## 3 Results and Discussion
### Sample Preparation  
As an accepted extraction method, solid-phase extraction (SPE) was widely used in the field of ZEN analysis. However, high labor-intensity and time-consuming limited the further application in large-scale analysis. To address such issues, a high-throughput sample preparation method using a 96-well \(\mu\)Elution plate was introduced to extract ZEN and its derivatives from human serum samples. The main parameters that affect extraction procedure were optimized including loading, washing and elution conditions.  
Phospholipids, carbohydrates, mineral salts and other metabolites in the serum may cause the endogenous interferences or the risk of cartridge clogging. Hence, all samples were 2-fold diluted with phosphate buffer or \(\beta\)-glucuronidase solvent before loading onto the plate.  
The washing and elution buffer directly affected the matrix effect and recovery. Spiked serum samples (both before and after enzymatic digestion) with the concentration of 10 ng mL\({}^{-1}\) for each analyte were used in the test of selection. To remove the salts and other polar interferences in serum, the plate was washed with 500 \(\mu\)L of water after loading as a weak wash step. For further cleanup, a selection of strong wash solvent was performed by washing the cartridge with methanol/water buffer (ranged from 5% to 100% methanol, \(v/v\)) and the effluent were collected for analysis of target compounds. All the analytes started to flow out at 50% methanol and were completely eluted by pure methanol. Hence, an additional wash with 500 \(\mu\)L of 50% methanol as a strong wash step was adopted before the final elution with 100% methanol. Varying elution volumes of 25, 50, 75, 100, 150 and 200 \(\mu\)L were evaluated as well to obtain high recoveries and less interference. The maximum recoveries were achieved for all analytes with the elution volume of 100 \(\mu\)L. To avoid the solvent effect and achieve sharp peaks, the collected eluent needed to be diluted with 100 \(\mu\)L of water before  
Figure 1: The overlaid chromatography of standard solution (10 ng mL\({}^{-1}\) of each analyte).  
instrumental analysis. Finally, the optimized extraction procedure was obtained as described in the method section.  
To determine the total amount of each analyte including the conjugated forms, enzymatic hydrolysis is necessary. Since ZEN biomarkers in body fluids commonly occurred in the low ng mL\({}^{-1}\) range, there was no need to worry about the adequacy of enzyme. On the other hand, the enzyme amount might should be optimized to minimize the exogenous interferences that influence analyte extraction and matrix effect. Spiked serum samples (10 ng mL\({}^{-1}\) for each analyte) together with varying levels of enzyme (100, 200, 500, 1000, 2000 and 5000 U mL\({}^{-1}\)) were incubated at 37 \({}^{\circ}\)C for 18 h. The recoveries of five analytes, except for \(\beta\)-ZAL, reduced in different degree while the enzyme concentration up to 2000 U mL\({}^{-1}\), as shown in Figure 2. Therefore, the concentration of 1000 U mL\({}^{-1}\) was chosen in further study.  
The sample preparation with the PRiME HLB \(\mu\)Elution plate eliminated laborious steps of evaporation and reconstitution. Under the optimized condition, this method drastically improved the efficiency, allowing 96 samples to be processed in 1 h with good recoveries and well controlled matrix interference.