Development of a NACE method for simultaneous measurement of three adenosine monophosphate isomers in biomimicking prebiotic synthesis without sample pretreatment

A practical NACE method was developed for simultaneous determination of three adenosine monophosphate (AMP) isomers. Separation of three AMP isomers was achieved using 200 mM Tris/H(3)BO(3) in acetontrile/water (2:1 v/v) at pH* 10.0 as the running buffer and +25 kV as the applied voltage over a bare fused-silica capillary of 50 microm id x 375 microm od x 54.5 cm (46 cm to the detector window). At 260 nm, the calibration curves were linear in the range of 1-100 microg/mL. The detection limits were less than 0.70 microg/mL. The recovery ranged from 94.5 to 106.4%. The intraday RSDs of the migration times were between 2.1 and 3.0%. The developed NACE method has been successfully applied for the determination of three AMP isomers in the real samples of biomimicking prebiotic synthesis reaction between N-(O,O-diisopropyl) phosphoryl amino acid and adenosine.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Analysis of metformin dosage formulations by capillary electrophoresis at nano scale detection

An inexpensive, rapid and reproducible capillary electrophoretic method has been developed and validated for the determination of metformin in pharmaceutical preparations. The method was developed utilizing a fused silica capillary (60 cm x 50 microm I.D.), phosphate buffer (50 mM, 3.0 pH)-acetonitrile (95:5, v/v) as background electrolyte (BGE), 20 kV applied voltage with UV detection at 254 nm and at a working temperature of 23 +/- 1 degrees C. Linearity was observed in the concentration range from 100 ng/L to 5 microg/L, with a correlation coefficient (R2) of 0.9998. The limits of detection and quantification achieved were 60 and 100 ng/mL, respectively. The recovery of metformin from pharmaceutical preparations was 99.1%. These validation parameters demonstrate the precision of the method and its suitability for the determination of metformin in pharmaceutical tablet formulations.     

Analysis of colistin sulfate by capillary zone electrophoresis with cyclodextrins as additive

A method for the quantitative analysis of colistin sulfate by capillary zone electrophoresis is described. Since colistin components have five free amino groups, they tend to adsorb onto the capillary wall and cause peak tailing. It was found that triethanolamine (TEA)-phosphate buffer at pH 2.5 was useful to reduce such adsorption. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found necessary for selectivity enhancement. In order to optimize the separation parameters and predict the method robustness, a central composite design was performed including three variables, namely concentration of M-beta-CD, TEA, and IPA. The effects of capillary length and applied voltage on separation were also investigated. The optimal conditions established were: 140 mM TEA-phosphate buffer containing 5 mM M-beta-CD and 6% v/v IPA, a capillary with 55 cm total length (50 microm inner diameter, 47 cm from inlet to detection window) and 24 kV applied voltage. The method was found to be robust when the variables were changed in the following range: 4-6 mM M-beta-CD, 5-7% v/v IPA, and 130-150 mM TEA. Further, the linearity, limit of detection (LOD), and limit of quantitation (LOQ), as well as repeatability for both colistin A and B were examined and three commercial samples were quantitatively analyzed.     

Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on-column field-amplified sample stacking and its application in Alzheimer's disease

Field-amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid-liquid extraction with isopropanol/n-hexane (v/v 3:97) and subsequent quantification by FASS-CE was used. Before sample loading, a water plug (0.5 psi, 6 s) was injected to permit FASS. Electrokinetic injection (7 kV, 90 s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60 mM, pH 4.0) with sodium octanesulfonate 40 mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28 kV and detected at 200 nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1-50 ng/mL. The limit of detection was 0.1 ng/mL (S/N=3, sampling 90 s at 7 kV). One female volunteer (54 years old) was orally administered a single dose of 10 mg donepezil (Aricept, Eisai), and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Simultaneous Determination of Atenolol and Chlorthalidone in Pharmaceutical Preparations by Capillary-Zone Electrophoresis

Abstract

A capillary zone electrophoresis (CZE) method for the simultaneous determination of the β-blocker drugs atenolol and chlorthalidone in pharmaceutical formulations has been developed. The CZE separation was performed under the following conditions: capillary temperature, 25°C; applied voltage, 25 kV; 20 mM H₃PO₄–NaOH running buffer (pH 9.0); and detection wavelength, 198 nm. Phenobarbital was used as internal standard. The method was validated and showed not only good precision and accuracy but also good robustness. The method has been successfully applied to the simultaneous determination of both atenolol and chlorthalidone in pharmaceutical tablets.     

