滇南草乌的化学成分研究(Ⅰ)

从著名民间药滇南草乌(Aconitum auslroyunnanense W. T. Wang)新鲜根中分得11个二萜生物碱,其中碱1通过光谱研究及化学方法证明其结构为14-acetyl sachaconitine,是一新化合物,并命名为南乌碱甲(austroconitine A)(1);碱2—8分别鉴定为:黄草乌碱乙(vilmorrianine B,即karakoline)(2),isotalatizidine(3),黄草乌碱丁(vilmorrianine D,即sachaconitine)(4),黄草乌碱丙(vilmorrianine C)(5),talatisamine(6),黄草乌碱甲(vilmorrianine A)(7),8-去乙酰滇乌碱(8-deacetyl yunaconitine)(8)。碱9—11的结构正在鉴定中。     

三尖杉中一种高刺桐类型的新生物碱——7-去氧福建三尖杉碱的分离和结构

从安徽产三尖杉(Cephalotaxus fortunes Hook.t)的弱碱中分到一种新生物碱,为7-去氧福建三尖杉碱(7-deoxycephalofortuneine)(1)和另外5种已知高刺桐类型生物碱:福建三尖杉碱(cephalofortuneine)(2),台湾三尖杉碱(wilsonine)(6),表台湾三尖杉碱(epiwilsonine)(7),可莫西明碱(comosimine),(Phellinc alkaloid 6)(3)和3-表福杉碱(3-epifortuneine)(4),其中4为首次从植物中得到的天然产物,3在三尖杉中系第一次分到。     

高效液相色谱法测定郁金香鳞茎中3种内源激素

利用Agilent高效液相色谱仪,建立了同时测定郁金香鳞茎中GA_3、IAA、ABA 3种植物内源激素的高效液相色谱检测方法和异丙醇提取方法。采用外标法,C_(18)反相柱,流动相A(甲醇)∶B(磷酸缓冲液pH=3. 5)=45∶55;流速1 mL·min~(-1);检测波长0～3. 2 min 265 nm,3. 0～4. 5 min 212 nm,4. 5～6. 5 min 218 nm,6. 5～13. 0 min265 nm;柱温20℃进行测定;以异丙醇提取剂和二氯甲烷低温摇床提纯鳞茎中内源激素。整个过程操作简单,只需2. 0～2. 5 h可完成激素提取测定,检测方法线性相关度均在0. 995以上,测出限GA_3200 ng·mL~(-1),IAA5 ng·mL~(-1),ABA 20 ng·mL~(-1);提取方法回收率为84. 812%～95. 679%,相对标准偏差为6. 432%～2. 831%。用此方法提取郁金香的内源激素做出的图基线平稳,准确度高,可得到满意的峰形,且操作简单,各环节激素的损失较少。     

RP-HPLC测定白屈菜药材中白屈菜碱的含量

选用超声波、热回流和索氏3种提取方法,甲醇、丙酮和氯仿3种提取溶剂提取白屈菜中的白屈菜碱,考察最佳提取方法和提取溶剂,采用高效液相色谱法测定白屈菜碱含量。采用Diamonsil C18反相色谱柱为分析柱,以乙腈-1%三乙胺溶液(20∶80)(三乙胺用磷酸调节p H至3.0)为流动相,流速为1 m L/min,检测波长为290nm。获得最佳提取方法为索氏提取法,最佳提取溶剂为丙酮,测得白屈菜中白屈菜碱的含量为0.118%。白屈菜碱在0.01~0.10 mg/m L范围内呈良好线性关系,r=0.9999(n=6),通过方法学考察,回收率为97.48%±1.46%,RSD为1.50%。实验结果表明丙酮索式提取法能较全面的提取白屈菜中的白屈菜碱,利用高效液相色谱法含量测定,简便快速,准确可靠,可用于白屈菜药材及其制剂的质量控制。     

刺异叶花椒的化学成分研究

从芸香科花椒属植物刺异叶花椒(Zanthoxylum dimorphophyllum var. spinifolium)中分得五个已知的化合物,其中四个为生物碱:氧化勒(木党)碱(oxyavicine)(Ⅰ);铁屎米酮(canthin-6-one)(Ⅱ);乙氧基白屈菜红碱(ethoxychelerythrine)(Ⅲ);勒(木党)碱(avicine)(Ⅳ);另一化合物为β-谷甾醇(β-sitosterol)(Ⅴ)。     

