## 2 Results and Discussion
CE conditions as in Figure 1. Legend: DA—dopamine; A—adrenaline; NA—noradrenaline; L-Trp—L-tryptophan; L-Tyr—L-tyrosine; 5-HT—serotonin; VMA—vanmillymaledic acid; HVA—homovanillic acid. To verify the influence of the changes in the SPE tunable parameters and the increase in the extraction efficiency of VMA and HVA, but also basic NTs, by SPE SCX, we changed the SPE eluent and applied pure MeOH or MeOH with sodium hydroxide (pH 9.3) for the elution of the analytes. In both cases, the efficiency was below our expectations, and the baseline was not stable because of additional signals coming from the matrix. Those signals interfered with the peaks of the studied compounds, which hindered the determination of basic NTs, as shown in Figure 2c (MeOH) and in Table 2. Indeed, pure MeOH as the SPE eluent of basic NTs demonstrated greater efficiency than MeOH with ammonium hydroxide (NH4 OH). Furthermore, the change in the SPE column type was studied towards the enhancement of the isolation of NTs from rat brain samples. The SPE SCX efficiency was based on the cation exchange mechanism, whereas the CSDAU columns utilized both a reverse (C8) phase and an ion exchange (benzenesulfonic acid) phase bonded to the same particle. The data obtained in our studies showed lower efficiency of CSDAU columns for the selective isolation of HVA and VMA. Additionally, the basic NTs, like A, NA, DA and amino acids (L-Tyr, L-Tryp), were not isolated efficiently enough using CSDAU columns to be detected by a diode array detector (DAD) coupled with the elaborated MEKC method. SPE CSDAU allowed 5-HT to be isolated, but the peak height was three times lower than that obtained when the clean-up step was omitted (Table 1). In the past five years, some authors applied DLLME for brain sample preparation for the determination of NTs but they used rat brain microdialysates, and the derivatization of the analytes was also necessary [17,18]. We applied the DLLME-based method for the isolation of NTs from brain samples which was previously tested and optimized for the isolation of NTs from urine samples [19]. However, for the brain samples, we carried out the procedure for the first time. The conditions that were optimized for urine samples worked relatively well for the isolation of DA, A, NA, L-Tyr, 5-HT, whereas, for L-Trp, HVA, and VMA, this protocol was not appropriate (Figure 3a, Table 1). The application of DLLME provided a simple and fast enrichment of the sample containing DA, A, NA, L-Tyr, 5-HT, since the MEKC-DAD method allowed those analytes to be determined without the need for derivatization or modification of the detector (there was no need for more sensitive laser-induced fluorescence (LIF) or mass spectrometry (MS) detectors). On the other hand, the sample matrix could have an influence on the determination of the analytes (Figure 3) because brain samples are much more complex than urine. Figure 3: Electropherogram of DLLME for the isolation of NTs from (**a**) the brain sample spiked with nine NTs (each analyte at the concentration of 10 \(\upmu\)g/g), (**b**) the blank brain control sample. Legend and CE conditions as in Figure 2.  
The last procedures that we have evaluated were based on the SPME technique with a polystyrene-divinylbenzene (PS-DVB) resin. [PERSON] et al. demonstrated the usefulness of the SPME procedure for the analysis of DA and 5-HT from 2.0 g of a rat brain sample [20]. Indeed, SPME is a powerful microextraction tool which cleans-up and concentrates up to 96 samples during one procedure. Thus, the time for the preparation of an individual sample decreases, and the entire experimental procedure consumes little time, which is relevant for easily degraded compounds such as NTs, their metabolites, and their precursor amino acids. Moreover, for this purpose, we applied, for the first time, IL, i.e., 1-ethyl-3-methylimidazolium tetrafluoroborate, to enhance the extraction efficiency of NTs from a 200 mg sample of rat brain.