A reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the simultaneous detection of zearalenone-14-glucoside (ZEN-14G) and its metabolite, zearalenone (ZEN), in the plasma, urine, and various tissues of rats. The performance of the developed method was validated by determining the selectivity, linearity (R2 > 0.99), sensitivity (lower limit of quantification, 0.1–1 μg/L), recovery (80.7 ± 3.0–112.3 ± 3.1%), precision (0.6–16.5%), and stability (81.7 ± 1.7–104.1 ± 3.9%). Through use of the methodological advances, the subsequent kinetics and distribution after administration of ZEN-14G by gavage were thoroughly investigated. ZEN-14G and ZEN exhibited similar trends in the plasma, and reached their peak concentrations at 10 min and then rapidly decreased. ZEN-14G could be quantified in the stomach, small intestine, and large intestine 24 h after administration, while ZEN was detectable in all tested tissues. Interestingly, ZEN-14G (7.6 ± 3.0 μg/L) and ZEN (977.5 ± 98.0 μg/L) were also detected in the urine 24 h after administration, indicating that ZEN-14G was prone to be slowly and continuously hydrolyzed into ZEN to be absorbed into the plasma and distributed to various tissues, thus leading to a cumulative exposure. Continuous attention should be paid to the co-exposure of ZEN and ZEN-14G, which might pose additional health risks to humans and animals.

A reliable ultra-high-performance liquid chromatography–tandem mass spectrometry method (UHPLC-MS/MS) was developed for the simultaneous determination of two mycotoxins, that is, zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) in formula feed, concentrated feed, and premixed feed products. An improved sample pretreatment was achieved with the hydrophilic–lipophilic balance (HLB) cartridges efficiently removing the impurities and enriching the target analytes in different feeds. The critical parameters affecting the performance of the solid-phase extraction (SPE) procedure were carefully optimized, and 20% acetonitrile in water as the loading solution, 50% methanol in water as the washing solvent, and 5 ml of methanol as the elution solvent yielded the optimal purification efficiencies. The established method was thoroughly validated in terms of linearity (R2 ≥ 0.999), sensitivity (limit of quantification in the range of 0.50–5.00 μg kg−1), recovery (89.35 ± 2.67% to 110.93 ± 1.56%), and precision (RSD, 3.00–14.20%), and it was then successfully applied to investigate a total of 60 feed samples. Among them, 50 samples were found to be contaminated with ZEN (an incidence of 83.3%) at levels ranging from 0.63 to 615.24 μg kg−1, whereas 22 samples were contaminated with ZEN-14G (an incidence of 36.7%) in the range of 0.89–15.31 μg kg−1. The developed method proved to be a specific and reliable tool for intensive monitoring of ZEN and ZEN-14G in complex feed matrices.