Analysis of hairy root culture of Rauvolfia serpentina using direct analysis in real time mass spectrometric technique

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the hairy root culture of Rauvolfia serpentina. The intact hairy roots were analyzed by holding them in the gap between the DART source and the mass spectrometer for measurements. Two nitrogen-containing compounds, vomilenine and reserpine, were characterized from the analysis of the hairy roots almost instantaneously. The confirmation of the structures of the identified compounds was made through their accurate molecular formula determinations. This is the first report of the application of DART technique for the characterization of compounds that are expressed in the hairy root cultures of Rauvolfia serpentina. Moreover, this also constitutes the first report of expression of reserpine in the hairy root culture of Rauvolfia serpentina.     

草乌中二萜类生物碱的电喷雾串联质谱研究

利用电喷雾质谱技术对传统中药草乌中的二萜类生物碱进行直接分析鉴定.通过实验数据并对比文献发现,草乌中含有单酯型、双酯型、三酯型和脂类等共4种类型生物碱,其中三酯型和脂类生物碱在草乌中为首次发现.由于这些生物碱的结构相似,在电喷雾串联质谱中碎裂方式相同,因此根据电喷雾串联质谱结果确定了这些生物碱的结构.     

An Protocol for Genetic Transformation of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> A4

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. To study and extract catharanthine and other metabolites from C. roseus, a technique was developed for producing hairy root cultures. In this study, the Agrobacterium rhizogenes A4 was induced in the hairy roots from leaf explants, and the concentration of antibiotics (100 mg/L kanamycin) was elucidated for selection after transformation. The polymerase chain reaction amplification of rol genes results revealed that transgenic hairy roots contained rol genes from the root induced (Ri)-plasmid. Catharanthine from C. roseus hairy roots was separated and analyzed using high-performance liquid chromatography. Over-expression of CrOrca3 (octadecanoid-responsive Catharanthus AP2/ERF domain), and cytohistochemical staining methods were used to validate transgenic hairy roots from C. roseus. Hairy root culture of C. roseus is a valuable approach for future efforts in the metabolic engineering of terpenoid indole alkaloids in plants.     

Alkaloid profiling of the Chinese herbal medicine Fuzi by combination of matrix-assisted laser desorption ionization mass spectrometry with liquid chromatography–mass spectrometry

A matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method was developed for the high throughput and robust qualitative profiling of alkaloids in Fuzi—the processed lateral roots of the Chinese herbal medicine Aconitum carmichaeli Debx (A. carmichaeli). After optimization, powdered roots – without any further sample preparation – could be used to screen for the presence of Aconitum alkaloids. Furthermore, the semi-quantitative potential of MALDI-MS was confirmed using liquid chromatography–mass spectrometry (LC–MS) as reference. In total over sixty alkaloids were detected by LC–MS and fifteen of them were tentatively identified. Both MALDI-MS and LC–MS analysis revealed significant variation in alkaloid content in different (commercial) samples. LC–MS analysis of three toxic alkaloids in 14 batches of Fuzi resulted in a variation of their concentrations expressed as RSDs of 138%, 99% and 221% for aconitine, hypaconitine and mesaconitine, respectively. The variation in concentrations (expressed as RSD) of about the ninety constituents detected were classified as follows: 13 constituents showed an RSD of 77–100%, 46 with an RSD of 100–150%, 21 with an RSD of 150–200% and 9 constituents with an RSD in concentration of 200–235%. These results demonstrate a strong difference in chemical composition of the various Fuzi and illustrate the necessity of adequate QA/QC procedures for both safety and efficiency of herbal medicine. The described analytical procedures for alkaloid profiling could play a role in these procedures.     

Challenging applications offered by direct analysis in real time (DART) in food-quality and safety analysis

Direct analysis in real time (DART) is an ambient ionization technique undergoing rapid development. With minimal sample pre-treatment, ionization of analyte molecules outside the mass spectrometry (MS) instrument in the ordinary atmosphere is feasible. This ionization approach relies upon the fundamental principles of atmospheric pressure chemical ionization.The current review highlights and critically assesses application of DART (and some related desorption/ionization techniques) coupled to various types of MS analyzers for both target and non-target analysis of complex food matrices. Based on existing studies, DART-MS is presented as a simple, high-throughput tool for:

(i)

qualitative confirmation of chemical identity;

(ii)

metabolomic fingerprinting/profiling; and,

(iii)

quantification of low-molecular-weight food components, including some trace organic contaminants.

