This doctoral thesis focuses on the development, optimization, and validation of an analytical method for the determination of pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) in plant-based matrices. Pyrrolizidine alkaloids represent an important group of naturally occurring toxins with well-documented hepatotoxic and carcinogenic effects. Their occurrence in food and feed has led to the establishment of maximum regulatory limits within the European Union. Reliable determination of these compounds remains analytically challenging, primarily due to the high number of isomeric forms that cannot be distinguished solely by mass spectrometric detection and therefore require efficient chromatographic separation.
Within this work, an LC-MS/MS method enabling the simultaneous determination of 36 legislatively regulated pyrrolizidine alkaloids and their N-oxides was developed. Through systematic optimization of chromatographic conditions, complete separation of all target analytes within a single analytical run was achieved. Full separation of the most challenging isomeric groups was accomplished using an Acquity UPLC CSH C18 column under alkaline mobile phase conditions containing 10 mM ammonium formate, in combination with an optimized gradient profile.
The thesis also included optimization of sample preparation. The highest recoveries were achieved using a QuPPe-based extraction method employing acidified methanol, without the need for an additional dispersive SPE clean-up step. The method was validated for cumin, chamomile, marjoram, and hay matrices. Recoveries ranged from 43–108 %, while method repeatability did not exceed 16 %. Limits of quantification were established between 2–10 µg/kg depending on the matrix and analyte.
The developed method was applied to the analysis of 50 real plant-based samples, including cumin, chamomile, caraway, and hay. Pyrrolizidine alkaloids were detected in 17 samples, with the highest total PA/PANO concentration found in chamomile (697.2 µg/kg), exceeding the current legislative limit. The obtained results confirm the occurrence of pyrrolizidine alkaloids in commonly consumed plant products and highlight the importance of reliable analytical methods for their systematic monitoring.