Bee pollen and Pyrrolizidine Alkaloids: A contamination profile Analysis using LC- MS/MS

Introduction Pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) contamination in foodstuffs and dietary supplements, such as honey and bee pollen, represents a potential health risk for consumers. This study aimed the development and validation of an LC-MS/MS method to quantify PAs/PANOs levels in bee pollen samples coming from local beekeepers (LB; Veneto/ Italy), great retail and web (GR). Methods The developed method was validated according to the Commission Decision 2002/657/EC. For this purpose, blank bee pollen samples were spiked with 17 PAs/PANOs standard solutions at three levels (2, 10 40 μg/Kg). PAs and PANOs were extracted from pollen using PLEXA cartridges (200 mg/6 mL, Agilent, USA) and the LC-MS/MS analysis was conducted on an LTQ-XL Ion trap operating in positive ion mode. Results All results obtained for recovery, linearity, repeatability and reproducibility in the validation study were in agreement with the limits reported by Decision 2002/657/EC. The limit of quantification was 0.4 μg/kg for all analytes. The validated method was applied to 81 bee pollen samples, of which 50 were positive: 20 were from LB and 30 from GR. The average total concentration of PAs/PANOs in samples from LB and GR were 27.8 μg/kg and 470.9 μg/kg, respectively. The identified PAs/PANOs could be categorized into three classes and their average concentrations were as follows: Eupatorium-Type 42.90 μg/kg (LB) vs. 162.76 μg/kg (GR); Echium-Type 7.46 μg/kg (LB) vs. 465.54 μg/kg (GR); Senecio-Type 16.24 μg/kg (LB) vs. 176.62 μg/kg (GR). Most samples (17) from LB presented Senecio-Type PAs/PANOs, whereas most samples (24) from GR presented Eupatorium-Type PAs/PANOs. Conclusions The validated LC-MS/MS method showed to be sensitive and suitable for the quantification of the 17 selected PAs/PANOs markers in bee pollen. Samples from LB and GR presented a very different PAs/PANOs contamination profile reflecting their different botanical and geographical origin. The average total concentration of PAs/PANOs was 17 times lower in samples from LB than in those from GR. Novel Aspect This study should highlight any hazard due to the import/export of pollen and suggest management strategies to beekeepers to ensure a healthy product. Acknowledgements: University of Padua for funding the Project CPTA 158894