Structural diversity of ecdysteroids of Lychnis flos-cuculi

Eleven ecdysteroids have been isolated from Lychnis floscuculi; we are the first who report eight ecdysteroids of the eleven compounds in this plant. Two of these ecdysteroids, dihydrorubrosterone and 20-hydroxyecdysone 3-acetate are newly discovered natural products. The success of isolation of these new ecdysteroids has been based on the use of separation methods in a proper order; these separation procedures were completing each others. At the beginning steps of isolation simple separation methods were used, such a solvent-solvent distribution and fractionated precipitation. Two third of the contaminants were removed thereby. High capacity low resolution methods were used then, such as classical adsorption column chromatography and preparative thin-layer chromatography. The major component (20-hydroxyecdyssone) and certain minor ecdysteroids (polypodine B and rubrosterone) were isolated in pure form here. Purification of the further minor components (poststerone, 2-deoxy-20-hydroxyecdysone, vitikosterone E, dihydrorubrosterone, makisterone A, taxisterone, 20-hydroxyecdysone 2-acetate, 20-hydroxyecdysone 3-acetate) required HPLC and other absorption chromatographic methods. Our recent separation scheme means a generally applicable guiding principle for isolation of any plant ecdysteroid, major and minor alike. Structural identification of the known ecdysteroids was based on their spectral data and that of their literature information. Structural elucidation of 20-hydroxyecdysone 3-acetate was done by the help of a standard component prepared by acetylation of 20-hydroxyecdysone. From the mixture of seven acetates the corresponding compound (20-hydroxyecdysone 3-acetate) was isolated, and used for identification. Structural diversity of ecdysteroids of Lychnis flos-cuculi is evaluated, and a tentative explanation is introduced for the formation and biosynthesis of the versatility of phytoecdysteroids.     

New Ecdysteroids from Serratula tinctoria

Six new ecdysteroids have been isolated from SERRATULA TINCTORIA; these are: the 2,22- and 3,22-diacetates of 20-hydroxyecdysone, 5beta-hydroxyrubrosterone, 3-epi-poststerone, 3-epi-rubrosterone, and 22-oxo-20-hydroxyecdysone. These minor compounds were found together with the known ecdysteroids, 20-hydroxyecdysone, its 2-, 3-, and 22-monoacetates, rubrosterone, poststerone, polypodine B (5beta,20-dihydroxyecdysone), pterosterone (25-deoxy-20,24-dihydroxyecdysone), and makisterone C (24-ethyl-20-hydroxyecdysone). All these ecdysteroids were isolated by a combination of several chromatographic techniques (liquid chromatography on alumina, DCCC, and HPLC), then identified using standard mass spectrometric and 2D (1)H-NMR techniques.     

Identification and determination of ecdysones and flavonoids in Serratula strangulata by micellar electrokinetic capillary chromatography

The simultaneous determination of 20-hydroxyecdysone ( 1), 25-deoxy-11,20-dihydroxyecdysone ( 2), kaempferol 4',7-dimethyl ether ( 3) and biochanin A ( 4) in the fruit, leaf and root of Serratula strangulata Iljin has been investigated by micellar electrokinetic capillary chromatography for the first time. With an electrolyte containing 15 mmol/L borate, 60 mmol/L SDS, 20 % (v/v) methanol, at pH 9.08 and 20 kV applied voltage, the four analytes were completely separated within 22.7 min. The effects of concentration of borate, electrolyte pH, the concentrations of SDS and organic modifier and the applied voltage on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients: 0.9986 - 0.9997) between the peak areas of the analytes and the corresponding concentrations. The levels of analytes in the different parts of S. strangulata were easily determined with recoveries ranging from 95.3 % to 104.0 %.     

Qualitative and quantitative analysis of four species of Curcuma rhizomes using twice development thin layer chromatography

The rhizomes of Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin and Curcuma longa are used as Ezhu or Jianghuang in traditional Chinese medicine for a long time. Due to their similar morphological characters, it is difficult to distinguish their origins of raw materials used in clinic. In this study, a simple, rapid and reliable twice development TLC method was developed for qualitative and quantitative analysis of the four species of Curcuma rhizomes. The chromatography was performed on silica gel 60F(254) plate with chloroform-methanol-formic acid (80:4:0.8, v/v/v) and petroleum ether-ethyl acetate (90:10, v/v) as mobile phase for twice development. The TLC markers were colorized with 1% vanillin-H(2)SO(4) solution. The four species of Curcuma were easily discriminated based on their characteristic TLC profiles, and simultaneous quantification of eight compounds, including bisdemethoxycurcumin, demethoxycurcumin, curcumine, curcumenol, curcumol, curdione, furanodienone and curzerene, in Curcuma were also performed densitometrically at lambda(scan)=518nm and lambda(reference)=800 nm. The investigated compounds had good linearity (r(2)>0.9905) within test ranges. Therefore, the developed TLC method can be used for quality control of Curcuma rhizomes.     

