Rapid determination of yunaconitine and related alkaloids in aconites and aconite‐containing drugs by ultra high‐performance liquid chromatography–tandem mass spectrometry

Yunaconitine (YAC) is a toxic aconite alkaloid that is considered to be a hidden aconite poison since it is frequently found in body fluids from aconite poisoning patients, but has not been well studied in commonly used herbal drugs. In this paper, a rapid and sensitive ultra high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) detection combined with microwave‐assisted extraction (MAE) was developed for high throughput simultaneous determination of YAC and six other toxic aconite alkaloids in 31 samples of crude, processed aconites and aconite‐containing drugs. The optimized method showed excellent linearity, precision, accuracy and recovery for all target compounds with short run time. YAC was detected in some samples with contents from 0.015 to 10.41 mg/g. This is the first report on the determination of YAC in Radix Aconiti, Radix Aconiti Kusnezoffii and aconite‐containing drugs. This newly developed method facilitates the rapid screening of YAC and related toxic aconite alkaloids and allows YAC to be used as a chemical marker for the quality control of aconites and aconite‐containing drugs. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry

This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in combination with multivariate data analysis provides a very flexible and reliable method for quality assessment of toxic herbal medicine.     

液相色谱-质谱法研究附子生物碱的煎煮减毒机理

建立了基于液相色谱-质谱分析的附子中生物碱指纹分析方法,并将其应用于附子煎煮减毒的机理研究。基于质谱特征鉴定了生物碱标准品在加热回流过程中产生的6种降解产物,确定了双酯型生物碱的煎煮条件降解规律。在此基础上研究了附子饮片煎煮不同时间条件下生物碱的指纹变化,结果表明,长时间煎煮可明显降低双酯型生物碱和生物脂碱的浓度,为附子的安全用药提供了科学依据。     

Influence of different processing times on the quality of Polygoni Multiflora Radix by metabolomics based on ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A metabolomics method based on ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed to evaluate the influence of processing times on the quality of raw and processed Polygoni Multiflora Radix . Principal component analysis and partial least‐squares discriminant analysis was used to screen the potential maker metabolites that were contributed to the quality changes. Then these marker metabolites were selected as variables in Fisher's discriminant analysis to establish the models that were used to distinguish the raw and processed Polygoni Multiflora Radix in the markets. Additionally, 36 compounds were identified. Twelve raw Polygoni Multiflora Radix samples and 23 processed Polygoni Multiflora Radix samples were distinguished. The results showed that the 12 raw Polygoni Multiflora Radix samples belonged to the group of processing time of 0 h, and two processed Polygoni Multiflora Radix samples were part of the group of processing times of 4 h, 12 samples belonged to group of processing times of 8 to 16 h, and nine samples were the group of processing times of 24 to 48 h. The results demonstrated that the method could provide scientific support for the processing standardization of Polygoni Multiflora Radix .     

Characterization and discrimination of raw and vinegar‐baked Bupleuri radix based on UHPLC‐Q‐TOF‐MS coupled with multivariate statistical analysis

Bupleuri Radix is a commonly used herb in clinic, and raw and vinegar‐baked Bupleuri Radix are both documented in the Pharmacopoeia of People's Republic of China. According to the theories of traditional Chinese medicine, Bupleuri Radix possesses different therapeutic effects before and after processing. However, the chemical mechanism of this processing is still unknown. In this study, ultra‐high‐performance liquid chromatography with quadruple time‐of‐flight mass spectrometry coupled with multivariate statistical analysis including principal component analysis and orthogonal partial least square‐discriminant analysis was developed to holistically compare the difference between raw and vinegar‐baked Bupleuri Radix for the first time. As a result, 50 peaks in raw and processed Bupleuri Radix were detected, respectively, and a total of 49 peak chemical compounds were identified. Saikosaponin a, saikosaponin d, saikosaponin b₃, saikosaponin e, saikosaponin c, saikosaponin b₂, saikosaponin b₁, 4′′‐O‐acetyl‐saikosaponin d, hyperoside and 3′,4′‐dimethoxy quercetin were explored as potential markers of raw and vinegar‐baked Bupleuri Radix. This study has been successfully applied for global analysis of raw and vinegar‐processed samples. Furthermore, the underlying hepatoprotective mechanism of Bupleuri Radix was predicted, which was related to the changes of chemical profiling.     

