## 3 Results and Discussion
### Validation of the Method  
The analytical performance of HPLC-MS/MS was evaluated by the selectivity, linearity, sensitivity, precision, and stability parameters. The method was selective for both ZEN and ZEN-14G in the plasma, different tissues, and urine, since no interference peaks appeared during the retention time in blank samples, also indicating that no ZEN or ZEN-14G existed in the regular rat feed (Figure 1). The SSE values for ZEN and ZEN-14G were 31-79% and 70-87%, respectively. The matrix-matched calibration curves were constructed to compensate for the matrix effects to accurately quantify the two analytes. Satisfactory linear relationships of the two target analytes were observed, with the coefficients of determination (\(R^{2}\)) greater than 0.99. The LLOD and LLOQ obtained were 0.03-0.3 ug/L and 0.1-1.0 ug/L, respectively (Table 2), which were obviously lower than those in plasma reported by [PERSON] et al. (ZEN, 7.4 ug/L) [48] and [PERSON] et al. (ZEN-14G, 1.7 ug/L) [49], but higher than those in plasma reported by [PERSON] et al. (ZEN, 0.1 ug/L;  
Figure 2: The extraction efficiencies of zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) by three candidate solvents (**A**) and acetonitrile containing different contents of formic acid (0.25-4%, _v/v_) (**B**) were compared. The concentration is 50 ug/L. Acceptable recovery rates are between 70% and 120%. ZEN-14G, 0.03 \(\upmu\)g/L) [24]. To date, there have been few studies on the simultaneous detection of ZEN and ZEN-14G in the plasma, different tissues, and urine of rats. The LLOQ values in this approach were obviously lower than or comparable with those in several tissues and urine reported (0.6-0.8 \(\upmu\)g/L) by [PERSON] et al. [50], proving that the sensitivity of the established method was sufficient for measuring both analytes in different biological matrices. Acceptable recovery rates of ZEN (80.7 \(\pm\) 3.0-112.3 \(\pm\) 3.1%) and ZEN-14G (85.0 \(\pm\) 4.9-109.3 \(\pm\) 2.4%) were obtained with the RSD values of intra-day and inter-day precision being in the range of 0.9-14.6% and 0.6-16.5%, respectively (Table 3). After a detailed study of the stability under different conditions, ZEN and ZEN-14G were fully verified to be stable (81.7 \(\pm\) 1.7-104.1 \(\pm\) 3.9%) in the plasma, different tissues, and urine of rats (Table S2). These satisfactory parameters proved that the developed method was selective, accurate, sensitive, and stable, and could be used for studies of the kinetics and distribution of ZEN and ZEN-14G in rats. To better understand the strengths and weaknesses of the developed HPLC-MS/MS method, we further compared it with previously reported methods of determining ZEN and ZEN-14G. In comparison with the rapid detection methods, namely immunoassay or aptasensors [51; 52], the current method exhibited comparable sensitivity and accuracy, and could be applied for the simultaneous determination of two compounds with different polarities in a variety of matrices. The method of ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) previously developed in our lab was also tested and showed high selectivity for the targets in different matrices (Figures S3-S8), but the sensitivity with the screening detection limits (SDLs) ranging from 50 \(\upmu\)g/L to 200 \(\upmu\)g/L seemed relatively lower (Table S3) [53]. A parallel analysis was carried out using the current method and a series of previously established LC-MS/MS methods for the analysis of ZEN and its glucoside in the plasma [54], liver [50], and urine [55] (Table S4). Accurate results were obtained independent of the matrix tested, proving it to be a promising tool for studies of the kinetics and distribution of ZEN and ZEN-14G in rats.