Škola MS: Challenges in Implementation of an LC/MS/MS Method for the Analysis of Pyrrolizidine Alkaloids in Food Matrices

Significance of the Topic

Analyzing pyrrolizidine alkaloids (PAs) in food matrices is critical due to their widespread occurrence in over 6,000 plant species and proven genotoxic and carcinogenic properties. Regulatory bodies, including the European Commission, have set maximum levels for PAs in foodstuffs under Regulation (EU) 2020/2040 to protect consumer health and ensure food safety.

Objectives and Study Overview

This study aims to develop and validate a robust LC–MS/MS method capable of separating and quantifying 28 relevant PA compounds, including their isomeric forms, in diverse food matrices (e.g., cumin seed, chamomile, marjoram, and hay). The method must achieve full chromatographic resolution, meet regulatory requirements, and offer low limits of quantification (LOQs) within a single analytical run.

Methodology and Instrumentation

Sample Preparation:

• QuPPe extraction: homogenized sample mixed with water and methanol containing 1% formic acid, shaken, centrifuged, and filtered.

Chromatographic Conditions:

• Column: High-performance C18 column with small particle size and extended length to enhance separation of PA isomers and matrix interferences.
• Mobile phase: Aqueous and organic solvents acidified with formic acid; pH carefully adjusted to optimize retention and resolution of stereo- and structural isomers.
• Column temperature: Elevated temperatures tested to improve peak shape and reduce coelution.
• Run time: Total chromatographic cycle of 23 minutes for simultaneous analysis of all regulated PAs.

Mass Spectrometry:

• Triple quadrupole LC–MS/MS operated in multiple reaction monitoring (MRM) mode to detect characteristic transitions of each PA.

Instrumentation:

• LC system: Ultra-high-performance liquid chromatography coupled to MS/MS.
• MS/MS detector: Triple quadrupole mass spectrometer optimized for sensitivity and selectivity of 1,2-unsaturated PAs.

Main Results and Discussion

Chromatographic Separation:

• All 28 targeted compounds, including pairs of stereoisomers and structural isomers, were baseline resolved under the optimized conditions.
• pH adjustments and temperature control were critical for separating groups such as intermedine/echinatine and their N-oxide forms, as well as senecionine derivatives.

Method Validation:

• Recovery rates across four matrices ranged from 74% (marjoram) to 99% (cumin seed), demonstrating robust extraction and quantification performance.
• Matrix effects were minimal, with signal suppression or enhancement within acceptable limits for all matrices.
• LOQs achieved were sufficiently low to comply with EU regulatory thresholds.

Benefits and Practical Applications

The developed LC–MS/MS method enables:

• Comprehensive monitoring of PAs in various food products to ensure consumer safety.
• Regulatory compliance with EU maximum levels for sum of PAs.
• High-throughput analysis due to single-run quantification of multiple analytes.
• Adaptation to routine QA/QC workflows in food testing laboratories.

Future Trends and Applications

Advancements may include:

• Integration of high-resolution mass spectrometry to further distinguish emerging PA analogues.
• Automation of sample preparation (e.g., online extraction) to increase throughput.
• Extension of the method to other complex matrices such as herbal supplements and honey.
• Development of rapid screening assays using ambient ionization techniques for on-site PA detection.

Conclusion

This work presents a validated LC–MS/MS protocol that fully resolves and quantifies all PAs regulated under EU legislation in a single analytical run. The method demonstrates robust performance across different food matrices, offering reliable recovery, minimal matrix effects, and low LOQs. Its adoption supports effective monitoring of food safety and regulatory compliance.

References

1. Commission Regulation (EU) 2020/2040 of 17 December 2020 setting maximum levels for pyrrolizidine alkaloids in foodstuffs.