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

Importance of the Topic

Pyrrolizidine alkaloids (PAs) are natural plant toxins with genotoxic and carcinogenic properties. Their presence in teas, herbs, spices, and honey poses health risks for consumers. Regulatory agencies in the EU have established maximum levels for 35 PAs to protect public health. A robust analytical approach is essential to ensure food safety and regulatory compliance.

Objectives and Study Overview

This study aims to develop and validate a simplified UHPLC-MS/MS protocol for simultaneous quantification of 35 EU-regulated PAs in various plant-based foods and honey. The method addresses chromatographic separation challenges of multiple isomeric PAs and meets stringent sensitivity requirements set by EU Regulation 2020/2040.

Methodology and Instrumentation

A streamlined sample preparation employs Oasis MCX strong cation-exchange SPE cartridges under acidic conditions to remove matrix co-extractives. Chromatographic separation was optimized on a UHPLC system with MS/MS detection, using RADAR acquisition to collect both multiple reaction monitoring and full-scan data concurrently. Baseline separation was achieved for 27 analytes, while four pairs of isomeric compounds are reported as summed values.

• Instrumental techniques
  • UHPLC-MS/MS with tandem quadrupole detection
  • RADAR acquisition mode
  • Oasis MCX SPE clean-up cartridges

Main Results and Discussion

Recovery experiments across six matrices showed mean recoveries between 77 % and 103 %, with most PAs exceeding 62 % recovery. Four N-oxide alkaloids in honey yielded recoveries of 50–60 %, but repeatability (RSD<10 %) allows for recovery correction or procedural calibration. LOD and LOQ values are well below EU maximum limits (e.g., 150 µg/kg for tea), enabling sensitive detection. The SPE clean-up significantly reduced total ion current from co-extractives, minimizing matrix interferences and system contamination.

Benefits and Practical Applications

• High sensitivity and selectivity for regulatory compliance
• Efficient sample clean-up reduces maintenance and extends instrument uptime
• Adaptable protocol for diverse food matrices including teas, spices, and honey
• Capability for monitoring food safety for vulnerable populations such as infants

Future Trends and Opportunities

The method can be expanded to additional PAs and food matrices, integrated into high-throughput screening workflows, and combined with emerging HRMS techniques for non-targeted analysis. Automation of SPE and sample handling will enhance laboratory efficiency and reproducibility. Ongoing regulatory updates may necessitate method adaptation for new analyte lists.

Conclusion

A robust UHPLC-MS/MS method with targeted SPE clean-up was developed and validated for 35 pyrrolizidine alkaloids in plant-based foods and honey. The protocol meets EU regulatory requirements, demonstrating excellent trueness, precision, and sensitivity. Baseline separation for most isomers and effective matrix removal support routine application in food safety laboratories.

References

• Dreolin N, Foddy H, Adams S, Hird S, Hancock P. Method Development and Validation for the Determination of Pyrrolizidine Alkaloids in a Range of Plant-Based Foods and Honey Using LC-MS/MS. Waters Corporation; 2022.
• European Commission Regulation (EU) 2020/2040 of 1 July 2022 amending Regulation (EC) No 1881/2006.