Enantiomeric determination of atropine in Datura Stramonium and Brugmansia Arborea seeds by LC-MS

Solanaceaes family plants can contains huge amounts of tropane alkaloids, which have anticholinergic activity. There are more than 200 tropane alkaloids but most studies are mainly focused on atropine and scopolamine determination. Although animals can also be affected by these compounds, they are less susceptible than humans to poison by alkaloids, because the presence of an esterase enzyme, which efficiently biotransformed the atropine. Besides, atropine are the mix of two enantiomers, (+,-) hyoscyamine, and only (-)-hyoscyamine has anticholinergic activity. The European Union have recommended the development of methods that allow for the enantioseparation of atropine. This feature is also very important for quality control in the pharmaceutical industry in order to achieve a better drug which should be enantiomerically pure, improving its efficiency.

Although information related to the racemization process is scarce, it is well-known that the extraction procedure can affect the ratio between both isomers, and therefore, the extraction conditions should be deeply studied in order to minimize this transformation process. Therefore, to achieve reliable results a chiral separation of these compounds must be created.

In this study a new chiral method for the determination of (+,-) hyoscyamine has been developed, applying a rapid QuEChERS method that prevent the racemization process in the extraction step and a Chiralpak AY3 column for the chromatographic resolution. The mobile phase used was ethanol containing 0.1% of diethanolamine (DEA) at flow rate 0.4 mL/min. The analysis was carried out by a mass spectrometry triple quadrupole analyser that provides low limits. Once the method has been developed, it was applied to the analysis of real samples of Solanaceaes seeds: Datura Stramonium and Brugmansia Arborea seeds, as well as contaminated buckwheat samples (mixing 1 g of Solanaceae seed with 1 kg of buckwheat), obtaining only the active enantiomer (Tab. 1).

Tab. 1. Concentrations (mg/kg) of (+,-) hyoscyamine in the tested samples
a Relative standard deviation is given in brackets (n = 4)

Then the racemization process was studied under pH and temperature conditions, noting that the combination of high temperatures and basic pHs provides the conversion to the inactive enantiomer, whereas when the only contribution of pH or temperature was studied, the interconversion rate reached until 80% (Fig. 1).

Fig. 1. pH and time influence in the enantiomerization of (-)-hyoscyamine in Stramonium seeds at 80 ºC.

In conclusion, due to the shortage of enantiomeric method for the determination of atropine, a new chiral method has been developed. This method has been applied to real Solanaceaes seeds and contaminated samples obtaining only the active enantiomers. Then, different pH and temperature conditions were probed only obtaining a complete racemization with basic pH and high temperatures, concluding that in a real contamination with Solanaceaes seeds, the enantiomer with pharmacology activity prevail against the inactive enantiomer.

Marín-Sáez Jesús
Department of Chemistry and Physics, Analytical Chemistry Area, University of Almería,
Research Centre for Agricultural and Food Biotechnology (BITAL),
Agrifood Campus of International Excellence ceiA3, Almería, Spain

 

Publication

Enantiomeric determination and evaluation of the racemization process of atropine in Solanaceae seeds and contaminated samples by high performance liquid chromatography-tandem mass spectrometry.
Marín-Sáez J, Romero-González R, Garrido Frenich A
J Chromatogr A. 2016 Nov 25

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