Components of Serratula species; screening for ecdysteroid and inorganic constituents of some Serratula plants]

Ecdysteroid and inorganic components were analyzed from several plant species belonging to Caryophyllaceae family, such as in the cases of Serratula tinctoria, Serratula wolffii, Serratula coronata (20-hydroxyecdysone and inorganic components) and Jurinea mollis, Serratula gmellinii and Leuzea carthamoides (inorganic components, only). The 20-hydroxyecdysone content was determined using thin-layer chromatography after a simple clean up that had been performed by solid-phase extraction. Inorganic constitutents were determined using either ICP or flame photometry. Vegetation dependence of both 20-hydroxyecdysone and inorganic elements was studied. Serratula coronata shows remarkable high 20-hydroxyecdysone (2.3%, in April) and the studied Serratula plants gave a minimum of 20-hydroxyecdysone in June. Favorable period for harvesting is suggested as July and August, through the blossoming of these plants. Potassium and 20-hydroxyecdysone content gave a similar tendency considering their vegetation dependence, while the magnesium content moved toward opposite direction. The calcium contents of Serratula tinctoria, Serratula wolffii and Serratula coronata were found between 2.2% and 3.8%, which values are high relative to the other medicinal plants. At the same time, the Cu content of the ecdysteroid producing (and screened) Caryophyllaceae plants is low. The Fe, Mn and Mg contents of Serratula coronata are high, even higher than that of the Leuzea carthamoides. Our results have suggested the importance of analysis and control of inorganic constituents of crude plant extracts used for medicinal and recreational purposes.     

高效液相色谱法测定去痛片中四种成分的含量

目的:建立高效液相色谱法测定去痛片中四种成分的含量。方法:采用C8柱,柱温为室温,以0.005mol/L己烷磺酸钠甲醇溶液-磷酸盐缓冲液(0.05mol/L磷酸二氢钾溶液,含1%三乙胺,用磷酸调节pH值至3.0)(75:25)为流动相,流速为1.3mL/min,检测波长220nm。结果:样品中四种成分完全分离且线性关系良好,氨基比林、非那西丁、咖啡因和苯巴比妥的加样回收率分别为99.4%、99.4%、99.6%和100.1%,RSD分别为0.70%、0.90%、0.78%和0.83%(n=9)。结论:本法结果准确,操作简便,可以有效地控制产品质量。     

Quantitative analysis and chromatographic fingerprinting for the quality evaluation of Scutellaria baicalensis Georgi using capillary electrophoresis

Quantitative analysis and chromatographic fingerprinting for the quality evaluation of a Chinese herb Scutellaria baicalensis Georgi using capillary electrophoresis (CE) technique was developed. The separation was performed with a 50.0cm (42.0cm to the detector window)x75mum i.d. fused-silica capillary, and the CE fingerprint condition was optimized using the combination of central composite design and multivariate analysis. The optimized buffer system containing 15mM borate, 40mM phosphate, 15mM SDS, 15% (v/v) acetonitrile and 7.5% (v/v) 2-propanol was employed for the method development, and the baseline separation was achieved within 15min. The determination of the major active components (Baicalin, Baicalein and Wogonin) was carried out using the optimized CE condition. Good linear relationships were provided over the investigated concentration ranges (the values of R(2): 0.9997 for Baicalin, 0.9992 for Baicalein, and 0.9983 for Wogonin, respectively). The average recoveries of these target components ranged between 96.1-105.6%, 98.6-105.2%, and 96.3-105.0%, respectively. CE fingerprints combined with the quantitative analysis can be used for the quality evaluation of S. baicalensis.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma

