## 3 Results and Discussion
### Linearity and Sensitivity  
Phytoestrogen levels present a wide variation between food and biofluids of individuals. Phytoestrogens quantification must be ensured below the part per billion, mainly when the method will be applied to individuals with a low intake of phytoestrogens. When human samples and food are meant to be analyzed at the same time, a dynamic range is the best way to ensure the flexibility of the analytical method. Calibration curves showed a linear response for all the 16 phytoestrogens. Figure 2: Representative MS chromatogram of the four MSD signals of a reagent blank (**A**) and a human blank serum (**B**) with internal standard. Linearity of the LC-MS system response was tested at a range of 0.16 to 16,000 ng/mL for phytoestrogens in solution, which showed correlation coefficients of \(\geq\)0.98. The analytical method demonstrated a linear response in the range of 0.02 to 2000 ng/mL for serum, and 0.008 to 1200 ng/mL for food and urine, with correlation coefficients of \(>\)0.97 in the samples, except for enterodiol (r\({}^{2}\) = 0.95) in food, biochanin A (r\({}^{2}\) = 0.95) and coumestrol (r\({}^{2}\) = 0.96) in urine (Table 2). Limits of detection (LOD), limits of quantification (LOQ), method linearity (r\({}^{2}\)) and recoveries  
Limits of detection and quantification are listed in Table 2. A comparative table with relevant methods is available as supplementary material for food, serum and urine (Supplementary Tables S1-S3). In food, the LODs and LOQs were lower for enterolactone (0.002 ng/mL and 0.008 ng/mL, respectively) and higher for equal (1.061 ng/mL and 3.541 ng/mL, respectively). The LODs were lower than the method reported by [PERSON] et al. (2004) with 2.3 ng/mL (0.2-10 ug/100 g, in bread) [33] and [PERSON] et al. (2007) with 1 ng/mL (1.5 ug/100 g) [28] but comparable with [PERSON] et al. (2015) with 0.03-0.3 ng/mL (legumes, LOQ of 0.01 ng/mL for biochanin A) [34]. The LODs correspond to 0.001 to 0.52 ug/100 g of food (final concentration depends on humidity), suitable for the analysis of food with low levels of phytoestrogens, and it is a useful method to develop a food database where phytoestrogens intake is characterized by a wide variety of food with low phytoestrogen levels. In serum, the LODs ranged from 0.001 to 0.53 ng/mL and LOQs from 0.003 to 1.8 ng/mL. These limits were lower than previous methods using different detectors, with LODs from 0.01 to 132.6 ng/mL [17; 14; 27; 32; 35; 36; 37], and comparable with commonetin (0.02 ng/mL), enterodiol (\(<\)0.01-0.06 ng/mL,) and glyciteina (0.01 ng/mL) [38; 39]. Therefore, the method presents an advantage for the analysis of serum in the ppb-levels. In urine, the LODs for the method ranged from 0.001 to 0.075 ng/mL, and LOQs from 0.003 to 0.25 ng/mL. Previous methods reported LODs between 0.05 and 65.1 ng/mL [14; 32; 40; 41], with similar limits for enterodiol (0.04 ng/mL), equal (0.06 ng/mL) and glycitein (0.0007 ng/mL) [40; 42]. The results indicate that the liquid-liquid extraction and HPLC-DAD-ESI-MS presented a high sensibility for the analysis of phytoestrogens in human urine. The sensitivity of the method could be explained by the addition of formic acid from the mobile phase in the injection solvent that improved the ionization efficiency, as well as the concentration of the sample during extraction and the injection volume (60% to 10-fold larger).