## 4 Discussion  
SCs are the second most seized NPS in Europe and one of the most abused drugs around the word after the \"classic\" ones. For this reason, novel miniaturized and green analytical methodologies are needed to screen these compounds in several biological matrices. In this regard, oral fluid can be an excellent source to monitor SC short-term consumption while being fast and easy to collect [8]. This is particularly important to police driving under the influence of drugs. While the sample can be analyzed on site using fast detection kits, this cannot be easily performed for recently introduced NPS on the market since they have distinct physico-chemical characteristics from classical drugs. For this reason, presumptive positive samples must be later confirmed using more  
Figure 2: Total ion chromatograms from assays performed on spiked (500.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\) L\({}^{-1}\)) and unspiked oral fluid samples obtained by \(\mathrm{BA\SIUnitSymbolMicro\text{g}}\)(Strata-X)-\(\mathrm{\SIUnitSymbolMicro\text{g}}\)L/GC-MS(SIM) under optimized experimental conditions. 1: \(\alpha\)-PVP; 2: \(\alpha\)-PVT; and 3: MDPV. advanced techniques such as LC-MS and GC-MS. While the former is a more frequently used instrumental system to analyze SCs in biological matrices, the latter is much cheaper, more comprehensive, and is ubiquitous in most analytical toxicology laboratories. Our results show that although the target SCs present partial thermal degradation during GC-MS(SIM) analysis, this phenomenon can be minimized by lowering injection temperatures and residence time at the inlet, and by avoiding active sites during chromatographic analysis (i.e., replacing liners more often) as previously suggested [25]. This data show that although reliable quantitation is limited using GC-MS(SIM), a suitable qualitative approach can be employed, especially taking into consideration the demand for alternative analytical methodologies for the trace detection of these compounds and the public health issues regarding its abuse. The validation assays showed that the reproducibility using pentaplicates is acceptable for this type of application (RSD < 15%) for samples presenting levels of the target compounds higher than 100 \(\mu\)g L\({}^{-1}\). The optimized sample preparation stage, using an \(N\)-vinylpyrrolidone sorbent phase as coating material for BA\(\mu\)E, showed very good performance for the detection of traces of \(\alpha\)-PVP, \(\alpha\)-PVT, and MDPV in oral fluid samples. This sorbent material combines small particle (33 \(\mu\)m) and pores sizes (85 A), high surface area (800 m\({}^{2}\) g\({}^{-1}\)), and the possibility to retain analytes by reverse-phase type interactions, i.e., hydrogen bonding, \(\pi\)-\(\pi\), and dipole-dipole and hydrophobic interactions, especially for compounds with mid-polar to non-polar characteristics such as the target SCs under study (log \(K_{\text{O/W}}\approx 3\); Table 1). In this particular case, the resulting efficiency may be attributed to dipole-dipole interactions between the pyrrolidone of the polymer and the electronegative groups in the target SCs. Furthermore, \(\pi\)-\(\pi\) and hydrophobic interactions promoted by the DVB groups may also contribute to the high retention of \(\alpha\)-PVP, \(\alpha\)-PVT, and MDPV. The \(\mu\)LD process showed that a mixture of the two solvents produced the best results, which is expected when analyzing compounds with different chemical structures. Although the optimized conditions showed that 16 h are needed to reach the microextraction equilibrium, it is not really a limitation using BA\(\mu\)E since it can be performed overnight with no special requirements. In regard to sample pH, since the target SCs presented weak base characteristics with p\(K_{\text{a}}\) values between 7.31 and 7.89 [34], it was expected that lower pH values would provide a loss in efficiency and a more basic solution the opposite.