A high-performance liquid chromatographic method was developed and validated for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma. The coumarin components and the internal standard isopsoralen were extracted from plasma samples with the mixture of tert-butyl methyl ether and n-hexane (4:1, v/v). Chromatographic separation was performed on a C(18) column (200 mm x 4.6mm, 5 microm) with the mobile phase acetonitrile-methanol-water-acetic acid (20:15:65:2, v/v/v/v) at a flow-rate of 1.0 ml/min. Only the peak of oxypeucedanin hydrate and byak-angelicin could be detected in dog plasma after oral administration of ethanol extracts of A. dahurica mainly containing xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate and byak-angelicin. The calibration curves of oxypeucedanin hydrate and byak-angelicin were linear over a range of 22.08-8830.00 and 6.08-2430.00 ng/ml in dog plasma, respectively. The quantification limit of oxypeucedanin hydrate and byak-angelicin in dog plasma was 22.08 and 6.08 ng/ml, respectively. The intra- and inter-day precision was less than 7.6% and 8.5% and the accuracy was from 91.9% to 106.1%. The lowest absolute recoveries of oxypeucedanin hydrate and byak-angelicin were 85.7% and 87.0%, respectively. The method was successfully applied to the pharmacokinetic studies of oxypeucedanin hydrate and byak-angelicin in dog plasma after oral administration of ethanol extracts from A. dahurica.     

Simultaneous Quantification of Six Major Flavonoids From Fructus sophorae by LC-ESI-MS/MS and Statistical Analysis

A new, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method has been developed for the determination of six major flavonoids including sophoricoside, genistin, genistein, rutin, quercetin, kaempferol in Fructus sophorae. Principal component analysis and hierarchical clustering analysis were used to classify and differentiate these samples. Chromatographic separation was performed on a C₁₈ column with linear gradient elution of methanol and 0.05% acetic acid (v/v) at a flow rate of 0.8 ml/min. The detection was accomplished in the negative mode using multiple-reaction monitoring. The total run time was 8.0 min. Full validation of the assay was carried out including linearity, precision, accuracy, recovery, limit of detection and limit of quantification. The validated method was successfully applied to the simultaneous determination of these active components in Fructus sophorae. The results demonstrated that the quantitative difference in content of six active compounds was useful for chemotaxonomy of many samples from different sources and the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by high-performance liquid chromatography–tandem mass spectrometric method coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of Fructus sophorae.     

麦白霉素的组分分析与质量控制

目的:考察国产麦白霉素组分含量,为提高中国药典2005年版麦白霉素质量标准提供依据。方法:采用 HPLC—MS 联用仪推测麦白霉素标准品中的8个主要组分,采用 HPLC 法测定12批样品的8个组分的含量情况。结果:12批样品中麦迪霉素 A_1和吉他霉素 A_6两个组分的含量分别为30%～50%和10%～20%,其它组分含量较低,各厂家产品组分的种类和含量不同。结论:建议修订麦白霉素组分质量标准,控制产品质量。     

补中益气丸的毛细管电泳指纹图谱研究

目的 建立补中益气丸(Buzhong Yiqi Wan,BZYQW)毛细管区带电泳指纹图谱,并采用内标法测定甘草次酸含量.方法 以50 mmol·L-1硼砂-150 mmol·L-1磷酸二氢钠-50 mmol·L-1磷酸氢二钠(体积比为1∶1∶1)为背景电解质(BGE)溶液,采用未涂层石英毛细管(75 cm×75 μm,有效长度63 cm),以色谱指纹图谱指数F为目标函数优化试验条件,优化出运行电压为11kV,紫外检测波长265 nm,重力进样20 s(高度10cm).以10批BZYQW为基础,以甘草次酸峰为参照物峰,确定15个共有指纹峰,建立了BZYQW-CEFP.结果 10批BZYQW质量由极好到劣依次可分为1、2、3、4、5、6、7、8级,采用系统指纹定量法评价出S4、S9和S10质量极好(1级),S5质量很好(2级),S3、S6、S7和S8质量好(3级),S1质量中(5级),S2质量次(7级).选用萘普生作内标,测定甘草次酸在10～100 μg (*)mL-1时线性关系良好,相关系数r=0.999 6,平均回收率为98.2％(n=6).结论 基于整体化学指纹综合定量分析与指标成分定量分析相结合能便捷有效地评价中药质量.     

