## 2 Results and Discussion  
### Optimization of the Solid-Phase Extraction Procedure  
Two solid-phase extraction (SPE) sorbents (Oasis HLB and Oasis MAX) were selected from previously published methods for other pharmaceuticals not included in these studies for evaluation [42; 43]. In addition, the C18 SPE sorbent was selected as an alternative sorbent for the evaluation, as SPE could be completed under neutral pH conditions, which reduced or eliminated the need for additives for pH adjustment of water or elution solvents. The addition of additives in solvents used in SPE such as acids (formic acid) or bases (ammonia) to alter pH was found to alter mass spectrometric sensitivity if not remove completely during the pre-concentration step. The pK\({}_{\text{a}}\) of the target analytes is high (pK\({}_{\text{a}}\) for bisphenol A (BPA), estrogens and surfactants, prednisone, and prednisolone were 9.8, 10.2-10.5, 12.4, and 12.5, respectively) such that the target analytes would not be ionized [38; 44]. BPA, 4-octylpphenol (4 OP), and 4-nonylphenol (4 NP) are also the most prone to degradation of the targeted analytes, and as such, these analytes were used for selection of solvents and best sorbent for SPE. After loading of the sample on the SPE sorbents such as C18 or Oasis HLB, a wash step with a volume of 2.0 mL of H\({}_{2}\)O was chosen as higher volumes resulted in earlier elution of the BPA into the F0 wash fraction. As can be seen from Figure 1, the first 1 mL of the elution of acetonitrile showed no detection of target analytes, so \(\sim\)0.8 mL of acetonitrile was also eluted into the F0 wash fraction to aid in the removal of more polar matrix components. Figure 1 shows that the low recoveries (\(<\)80%) for BPA, 4 NP, and 4 OP were obtained when the elution from C18 was completed with 20 mL of methanol or 50/50 \(v\)/% methanol/acetonitrile. Furthermore, BPA, 4 OP, and 4 NP observed stability issues when the sample preparation took over one week even if the extracts were stored in the refrigerator overnight. To pre-concentrate the target analytes, \(>\)30 h was required for the drying of the extracts to 0.8 mL at 0.5 mL/h in the SPE apparatus. Faster drying rates led to the loss of target analytes. The degradation of BPA and 4 NP analytes in methanol has been previously reported to occur within seven days [29]. The precipitation of the injected samples occurred in the LC-MS/MS system when methanol was used as the elution solvent for SPE and dilution of samples before LC-MS/MS analysis. This precipitation was evident on the pre-column over time and deterioration in peak shapes was observed. Retention time shifts and reduced MS sensitivity were also observed if samples were stored for more than one week from the time of elution from the SPE cartridge. Consequently, the time to completion of analysis from the elution step was reduced to five days or less, and under these conditions no degradation of the sample was evident. An improved stability of target analytes through the sample preparation procedures was also observed when acetonitrile was used as the elution solvent. BPA eluting within the first 5 mL of acetonitrile, while 4 OP and 4 NP required 10 mL and 15 mL of acetonitrile to adequately recover the target analytes from the C18 SPE sorbent, respectively (see Figure 1C). Consequently, an elution volume of 15 mL of acetonitrile was chosen to obtain recoveries \(>\)80%. Recoveries of a larger range of target analytes on the three SPE sorbents (Oasis HLB, Oasis MAX, and C18) were then evaluated. A volume of the F1 fraction (1 st eluted fraction where target analytes are detected) was selected to be 15 mL of acetonitrile as this volume was considered the maximum volume practical, as subsequent drying steps required \(\sim\)30 h (0.5 mL/h drying rate). As noted previously, longer sample preparation times could lead to problems with the stability of the target analytes.