## 3 Results and Discussion
### Recovery  
Recoveries (REs) of phytoestrogens (Table 2) in food were in the range of 66 to 113%, except for kaempfer, luteolin and resveratrol (15 to 44%). Previous methods (Supplementary Table S1) have reported recoveries between 70 to 110% and others as low as 30% [12; 28; 33; 34; 43]. Recovery in food could vary between samples due to the intrinsic composition of the food matrix. Potential interfering substances, such as proteins, lipids, carbohydrates and fiber could result in decreased hydrolysis and RE% of phytoestrogens [28]. Also, the matrix effect is challenging to establish in food and could differ significantly due to the variety of matrices and the lack of quality control for foodstuff samples (as opposed to single matrix sample like serum) [28].  
REs in serum were from 63 to 104% except for coumestrol (30%). Compared to previous methods (Supplementary Table S2, an improvement in recovery was observed for equal (88%) [37], coumestrol (20%) [32], and secoisolariciresinol (86-96%) [32; 44], while the rest presented comparable recoveries [14; 27; 36]. Despite the advances in the method extraction, RE of coumestrol was below 60%. Coumestrol with a relatively non-polar characteristic may yield low recoveries, which enhances high retention on the sorbent, low solubility in the elution solvent and low RE% [32]. Although a higher recovery can be achieved by liquid-liquid extraction (77%) [36], the present method represents an advance in coumestrol analysis given that other methods excluded its analysis in serum [14; 36]. A minor matrix effect was observed in pooled blank serum samples, with ion suppression for biochanin A, coumestrol and formonentin and enhanced ionization for matairesinol and secoisolariciresinol (Supplementary Table S4).  
For urine, liquid-liquid extraction presented REs within the expected levels. The REs of the spiked samples were between 76 to 111%, except for resveratrol (50%) and secoisolariciresinol (59%). Acceptable recoveries of phytoestrogens in urine have been observed in previous methods (Supplementary Table S3) but these have focused mainly on isoflavones (65-110%) [14; 32; 37; 40; 41; 44]. Matrix effect was not considered as urine could be described as a relatively \"clean\" sample (as opposed to serum) [29].  
Acceptable RE% for all compounds is a difficult task when a wide variety of phytoestrogens are being analyzed per chromatographic run. The pretreatment of the sample and the washing solution used in the solid-phase extraction resulted in an overall improvement in recovery of kaempfer, biochanin A and luteolin (increasing up to 30-50%, data not shown). The recoveries of equal and glycitein were lower when using 100% methanol as the injection solvent (data not shown). In the same way, REs decreased for some of the phytoestrogens when changing the solvent to the mobile phase. The injection solvent was varied to increases the solubility of the phytoestrogens while maintaining the strength of the mobile phase from methanol to methanol/initial mobile phase (\(\gamma\)/\(v\), 40/60). Increasing the methanol percentage in the injection solvent and maintaining a percentage of mobile phase allowed acceptable recoveries (60-110%). [PERSON] et al. (2010) and [PERSON] et al. (2010) presented high RE (65-105%) using methanol/mobile phase as the injection solvent [35; 36]. Although aqueous injection solvent could be used [14; 17], it should be mentioned that the present study analyzes a wide variety of phytoestrogens, with different polarities.