Separation and determination of flavonoids in Lamiophlomis rotata by capillary electrophoresis using borate as electrolyte

A capillary electrophoresis method was developed for the simultaneous determination of five flavonoids such as luteolin-7-O-glucoside, isorhamnetin, apigenin, luteolin and quercetin in Lamiophlomis rotata (Benth.) Kudo. Optimal conditions were obtained at pH 9.0 with 30 mM borate as buffer containing 8% (v/v) acetonitrile, 20 kV as driving voltage and 210 nm as detection wavelength. The association constant K and the change in Gibbs free energy (DeltaG) of the interaction of flavonoids with borate anion ion (a typical ion-dipole or ion-induced dipole interaction) were calculated for the quantitative evaluation and characterization of the interaction. The described method was successfully applied for the rapid and efficient quality control by quantifying flavonoids in L. rotata. Repeatability tests showed that the R.S.D.s of both intra- and inter-day migration times and peak areas were less than 5%. The LOD of the five flavonoids was less than 3.75 mg/l. Recovery results ranged from 94.2% to 105.1%.     

毛细管电泳法测定丹参水溶性有效成分

目的建立高效毛细管电泳快速分离和测定丹参水溶性有效成分的方法.方法以pH为7.0,含0.5mmol*L-1十六烷基三甲基溴化铵,浓度为180mmol*L-1的磷酸盐缓冲液与乙腈按40∶11(v/v)混合配制电泳缓冲液,在75μm(id)×34.5(eff.30)cm的毛细管柱上,10kV(6s)电迁移进样,运行电压为8kV,紫外检测波长为200nm,柱温控制于20℃.结果在8min内能将以乙腈-水(20∶80)为溶剂的样品溶液,即内标(对羟基苯甲酸)、丹参素、原儿茶醛和原儿茶酸完全基线分离,柱效达40万/m理论塔板数,以丹参素、原儿茶醛和原儿茶酸与内标的峰面积比值为定量指标,定量检测限为0.1μg*ml-1,线性范围为5.0～0.1μg*ml-1,方法学的日内和日间变异均小于6%.结论本法快速、高效、简便,结果准确可靠.     

Determination of kava lactones and flavonoid glycoside in Scorzonera austriaca by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the quantitative analysis of three active comppounds, 12-hydroxy-desmethoxyyangonin (HD), 12-beta-d-glucopyranoside-desmethoxyyangonin (GD) and luteolin 3'-(6-E-p-coumaroyl-beta-d-glucopyranoside) (LG) in Scorzonera austriaca with UV detection at 254 nm. The applied voltage was 25 kV and the capillary temperature was kept constant at 25 degrees C. The effect of buffer pH, the concentration of electrolyte and organic modifier on migration were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 10% (v/v) methanol. Daphnetin was used as internal standard for quantification. Regression equations revealed good linear relationship between the ratios of the peak area of each compound and its the ratios of concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <1.46% and 5.13% (inter-day), and <1.65% and 5.16% (intra-day), respectively. The contents of the three compounds in S. austriaca were successfully determined with satisfactory repeatability and recovery.     

非水毛细管电泳法测定延胡索中延胡索乙素的含量

目的：建立非水毛细管电泳法,分离测定延胡索中延胡索乙素的含量。方法：以５０ｍｍｏｌ／Ｌ醋酸钠甲醇液含２ｍｏｌ／Ｌ醋酸为分离缓冲液。结果：本法在延胡索乙素浓度为５－４０μｇ／ｍｌ范围内具有良好的线性关系和重现性,且无需预处理。回收率为９７．４２％,ＲＳＤ＝１．６８％,结论：本法准确、快速、简便。     

Determination of oregonin in Alnus plants and biological samples by capillary electrophoresis

Oregonin, existing primarily in the Alnus plants, displayed anti-inflammatory and antioxidative activities. The capillary zone electrophoresis (CZE) method was developed in this study to quantitatively determine oregonin content in the Alnus plants for the first time. Various parameters, including buffer concentration, pH and applied voltage, were evaluated for their optimum analytical conditions. The optimized buffer was composed of 30 mM sodium tetraborate at pH 8.0. The separation voltage was set at 30 kV and the UV detection wavelength was set at 220 nm. Oregonin could be determined within 6 min under such optimized conditions. Relative standard deviation (R.S.D.) of the run-to-run repeatability and intermediate precision of the retention time of oregonin was within 1.36%. Run-to-run repeatability and intermediate precision of the peak area ratios of oregonin to internal standard, theophylline, were both within 1.55% R.S.D. The presented method was applied to analyze oregonin in leaves of Alnus formosana, seeds of various Alnus plants as well as biological samples. The stability of oregonin in biological system was indicated in this study. It demonstrates the potential of this developed method in natural product research.     

