## 3 Results and Discussion
### Selectivity  
Only a few peaks were observed in the reagent blanks and in the matrix blanks but did not interfere with the analysis (Figure 2). These peaks were due to the enzyme _Helix pomatia_, which has been reported to contain levels of some phytoestrogens. The enzyme H1 contained naringenin (0.47 ng/mg), kaempfer (0.02 ng/mg), biochanin A (0.01 ng/mg), and luteolin (0.517 ng/mg) but not secoisolariciresinol and genistein (as previously reported for the HP-2 type enzyme [29; 30]). [PERSON] et al. 2006 [30] showed that _Helix pomatia_ could be purified to remove phytoestrogen contamination by SPE. However, this additional purification step could affect the enzymatic activity, remove or reduce components involved in the deconjugation process, and lower the levels of free phytoestrogens. In particular, the concentration of coumestrol has been found to be reduced (by 17%) when the  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**MSD ESI (-)** & **Analyte** & **Retention Time (min)** & **Ion (\(m\)/\(z\))** & **Fragmentor Voltage (V)** \\ \hline \multirow{4}{*}{1} & Resveratrol & 3.7 & 227.1 & 180 \\  & Glycitein & 6.9 & 283.0 & 160 \\  & Naringenin & 8.0 & 271.0 & 180 \\  & Kaempfer & 9.5 & 285.0 & 200 \\  & Biochanin A & 12.8 & 283.1 & 180 \\ \hline \multirow{4}{*}{2} & Secoisolariciresinol & 4.5 & 361.1 & 100 \\  & Luteolin & 7.2 & 285.1 & 180 \\  & Equiol & 8.3 & 241.1 & 160 \\  & Coumetrol & 9.7 & 267.1 & 200 \\ \hline \multirow{4}{*}{3} & PhP \(\beta\)-glu \({}^{\ddagger}\) & 4.7 & 493.1 & 100 \\  & Daidzein & 5.9 & 253.1 & 180 \\  & Enterodiol \({}^{\lx@sectionsign}\) & 7.0 & 253.1 & 180 \\  & Mataitesinol & 7.8 & 357.0 & 130 \\  & Genistein & 8.6 & 269.0 & 180 \\  & PhP \({}^{\ddagger}\) & 9.2 & 317.1 & 100 \\  & IS \({}^{\star}\) & 10.0 & 197.1 & 100 \\ \hline \multirow{4}{*}{4} & 4 MU \({}^{\ddagger}\) & 4.0 & 175.1 & 100 \\  & Quercetin \({}^{\lx@sectionsign}\) & 6.6 & 151.1 & 200 \\ \cline{1-1}  & Enterolactone & 9.0 & 297.1 & 180 \\ \cline{1-1}  & Formononetin & 11.3 & 267.1 & 200 \\ \cline{1-1}  & 4 MUS \({}^{\ddagger}\) & 13.8 & 293.2 & 100 \\ \hline \hline \end{tabular}  
* Internal standard (IS); 4-hydroxybenzophenone and \({}^{\ddagger}\) deconjugation standards; 4-methylumbelliferonyl sulfate (4 MUS), 4-methylumbelliferone (4 MU), phenolphthalein \(\beta\)-D-glucuron (PhP-\(\beta\)-glu) and phenolphthalein (PhP). The [M-H]\({}^{\star}\) were chosen for quantification except for \({}^{\lx@sectionsign}\) quercetin and enterodiol. ESI, electrospray ionization; MSD, mass-selective detector.  
\end{table}
Table 1: Target ion (\(m\)/\(z\)), retention time (min) and fragmentor voltage (V) for phytoestrogens quantification in the four MSD signals.  
purified enzyme was used compared to the crude enzymatic solution. In serum, changing the enzyme concentration [29] could reduce interferences, however, complete hydrolysis should be ensured and awaits evaluation. The low content of phytoestrogens in the enzyme were corrected for the presence of phytoestrogens in each sample analyzed using the reagent blank [31]. Coleuition of seciosolaricinesinol with matrix components at the beginning of the gradient can interfere with the analysis in serum [32]. However, solid-phase extraction and liquid-liquid extraction conditions reduced endogenous substances in serum, urine and food. Additionally, changes in the chromatographic method and using single ion monitoring allowed the reduction of interferences and maximized selectivity.