Quantification of Atropine in Leaves of Atropa belladonna: Development and validation of method by high-perfomance liquid chromatography (HPLC)

Abstract

The leaves of Atropa belladonna (L) are characterized by the presence of the alkaloid atropine, known for the
antimuscarinic activity. They are used in therapy, mainly in homeopathic preparations. An HPLC method was developed
and validated to quantify atropine in belladonna leaves. The samples were extracted with methanol, followed by acidbase
extraction with 5% HCl and dichloromethane. Analysis by HPLC was performed on C18 column, in a linear
gradient system using two mobile phases (water and acetonitrile), both acidified with trifluoroacetic acid. The
determinations were performed using a reference standard and a diode array detector at 210 nm. The method was
validated and proved to be specific/selective, comparing the UV profiles and the purity of the atropine peaks in reference
and sample solutions and analyzing sample solutions with and without addition of standard, which produced an increase
only of the area of peak of the sample, without changing the area of the adjacent peaks. The linearity (50 - 200 μg/mL)
was provided by analysis of the analytical curves of atropine, with r2 = 0.9996. LOD and LOQ were 3.75 and 11.4
μg/ml, respectively. The method was precise, reproducible and accurate, and presented recovery equal to 103.0%. The
method was considered robust for the analyzed parameters. Four commercial samples were analyzed and the mean levels
of atropine found ranged from 0.16 - 0.27 %. Thus, the method developed is effective for atropine quantification in A.
belladona leaves, because meets validation requirements of current legislation, besides presenting itself as a useful tool in
the control of the quality of this plant material.