Flavonol aglycones as chemical markers for pollen quality assessment: a validated HS-US/UHPLC-PDA study with chemometric differentiation of bee-collected and Pinus pollens.

BACKGROUND: Pollen represents a pivotal resource at the agriculture-food nexus, valued for its dual functionality as a nutraceutical and food ingredient. However, the lack of standardized quality markers impedes its reliable characterization and broader industrial application. This study aimed to establish a validated analytical framework for the comparative profiling of flavonol aglycones in pollen, addressing the critical need for objective indicators to ensure standardization and traceability within agricultural supply chains. RESULTS: Thirty-one pollen samples, comprising 19 bee-collected and 12 Pinus varieties from 15 Chinese provinces, were analyzed via high-speed shear homogenization coupled with ultrasonication (HS-US) extraction, acid hydrolysis, and ultrahigh-performance liquid chromatography-photodiode array (UHPLC-PDA) quantification. The HS-US method yielded a 2.9-fold increase in flavonol aglycone recovery compared to conventional protocols, satisfying rigorous validation criteria (linearity R ≥ 0.999; accuracy 90-106%; precision relative standard deviation < 5%). Quercetin and kaempferol were identified as robust quality indices. Bee-collected pollen exhibited significantly higher total flavonol aglycone content (mean: 0.825 g kg-1) compared to Pinus pollen (mean: 0.156 g kg-1; P < 0.001). Furthermore, chemometric analysis revealed biogeographic clustering, supporting the utility of these markers for quality assessment. CONCLUSION: This study presents a robust analytical approach that effectively surmounts the sporopollenin extraction barrier, enabling reliable quantification of flavonol aglycones. The validated markers provide objective tools for quality control, establishing a chemical foundation for linking standardized pollen products to their functional applications. Consequently, the developed HS-US/UHPLC-PDA platform is readily adaptable for routine quality assessment in food and agricultural settings. © 2026 Society of Chemical Industry.