With regard to regulatory requirements, we mention practical aspects of DART-MS use, as well as performance characteristics that can be attained.     

Direct mass spectrometric analysis of flavors and fragrances in real applications using DART

DART (Direct Analysis in Real Time) is an innovative technology to analyze complex solid samples at atmospheric pressure and ground potential by simply placing them between a DART ion source and a mass spectrometer. The analytes are ionized by a gun of neutral metastable species. The first examples of the application of DART to the analysis of flavor and fragrance raw materials in real, complex applications are reported here. A remarkably high potential of the technique is demonstrated. DART was applied to semi-quantitative analyses of perfumery raw materials deposited on smelling strips. In optimal cases, limits of detection around 100 pg were achieved. DART also allowed the assessment of the deposition and release of fragrance on surfaces such as fabric and hair. Finally, DART permitted the screening of twelve chewing gum samples for the possible presence of taste-refreshing compounds in less than 30 min.     

Analysis of cell cultures of Taxus wallichiana using direct analysis in real-time mass spectrometric technique

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the calli of Taxus wallichiana. The intact callus samples were directly analyzed by holding them in the gap between the DART source and mass spectrometer for measurements. Five C-14 oxygenated taxoids were characterized from the analysis of the calli of the Taxus wallichiana almost instantaneously. The confirmation of the structures of the identified taxoids was made through their accurate molecular formula determinations.     

Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

The comparative research on constituents of Radix Aconiti and its processing by HPLC quadrupole TOF-MS

Based upon the regulations stipulated by the State Food and Drug Administration of China, only the processed, detoxified tubers and roots of Aconitum are allowed to be administered orally, used in clinical decoctions and adopted as raw materials for pharmaceutical manufacturing, so the processing principle of preparation of Radix Aconiti is important for ensuring the Radix Aconiti praeparata quality. A simple approach was described for HPLC‐Q‐TOF‐MS screening and identification of many of the aconitine alkaloids present in unprocessed Radix Aconiti and Radix Aconiti praeparata. To compare their fingerprints, the processing principle of preparation of Radix Aconiti was developed. Twenty‐nine compounds and 26 compounds were assigned to aconitine alkaloids and tentatively identified by comparing accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literature. The nonester alkaloids were almost the same. The diester diterpene alkaloids were decreased, the monoester‐diterpene alkaloids were increased and lipo‐alkaloids decreased obviously in the processing of the preparation. These transformed components could be regarded as potential chemical markers that can be used to distinguish between raw and processed herbs. Copyright © 2012 John Wiley & Sons, Ltd.     

The coupling of direct analysis in real time ionization to Fourier transform ion cyclotron resonance mass spectrometry for ultrahigh‐resolution mass analysis

Direct Analysis in Real Time (DART) is an ambient ionization technique for mass spectrometry that provides rapid and sensitive analyses with little or no sample preparation. DART has been reported primarily for mass analyzers of low to moderate resolving power such as quadrupole ion traps and time‐of‐flight (TOF) mass spectrometers. In the current work, a custom‐built DART source has been successfully coupled to two different Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometers for the first time. Comparison of spectra of the isobaric compounds, diisopropyl methylphosphonate and theophylline, acquired by 4.7 T FT‐ICR MS and TOF MS, demonstrates that the TOF resolving power can be insufficient for compositionally complex samples. 9.4 T FT‐ICR MS yielded the highest mass resolving power yet reported with DART ionization for 1,2‐benzanthracene and 9,10‐diphenylanthracene. Polycyclic aromatic hydrocarbons exhibit a spatial dependence in ionization mechanisms between the DART source and the mass spectrometer. The feasibility of analyzing a variety of samples was established with the introduction and analysis of food products and crude oil samples. DART FT‐ICR MS provides complex sample analysis that is rapid, highly selective and information‐rich, but limited to relatively low‐mass analytes. Copyright © 2010 John Wiley & Sons, Ltd.     

Hyoscyamine Biosynthesis in <Emphasis Type="Italic">Datura stramonium</Emphasis> Hairy Root In Vitro Systems with Different Ploidy Levels

Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for <1% of the total ion current of the alkaloid mixture in gas chromatography–mass spectrometric analyses). Significant differences in the effects of the main nutrients on the growth of the hairy roots obtained from diploid and tetraploid plants and their hyoscyamine contents were observed. The maximal yield of hyoscyamine (177 mg/L) was obtained when hairy roots from tetraploid plants were cultivated in Murashige–Skoog nutrient medium supplemented with 6% sucrose. Time courses of utilization of the main nutrients in the medium during cultivation of D. stramonium hairy root cultures are also presented.     

