## 4 Conclusions  
We described a comprehensive HPLC-MS validated method for phytoestrogen analysis in human samples and foodstuffs, which represents an advance in the quantification of phytoestrogens. The method allowed the simultaneous quantification of 16 phytoestrogens, expanding the number of phytoestrogens analyzed by chromatographic run in a short time. The evaluated parameters demonstrated that linearity, selectivity, accuracy, precision and recovery are within the acceptable range, with high sensitivity in the lower ppb-level. The method was successfully applied to food, urine and serum samples. Therefore, this method is flexible and suitable for phytoestrogens quantification that could be used to analyze human samples in large-scale epidemiological studies investigating the association between phytoestrogen exposure and human health. The method described here could be used to determine phytoestrogens' content in regional food and create a phytoestrogens database in (North) Mexico.  
The following are available online at [[http://www.mdpi.com/2076-3417/10/22/8147/s1](http://www.mdpi.com/2076-3417/10/22/8147/s1)]([http://www.mdpi.com/2076-3417/10/22/8147/s1](http://www.mdpi.com/2076-3417/10/22/8147/s1)), Supplementary Table S1. Comparison of relevant methods for the quantification of phytoestrogens in food, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analyzed, limits of detection/limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter/intra- precision (%). Supplementary Table S2. Comparison of relevant methods for the quantification of phytoestrogens in serum, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analyzed, limits of detection/limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter/intra- precision (%). Supplementary Table S3. Comparison of relevant methods for the quantification of phytoestrogens in urine, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analyzed, limits of detection/limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter/intra- precision (%). Supplementary Table S4. Matrix Effect (%) at low and high quality control level in four different blank serums. Supplementary Table S5. Comparison of phytoestrogens content (ug/100 g) found in this study with relevant previously studies and public databases.  
Conceptualization, G.C.-J. and A.I.V.-Q.; methodology, G.C.-J., A.I.V.-Q., S.A.P.-D., K.M.C.-S., M.M.C.-S., M.M.C.-S., M.d.C.B.-A. and M.d.R.R.-B.; validation, G.C.-J., A.I.V.-Q., S.A.P.-D., K.M.C.-S., M.M.C.-S., M.M.C.-S., M.d.C.B.-A. and M.d.R.R.-B.; formal analysis, S.A.P.-D., K.M.C.-S., M.M.C.-S. and P.G.-C.; investigation, S.A.P.-D., K.M.C.-S., M.M.C.-S., M.d.S.-T., G.C.-J., A.I.V.-Q. and P.G.-C.; data curation, G.C.-J., S.A.P.-D., M.d.S.S.-T., M.C.B.-A., M.R.R.-B., M.L.G.-C. and A.I.V.-Q.; writings-original draft preparation, S.A.P.-D.; review and editing, S.A.P.-D., G.C.-J., M.d.R.R.-B., M.L.G.-C., and A.I.V.-Q.; supervision, G.C.-J. and A.I.V.-Q.; project administration, G.C.-J.; funding acquisition, G.C.-J. All authors have read and agreed to the published version of the manuscript.  
This research was supported by the National Council of Science and Technology (CONACyT) in Mexico and was conducted with register grant number #CB-2008-01-106028.  
The authors would like to thank the women for their time and contribution to the research and for providing samples for analysis. We thank Imperial College London Open Access Fund for covering the open access publication fees and Agilent Technologies for providing the mass spectrometer. The authors would like to thank [PERSON], [PERSON], [PERSON] and [PERSON] for their technical assistance.  
The authors declare no conflict of interest.