HPLC-DAD-ELSD法同时测定小柴胡汤正丁醇有效部位中7种成分的含量(英文)

本文建立了同时测定小柴胡汤正丁醇有效部位中甘草苷、黄芩苷、汉黄芩苷、黄芩素、汉黄芩素、人参皂苷Re和人参皂苷Rb1含量的HPLC-DAD-ELSD法。采用Diamonsil C18(2)色谱柱(4.6 mm×250 mm,5.0μm),以乙腈–0.05%甲酸水溶液为流动相,梯度洗脱,流速1 mL/min,二极管阵列检测器的检测波长为276 nm,蒸发光散射检测器的漂移管温度为110 oC,载气流速氮气2.1 L/min,柱温为30 oC。结果表明:7种有效成分在一定范围内线性关系良好。该方法简便、快速、重复性好,可用于同时测定小柴胡汤正丁醇有效部位中7种有效成分的含量。     

胶束电动毛细管色谱法测定金果榄中古伦宾和蝙蝠葛任碱的含量

目的建立测定金果榄药材中古伦宾和蝙蝠葛任碱含量的胶束电动毛细管色谱法。方法采用未涂层熔融石英毛细管柱(35.2 cm×50μm I.D.,有效长度为26.9 cm),运行缓冲液为20 mmol.L-1硼酸溶液(含200 mmol.L-1十二烷基硫酸钠,pH值8.0)-异丙醇-甲醇(体积比70∶20∶10),分离电压为18 kV,检测波长为210 nm,压力自动进样。结果古伦宾和蝙蝠葛任碱的线性范围分别为0.210～1.68 g.L-1和0.026～0.21 g.L-1,线性关系良好。平均加样回收率分别为100.5%和96.1%,RSD分别为3.4%和3.6%(n=6)。应用该法测定10批金果榄药材中古伦宾和蝙蝠葛任碱的含量。结论该方法准确、可靠、环保,适用于金果榄中古伦宾和蝙蝠葛任碱的同时含量测定。     

Quantitative determination of eight components in rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longa using pressurized liquid extraction and gas chromatography-mass spectrometry

Curcuma longa (Zingiberaceae) is a native plant of southern Asia and is cultivated extensively throughout the warmer parts of the world. Jianghuang and Yujin are rhizome and tuberous root of C. longa, respectively, which were traditionally used as two Chinese medicines. In this paper, pressurized liquid extraction (PLE) and gas chromatography-mass spectrometry (GC-MS) were developed for quantitative determination/estimation of eight characteristic compounds including beta-caryophyllene, ar-curcumene, zingiberene, beta-bisabolene, beta-sesquiphellandrenendrene, ar-turmerone, alpha-turmerone and beta-turmerone in Jianghuang and Yujin. A HP-5MS capillary column (30 m x 0.25 mm i.d.) coated with 0.25 microm film 5% phenyl methyl siloxane was used for separation and selected ion monitoring (SIM) method was used for quantitation. Hierarchical cluster analysis based on characteristics of eight identified peaks in GC-MS profiles showed that 10 samples were divided into two main clusters, Jianghuang and Yujin, respectively. Four components such as ar-curcumene, ar-turmerone, alpha-turmerone and beta-turmerone were optimized as markers for quality control of rhizome (Jianghuang) and tuberous root (Yujin), which are two traditional Chinese medicines, from Curcuma longa.     

HPLC-DAD-ELSD法同时测定复方贞术调脂胶囊15个活性成分(英文)

复方贞术调脂胶囊(FTZc)是一种用于治疗高脂血症的专利药物,由八味中药组成,含有多活性成分的复方制剂。本文建立了可行的HPLC-DAD-ELSD方法用于FTZc的质量控制,同时测定15个活性成分(红景天苷、特女贞苷、木兰碱、迷迭香酸、丹酚酸B、非洲防己碱、表小檗碱、药根碱、黄连碱、巴马汀、小檗碱、5,7-二甲氧基香豆素、人参皂苷Rg1、人参皂苷Rb1和齐墩果酸)。色谱柱采用Ultimate XB C18,流动相为乙腈–水(0.25%乙酸,0.13%三乙胺)(梯度洗脱),流速为0.8mL/min,检测方法采用光电二极管阵列检测器的多波长检测模式和蒸发光散射检测器联合应用。方法学验证表明该方法精密度、准确度良好,成功用于不同批次FTZc的测定。结果表明,本文建立的测定多种生物活性成分HPLC-DAD-ELSD方法切实可行、方法可靠,特别适用于复方贞术调脂胶囊的质量评价。     

Simultaneous determination of six major stilbenes and flavonoids in Smilax china by high performance liquid chromatography

A simple, sensitive and specific HPLC method was developed for simultaneous determination of the six major active constituents in Smilax china, namely taxifolin-3-O-glycoside (1), piceid (2), oxyresveratrol (3), engeletin (4), resveratrol (5) and scirpusin A (6), respectively. The samples were separated on an Aglient Zorbax XDB-C18 column with gradient elution of acetonitrile and 0.02% phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detected at 300 nm. The six target compounds were completely separated within 35 min. All calibration curves showed good linearity (r2>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.7%. The recoveries were between 93.7 and 103.0%. The method was successfully applied to the analysis of six constituents in 15 commercial samples of S. china. The results indicated that the developed HPLC assay was readily utilized as a quality control method for S. china.     

