Exploring the limits of UHPLC-MS/MS with polarity switching towards the quantification of 931 mycotoxins and other secondary metabolites in cereal-based food

High-throughput LC–MS/MS methods are increasingly essential for regulatory screening of mycotoxins in food as the number of relevant analytes grows. We evaluated the limits of such quantitative methods covering 931 mycotoxins and other secondary metabolites in cereal-based food (muesli), by comparing ultra-high-performance liquid chromatography (UHPLC) with high-performance liquid chromatography (HPLC), polarity switching with separate injections, and short vs. long gradients to optimize speed without compromising performance. To manage validation workload, 52 representative analytes—including all regulated mycotoxins—distributed across the chromatogram were selected for assessment. The final multi-class LC–MS/MS quantitative method covers 931 mycotoxins and secondary metabolites in an 11-minute run time, providing a scalable solution for routine testing, regulatory and emergency response. Across methods, no significant differences were observed in matrix effects, repeatability, or within-laboratory reproducibility, except at the lowest spiking levels. Reduced repeatability increased limits of quantification (LOQs) by a factor of 3–4 compared to the original 2 × 20.5-minute method, highlighting the main trade-off when accelerating analysis, despite the nearly fourfold gain in throughput. This ultra-fast approach meets the required sensitivity and measurement uncertainty for all 14 regulated mycotoxins across five muesli varieties, supporting regulatory control. Replacing the conventional dual-injection workflow with this single-injection protocol could reduce laboratory CO₂ emissions by ~22 tons annually. • First 11-min UHPLC-MS/MS method covering 931 secondary metabolites • Fast polarity switching enabled single-run detection of diverse toxin classes • Regulatory-level sensitivity achieved for all 14 EU-regulated mycotoxins