Micellar electrokinetic capillary chromatography for selected tropane alkaloid analysis in plant extract

Summary Micellar electrokinetic capillary chromatography was applied to the simultaneous analysis of six tropane alkaloids, including hyoscyamine and scopolamine. Successful results were obtained using a 30 mM boratephosphate buffer at basic pH (8.5) in the presence of 50 mM sodium dodecyl sulfate. The operating conditions, such as buffer concentration and pH, micelle concentration and organic modifier type and percentage were also discussed on the basis of the results given with a tropane alkaloid mixture. Addition of organic modifiers showed an improvement in separation efficiency and resolution. Moreover, hyoscyamine and littorine, two positional isomers, were only resolved by the addition of organic solvents such as methanol or acetonitrile. The optimized conditions were finally applied to the analysis of tropane alkaloids found in genetically transformed root cultures ofDatura candida x D. aurea. Dedicated to Professor Werner Haerdi on the occasion of his 70^th birthday.     

A non-aqueous solid phase extraction method for alkaloid enrichment and its application in the determination of hyoscyamine and scopolamine

A non-aqueous solid phase extraction (SPE) method utilizing silica based strong cation exchange (SCX) was developed and optimized for the enrichment of alkaloids. In this method, silica based SCX SPE columns were used for the elimination of non-alkaloid compounds and the preconcentration of alkaloids from the extracts. Mass spectrometry was employed to analyze the alkaloid-enriched fraction, and results showed that the SPE method developed in this study was effective for the removal of non-alkaloids. Then, this pretreatment method was combined with high performance liquid chromatography for the quantification of scopolamine and hyoscyamine from Scopolia tangutica Maxim. The recoveries of scopolamine and (-)-hyoscyamine were 98.51% and 91.12%, respectively. Relative standard deviation values were 1.4% for scopolamine and 1.6% for (-)-hyoscyamine. The linearity was good in the 0.01-0.8 mg mL(-1) range for hyoscyamine and 0.01-0.4 mg mL(-1) range for scopolamine.     

Capillary electrophoresis-diode array detection--electrospray mass spectrometry for the analysis of selected tropane alkaloids in plant extracts

Capillary zone electrophoresis, coupled to UV and interfaced with electrospray ionization mass spectrometry (ESI-MS), is described for the simultaneous analysis of hyoscyamine and scopolamine. On-line UV detection occurred at 22 cm from the inlet of the capillary and ESI-MS monitoring was performed along the entire length of the capillary (85 cm). An alkaline solution of 40 mM ammonium acetate at pH 8.5 was suitable for the analysis of the alkaloids under consideration. Under the optimized conditions, including CE and ESI-MS parameters, the two alkaloids were resolved within a short time and with very high sensitivity. The differentiation of hyoscyamine and its positional isomer littorine, commonly encountered in plant material, is also presented using up-front collision-induced dissociation. Finally, the developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura candida x D. aurea hairy root extract.     

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.     

Nonaqueous capillary electrophoresis for the analysis of selected tropane alkaloids in a plant extract

Summary The potential of nonaqueous capillary electrophoresis has been investigated for the separation of structurally similar tropane alkaloids. The effects of the organic solvent and of electrolyte composition on separation selectivity, migration times, and efficiency are described. The addition of trifluoroacetic acid to the separation buffer was found beneficial for manipulation of the order of migration of the two positional isomers littorine and hyoscyamine. Replicate injections under nonaqueous conditions gave migration time and peak area data of excellent precision. The application of the optimized conditions to the analysis of hyoscyamine and scopolamine in genetically transformed root cultures ofDatura candida x D. aurea is presented.     

Identification and estimation of the levo isomer in raw materials and finished products containing atropine and/or hyoscyamine