用高效毛细管电泳法鉴别冬虫夏草及其伪品

目的:应用高效毛细管电泳法鉴别冬虫夏草及其伪品。方法:石英毛细管70μm×60 cm,电解缓冲液30mmol/L硼酸盐溶液(pH 8.5)、电压20 kV,检测波长200 nm。结果:在该分离条件下,15 min可获得冬虫夏草及其伪品的特征性电泳图谱,经电泳图谱解析可鉴别两者。结论:该法简便、快速、准确,可作为中药冬虫夏草及其伪品的有效鉴别方法。     

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined.     

Separation and determination of four ganoderic acids from dried fermentation mycelia powder of Ganoderma lucidum by capillary zone electrophoresis

Ganoderic acids (GAs) were bioactive secondary metabolites produced by a traditional mushroom Ganoderma lucidum. We describe a simple and efficient method for the separation and quantitative determination of four GAs, namely Ganoderic acid T (GA-T), Ganoderic acid Mk (GA-Mk), Ganoderic acid Me (GA-Me) and Ganoderic acid S (GA-S) from dried triterpene-enriched extracts of G. lucidum mycelia powder by capillary zone electrophoresis (CZE). Under the optimum conditions, the four GAs reached the baseline separation in 9 min with Glycyrrhetinic acid (GTA) as internal standard. The four GAs and internal standard (GTA) were detected at a wavelength 245 nm. All calibration curves showed good linearity (r² > 0.9958) within test ranges. Limit of detection (LOD) and limit of quantification (LOQ) were less than 0.6 and 1.8 μg/mL, respectively. The relative standard deviation (R.S.D.) values of precision and recoveries were less than 5% and recoveries ranged from 91.4% to 103.6%. This was the first report on simultaneous determination of the four GAs and the results provided a firm basis for the trace analysis of GAs in dried fermentation mycelia powder of G. lucidum with high accuracy.     

Enantiomeric purity determination of tamsulosin by capillary electrophoresis using cyclodextrins and a polyacrylamide-coated capillary

The chiral separation of racemic tamsulosin hydrochloride (TH) was carried out using cyclodextrin (CD)-mediated capillary electrophoresis (CE) with DAD at 200 nm. The best separation of enantiomers of the studied compound was achieved at 20 kV with 30 cm x 50 microm I.D. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm) and running buffer with sulfated-beta-CD (S-beta-CD) as chiral selector. Other selected native or derivatized CDs were also tested: beta-CD (5, 15 mmol l(-1)), carboxymethyl-beta-CD (5, 30 mmol l(-1)), dimethyl-beta-CD (15 mmol l(-1)) and hydroxypropyl-beta-CD (5, 30 mmol l(-1)). Several parameters such as capillary pretreatment, buffer type and concentration, pH of background electrolyte, methanol content, separation temperature and voltage, were optimized. The excellent baseline separation of chiral TH was successfully achieved within 12 min using 100 mmol l(-1) phosphate buffer with pH 2.5 containing 1.7 mmol l(-1) S-beta-CD. Rectilinear calibration range was 50.0-500.0 mumol l(-1) of each enantiomer (r = 0.9993-0.9996). The method was applied to the assay of R-TH in Omnic, capsules (nominal content 0.4 mg per capsule) with R.S.D. 2.75% (n = 6), recovery 99.3-101.7% and it was suitable for the chiral purity control of the active enantiomer in the pharmaceutical.     

Isolation and analysis of bioactive diterpenoids in Salvia species (Salvia chionantha and Salvia kronenburgiii) by micellar electrokinetic capillary chromatography

In the present work, we isolated two bioactive diterpenoids, horminone and 7-O-acetylhorminone and developed a micellar electrokinetic chromatography (MEKC) method for the simultaneous quantitative analysis of them in Turkish Salvia species. The optimal separation electrolyte was 50 mmol/L SDS and 25% methanol at pH 11.5. The limits of detection (S/N = 3) were 3.269 and 4.518 μg/mL for horminone and 7-O-acetylhorminone, respectively. The method has been applied successfully to analyze these two components in Salvia chionantha and Salvia kronenburgii acetone extracts.     

