刺五加及短梗五加根皮有效成分含量对比研究

目的:建立刺五加及短梗五加根皮刺五加苷B、刺五加苷E及异嗪皮啶的高效液相测定方法,测定刺五加及短梗五加中上述成分的含量,对比研究刺五加及短梗五加根中的成分含量差异。方法:采用高效液相色谱法,Agilent TC-C18色谱柱(150 mm×4.6 mm,5μm),以甲醇(A)-水(B)为流动相,梯度洗脱:(0～15 min:20A;15～20 min:21A;20～35 min:30A),流速0.8 mL.min-1,柱温30℃,检测波长210 nm。结果:样品进样量在各自浓度范围内呈良好的线性关系,加样回收率分别为99.1%、97.3%和99.9%,RSD为0.7%和1.3%和0.6%。结论:本法操作简便,分离效果好,可用于刺五加及短梗五加根皮刺五加苷B、刺五加苷E及异嗪皮啶的含量测定。     

反相高效液相色谱法同时测定车前子中的毛蕊花苷和异毛蕊花苷

采用反相高效液相色谱法在Waters Symmitry C18(5 μm,250 mmm×4.6 mm)色谱柱上以甲醇:体积分数0.1%甲酸溶液(40:60,v:v)为流动相同时测定车前子中毛蕊花苷和异毛蕊花苷的含量,流动相流速为0.6 mL/min,检测波长为330 nm.毛蕊花苷线性范围为0.2～2.0 μ,r=0.9982.异毛蕊花苷线性范围为0.2～2.0 μ,r=0.9957.样品的平均回收率分别为99.70%,101.18%.测得大粒车前子中毛蕊花苷含量为8.65 mg/g,异毛蕊花苷含量为1.36 mg/g.此方法准确、快速,适用于车前子中毛蕊花苷和异毛蕊花苷的定量分析.     

高效液相色谱法同时测定17种磺胺类药物的研究

建立了高效液相色谱-紫外法同时测定17种磺胺类药物的分析方法.主要研究了色谱柱、流动相配比对磺胺类药物分离的影响.通过研究,确定了最佳液相色谱分析条件.分离条件为:YMC ODS-C18柱;以2%乙酸溶液、乙腈和甲醇作为流动相,进行梯度洗脱;检测波长为270 nm.该方法的检测限为:磺胺胍和磺胺为2.0 ng/ml,磺胺二甲氧哒嗪、磺胺二甲基嘧啶、磺胺多辛、磺胺胍、磺胺甲基嘧啶、磺胺甲噻二唑、磺胺甲基异噁唑、磺胺甲氧哒嗪、磺胺6-甲氧嘧啶、磺胺二甲基噁唑、磺胺、磺胺吡啶、磺胺喹噁啉、磺胺噻唑和磺胺异噁唑均为5.0 ng/ml.各组分的回收率在81.3%～97.9%,相对标准偏差在0.1%～4.3%.     

褐飞虱对噻虫嗪和烯啶虫胺的抗性风险评估

采用稻茎浸渍法,测定了广州市本地褐飞虱种群对噻虫嗪和烯啶虫胺的室内毒力,评估褐飞虱对其的抗性风险。结果表明：广州本地褐飞虱种群对噻虫嗪和烯啶虫胺的LC50分别为02857mg/L和05022 mg/L,分别是敏感品系LC50的267倍和106倍,仍属敏感水平。室内抗性筛选结果表明：经过30代的连续筛选后,噻虫嗪的抗性上升82980倍,达到极高抗性水平,烯啶虫胺的抗性上升3170倍,达到中等抗性水平,表明褐飞虱对噻虫嗪和烯啶虫胺存在抗性风险的可能。根据试验结果,对褐飞虱噻虫嗪和烯啶虫胺抗性的预防治理提出了应用策略。     

异槲皮苷对Aβ25-35导致的PC12细胞损伤的保护作用及机制研究

探讨异槲皮苷对β-淀粉样蛋白(Aβ25-35)导致的PC12细胞氧化损伤的保护作用。首先通过分子对接技术分析异槲皮苷与AMPK的结合情况。采用Aβ25-35(20μmol/L)损伤PC12细胞建立细胞氧化损伤模型,采用甲基噻唑蓝(MTT)法检测细胞活力,通过试剂盒检测乳酸脱氢酶(LDH)漏出量、活性氧(ROS)含量、丙二醛(MDA)含量以及抗氧化物酶超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,采用Western blot法检测磷酸化腺苷酸活化蛋白激酶(p-AMPK)、过氧化物增殖体受体辅激活子-1α(PGC-1α)、沉默信息调节因子3(Sirt3)和异柠檬酸脱氢酶(IDH2)的蛋白表达。结果显示异槲皮苷与AMPK的结合力为-9.48 kJ/mol,提示AMPK可能为异槲皮苷的潜在作用靶点。异槲皮苷(1、10和100μmol/L)能够浓度依赖性的显著抑制Aβ25-35导致的PC12细胞死亡,减少ROS和MDA含量,升高SOD和GSH-Px活力。异槲皮苷抑制Aβ25-35导致的细胞氧化损伤并上调p-AMPK、PGC-1α、Sirt3和IDH2的蛋白表达。以上结果表明异槲皮苷可能通过调控AMPK/Sirt3信号通路发挥抗Aβ25-35导致的PC12细胞氧化损伤作用。     

