MS/MS Acquisition Parameters Used for the Analysis of Isoflavones and Iss
Fig. S1.Peak area of isoflavones and ISs when solubilized in different methanol/water mixtures. Analytical concentration was set at 50 ngmL-1 (n=3, m=3). Values marked by the same letter are not significantly differentwith the Tukey’s HSD test (p-value > 0.05). The arrow represents the selected solvent.
Fig.S2.Residual of isoflavones and ISs when re-solubilized solutions with different methanol/water mixtures are used. The starting concentration was set at 500 ngmL-1 for all compounds (a Residual [%] = [Arearesidual/AreaMeOH 60%]*100) (n=3, m=2).Values marked by the same letter are not significantly differentwith the Tukey’s HSD test (p-value > 0.05). The arrow represents the selected solvent.
Fig. S3. Sum of isoflavone concentrations found in a representative sample of cows feed after three consecutive extractions under selected UAE conditions.
Fig.S4. Effect of each enzyme on the isoflavone hydrolysis process from the forage mix (n=3). The extraction conditions were: temperature 80°C; duration 10 min; MeOH concentration 55%; and solid-liquid ratio 0.5g25mL-1. The hydrolysis conditions were: temperature 37°C; hydrolysis time 18 h; and pH 5. Values marked by the same letter are not significantly differentwith the Tukey’s HSD test (p-value > 0.05).
Fig.S5. Effect of enzyme mix concentration on the isoflavone hydrolysis process from the forage mix (n=3).The extraction conditions were: temperature 80°C; duration 10 min; MeOH concentration 55%; and solid-liquid ratio 0.5g25mL-1. The hydrolysis conditions were: temperature 37°C; hydrolysis time 18 h; and pH 5. ‘Mix Enz. No Dil’contained ≥ 12, ≥ 24, ≥ 10200 unitsmL-1 of cellulase, β-glucosidase and β-glucuronidase, respectively, and was then diluted by a factor of 2 ‘Mix Enz. Dil 2’, 4 ‘Mix Enz. Dil 4’ and 8 ‘Mix Enz.Dil 8’.Values marked by the same letter are not significantly differentwith the Tukey’s HSD test (p-value > 0.05).
TableS1
MS/MS acquisition parameters used for the analysis of isoflavones and ISs.
Compound / Ionization / Cone voltage (V) / Precursor ion (m/z) / Collision energy (V) / Product ions (m/z) / Dwell time (s)DA / ESI+ / 35 / 255 / 25 / 199 Q / 0.03
25 / 137 q / 0.03
GE / ESI+ / 50 / 271.1 / 25 / 153 Q / 0.05
25 / 215 q / 0.05
FO / ESI+ / 40 / 269.2 / 35 / 197 Q / 0.35
25 / 237.1 q / 0.35
BA / ESI+ / 40 / 285.2 / 35 / 213 Q / 0.05
30 / 269.1 q / 0.05
Flavone (IS) / ESI+ / 45 / 223.1 / 25 / 121 Q / 0.07
25 / 178 q / 0.07
Daidzein-d4 (IS) / ESI+ / 45 / 259.2 / 25 / 203 Q / 0.05
25 / 137 q / 0.05
Q product ion for quantification, q product ion for identification.
TableS2
Intra-/inter-assay precision (RSD%) and accuracy (bias%) for each isoflavone.
Injected concentration / Instrumentation parameter / Day / DA / GE / FO / BA15 ngmL-1 / Precision a (RSD%) / Intra-assay c / 1 / 4.0 / 3.4 / 2.3 / 4.7
2 / 3.0 / 2.3 / 2.0 / 8.2
3 / 3.4 / 1.8 / 3.4 / 4.6
Inter-assay d / 4.5 / 3.7 / 5.5 / 7.6
Accuracy b (bias%) / Intra-assay c / 1 / 5.0 ± 3.79 / 4.1 ± 3.29 / 4.4 ± 2.18 / 3.6 ± 4.52
2 / 2.7 ± 1.88 / 3.1 ± 1.45 / 7.6 ± 2.11 / 8.8 ± 6.22
3 / 2.2 ± 2.68 / 1.3 ± 1.15 / 4.1 ± 0.88 / 7.7 ± 4.90
Inter-assay d / 3.3 ± 2.96 / 2.8 ± 2.35 / 5.4 ± 2.35 / 6.7 ± 5.41
30 ngmL-1 / Precision a (RSD%) / Intra-assay / 1 / 1.9 / 2.2 / 2.8 / 6.0
2 / 3.7 / 2.2 / 4.0 / 4.2
3 / 1.9 / 1.5 / 1.3 / 4.6
Inter-assay / 3.6 / 3.1 / 4.1 / 7.4
Accuracy b (bias%) / Intra-assay c / 1 / 3.0 ± 1.86 / 1.8 ± 1.29 / 2.4 ± 2.35 / 5.4 ± 2.84
2 / 3.9 ± 2.28 / 6.9 ± 2.37 / 9.5 ± 4.35 / 11.4 ± 4.71
3 / 1.9 ± 1.57 / 2.8 ± 1.59 / 6.5 ± 1.39 / 4.5 ± 2.61
Inter-assay d / 2.9 ± 1.98 / 3.8 ± 2.27 / 6.1 ± 3.20 / 7.1 ± 4.24
50 ngmL-1 / Precision a (RSD%) / Intra-assay / 1 / 2.2 / 3.7 / 4.4 / 5.9
2 / 2.3 / 2.2 / 3.0 / 4.6
3 / 1.4 / 3.9 / 1.6 / 5.9
Inter-assay d / 3.1 / 3.6 / 4.4 / 7.0
Accuracy b (bias%) / Intra-assay c / 1 / 4.1 ± 2.13 / 2.6 ± 2.57 / 3.4 ± 2.15 / 5.6 ± 4.48
2 / 1.8 ± 1.42 / 4.6 ± 2.32 / 8.1 ± 3.21 / 6.6 ± 4.96
3 / 1.5 ± 1.10 / 3.5 ± 1.16 / 5.9 ± 1.74 / 5.5 ± 3.27
Inter-assay d / 2.5 ± 1.91 / 3.6 ± 2.14 / 5.8 ± 3.01 / 5.9 ± 4.01
80 ngmL-1 / Precision a (RSD%) / Intra-assay / 1 / 3.2 / 2.6 / 1.8 / 2.9
2 / 2.3 / 1.5 / 2.0 / 5.0
3 / 3.3 / 2.9 / 3.2 / 4.3
Inter-assay d / 2.8 / 2.4 / 2.5 / 4.9
Accuracy b (bias%) / Intra-assay c / 1 / 2.6 ± 1.84 / 2.1 ± 1.37 / 1.3 ± 0.96 / 3.3 ± 0.98
2 / 2.0 ± 0.78 / 1.9 ± 1.53 / 2.6 ± 1.52 / 4.6 ± 2.62
3 / 2.6 ± 1.68 / 2.6 ± 0.47 / 3.8 ± 1.37 / 5.4 ± 3.88
Inter-assay d / 2.4 ± 1.43 / 2.2 ± 1.16 / 2.6 ± 1.58 / 4.4 ± 2.71
a, b, c n=5, d n=15.