## 2 Materials and Methods
### Calibration and Preparation Procedures  
Thirteen separate starting solutions were prepared by dissolving 10 mg of each metabolite compounds, which were 3-PBA, _cis/trans_-DCCA, _cis_-DBCA, _trans_-CDCAPBA, DMP, DMTP, DMDTP, DEP, DETP, DEDTP, and 7-phenol, with acetonitrile in thirteen separate 10 mL glass volumetric flasks (1 g/L). The multicomponent stock solution was prepared by adding 100 \(\upmu\)L of each starting solution into a 10 mL glass volumetric flask and filling with acetonitrile to the mark (10 mg/L). Two working solutionsof different concentrations were prepared by this multicomponent stock solution with acetonitrile, and other solutions used in this study were prepared following the methods shown as follows:  
**Working solution I**  
One milliliter of the multicomponent stock solution was diluted in a 20 mL glass volumetric flask (500 \(\mathrm{\SIUnitSymbolMicro g}\)/L). **Working solution II**  
One hundred microliter (100 \(\mathrm{\SIUnitSymbolMicro L}\)) of the multicomponent stock solution was diluted in a 20 mL glass volumetric flask (50 \(\mathrm{\SIUnitSymbolMicro g}\)/L). **Blank I**  
We collected urine from 10 volunteers who were not exposed to any insecticides in Hualien city (eastern Taiwan, ROC), pooled them together followed by dilution with distilled deionized water (1:1 (v/v)), and filtered the diluted pooled urine through a 0.2 \(\mathrm{\SIUnitSymbolMicro m}\) syringe filter. The filtered sample was extracted and screened for the target metabolites using the previously described method to confirm the blankness. The Blank I sample was dispensed into several 100 mL glass volumetric flasks and stored at \(-20\)\({}^{\circ}\)C.  
**Blank II**  
We also collected urine from 15 other non-exposed volunteers residing in Hsinchu city, Taichung city, and Changhua city (western Taiwan, ROC), pooled them together followed by dilution with distilled deionized water (1:1 (v/v)), and filtered the diluted pooled urine through a 0.2 \(\mathrm{\SIUnitSymbolMicro m}\) syringe filter. The filtered sample was confirmed to be blank, dispensed into different 100 mL glass volumetric flasks, and stored at \(-20\)\({}^{\circ}\)C.  
**Calibration standards (concentration range: 0.5 to 150 \(\mathrm{\SIUnitSymbolMicro g}\)/L)**  
The standards were prepared from two working solutions. Nine calibration concentrations were prepared in acetonitrile, between 0.5 and 150 \(\mathrm{\SIUnitSymbolMicro g}\)/L (i.e., 0.5, 1, 2, 5, 10, 20, 50, 100, and 150 \(\mathrm{\SIUnitSymbolMicro g}\)/L). Aliquots of 2 mL Blank I were processed with acid hydrolysis and LLE, and the residues after concentration were added separately to standards at different concentrations. The prepared standard solutions were processed with derivatization prior to GCMS analysis. Samples for various tests were prepared in low (L), medium (M), and high (H) concentrations. The detailed preparation methods are described as follows. **Group A**  
Multicomponent testing samples of two concentrations were prepared by spiking working solution II with Blank I (Group-AL: 0.5 \(\mathrm{\SIUnitSymbolMicro g}\)/L, \(n\) = 10; Group-AH: 5 \(\mathrm{\SIUnitSymbolMicro g}\)/L, \(n\) = 10). **Group B**  
Group B is designed for testing the extraction effectiveness as compared to Group A, serving as control. The concentrations were prepared to be as same as in Group A (0.5 and 5 \(\mathrm{\SIUnitSymbolMicro g}\)/L), but with a different preparation method that was the same as the preparation of standards. The urinary residues of Blank I after acid hydrolysis, LLE, and concentration were supplemented with 0.5 and 5 \(\mathrm{\SIUnitSymbolMicro g}\)/L in acetonitrile to become Group BL (0.5 \(\mathrm{\SIUnitSymbolMicro g}\)/L, \(n\) = 10) and Group BH (5 \(\mathrm{\SIUnitSymbolMicro g}\)/L, \(n\) = 10), respectively. The Group B solutions were then processed with derivatization, followed by GCMS analysis. **Group C**  
Group C was used for testing the within-day precision.