LC-MS/MS法测定人血浆中加巴喷丁的浓度及其药动学研究

建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中加巴喷丁的浓度并将其应用于人体药动学研究。取血浆样品经甲醇沉淀蛋白后,以甲醇0.2%甲酸水溶液(80∶20)为流动相,用Inertsil ODS-3 C18柱(50 mm×2.1 mm ID,3μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 172→m/z 154(加巴喷丁)和m/z 130→m/z 71(内标二甲双胍)。加巴喷丁线性范围为40.8～8.16×103 ng.mL 1,定量限为40.8 ng.mL 1,每个样品测试时间仅2.2 min,日内、日间精密度(RSD)均小于12%,准确度(RE)在±6.4%范围内。应用此法研究了20名健康志愿者单剂量口服加巴喷丁胶囊600 mg后的药动学特点。该方法快速、专属、灵敏、适用性强,可应用于加巴喷丁的人体药动学研究。     

Determination of N3-methyl-5′-deoxy-5-fluorouridine,a novel metabolite of doxifluridine,in body fluids by high performance liquid chromatography

A high performance liquid chromatography method for the determination of N³-methyl-5′-deoxy-5-fluorouridine, a possible metabolic product of the anticancer pro-drug 5′-deoxy-5-fluorouridine, in human serum and urine is described. Sample treatment involved addition of internal standard (5-bromouracil) and protein precipitation with ammonium sulphate (serum samples) followed by liquid–liquid extraction with ethyl acetate–isopropanol (90:10, v/v). The average recovery at 0.5 mg ml⁻¹ level was (80±4%). A linear response extending over two decades of concentration was observed. Detection limits of 50 and 100 ng ml⁻¹ were obtained in serum and urine, respectively.     

反相高效液相色谱法测定连翘不同药用部位有效成分的含量

目的比较青翘、老翘不同产地不同药用部位（果壳、种子、果柄）中连翘苷、连翘酯苷、槲皮素的含量。方法采用高效液相色谱法,Ameritech酷瑞C18（250mm×4．6mm,5μm）色谱柱,流动相为乙腈（A）-0．1％甲酸（B）（0～10min,18％～20％A;10～20min,20％～42％A;20～25min,42％A）,流速为1．0mL·min^-1,柱温30℃,检测波长连翘酯苷A、连翘苷为280nm,槲皮素为254nnl。结果青翘、老翘种子中均以连翘酯苷A为主,且其含量远高于连翘苷和槲皮素,果壳中以连翘酯苷A和连翘苷为主,而果柄中3种成分含量差异较小,此外,3种成分在青翘中的含量均高于老翘。结论本研究为连翘药材的质量评价和资源的合理利用提供了科学依据。     

Development and validation of a liquid chromatographic method for the simultaneous determination of aniracetam and its related substances in the bulk drug and a tablet formulation

Simultaneous determination of aniracetam and its related impurities (2-pyrrolidinone, p-anisic acid, 4-p-anisamidobutyric acid and (p-anisoyl)-4-methyl-2-pyrrolidinone) was accomplished in the bulk drug and in a tablet formulation using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS-CN column (150 mm × 4.0 mm, 5 μm) using a gradient elution program with solvent A composed of phosphate buffer (pH 4.0; 0.010 M) and solvent B of acetonitrile-phosphate buffer (pH 4.0; 0.010 M) (90:10, v/v). The flow rate of the mobile phase was 1.0 mL min⁻¹ and the total elution time, including the column re-equilibration, was approximately 20 min. The UV detection wavelength was varied appropriately among 210, 250 and 280 nm. Injection volume was 20 μL and experiments were conducted at ambient temperature. The developed method was validated in terms of system suitability, selectivity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities, short term and long term stability of the analytes in the prepared solutions and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of aniracetam and its studied related impurities.