Analysis of metacycline by capillary electrophoresis

The development and validation of an optimized capillary electrophoresis method for the determination of metacycline in the presence of its related substances by capillary electrophoresis is shown. The influence of methanol as organic modifier, buffer pH, buffer concentration, capillary length, column temperature, Triton X-100 and methyl-beta-cyclodextrin was investigated. A central composite design was performed in order to optimize the method. The optimal separation conditions were: uncoated fused-silica capillary (39 cm total length, 31 cm effective length, 50 microm ID); as background electrolyte a solution of 160 mM sodium carbonate and 1 mM EDTA (pH 10.35)/methanol (89:13 v/v); temperature, 15 degrees C; voltage, 12 kV. The method showed good selectivity, repeatability, linearity, and sensitivity. The limits of detection and quantitation are 0.024% and 0.06%, respectively, relative to a 2.5 mg/mL solution. Six commercial samples were analyzed quantitatively.     

Analysis of the aconitine alkaloids in traditional Chinese medicines by nonaqueous capillary electrophoresis using a new recording mode

The determination of three aconitine alkaloids (hypaconitine, aconitine, mesaconitine) in five traditional Chinese medicines including two Tibetan medicines, Chuanwu, Caowu, Fuzi, Aconitum Tanguticum Maxim and Aconitum Gymnandrum Maxim by non-aqueous capillary electrophoresis using a new recording mode is described. The dissociation constants of aconitine, mesaconitine and hypaconitine have also been determined by CZE and were 7.71, 6.60 and 6.25, respectively. The separation was achieved by optimizing the applied voltage, the pH and the concentration of the buffer. The electrophoretic medium was 20 mM borax-70% (v/v) methanol (pH 8.5) and an uncoated capillary (50 cm x 75 microm i.d.) was used. Detection was carried out with a UV monitor at 214 nm. The total time for separation and determination was under 13 min.     

Analysis of aromatic and terpenic constituents of pepper extracts by capillary electrochromatography

An original method based on CEC has been developed for the determination of aromatic and terpenic compounds in extracts of spices obtained from Piper nigrum. The method is based on the use of a fused silica capillary (effective length: 23.5 cm, internal diameter: 100 microm) packed with a C18 sorbent (packing length: 23 cm, particle size: 5 microm). The mobile phase is a 50 mM, pH 6.0 ammonium acetate/ACN (10:90 v/v) mixture. Applying a 30 kV voltage, the following 11 compounds were separated and analysed: terpinen-4-ol, caryophyllene oxide, limonene, alpha-pinene, 3-carene, beta-pinene, alpha-humulene, beta-caryophyllene, alpha-phellandrene, eugenol and piperine. Compound determination is carried out using a diode-array detector set at 265 and 338 nm for alpha-phellandrene and piperine, respectively, and at 210 nm (reference subtraction at 282 nm) for all the other analytes. The optimised method has been validated with good results in terms of linearity, limits of quantitation, detection and precision. The CEC method was successfully applied to the analysis of essential oils and methanolic extracts of 'black', 'white' and 'green' pepper.     

Validated Assay for the Determination of Mitoxantrone in Pharmaceuticals Using Capillary Zone Electrophoresis

Abstract

The first capillary zone electrophoretic (CZE) method for the determination of mitoxantrone (MTX) in pharmaceutical formulations was developed. The influence of background electrolyte (BGE) species, pH, concentration (c _BGE), organic modifier, capillary temperature, applied voltage, and injection time was investigated. Optimum results were achieved with 25 mM ammonium acetate at an apparent pH value of 5.0 in 50% v/v acetonitrile, applied voltage of +30 kV, and capillary temperature of 25°C. The samples were introduced into the capillary hydrodynamically for 2 s at 33.5 mbar. Mitoxantrone was detected at a wavelength of 242 nm. Mitoxantrone and doxorubicin (DOX) (used as internal standard, ISTD) were completely separated in less than 7 min. The method was suitably validated with respect to linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision, selectivity, and robustness. The proposed method was applied successfully for the determination of MTX in its injectable pharmaceutical formulation.     

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography 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.     

Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis.

A capillary electrophoresis method was developed for the separation and determination of tropane alkaloids in Flos daturae plants. Separation was performed on a fused silica capillary(42.1 cm x 50 microm i.d.) at an applied voltage of 20 kV. Scopolamine, atropine and anisodamine were well separated in the buffer of 50 mmol/L phosphate buffer (pH 5.0) containing 20% (v/v) tetrahydrofuran (THF). Beer's law was obeyed in the range of concentration of 2.4-21.8 microg/mL for scopolamine, 4.0-36.0 microg/mL for atropine and 2.6-23.7 microg/mL for anisodamine, respectively, and the correlation coefficients were over 0.999 (n = 6). The developed method was applied for the analysis of herb samples.     

Separation of sympathomimetic amines of abuse and related compounds by micellar electrokinetic chromatography.