滤光膜对黄檗(Phellodendron amuranse)幼苗三种生物碱含量的影响

以滤光膜遮光处理1年生黄檗(Phellodendron amuranse)幼苗,测定了幼苗生物量及根、茎外皮中的小檗碱、药根碱和掌叶防己碱含量.红色、黄色、蓝色和绿色滤光膜对黄檗幼苗的根和茎生物量都有不同程度的抑制作用,其中黄膜的抑制作用最小,而蓝膜和绿膜的抑制作用最强.滤光膜处理也不同程度地抑制了小檗碱、药根碱和掌叶防己碱的合成和积累,红膜的抑制作用最小,蓝膜和绿膜的抑制作用最强.滤光膜处理后,3种生物碱的单株产量都低于对照,红膜处理小檗碱的产量显著高于黄膜处理,但红膜和黄膜处理下的药根碱和掌叶防己碱的产量差异不显著,蓝膜和绿膜处理的3种生物碱的产量都始终最低.滤光膜处理不利于黄檗幼苗的生长和上述3种生物碱的积累.     

高效液相色谱法测定附子及其炮制品中三种双酯型生物碱

本文建立了一种能同时检测附子及其炮制品中乌头碱、新乌头碱和次乌头碱含量的HPLC-DAD检测方法。采用的色谱柱为Hepersil BDS C18分析柱(150 mm×4.6 mm ID,5μm),流动相为40 mmol/L乙酸铵(浓氨水调pH10.0)(A)-乙腈(B)梯度洗脱,检测波长为240nm,流速为1 mL/min。乌头碱、新乌头碱、次乌头碱的线性范围分别为0.0232-2.32μg、0.0216-2.16μg、0.0214-2.14μg(r=0.999976、0.999992、0.999994,n=6);加样回收率为96.4-103.5%(n=6),RSD均小于2.3%。测定结果表明7批不同产地附子生品及16批由同一产地生附子制得的炮制品中三种双酯型生物碱的含量差异悬殊,该方法为附子质量标准和炮制工艺规范的制定及进一步的药效学研究提供了可靠的数据和方法学平台。     

光强对黄檗幼苗三种生物碱含量的影响

设置光强分别为全光照的100%(不遮荫)、75%、50%和25%的4种光照处理,比较了不同光强下1年生黄檗幼苗主要药用成分小檗碱、药根碱和掌叶防己碱含量的变化.结果表明,全光照有利于小檗碱、药根碱和掌叶防己碱在茎外皮和根中的合成和积累,总体上随光强减弱,3种生物碱含量降低.相对光强为75%时黄檗幼苗生物量最大,而75%相对光强下黄檗幼苗3种生物碱的单株产量(生物碱含量与生物量之积)最高.     

单面针的生物碱研究

自芸香科(Rutaceae)花椒属植物单面针(Zanthoxylum nitidum var. fastuosum How ex Huang)的根皮中分得五种已知生物碱:乙氧基白屈菜红碱(ethoxychelerythrine)(Ⅰ);氯化光花椒碱(nitidine chloride)(Ⅱ);去甲基白屈菜红碱(des-N-methychelerythrine)(Ⅲ);α—别隐品碱(α-allocryptopine)(Ⅳ);鹅掌揪宁(liriodenine)(Ⅴ).     

