Anisodamine

TAs were extracted from approximately 200 mg of dried plant material, according to a previously reported method [34 (link)]. The mobile phase was composed of acetonitrile and ammonium acetate (acetonitrile:50 mM ammonium acetate = 89:11). The flow speed was 1 mL/min. The standard samples of hyoscyamine, anisodamine, and scopolamine were purchased from Sigma-Aldrich (Sigma, LA, USA). The standard samples were dissolved in methanol at a final concentration of 2000 mg/mL and were gradually serially diluted to 1000, 750, 500, 250, 100, 50, 25, 10, and 5 mg/mL. The HPLC system was an LC-20A from Shimadzu; the detector was a photo-diode array. The detecting wavelength was 226 nm. The temperature of the CTP-ODS column (150 × 4.6 mm) was 40 °C. There were at least three independent biological replicates. The sample solution was injected at 20 μL each time. The method for the analysis of the TAs content was the same as that described in our recent work [26 (link),28 (link),29 (link),35 (link),36 (link)].

Atropine ((±)-hyoscyamine), (±)-scopolamine hydrochloride, anisodamine, and homatropine hydrobromide were bought from Sigma-Aldrich as standards for tropane alkaloids (Madrid, Spain). For internal standards (IS), atropine-d5 and scopolamine-13C, d3 Hydrobromide were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Before use, all standard solutions were maintained at room temperature for 30 min before being stored in amber vials in the dark at −20 °C.
Methanol (MeOH), acetonitrile (ACN), both HPLC-gradient grade, and formic acid were purchased from Merck (Darmstadt, Germany). Milli-Q plus system from Millipore (Molsheim, France) was used to purify the water, and it had a resistivity of 18.2 M cm.
For QuEChERS, magnesium sulfate and trisodium citrate dihydrate were purchased from PanReac (Barcelona, Spain), sodium chloride from Fluka (Seelze, Germany), and sodium citrate dibasic sesquihydrate from Sigma-Aldrich (Madrid, Spain). Agilent Technologies (Santa Clara, CA, USA) supplied the primary secondary amine-bonded silica (PSA) that was used for clean-up, in combination with anhydrous magnesium sulfate acquired from PanReac (Barcelona, Spain).