离心超滤质谱筛选川乌中与人血清白蛋白相互作用的乌头类生物碱

采用离心超滤质谱技术从川乌总生物碱提取物中筛选与人血清白蛋白相互作用的乌头类生物碱成分,并用LC-MSn技术对筛选出的活性成分进行了分离鉴定.结果表明,从川乌总生物碱中筛选并鉴定出9种与人血清白蛋白存在相互作用的乌头类生物碱:苯甲酰新乌头原碱、苯甲酰乌头原碱、10-OH-中乌头碱、中乌头碱、10-OH-乌头碱、乌头碱、次乌头碱、脱氧乌头碱和3,13-脱氧乌头碱.     

复方中药四逆汤中乌头碱类二萜生物碱的电喷雾串联质谱研究

利用电喷雾串联质谱方法不经柱分离而直接分析复方中药四逆汤中二萜生物碱的组成,乌头碱类二萜生物碱在质谱条件下形成质子化分子,一种生物碱对应一个分子离子峰.检测到了苯甲酰单酯型、双酯型和脂类生物碱等18种二萜生物碱,其中苯甲酰中乌头原碱等单酯型生物碱是四逆汤中的主要生物碱成分.     

乌头与贝母配伍化学成分变化的UPLC/Q-TOFMS研究

利用UPLC/Q-TOFMS对中药十八反药对——乌头贝母配伍的合煎液与合并液进行检测,经过MassLynx 4.1分析其化学成分存在差异,发现合煎后次乌头碱水解受到部分抑制,而其他主要的双酯型二萜生物碱溶出较少且水解较彻底,乌头与川贝配伍合煎的毒性相对较小,因而可能通过合理炮制减毒后应用于临床治疗.     

近红外光谱法快速测定附子中6个生物碱的含量

应用近红外光谱(NIRS)技术结合偏最小二乘(PLS)和最小二乘支持向量机(LS-SVM)建立了附子中多指标成分的快速无损检测方法。选取38批样品建立了同时测定附子样品中6种成分含量的高效液相色谱(HPLC)方法;通过采集附子样品的NIRS图,分别采用PLS和LS-SVM建立了各个成分HPLC测定值与NIRS图的定量校正模型。所建立的苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱、单酯型生物碱总量和双酯型生物碱总量LS-SVM模型的相对预测偏差(RPD)分别为3.3、3.2、4.1、7.7、8.8、7.6、4.0和8.6;验证集相关系数(rpre)分别为0.9486、0.9475、0.9668、0.9909、0.9946、0.9969、0.9669和0.9927,且LS-SVM模型优于PLS模型,说明NIRS模型验证集与HPLC测定值具有良好的非线性关系,模型预测效果良好。采用NIRS技术结合LS-SVM模型可以快速对附子中的上述6个生物碱含量以及单酯型生物碱总量和双酯型生物碱总量进行检测,方法操作简便,对控制附子中的生物碱含量具有一定的指导作用。     

大孔树脂对麻黄细辛附子汤生物碱成分静态吸附及解吸附性能研究

采用高效液相色谱法(HPLC)测定麻黄细辛附子(MXF)汤复方水提液中盐酸麻黄碱、盐酸伪麻黄碱、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱含量,考察了4种大孔树脂(HPD–100,HPD–722,HPD–400,HPD–600型)对5种生物碱的静态吸附、解吸附性能,筛选出分离纯化麻黄细辛附子汤中生物碱的最佳树脂。结果显示,HPD–722型树脂对5种生物碱的吸附过程稳定且吸附量大,具有较高的解吸附率(87.26%),优于其它3种树脂。HPD–722型大孔树脂适用于富集麻黄细辛附子汤中的生物碱类化合物。     

电喷雾串联质谱分析附子炮制中的化学成分变化

利用电喷雾质谱方法(ESI-MS)分析了附子加辅料(甘草)炮制前后水煎液中二萜类生物碱在种类和含量方面的变化,通过加入内标化合物,建立了电喷雾质谱的半定量分析方法。此方法具有快速、准确、灵敏的特点,能够更加全面地反映中药配伍炮制过程中多种化学成分的含量变化,并能根据电喷雾串联质谱的分析结果鉴定配伍后产生的新的化学成分,在共煎液中的次乌头碱、中乌头碱和乌头碱的相对含量分别是单煎液中的5.67%、4.05%和4.88%。通过研究附子与甘草的单煎液、共煎液以及药渣中化学成分的变化,揭示了甘草作为辅料,在炮制过程中对附子减毒作用机理。     

