## 3 Results and Discussion  
### Multiclass Pesticide Extraction by Immersion SPME  
The pesticide selection chosen for the method development included several pesticide classes: Chloroacetamides (metazachlor and metolachor), organochlorines (aldrin, lindane and pentachloroanisole), organophosphates (malathion and chlorpyrifos), triazoles (tetraconazole, penconazole and tebuconazole), as well as one pyrethroid, thiocarbamate, and unclassified pesticides, namely, felthurin, prosulfocarb, and buprofezin. The aim was to gauge the applicability of the developed method towards different chemical properties. The original multiclass extraction method which was adapted used 6% methanol in water (\(v/v\)) as a solvent for immersion SPME, followed by desorption of the fiber in methanol and subsequent injection [10]. In the present work, ethanol was used instead, mainly because it has a much smaller toxicity (negligible for humans considering normal laboratory exposure levels), can be produced more easily from renewable feedstocks, and is slightly less polar, which is relevant for the extraction of organochlorines, especially. Furthermore, most salts have lower solubility in ethanol than methanol [16], which is important if in the future this method is applied to soils with greater salt content, where the use of methanol for redissolution of the derivatized glyphosate extract might introduce salts into the chromatographic system. From the two fiber chemistries available, PDMS/DVB and C18, the latter was chosen because the method introduces the analysis of organochlorines, not contemplated in the previous work [10]. Initially, a three factor Box-Behnken design was used as an attempt to optimize the immersion SPME extraction It modeled the percentage of ethanol in the extraction solvent (2%, 6%, and 10%, \(v/v\)), the extraction time (60, 90, and 120 min), and the retroextraction volume (100, 120, and 140 \(\upmu\)L). However, the results showed that for most analytes, the extraction time was by far the most relevant factor, as it seemed that even 120 min were insufficient to attain equilibrium. Thus, this factor was far outside the values at which a response surface might show a maximum. As a result, the retroextraction volume was fixed to 120 \(\upmu\)L (as a compromise between obtaining a good concentration factor and enough working volume), and a two-factor, three-level full-factorial experimental design was constructed (nine experiments), where the percentages of ethanol in the extraction solvent  
Figure 2: Schematic representation of the full method. SPME tip retroextraction happens at the same time as the extraction with ammonia solution. were 2, 6, and 10%, and the extraction times were 120, 150, and 180 min. Each experiment was repeated three times, for a total of 27 runs. The results (in terms of maximum average signal) are presented in Table 1.  
From the results, 180 min was selected as the extraction time. A long extraction time is not ideal, because the full method including glyphosate extraction and derivatization takes several hours, and at worst can be longer than a single 8 h shift, which would compromise its applicability in routine laboratories without the implementation of process automation. The results for just 180 min of extraction can be seen in Figure 3.  
From these results, 2% ethanol in water was chosen as the extraction solvent. Although the more nonpolar compounds were favored by the greater percentage of ethanol, most had the highest signals at 2%. Furthermore, a smaller amount of ethanol would be favorable for the subsequent glyphosate extraction, as the organic modifier would not help in the dissolution of glyphosate. Interestingly, no compound had a maximum at 6% ethanol, which seems to suggest that the ideal percentage for each pesticide lies either closer to 2% or 10%, or ou