Determination of gallic acid and salidroside in Rhodiola and its preparation by capillary electrophoresis

A new, simple, and rapid capillary electrophoresis (CE) method employing hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier was developed for the identification and quantitative determination of two pharmaceutically active constituents—gallic acid (GA) and salidroside (S)—in extracts of Rhodiola root and its medicinal preparation. The optimum separation was achieved at pH 11.00 with the use of 10 mM borate buffer containing 0.001% (w/v) of HDB. The applied voltage was ∼15 kV and the capillary temperature was kept constant at 25°C. m-Phthalic acid was used as an internal standard for quantification. The calibration dependences exhibited good linearity for the ratios of the concentrations of standard samples and internal standard and the ratios of the peak area of samples and internal standard over the concentration range from 24 to 1200 μg/mL for GA and 2.4 to 72 μg/mL for S. The correlation coefficients were 0.9999 and 0.9997, and the detection limits of the CE method corresponding to a signal-to-noise ratio of three were 6 and 2 μg/mL for GA and S, respectively. The relative standard deviations of the relative migration time and the relative peak area of samples were 0.5 and 4.0% for GA and 1.9 and 5.3% for S. The effects of buffer pH and the concentration of HDB on the resolution were studied systematically. The contents of these two active compounds in Rhodiola root and its preparation were successfully determined over 6 min with satisfactory repeatability and recovery. The text was submitted by the authors in English.     

Direct Determination of Barbiturates in Urine by Capillary Electrophoresis Using a Capillary Coated Dynamically with Polycationic Polymers

A new, simple and rapid capillary electrophoresis method, using hexadimethrine bromide (HDB) as electro-osmotic flow modifier, has been developed for the identification and determination of nine barbiturates, barbital acid, barbital, phenobarbital, pentobarbital, amobarbital, thiobarbituric acid, butobarbital, N-methyl-5-phenyl-ethyl barbital acid and 5-cyclohexenyl-5-ethyl barbital acid in urine with UV detection at 200 nm. The applied voltage was ?25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of HDB and the concentration of α-cyclodextrin were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.04% (w/v) HDB and 2.06 mM α-cyclodextrin. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. The correlation coefficients were from 0.9990 to 0.9997. The relative standard deviations of migration times and peak areas were <3.84 and 5.45% (intra-day). The nine barbiturates in urine were successfully determined within 7 min, without a prior preparation step and the method is useful for the investigation of intoxication.     

Quantification in capillary electrophoresis-mass spectrometry: long- and short-term variance components and their compensation using internal standards

Different approaches were chosen to examine ionization reproducibility of analytes after separation by capillary electrophoresis-mass spectrometry (CE-MS) in a commercially available sheath-flow electrospray interface. For this task three different standard samples were examined. Sample 1 contained neostigmine bromide (cationic), paracetamol (PCM) (neutral) and nicotinic acid (anionic component). Results were evaluated using internal standard (IS) calculations. Sample 2 represented an isotopically labelled IS of the quantified substance (PCM/D4-PCM), while sample 3 (neostigmine bromide/scopolamine hydrobromide) provided an IS closely migrating to the tested substance. Furthermore, short-time variations inside the interface were examined by multiple injections of the same substance. For sample 1, the relative standard deviations (RSD%s) were between 8 and 25% (n at least 58) for the peak area ratios. Multiple injected samples gave 5.5-19.4% (n = 25) for peak area RSD%. Using a closely migrating IS, sample 3, RSD%s between 6.5 and 10% (n at least 63) were achieved. With isotopically labelled IS, sample 2, an RSD% of 3-4% was achieved for peak area ratios over long periods (n = 25), for shorter periods (n = 9) even 1-2% RSD% was obtained. Keeping the instrument settings constant, the influence on the ionization efficiency and reproducibility was tested, varying the buffer pH, the organic buffer modifier and the sample concentration. Repeatabilities of migration time and peak area were measured and compared. Two 10 mM ammonium acetate buffers with pH 4.0 and 8.5 were investigated. No influence of buffer pH on peak area reproducibility was found. Isopropanol as organic buffer modifier significantly improved the ionisation leading to larger peak areas, but reduced reproducibility. The basic buffer produced slightly better RSD%s for migration times (2.5-4.0%) (n = 180) and faster analysis for the different test analytes of sample 1, while with the acetic buffer, RSD%s from 3.9 to 6.0% were obtained (n at least 163). The positioning of the capillary turned out to be the crucial parameter to ensure reproducible results. Thus, a procedure was established to ensure a defined ion-intensity level after capillary changes. The investigation of the different sample concentrations gave negligible differences in RSD%, showing that the signal-to-noise ratio was not the crucial parameter for reproducibility here, in contrast to CE-UV detection.     

