Mass Spectrometry & Spectroscopy
Are Your Herbal Teas Safe? Tropane Alkaloid Analysis Using SPE Combined With LC-MS/MS
SPE Method: James Edwards, Porvair Sciences Ltd
A 10 mg 96 well plate (Microlute CP 10 mg SCX) with a positive pressure manifold (UltraPPM Lite)was used for processing samples. Tea infusion samples were pH adjusted with formic acid to a concentration of 0.1% (v/v) to prepare them for loading onto the SPE plate.
Tropane alkaloids are toxic compounds and are most abundant in the Solanaceae family of plants (deadly nightshade, henbane, mandrake and Jimson weed) [1]. Atropine and scopolamine are the most common tropane alkaloids found in food samples [2]. These alkaloids can contaminate herbal teas through plant debris [3] or soil migration [4]. They cause symptoms of reduced salivation, skin dryness, pupil dilation and with higher doses, drowsiness, visual disturbances, palpitations, disorientation and hallucinations [5]. The European Union (EU) has set a regulatory limit of 0.2 ng/mL [6] for the sum of atropine and scopolamine in herbal infusions.
HPLC-MS/MS Method:
• Reconstitution: Eluate evaporated using a nitrogen blowdown evaporator (Ultravap Mistral) at 30°C and reconstituted with 0.2 mL of 0.1% formic acid in ultrapure water
HPLC-MS/MS Method: HPLC: ACQUITY Premier UPLC
Solvent AH2
Table 1: The HPLC and Mass Spectrometer conditions used for analysis and the atropine and scopolamine MRM transitions.
Mass Spectrometer: Xevo TQ-S Micro O + 0.1% formic acid
Solvent BMeOH + 0.1% formic acid Gradient
Figure 1: Chemical structures of tropane alkaloids, including the tropane bicyclic ring structure, atropine and scopolamine.
Current methods for analysis of tropane alkaloids in food use large bed weight solid phase extraction (SPE) products (>150 mg) [1], which are time consuming and less sustainable due to large solvent usage. This study validates the extraction of atropine and scopolamine with a 10 mg polymeric SCX 96 well SPE plate followed by HPLC-MS/ MS analysis.
Following validation of the method, a range of herbal teas (chamomile, peppermint and green tea) sold on the UK market were analysed to assess the presence of atropine and scopolamine within the fi nal herbal tea infusions.
Materials and Methods
Tea infusion, according to ISO 3103:1980 [7]: 2.00 g of tea was added to a stainless-steel tea strainer and placed in a beaker. 150 mL of boiling ultrapure water was poured through the tea strainer into the beaker and steeped for fi ve minutes. The fi nal tea solution was allowed to cool to room temperature and then fi ltered through a 0.22 µm PES fi lter.
SPE Method:
A 10 mg 96 well plate (Microlute CP 10 mg SCX) with a positive pressure manifold (UltraPPM Lite) was used for processing samples. Tea infusion samples were pH adjusted with formic acid to a concentration of 0.1% (v/v) to prepare them for loading onto the SPE plate.
• Conditioning: 1 mL of methanol • Equilibration: 1 mL of 0.1% formic acid in ultrapure water • Loading: 1 mL of acidifi ed tea infusion sample (0.1% formic acid) • Wash 1: 1 mL of 0.1% formic acid in ultrapure water • Wash 2: 1 mL of 0.1% formic acid in methanol • Drying step: Dried at 20 PSI for 2 minutes with the positive pressure manifold
• Elution: 2 x 500 µL of 0.5% ammonia in methanol (dried at 20 PSI for 2 minutes after each elution with the positive pressure manifold)
• Reconstitution: Eluate evaporated using a nitrogen blowdown evaporator (Ultravap Mistral) at 30°C and reconstituted with 200 µL of 0.1% formic acid in ultrapure water
Time(min) 0.00 0.65 4.15 4.17 5.15 5.17 6.75
Flow Rate 400 µL/min Seal Wash On (5 min)
Sample Temperature 13°C
MRM Transitions: Analyte
Atropine Scopolamine
Precursor ion (m/z)
289.83 302.82
Product ion (m/z) Cone voltage (V) Collision energy (V)
124.06 (Quan ion) 92.95 90.99
138.05 (Quan ion) 156.03 102.94
Validation guidelines:
Testing followed pesticide validation guidelines for food - SANTE/11312/2021 [8] and the Limit of Quantifi cation (LOQ) was set by Regulation 2023/2783 [9]: • Recovery: 70-120% at three concentrations (0.2, 1.0 and 5.0 ng/mL) • Repeatability: ≤20% at each level • Linearity: R2
≥0.99 with residuals ≤±20% • LOQ: <0.05 ng/mL
Results and Discussion Method validation for SPE followed by HPLC-MS/MS
Table 2 shows the linearity, LOQ and matrix effect (ME) for atropine and scopolamine for each of the herbal infusions validated.
30 28
22 30 42 18 15 40
A (%) 90.0 90.0 78.0 0.0 0.0
90.0 90.0
B (%) 10.0 10.0 22.0
100.0 100.0 10.0 10.0
Needle WashMeOH + 0.1% formic acid (3 sec) Syringe Draw Rate 30 µL/min
Needle Placement 4mm Injection Volume 5 µL
ColumnWaters ACQUITY UPLC-BEH C18 (2.1 x 50, 1.7 µm) Column Temperature 30°C Source Type ESI Capillary Voltage 1000 V Desolvation Temperature 600°C Source Temperature 150°C Desolvation Gas Flow1000 L/hr
Cone Gas Flow 0 L/hr Detector Gain 1.00
Minimum Points Per Peak 15
• Conditioning:1 mL of methanol • Equilibration:1 mL of 0.1% formic acid in ultrapure water • Loading:1 mL of acidified tea infusion sample (0.1% formic acid) • Wash 1:1 mL of 0.1% formic acid in ultrapure water • Wash 2:1 mL of 0.1% formic acid in methanol • Drying step:Dried at 20 PSI for 2 minutes with the positive pressure manifold • Elution: 2 x 500 µL of 0.5% ammonia in methanol (dried at 20 PSI for 2 minutes after each elution with the positive pressure manifold)
INTERNATIONAL LABMATE - JULY 2025
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