The belladonna alkaloids atropine sulfate and hyoscyamine sulfate, occasionally used as anticholinergic and antimuscarinic agents, have identical molecular formulas but different stereo configurations. Hyoscyamine sulfate contains almost 100% of the levo isomer, whereas atropine sulfate is composed of equal parts of dextro and levo isomers. It is believed that the therapeutic properties of these alkaloids are due exclusively or primarily to the levo isomer. Currently available methods determine only the total amount of atropine (hyoscyamine) sulfate. A method has been developed and is reported for the identification and estimation of the levo and dextro isomers of atropine and hyoscyamine. Reference solutions are prepared in methanol at the following weights per 100 mL: 8.0 mg atropine sulfate; 4.0 mg hyoscyamine sulfate; 7.0 mg scopolamine hydrobromide; and 10.0 mg homatropine methylbromide. Samples of raw materials are similarly prepared in methanol, commercial products are also extracted or diluted with methanol, and solutions are filtered. Liquid chromatography is used for separations on a 25 cm Chirobiotic T2 column. The mobile phase is prepared by mixing 3.0 mL acetic acid and 2.0 mL triethylamine with 1000 mL methanol. The injection volume is 100 or 200 microL; the flow rate is about 0.35 mL/min. Fluorescence detection is at 255 nm excitation and 285 nm emission. Scopolamine hydrobromide and hyoscyamine eluted after 20 and 60 min, respectively. Atropine sulfate generated 2 peaks after 60 and 65 min. Homatropine methylbromide also produced 2 peaks after 70 and 85 min. Samples tested in this study included raw materials and commercial tablets or injections containing belladonna alkaloids. In all cases, the percentage calculated was that of the levo isomer relative to the total amount of atropine (hyoscyamine) present.     

Analysis of tropane alkaloids with thermospray high-performance liquid chromatography-mass spectrometry

A thermospray high-performance liquid chromatography-mass spectrometry method for analysis of hyoscyamine and scopolamine in plant cell culture samples is described. The alkaloids were separated on a polymeric reversed-phase column with an alkaline ammonium acetate buffer-acetonitrile eluent. Selected-ion recording of the protonated molecular ions was used for quantitation of the compounds. The compounds were fragmented by discharge-assisted ionization and elevated thermospray capillary temperatures or ion repeller potentials.     

How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhy, drazone (MGBG) and the phenylalamine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250–1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.     

Automated pre-column derivatisation HPLC

Summary An automated pre-column derivatisation system is presented. As an example of its application we have chosen ion-pair partition chromatography. Ion-pairs of a mixture of some alkaloids (apoatropine, hyoscyamine, scopolamine and ergotamine) were formed with picric acid in an aqueous phase, extracted into chloroform and injectedin-situ onto a silica gel column for separation within 15 minutes.This paper shows the possibility of making an automated pre-column derivatisation system with a relative standard deviation of less than 3% per injection (n=6). The linearity check was made for hyoscyamine and ergotamine solutions in the concentration range of 80 to 200 ng respectively 240 to 720 ng per injection and found to be linear. The information obtained is of general validity and could generate more studies in this area.     

Biogenesis of the alkaloids ofDatura innoxia

Summary 1. It has been shown that acetone, acetate, and methylamine are utilized in the biosynthesis of the tropane alkaloids. The most specific precursor of the alkaloids was acetone.2. Acetate and methylamine are implicated in alkaloid biosynthesis in the roots, and acetone in the leaves. The biosynthetic process does not have a smooth nature but one with a number of maxima.3. The accumulation of hyoscyamine takes place predominantly in the roots and that of scopolamine in the generative organs. The content of scopolamine is higher.Khimiya Prirodnykh Soedinenii, Vol. 3, No. 2, pp. 188–191, 1967     

超临界流体萃取在天然药物分析中的应用

按物质的不同性质综述了１９９５年以来超临界流体萃取技术在天然药物分析中的一些进展情况,常见的分析主要包括萜类、生物碱、黄酮类、挥发油及苯丙素类。超临界流体萃取作为一门新型的样品前处理技术,在天然药物的分离分析中展示了其特有的优点。该技术操作温度低,不会引起热敏性的分解变质;使用的有毒溶剂少,从而减少化学药品对药物的污染。     

Determination of the enantiomeric purity of scopolamine isolated from plant extract using achiral/chiral coupled column chromatography

The optical purity of scopolamine derived from Datura sanguinea was determined using coupled column chromatography. A C18 column was used to separate scopolamine from the additional alkaloids and other biological material present in the vegetal extract. The C18 column was coupled through a six-port switching valve to two beta-cyclodextrin columns in series which were used to resolve the scopolamine enantiomers. A single acetylated beta-cyclodextrin column gives equivalent results to the native cyclodextrin columns because of slightly higher enantioselectivity for scopolamine. A multistep extraction procedure is used to isolate scopolamine from the vegetal material. 4-6% of the scopolamine in the final extract was found to be the d enantiomer. Sample extracts as well as commercial scopolamine hydrobromide were treated under various conditions commonly encountered during typical commercial extraction procedures and analyzed in order to determine if the d enantiomer was present in the original material or if it was produced during the extraction process and, if so, determine which step and conditions contribute to racemization. Both the salt and the extract were found to be susceptible to racemization under basic conditions (greater than or equal to pH 9) although the extract appeared to be more susceptible than the salt. Tropic acid formed from the hydrolysis of scopolamine seemed to be completely racemized even though the remaining scopolamine was only partially racemized. Within experimental error, no d enantiomer was found in the original fresh plant material.     

