Development of a capillary electrophoresis method for the enantioselective estimation of primaquine in pharmaceutical formulations

A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).     

毛细管电泳法快速检测消毒剂中的醋酸氯己定

建立了消毒剂中活性成分醋酸氯己定(又名:醋酸洗必泰)的毛细管电泳快速检测法。采用15 mmol/L磷酸盐-乙腈( 体积比为60∶40)缓冲体系,将醋酸氯己定在50 cm×75 μm i.d.的石英毛细管柱中进行电泳分离,电泳电压为15 kV,检 测波长为254 nm。同时,对毛细管电泳分析醋酸氯己定的条件(如缓冲液的种类、pH值、浓度及电泳电压等)进行了优化 。用该方法对消毒剂样品进行测定,在4 min内可完成分析。醋酸氯己定在质量浓度为0.01～0.10 g/L时线性良好,检测 限为0.004 mg/L,吸光度值的相对标准偏差为3.97%,迁移时间的相对标准偏差为2.99%,样品加标回收率为91.4%～116.6%。将该方法 与高效液相色谱法进行比较,两种方法测定结果的相对误差≤4%。所建立的检测醋酸氯己定含量的毛细管区带电泳法简单 、快速,适用于消毒剂样品的测定。     

Simultaneous capillary electrophoretic separation and detection of P(V) and As(V) As heteropoly-blue complexes

A capillary electrophoretic (CE) method was developed for the simultaneous determination of P(V) and As(V). A Mo(VI)-ascorbic acid reagent reacted with a mixture of trace amounts of P(V) and As(V) to form the corresponding heteropoly-blue complexes in 0.05 M acetate buffer (pH 3.5). When 0.05 M malonate buffer was used as a migration buffer, the peaks due to their migrations were well separated in the electropherogram, and the pre-column complex-formation reaction was applied to the simultaneous CE determination of P(V) and As(V) with direct UV detection at 220 nm. With the proposed method, the calibration curves were linear in the concentration range of 5 x 10(-7) - 1 x 10(-4) M, with a detection limit of 1 x 10(-7) M (a signal-to-noise ratio of 3). Interference from foreign ions was also discussed.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1x10(-9) M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE-LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5-4.1x10(-7) M.     

Separation and determination of vanadium in fertiliser by capillary electrophoresis with a light-emitting diode detector

A method has been developed for determination of vanadium, as an anionic ternary complex of vanadium(V) with 4-(2-pyridylazo) resorcinol (PAR) and hydrogen peroxide, after separation by capillary electrophoresis (CE). The optimum conditions for the formation of the ternary complex were acetate buffer (3 mmol L(-1)) at pH 6 containing 0.15 mmol L(-1) PAR and 7.1 mmol L(-1) H(2)O(2). The CE separation was conducted using 15 mmol L(-1) acetate buffer at pH 6 as the background electrolyte; the separation potential was -30 kV and the injection time 100 s. The vanadium complex was detected photometrically at 568 nm, by use of a light-emitting diode (LED); the detection limit was 19 ppb. The method was applied to the analysis of vanadium in fertilisers. Clean-up of the digested fertiliser sample, with Sep-Pak C(18) coated with tetrabutylammonium hydroxide, before analysis was used to remove matrix ions which otherwise caused electrophoretic de-stacking. Vanadium levels found in the fertiliser samples by use of the CE method were found to be comparable with results obtained by HPLC and ICP-MS.     

Non-aqueous capillary electrophoresis with diode array and electrospray mass spectrometric detection for the analysis of selected steroidal alkaloids in plant extracts

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.     

荧光猝灭法测定壳聚糖含量

在pH 6.3的NaH2PO4-Na2HPO4缓冲溶液中,壳聚糖对荧光素的荧光强度具有明显的猝灭作用,且在一定浓度范围内,其猝灭程度与加入的壳聚糖浓度成线性关系,据此建立了一种新的测定壳聚糖含量的荧光猝灭分光光度法。该方法的回归方程为ΔF=64.02+42.28ρ(mg/L),R2=0.9942,线性范围为0.50~10.0 mg/L,检出限为0.27 mg/L。样品测定的RSD为4.5%(n=6),平均回收率为99.3%。采用该方法可测定复杂样品中的壳聚糖含量。     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE-EC). CE was performed in 0.20 mol L(-1) phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-microm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 micromol L(-1); detection limits (S/N = 3) were 0.03-0.13 micromol L(-1). The proposed method has been successfully used to analyze real biological samples.     