复方乌头汤配伍贝母化学成分变化研究

将超高效液相色谱-质谱联用(UPLC-MS) 及UPLC-MSⁿ技术与偏最小二乘法判别分析(PLS-DA)结合, 对乌头汤分别与川贝、 浙贝按照制川乌与贝母生药质量比1: 1配伍前后化学成分的变化情况进行了研究. 在PLS-DA模型中, 乌头汤与川贝、 浙贝配伍前后的煎煮液均可明显区分. 乌头汤与川贝共煎后获得8个化学标志物, 可鉴定出7个化合物, 其中尼奥灵、 附子灵及苯甲酰基新乌头原碱在共煎后含量上升, 塔拉定、 10-OH苯甲酰基乌头原碱、 10-OH苯甲酰基新乌头原碱和苯甲酰基去氧乌头原碱在配伍后含量下降. 乌头汤与浙贝共煎后获得7个化学标志物, 分别为Chuanfumine、 Songorine、 塔拉定、 尼奥灵、 附子灵、 苯甲酰基新乌头原碱和10-OH苯甲酰基新乌头原碱, 所有化学标志物的含量在共煎后均降低.     

电喷雾质谱半定量方法研究制川乌配伍后的大鼠肠内菌生物转化

利用建立的代谢前后生物碱成分的电喷雾质谱半定量分析方法,通过分析各药对共煎液经大鼠肠内菌群代谢后主要生物碱的含量变化,对制川乌与浙贝母、法半夏、白蔹分别配伍后共煎液中双酯型、单酯型及脂型生物碱的肠内菌生物转化进行了深入研究,研究结果表明,配伍后中药浙贝母增加共煎液中双酯型生物碱含量,法半夏降低共煎液中双酯型生物碱含量,而白蔹对共煎液中双酯型生物碱含量影响不大;在代谢过程中,大鼠肠内菌群能够将复方中双酯型生物碱转化为脂型生物碱,从而达到中药配伍的减毒增效目的。本文通过化学方法和肠内菌代谢研究证明了制川乌配伍及代谢的机理。     

柱切换液相色谱法快速定量检测参附注射液中乌头碱类生物碱和人参皂苷

利用柱切换液相色谱,建立了参附注射液中苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱和乌头碱6种乌头碱类生物碱,以及Rg1、Re、Rf、Rb1、Rc、Ro、Rb2、Rb3、Rd 9种人参皂苷的分析方法。首先利用强阳离子交换的在线固相小柱选择性富集和净化样品中生物碱类成分,优化了色谱条件;并采用EC－C18柱作为人参皂苷的分析柱,通过优化实验条件,结合柱切换方式,去除了样品中辅料等大极性基质成分对色谱柱的污染,实现了生物碱分析和人参皂苷分析的自动切换。结果显示,样品中的生物碱和人参皂苷分离良好,线性相关系数（r2）均大于0.999,连续进样精密度的相对标准偏差（RSD） < 2.0%,重复性的RSD < 2.0%;其中6种生物碱的平均回收率为95.1%～98.6%,检出限为4.0～8.2 ng/mL;9种人参皂苷的平均回收率为91.7%～104%。所构建的基于柱切换液相色谱技术的在线固相萃取方法能够有效去除样品中的基质干扰,快速完成参附注射液中3种单酯型生物碱和9种人参皂苷的快速定量,同时也可对3种双酯型生物碱进行限量检测,可应用于药物的质量评价。     

乌头碱水解产物的研究

乌头碱为C_(19)二萜类双酯型生物碱,是乌头类中药的主要毒性成分和活性成分,乌头碱具有很强的抗炎和镇痛活性~([1]),在临床上被用于抗肿瘤药物,由于其治疗剂量与中毒剂量接近,必须经炮制后使用.乌头碱水解产物已有报道,但主要集中在苯甲酰乌头原碱和乌头原碱上,焦乌头碱很少提及,本研究利用硅胶柱色谱法对乌头碱的水解产物进行了分离和纯化,利用质谱和核磁共振谱确定了3种主要产物的结构,为研究乌头碱类生物碱的水解产物提供了新的数据.     

