Electrophoretic behavior study and determination of some active components in Chinese medicinal preparations by capillary electrophoresis

The determination of icariin (IC), rhein (RH), chrysophanol (CH), physcion (PHY), glycyrrhetic acid (GE), and glycyrrhizic acid (GI), in traditional Chinese preparations, Anshen Bunao oral liquid and Maren pill, has been investigated by micellar electrokinetic capillary electrophoresis. With borate buffer (10 mM), SDS (20 mM) and acetonitrile (10%) as background electrolyte (pH 9.55), 20 kV applied voltage and 254 nm UV detection, the six active compounds were completely separated within 10 min. The effects of buffer pH, concentration of borate, SDS and modifier on electrophoretic behavior and separation are discussed. Regression equations revealed linear relationships (correlation coefficients: 0.9960-0.9999) between the peak-area of each component and the content. In addition, the levels of the six active compounds in two kinds of traditional Chinese medicinal preparations were easily determined with recoveries of from 94.7% to 106.4%.     

Determination of active components in Chinese medicinal preparations by capillary electrophoresis

The determination of paeoniforin, paeonol, and censenoside Rg₁ in traditional Chinese medicinal preparations, Tze Po San Pien pills and Liuwen Dihuang pills has been investigated by micellar electrokinetic capillary electrophoresis with borate buffer (20 mM), sodium dodecylsulfate (30 mM) and acetonitrile (20%) as background electrolytes (pH 9.30), 20 kV applied voltage and 203 nm UV detection. The effects of SDS concentration, borate, buffer pH, and organic modifier on electrophoretic behavior and separation are discussed. Regression equations revealed linear relationships between the peak-area of each component and the content with the correlation coefficients from 0.9982 to 0.9999. In addition, the levels of the active compounds in two kinds of traditional Chinese medicinal preparations were easily determined with the recoveries from 93.1% to 108.2%.      

毛细管电泳法分离测定芦丁、槲皮素和连翘苷

用毛细管电泳紫外检测法同时测定了芦丁、槲皮素和连翘苷，研究了各种条件的影响，得到了优化的实验条件，在20mmol/L的Na2B4O7(H3B03调节至pH8．40)-30mmol/L十二烷基硫酸钠-10％乙腈(1 9)的缓冲溶液中，分离电压为12kV时，芦丁、槲皮素和连翘苷在10min内得到了良好的分离，检测波长为254nm，芦丁、槲皮素和连翘苷分别在0．01-1．0mg/mL，0．01-1．0mg／mL和0．05-1．0mg/mL质量浓度范围内与电泳峰高呈现良好线性关系，检测下限分别为0．005mg/mL，0．005mg/mL和0．01mg／mL，应用于实际样品的测定。     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.

     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Determination of baicalin, chlorogenic acid and caffeic acid in traditional chinese medicinal preparations by capillary zone electrophoresis

Summary A capillary zone electrophoresis (CZE) method was developed for the simultaneous assay of three bioactive components—baicalin, chlorogenic acid and caffeic acid—in seven traditional Chinese medicinal preparations. The analytes were separated successfully within 3.5 min using 10 mM borate buffer (pH8.6). Regression equations revealed linear relationships (correlation coefficients 0.9942–0.9996) between the peak area and concentration of the three analytes. The relative standard deviations of the migration times and the peak areas of the three constituents were 1.12–2.68% and 1.62–5.73%, respectively. Recovery of the three constituents ranged from 89 to 107%. The extraction efficiencies of different extraction solutions are also discussed. The contents of the three components in seven different Chinese medicinal preparations containing Honeysuckle flower and/orScutellariae radix were determined by the CZE method with satisfactory results.     

Assay of the Related Compounds Thiamphenicol, Florphenicol, and Chloramphenicol by CE

The purpose of this study was to examine the migration behaviour of a mixture of thiamphenicol, its two analogues florphenicol and chloramphenicol, and four impurities found in thiamphenicol. Capillary zone electrophoresis was performed with phosphate–borate or borate running buffers, pH 8.5, with or without addition of sodium dodecylsulphate (SDS). Although the compounds could not be resolved in the absence of SDS, supplementation of both types of buffer, at concentrations of 100 mM and higher, with 50 mM SDS enabled separation. On the basis of migration time and resolution, 100 mM borate buffer containing 50 mM SDS was identified as the best for the study. The accuracy and precision of the method were good under these conditions.     

High‐performance liquid chromatography with photodiode array detection and chemometrics method for the analysis of multiple components in the traditional Chinese medicine Shuanghuanglian oral liquid

Shuanghuanlian oral liquid, a traditional Chinese medicine preparation, is a mixture of three herbs (Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae). In this study, the quantitative analysis of three main active compounds, chlorogenic acid, forsythin and baicalin in samples from different manufacturers was performed rapidly by high‐performance liquid chromatography coupled with photodiode array detection followed by Contour Projection coupled to stepwise regression treatment of the obtained three‐dimensional spectra in which the partial overlap between adjacent target components existed. The method was validated for linearity (R>0.9940), precision (RSD<1.25%), recovery (92.20–102.50%), limit of detection (0.01–0.02 μg/mL) and limit of quantification (0.03–0.07 μg/mL). The results indicated that the combination of the three‐dimensional spectra of traditional Chinese medicine and Contour Projection‐stepwise regression offered an accurate, simple, low‐cost and eco‐friendly way for the rapid quantitative analysis of Shuanghuanlian oral liquid samples.     