Application of metabolic engineering to the production of scopolamine

Scopolamine is an alkaloid widely used in medicine for its anticholinergic activity. The aim of this review is to show that metabolic engineering techniques constitute a suitable tool to improve the production of tropane alkaloids, focusing in particular on scopolamine. We present an overview of results obtained by various research groups, including our own, who have studied the overexpression of genes involved in the biosynthesis of scopolamine in different plant species that produce tropane alkaloids. Experiments carried out to improve production in hairy root cultures will also be described, as well as those attempting to biotransform hyoscyamine into scopolamine in roots and transgenic tobacco cells.     

实时直接分析-串联质谱法快速筛查火锅底料、牛肉面汤及调味料中罂粟壳生物碱

建立了采用实时直接分析-串联质谱(DART-MS/MS)对火锅底料、牛肉面汤及调味料中5种非法添加的罂粟壳生物碱进行快速筛查的方法。样品经乙腈提取净化后,在离子化温度为300℃、栅极电压为150 V、进样速率为0.8 m m/s的DART离子源正离子模式条件下进样,以电喷雾离子源正离子多反应监测(MRM)模式进行检测,实现了样品经简单预处理后使用DART-MS/MS进行检测的新方法。该方法简便、快速,能满足大批量非法添加样品的快速筛查分析要求。     

Rapid determination of 5-hydroxymethylfurfural by DART ionization with time-of-flight mass spectrometry

DART (direct analysis in real time), a novel technique with wide potential for rapid screening analysis, coupled with high-resolution time-of-flight mass spectrometry (TOF-MS) has been used for quantitative analysis of 5-hydroxymethylfurfural (5-HMF), a typical temperature marker of food. The DART/TOF-MS method was optimised and validated. Quantification of 5-HMF was achieved by use of a stable isotope-labelled 5-HMF standard prepared from glucose. Formation of 5-HMF from saccharides, a potential source of overestimation of results, was evaluated. Forty-four real samples (honey and caramelised condensed sweetened milk) and 50 model samples of heated honey were analysed. The possibility of using DART for analysis of heated samples of honey was confirmed. HPLC and DART/TOF-MS methods for determination of 5-HMF were compared. The correlation equation between these methods was DART?=?1.0287HPLC?+?0.21340, R ²?=?0.9557. The DART/TOF-MS method has been proved to enable efficient and rapid determination of 5-HMF in a variety of food matrices, for example honey and caramel.     

Methyl jasmonate influence on silymarin production and plant stress responses in Silybum marianum hairy root cultures in a bioreactor

In this article our aim was to evaluate mass cultivation of S. marianum hairy roots in a bioreactor to produce silymarin. The effects of methyl jasmonate (MJ) elicitation on the accumulation of silymarin and the extent of the MJ-induced oxidative damage were investigated in bioreactor hairy root cultures of S. marianum. The growth rate of the bioreactor hairy root cultures was higher than that of those in a shake flask after 3 weeks. Silymarin accumulation was increased from 0.13?mg?g?1 DW in non-treated hairy roots to 0.22?mg?g?1 DW in hairy roots 72?h after 100?μM?MJ treatment. Guaiacol peroxidase and ascorbate peroxidase were activated by MJ?72?h after treatment, being 3.2- and 1.3-fold higher, respectively, than that of the control. An increase in enzymatic activity suggests increased scavenging of reactive oxygen species, indicating the tolerance to MJ stress. These results suggest that MJ elicitation is beneficial for silymarin production using bioreactor hairy root cultures.     

Metabolomics based on liquid chromatography with mass spectrometry reveals the chemical difference in the stems and roots derived from Ephedra sinica

To better understand different traditional uses of the stems (known as Mahuang) and roots (known as Mahuanggen) of Ephedra sinica, their chemical difference should be investigated. In this study, an ultra‐fast liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry untargeted metabolomics approach was established to reveal global chemical difference between Mahuang and Mahuanggen. Clear separation was observed in scores plots of principal component analysis and orthogonal partial least squares‐discriminant analysis. Twenty two chemical markers responsible for such separation were screened out and unambiguously/tentatively characterized. Then chemical markers of pharmacologically important ephedrine and pseudoephedrine were absolutely quantified using liquid chromatography coupled with tandem mass spectrometry under multiple reaction monitoring mode. The results showed that Mahuang was rich in ephedrine‐type alkaloids, while Mahuanggen was rich in macrocyclic spermine alkaloids. Additionally, different types of flavan‐3‐ols and flavones exist in Mahuang and Mahuanggen extracts. This research facilitates a better understanding of different traditional uses of Mahuang and Mahuanggen and provides references for chemical analysis of other medicinal plants.     

