Determination of dipicolinic acid, a bacterial spore biomarker, in food matrices using mixed-mode solid-phase extraction coupled with fluorescence spectroscopy

Dipicolinic acid (DPA) is a unique molecular biomarker of bacterial spores, playing a pivotal role in spore resistance to processing. DPA determination can guide food safety risk assessment but remains a challenge due to food matrix interference. In this study, a mixed-mode (ion-exchange/reversed-phase) solid-phase extraction (SPE) method, coupled with terbium-DPA fluorescence spectroscopy, was developed and evaluated for DPA determination in six liquid food matrices. Reversed-phase (PRiME HLB, HLB, and C18) and mixed-mode (WAX and MAX) SPE sorbents were screened for suitability to recover DPA from foods. Among them, a mixed-mode polymeric sorbent combining reversed-phase and strong anion-exchange retention (MAX) exhibited the most favorable balance between high DPA recovery and minimal matrix effects. After spiking foods (5 μg/mL), DPA recovery was evaluated under controlled sample loading pH (1.0, 3.0, or 7.0). The developed SPE method incorporated sample loading pH 1.0, loading flow rate of 1.0 mL/min, vacuum-drying of the sorbent for 15 min, and elution of DPA with acidified ethyl acetate (20 mL); these were key determinants of method performance. The developed method achieved the following recoveries: infant formula (105.2 ± 3.2%), carrot juice (87.2 ± 6.0%), vegetable broth (99.3 ± 4.8%), mushroom broth (92.1 ± 1.6%), chicken broth (92.0 ± 2.5%), and beef broth (95.5 ± 3.3%). Additionally, the method resulted in low matrix effects (−26% to +23%). Overall, the developed method has the potential to enable same-day determination of DPA in liquid food matrices and provides a foundation for future applications involving bacterial spores.