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. The leaves 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 acid-base extraction with 5% HCl and dichloromethane. Analysis by HPLC was performed on C18 column, in a linear gradient system using a system with 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 developed method is effective for quantification of atropine in beladona leaves and meets the validation requirements of current legislation and ensuring the quality control of the plant material.

Quantification of Atropine and Scopolamine in Different Plant Organs of Datura Metel: Development and Validation of High-Performance Liquid Chromatography Method

“…The average % RSD was 1.1 for SC and 1.5 for AT. % Recovery and % RSD were within the accepted limits from 80 % to 120 % [23], and not more than 5 % [13], respectively, which indicates the applicability of the method for scopolamine and atropine analysis.…”

“…Many healthy people have died from ingesting Datura, usually as a result of respiratory paralysis or heart failure [13]. For this reason, the assessment of tropane alkaloids concentration in plants of the Solanaceae family is highly important for toxicological and forensic purposes [14].…”

Scopolia carniolica var. hladnikiana: Alkaloidal Analysis and Potential Taxonomical Implications

“…The methods used for extraction and analysis were based upon previously published research [ 29 ] with minor alterations.…”

Pipes and Potions: Testing the Efficacy of European Folk Preparation Methods for Anticholinergic Solanaceae Plants

“…The control samples for both root and leaf powder were extracted using a modified procedure based on our previous project [ 14 ], which followed a process modified from previously published research [ 15 ].…”