Separation of twelve sympathomimetic amines and related compounds by micellar electrokinetic chromatography (MEKC) with UV absorbance detection is described. These amines were well separated within 25 min using 50 mM sodium tetraborate solution containing 15 mM sodium dodecylsulfate (SDS) of pH 9.3 as a running solution and detected at 210 nm. MEKC was performed with an applied voltage of 13 kV at 25 degrees C using a fused-silica capillary (50 cm x 75 mm i.d.) with effective length of 37.5 cm. The detection limits of these compounds were in the range from 4 to 97 fmol/injection at a signal-to-noise ratio (S/N) of 3. The reproducibility of the method expressed as relative standard deviation (RSD) for within-day (n=6) and between-day (n=5) assays was less than 4.8 and 8.8%, respectively. The proposed method could be applied to the determination of an anorectic drug, phentermine, in Chinese tea with a detection limit of 99 microg/g (105 fmol/injection, S/N=3).     

Determination of brompheniramine enantiomers in rat plasma by cation‐selective exhaustive injection and sweeping cyclodextrin modified electrokinetic chromatography method

A method consisting of cation‐selective exhaustive injection and sweeping (CSEI‐sweeping) as online preconcentration followed by a cyclodextrin modified electrokinetic chromatography (CDEKC) enantioseparation has been developed for the simultaneous determination of two brompheniramine enantiomers in rat plasma. In this method, analytes were electrokinetically injected at a voltage of 8 kV for 80 s in a fused‐silica capillary. Prior to the injection, the capillary was rinsed with 50 mM phosphate buffer of pH 3.5, followed by a plug of a higher conductivity buffer (150 mM phosphate pH 3.5, 20 psi, 6 min) and a plug of water (0.5 psi, 5 s). Separation was carried out applying –20 kV in 50 mM phosphate buffer, pH 3.5, containing 10% v/v ACN and 30 mg/mL sulfated‐β‐cyclodextrin (S‐β‐CD). Analytical signals were monitored at 210 nm. The detection sensitivity of brompheniramine enantiomers was enhanced by about 2400‐fold compared to the normal injection mode (hydrodynamic injection for 3 s at 0.5 psi, with a BGE of 50 mM phosphate buffer containing 20 mg/mL S‐β‐CD at pH 3.5), and LLOQ of two enantiomers were both 0.0100 μg/mL. In addition, this method had fairly good repeatability and showed promising capabilities in the application of stereoselective pharmacokinetic investigations for brompheniramine enantiomers in rat.     

Determination of ticagrelol in rat plasma and tablets by micellar electrokinetic chromatography coupled with large volume sample stacking: Application to a pharmacokinetic study

A method using micellar electrokinetic chromatography coupled with large-volume sample stacking for the determination of ticagrelol was developed and validated. The analysis was performed in a fused silica capillary (41.5 cm effective length, 50 μm diameter) with ultraviolet detection at 195 nm. The background electrolytes were 30 mM phosphate buffer of pH 3.0 with 120 mM sodium dodecylsulfate and 10 % (v/v) acetonitrile (120 s X 50 mbar; 20°C; -18 kV) and 30 mM borate buffer of pH 8.5 with 75 mM sodium dodecylsulfate (120 s X 50 mbar; 20°C; 25 kV); under acidic and alkaline conditions, respectively. The method was found to be reliable with respect to specificity, linearity of the calibration line (R² > 0.99), repeatability (relative standard deviation 2.56%–3.34%), and accuracy (recovery in the range 101.21%–102.67%). The limits of detection and quantitation were 0.032, 0.071, and 0.087, 0.188 μg/mL, respectively. The method was successfully applied for the determination of ticagrelol concentrations in rat plasma and tablets with good recoveries and reproducibility. The presented method proved to be suitable for monitoring ticagrelor in rat plasma.     

逍遥丸的毛细管电泳指纹图谱的建立

采用毛细管区带电泳法建立了逍遥丸(Xiaoyao Pill, XYP)的毛细管电泳指纹图谱(CEFP)。运用正方形优化法,以色谱指纹图谱分离量指数(RF)为优化的目标函数,对建立指纹图谱的实验条件进行了优化,确定了最佳背景电解质(BGE)溶液50 mmol/L硼砂-50 mmol/L磷酸氢二钠-150 mmol/L磷酸二氢钠-50 mmol/L碳酸氢钠(1:1:1:5, v/v/v/v; pH 7.40)、紫外检测波长228 nm、运行电压12 kV、重力进样25 s (高度14 cm)的分离检测条件。采用未涂层石英毛细管(70 cm×75 μm,有效分离长度57 cm)分离,以咖啡酸色谱峰为参照,确定13批逍遥丸样品的21个共有指纹峰。通过聚类分析确定用其中10批样品生成对照CEFP,以此为标准用系统指纹定量法鉴别13批逍遥丸的质量,结果显示: S3号样品的化学成分数量和分布比例不合格,S10和S12号样品含量明显偏高,其余批次质量均合格。所建立的正方形优化法操作简便,适用于中药的毛细管区带电泳BGE的选择;所建立的逍遥丸CEFP具有较好的精密度和重现性,可以为逍遥丸的质量控制提供新的参考。     

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.