## 3 Results
### BA\(\mu\)E-\(\mu\)LD Optimization Assays  
The proposed analytical approach, although user friendly and environmentally more favorable than conventional techniques, requires the optimization of several important parameters that can contribute to its effectiveness. To achieve this, the BA\(\mu\)E efficiency was evaluated using a univariate optimization strategy to optimize several parameters. We started by choosing the best sorbent coating (activated carbon or polymer) to microextract the target NPS from aqueous media. Afterwards, we optimized the desorption solvent (ACN, MeOH, and mix (MeOH/ACN, 1/1, \(v/v\))) and time (15, 30, 45, and 60 min). Next, the magnetic stirring rate (750, 1000, and 1250 rpm) and equilibrium time (1, 2, 3, and 16 h) were also evaluated. Finally, sample pH (2.0, 5.5, and 12.0) as well as the addition of NaCl (0, 10, and 20%, \(w/v\)) and MeOH (0, 10, and 20%, \(v/v\)) to the matrix were optimized. Except when specified, all assays were performed in triplicate.  
Starting with the sorbent selection, three activated carbon materials (N2, SX1, and R) and four polymers (Strata-X, LiChroult EN, Strata-DVB, and WCX) were tested as coating phases for enrichment purposes under standard conditions; microextraction stage: 16 h (1000 rpm), pH 5.5 (25 mL), and liquid desorption stage: MeOH (100 \(\upmu\)L), 30 min under sonication treatment. By observing Figure 1a, it was found that Strata-X (N-vinylpyrrolidone-based polymer) presented higher extraction efficiencies when compared to the other sorbents tested, resulting in average recoveries between 66.4% (\(\alpha\)-PVP) and 85.4% (\(\alpha\)-PVT).  
The sensitivity of the methodology was also checked through the determination of the LODs by measuring the S/N ratio of 3/1 for all the target analytes. The data show that LODs of 100.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\) L\({}^{-1}\) were achieved for all three SCs under study, which shows that some matrix effects are noticed. To assess the selectivity, ten oral fluid samples were analyzed using the optimized methodology without spiking. The results show that no detectable compounds (<LOD) eluted at the same RT of \(\alpha\)-PVP, \(\alpha\)-PVT, and MDPV (Figure 2). Despite all these data, the thermal degradation still continued to occur, probably due to oral fluid interferents, although all care to minimize this phenomenon were taken into consideration. For this reason, we decided that the GC-MS(SIM) approach was not suitable for the quantitative analysis of the three SCs. Despite this drawback, the optimized methodology could still be used as an alternative methodology to screen these three NPS in oral fluids. For this purpose, several assays were performed in duplicates by spiking five oral samples, to a final concentration of 500.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\) L\({}^{-1}\), under optimized experimental conditions. Figure 2 exemplifies the total ion chromatograms from spiked (500.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\) L\({}^{-1}\)) and unspiked oral fluid samples, where good selectivity was noticed.  
As was stated before, although the developed methodology cannot be accurately used for quantitative purposes, it can still be used to detect and confirm the presence of the three SCs in oral fluids since these compounds have previously been detected in this type of sample matrix in the ranges proposed by this approach. For instance, other researchers have detected MDPV and \(\alpha\)-PVP in the range of 20.5-831.7 and 81.9-935.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\) L\({}^{-1}\), respectively [10]. It must also be emphasized that the application of an analytical methodology using the GC-MS system without derivatization, even under all these limitations, can be a simple alternative for screening types of compounds in oral fluids.