# Antinociceptive Effect of an Aqueous Extract and Essential Oil from _Baccharis heterophylla_
## 2 Results and Discussion
### Method Validation  
In the present study, chlorogenic and di-caffeoylquinic acids were selected as markers for the quantitative analysis of _B. heterophylla_. The optimal chromatographic separation was achieved with a Waters HPLC equipped with an XBridge BEH Shield RP18 column and a mixture of water containing 0.1% formic acid and acetonitrile as mobile phase, which was chosen after several trials; the PDA detection wavelength (\(\uplambda\)) was set at 327 nm; under these optimized conditions, an effective resolution was achieved for compounds **1-4**. Compounds **1-4** were identified by their retention time and coelution with AE; a representative chromatogram of the AE of _B. heterophylla_ (BH-1) is shown in Figure 4. The developed method was validated using compounds **1**, **3** and **4** according to the International Conference on Harmonization guideline (ICH) [37], and was successfully applied to determine the content of caffeoylquinic acids in AE prepared from different batches of the plant. The overall results are summarized in Tables 2 and 3. Figure 3: Total ion current chromatogram of the components of the essential oil from _B. heterophylla_ extracted using hydrodistillation. Peaks are: \(\upalpha\)-pinene (9), \(\upbeta\)-pinene (**10**), myrcene (**11**) and D-limonene (**12**). The method's selectivity was determined by comparing the chromatographic profile of AE with the data obtained for the standards, considering the retention time and UV spectra (Figures S4-S6, Supplementary Materials). The linearity was tested by analyzing a series of different concentration ranges: 5 to 150 \(\upmu\)g/mL for **1**, 20 to 200 \(\upmu\)g/mL for **3**, and 13.4 to 133.4 \(\upmu\)g/mL for **4**. In all cases, the value of the determination coefficient (\(R^{2}\)) was greater than 0.99. The limits of detection (LOD) and quantification (LOQ) values were 0.5 \(\upmu\)g/mL and 1.5 \(\upmu\)g/mL for compound **1**, 2.2 \(\upmu\)g/mL and 6.6 for compound **3** and 1.4 \(\upmu\)g/mL and 4.1 \(\upmu\)g/mL for compound **4**. The intraday and interday precision RSDs were no more than 2% while the repeatability variation was no more than 2% (Table 2). Percentage recoveries of the standards are also indicated in Table 2; in each case, a good accuracy was obtained in the range from 99.15% to 100.83% (RSD \(\leq\) 2.0%). Altogether the described previous data revealed that the method was linear, precise and accurate in the range of concentrations evaluated. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{\(\mathbf{N}^{\circ}\)} & \multirow{2}{*}{\begin{tabular}{c} **Linear Range** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} } & \multirow{2}{*}{**Calibration Equation**} & & \multirow{2}{*}{\(\mathbf{R^{2}}\)} & \multirow{2}{*}{\begin{tabular}{c} **LOQ** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **LOD** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} } & \multicolumn{2}{c}{**Precision**} & \multirow{2}{*}{
\begin{tabular}{c} **Recovery** \\ **Intraday** \\ **(\% RSD)** \\ \end{tabular} } \\ \hline
**1** & 5–150 & y = 84,372x + 67,400 & 0.999 & 1.5 & 0.5 & 0.91 & 1.32 & 100.83 \\
**3** & 20–200 & y = 93,446x \(-\) 216,607 & 0.997 & 6.6 & 2.2 & 1.22 & 1.8 & 99.15 \\
**4** & 13.4–133.4 & y = 72,602x \(-\) 156,669 & 0.996 & 4.1 & 1.4 & 1.96 & 2.0 & 100.4 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Parameters of validation of method for determination of constituents in _B. heterophylla_. Figure 4: HPLC-PDA chromatogram of _B. heterophylla_ aqueous extract under optimized conditions; detection wavelength at 327 nm. Peak identification: **1**: \(R_{T}\) 6 min; **2**: \(R_{T}\) 14.6 min; **3**: \(R_{T}\) 15.6 min; **4**: \(R_{T}\) 16.2 min; **5**: \(R_{T}\) 17.2 min; **6**: \(R_{T}\) 23.6 min.