## 4 Conclusions  
An effective HPLC-MS/MS method was established for the analysis of ZEN-14G and its metabolite, ZEN, in the plasma, various tissues, and urine of rats. After careful validation, the developed method was proved to be selective, accurate, sensitive, and stable, and was subsequently applied to explore the kinetics and tissue distribution after the administration of ZEN-14G by gavage to rats. ZEN-14G could be partially absorbed into the plasma and partially converted into ZEN, followed by sustained absorption and excretion. After 24 h, ZEN-14G could still be detected in the digestive tract and urine, while its metabolite, ZEN, could be found in all biosamples at high concentrations, verifying the cumulative health risks to humans and animals. Therefore, it is recommended to include masked mycotoxins in monitoring and control programs of agricultural practices and food safety authorities, with the established method as a powerful tool. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13084990/s1](https://www.mdpi.com/article/10.3390/app13084990/s1)]([https://www.mdpi.com/article/10.3390/app13084990/s1](https://www.mdpi.com/article/10.3390/app13084990/s1)). Figure S1. Comparison of the ionization efficiencies of zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) among four candidate mobile phases. (A) a methanol-water mixture, (B) a methanol-water mixture containing 0.1% formic acid, (C) a methanol-water mixture containing 5 mmol/L ammonium acetate, and (D) a methanol-water mixture containing 5 mmol/L ammonium acetate and 0.1% formic acid. The concentration of ZEN and ZEN-14G was 50 ug/L. Figure S2. Ionization efficiencies of ZEN and ZEN-14G compared among different mobile phases. (A) a methanol-water mixture containing 1 mmol/L ammonium acetate,  
Figure 5: The concentrations of zearalenone-14-glucose (ZEN-14G) and zearalenone (ZEN) in urine collected 6–24 h after oral administration to rats (dose of 4 mg/kg body weight, \(n=3\)). Figure 6: The supposed biotransformation characteristics in the plasma, tissue distribution, and urinary excretion after the administration of ZEN-14G by gavage to rats. (B) a methanol-water mixture containing 3 mmol/L ammonium acetate, (C) a methanol-water mixture containing 5 mmol/L ammonium acetate, and (D) a methanol-water mixture containing 7 mmol/L ammonium acetate. The concentration of ZEN and ZEN-14G was 50 ug/L. Figure S3. Detection and identification of ZEN-14G by the established UHPLC-Q-TOF/MS method in (A) the blank small intestine sample, (B) the neat solution (200 ug/L), (C) the spiked small intestine sample (200 ug/L), and (D) the small intestine samples gathered at 24 h after the administration of ZEN-14G by gavage (4 mg/kg body weight) to rats. Figure S4. Detection and identification of ZEN by the established UHPLC-Q-TOF/MS method in (A) the blank small intestine sample, (B) the neat solution (200 ug/L), (C) the spiked small intestine sample (200 ug/L), and (D) the small intestine samples gathered at 24 h after the administration of ZEN-14G by the established UHPLC-Q-TOF/MS method in (A) the blank large intestine sample, (B) the neat solution (200 ug/L), and (D) the large intestine samples gathered at 24 h after the administration of ZEN-14G by gavage (4 mg/kg body weight) to rats. Figure S7. Detection and identification of ZEN-14G by the established UHPLC-Q-TOF/MS method in (A) the blank urine sample, (B) the neat solution (200 ug/L), (C) the spiked large intestine sample (200 ug/L), and (D) the large intestine samples gathered at 24 h after the administration of ZEN-14G by gavage (4 mg/kg body weight) to rats. Figure S8.