利用超高效液相色谱和离子色谱法测定注射用磷酸川芎嗪的含量

目的:建立超高效液相色谱法(UPLC)和离子色谱法(IC)测定磷酸川芎嗪中川芎嗪和磷酸的含量,为质量评价提供依据。方法:UPLC测定川芎嗪的色谱柱为Waters Acquity BEH C18 (2.1 mm×50 mm,1.7μm);检测波长:300 nm检测川芎嗪,274 nm检测有关物质邻苯二甲酸二甲酯;流动相为0.1%甲酸水溶液(A)-0.1%甲酸乙腈(B),梯度洗脱(0.0～0.8 min,10%B→90%B;0.8～0.81 min,90%B→10%B;0.81～1.00 min,10%B),流速:0.7 m L/min。IC测定磷酸的离子交换色谱柱为Dionex IonPac AS11-HC-4μm (4×250 mm),流动相为30 mmol/L KOH溶液等度洗脱15 min,流速1.0 m L/min,柱温35℃;电导检测器;抑制器电流为50 m A。结果:川芎嗪和磷酸在10～100 g/m L内具有良好的线性关系,相关系数均为1.0,UPLC法测定川芎嗪的回收率为102.0%。IC测定磷酸的回收率为99.8%。7个公司生产的注射剂中川芎嗪的含量均在药典规定的范围90%～110%内。但是其中3个公司生产的注射剂磷酸超出药典规定范围90%～110%。结论:与常规HPLC/UPLC测定磷酸川芎嗪含量方法比较,本文所用方法测定结果更加准确、全面、且重复性好,能够真实反应注射用磷酸川芎嗪的实际含量,对于注射用磷酸川芎嗪的安全性和有效性评估提供了一定的依据。     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

HPLC法同时测定草苁蓉中草苁蓉纳拉苷和草苁蓉苷B

为建立高效液相色谱法同时测定草苁蓉干燥全草中草苁蓉纳拉苷和草苁蓉苷B的含量的分析方法,采用色谱柱:Klimail 100-5 C₁₈柱(250 mm×4.6 mm,5μm);流动相:以乙腈为流动相A,以0.5%甲酸水溶液为流动相B,梯度洗脱条件:0~12 min,15%A;12~30 min,15%~20%A;30~40 min,20%~25%A;40~45 min,25%~30%A;45~60 min,30%~100%A;流速:1.0 mL·min^-1;检测波长:260 nm;柱温:30℃;进样量:20μL。得到草苁蓉纳拉苷的线性范围为4.688~150μg·mL^-1(R²=0.999 5);草苁蓉苷B的线性范围为3.438~110μg·mL^-1(R²=0.999 1);平均回收率分别为97.86%、96.55%;RSD分别为1.01、1.23(n=9)。本研究利用高效液相色谱法建立了同时测定草苁蓉全草中草苁蓉纳拉苷和草苁蓉苷B两种组分的方法。方法学的验证结果证明,该方法简便、快捷,重现性好,可以用于草苁蓉中草苁蓉纳拉苷和草苁蓉苷B两种组分的含量测定。     

白刺花中3种生物碱的含量及HPLC指纹图谱

为建立白刺花高效液相指纹图谱,并测定白刺花不同部位(根、茎、叶)苦参碱、槐果碱和氧化苦参碱的含量。实验采用AgilentTC-C₁₈(2)色谱柱(250×4.6 mm,5μm),以乙腈-0.09%三乙胺水溶液(4∶96,磷酸调pH3.5)为流动相,流速为1.0 mL/min,紫外检测波长205 nm,柱温35℃。建立了白刺花的高效液相指纹图谱,确定了14个共有峰,样品的相似度在0.9以上。在该色谱条件下,各成分有较好的分离度,苦参碱、槐果碱和氧化苦参碱分别在6.34～101.5、6.47～103.5、8.38～100.5μg/mL范围内呈良好的线性关系(r>0.999)。用该方法测得白刺花根中苦参碱、槐果碱、氧化苦参碱的平均含量分别为8.204 8、1.237 1、66.147 6 mg/g;茎中分别为4.246 3、1.549 6、25.035 1 mg/g;叶中分别为9.314 8、6.947 4、1.573 4 mg/g。该方法简单、准确,可用于白刺花的质量控制。     

Supercritical carbon dioxide extraction of colchicine and related alkaloids from seeds of Colchicum autumnale L

A method for the extraction of the alkaloids colchicine, 3-demethylcolchicine and colchicoside from seeds of Colchicum autumnale by supercritical carbon dioxide has been established. Several parameters such as pressure, temperature, percentage of modifier and extraction time have been examined. Two extraction steps with constant carbon dioxide density (0.90 g/mL) and flux (1.5 mL/min) were required to extract the alkaloids in 110 min using 3% methanol as modifier. The quantitative determination of the alkaloids was performed by HPLC; the percentages of recovery were higher than 98% for the three alkaloids. This extraction procedure was compared with a conventional method involving maceration and sonication, and the same levels of alkaloids were obtained in each case. The supercritical carbon dioxide method is, however, very efficient, more rapid and more environmentally friendly than conventional methods.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography.

