Separation and determination of anthraquinones in Cassia obtusifolia (Leguminosae) by micellar electrokinetic capillary electrophoresis

An MEKC method was developed for the determination of the five pharmaceutically important anthraquinones: chrysophanol (1), physcion (2), emodin (3), aloe-emodinin (4), and rhein (5) in Cassia obtusifolia (Leguminosae). A buffer solution (pH 9.00) composed of 20 mM sodium borate, 20 mM sodium deoxycholate (DOC), and 15% ACN was found to be the most suitable electrolyte for this separation. Regression equations revealed linear relationships (correlation coefficients: 0.9993, 0.9992, 0.9996, 0.9989, and 0.9991) between the peak area of each compound (1, 2, 3, 4, and 5) and its concentration. The RSDs of migration times and peak areas were <1.23 and 2.72% within 1 day, respectively. The effects of pH value, surfactant (DOC) concentration, and organic modifier on the migration were also studied. By this way, the contents of five anthraquinones in the extracts of the seed of C. obtusifolia (Leguminosae) from different sources were successfully determined within 14 min.     

Determination of Flavonoids from Paulownia tomentosa (Thunb) Steud by Micellar Electrokinetic Capillary Electrophoresis

A micellar electrokinetic capillary electrophoresis (MEKC) method was developed for the determination of three flavonoids: diplacone (DI), mimulone (MI) and apigenin (AP) in the flowers of Paulownia tomentosa (Thunb) Steud. A buffer solution (pH 10.00) of 20 mM sodium borate, 10 mM sodium dodecyl sulfate (SDS) and 5% methanol was found to be the most suitable electrolyte for this separation. Regression equations revealed linear relationships (correlation coefficients: 0.9997 and 0.9998) between the peak area of each compound (DI, MI and AP) and its concentration. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of pH, surfactant (SDS) concentration and organic modifier (methanol) on migration were also studied. The contents of three flavonoids in the extracts from the flowers of P. tomentosa applying different solvents were successfully determined within 12 min.     

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 coumarins from Cacalia tangutica by capillary zone electrophoresis

Capillary zone electrophoresis (CZE), using a 20 mmol/L borate buffer at pH 10.5, was developed for the identification and determination of three coumarins--7-hydroxy-coumarin (HC), 7-hydroxy-8-methoxy-coumarin (HMC) and 7-O-beta-D-glucosyl-coumarin (GC)--in the extracts of the flower of Cacalia tangutica. Regression equations revealed linear relationships (correlation coefficient 0.9986-0.9990) between the corrected peak area (the ratio of peak area to migration time) of each constituent and its concentration. The relative standard deviations (RSD) of the migration time and peak area were 1.45-1.52 and 2.60-3.84% (intra-day), and 1.75-2.22 and 2.90-4.04% (inter-day), respectively. The recoveries of three constituents ranged between 94.5 and 105.6%. The effects of pH value, buffer concentration and applied voltage on the migration behavior of HC, HMC and GC were investigated. The contents of the three active constituents in the flower of Cacalia tangutica were successfully determined within 6 min under the optimum conditions chosen. Moreover, the dissociation constants for three coumarins were also determined by CE.     

Simultaneous analysis of polyhydroxylated alkaloids by capillary electrophoresis using borate complexation and evaluation of sweeping technique for sensitivity improvement

Summary Capillary zone electrophoresis coupled to UV detection was used for the simultaneous analysis of naturally occurring polyhydroxylated alkaloids. This separation was based on anin-situ complexation with borate ions. The effect of parameters such as borate concentration, capillary temperature and analyte molecular structure on migration times and selectivity were discussed. The best separation was obtained with a fused silica capillary (48.5 cm total length ×50 μm I.D., with a bubble factor of 3), 80 mM sodium tetraborate aqueous solution at pH 9.2 and temperature of 20°C. The method was validated and showed good data in terms of migration time and peak area reproducibility, selectivity, linearity and accuracy. The validated method was applied to determine miglitol in commercially available pharmaceutical tablets. To further improve method sensitivity, a sweeping technique involving borate ions was evaluated. This technique was found very sensitive to the analyte complexation with borate, borate concentration, and temperature as well as sample matrix. In the case of miglitol, a 35-fold improvement in peak height was achieved.     