利用液质联用技术分析干姜对乌头类生物碱在大鼠肠内吸收的影响

为从吸收的角度考察干姜对乌头类双酯型生物碱的解毒机理, 采用外翻肠囊法展开实验. 利用超高液相与三重四极杆质谱联用技术定量检测双酯型生物碱成分, 采用标准曲线法计算乌头碱、中乌头碱、次乌头碱在肠囊内吸收的绝对含量, 采用质谱峰面积直接分析其它双酯型生物碱的相对变化, 结果加入干姜提取液后, 乌头碱、中乌头碱、次乌头碱的单位肠管面积累计吸收量均降低, 10-羟基中乌头碱的的累积峰面积降低; 加入维拉帕米后, 双酯型生物碱的单位肠管面积累计吸收量及累积峰面积均增加; 向含有地高辛的肠营养液中加入干姜提取液后, 地高辛在各实验时间点的单位肠管面积累计吸收量均降低, 根据以上结果推测干姜抑制乌头类双酯型生物碱在大鼠肠囊内吸收的可能机制是通过诱导肠内P-葡糖蛋白, 从而抑制作为P-葡糖蛋白底物的双酯型生物碱的吸收, 最终起到减毒作用.     

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。     

基于新型介质阻挡放电离子源的药物快速检测方法研究

本研究将单电极放电技术的DBDI与质谱(MS)联用,快速检测了4种低极性的合成药物,结果表明,4种合成药物主要生成[M+H]+分子离子.此外还利用DBDI-MS对草乌、制草乌切片进行快速分析,在草乌中检测到乌头碱、中乌头碱、脱氧乌头碱的[M+H]+离子,以及[M+H-60]+碎片离子;在制草乌中只检测到乌头碱、中乌头碱、脱氧乌头碱的[M+H-60]+碎片离子.所测草乌中的标志性药效成分主要为双酯类生物碱,制草乌中的标志性药效成分主要为单酯类生物碱.新型DBDI为药物研究提供了一种新的、快速检测方法,具有十分重要的理论和实际应用意义,在药物研究领域具有极大的应用潜力.     

Screening out Potential Cardio-Toxic Components of Chinese Herb Radix Aconiti Lateralis in Rat Dosed Plasma by High Performance Liquid Chromatography/Electrospray Ionization Quadrupole Time-Of-Flight Mass Spectrometry

This study established a new, rapid, and accurate method for bioactive compound screening by the combination of serum pharmacochemistry and serum pharmacology based on a spectrum-effect relationship model. HPLC/ESI-Q-TOF-MS was used for the chemical profiling of samples of dosed plasma, control plasma, and Radix Aconiti Lateralis decoction. A comparison was made of the resulting chemical profiles, which totaled 20 compounds including 16 prototype compounds and 4 metabolites derived from SF decoction that were detected as potential bioactive components. Using MS/MS analysis and accurate molecular weight assessments of TOF, 17 components in 20 potential bioactive compounds were structurally identified. Moreover, the pretreated plasma samples were injected into venous sinus of isolated toad hearts to investigate the cardio-toxicity according to heart ratio (HR) situation and electrocardiograph (ECG) parameters, including PR interval, QRS interval, and QT interval. Finally, 4 compounds in these 20 potential bioactive components that had closest correlation with 4 cardio-toxic experimental indexes respectively were investigated by spectrum-effect relationship model for the first time. The 4 compounds were considered as the real cardio-toxic components. This study proposed a series of potential bioactive components in Radix Aconiti Lateralis and provided a general research pattern to identify the potential bioactivity compounds in Chinese herbs or natural pharmacy.     

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...     

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.     