毛细管离子电泳法同时测定腌菜中硝酸根和亚硝酸根

以溴离子(Br-)为内标,建立了毛细管离子电泳同时测定腌菜中的硝酸根和亚硝酸根的方法。讨论了缓冲液pH、样品和缓冲液中氯化钠浓度、分离电压对分离的影响。结果表明:以含1mol LNaCl的40mmol LH3PO4 NaOH缓冲-、-得到基线分离。NO3-和NO2液(pH3.5)为背景电解质,4min内Br-、NO3-检出限分别为0.1g L和0.3g L,峰面积相对标准偏差分别为4.6%和NO25 8%。     

Determination of active constituents in Lonicera confusa DC. by capillary electrophoresis with amperometric detection

A method based on capillary electrophoresis with amperometric detection has been developed for the determination of luteolin, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid in the dried flower buds, leaves and stems (three medicinal parts) of Lonicera confusa DC., respectively. The effects of several important factors such as detection potential, the concentration of the running buffer, separation voltage and injection time were investigated to acquire the optimum conditions. The detection electrode was a 300 microm diameter carbon disc electrode at a working potential of + 0.90 V (vs saturated calomel electrode). The four analytes can be well separated within 10 min in a 40 cm-long fused silica capillary at a separation voltage of 12 kV in a 50 mM borate-25 mM phosphate buffer (pH 8.0). The relationship between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N = 3) ranging from 0.35 to 0.52 microM for all analytes. The proposed method has been successfully applied to the monitoring of bioactive constituents in the real plant samples with satisfactory assay results.     

Determination of six bioactive components of Saussurea katochaete by capillary electrophoresis

The simultaneous determination of 3,4-dimethoxy-3'-hydroxy propiophenone, 5-hydroxy-7-methoxy coumarin, 7-hydroxy coumarin, 3',5'-dimethoxy apigenin, apigenin and 4-hydroxy cinnamic acid in the extract of S. katochaete has been investigated by capillary electrophoresis for the first time. The six active components were completely separated within 10 min in 20 mM Na(2)HPO(4) buffer at pH 11.00 with 10% (v/v) methanol and detected at 214 nm. The applied voltage was 15 kV and the temperature was kept at 25 degrees C. The effects of buffer pH, the concentration of Na(2)HPO(4) and the concentration of methanol on the separation efficiency were studied systematically. The regression equations revealed good linear relationships (correlation coefficients were 0.9987-0.9998) between the peak area of each analyte and its concentration. The relative standard deviations (RSD) of migration time and peak areas were <1.63 and <4.03%, respectively. The application of this method for the separation and determination of the six bioactive components in S. katochaete was reported. The contents of the six analytes ranged from 0.023 to 0.131 mg/g and recoveries ranged from 94.7 to 104.8%.     

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25 degrees C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.     

