## 3 Results and Discussions
### OA Derivatization Step and GC-MS Analysis
For this, the cartridge was activated and conditioned with 250 uL of MeOH and H\({}_{2}\)O, respectively, and the loading step was performed with 250 uL of a 5 mg/L standard solution in water in a 3-fold draw-effect cycle. The first studies were performed at a higher concentration for the target analytes to be detected by GC-MS. Then, the analytes were eluted with 50 uL of MeOH twice (50 uL \(\times\) 2). Figure 2: Total ion chromatography (TIC) obtained for each opium alkaloid using the SIM mode for a poppy seed tea extract spiked at a low level of validation (100 ug/L). As shown in Figure 3a, the areas obtained were generally larger when derivatization was performed after the \(\upmu\)SPEed(r) procedure. Thus, it was decided to perform derivatization after the \(\upmu\)SPEed(r) procedure. Subsequently, as no cartridge was shown to be clearly better than the others for all the analytes, due to the different chemical properties of OAs, in addition to testing all the cartridges by performing the loading step at pH 7, they were tested by acidifying the 5 mg/L standard solution with HCl (pH 3) and basifying it with NaOH (pH 9). Comparing the areas obtained at pH 7 (Figure 3b) and those obtained at pH 3 (Figure 3c) and 9 (Figure 3d), the structural and polarity differences between the analytes were even more evident. Some cartridges showed larger areas with lower pHs, such as C4, and others showed higher pHs, such as C8 or PS/DVB-SAX. However, the areas obtained with pH 3 and 9 for morphine were very small for all cartridges. Therefore, pH 7 was selected, and, finally, it was decided to select the silica and PS/DVB-RP cartridges, which showed the best responses for morphine and codeine, and for the rest of the analytes there was little difference between the cartridges with respect to the size of the areas. Afterwards, the two selected cartridges were studied by increasing the draw-effect cycles in the loading step to obtain the maximum possible adsorption of 5 mg/L of standard solution and to be able to select the cartridge that could adsorb the most. As shown in Figure 4a, after comparing the areas obtained, it was seen that the PS/DVB-RP cartridge, after 10 cycles, achieved a considerably larger area. Figure 3: Normalized areas (%) obtained for OAs in the optimization of the \(\upmu\)SPEed® procedure with nine cartridges: derivatization before (**a**) and after (**b**), acidifying to pH 3 (**c**) or basifying to pH 9 (**d**) in the loading step. All assays were performed with 5 mg/L standard solutions in triplicate; error bars indicate the relative standard deviations (RSDs %). Later, different elution solvents (MeOH unmodified, MeOH with 10% formic acid, MeOH with 10% NaOH, and CHCl\({}_{3}\)) were evaluated with the other optimal conditions, with 5 mg/L of standard solution in the loading step. As shown in Figure 4b, MeOH with 10% formic acid resulted in the largest area shown for all analytes. Finally, as one of the advantages of the \(\upmu\)SPEed(r) technique is the possibility of precontrating sample extracts to obtain lower instrumental limits, the studies were carried out with the optimal conditions, but with 10, 12 and 15 extract-discard cycles in the loading step. For this purpose, this study was carried out at the low validation level (100 \(\upmu\)g/L) to determine whether a preconcentration could be carried out with the \(\upmu\)SPEed(r) to allow the desired concentration levels to be quantified in the equipment. The recovery values were calculated with their respective simulated values. The values obtained by performing 10 cycles were 91 \(\pm\) 8% for morphine and around 100% recovery for the rest of the OAs; performing 12 cycles, codeine, thebaine and papaverine showed around 100% recovery, but morphine showed 37 \(\pm\) 12% and noscapine 85 \(\pm\) 9%; and, performing 15 cycles, morphine and noscapine showed close to 15% recovery, codeine 40 \(\pm\) 9%, papaverine 65 \(\pm\) 9% and thebeaine 82 \(\pm\) 9%.