川乌与半夏、瓜蒌、贝母、白蔹、白芨配伍禁忌的化学研究

运用高效液相色谱方法,定量分析了川乌单煎液及其与生半夏、法半夏、全瓜蒌、瓜蒌皮、瓜蒌籽、浙贝母、川贝母、白蔹、白芨共煎液的双酯型生物碱含量.并利用电喷雾质谱通过加入内标的半定量分析方法研究生川乌配伍前后生物碱成分和含量的变化.结果显示生川乌与生半夏、瓜蒌籽、全瓜蒌、瓜蒌皮、浙贝母、白芨的共煎液中双酯型生物碱含量高于生川乌单煎液而生川乌与法半夏、川贝母、白蔹的共煎液双酯型生物碱含量变化微弱或有所减少.电喷雾质谱半定量分析方法与高效液相色谱方法的分析结果一致,并且与上述药对的LD50值结果也基本一致.而毒性成分的变化趋势与共煎前后溶液的pH变化相关.     

Ultra‐fast LC–ESI‐MS/MS method for the simultaneous determination of six highly toxic Aconitum alkaloids from Aconiti kusnezoffii radix in rat plasma and its application to a pharmacokinetic study

A fast, sensitive, and efficient ultra‐fast LC–ESI‐MS/MS method was developed for the simultaneous quantitation of six highly toxic Aconitum alkaloids, that is, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, in rat plasma after oral administration of crude ethanol extracts from Aconiti kusnezoffii radix by ultrasonic extraction, reflux extraction for 1 h, and reflux extraction for 3 h, respectively. The separation of six Aconitum alkaloids and aminopyrine (internal standard) was performed on an InertSustain® C₁₈ column, and the quantification of the analytes was performed on a 4000Q ultra‐fast LC–MS/MS system with turbo ion spray source in the positive ion and multiple‐reaction monitoring mode. Absolute recoveries ranged within 65.06–85.1% for plasma samples. The intra‐ and interday precision and accuracy of analytes were satisfactory. The methods were validated with sensitivity reaching the lower LOQ for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, which were 0.025, 0.025, 0.050, 0.025, 0.025, and 0.100 ng/mL, respectively. The method was successfully applied to a pharmacokinetic study of six Aconitum alkaloids in rat plasma after oral administration of crude ethanol extracts from the raw root of Aconitum kusnezoffii Reichb. by three different extraction processes.     

Studies on the intestinal absorption of the alkaloids in the Gancaofuzi decoction in a Caco-2 cell culture system by UPLC–MS/MS analysis

In this study, the Caco-2 cell monolayer model was used to research the characteristic absorption and efflux of five diterpenoid alkaloids in Gancaofuzi decoction. An ultra performance liquid chromatography coupled with tandem mass spectrometry(UPLC–MS/MS) method was developed for the determination of the simulated intestinal transport of five diterpenoid alkaloids with reserpine as internal standard. The use of the apparent permeability coefficient(Papp) and efflux rate(Er) was instituted to evaluate the intestinal absorption of the alkaloids. Transport of the five alkaloids in Caco-2cell monolayer model was observed to better understand whether the intestinal absorption of alkaloids was influenced by the compatibility of four herbs in Gancaofuzi decoction. The results show that the Papp values of the five diterpenoid alkaloids were all more than 1 * 10~(-6)cm/s, confirming that the processes of permeability were valid. The flux of the alkaloids was time-dependent, and the intestinal absorption mechanism of the five alkaloids was mainly based on passive transport. The compatibility of Heishunpian, Baizhu, Guizhi and Gancao can reduce the intestinal absorption of alkaloids, especially the most toxic hypaconitine, and the attenuated effect of mixed herbal water extracts was better than that of different herbs' water extracts combination. The results prove that compatibility of four herbs in Gancaofuzi decoction is rational.     

