*3.5. Method Validation Results*

There was a good linear relationship between the concentration of gelsemine in honey and koumine in honey in the range of 5–1000 ng/g, and humantenmine in the range of 20–1000 ng/g. The linear correlation coefficients R2 were all higher than 0.998 when the injection amount was 500 μL. The extraction recoveries were in the range of 81%–94.2% with an intraday RSD, and the interday RSD were all less than 5%. The accuracy and precision parameters met the methodological requirements. The LOQ of gelsemine and koummine was 5 ng/g, and the LOQ of humantenmine was 20 ng/g (Figure 4). The LOD of gelsemine, koummine and humantenmine was 2 ng/g. In 2D-LC, the retention times of the three alkaloids were approximately 7.28 min, 8.30 min and 11.30 min. The peak shapes of the three alkaloids were good, and there was no interference near the target peak. The sample stability results were in the range of 1.1%–10.16% (Table 2). The standard stock solution stability results were in the range of 0.2%–2.3% (Table 3). The method has good linearity, recovery, accuracy, precision, durability and specificity. These results indicate that this method can be used for the detection of snout toxicity in honey samples, reduce the risk of snout poisoning and can also be applied to the clinical detection of honey poisoning events. What's more, it also provides a theoretical basis and technical reference for the establishment of detection methods for other Chinese herbal medicines or toxic ingredients in honey.

**Table 2.** Results of linear range, correlation coefficient, LOD, recovery, accuracy, precision and sample stability.



**Table 2.** *Cont.*

**Figure 4.** 2D-LC of blank samples (red) and added sample (bule-20 ng/g) (**a**) gelsemine, (**b**) koumine and (**c**) humantenmine.


**Table 3.** Results of standard stock solution stability.
