: 未涂层弹性石英毛细管柱(46 cm×50 μm),缓冲溶液为70 mmol/L硼酸(pH=9.5)(含体积分数为4%的乙腈),检测波长220 nm,电泳电压30 kV,进样时间5 s,电泳温度25 ℃。该法用于测定市售饮料样品得到满意结果: 在1～250 mg/L范围内线性关系良好,相关系数不小于0.9938,回收率在95.8%与108.7%之间。该法简便、准确,能够满足食品添加剂的常规检测要求。     

HPLC Determination of Fexofenadine in Human Plasma For Therapeutic Drug Monitoring and Pharmacokinetic Studies

A simple and sensitive method was developed for fexofenadine determination in human plasma by liquid chromatography with ultraviolet detection. Satisfactory separation was achieved on a Hypersil® BDS C₁₈ column (250 × 4.6 mm, 5μm) using a mobile phase comprising 20 mm sodium dihydrogen phosphate‐2 hydrate (pH adjusted to 3 with phosphoric acid)–acetonitrile at a ratio of 52:48, v/v. The elution was isocratic at ambient temperature with a flow rate of 1.0 mL/min. The UV detector was set at 215 nm for the drug and 330 nm for the internal standared (tinidazole). The total time for a chromatographic separation was ~6.5 min. Linearity was demonstrated over the concentration range 0.01–4 μg/mL. The observed within‐ and between‐day assay precision ranged from 0.346 to 13.6%; accuracy varied between 100.4 and 111.2%. This method was successfully applied for therapeutic drug monitoring in patients treated with clinical doses of fexofenadine and for pharmacokinetic studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Method development and validation for the simultaneous determination of four coumarins in Saussurea superba by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the determination of four coumarins--skimmin, scopolin, scopoletin, and umbelliferone-in Saussurea superba with UV detection at 254 nm. The capillary temperature was kept constant at 25 degrees C. Effects of buffer pH, electrolyte concentration, organic modifier, and applied voltage on migration behavior were studied systematically. The optimum conditions for separation were achieved by using 30 mM borate buffer at pH 9.02 containing 15% (v/v) methanol as the electrolyte and 25 kV as the applied voltage. For all analytes a good linear regression relationship (r > 0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, and accuracy. The validated method was successfully applied to the simultaneous determination of the four analytes in S. superba.     

Simultaneous determination of myocardial creatine phosphate and adenine nucleotides by reversed-phase HPLC

An isocratic HPLC system has been developed which allows for the rapid (single run of 20 min) measurement of creatine phosphate (PCr) and adenine nucleotides (ATP, ADP and AMP) in extracts from freeze-clamped and freeze-dried myocardial tissues. The separation was achieved at room temperature by using a RP18 column and a dual variable wavelength spectrophotometer, set at 210 and 254 nm. The solvent was 30 mM potassium dihydrogen phosphate, 15 mM tetrabutylammonium hydrogen sulfate, pH 6.7, 19% (v/v) acetonitrile. A distinct separation (confirmed with the retention time of standard sample) of these high energy compounds was achieved. Standard curves were linear. In isolated rat hearts the following values were obtained (mumol/g dry wt, mean +/- SEM): ATP 21.5 +/- 1.3, ADP 4.6 +/- 0.2, AMP 1.5 +/- 1.1 and PCr 32.5 +/- 1.3; which are consistent with previously published values for high energy compounds in this tissue.     

Separation of Hydroxybenzoic Acid Isomers by Capillary Zone Electrophoresis

Abstract

Capillary zone electrophoresis is a highly efficient analytical technique that has been shown to be particularly useful for the analysis of isomers. Using a cathodic injection and anodic detection scheme, ortho-, meta- and para-hydroxybenzoic acids were separated in a fused-silica capillary column with a phosphate buffer at pH 10.3 (25.0 mmol/1 phosphate + 0.20 mmol/1 cetyltrimethylammonium bromide (CTAB) + 10% (v/v) 1-propanol with an applied voltage of 10 KV followed by direct UV detection. The use of CTAB as electroosmotic flow modifier allows the rapid separation of the three isomers by reversing the direction of electro-osmotic flow. The influence of pH, CTAB concentration and organic solvents on the migration behaviour of the solutes were also studied.     

Different sample stacking strategies for the determination of ertapenem and its impurities by micellar electrokinetic chromatography in pharmaceutical formulation

Ertapenem, a Group 1 carbapenem, is most recently introduced into the market. It is a beta-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens, but low activity against some nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter spp., enterococci and methicillin-resistant staphylococci. The elaborated method of micellar electrokinetic chromatography (MEKC) of ertapenem separation from its impurities was successfully performed using normal stacking mode (NSM) and stacking with reverse migrating micelles (SRMM), followed by UV absorption detection at 214 nm. The best results were obtained with 60mM sodium dihydrogen phosphate and 20mM boric acid buffer pH 6.0, as background electrolyte. Uncoated fused-silica capillary and neutral-coated capillary with normal and reverse polarity, and voltage values of +18 and -12 kV, respectively, were used throughout the investigation. Sodium dodecyl sulfate was employed as the pseudostationary phase. A comparison of applied techniques, including sensitivity enhancement factors and limits of detection (LOD), is presented. The optimized method was validated in terms of linearity, accuracy and precision. Comparable LOD was obtained using both stacking methods (0.3 microg/mL) but better efficiency of ertapenem peak was obtained using NSM. Under the optimum stacking conditions, about 183-4.75-fold and 1289-4.07-fold improvements in peak areas were obtained for NSM and SRMM, respectively, compared to the usual hydrodynamic sample injection (10s). The reproducibility, expressed by relative standard deviations (RSD) of the migration times, for NSM was about 0.96-1.25 and for SRMM was 0.32-0.45. The RSD of corrected peak areas, for NSM was about 1.07-8.14 and for SRMM was 0.74-8.12. The difference in separation time between the two techniques was not obvious. Satisfactory separation was possible after less than 11min of electrophoresis. The evaluated MEKC method was applied in the analysis of medicinal product containing ertapenem: Invanz-ertapenem for injection.     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.