Optimization of the separation of a group of antifungals by capillary zone electrophoresis

Two simple, rapid, and efficient methods for the analysis of seven antifungal compounds have been developed by capillary zone electrophoresis. Resolutions higher than 1.5 were obtained using 0.025 M phosphate buffer (pH 2.30) (analysis time close to 9 min) or 0.2 M formic acid (pH 2.15) (analysis time close to 6 min), with an applied voltage of 20 kV and a temperature of 30 degrees C. The highest sensitivity and selectivity can be obtained using phosphate buffer but the shortest analysis times are achieved in the formic system. The analytical characteristics of the optimized methods were investigated. The reproducibility obtained for migration times (RSD(n = 10) < or = 1.0%) and peak areas (RSD(n = 10) < or = 4.3%) was acceptable, but better reproducibilities were obtained when verapamil was used as internal standard (RSD(n = 10) < 0.4% for relative migration times and RSD(n = 10) < or = 2.2% for peak area ratios). The lowest limit of detection was obtained for clotrimazole (0.12 microg/ml) and the highest for fluconazole and voriconazole (0.90 microg/ml). The lowest and the highest limits of quantitation were, respectively, 0.40 microg/ml for clotrimazole and 3.00 microg/ml for fluconazole and voriconazole.     

毛细管电泳-电感耦合等离子体质谱法测定藻类中6种不同形态的砷化合物

建立了一种利用毛细管电泳与电感耦合等离子体质谱联用技术(CE-ICP-MS)分析检测6种不同形态砷化合物的方法。详细研究了缓冲溶液的种类、pH值和浓度,分离电压以及进样时间等因素对6种砷化合物的分离度、灵敏度和重现性等的影响。结果表明,在最佳条件下,三价砷(As3+)、一甲基砷(MMA)、二甲基砷(DMA)、五价砷(As5+)、砷胆碱(AsC)和砷甜菜碱(AsB)6种化合物在25 min内得到完全分离。6次平行测定中,6种砷化合物峰面积的相对标准偏差(RSD)为3%～5%,检出限(以As计)(3倍信噪比)为0.08～0.12 μg/L。应用该方法成功地对海带中6种砷化合物进行了分析,回收率为90%～103%。该方法具有耗时短、灵敏度高、样品消耗量少、稳定性好等优点,可用于藻类样品中不同形态砷化合物的分析。     

Co-electroosmotic capillary electrophoresis determination of phenolic acids in commercial olive oil

Fourteen phenolic acids have been selectively determined in olive-oil samples using the co-electrosmotic capillary electrophoresis mode with UV detection after the LLE extraction system. A polycationic surfactant (hexadimetrine bromide, HDB), which dynamically coats the inner surface of the capillary and causes a fast anodic electroosmotic flow, was added to the electrolyte. The main factors affecting co-electroosmotic flow (EOF) such as type of modifier, concentration, and influence of organic solvents have been studied. Other parameters such as pH, type, and concentration of buffer, applied voltage, and injection time were also optimised using hydrodynamic injection for 8 s and UV detection at 210 nm. The composition optimum of the running buffer used was a 20% 2-propanol, 0.001% HDB, and 50 mM sodium borate at a pH value of 9.6. The method has been applied to determination and quantification of fourteen phenolic acids at ppb levels in olive oil samples after a liquid-liquid extraction.     

Separation of iota-, kappa and lambda-carrageenans by capillary electrophoresis

The present study reports a novel method for the separation of the high-molecular-weight anionic polysaccharides, iota, kappa, and lambda carrageenans, in capillary electrophoresis (CE). Carrageenan samples are first derivatised with 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated in an ammonium acetate background electrolyte (BGE) and detected with laser-induced fluorescence (LIF). The effects of changes of instrumental parameters (temperature, injection mode, field strength) and the composition of the BGE (concentration and pH) are reported, and are explained in terms of the physical chemistry of the BGE and the biopolymers. Optimal separation conditions for kappa, iota, and lambda carrageenans, including an APTS internal standard, were found in a polyvinyl alcohol coated capillary with an ammonium acetate BGE of low concentration (25 mM) and moderate pH (8.0). This BGE gave the best reproducibility in tests on iota/kappa mixtures, with relative standard deviations (RSDs) in migration times and normalised peak areas (relative to the APTS internal standard) of less than 0.1% and 1%, respectively. Using this BGE at 50 degrees C and a voltage of 30 kV, all three carrageenan subtypes were separated in a run time of 3 min.     

Assessment of the capabilities of capillary zone electrophoresis for the determination of hippuric and orotic acid in whey.