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairing HPLC with uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate-buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2. 5 x 10(6) cells/100 microl) were centrifuged at 13,000g for 10 min at 4 degrees C. The resulting supernatants were stored at -80 degrees C prior to analysis. Aliquots (100 microl) were neutralized with 4.3 microl saturated Na2CO3 solution prior the injection of 40 microl onto the HPLC column (injection speed 250 microl/min). Chromatographic separations were performed using two Symmetry C18 3. 5-microm (2 x 3.9 x 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 x 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30 degrees C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min --> 100/0 at 1 min --> 36/64 at 5 min --> 31/69 at 90 min --> 31/69 at 105 min --> 0/100 at 106 min --> 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH2PO4, 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7. 0 (550 microl 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 M KH2PO4, 0.028 M tetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r2 > 0.99) established with the addition-calibration methods up to 120 pmol/40-microl injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/antiviral drugs and diseases.     

天可力氨糖胶囊中氨基葡萄糖硫酸钾测定方法比较

目的:选择一种能准确测定天可力氨糖胶囊中氨基葡萄糖硫酸钾含量的方法。方法:参照GB/T 20365-2006硫酸软骨素和盐酸氨基葡萄糖含量的方法进行液相色谱法测定以及利用盐酸氨基葡萄糖Fdson—Morgan反应后在525 nm左右波长处有吸收峰,测定其吸收值与标准品比较进行比色定量。结果:液相色谱法测定时胶囊中的其它中药成分干扰很大,数据偏差太大;而比色法测定时,氨基葡萄糖盐酸盐含量在175μg/mL浓度范围内,呈良好线性关系:Y(μg/mL)=0.196X-O.004,R=0.997,平均回收率102.09%。结论:该比色法测定方法灵敏、准确、简便,可供天可力氨糖胶囊中氨基葡萄糖硫酸钾含量测定。     

An improved HPLC method for determination of carotenoids in human serum

An HPLC method was developed to determine the various carotenoids in human serum. A C-30 column and a mobile phase of 100% methanol (A) and 100% methylene chloride (B) with the following gradient elution were used: 90% A and 10% B in the beginning, maintained for 5 min, decreased to 78% A at 15 min, 62% A at 30 min, 52% A at 40 min, 41% A at 50 min, 38% A at 55 min, maintained for 3 min, and returned to 100% A at 65 min. A total of 21 carotenoids, including all-trans forms of lutein, zeaxanthin, alpha-cryptoxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and lycopene, as well as their 14 cis-isomers were resolved within 51 min at a flow rate of 1.0 mL/min and detection at 476 nm. all-trans-beta-Carotene was found to be present in highest amount (256.3-864.2 ng/mL), followed by all-trans-lycopene (64.4-569.2 ng/mL), all-trans-lutein (137.9-450.3 ng/mL), all-trans-alpha-cryptoxanthin (55.7-188.2 ng/mL), all-trans-beta-cryptoxanthin (43.1-134.5 ng/mL), all-trans-alpha-carotene (20.0-122.1 ng/mL) and all-trans-zeaxanthin (9.1-21.3 ng/mL). Similar trend was observed for cis-isomers of carotenoids.     

Simultaneous determination of four active alkaloids from a traditional Chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS

A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

HPLC测定葛花中6″-O-木糖鸢尾苷的含量

本文建立了葛花中6″-O-木糖鸢尾苷含量测定的HPLC方法。采用GRACE C18(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-水梯度洗脱,流速为0.8 mL/min,紫外检测波长为265 nm,柱温为室温。6″-O-木糖鸢尾苷的峰面积(Y)与浓度(X)在10.33～185.99μg/mL范围内线性关系良好,Y=30731X–216635,r=0.9992(n=7);平均回收率为100.2%,RSD<2%(n=9);测得8批葛花药材中6″-O-木糖鸢尾苷含量为11.08～48.23 mg/g。方法学验证结果表明该法准确、可靠,可用于葛花中主要成分6″-O-木糖鸢尾苷的含量测定。