高效液相色谱法同时测定青钱柳中9种指标成分的含量

建立高效液相色谱法同时测定青钱柳中绿原酸、香草酸、逆没食子酸、异槲皮苷、阿福豆苷、槲皮苷、山奈酚、山奈素和乌苏酸含量的方法。采用Agilent ZORBAX Eclipse XDB-C18色谱柱(150 mm×4.6 mm,3.5μm);流动相为0.2%磷酸乙腈-0.2%磷酸水,梯度洗脱;流速为0.7 m L/min,柱温35℃;检测波长:香草酸、逆没食子酸和乌苏酸为210 nm;绿原酸、异槲皮苷、阿福豆苷、槲皮素、山奈酚和山奈素为360 nm。青钱柳在该色谱条件下9种成分分离较好,平均加样回收率为95.86%~103.30%,RSD2.5%。该方法简便、准确、重复性好,适用于青钱柳中绿原酸、香草酸、逆没食子酸、异槲皮苷、阿福豆苷、槲皮苷、山奈酚、山奈素和乌苏酸含量的同时测定。     

中药防风抗流感病毒活性及不同极性部位的谱效相关性分析

研究防风不同极性部位指纹图谱与抗流感病毒活性的谱效关系,推断防风中具有抗流感病毒活性的有效成分。本实验采用细胞病变效应(cytopathic effect,CPE)法检测防风不同极性部位的抗流感病毒活性;建立防风不同极性部位的UPLC-Q-Exactive Orbitrap/MS指纹图谱;采用Spearman分析、灰色关联度分析(GRA)及偏最小二乘回归(PLSR)分析法分析防风不同极性部位的UPLC特征指纹峰与其抗流感病毒活性之间的谱效关系。在标定的28个特征指纹峰中,确定了1、5、7、11、13、20、24号峰与抗流感病毒活性呈正相关,其对应的物质依次是焦谷氨酸、divaricatacid、升麻苷、亥茅酚苷、补骨脂素、异嗪皮啶、5-羟基-8-甲氧基补骨脂素,为深入研究防风抗流感病毒活性物质基础提供实验基础。     

HPLC法分析刺五加茎中原儿茶酸及苯丙素类成分动态累积规律研究

本研究采用高效液相色谱建立了一种简便、灵敏可同时测定刺五加茎中原儿茶酸、L-苯丙氨酸、刺五加苷B、绿原酸、咖啡酸、刺五加苷E和异嗪皮啶含量的方法。采用Welchrom C18色谱柱(250×4.6 mm,5μm),以乙腈-0.1%磷酸水为流动相,流速为1 mL·min~(-1),检测波长为210 nm,柱温为30℃。在55 min内,刺五加茎中7种成分完全分离,样品含量与其对应峰面积Y呈现良好的线性关系(r=0.999 3~0.999 9);平均加样回收率为99.2%~100.7%,RSD为0.72%~1.14%。应用该方法,我们对采收于不同日期的刺五加茎中的七种成分进行了测定,测定结果表明,7种功效成分含量的综合评价指标在11月份较高。本研究建立的多成分HPLC测定方法简便、灵敏、稳定,适合用于刺五加多成分的定量分析和刺五加药材质量控制。     

