## 3 Results and Discussions
### Validation of the Proposed Method Based on \(\upmu\)SPEed(r) Followed by GC-MS  
The results of the validation of the proposed method based on \(\upmu\)SPEed(r) followed by GC-MS for the quantification of OAs in poppy seed teas are shown in Table 2. The regression lines were obtained by least-squares linear regression analysis of the data and provided excellent correlation coefficient (R\({}^{2}\)) values between 0.998 and 1.000 for all analytes. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Analyles**} & **Linear Range** & \multirow{2}{*}{**Matrix-Matched Calibration (R\({}^{2}\))**} & **MDL** & **MQL** & **Accuracy\({}^{*}\)** & **Precision (\(\upmu\)**RSD) \({}^{*}\)** \\  & **(\(\upmu\)g/L)\({}^{*}\)** & & **(\(\upmu\)g/L)\({}^{*}\)** & **(\(\upmu\)g/L)\({}^{*}\)** & **ME** & **Accuracy (\(\upmu\)g/L SD)** & **Intra-Day** & **Inter-Day** \\ \hline \multirow{3}{*}{Codeine} & \multirow{3}{*}{5-800} & \multirow{3}{*}{y = 37 \(\times\) 10\({}^{6}\times\) - 4.1 \(\times\) 10\({}^{2}\) (0.999)} & \multirow{3}{*}{0.3} & \multirow{3}{*}{1} & \multirow{3}{*}{8} & LL & \(91\pm 8\) & 9 & 11 \\  & & & & & & ML & \(101\pm 4\) & 4 & 7 \\ \cline{1-1}  & & & & & HL & \(99\pm 1\) & 1 & 4 \\ \cline{1-1}  & & & & & LL & \(90\pm 8\) & 8 & 10 \\ \cline{1-1}  & & & & & ML & \(90\pm 7\) & 7 & 13 \\ \cline{1-1}  & & & & HL & \(90\pm 3\) & 3 & 7 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Validation parameters for the proposed method based on \(\upmu\)SPEed® followed by GC-MS for the quantification of opium alkaloids in poppy seed teas. Figure 4: Normalized areas (%) obtained with the silica and PS/DVB-RP cartridge with 3, 5 and 10 draw–effect cycles in the loading step at pH 7 (**a**) and with the PS/DVB-RP cartridge with a different elution solvent with a pH of 7 in the loading step (**b**). All assays were performed with 5 mg/L standard solutions in triplicate; error bars indicate the relative standard deviations (RSDs %). The method showed low MDL and MQL values expressed in ug/L of poppy seed tea (Table 2): 0.06 and 0.2 ug/L for papaverine, 0.07 and 0.2 ug/L for noscapine, 0.2 and 1 ug/L for thebeaine, 0.3 and 1 ug/L for codeine, and 0.5 and 1.6 ug/L for morphine, respectively. The MEs were calculated by comparing the slopes of the matrix and solvent calibration curves. As shown in Table 2, the MEs of the proposed methodology were negligible, as all values were within \(+/-20\%\). This means that the ugSPEC(r) procedure was able to eliminate all possible matrix effects. For this reason, solvent regression lines could be used to quantify the samples, simplifying the analysis [35]. Accuracy and precision were evaluated at three different concentration levels: 100 ug/L (LL), 400 ug/L (ML) and 800 ug/L (HL). Accuracy was expressed as the average recovery obtained comparing six samples (\(n=6\)) spiked with the corresponding values with their simulated samples. As shown in Table 2, recovery values were adequate according to the guidelines [35] for all validation levels (between 89 and 108%). In addition, satisfactory results were obtained for intra-day and inter-day precision at the three concentration levels, since the RSD values were lower than 20%, according to the guidelines [35], and the lowest value was 14% (Table 2). Furthermore, as shown in Figure 2, good selectivity for the method was demonstrated, since no interfering peaks were found at the retention time for the target analytes. The most significant advantages of the proposed method over previously published ones were that this method, in addition to extending to morphine, codeine and thebeaine, also extends to papaverine and noscapine, which can be even more toxic [17]. In addition, by performing the ugSPEC(r) procedure, it was possible to preconcentrate the extract 10 times more, such that the present method has 10-fold lower limits than those of the work of [PERSON] et al.