Enhancement by Glycyrrhizae Radix of hepatic metabolism of hypaconitine,a major bioactive and toxic component of Aconiti Laterlis Radix,evaluated by HPLC‐TQ‐MS/MS analysis

Glycyrrhizae Radix (GR) is often prescribed together with Aconiti Laterlis Radix (ALR) (a so‐called compatible drug pair) in traditional Chinese medicinal practice to reduce toxicity of ALR. However, the mechanisms involved remain to be addressed. In this study, the metabolic interactions between GR–ALR drug pair were investigated for the first time. First, an HPLC‐TQ‐MS/MS method was developed to analyze hypaconitine, a major bioactive and toxic component of ALR, in rat liver S9. Then the in vitro metabolic rates of hypaconitine by different rat liver S9 were compared using the established method. The experiments were designed in four groups: pure hypaconitine (group I) and ALR extract (group II) incubated with liver S9 of normal rats, and pure hypaconitine (group III) and ALR extract (group IV) incubated with liver S9 of GR‐pretreated rats. When incubated for more than 4 h, the metabolic rates of hypaconitine in group III were significantly higher than those in group I, and when incubated for more than 2 h, the metabolic rates of hypaconitine in group IV were significantly higher than those in group II, suggesting that GR can enhance metabolic rate of hypaconitine, the mechanism of which might be related to hepatic metabolizing enzyme induction by GR. Copyright © 2012 John Wiley & Sons, Ltd.     

基于UPLC/Q-TOF-MS分析附子半夏配伍相反的物质基础

利用超高效液相色谱-飞行时间质谱联用技术(UPLC/Q-TOF-MS)分析附子半夏药对配伍相反的物质基础,从化学成分层次阐释其配伍相反机制.基于UPLC/Q-TOF-MS建立附子半夏药对配伍后生物碱类成分的化学指纹图谱,通过主成分分析法和正交偏最小二乘判别法分析药对配伍在合煎过程中的生物碱类成分的含量变化,找出差异变化显著的化学成分.结果表明正离子模式时附子半夏药对合煎液中次乌头碱,中乌头碱,乌头碱,去氧乌头碱,10-OH-中乌头碱,10-OH-乌头碱等的含量明显增高,而中乌头原碱,去乙酸中乌头原碱,去乙酸次乌头原碱,苯甲酰乌头原碱,苯甲酰次乌头原碱,10-OH-苯甲酰中乌头原碱等含量降低.附子半夏药对配伍应用时双酯型二萜生物碱的含量明显增高,而单酯型二萜生物碱的含量明显降低,这可能是附子半夏药对配伍相反作用的物质基础.     

In Situ Analysis for Herbal Pieces of Aconitum Plants by Using Direct Analysis in Real Time Mass Spectrometry

In this study, an extend application was developed to in situ analyze the herbal pieces of Aconitum plants by Direct Analysis in Real Time Mass Spectrometry (DART‐MS). Nearly all aconitine‐type alkaloids can be desorbed and ionized in this method, including diester diterpenoid aconitines (DDAs), monoester diterpenoid aconitines (MDAs) and some other diterpenoid aconitines. The spectra of in situ analysis for the herbal pieces of aconitum plants are similar with that of their extracts. Radix Aconiti and Radix Aconiti Kusnezoffii can be distinguished from each other by the intensity differences of character fragment ions from MDAs, such as m/z 586, 572, and 556. The qualified and unqualified herbal pieces can be also identified by the relative abundances of DDAs. The RSD of the relative abundances of some character ions at m/z 556, 586, and 590 were 13.53%, 4.03%, and 12.03%, respectively. So this in situ analytical method can identify both the types of Aconitum preparata and their quality. It provides the following advantages in the analysis of Chinese herbs: fast detection without much pretreatment, high‐throughput analysis, and reduction of pollution without any organic solvent.     