三种杀虫剂对烟粉虱优势寄生蜂海氏桨角蚜小蜂的安全性评价

为评价噻虫嗪、阿维菌素和氟啶虫胺腈对烟粉虱优势寄生蜂海氏桨角蚜小蜂Eretmocerus hayati的安全性,采用琼脂保湿浸叶法分别测定了3种杀虫剂对烟粉虱成虫和海氏桨角蚜小蜂成蜂的室内毒力以及对海氏桨角蚜小蜂蛹羽化率的影响.结果表明,噻虫嗪、阿维菌素和氟啶虫胺腈对烟粉虱的LC50分别为453.76 mg/L、2.00 mg/L和29.47 mg/L,对海氏桨角蚜小蜂成蜂的LC50分别为0.23 mg/L、1.07 mg/L和0.64 mg/L.通过风险系数评估,表明阿维菌素对海氏桨角蚜小蜂成蜂安全,而噻虫嗪和氟啶虫胺腈对该蜂成蜂具有轻微到中度毒性.3种杀虫剂在烟粉虱和寄生蜂之间的选择性毒力指数表明噻虫嗪对海氏桨角蚜小蜂的负向选择性最强,其次是氟啶虫胺腈,阿维菌素最弱.3种杀虫剂均可显著降低海氏桨角蚜小蜂蛹的羽化率,对蛹的毒性为轻微有害,风险等级为2级.本研究结果将为烟粉虱综合治理中协调使用寄生蜂和化学药剂奠定理论基础.     

培育年限对刺五加主要药用活性成分的影响

为评价不同培育年限对刺五加根、茎部多种活性成分积累的共同影响,对提高刺五加培育和高值利用具有重要作用,可为规范化种植和合理开发利用刺五加提供理论依据。采集同一产地三年生、五年生、九年生刺五加为实验样本,运用超高效液相色谱系统（Ultra-Performance LC,Waters,Japan）分析不同培育年限对刺五加根、茎中槲皮苷、金丝桃苷、芦丁、紫丁香苷、刺五加苷E、异嗪皮啶6种活性成分含量的同时影响。结果表明,五年生刺五加根、茎中6种主要活性成分的综合得分最高。其中,黄酮类成分槲皮苷在三年生根中含量最高,在五年生茎中含量最高;芦丁在五年生根及茎中含量均最高,在根中三年生含量最低,九年生茎中含量最低;金丝桃苷在五年生根及三年生茎中含量最高。苯丙素类成分异嗪皮啶在三年生根及茎中含量最高,在五年生根和茎中含量最低;紫丁香苷在九年生根中含量最高,在五年茎中含量最高;刺五加E在五年生根中含量最高,在三年生茎中含量最高。不同药用成分在不同生长阶段的刺五加根和茎中积累不同,定向培育可根据目的活性成分选择适合的采收年限。     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Determination of free amino acids in burley tobacco by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method was developed for determining free amino acids in burley tobacco. The test was done by OPA/3-mercaptopropionic acid as the pre-column derivatizing reagent. Chromatographic column was Elitte C¹⁸ column (4.6 mm × 250 mm i.d., 5 μm). Mobile phase A was 18 mol/l NaAc (pH7.2) including 0.002%(v/v) triethylamine and 0.3%(v/v) furanidine. Mobile phase B was 100 mol/l NaAc (pH7.2)–acetonitrile–methanol (v/v = 1:2:2). The column temperature was 40 °C and the flow rate was 1.0 ml/min. The fluorescence detector was used with 350 nm excitation wave length and 450 nm emission wave length. The average recoveries of the method ranged from 95.3–100.7% with the relative standard deviation of 2.32–9.24%. The method is simple, accurate and has good repeatability. The results of the determination of seventeen kinds of free amino acids in burley leaves were produced by the way of different ratios of cake fertilizer and inorganic fertilizer. The results show that Aspartic acid has the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents of most of the free amino acids are increased and then gradually decreased with the increase in organic manure. The contents of most of the free amino acids are very close at 15:85% ratio and 30:70% ratio of cake fertilizer and inorganic fertilizer. The total amount of free amino acids is the highest at 30:70% ratio of cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at 30:70% ratio of cake fertilizer and inorganic fertilizer.     

Simultaneous determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxo-metabolites in plasma by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C₁₈ steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until 30 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

小鼠脑组织中溶血卵磷脂和卵磷脂含量的简便分析方法

目的建立快速准确测定小鼠脑组织中卵磷脂（PC）和溶血卵磷脂（LPC）含量的简便方法。方法小鼠脑组织经氯仿和甲醇抽提总脂后,以氯仿/甲醇/乙酸/丙酮/水（40/25/7/4/2,v/v）为展层剂,采用单相薄层层析（TLC）并结合钼蓝定磷法测定两种磷脂的含量。结果该法测定PC和LPC的回收率分别为94.91%±6.74%和104.00%±5.38%,相对标准偏差分别为5.05%和7.10%,最低检测限为4.80nmol。用此法测得小鼠脑组织中PC和LPC的含量分别为20257.14±1022.88nmol/g脑重、420.91±29.87nmol/g脑重。结论TLC法用于小鼠脑组织中LPC等磷脂的测定,简便易操作、重现性好,不仅能够分离测定PC和LPC,而且能够分离测定小鼠脑组织中的另外其它5种磷脂。     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.