Simultaneous quantitation of seven alkaloids in processed Fuzi decoction by rapid resolution liquid chromatography coupled with tandem mass spectrometry

A rapid analytical method based on rapid resolution LC coupled with MS/MS was first established to quantify seven alkaloids in processed Fuzi decoction. The chromatographic method was optimized to allow simultaneous analysis of all analytes in 5 min and demonstrated good linearity (r > 0.9995), repeatability (RSD < 4.36%), intra‐ and interday precisions (RSD < 5.07%) with good accuracies (97.76–105.08%) and good recovery (95.0–107.5%) of seven alkaloids, namely higenamine, benzoylhypaconine, benzoylmesaconine, benzoylaconine, aconitine, hypaconitine, and mesaconitine. The LODs for these markers were in the range of 2.30–17.00 pg/mL. Quantitative analysis of the seven alkaloids in Baifupian decoction and Heishunpian decoction showed that the content of the seven marker chemicals varied significantly and concluded that the quality of Fuzi was greatly affected by different processed methods. The developed method could be used as a rapid, sensitive, and reliable approach for assessment of the quality of processed Fuzi and related decoction.     

Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C₁₈ column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L⁻¹ ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.     

Chemical composition database establishment and metabolite profiling analysis of Yangyin qingfei decoction

The chemical fingerprinting and metabolite profile in a rat plasma sample after intragastric administration of Yangyin qingfei decoction (YYQFD, 14 g/kg) were investigated. First, YYQFD was analyzed by UPLC/Q‐TOF MS to establish the chemical composition database by comparing their retention behavior, accurate molecular mass and MS² data with those of references or known compounds in the literature. In this database, 100 chemical constituents with information on retention time, molecular mass, molecular formula, MS² data and compound name were identified, which can provide compound information for further metabolite profiling studies. Furthermore, 64 compounds including 37 prototypes and 27 metabolites were detected in the dosed rat plasma sample, and the metabolic pathways of YYQFD were hydrolyzation, hydroxylation, dehydrogenation, glucuronidation, glucosylation, sulfation and mixed modes. Among the five component herbs in the YYQFD, Glycyrrhizae Radix et Rhizome and Fritillariae Thunbergii bulbs were actively metabolized, contributing 16 and 7 metabolites, respectively. It is suggested that chemical characterization and metabolite profiling studies are valuable to elucidate the material basis of herbal preparations.     

UPLC‐ESI/MS determination of 17 active constituents in two categorized formulas of traditional Chinese medicine,Sanhuang Xiexin Tang and Fuzi Xiexin Tang: application in comparing the differences in decoctions and macerations

A rapid and sensitive UPLC‐ESI/MS method was established and validated to determine 17 active constituents (aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, berberine, palmatine, jatrorrhizine, coptisine, baicalein, wogonin, baicalin, wogonoside, emodin, aloe‐emodin and rhein) in Sanhuang Xiexin Tang (SXT) and Fuzi Xiexin Tang (FXT), which are two classic compound recipes from Xiexin Tang categorized formulas in traditional Chinese medicien. The separation was performed on a UPLC BEH C₁₈ column gradient eluted using acetonitrile and 0.1% formic acid as mobile phase. ESI/MS was operated in positive ([M + H]⁺) in selected ion recording mode for analysis of alkaloids and flavones, while in negative ([M ? H]^?) selected ion recording mode for anthraquinones. All of the 17 constituents exhibited good linearity in a relatively wide concentration ranges with the lowest limits of detection of 0.38 ng/mL. All of the relative standard deviation values of intra‐ and inter‐precisions and stabilities of 17 constituents were within 5%. The method was successfully applied to determine 17 active constituents in decoctions and macerations of SXT and FXT. The results indicated that different preparative methods resulted in significant diversity in concentrations of the 17 analytes. Herb–herb interaction appeared between aconitum alkaloids in Aconiti Lateralis Radix Preparata and another three herbs. Copyright © 2013 John Wiley & Sons, Ltd.     

Exploring the ester-exchange reactions of diester-diterpenoid alkaloids in the aconite decoction process by electrospray ionization tandem mass spectrometry

The chemical components in the decoctions of Chinese herbal medicines are not always the same as those in the crude herbs because of the insolubility or instability of some compounds. In this work electrospray ionization tandem mass spectrometry was used to explore the ester-exchange reactions for aconitine-type diester-diterpenoid alkaloids occurring during the process of decocting aconite root. The aconitines were screened in a diverse range of samples, including crude aconite, decoction of crude aconite, residues from decoction of crude aconite, prepared aconite, decoction of prepared aconite, decoction of prepared aconite with added palmitic acid, and decoction of a mixture of mesaconitine and hypaconitine standards with liquorice root. It was found that diester-diterpenoid aconitines were converted into lipo-alkaloids as well as monoester alkaloids by the decoction of aconite.