Lidia Mateus

Enantioseparation of atropine by capillary electrophoresis using sulfated β-cyclodextrin: application to a plant extract.

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.

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.

Optimisation of accelerated solvent extraction of cocaine and benzoylecgonine from coca leaves

Accelerated solvent extraction (ASE) was developed for the rapid extraction of cocaine and benzoylecgonine from coca leaves. Several parameters such as the nature of the extracting solvent, the pressure, temperature, extraction time, addition of alkaline substances, and sample granulometry were investigated to find the best extraction conditions. A central composite design was used to optimise critical parameters (pressure, temperature, and extraction time) and to assess the robustness of the extraction method. It was demonstrated that the extraction method was perfectly robust around optimal conditions (20 MPa, 80°C, and 10 min). Determination of both cocaine and benzoylecgonine in coca leaves was carried out by means of gas chromatography with flame ionisation detection (GC-FID) or capillary electrophoresis with UV detection (CE-UV). With the latter, separation of both compounds was achieved in less than 4 min with the use of a short-end injection procedure.

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.

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

SummaryThe 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.

Capillary electrophoresis for the analysis of tropane alkaloids : pharmaceutical and phytochemical applications

Three capillary electrophoresis methods, using UV detection, were developed for the simultaneous determination of several tropane alkaloids, including atropine, scopolamine and synthetic derivatives. After optimization, the validated capillary zone electrophoresis methods were applied to the determination of these compounds in various pharmaceutical forms, such as ophthalmic and injection solutions, tablets, suppositories and aerosols. Capillary electrophoresis in the micellar mode was found to be more appropriate for the analysis of hyoscyamine and scopolamine in plant material. These two compounds are generally found together with other tropane alkaloids which present similar structures and charge to mass ratio. Furthermore, the separation of positional isomers, such as hyoscyamine and littorine generally encountered in plant extracts, was also considered. The developed method was applied to the analysis of hairy root extracts of Datura candida x Datura aurea, Datura quercifolia and Hyoscyamus albus.

Use of a Doehlert design in optimizing the analysis of selected tropane alkaloids by micellar electrokinetic capillary chromatography

The Doehlert design was used in optimizing the analysis of selected tropane alkaloids (including hyoscyamine, scopolamine and littorine) by micellar electrokinetic capillary chromatography. Three variables, i.e., pH, SDS concentration and organic modifier percentage were investigated. Resolutions as well as analysis time, generated power and current were established as responses. A model was obtained by linear multiple regression of a second-degree mathematical expression. The Doehlert design structure allows to study the response surfaces with a good quality of the parameters estimation of the quadratic model. Thus, it is possible to obtain the region in which the optimum values of such variables are simultaneously achieved. From the models, the most favorable conditions were determined by optimizing the resolution between hyoscyamine and littorine –two positional isomers– and by setting the other responses at threshold values. Successful results were obtained using a 30 mM borate–phosphate buffer at basic pH (8.7) in the presence of 40 mM sodium dodecyl sulfate and 16.5% acetonitrile. Results were compared with a previous study in which a systematic investigation of the operating parameters was carried out.

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

SummaryMicellar 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.

Nonaqueous versus aqueous capillary electrophoresis for the dosage of N-butylscopolamine in various pharmaceutical formulations

A simple nonaqueous capillary electrophoresis method is described for the separation of several atropine and scopolamine related drugs. The analysis of these pharmaceutical compounds was performed in a methanol-acetonitrile (25/5, v/v) mixture containing 25 mM ammonium acetate and 1 M acetic acid. The robustness was proved using a full factorial design at two levels. The method was validated and successfully applied for the determination of N-butylscopolamine in different pharmaceutical preparations. Results were compared to those obtained by a capillary electrophoresis method based on aqueous media.

Validated capillary electrophoresis method for the determination of atropine and scopolamine derivatives in pharmaceutical formulations

The simultaneous determination of atropine and scopolamine derivatives, which have similar structures, was investigated by using capillary zone electrophoresis. The effects of buffer pH, buffer concentration and hydroxypropyl-beta-cyclodextrin concentration on migration time and resolution of the investigated compounds were systematically studied. The selected electrophoretic buffer consisted of a 80 mM sodium citrate pH 2.5, containing 2.5 mM hydroxypropyl-beta-cyclodextrin as the complexing agent. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 1 and 2% for the intermediate precision of migration times and peak area ratios, respectively. The linearity of the method was assessed between 50 and 150% of the theoretical content (coefficient of correlation greater than 0.99). The proposed method was found to be suitable and accurate for the determination of these basic drugs in pharmaceutical preparations.