Isolation of atropine and scopolamine from plant material using liquid-liquid extraction and EXtrelut® columns.

Tropane alkaloids are toxic secondary metabolites produced by Solanaceae plants. Among them, plants from Datura genus produce significant amounts of scopolamine and hyoscyamine; the latter undergoes racemization to atropine during isolation. Because of their biological importance, toxic properties and commonly reported food and animal feed contamination by different Datura sp. organs, there is a constant need for reliable methods for the analysis of tropane alkaloids in many matrices. In the current study, three extraction and sample-clean up procedures for the determination of scopolamine and atropine in plant material were compared in terms of their effectiveness and repeatability. Standard liquid-liquid extraction (LLE) and EXtrelut® NT 3 columns were used for the sample clean-up. Combined ultrasound-assisted extraction and 24h static extraction using ethyl acetate, followed by multiple LLE steps was found the most effective separation method among tested. However, absolute extraction recovery was relatively low and reached 45-67% for atropine and 52-73% for scopolamine, depending on the compound concentration. The same method was also the most effective one for the isolation of target compounds from Datura stramonium leaves. EXtrelut® columns, on the other hand, displayed relatively low effectiveness in isolating atropine and scopolamine from such a complex matrix and hence could not be recommended. The most effective method was also applied to the extraction of alkaloids from roots and stems of D. stramonium. Quantitative analyses were performed using validated method based on gas chromatography with flame ionization detector (GC-FID). Based on the results, the importance of the proper selection of internal standards in the analysis of tropane alkaloids was stressed out.

Evaluation of four derivatization methods for the analysis of fatty acids from green leafy vegetables by gas chromatography.

Green leafy vegetables are valuable secondary sources of nutrients, including lipids, commonly consumed in developing countries. However, method development for the analysis of fatty acids is usually focused on the animal lipid samples, rarely including natural plant extracts. Hence, the usefulness of four derivatization methods for the gas chromatographic analysis of plant lipids was studied. Methylation using 10% solution of BF3 in methanol and 2.0M solution of (trimethylsilyl)diazomethane (TMSD) in hexane, trimethylsilylation and tert-butyldimethylsilylation were compared using lipid standards and extracts from the leaves of Solanum macrocarpon and S. melongena after saponification. While silylation was found effective and precise using lipid standards, it initially did not perform well in the analysis of plant lipids due to the presence of transesterification products in samples. Optimization of the hydrolysis conditions resulted in an effective analysis of these derivatives, but poor separation of FA(18:0) from unsaturated FA(18:X) compounds and the presence of larger amounts of interferences disqualified the use silylation for the analysis of plant fatty acids in applied analytical conditions. Methylation using TMSD gave more precise quantitative results when compared to BF3/MeOH method. Also, it produced a significantly lower amount of interferences when applied to plant lipid samples. Additionally, the TMSD-based method is simple, safe and less time-consuming when compared to other procedures. Thus, we suggest using TMSD-based methylation as a method of choice in the GC analysis of plant-derived fatty acids.