Method Development and Validation for the Determination of Pyrrolizidine Alkaloids in a Range of Plant-Based Foods and Honey Using LC-MS/MS

Importance of the topic

Pyrrolizidine alkaloids (PAs) are plant-derived toxins with genotoxic and carcinogenic effects. Approximately 6000 flowering plants can biosynthesize PAs, and cross-contamination during harvesting can lead to their occurrence in teas, herbs, spices, and honey. Stringent EU maximum levels and health concerns require sensitive, reliable methods for routine monitoring.

Objectives and study overview

This work aimed to develop and validate a single UPLC-MS/MS method for the quantitative determination of 35 EU-regulated PAs in various plant-based food matrices and honey. Key goals included resolving critical isomeric pairs, complying with CEN single-laboratory validation criteria, and ensuring low-level quantification suitable for foods intended for infants and young children.

Methodology

Samples (1 g plant material or 2 g honey) were extracted with 50 mM sulfuric acid in water:methanol (1:1), followed by cleanup on Oasis MCX cation-exchange SPE cartridges. Eluates were evaporated and reconstituted in water:methanol (95:5). UPLC separation used a BEH C8 column (2.1 × 100 mm, 1.7 µm) with a gradient of ammonium formate/formic acid in water and formic acid-acidified acetonitrile. Detection was carried out on a Xevo TQ-S micro triple quadrupole in positive ESI mode using MRM; RADAR mode combined MRM and full-scan acquisition to monitor matrix interferences.

Instrumentation

• UPLC: ACQUITY UPLC I-Class PLUS with Flow-Through Needle injector
• Column: BEH C8, 2.1 × 100 mm, 1.7 µm
• Mass spectrometer: Xevo TQ-S micro, ESI+, MRM and RADAR modes
• Sample cleanup: Oasis MCX SPE cartridges

Main results and discussion

Baseline separation was achieved for 27 of 35 PAs; four isomeric pairs were quantified as combined sums. Method limits of quantification were 0.6 µg/kg for individual PAs and 1.2 µg/kg for coeluting sums, well below regulatory limits. Recoveries ranged from 77% to 103% with RSDr ≤ 10%, and matrix effects varied from –76% to +282%, highlighting the need for matrix-matched calibration.

Benefits and practical applications

• Validated quantitation of 35 PAs in compliance with EU Regulation 2020/2040
• Effective removal of co-extractives via MCX SPE reduces isobaric interference
• Rapid method optimization using RADAR enhances robustness
• Sensitivity suitable for infant and young children food testing

Future trends and potential applications

Future developments may include integration of high-resolution MS for broader screening, automation of sample preparation, expansion to additional food commodities, and creation of multi-class toxin panels to address emerging contaminants.

Conclusion

A robust UPLC-MS/MS workflow has been established for monitoring 35 PAs across diverse matrices, achieving high sensitivity, reproducibility, and regulatory compliance. The approach simplifies isomer separation and supports routine food safety analysis.

References

• The Plant List Version 1.1 (2013)
• Schrenk et al. Food Chem. Toxicol. 136 (2020) 111107
• EFSA Journal 9(11):2406 (2011)
• EFSA Journal 15(7):4908 (2017)
• Commission Regulation (EU) 2020/2040 (2020)
• BfR-PAs-Tea-2.0/2014; BfR-PAs-Honey-1.0/2013; EURL-MP-method_002 (2019)