Direct analysis in real time—a critical review on DART-MS

Direct analysis in real time mass spectrometry (DART-MS) has become an established technique for rapid mass spectral analysis of a large variety of samples. DART-MS is capable of analyzing the sample at atmospheric pressure, essentially in the open laboratory environment. DART-MS can be applied to compounds that have been deposited or adsorbed on to surfaces or that are being desorbed therefrom into the atmosphere. This makes DART-MS suitable and well-known for analysis of ingredients of plant materials, pesticide monitoring on vegetables, forensic and safety applications such as screening for traces of explosives, warfare agents, or illicit drugs on luggage, clothes, or bank notes, etc. DART can also be used for analysis of either solid or liquid bulk materials, as may be required in quality control, or to quickly investigate the identity of a compound from chemical synthesis. Even living organisms can be subjected to DART-MS. Driven by different needs in analytical practice, the combination of the DART ionization source and interface can be configured in multiple geometries and with various accessories to adapt the setup as required. Analysis by DART-MS relies on some sort of gas-phase ionization mechanism. In DART, initial generation of the ionizing species is by use of a corona discharge in a pure helium atmosphere which delivers excited helium atoms that, upon their release into the atmosphere, will initiate a cascade of gas-phase reactions. In the end, this results in reagent ions created from atmospheric water or (solvent) vapor in the vicinity of the surface subject to analysis where they effect a chemical ionization process. DART ionization processes may generate positive or negative ions, predominantly even-electron species, but odd-electron species do also occur. The prevailing process of analyte ion formation from a given sample is highly dependent on analyte properties.     

Development of a Mathematical Model for Growth and Oxygen Transfer in In Vitro Plant Hairy Root Cultivations

Genetically transformed, "Hairy roots" once developed can serve as a stable parent culture for in vitro production of plant secondary metabolites. However, the major bottleneck in the commercial exploitation of hairy roots remains its successful scale-up due to oxygen transfer limitation in three-dimensionally growing hairy root mass. Mass transfer resistances near the gas-liquid and liquid-solid boundary layer affect the oxygen delivery to the growing hairy roots. In addition, the diffusional mass transfer limitation due to increasing size of the root ball (matrix) with growth also plays a limiting role in the oxygen transfer rate. In the present study, a mathematical model is developed which describes the oxygen transfer kinetics in the growing Azadirachta indica hairy root matrix as a case study for offline simulation of process control strategies ensuring non-limiting concentrations of oxygen in the medium throughout the hairy root cultivation period. The unstructured model simulates the effect of oxygen transfer limitation in terms of efficiency factor (η) on specific growth rate (μ) of the hairy root biomass. The model is able to predict effectively the onset of oxygen transfer limitation in the inner core of the growing hairy root matrix such that the bulk oxygen concentration can be increased so as to prevent the subsequent inhibition in growth of the hairy root biomass due to oxygen transfer (diffusional) limitation.     

Mass‐spectrometry‐directed analysis and purification of pyrrolizidine alkaloid cis/trans isomers in Gynura japonica

Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass‐directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including (E)‐seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid‐induced toxicity.     

生附子中C19型二萜生物碱的高速逆流色谱分离及其结构鉴定

应用高速逆流色谱法分离制备了生附子中的3个C19型二萜生物碱类化合物。以正己烷-乙酸乙酯-甲醇-水(3:5:4:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2.0 mL/min、检测波长235 nm条件下进行分离制备;一次性从90 mg附子总碱粗提物中分离制备得到15.3 mg北草乌碱,35.1 mg中乌头碱和22.7 mg次乌头碱,经高效液相色谱分析,测得它们的纯度分别为97.9%、96.2%和99.2%。并应用波谱(电喷雾离子质谱、核磁共振氢谱和核磁共振13C谱)解析法确定了它们的结构。利用该方法可以对生附子中的二萜类生物碱成分进行快速的分离和纯化