Determination of pyrrolizidine alkaloids in plant material using on-line SPE coupled to UHPLC-MS/MS

Significance of the Topic

Pyrrolizidine alkaloids (PAs) are plant-derived toxins with carcinogenic and genotoxic potential.
They occur in common dietary and herbal products such as tea, honey and botanical supplements.
Contamination of animal feed and plant‐based foods with PAs poses a public health concern and drives regulatory evaluation worldwide.

Study Objectives and Overview

This work aimed to develop a streamlined on-line SPE coupled to UHPLC-MS/MS method for sensitive quantification of PAs and their N-oxides in tea samples.
The approach sought to minimize manual cleanup, maintain high chromatographic resolution and achieve low µg/kg limits of quantification across multiple tea matrices.

Methodology and Instrumentation

Sample Preparation:

• Green, black and herbal teas sonicated twice in 0.05 M H2SO4.
• Combined extracts neutralized with NH4OH and centrifuged.

On-line SPE–UHPLC Workflow:

• Automated loading of aqueous tea extract onto SPE cartridge.
• Washing of matrix interferences with polar solvents.
• Elution of PAs into a loop (10 µL) and transfer to UHPLC column.
• Binary UHPLC gradient delivering high backpressure separation.

Used Instrumentation

• UHPLC System: Shimadzu Nexera
• Analytical Column: Shim-pack XR-ODS III (150 × 2.0 mm, 2.2 µm)
• SPE Cartridge: EVOLUTE® EXPRESS ABN (30 × 2.1 mm)
• Mass Spectrometer: Shimadzu LCMS-8060 with positive ESI

Main Results and Discussion

The method achieved simultaneous analysis of 16 PAs and 14 N-oxides with linear calibration (r² > 0.99, 1/C weighting).
Limits of quantification ranged from 1 to 10 µg/kg in all tea types.
Representative LLOQs of 0.05–0.5 ng/mL correspond to 1–10 µg/kg in solid tea.
Analysis of 29 commercial tea samples revealed PA contamination in 59% of cases, underscoring widespread occurrence.

Benefits and Practical Applications

The integrated on-line SPE–UHPLC-MS/MS platform offers:

• Reduced manual handling and solvent use.
• Enhanced throughput and reproducibility.
• Low matrix interference and high sensitivity suitable for QA/QC.

Future Trends and Potential Applications

Further developments may include ruggedization for routine regulatory monitoring and expansion to other plant-derived matrices.
Advances in column technology and MS data processing will support wider screening of emerging plant toxins.

Conclusion

The developed on-line SPE–UHPLC-MS/MS method delivers high-sensitivity, high-throughput analysis of pyrrolizidine alkaloids in tea.
It significantly reduces sample preparation while maintaining excellent quantitative performance.

Reference

S. Vedder, A. Grüning, J. Sander, "Determination of pyrrolizidine alkaloids in plant material using on-line SPE coupled to UHPLC-MS/MS," ASMS 2018, WP-249.