## 3 Results and Discussions  
### OA Derivatization Step and GC-MS Analysis  
First, derivatization was performed following the protocol of [PERSON] and [PERSON] (1987)[11], using 100 \(\upmu\)L of CHCl\({}_{3}\) and 100 \(\upmu\)L of TFA for 20 min at 60 \({}^{\circ}\)C. A quantity of 1 mL of a 50 \(\upmu\)g/mL standard solution was injected into the GC-MS equipment, and the MS acquisition was carried out in FS mode with a mass-to-charge ratio ranging from 50 to 500 (m/z) to identify each OA based on its mass spectrum. Considering the low levels at which OAs can occur, the full-scan MS detection is not the most suitable approach for this purpose. The low sensitivity and selectivity of this MS acquisition mode were overcome using the SIM mode, in which the most abundant OA fragment ions seen in the full scan were selected for quantitative purposes (Table 1). This MS acquisition mode presents high selectivity for every single OA in the sample as well as high sensitivity and allowsthe confirmation of analyte identity and accurate quantification of analytes. This allows the achievement of lower detection and quantification limits by improving the selectivity and sensitivity of the instrumental analysis. A chromatographic method with a starting temperature of 80 \({}^{\circ}\)C and rising to 300 \({}^{\circ}\)C with a ramp of 4 \({}^{\circ}\)C/min, corresponding to an analysis time of 45 min, was used. Thus, it was possible to separate and identify each of the analytes with their respective ions to create the SIM method that allows increasing specificity and the elimination of interference. Once the SIM method was created, the analysis time was reduced by modifying the oven temperatures. For this, the first step was to increase the starting temperature from 80 \({}^{\circ}\)C to 180 \({}^{\circ}\)C, since the retention time of the first analyte was 24.14 min. This decreased the retention time of the first analyte to 12.10 min and the analysis time to 31 min. Then, to shorten it further, the starting temperature was raised to 200 \({}^{\circ}\)C, achieving a codeine retention time of 7.30 min and a total time of 21 min. Finally, the starting temperature was raised to 220 \({}^{\circ}\)C and the ramp was made faster--instead of 4 \({}^{\circ}\)C/min, it was set to 6 \({}^{\circ}\)C/min; thus, the retention times for each of the analytes were: codeine, 5.06 min; morphine, 5.46 min; thebaine, 6.31 min; papaverine, 9.44 min; and noscapine, 12.77 min, with a total time of 14.33 min, as shown in Figure 2. In addition, 1 min of hold time at the end of the method was increased to ensure the cleanliness of the column between injections. The spectra of each compound can be used for the rapid identification of compounds before quantification, i.e., from the TIC (Figure 2) they can be extracted and quickly checked and identified. Once the chromatographic method was optimized, some tests were carried out to optimize the derivatization step. For this, different derivatization solvents were tested (CHCl\({}_{3}\), MeOH, ethyl acetate and hexane) at different temperatures (40 and 60 \({}^{\circ}\)C) and times (10, 20 and 40 min). Finally, the optimum conditions selected were: CHCl\({}_{3}\) at 60 \({}^{\circ}\)C for 20 min, thus confirming that the protocol used in the work of [PERSON] and [PERSON] (1987) [11] to derivatize morphine and codeine was also the most suitable for the derivatization of all OAs.3.2. uSPefed(r) Procedure Optimization. To optimize the uSPefed(r) procedure, nine cartridges were tested due to the different chemical properties of the OAs, papaverine and noscapine having the lowest water solubilities (0.013 and 0.18 g/L, respectively) and morphine having the highest (10.20 g/L) [31]. First, nine cartridges were evaluated by performing the derivatization step before and after to see when it was more efficient to perform it.