Analysis and enantioresolution of donepezil by capillary electrophoresis

The analysis of donepezil, a centrally acting acetylcholine esterase inhibitor, is described by a CZE method suitable for applications in pharmaceutical field. A rapid migration of the analyte was obtained under acidic conditions (pH 3.0); with detection wavelength of 320 nm a LOD of 0.8×10⁻³ mg/ml was provided. Applications on real sample (pharmaceuticals) were carried out using two different instruments with comparable results in terms of reproducibility and accuracy. The use of chiral selectors in the running buffer allowed the enantioseparation of donepezil; charged cyclodextrins (carboxymethyl-β-cyclodextrin and sulfated-β-cyclodextrin) were suitable for the chiral resolution of the analyte. Interesting results were also obtained using human serum albumin. The protein-based CE enantioseparation was carried out at pH 7.4 avoiding the partial filling technique due to the good absorptivity of donepezil at 320 nm. Interestingly, the use of bicine as BGE provided a significative improvement in the enantioresolution compared to that obtained by phosphate buffer.     

毛细管电泳法分离测定板蓝根中的活性有机酸

目的 建立一种用毛细管区带电泳法测定板蓝根中有机酸含量的方法。方法 用含有15 mmol·L-1的β-环糊精的硼砂缓冲液(50 mmol·L-1,pH 10.5)-甲醇20∶5作为电泳缓冲液分离,以肉桂酸为内标,202 nm为测定波长,运行电压20 kV,柱温25℃。结果 邻氨基苯甲酸、苯甲酸、水杨酸与丁香酸在10 min内完全分离且呈良好的线性关系,其平均回收率分别为100.1％、97.9％、99.8％、99.1％(n=6)。结论 该方法快速、高效、简便,结果准确可靠。     

Profiling of levoamphetamine and related substances in dexamphetamine sulfate by capillary electrophoresis

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity of dexamphetamine as well as the analysis of 1R,2S-(−)-norephedrine and 1S,2S-(+)-norpseudoephedrine as potential impurities has been developed and validated. Heptakis-(2,3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin was chosen as chiral selector upon a screening of neutral and charged cyclodextrin derivatives. Separation of the analytes was achieved in a fused-silica capillary at 20 °C using an applied voltage of 25 kV. The optimized background electrolyte consisted of a 0.1 M sodium phosphate buffer, pH 2.5, containing 10 mg/ml of the cyclodextrin. The assay was linear in the range of 0.06–5.0% of the impurities based on a concentration of 2.0 mg/ml dexamphetamine sulfate in the sample solution. Analysis of commercial dexamphetamine sulfate samples revealed the presence of 3–4% of levoamphetamine while norephedrine or norpseudoephedrine could not be detected, indicating that the compound was prepared by fractionated crystallization of racemic amphetamine. Comparison with polarimetric measurements indicated that dexamphetamine with an enantiomeric excess as low as 80% still passes the pharmacopeial test of specific rotation while an amount of 0.06% of levoamphetamine can be detected by capillary electrophoresis.     

Quantifying trifluoroacetic acid as a counterion in drug discovery by 19F NMR and capillary electrophoresis

Drug discovery compounds are often isolated as salts of trifluoroacetate from preparative high performance liquid chromatography, which are then used for biological assays in order to assess their efficacy against the biochemical target of interest. It is, therefore, imperative to determine the TFA content in order to ascertain the correct formula weight and when required, to ensure that the TFA has been completely exchanged for another counterion in order to have superior pharmacokinetic properties and to avoid potential toxicity effects. In this paper, we present capillary electrophoresis and (19)F nuclear magnetic resonance methods for determining the TFA content of drug discovery compounds. Furthermore, these methods have been successfully applied in a high-throughput fashion, which is a key feature for general applicability in a pharmaceutical setting.     

Enantiomeric separation of denopamine by capillary electrophoresis and high-performance liquid chromatography using cyclodextrins

Direct separation of enantiomers of denopamine was investigated by two separation methods. One is capillary zone electrophoresis (CZE) using cyclodextrins (CDs) (CD-CZE) and the other is high-performance liquid chromatography (HPLC) using CD immobilized chiral stationary phases (CD-CSPs). Enantiomers of denopamine were successfully resolved by employing heptakis (2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) and acetyl-β-cyclodextrin (AC-β-CD), and partially resolved with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and β-CD polymer under acidic conditions. Separation of enantiomers of denopamine by HPLC was also successful with one of the CD-CSPs, where perphenylated β-CD (Phβ-CD) was immobilized. In CD-CZE, some polymeric additives, such as hydroxypropylmethylcellulose (HPMC) and polyvinylalcohol (PVA), and a coated capillary were used to improve the enantioseparation of denopamine. Method validations such as linearity, recovery and repeatability, etc., were investigated for HPLC, and the method was applied to the optical purity testing of the drug substances and in tablet form.     