A rapid method was developed for the simultaneous determination of hippuric and orotic acid in rennet whey by capillary zone electrophoresis using an uncoated capillary utilizing a 0.04 M amino-2-methyl-1,3-propanediol (AMPD)-N,N-bis(2-hydroxyethyl) glycine (BICINE) buffer (pH 8.8) with UV detection at 254 and 280 nm. Whey proteins were removed by ultrafiltration. The method was evaluated for external, internal and standard addition procedures for both peak areas and peak heights. The use of an internal standard (sorbic acid) eliminated injection errors and gave, when applied to peak areas, the same levels for hippuric and orotic acid in those obtained with high-performance liquid chromatography. Relative standard deviations were 1-2%. Peak heights gave erratic results owing to sample matrix effects on peak widths.     

Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers

A cyclodextrin-modified capillary zone electrophoresis method was developed for the separation and determination of three isomeric compounds (ursolic acid, oleanolic acid and betulinic acid), caffeic acid, p-coumaric acid, rosmarinic acid, rutin and quercetin. Without the addition of beta-cyclodextrin (beta-CD) and methanol, the separation of these analytes was poorly resolved. These eight compounds, however, were well separated from each other within 20 min with a borax running buffer (40 mM of borax, pH 9.4) containing 2mM beta-CD and 4% (v/v) methanol at the voltage of 25 kV, temperature of 25 degrees C and detection wavelength of 210 nm. The relative standard deviations (RSDs) of migration time ranged from 0.16 to 0.74% while those of the peak area ratios ranged from 2.17 to 4.61% for six determinations of the analytes at concentration of 10 and 25 microg mL(-1). The correlation coefficients of the calibration curves of the analytes were all >0.998, and the recoveries were from 96.8 to 103.6%. The method was successfully applied to determine these bioactive components in the samples of Prunella vulgaris L. and its beverage drink products. Our results reveal that only the isomeric compounds and rosmarinic acid could be detected in the spikes of P. vulgaris L.; other components were either too low to be detected or not present while only rosmarinic acid was detected in the beverage products.     

毛细管电泳-电化学检测法测定黄芪及其制剂中的活性成分

采用毛细管电泳-电化学检测法(CE-ED)对中药黄芪的主要活性成分芦丁、阿魏酸、香草酸、绿原酸、槲皮素和咖啡酸进行了分离和测定。分别考察了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间等实验参数对实验结果的影响。在优化的实验条件下,以直径300 μm的碳圆盘电极为工作电极,检测电位为+0.95 V(相对于饱和甘汞电极),在10 mmol/L硼酸盐(pH 8.2)的运行缓冲溶液中,上述6种活性成分能在17 min内实现很好的基线分离,被测物浓度与峰电流在3个数量级范围呈良好的线性关系,检出限(S/N=3)范围为78～110 μg/L。在不同的加标水平下,6种活性成分的平均回收率为96.0%～103.0%,相对标准偏差为1.9%～3.6%(n=3)。该方法样品处理简单,无需预富集,已应用于实际样品的分析,并获得了令人满意的结果。     

Separation of human fibrinopeptides by capillary zone electrophoresis

We describe a method for the simultaneous determination of the five fibrinopeptide forms derived from the thrombin-promoted activation of human fibrinogen by capillary zone electrophoresis (CZE). The fibrinopeptide mixture was first desalted by a solid-phase extraction (SPE) step. The analysis was performed in reversed polarity in a highly cross-linked polyethylene glycol (PEG)-coated capillary with UV-light absorption detection at 200 nm. Several parameters including buffer concentration and pH, presence of an organic modifier, temperature, and applied voltage, have been tested. The best separations were obtained within 20 min, utilizing a 20 mM sodium phosphate buffer without organic modifier, in the narrow 6.1-6.2 pH range, at 25 degrees C, with an applied voltage of 20 kV. Quantitative analysis is made possible by the use of sheep fibrinopeptide A as an internal standard to correct for both extraction and injection errors.     

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 microg/mL (0.2-1.0 microg/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.     