毛细管电泳-激光诱导荧光用于中药制剂中氨基酸成分的分析

建立了一种用于测定中药制剂中氨基酸成分的毛细管电泳-荧光检测方法. 用含有α-环糊精(α-CD)的硼砂缓冲溶液为背景电解质, 经异硫氰酸荧光素(FITC)衍生的5种氨基酸在50 min内可以得到很好的分离和测定. 考查了各个分离参数对分离的影响, 得到的优化条件为: 含45 mmol/L的α-环糊精的80 mmol/L硼砂缓冲溶液(pH值9.2)作为背景电解质, 分离电压20 kV; 柱温22 ℃. 衍生试剂FITC与单个氨基酸的化学计量比为4∶1时, 能够获得稳定荧光强度的氨基酸衍生物. 在优化条件下, 各氨基酸成分在73.5～2900 nmol/L 的浓度范围内呈良好的线性关系(相关系数r2为0.9906～0.9998). 保留时间和峰面积的相对标准偏差分别为0.8%～3.0%和0.7%～5.7%, 检测限(3倍信噪比)为3.5～35 nmol/L. 该方法准确可靠, 可用于质量控制为目的的中药制剂中氨基酸成分的定量测定.     

CE coupling with end-column electrochemiluminescence detection for chiral separation of disopyramide

CE with electrochemiluminescence (ECL) detection technique was successfully applied for the chiral separation of a kind of class IA antiarrhythmic racemic drug. To the best of our knowledge, this is the first report of ECL detection used in chiral CE. To get better detection sensitivity and good enantioresolution at the same time, the conditions of capillary inlet and outlet buffer were systematically optimized. Unlike the traditional chiral separation method, the buffers we used in the capillary inlet and outlet differed from each other in terms of buffer pH, ionic strength, type of BGE as well as buffer composition. Under the optimum conditions, baseline enantioseparation and highly sensitive detection of the enantiomers were achieved. Wide linear relationship of each enantiomer was achieved in the range of 5 x 10(-7) to 2 x 10(-5) mol/L with relative coefficients of 0.996 and 0.997, respectively. The detection limits were estimated to be 8 x 10(-8) and 1.0 x 10(-7) mol/L (S/N = 3) for the enantiomers, respectively. In addition, a successful application of this new method to the chiral separation of the racemic drug in spiked plasma samples confirmed the validity and applicability of the chiral CE-ECL method.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.     

Validation of a capillary electrophoresis method for the simultaneous determination of amlodipine besylate and valsartan in pharmaceuticals and human plasma

A rapid, simple, and sensitive capillary electrophoresis (CE) method was developed and validated for the simultaneous determination of amlodipine (AML) and valsartan (VAL) in pharmaceuticals and human plasma using a UV photodiode array detector. Electrophoretic conditions were optimized to improve separation, sensitivity, and rapidity. The optimal conditions were 25 mM phosphate buffer at pH 8.0, injection time 10.0 s, voltage 25 kV, and column temperature 25 degrees C, with detection at 214 nm. The method was found to be linear in the range of 1.0-35 and 1.0-350 mg/L, with weighted regression 0.9999 and 0.9994, for AML and VAL, respectively. Validation of the method showed acceptable intraday and interday accuracy (85.5-95.3%) and precision (RSD 1.64-4.2%) in pharmaceutical formulation and human plasma analysis. The sensitivity of the method was enhanced by both optimization of the CE procedure and preconcentration performed by liquid-liquid extraction. The LOD for both AML and VAL was 0.03 mg/L, which allows analysis at the level of the drugs possibly found in human plasma. Therefore, the proposed method is suitable for QC in pharmaceutical laboratories and therapeutic drug monitoring in clinical laboratories.     