Development and validation of a quantitative assay for raloxifene by capillary electrophoresis

The migration behavior of raloxifene was investigated by capillary electrophoresis (CE). The influence of different parameters (nature and concentration of the running buffer, pH and applied voltage) on migration time, peak symmetry and efficiency was systematically investigated. A buffer consisting of 20 mM acetate buffer of pH 4.5 was found to provide a very efficient and stable electrophoretic system for the analysis of raloxifene. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing this drug in human plasma and pharmaceutical preparations.     

Development of new formulation and its evaluation by capillary electrophoresis of tablets containing tramadol hydrochloride and paracetamol

Abstract

Context: A quick and efficient method to eliminate pain is combining complementary action and substances with synergic effect. Such an association is the one between tramadol hydrochloride and paracetamol, in which the rapid reaction time of the paracetamol is combined with the long-term effect of the tramadol hydrochloride.

Objective: Our aim was to develop and characterize alternative tablet formulations containing tramadol hydrochloride 37.5?mg and paracetamol 325?mg with different excipients in different ratio, and also the development of a new electrophoretic method for the quantitative determination of the active substances.

Materials and methods: The tablets were evaluated for physical characteristics (weight, thickness, diameter, mechanical strength, friability, disintegration time), drug content and dissolution following Ph.Eur.7 and USP 35 guidelines. A new capillary electrophoretic method was developed for the determination of the two active substances.

Results: Capillary electrophoresis proved to be an efficient method for the determination of drug content and also for the dissolution profile. The two substances were separated based on the differences between their own electrophoretic mobilities, and determined in UV at two different wavelengths.

Conclusions: Tablet formulation proved to be a significant factor in quality of tramadol hydrochloride/paracetamol tablets, as considerable differences between tablets containing different excipients were found; while capillary electrophoresis can be considered a useful alternative for the quantitative determination of tramadol hydrochloride/paracetamol combinations.     

Determination of free isomeric oleanolic acid and ursolic acid in Pterocephalus hookeri by capillary zone electrophoresis

A rapid capillary zone electophoresis (CZE) method for the quantification of two bioactive terpenes in Pterocephalus hookeri was developed. With beta-cyclodextrin as an additive, excellent resolution for these hydrophobic isomers can be obtained in less than 11 min. Linear calibration range for oleanolic acid was between 15.6 and 1000 microg/ml (r=0.9998), for ursolic acid between 31.2 and 1000 microg/ml (r=0.9992). The limits of detection for oleanolic acid and ursolic acid were 3.4 and 3.8 microg/ml, respectively. The contents of the free oleanolic acid and ursolic acid in P. hookeri were determined with recoveries ranging from 95.2 to 106.0%. The contents of oleanolic and ursolic acids were found to be 0.21 mg/g (RSD 5.49%) and 0.53 mg/g (RSD 3.37%), respectively. After hydrolysis by acid, these values were 1.12 mg/g (RSD 3.29%) and 0.43 mg/g (RSD 3.42%).     

毛细管电泳法测定氧氟沙星及左氧氟沙星在其制剂中的含量

建立制剂中氧氟沙星及左氧氟沙星毛细管电泳高频电导分析法，并用于片剂、滴眼液中氧氟沙星及左氧氟沙星含量的测定。对电泳介质的种类、浓度以及操作电压和进样量等影响因素进行了优化。实验采用5mmol／L乳酸为缓冲溶液，分离电压20．0kV，可在7min内实现对氧氟沙星及左氧氟沙星的分离和检测。在优化实验条件下，氧氟沙星的线性范围0．4～220μg／ml，检出限0．2μg／ml，回收率94．4％～102％；左氧氟沙星的线性范围0．6-200μg／ml，检出限0．3μg／ml，回收率95.4％～102％。     

毛细管电泳法测定(S)-2-(6-羟基-2,3-二氢苯并呋喃-3-基)乙酸甲酯中R异构体

目的建立毛细管电泳法测定(S)-2-(6-羟基-2,3-二氢苯并呋喃-3-基)乙酸甲酯中R异构体的方法。方法采用毛细管电泳法。以磺酸-β-环糊精(S-β-CD)为选择剂;25 mmol/L硼酸盐缓冲液(pH 8.9,含S-β-CD 1.8%)为运行缓冲液;运行电压为25 kV;柱温为15℃;检测波长:214 nm;3.4 kPa压力进样10 s。结果 (S)-2-(6-羟基-2,3-二氢苯并呋喃-3-基)乙酸甲酯与R异构体的分离度为2.9。R异构体在2~30μg/m L与峰面积线性关系良好(r=0.999 5),平均回收率为96.6%,RSD值为4.9%(n=6)。结论建立的毛细管电泳法操作简便,结果准确可靠,可用于(S)-2-(6-羟基-2,3-二氢苯并呋喃-3-基)乙酸甲酯中R异构体的控制。