## 3 Results and Discussions
### Transfer Study of OAs from Poppy Seeds to Tea Infusions  
To determine the level of transfer of OAs from poppy seeds to tea infusions, three factors (water temperature, infusion time and seed amount) were studied at two levels each (n = 8), each condition being studied in triplicate. For this, the seeds (S-1) were spiked to the maximum legislated limit (20 mg/kg) [23], having previously been washed and dried. Each of the studies was performed with 100 mL of deionized water, and, after cooling to room temperature (25 \(\pm\) 1 \({}^{\circ}\)C), the preparations were filtered through a nylon syringe filter; finally, the proposed uSPFed(r) procedure was applied, followed by the GC-MS analysis.  
The areas obtained were interpolated on the respective matrix-matched calibrations to calculate the concentrations of OAs in the teas. The concentrations obtained were compared, as shown in Figure 5a. ANOVA tests were performed to determine the statistically significant differences between each of the studies performed, and the transfer ratios (%) were calculated to determine the conditions under which the highest concentrations of OAs could be obtained in the teas. By spiking the seeds with the maximum limit established in the legislation and using two quantities of seeds (2 and 4 g), the concentrations that could be obtained in these teas were 0.4 and 0.8 mg/L, respectively, if 100% transfer occurred. As can be seen in Figure 5a, with 4 g of seeds at 90 \({}^{\circ}\)C for 5 min, statistically significantly higher concentrations of codeine, morphine and thebaine in teas were obtained.  
This was because, the greater the amount of seeds, the greater the concentration of OAs obtained, i.e., 100 mL of water was still sufficient for the extraction of all opiates from the seeds. Therefore, transfer ratios (%) lower than 100% should not be due to a lower transfer but rather to thermal degradation, as confirmed by some published works [7; 33; 38; 39]. This degradation is mainly observed in codeine and morphine, which are the most thermally sensitive analytes [38]. Thus, between the results of the studies performed at 90 \({}^{\circ}\)C and those performed at 100 \({}^{\circ}\)C, statistically significant differences were  
Figure 5: Mean concentrations (\(n=3\)) obtained in each transfer study with their transfer rates (%). The same letters mean that there were no statistically significant differences; different letters mean that there were such differences (\(p\leq 0.05\)) (**a**). Pareto Chart of the standardized effect of each of the responses (concentration of each analyte), showing the three factors: (A) poppy seed amount (g), (B) infusion time (min) and (C) temperature (\({}^{\circ}\)C) (**b**).  
observed for all analytes, although they were much lower in the cases of papaverine and noscapine, in which thermal degradation was affected less. Regarding infusion time, higher concentrations could be expected with longer times, as the contact time between water and seeds was increased. However, it was observed that increasing the time could in some cases decrease the concentration of OAs in the tea. This may have been due to increased degradation with increasing exposure time of the OAs at high temperatures. All of this is confirmed by the Pareto charts shown in Figure 5b, in which effect A (amount of seeds) has a very significant positive effect on all analytes, effect B (infusion time) has a negative effect on all analytes (the effects being much higher in the cases of codeine and morphine) and effect C (temperature) has a negative effect on all analytes except noscapine, for which no statistically significant difference was observed.  
Therefore, the conditions selected for the highest rate of transfer or lowest rate of degradation were 4 g at 90 \({}^{\circ}\)C for 5 min, obtaining 90% transfer in codeine, 75% in morphine, 96% in thebaine, and 100% in papaverine and noscapine.