*3.3. Optimization Flush Solvent*

On the one hand, honey is a highly complex sugar mixture, and approximately 95% of the ingredients are sugar. On the other hand, the SPE column we used would adsorb substances with a larger polarity, so we chose to wash out the adsorbed impurities with water or methanol. Because the material is alkaloids, methanol leaching can lead to the loss of the target substance, reducing the extraction of the recovery. The sugar in honey is easily soluble in water. Therefore, water was selected as the flush solvent first, and then the amount of flush solvent was investigated. The results of rinsing with 5, 10 and 15 mL of water were investigated. The results are shown in Figure 2. The impurity removal effect of flushing with 15 mL of water is good, which is conducive to the recovery of gelsemine but will cause the loss of koumine and humantenmine, while flushing with 10 mL of water could completely elute saccharides and achieve a good recovery of the target components. However, the flushing effect of the 5 mL volume was significantly lower than that of the 10 mL volume. Combining the above results, 10 mL of water was selected to wash away the interference.

**Figure 2.** Comparison of the effects of 5 mL, 10 mL and 15 mL of flush solvent.

#### *3.4. Optimization Elution Solvent*

Because the target substance is alkaloid, it was converted into a free alkaloid under alkaline conditions. The organic reagent can be made alkaline by adding the appropriate amount of ammonia water. We compared 1% ammoniated methanol with 5% ammoniated methanol. The eluting effect of the 1% ammoniated methanol was no different from that of the methanol. The eluting effect of the 5% aminoacylated methanol was obvious due to the 1% aminoacylated methanol. To better dissolve alkaloids in organic solvents, the 5% ammoniated methanol was selected for elution, and then the amount of elution solvent was investigated [30]. The target substance was eluted with 3 mL, 5 mL and 8 mL solvents. The effect of the elution solvent amount on the extraction recovery of the target substance was observed. With the increase in the amount of elution solvent, the recovery rate improved significantly. When the elution solvent was 3 mL, the recovery rate of leucosin and leucosin

was about 65%, when the elution solvent was 5 mL, the recovery rate of leucosin and leucosin increased to 70%–75% and when the elution solvent volume reached 8 mL, the recovery rate was over 80%, which met the experimental requirements. The results are shown in Figure 3. More elution solvent can elute the target substance completely, but organic reagents are harmful to the body, so, after comprehensive consideration, 8 mL of the 5% ammoniated methanol was finally selected as the elution solvent in the spirit of cost savings and on the premise that the extraction recovery meets the experimental requirements.

**Figure 3.** Comparison of elution effects with 3 mL, 5 mL and 8 mL of elution solvent.
