*3.2. SPE Column Selection*

We chose the solid phase extraction column (e.g., the HLB column and PRS cation exchange column) commonly used in alkaloid detection methods [33,34]. The HLB column is filled with a synthetic ultra-low pressure rapid reverse-phase chromatography filler, like the C18 column, which is suitable for the extraction of non-polar to moderately polar acidic, neutral and basic compounds. HLB columns are often used in the detection of complex substrates such as blood, urine and food. The PRS strong cation exchange column is based on the strong cation exchange being adsorbent. The main functional group is sulfonyl propyl, which has two ways of cation exchange and reverse phase retention. The retention mechanism is mainly related to strong cation exchange, while polarity is a secondary effect. It is suitable for the extraction of basic compounds. Therefore, we chose an HLB column and a PRS cation exchange column to investigate the effect of the pretreatment. Under the same pretreatment conditions (5 mL of methanol and 5 mL of Milli-Q water.), the HLB column showed poor retention of koumine and humantenmine, while the PRS column showed a high sensitivity and retention of koumine and humantenmine. The reason is that the cationic group of alkaloids can exchange with the positive ion [H] + of the PRS column, while the retention of the HLB column to the substance with greater polarity was poor. As shown in Figure 1, a cation exchange column was selected, and a solid phase extraction column (PRS) was used for sample extraction and purification; thus, a good peak shape and separation will be obtained.

**Figure 1.** Comparison between the PRS cation exchange column (blue) and HLB solid phase extraction column (red). (**a**) Gelsemine, (**b**) koumine and (**c**) humantenmine.
