Simultaneous and Quantitative Determination of Pyrrolizidine and Tropane Alkaloids in Food by LC-MS/MS, First Action 2025.02.

Background: Pyrrolizidine alkaloids (PAs) are natural toxins produced by > 6,000 flowering plant species. Some PAs are hepatotoxic and genotoxic carcinogens. Reported cases of direct poisoning in humans have already been documented, but the highest risk of human exposure occurs through the consumption of crops contaminated with PA-producing weeds. The European Commission Regulation EU 2023/915 set maximum levels (MLs) for the sum of 35 PAs in tea, herbal teas, herbs, and spice seeds at levels ranging from 75 to 1000 µg/kg (dry matter basis).

Objective: The study describes the performance characteristics of a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the 35 PAs mentioned in AOAC INTERNATIONAL Standard Method Performance Requirements (SMPRs®) 2023.002 in tea, herbal infusions, herbs, seed spices, cereals, and honey. The analyte scope also comprised of nine additional PAs along with two tropane alkaloids (TAs, namely atropine and scopolamine) for which MLs are also set in plant-based products in European Union (EU 2023/915).

Method: The protocol involves an extraction of toxins from food using aqueous sulfuric acid followed by solid-phase extraction purification. After elution and evaporation to dryness, the residue is reconstituted in water-methanol solution prior to LC-MS/MS analysis. Alkaline mobile phases are used to favor the separation of isomers. Identification of the analytes is conducted according to the confirmation criteria defined in EU 2023/2783 and SANTE 11312/2021v2 documents. Quantification is performed by isotopic dilution using four isotopically labelled PAs and two isotopically labelled TAs as internal standards.

Results: Method validation workload was shared by three different laboratories on representative matrices from the following commodities: dried herbs, dried tea, dried herbal infusions, seed spices, cereals, and honey. The 35 target analytes specifically listed in the AOAC SMPR 2023.002 have been successfully validated (recovery 70-120%, repeatability < 20%, intermediate reproducibility < 20%), allowing LOQs down to 1 µg/kg.

Conclusions: The performances of the method are in agreement with criteria defined in AOAC SMPR 2023.002.

Highlights: The Expert Review Panel for Pyrrolizidine Alkaloids approved the present method as AOAC Official First Action 2025.02.