Multi-development-HPTLC method for quantitation of hyoscyamine, scopolamine and their biosynthetic precursors in selected solanaceae plants grown in natural conditions and as in vitro cultures.

INTRODUCTION: Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors. OBJECTIVE: To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆(1) -pyrrolium cation) with tropane alkaloids. METHODS: Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated. RESULTS: Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples. CONCLUSION: This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures).

Simultaneous analysis of hyoscyamine, scopolamine, 6beta-hydroxyhyoscyamine and apoatropine in Solanaceous hairy roots by reversed-phase high-performance liquid chromatography.

A new high-performance liquid chromatographic method is described for tropane alkaloid analysis in genetically transformed root cultures of Datura innoxia Mill. and Atropa belladonna L. Sample preparation, tropane alkaloid extraction with chloroform-methanol-concentrated ammonia 15:5:1 (v/v/v), was followed by solid-phase extraction on Supelclean LC-18 cartridges. Optimized conditions and careful pH control resulted in high recovery and reproducibility. Simultaneous determination of apoatropine, 6beta-hydroxyhyoscyamine, hyoscyamine and scopolamine was performed by HPLC on C18 (2) reversed-phase column. The application of Luna new-generation silica-based stationary phase resulted in excellent peak shapes using an ion-pair reagent and triethanolamine free mobile phase and allowed to exploit the full power of pH-dependent selectivity. Simplicity and improved selectivity make this method a preferred alternative of published ion-pair chromatographic methods. Validation studies proved that the global method has good repeatability and satisfactory recovery. Absolute limits of detection were 0.6, 0.6, and 0.8 ng for hyoscyamine, 6beta-hydroxyhyoscyamine, and scopolamine respectively.

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.

Unit-dose assay of tropine alkaloids and their synthetic analogs.

A charge-transfer spectrophotometric method was developed for unit-dose assay of the tropine alkaloids and some of their synthetic analogs. The high molar absorptivities of the charge-transfer bands of the alkaloids with iodine in ethylene dichloride resulted in improved recoveries and good precision, particularly at the low dose levels of pediatric and hypodermic tablets.

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.

Distribution of hyoscyamine and scopolamine in Datura stramonium.

The production of hyoscyamine and scopolamine in Datura stramonium has been investigated in the different plant parts, at different stages of their life cycle. Maximum contents were found in the stems and leaves of young plants, hyoscyamine being always the predominant component.

Molecular and crystal structure of flutropium bromide.

(8r)-8-(2-Fluoroethyl)-3 alpha-hydroxy-1 alpha H, 5 alpha H-tropanium bromide benzilic acid ester (flutropium bromide, Ba 598 BR) crystallizes in the monoclinic space group P 2(1)/n and shows disorder. Nevertheless the averaged crystal structure could be solved and refined to R = 0.113.