## 3 Results and Discussion  
### Chromatography and Mass Spectrometry Optimization  
In order to optimize the separation and detection of the 16 phytoestrogens of interest, variables such as column length, column temperature, mobile phase, gradient and flow were evaluated. The final parameters presented in Section 2.6 Liquid chromatography/mass spectrometry (column description, column temperature and mobile phase gradient flow) allowed the resolution of the phytoestrogens by HPLC-MS. Phytoestrogens identification was achieved by retention time, UV and mass spectra (Figure 1). From the absorption spectra, flavonoids presented their two maximum wavelengths of absorbance in the range of 305 to 375 nm (band I) and 200 to 280 nm (band II) [24]. The wavelengths of maximum absorbance for resveratrol were found at 215 and 310 nm, which are characteristic of its alkene and m-dihydroxibencenalkene [25]. For lignans, the maximum wavelengths were at200, 220 and 280 nm, which represent the furan, pencil and phenilaril structures, characteristic of these compounds [26].  
Figure 1: Ultraviolet (UV) spectra, high-performance liquid chromatography (HPLC) and normalised-mass spectrometry (MS) chromatograms of the four mass-selective detector (MSD) signals of pure phytoestrogens standards (160 ng/mL) and internal standard (IS) in the injection solvent (methanol and initial mobile phase; \(v/v\), 40/60). Bio A, biochanin A; Cou, coumestrol; Dai, daidzein; Ediol, enterodiol; Elac, enterolactone, Eq, equal; For, formononetin; Gen, genistein; Gly, glycitein; Kae, kaempfer; Lut, luteolin; Mat, matizesinol; Nar, naringenin, Que, quercetin; Sec, secoisolariciresinol; and Res, resveratrol. The overlapping peaks are due to overlay of the m/z values used in multiple MSD signals and the different ion types of the phytoestrogens. Distortion of peaks is due to the solvent strength of the injection solvent. The ion type, \(m/z\) and MSD used for quantification are describe in Table 1.  
Different mass parameters, such as fragmentation voltage were tested to increase predominant ions, to reduce noise and to increase the sensitivity of the method. Spray chamber parameters were set according to the column flow with a gas temperature of 350 \({}^{\circ}\)C, nebulizer pressure of 60 psi and drying gas flow of 11.0 L/min. Phytoestrogens were evaluated in positive and negative ion mode using ESL Negative mode was selected due to high sensitivity, minor noise, and lower fragmentation. A scan analysis was undertaken to all the phytoestrogen standards to identify and confirm each phytoestrogen chemical structure. The predominant ions were [M-H]\({}^{\star}\), while fragment ion formation presented a low relative abundance (by Retro-Diels-Alder, neutral and radical fragmentation). Each ionization pattern ion was characteristic for each phytoestrogen which has been observed previously, using different interfaces and masses [17; 24; 27; 28]. The [M-H]\({}^{\star}\) were chosen for quantification in SIM mode (Table 1). Since quercetin and enterodi present the similar molar masses (302 g/mol) and a close elution, the ions with \(m\)/\(z\) of 151.1 and 253.1 were selected for their quantification, respectively. Phytoestrogens were analyzed using the four MSD signals.