High-speed ion-pair partition chromatography in pharmaceutical analysis.

Ion-pair chromatography offers attractive possibilities in pharmaceutical analysis. The specificity of the separation systems can be varied over a wide range by appropriate selection of the stationary phase. The choice of a suitable counter-ion can also drastically improve the detection limit, permitting the determination of drug substances in low dosage and possibly of by-products or breakdown products. Ion-pair chromatography of tropane and ergot alkaloids has been investigated using picrate as counter-ion. Alumina, Kieselguhr and various grades of silica gel have been tested as supports. Partition properties studied in a batch procedure have been compared with the actual chromatographic conditions. Columns (10 cm) filled with silical gel (particle size, 5 mum; pore size, 1000 A) show the best performance in the separation of hyoscyamine, scopolamine and ergotamine as picrate ion-pairs. Close control of pH and temperature is essential for reproducible separations. Improvements in detection limits between 100 and 300 times have been observed with these systems. Ion-pair extractions of these alkaloids from dosage forms can be used for sample preparation prior to injection on the the column. This provides an added degree of selectivity and sensitivity.     

Orthogonal array design for the optimization of supercritical fluid extraction of baicalin from roots of Scutellaria baicalensis Georgi

Supercritical fluid was used to extract baicalin from the roots of Scutellaria baicalensis Georgi. An orthogonal array design (OAD), OA(9)(3(4)), was employed as a chemometric method for the optimization of the supercritical fluid extraction of baicalin from the herbal medicine. Four parameters, namely, modifiers, temperature and pressure of supercritical fluid, and the dynamic extraction time, were studied and optimized by a three-level OAD in which the interactions between the parameters were neglected. Determinations of the extracts were performed by high-performance liquid chromatography. The effects of parameters were studied using analysis of variance. The results showed that selection of the modifier was the main factor in attaining higher yields of baicalin. While the other three factors had some effect on the extraction of the compound, the effect was much less than that of the modifiers. 1,2-Propanediol-modified supercritical fluid was more effective than 95% ethanol-modified supercritical carbon dioxide or methanol-modified supercritical carbon dioxide for the extraction of baicalin from the solid matrix. Finally, experimental conditions were proposed which can provide the highest extraction yield with respect to the considered factors.     

HPLC-electrospray ionization-MS-MS analysis of Cephalotaxus harringtonia leaves and enhancement of the extraction efficiency of alkaloids therein by SFE

A high-performance liquid chromatographic method is developed for use in the electrospray mass spectrometric (MS)-MS analysis of alkaloids contained in Cephalotaxus harringtonia leaves. Nine alkaloids having ester groups can be separated and detected with good sensitivity. The MS and MS-MS spectra obtained provides information on their chemical structures. Supercritical fluid extraction is also applied in order to improve the extraction efficiency of Cephalotaxus alkaloids such as cephalotaxine, harringtonine, homoharringtonine, and isoharringtonine. When carbon dioxide-methanol-water (80:18:2, v/v) is used the extraction yield is found to be higher than that using the other supercritical solvents evaluated and conventional organic solvent extraction.     

Solid-liquid extraction and cation-exchange solid-phase extraction using a mixed-mode polymeric sorbent of Datura and related alkaloids