Separation of organic and inorganic arsenic species by capillary electrophoresis using direct spectrophotometric detection

Capillary electrophoresis (CE) with UV detection was used for the determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, p-aminophenylarsonic acid, 4-hydroxy-3-nitrobenzenearsonic acid, 4-nitrophenylarsonic acid, phenylarsonic acid, and phenylarsine oxide. The electrophoretic mobilities of these anionic species were determined in a 20 mM phosphate buffer in a pH range from 4 to 11, which established pH 10 as the optimum for the separation. The target analytes were then separated in a fused-silica capillary using 20 mM NaHCO(3)-Na(2)CO(3) buffer, pH 10, as electrolyte and detected at 192 nm. Both normal- and reversed electroosmotic flow (EOF) separation modes were investigated and in the latter case, poly(diallydimethylammonium chloride) (PDDAC), was used for dynamic coating of the capillary and to provide a stable and reproducible reversed EOF (relative standard deviation RSD, 0.39%). The influence of electrolyte pH and composition, applied voltage, as well as EOF reversal protocols upon the method performance criteria were investigated. The optimised method provided limits of detection for the target analytes of 1.62, 6.22, 1.45, 1.83, 0.34, 0.40, 0.40, 0.18, and 0.30 mg/L As, respectively. Linearity was obtained in the range of 0.5-40 mg/L As (for aryl compounds) and from 5-100 mg/L As (for the remaining analytes). Reproducibility of peak areas was in the range of 0.8-5.5% RSD. The method was applied to the determination of four aryl arsenic compounds used as additives in animal feed. Analytes were extracted with 40 mM hydrochloric acid - acetonitrile 4:1 v/v, and then cleaned up by passing through a C(18) solid-phase extraction cartridge before analysis by CE with detection at 200 nm. Recoveries for the four analytes were in the range of 78.8-108.3%.     

Development of a capillary electrophoresis method for the characterization of collagens in cartilage tissue

The use of capillary electrophoresis (CE) for the separation of peptides specific to type I and type II collagen is evaluated. The aim of this work is to develop a method to characterize cartilage, cartilage repair tissue, and tissue engineered cartilage. The analysis is dependent on the cleavage of collagen into constituent peptides by cyanogen bromide. A number of these peptides are specific to the collagen type. CE is evaluated for the separation of these specific peptides using uncoated and coated capillaries over a wide range of pH and buffer concentrations. Separation of peptides specific to type I and type II collagen is achieved using a Supelco CElect N capillary and a 100mM phosphate buffer at pH 6. Meniscal cartilage is characterized using this method. The proportion of type I collagen to type II collagen corresponds well with that reported by others and indicates the potential of this method for the characterization of cartilage.     

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser-induced fluorescence detector

A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na?B?O?/NaH?PO? buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.     

Simultaneous determination of phosphonate, phosphate, and diphosphate by capillary electrophoresis using in-capillary complexation with Mo(VI)

This study describes the simultaneous determination of phosphonate, phosphate, and diphosphate by CE with direct UV detection, based on in-capillary complexation with Mo(VI). When a mixture of phosphonate, phosphate, and diphosphate was injected into a capillary containing 3.0 mM Mo(VI), 0.05 M malonate buffer (pH 3.0) and 45% v/v CH3CN, three well-defined peaks, due to the migration of the corresponding polyoxomolybdate anions, were separated. The respective calibration graphs were linear in the concentration range of 2 x 10(-6)-2 x 10(-4) M for phosphonate, 1 x 10(-6)-5 x 10(-5) M for phosphate, and 1 x 10(-6)-2 x 10(-4) M for diphosphate; the correlation coefficients were better than 0.9990. The present CE method is successfully applied to the simultaneous determination of phosphonate, phosphate, and diphosphate in tap water.     

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.     

高效毛细管电泳安培法测定肉苁蓉中松果菊苷的含量

采用毛细管电泳安培法建立了肉苁蓉中松果菊苷含量的检测方法,探讨了缓冲溶液种类、工作电位、分离电压、进样时间等对分离和检测的影响.在15 mmol/L硼砂缓冲溶液(pH 9.5),25 kV电压,0.6 V工作电位的条件下,松果菊苷的质量浓度在0.5～50.0 mg/L范围内与峰面积呈良好线性,相关系数为0.9994,检...     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE–EC). CE was performed in 0.20 mol L^–1 phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-μm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 μmol L^–1; detection limits (S/N = 3) were 0.03–0.13 μmol L^–1. The proposed method has been successfully used to analyze real biological samples.