Separation and determination of alantolactone and isoalantolactone in traditional Chinese herbs by capillary electrophoresis

A simple micellar electrokinetic chromatography (MEKC) method, using a 20 mM borate buffer (pH 8.0) with 25 mM SDS in the presence of 10% (V/V) methanol, was established for the identification and determination of two isomers, alantolactone (AL) and isoalantolactone (IAL), in the extracts of Inula helenium L. and Inula racemosa Hook f. Regression equations revealed linear relationships (correlation coefficients: 0.9990 for AL, 0.9991 for IAL) between the peak area of AL and IAL and their concentration. The relative standard deviations of the migration time and peak area of the two constituents were 1.51, 1.62 and 2.01, 1.98%, respectively. The recoveries of the two constituents ranged between 95–105% for AL and 93–108% for IAL.     

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.     

Separation and determination of isoflavones in red clover by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method has been developed for the determination of the four isoflavones, i.e. biochanin A, formononetin, genstein and daidzein in red clover (Trifolium Pratense L.). The effect of running buffer pH and concentration were investigated. An electrolyte composed of 30 mm borate, 20 mm sodium dodecyl sulfate (SDS) and 4 mg/mL HP-beta-CD containing 5% (v/v) ethanol at pH 10.1 provides a satisfactory separation for all the analytes. The applied voltage was 25 kV, and the capillary temperature was kept constant at 25 degrees C with a UV detection at 254 nm. The relative standard deviations (RSD) of the migration time and peak area were less than 1.73 and 3.94% (intra-day), and 2.29 and 4.38% (inter-day), respectively, under the optimized separation conditions. Regression equations revealed a good linear relationship between the peak area of each compound and its concentration. The contents of the four compounds in red clover were successfully determined with satisfactory repeatability and recovery.     

Rapid Determination of Diterpenoids in Andrographis paniculata by Microemulsion Electrokinetic Capillary Chromatography with Short-End Injection

A rapid, simple, and reliable method has been developed for routine capillary electrophoretic analysis of two diterpenoids, andrographolide and dehydroandrographolide, in Andrographis paniculata. For this system, using ethyl acetate oil-based microemulsion electrokinetic chromatography (MEEKC) and the short-end injection technique, analysis was complete in less than 2.5 min. In method validation the relative standard deviations of migration time and peak area of the two constituents were, respectively, 0.54% and 1.70% for andrographolide and 0.45% and 2.11% for dehydroandrographolide. Regression equations revealed linear relationships (correlation coefficients 0.9994 for andrographolide and 0.9993 for dehydroandrographolide) between peak area and concentration. The effects of buffer pH, borate concentration, SDS concentration, co-surfactant type and concentration, injection time, and running potential were systematically investigated. The method can be successfully implemented in routine quality-control testing.     

Separation and determination of phenylpropanoid glycosides from Pedicularis species by capillary electrophoresis

Capillary zone electrophoresis, using 30 mM borate buffer (pH 9.00) with 10% (v/v) methanol, was established for the identification and determination of four phenylpropanoid glycosides (PPGs)--echinocoside (ECH), verbascoside (VER), pedicularioside M (PED-M) and pedicularioside A (PED-A)--in extracts of Pedicularis longiflora var tubiformis, Pedicularis longiflora and Pedicularis Kansuensis. Regression equations revealed linear relationships (correlation coefficients: 0.9993-0.9999) between the peak area of each compound (ECH, VER, PED-M and PED-A) and its concentration. The relative standard deviations of the migration times and peak areas were <1.93 and 4.54%, respectively. The recoveries of four PPGs ranged between 95.6 and 108.4%. The effects of several CE parameters on the resolutions were studied systematically.     

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.     

Enantiomeric separation of synthetic 2,3-dihydroxy-3-phenylpropionate compounds by beta-cyclodextrin-modified capillary electrophoresis

A new capillary electrophoretic method was developed for enantiomeric separation and optical impurity analysis of three synthetic 2,3-dihydroxy-3-phenylpropionate compounds using native beta-cyclodextrin (beta-CD) as chiral selector and borate as a background electrolyte. The separation was carried out in uncoated capillary (58.5 cm x 75 microm I.D., effective length 48.5 cm). The results showed that beta-CD as the chiral selector exhibited good enantioselectivity and the baseline separation was obtained at pH 9.8, 200 mM borate buffer containing 1.7% beta-CD at applied voltage 15 kV and capillary temperature 20 degrees C within 15 min. The precision of each tested compound was less than 1.0% at migration time and 5.0% in corrected peak area and the accuracy of the method was in the range of 98.7-105%. Furthermore, the developed method was successfully applied to the determination of the undesirable trace (2S,3R)-(+)-form impurity in the synthetic (2R,3S)-(-)-2,3-dihydroxy-3-phenylpropionate samples.     

