## 3 Results and Discussions
### Occurrence of OAs in Tea Infusions from Different Poppy Seeds  
To determine the occurrence of OAs in poppy seed teas prepared using different samples of poppy seeds, the conditions with greater rates of transfer were used. For this reason, 4 g of each of the poppy seed samples were infused with 100 mL of water at 90 \({}^{\circ}\)C for 5 min. The studies were performed in triplicate, taking a fresh 4 g of each sample for each infusion to determine the extent of the variation for each seed sample. After cooling to room temperature, the preparations were filtered through a nylon syringe filter, and, finally, the \({}_{\mathrm{t}}\)SPEed(r) procedure was applied, followed by the proposed GC-MS analysis. The areas obtained were interpolated on the respective matrix-matched calibrations to calculate the concentrations of OAs obtained for each of the teas. As shown in Table 3, all OAs were quantified for all the poppy seed teas analyzed. Furthermore, as can be seen, the calculated standard deviations of the three replicates were in some cases high. This may have been due to the heterogeneous contamination that seeds suffer, external contamination depending on multiple factors, such as climate, time of harvest, and variety, among others [5; 6; 8]. Of the four poppy seed teas analyzed, S-1 and S-2 showed the lowest OA contents, morphine and noscapine being the analytes with the highest concentrations in both teas (43 \(\mathrm{\SIUnitSymbolMicro g}\)/L and 23 \(\mathrm{\SIUnitSymbolMicro g}\)/L, respectively) and thebaine the one with the lowest (below the MQL). These samples were labelled _Papaver rheas_ L. (Table S1), which does not contain OAs in its latex, such that its seeds cannot be contaminated. Therefore, the determinations of OAs in these infusions confirmed the mislabeling of the products, as observed in previous studies [5]. S-3 showed a higher content of all OAs, especially morphine 227 \(\mathrm{\SIUnitSymbolMicro g}\)/L, and S-4 was the sample with the highest concentration of all OAs, giving up to 1563 \(\mathrm{\SIUnitSymbolMicro g}\)/L of morphine, 254 \(\mathrm{\SIUnitSymbolMicro g}\)/L of codeine, 71 \(\mathrm{\SIUnitSymbolMicro g}\)/L of thebaine, 56 \(\mathrm{\SIUnitSymbolMicro g}\)/L of noscapine and 29 \(\mathrm{\SIUnitSymbolMicro g}\)/L of papaverine. This sample was diluted 1:2 to quantify it within the calibration line and then a recalculation was performed to give the final concentration. Considering the transfer rates of 75% for morphine and 90% for codeine, the estimated quantity for S-4 poppy seeds expressed in morphine equivalents is 54 mg/kg, which is higher than the legislated \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Code Sample** & **Morphine** & **Codeine** & **Thebaine** & **Papaverine** & **Noscapine** & **ME a** \\ \hline S-1 tea & 25 \(\pm\) 3 & 4 \(\pm\) 1 & \(<\) MQL & 8.13 \(\pm\) 0.02 & 20.6 \(\pm\) 0.2 & 26 \\ S-2 tea & 43 \(\pm\) 25 & 1.8 \(\pm\) 0.4 & \(<\) MQL & 8.2 \(\pm\) 0.2 & 23 \(\pm\) 2 & 43 \\ S-3 tea & 227 \(\pm\) 39 & 58 \(\pm\) 42 & 56 \(\pm\) 36 & 11 \(\pm\) 6 & 29 \(\pm\) 6 & 239 \\ S-4 tea & 1563 \(\pm\) 33 & 254 \(\pm\) 35 & 71 \(\pm\) 13 & 29 \(\pm\) 2 & 56 \(\pm\) 1 & 1614 \\ \hline \end{tabular} a ME: morphine equivalents = morphine + 0.2 \(\times\) codeine; \(<\)MQL: below the method quantification limit.  
\end{table} 
Table 3: Occurrence of opium alkaloids (main concentration (\(\mathrm{\SIUnitSymbolMicro g}\)/L) of three replicates \(\pm\) standard deviation) in poppy seed teas prepared with four different samples of poppy seeds (S-1, S-2, S-3 and S-4). limit (20 mg/kg). It should be noted that the seeds were purchased in 2021 and therefore could not have been expected to comply with current legislation, according to which, if the seeds were marketed before 1 July 2022, they can remain in trade until the best-before date is reached [23]. In addition, the acute dose of morphine equivalents set by the EFSA in 2018 is 10 ug per kg body weight.