Determination of Pyrrolizidine Alkaloids and Their N‑Oxides in Bee Pollen by Accelerated Solvent Extraction Followed by Magnetic Covalent Organic Framework-Based Offline Purification and Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) are naturally occurring toxic plant metabolites that have attracted widespread attention due to their potential food safety hazards. In this study, an efficient analytical method for determining PAs/PANOs in bee pollen was established by combining accelerated solvent extraction (ASE) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A magnetic covalent organic framework material (Fe₃O₄@COF-TAPB-TFP) was synthesized and used as an adsorbent for offline purification to reduce matrix interference and improve detection sensitivity. Under optimal conditions, the method showed excellent linearity (R² ≥ 0.99) in the range of 1–200 µg·L⁻¹, with recoveries of 70.1%–95.6% for spiked blank samples at three levels (10, 50, and 100 µg·kg⁻¹), relative standard deviations below 15%, and limits of quantification of 1.2–9.9 µg·kg⁻¹. This method was successfully applied to the analysis of local market bee pollen samples, with detected PAs/PANOs levels ranging from 25.6 to 178.6 µg·kg⁻¹. Overall, the Fe₃O₄@COF-TAPB-TP-assisted ASE-UHPLC-MS/MS method provides an accurate, rapid, highly automated, and high-throughput platform for monitoring PAs/PANOs contamination in food products.