Determination of low molecular weight organic acids in soil,plants, and water by capillary zone electrophoresis

Determination of low molecular weight organic acids in soils and plants by capillary zone electrophoresis was accomplished using a phthalate buffer and indirect UV detection mode. The influence of some crucial parameters, such as pH, buffer concentration and surfactant were investigated. A good separation of seven organic acids was achieved within 5 min using an electrolyte containing 15 mmol L(-1) potassium hydrogen phthalate, 0.5 mmol L(-1) myristyltrimethylammonium bromide (MTAB), and 5% methanol (MeOH) (v/v) at pH 5.60, separation voltage -20 kV, and temperature 25 degrees C. The relative standard deviation (n=5) of the method was found to be in range 0.18-0.56% for migration time and 3.2-4.8% for peak area. The limit of detection ranged between 0.5 micro mol L(-1) to 6 micro mol L(-1) at a signal-to-noise ratio of 3. The recovery of standard organic acids added to real samples ranged from 87 to 119%. This method was simple, rapid and reproducible, and could be applied to the simultaneous determination of organic acids in environmental samples.     

Determination of low-molecular-mass organic acids in any type of beer samples by coelectroosmotic capillary electrophoresis

A separation and determination of a mixture of 19 low-molecular-mass organic acids usually present in beer samples was developed using coelectroosmotic capillary zone electrophoresis. A polycation (hexadimetrine bromide, HDB) has been added to the electrolyte, which dynamically coats the inner surface of the capillary and causes a fast anodic electroosmotic flow. The main factors affecting reversal of the EOF such as type of modifier and concentration and influence of organic solvents were studied. Three types of modifiers, two alkylammonium salts (cethyltrimethylammonium bromide and tetradecyltrimethylammonium bromide) and a polycation (HDB) were investigated. The composition of the running buffer results on a 25% 2-propanol, 0.001% HDB and 50 mM sodium phosphate. The different instrumental parameters affecting the capillary electrophoretic separation were also optimized resulting on a -15 kV voltage with a hydrodynamic injection for 7 s with a UV detection at 210 nm. The applicability of the present method has been demonstrated for the determination of organic acids in different beer samples.     

高效毛细管电泳测定虎耳草属植物中虎耳草素含量

以苯乙胺为内标，建立了高效毛细管电泳法(HPCE)测定虎耳草属植物中虎耳草素含量的方法．讨论了缓冲溶液体系、pH值及缓冲液浓度对分离的影响．结果表明，以10mmol／L硼砂-盐酸缓冲液(pH9．00)为分离介质，虎耳草素标准品与内标实现基线分离，且实验结果线性关系良好(r=0．9989)，日内和日间相对标准偏差分别为1．7％和4．6％．     

Urtica dioica agglutinin: separation, identification, and quantitation of individual isolectins by capillary electrophoresis and capillary electrophoresis-mass spectrometry

With benign prostatic hyperplasia (BPH) being a major health problem in ageing men, alternative therapeutic approaches (e.g., with phytopharmaceuticals) are of great interest. Based on pharmacological evidences, one of the most promising options in that respect are the lectins found in Urtica dioica (stinging nettle) roots. In this study the qualitative and quantitative analysis of individual isolectins in U. dioica extracts is described, which is the first report on using capillary electrophoresis (CE) for the analysis of lectins in plant material at all. By utilizing a 200 mM sodium acetate buffer (pH 3.75) a baseline separation and determination of four closely related isolectins was feasible within 20 min in the aqueous plant extracts. The individual compounds were identified based on reference compounds as well as data obtained from CE-mass spectrometry (MS) experiments. After modifying the optimized CE conditions to 100 mM ammonium formate buffer with pH 3.75 and a voltage of 15 kV, the isolectins were clearly assignable in positive electrospray ionization (ESI) mode. The quantitative results obtained by CE (the total lectin content varied from 0 to 0.42% in the samples) were accurate (recovery rates of spiked samples between 92.5 and 96.2%), precise (relative standard deviation < 5%) and in good agreement to those obtained by High-performance liquid chromatography (HPLC). As for peak resolution, assignable compounds and required separation time the newly developed CE method was clearly advantageous over the determination achieved by LC.