## 2 Materials and Methods
### Analytical Method Validation  
There is no official regulation regarding analytical performance requirements for quantifying OAs in food or other matrices. Therefore, the validation of the method was performed following the criteria described in SANTE/11312/2021 for pesticides, in EC Regulation No. 401/2006 and in the ICH Q\({}_{2}\)(R1) guidelines [35; 36; 37]. The validation method was performed for linear dynamic range (LDR), matrix effect (ME), method detection and quantification limits (MDL, MQL), accuracy, precision and selectivity. The validation was carried out with a spiked sample previously washed and dried (S-1). Accordingly, LDR was evaluated at six concentration levels with standard solutions prepared and analyzed using the proposed \(\upmu\)SPead(r) procedure. Concentration ranges were selected according to the sensitivity of the GC-MS system to each target analyte, as \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compound Name** & **Quantification 1 (m/z)** & **Ion 2 (m/z)** & **Ion 3 (m/z)** \\ \hline Codeine & 299 & 229 & 162 \\ Morphine & 285 & 215 & 162 \\ Thebaine & 311 & 296 & 242 \\ Papaverine & 338 & 324 & 154 \\ Noscapine & 220 & 215 & 205 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Retention time and m/z values of fragment ions selected for the quantification and confirmation of the five opium alkaloids. well as the amounts expected in the tea infusions. Matrix-match calibration lines were prepared in the same way, but instead of using standard solutions, the tea obtained from the poppy seeds (S-1) that had been previously washed and dried was spiked. All calibration curves were obtained with the mean peak area of each analyte versus analyte concentration and fitted by least-squares linear regression. Matrix effects were determined by comparing the slopes of the calibration equations obtained from matrix-matched and solvent-based calibration curves for each analyte, calculated using the following formula: (slope matrix-matched/slope solvent-based \(-1\)) \(\times\) 100. The ME is negligible when it is lower than \(+/-20\%\)[35], representing signal enhancement when ME values are greater than 20% and signal suppression when values are greater than \(-20\%\). The sensitivity of the method for each sample was determined according to the MDLs and MQLs of the OAs from the analysis of the lowest concentration analyzed (5 \(\upmu\)g/L), which yielded signal-to-noise (S/N) ratios of 3 and 10 (when the quantification ion was monitored), respectively. The accuracy, expressed as recovery percentage (%), was assessed by comparing the areas obtained for spiked samples (\(n\) = 6) with known concentrations of analytes and by performing the \(\upmu\)SPEed(r) procedure with those areas obtained for simulated samples (samples spiked at the same concentration but at the end of the \(\upmu\)SPEed(r) procedure, prior to the derivatization step for GC-MS analysis). The recovery assays were performed by spiking the samples at three concentration levels: 800 \(\upmu\)g/L (high level, HL), 400 \(\upmu\)g/L (medium level, ML) and 100 \(\upmu\)g/L (low level, LL). In the absence of legislation setting a maximum limit for OAs in poppy seed tea infusions, the Regulation (EU) 2021/2142 maximum limit of 20 mg/kg in seeds was followed, and the concentrations of OAs in the teas were estimated assuming complete transfer. Thus, for the seeds with 20 mg/kg of OAs, the concentration in tea (2 g of seed in 100 mL of water) was 400 \(\upmu\)g/L, this being the intermediate level of validation. The recovery values should be between 70 and 120%. On the other hand, method precision was evaluated in terms of repeatability (intraday) and reproducibility (inter-day), using the same validation levels as for the accuracy assays. For repeatability (expressed as a percentage of the relative standard deviation, RSD %), six replicates (\(n\) = 6) of the whole procedure were performed on the same day.