在线扫集-毛细管胶束电动色谱法对石榴与石榴叶中没食子酸含量的测定

建立了一种在线扫集-胶束电动色谱法测定没食子酸的新方法。考察了背景溶液pH值、十二烷基硫酸钠(SDS)浓度、样品基体组成和进样时间对富集效果的影响。使用未涂层的毛细管柱(48.5 cm×75μmi.d.,有效柱长40 cm),pH9.0的20 mmol/L硼酸盐+50 mmol/LSDS为背景溶液,在紫外检测波长272 nm、运行电压18 kV的条件下,200 s内的富集倍数可达20倍。线性范围为0.62~10.30 mg/L(r=0.999),检出限(S/N=3)为0.08 mg/L,平均回收率为104%。没食子酸迁移时间和峰面积的相对标准偏差分别为1.2%和1.6%。方法快速、准确可靠、灵敏度高、重复性好,可检测石榴不同部位和石榴叶以及饮料中没食子酸的含量。     

Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization

An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples.     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

Identification and determination of aesculin and aesculetin in ash barks by capillary zone electrophoresis

Summary A capillary zone electrophoresis method for identification and determination of aesculin and aesculetin has been established using borate-phosphate buffer containing 30% ethanol with on-column UV detection. A detailed investigation of the influence of changes in borate concentration, pH, applied voltage, temperature and organic modifier was then carried out. For both aesculin and aesculetin, a linear plot of migration time (MT) against borate concentration was obtained, and ln[measured peak area (MA)] and lnMT both gave linear plots against ln(applied voltage) with correlation coefficient r>0.999, which also resulted in a linear correlation between MA and MT (r≥0.9998) under varied voltage. Ethanol as organic modifier to the background electrolytes helped in separating aesculin and aesculetin from other components in ash barks. The reproducibility with relative standard deviation in MT and in normalized peak area(NA) and linearity based on NA against concentration were evaluated. Finally, the method was successfully applied to monitor the quality of different ash barks and to compare the effect of sample preparation on content of bioactive components in ash bark. Results indicate that CZE promises to be applicable to quality control of traditional Chinese medicines containing aesculin and aesculetin.     

Identification and determination of effective components in Euphrasia regelii by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for simultaneous determination of eukovoside, cinnamic acid and ferulic acid in Euphrasia regelii for the first time. The electrophoresis buffer was 20 mmol/L sodium borate containing 10% (v/v) methanol (pH 8.50). The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9995-0.9998) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Euphrasia regelii were easily determined with recoveries ranging from 95.5 to 104.2%.     

A simple and sensitive CE method for the simultaneous determination of catecholamines in urine with in-column optical fiber light-emitting diode-induced fluorescence detection

A simple and sensitive method has been developed for simultaneous analysis of three catecholamines: dopamine (DA), epinephrine (EP) and norepinephrine (NE) in urine by capillary electrophoresis (CE) coupled with in-column fiber-optic light-emitting diode-induced fluorescence detection (ICFO-LED-IFD). Fluorescein isothiocyanate was used as the fluorescence tagged reagent for derivatization of DA, EP and NE. The CE conditions for separation of these catecholamines were systematically investigated. It was found that catecholamines could be more effectively separated by adding β-cyclodextin (β-CD) and acetonitrile (ACN) to a background electrolyte (BGE) of sodium borate. The migration times are 10.61, 10.83 and 11.14 min for DA, EP and NE, respectively and the catecholamines are completely separated within 11.5 min under the optimal condition of a BGE containing 10% v/v ACN, 20 mM β-CD and 20 mM sodium borate (pH 9.5), and an applied voltage of 13 kV. The relative standard deviations of migration time and peak area for these catecholamines are less than 0.16 and 2.0%, respectively. The limit of quantifications (LOQs) for DA, EP and NE are 3.5, 1.0 and 3.1 nM whereas the limit of detections (LODs) for DA, EP and NE are 1.0, 0.3 and 0.9 nM, respectively. Our proposed CE method provides low LOQ and LOD values. This CE-ICFO-LED-IFD methodology has been successfully applied to analyze catecholamines in human urine samples with good accuracy and satisfactory recovery.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

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.