An enzyme-linked immunosorbent assay for aconitine-type alkaloids using an anti-aconitine monoclonal antibody

3-Succinylaconitine was conjugated with bovine serum albumin (BSA) for use as an immunogen for the preparation of a monoclonal antibody (MAb) against aconitine (Aco). Splenocytes from mice immunized with the Aco-BSA conjugate were fused with an aminopterin-sensitive mouse myeloma cell line, P3-X63-Ag8-653, and a hybridoma secreting a MAb against Aco was successfully obtained. The MAb cross-reacted with mesaconitine, hypaconitine and jesaconitine, which are Aco-type alkaloids, but not with any other compounds examined. The full measurement range of an enzyme-linked immunosorbent assay (ELISA) developed using the new MAb extended from 100 ng mL⁻¹ to 1.5 μg mL⁻¹ of Aco. The concentrations of Aco-type alkaloids in various Aconiti radixes assayed using the new ELISA method showed good agreement with previous reports.     

A metabolomic analysis of the toxicity of Aconitum sp. alkaloids in rats using gas chromatography/mass spectrometry

A metabolomic investigation of intoxication with Aconitum sp. alkaloids was carried out. Plasma obtained from Wistar rats administered these alkaloids was analyzed using gas chromatography/time‐of‐flight mass spectrometry. Samples were analyzed following protein precipitation, liquid‐liquid extraction, and derivatization. Thirty‐six metabolites were identified among the detected compounds. Subsequent data analysis using the t test and principal component analysis revealed metabolic differences between the control rats and treated groups as well as between the groups of rats administered different alkaloids. Twenty‐seven metabolites were significantly different between plasma samples from the controls and treated groups. The significant decreased plasma levels of glutamine and creatinine in all treated groups suggested impaired heart and muscle function caused by alkaloids. The plasma levels of 22 metabolites in the hypaconitine group were significantly decreased. In contrast, only 8 and 13 metabolites were observed with significantly decreased levels in the aconitine and mesaconitine groups, respectively.These results indicated that Aconitum sp. alkaloids can cause metabolic disorders in rats. The toxicity and corresponding mechanism of hypaconitine was different from those of aconitine and mesaconitine, based on the differences of perturbed metabolic patterns between groups. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

C19 and C20 Diterpene Alkaloids from Aconitum toxicum Rchb.

Acotoxinine ( 1 ), a new norditerpene alkaloid, was isolated from the roots of Aconitum toxicum Rchb ., together with the structurally related C₁₉ diterpene alkaloids neoline ( 2 ) and aconitine ( 3 ) and C₂₀ diterpene alkaloids songorine ( 4 ) and songoramine ( 5 ). The structures were elucidated by HR‐MS and advanced NMR methods, including ¹H‐NMR, JMOD, ¹H,¹H‐COSY, HSQC, and HMBC experiments. This is the first report of C₂₀ diterpenoid alkaloids in Aconitum toxicum.     

A liquid chromatography–tandem mass spectrometry approach for study the tissue distributions of five components of crude and salt‐processed Radix Achyranthes in rats

This study developed a robust and reliable approach using liquid chromatography– tandem mass spectrometry for the simultaneous determination of five saponins in rat tissues: β‐ecdysterone, chikusetsusaponin IV, ginsenoside Ro, 25S‐inokosterone and chikusetsusaponin IVa. This is the first report on a comparative tissue distribution study of crude and salt‐processed Radix Achyranthes in rats. After one‐step protein precipitation by acetonitrile, the tissue samples were sent to LC–MS/MS for multiple reaction monitoring. The retention times of the five saponins and internal standard were 1.77, 3.14, 3.01, 1.83, 3.26 and 4.77 min. The standard curves showed good linear regression (r² > 0.9991) in the range of 10.3–1562.5 ng/mL. The intra‐ and inter‐day accuracy and precision were within 15% of the nominal concentration. The recoveries of the five saponins were 92.0–99.9%. Finally, this approach was successfully applied to tissue distribution analysis of the five saponins after oral administration of crude and salt‐processed Radix Achyranthes in rats. The largest concentration of the five saponins was observed in kidney after salt‐processing, which indicated that processing could enhance the bioavailability.