Tropane alkaloids solid-liquid extraction methods were developed and comprised ambient pressure ones: extraction with hot solvent, extraction at room temperature, on ultrasonic bath as well as pressurised liquid extraction (PLE) techniques. The highest yields of l-hyoscyamine in methanol PLE method (3 x 5 min, 110 degrees C) and scopolamine extracted with 1% tartaric acid in methanol (15 min, 90 degrees C) were determined. A mixed-mode reversed-phase cation-exchange solid-phase extraction (SPE) procedure was optimised for simultaneous recoveries of L-hyoscyamine, scopolamine, scopolamine-N-oxide from plant extracts as well as quaternary alkaloid representative: scopolamine-N-methyl bromide. First three alkaloids were efficiently eluted (recoveries 80-100%) from an Oasis MCX cartridge with methanol-10% ammonia (3:1, v/v) solution, whereas for the quaternary salt tetrahydrofuran-methanol-25% ammonia (6:1:3, v/v) was used with recoveries 52-6%. HPTLC-densitometric assay on silica gel plates was elaborated at 205 nm without derivatization and included: single development (over a distance 9.5 cm) with acetone-methanol-water-25% ammonia (85:5:5:8, v/v) mobile phase for L-hyoscyamine and scopolamine separation, whereas for scopolamine-N-oxide and scopolamine-N-methyl bromide a second development (to a distance 5.5 cm) with acetonitrile-methanol-85% formic acid (120:5:5, v/v) was applied. Newly elaborated RP-HPLC-diode array detection method was performed on Waters XTerra RP-18 column with gradient of acetonitrile in 15 mM ammonia solution and alkaloids were baseline separated within 20 min. Both chromatographic methods were validated and their quantitative results were compared. Good correlation between HPLC and HPTLC quantitative results was measured (correlation coefficients of mean values were 0.92086 and 0.99995 for L-hyoscyamine and scopolamine, respectively). In the RP-HPLC method, which was from 1.5- up to 7-fold more sensitive than HPTLC, limits of detection (LOD) and limits of quantitation (LOQ, in bracket) were (in ng/microl) as follows: 0.25 (0.82) for L-hyoscyamine, 0.29 (0.97) for scopolamine, 0.13 (0.45) for scopolamine-N-oxide and 0.58 (1.91) for scopolamine-N-methyl bromide. By the use of the optimised chromatographic methods, 14 various samples from the leaves and fruits of Datura sp. were screened for L-hyoscyamine and scopolamine contents and the most promising samples were established.     

Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.     

超临界CO2络合萃取金属离子的研究与应用

本文综述了国内外对超临界CO2络合萃取技术的研究与应用现状.总结并分析了超临界CO2流体萃取金属离子的原理、方法,动力学机理,络合剂和改性剂的选择,络合萃取过程的影响因素,并着重介绍了其在环境保护、核工业、冶金工业、化学分析、中药产业、保健品工业中的应用.最后指出了它目前存在的问题,并对未来研究方向进行了展望.     

Alkaloids of the Genus Datura: Review of a Rich Resource for Natural Product Discovery

The genus Datura (Solanaceae) contains nine species of medicinal plants that have held both curative utility and cultural significance throughout history. This genus’ particular bioactivity results from the enormous diversity of alkaloids it contains, making it a valuable study organism for many disciplines. Although Datura contains mostly tropane alkaloids (such as hyoscyamine and scopolamine), indole, beta-carboline, and pyrrolidine alkaloids have also been identified. The tools available to explore specialized metabolism in plants have undergone remarkable advances over the past couple of decades and provide renewed opportunities for discoveries of new compounds and the genetic basis for their biosynthesis. This review provides a comprehensive overview of studies on the alkaloids of Datura that focuses on three questions: How do we find and identify alkaloids? Where do alkaloids come from? What factors affect their presence and abundance? We also address pitfalls and relevant questions applicable to natural products and metabolomics researchers. With both careful perspectives and new advances in instrumentation, the pace of alkaloid discovery—from not just Datura—has the potential to accelerate dramatically in the near future.     

Supercritical fluid extraction and liquid chromatography-electrospray mass analysis of vinblastine from Catharanthus roseus

Supercritical fluid extraction using carbon dioxide modified with methanol, methanol-diethylamine, or methanol-triethylamine was used to extract vinblastine from the aerial portions of Catharanthus roseus. An HPLC-electrospray ionization (ESI)/MS analysis method was also developed to quantify the alkaloids in these extracts. Of the supercritical solvents evaluated, carbon dioxide-methanol-triethylamine (80 : 18 : 2) at 80 degrees C and 34.0 MPa greatly improved the supercritical fluid extraction (SFE) yield of vinblastine by as much as 76.4% over methanol extraction, while the other solvent conditions extracted the compound at yields less than 25% that of a methanol extraction. These results were confirmed by the robust HPLC-ESI/MS analytical method developed in this study.