## 3 Results and Discussion
### Optimization of Sample Preparation
#### 3.2.3 Optimization of Purification Conditions of Solid-Phase Purification Column  
Both the C18 purification column and the HLB (Hydrophilic-Lipophilic Balance) are classified as non-polar adsorption columns, designed to retain non-polar target analytes in polar aqueous solvents through non-polar interactions ([PERSON], 1999) [22]. The HLB purification column was developed as an advanced alternative to the traditional silica gel C18 column. In previous studies, the purification of macrolides predominantly utilized C18 columns ([PERSON] et al., 2017) [23]. However, the HLB column, which incorporates a styrene-divinylbenzene copolymer, offers several advantages over the C18 column, including a higher tolerance to loading solutions, simpler activation procedures, and greater resistance to drying ([PERSON] et al., 2003) [24]. While both columns serve similar functions, the HLB column is more user-friendly and efficient. Therefore, the HLB purification column was selected as the solid-phase extraction (SPE) column for this method.  
In previous studies, acetonitrile, methanol, and ethyl acetate were evaluated as elution solvents for the HLB purification column ([PERSON] et al., 2005) [25]. While ethyl acetate demonstrated the strongest elution capability and yielded the highest recovery rates, it also introduced significant matrix interference and extraneous peaks during sample analysis ([PERSON] et al., 2011) [26]. In contrast, acetonitrile and methanol exhibited comparable elution efficiency with minimal differences in performance. Based on these findings, methanol was selected as the elution solvent for this method. To ensure the complete elution of the target analytes, 6 mL of methanol was used for the elution process.  
After determining the drug elution solvent for the HLB purification column, the elution conditions were further optimized. The HLB column was eluted with pure water, 0.1% formic acid, and methanol solutions at concentrations of 5%, 10%, 15%, and 20% to identify the optimal elution conditions. Preliminary results indicated that the average recovery rates for drugs eluted with 20% methanol ranged between 50% and 90%, with significant losses observed for certain individual analytes.  
To further optimize the elution conditions, 17% methanol, 17% acetonitrile and 18% methanol, 18% acetonitrile were evaluated as eluents. The effects of these elution conditions on the recovery rates of the target drugs and the interference from feed matrix impurities were investigated. The average recovery rates under the two elution conditions are illustrated in Figure 5a, while the matrix effect rates are shown in Figure 5b. The results demonstrated that both elution methods had a minimal impact on the recovery rates of the target drugs when using the HLB purification column. However, the 18% methanol/18% acetonitrile eluent significantly improved the average recovery rate of IVM (ivermectin). This enhancement is likely attributed to the more efficient elution, better removal of impurities, and improved chromatographic response at this concentration. Additionally, the matrix effect observed in chromatograms treated with the 18% eluent was lower compared to those treated with the 17% eluent, indicating superior sample purification efficiency. To ensure the complete removal of impurities, the HLB purification column was sequentially eluted with 18% methanol followed by 18% acetonitrile.