Atropine is an antimuscarinic alkaloid identified in Atropa belladonna. In pharmacopeias, percolation is standardized as an extraction method for A. belladonna leaves, along with liquid-liquid extraction as a cleanup procedure and titration as an analytical method for assaying the atropine in the leaves. In this study, a faster, solvent-saving, and more reliable method for quality control of A. belladonna samples was developed. Ultrasound-assisted extraction was proposed and optimized by fractional factorial design followed by Box-Behnken design. For modeling atropine content, the following optimal conditions were established: particle size, 180 μm; percentage methanol in water, 50%; volume of solvent, 15 ml; time of extraction, 60 min; and number of extractions, two. This led to a significant improvement in atropine extraction (P < 0.001). For cleanup, solid-phase extraction was used as an alternative to liquid-liquid extraction, giving similar results, with higher reproducibility. Finally, for the atropine assay, a UPLC method was validated as a substitute for the classic titration method. Taken together, the development of an ultrasound-assisted extraction-solid-phase extraction-UPLC approach allowed the determination of atropine content in A. belladonna leaves in a time- and solvent-saving manner, with high reliability.
Keywords: alkaloid; design of experiment; extraction; method validation; response surface methodology.
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