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.     

Separation and determination of lignans from seeds of Schisandra species by micellar electrokinetic capillary chromatography using ionic liquid as modifier

In this paper, a micellar electrokinetic chromatographic (MEKC) method using ionic liquid as modifier for the quantification of the active components of lignans found in the medicinal herbs Schisandra species was developed for the first time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds, the addition of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as BMIM-BF4 and SDS concentration, applied voltage, background electrolyte pH and concentration, were evaluated. Under the optimal conditions (5 mM borate-5 mM phosphate buffer in the presence of 20 mM SDS and 10 mM BMIM-BF4, pH 9.2, applied voltage 25 kV and detection at 254 nm), the method successfully applied to the determination of lignans in extracts of Schisandra chinensis (Turcz.) Baill. and Schisandra henryi C.B. Clarke in less than 13 min. The separation mechanism was also discussed.     

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.     

Separation and determination of rutin and quercetin in the flowers ofSophora japonica L. by capillary electrophoresis with electrochemical detection

Summary Capillary electrophoresis with amperometric detection has been evaluated for the simultaneous determination of rutin and quercetin. The cyclic voltammogram, hydrodynamic voltammogram, and the effects of pH, concentration of buffer and sodium dodecyl sulfate (SDS), and amount of organic modifier on the separation and the detection were studied. The optimized conditions were: detection potential 1.2V, separation at 12 kV, 5 s at 15 kV for sample injection, running electrolyte 20 mmol L⁻¹ borate buffer, pH 8.8, containing 40 mmol L⁻¹ SDS and 10% acetonitrile. The detection limit of the method was low, 0.001 and 0.0005 mg mL⁻¹, for rutin and quercetin, respectively; the linear ranges were wide −0.005–0.5 and 0.005–0.4 mg mL⁻¹, respectively. The variations in peak current and migration time for eight consecutive injections of a standard solution containing 0.1 mg mL⁻¹ of each compound were 4.78 and 3.63%, and 6.50 and 2.59% for rutin and quercetin, respectively. The levels of the two compounds in traditional Chinese herbal drugs were easily determined.     

市售大黄及三黄片中蒽醌类活性成分的胶束电动毛细管色谱含量测定

建立了胶束电动毛细管色谱分离和测定大黄及其制剂三黄片中蒽醌类活性组分的方法．考察了背景电解质pH、表面活性剂浓度、有机改性剂种类和浓度对分离的影响．实验结果表明：在缓冲液pH为9．5、SDS浓度为25mmol／L、乙氰浓度为20％时的优化条件下,大黄及三黄片中蒽醌类活性组分得到基线分离且方法具有较好的重现性．     

Separation and determination of chalcones from Carthamus tinctorius L. and its medicinal preparation by capillary zone electrophoresis

A new capillary zone electrophoresis (CZE) method was developed for simultaneous assay of four chalcones, hydroxysafflor yellow A, safflor yellow A, safflamin C, and safflamin A, in the Chinese herbal extract from Carthamus tinctorius L. The optimum buffer system was 30 mM borate buffer (Na2B407/HCl, pH 9.00) with 10% (v/v) methanol. The voltage was 15 kV and detection was at 270 nm. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992, 0.9989, and 0.9996) between the peak area of each compound and its concentration. The within-day 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 four chalcones in Carthamus tinctorius L. were successfully determined with satisfactory repeatability and recovery. The possibilities of using this method for the determination of chalcones in Chinese medicinal preparation was also tested.     

Micellar electrokinetic capillary chromatography as an alternative method for the determination of ethinylestradiol and levo-norgestrel

A method for quantifying of ethinylestradiol (ETE) and levo-norgestrel (LEV) in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 25 degrees C and 25 kV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate (SDS) in 30% acetonitrile/water (v/v). Under these conditions the analysis takes about 7 min. The method has been applied for quantifying both compounds in six different commercial contraceptives and the proposed method gave good results when compared with a reference liquid chromatographic (LC) method.     

Far infrared-assisted extraction followed by MEKC for the simultaneous determination of flavones and phenolic acids in the leaves of Rhododendron mucronulatum Turcz

A method based on micellar electrokinetic chromatography with amperometric detection and far infrared‐assisted extraction has been developed for the simultaneous determination of two flavones (rutin and farrerol) and three phenolic acids (syringic acid, vanillic acid, and 4‐hydroxybenzoic acid) in the dried leaves of Rhododendron mucronulatum Turcz., a commonly used traditional Chinese medicine. The effects of some important factors such as the voltage applied on the infrared generator, irradiation time, the concentration of borate and sodium dodecylsulfate (SDS), separation voltage, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300‐μm diameter carbon disc electrode. The five analytes could be well separated within 8 min in a 40 cm‐long capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2) containing 50 mM SDS. The relationship between peak current and analyte concentration was linear over about three orders of magnitude with the detection limits (S/N=3) ranging from 0.20 to 0.46 μM. The results indicated that far infrared irradiations significantly enhanced the extraction efficiency. The extraction time was substantially reduced to 6 min compared with 3 h for conventional hot solvent extraction.