Matrix E ffect and Recovery

The recoveries after SPE were 93.2% and 97.4% for sakuranetin and 7-methoxyaromadendrin respectively, which means that 6.8 and 2.6% were lost in the solid phase extraction process. The ratios (A/B × 100)% were 88.3% for sakuranetin and 94.7% for 7-methoxyaromadendrin, so the presence of the matrix decreases the recovery of sakuranetin and 7-methoxyaromadendrin by 4.9% and 2.7% respectively. These results showed that all values were within acceptable ranges.

### Precision and Accuracy of the System

In this method, injection errors, dilution, adduct formation etc. can occur. Therefore, precision and accuracy of the system were evaluated. The results sugges<sup>t</sup> that both parameters were acceptable for the quantification method developed. In the case of precision, very similar % RSD were observed. The results of sakuranetin were less than 2.5% in the experiments intra-day and inter-day whereas for 7-methoxyaromadendrine were less than 3.5%. Accuracy showed results above 98% intra-day and inter-day in both standards for the concentrations tested, Table S2.

#### 2.3.2. Concentration of Standards in the Extracts

The linear calibration curves were used for quantitative analyses of the standards in the extracts, Table 3. In general, the ethyl acetate extracts showed higher concentration of the analyzed patterns. The concentration of sakuranetin is approximately three times the concentration of 7-methoxyarycomadendrine. Extracts from the leaves of the vegetative season present a higher concentration of analyzed flavonoids than those from the flowering season.


**Table 3.** Concentration of sakuranetin and 7-methoxyaromadendrin in the extracts by UPLC/ESI/TQD/MSn.

#### 2.3.3. Principal Component Analysis of Extracts

Principal component analysis (PCA) allowed comparing extracts of *Ageratina havanensis* according their chemical composition. The original variables were reduced to the two principal components PC1 (77.9%) and PC2 (5.6%) representing an 83.6% of the total data variance (Table S3). According to eigenvalues, peaks with *m*/*z* 447 gave principal contribution to PC1. Also, sakuranetin ( *m*/*z* 285, [M − H]−), 7-methoxyaromadendrin ( *m*/*z* 301, [M − H]−) and peaks with *m*/*z* 463 had grea<sup>t</sup> influence on the variability of the data in this component. From the extracts of *Ageratina havanensis,* were isolated and identified in previous studies three glycosides with *m*/*z* 447 and the same fragmentation ([286 + 162 − H]−) in its MS<sup>2</sup> spectrum [17,18]. Because of that, it was concluded that the most influential variables in PC1 were the concentration of sakuranetin and 7-methoxy-aromadendrin. On the other hand, the glycosides ( *m*/*z* 447 and *m*/*z* 463) were the variables that most influenced on PC2, Table S4.

Figure 2 shows that the ethyl acetate extract from the leaves collected in vegetative state (Lv-EtOAc) differs from the rest of the extracts with respect to PC1 followed by the ethyl acetate extract from the leaves collected in flowering state (Lf-EtOAc). The rest of the extracts showed similarity in PC1. According to this behavior, it was concluded that sakuranetin, 7-methoxyaromadendrin and the glycosides (the original variables that most influence CP1) were higher in the vegetative stage, mainly in the ethyl acetate extract of leaves. With respect to PC2, the *n*-butanol extract of the leaves collected in flowering state (Lf-*n*-BuOH) is different from the rest of the extracts. Sakuranetin and 7-methoxyaromadendrin in this case do not influence the total variability of the data but the glycosides have a significant influence. These results were expected since in the quantification, the concentration of both sakuranetin and 7-methoxyaromadendrin resulted higher in the extracts of ethyl acetate of the leaves, being greater in the vegetative state, whereas in the ethanolic and *n*-butanolic extracts, these flavonoids were below the limit of quantification.

**Figure 2.** Scatter plot of PCA of the extracts on the first two principle components.
