## 3 Results and Discussion  
### LC Separation-LTQ/Orbitrap MS Determination  
Analytes of interest were satisfactorily separated using a methanol based gradient elution program in 9 min. It is noteworthy that injection of the analytes dissolved in methanol: water (1:1) allowed us to obtain better-defined chromatography peaks while the addition of 0.1% ammonia solution to all samples prior to analysis produced the best result concerning peak intensity and morphology (Figure S1). Identification of the target EDCs was based on their retention time in combination with the identified parent deprotonated [M-H]\({}^{-}\) ion with excellent mass accuracy (<2 ppm at 50 ng mL\({}^{-1}\), Table S2) at high resolution. The LC system proved indispensable for accurate mass measurements since discrimination of the two nonylphenol isomers is mandatory. In this case, structural isomers with the same accurate mass required a satisfactory separation, based on their different retention time rather than the exact mass (NP: 4.98 min and 4 NP: 5.17 min; both accurate masses at m/z = 219.1754). Moreover, a resolution of 60,000 proved to be the best compromise between peak shape and width, mass deviation and data points over the chromatographic peak for this application. In the case that concerns complex matrices such as food of animal origin, an increased resolution power is suggested due to the reduction in the chemical noise [26]. As already mentioned, many parameters have to be considered for tuning when a new LC/MS method is developed for the quantitative analysis of known compounds. In the first set of screening experiments, as many parameters of the acquisition method as possible were optimized in the tuning sections of the instrument by direct infusion of known concentrations of the target EDCs, in order to achieve the maximum sensitivity and selectivity. The common tuning parameters for an ESI interface, i.e., tube lens and ion optics voltages, which depend mainly on the molecular structure of the analytes, as well as the capillary voltage, were optimized automatically. In order to simplify the statistical design and minimize the number of experiments, variables that were already automatically tuned or those deemed to have a negligible impact on tuning optimization for the target analytes were held constant (e.g., sheath and auxiliary gas flow rates that depend on the column flow rate and parameters already set such as tube lens). However, it was observed that the value of some parameters, such as the spray voltage, considerably contributed to the signal of the monitoring ions, thus it was selected as a parameter requiring further optimization. The ionization by ESI source occurs at the solution state; therefore, the mobile phase composition may affect the sensitivity of the analyte. To achieve the highest sensitivity, different modifiers were assayed in mobile phase such as ammonia and ammonium formate at pH values from 6.3 to 10.8. In this study, the best option proved to be ammonia rather than ammonium formate addition. Therefore, the percentage content of ammonia in the mobile phase was also subjected to further optimization. Along with spray voltage and ammonia content in the mobile phase, injection volume and oven temperature values were found to be of critical concern for the analytes' chromatographic performance in terms of their peak signal-to-noise ratio (S/N). Then, a fractional factorial design 3\({}^{(4-1)}\) was employed in order to efficiently optimize the four most pronounced parameters affecting the LC-LTQ/Orbitrap MS analysis of BPA, OP and NP isomers and to determine whether a global maximum for S/N was within the experimental design region. In this design, 27 runs were randomly carried out (Table S3). The signal-to-noise ratio (S/N) of [M-H]pseudomolecular ions obtained from each run was used as the response, as a measure of sensitivity. The normality assumption was satisfied as the residual plot approximated along a straight line (Figure S2).