Quantitation of Pyrrolizidine Alkaloids in Honey and Herbal Teas by UHPLC/MS/MS

Significance of the topic

Quantitation of pyrrolizidine alkaloids (PAs) in food products such as honey and herbal teas is critical for consumer safety due to the hepatotoxic and genotoxic potential of these natural plant metabolites. PAs occur in over 6,000 plant species and may contaminate nectar, pollen, or herbal ingredients, leading to inadvertent dietary exposure. Regulatory bodies recommend limiting daily intake according to the ALARA principle (as low as reasonably achievable) to mitigate health risks. A robust analytical approach is therefore essential for reliable surveillance and risk assessment.

Objectives and overview

This study aimed to develop a fast, sensitive, and user-friendly method for simultaneous quantitation of 28 PAs and their N-oxides in honey and herbal tea matrices. The scope included method optimization, validation, and real-world application to rooibos tea samples purchased on the market.

Methodology and instrumentation

• Sample preparation: Honey (5 g) and ground tea (2 g) were extracted with 0.05 M sulfuric acid using heating or ultrasonication, followed by centrifugation.
• SPE cleanup: Agilent Bond Elut strong cation exchange cartridges were conditioned, loaded with sample extract, washed with water and methanol, and eluted with ammonia/methanol.
• Reconstitution: Dried eluates were reconstituted in methanol/water (5:95, v/v) and diluted prior to analysis.
• UHPLC separation: Agilent 1260 Infinity LC with InfinityLab Poroshell 120 EC-C18 column (2.1×100 mm, 2.7 µm) at 25 °C; mobile phases of 5 mM ammonium formate/0.025 % formic acid in water and methanol; 23.5 min gradient, 0.3 mL/min.
• MS detection: Agilent 6490 iFunnel triple quadrupole in positive ESI Jet Stream mode with dynamic MRM (three transitions per compound), unit resolution.

Main results and discussion

• Chromatography achieved baseline separation of key isomers (e.g., intermedine/lycopsamine and Senecionine family), reducing interference.
• Matrix effects ranged from moderate suppression (up to 60 % in honey, 90 % in tea); compensated by matrix-matched calibration.
• Linear ranges: 1–100 µg/kg in honey, 5–500 µg/kg in tea; correlation coefficients R2>0.996.
• LOQs: 1 µg/kg (honey) and 5–10 µg/kg (tea) for most analytes; LODs set at 50 % of LOQs.
• Recovery at 20 µg/kg: 81–135 % in honey; at 100 µg/kg: 59–85 % in tea, reflecting more complex tea matrix.
• Application: Analysis of 24 rooibos teas revealed total PA levels from 143 to 2,300 µg/kg, dominated by Retrorsine, Senecionine, Seneciphylline and their N-oxides.

Benefits and practical applications

• The method offers a balance of sensitivity, selectivity, and throughput suitable for routine monitoring of regulatory compliance.
• Small sample amounts and a simple SPE workflow reduce solvent consumption and processing time.
• Use of dynamic MRM and three transitions per analyte improves confidence in identification in complex matrices.

Future trends and opportunities

Further developments may include incorporation of isotopically labeled internal standards to enhance quantitation accuracy, extension to additional food and feed matrices, automation of SPE processes, and high-resolution MS workflows to detect emerging PA analogs. Integration with risk assessment models and open-access databases could streamline monitoring across supply chains.

Conclusion

A validated UHPLC-MS/MS method for 28 PAs and N-oxides in honey and herbal teas was established, exhibiting low LOQs, satisfactory recoveries, and reliable isomer separation. The approach supports regulatory and quality control laboratories in safeguarding against PA exposure.

Reference

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8. BfR Method protocol BFR-PA-Tea-2.0/2014 for determination of PAs in plant material by SPE-LC-MS/MS.