A comprehensive HPLC‐DAD‐ESI‐MS validated method for the quantification of sixteen phytoestrogens in food, serum and urine Supporting Information

Susana Alejandra Palma‐Duran1,2, Graciela Caire‐Juvera3, Melissa María Campa‐Siqueiros3, Karina María Chávez‐Suárez3, María del Refugio Robles‐ Burgueño2, María Lourdes Gutiérrez‐Coronado2, María del Carmen Bermúdez‐Almada2, María del Socorro Saucedo‐Tamayo3, Patricia Grajeda‐Cota2 and Ana Isabel Valenzuela‐Quintanar2*

1 Biomolecular Medicine, Division of Systems Medicine, Department of Metabolism, Digestion, and Reproduction, Imperial College London, London, United Kingdom. Department of Food Science, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora, México; 83004. Tel: +52 (662) 2892400 ext 344. 2 Department of Food Science, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora, México; 83004. Tel: +52 (662) 2892400 ext 344. 3 Department of Nutrition, Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora, México; 83004. Tel: + 52 (662) 2892400 ext 395. * Correspondence: [email protected], Tel: + 52 (662) 2892400 ext 344.

Supplement Table 1. Comparison of relevant methods for the quantification of phytoestrogens in food, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analysed, limits of detection / limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter / intra‐ precision (%) Author, Matrix System Extraction Phytoestrogens LOD/LOQ RE% Accuracy Inter/Intra‐assay year Palma‐ Boiled rice and LC‐DAD‐ESI‐MS SPE (200 µL, Resveratrol 0.025/0.084 44 4 8.36/7.11 Duran et potato (100 mg F‐D) (50 µL) MeOH/mobile Biochanin A 0.006/0.022 74 7 3.13/1.97 al. 2020 phase, 40/60, v/v) Secoisolariciresinol 0.023/0.078 66 6 1.5/2.9 Luteolin 0.009/0.033 20 5 5.53/11.56 Coumestrol 0.003/0.117 88 10 9.83/5.99 Formononetin 0.007/0.023 78 1 7.24/4.08 Daidzein 0.011/0.037 67 12 2.86/6.93 0.043/0.144 72 18 3.12/5.96 Equol 1.061/3.541 94 6 8.47/6.34 Kaempferol 0.008/0.029 15 9 11.16/13.16 Glycitein 0.212/0.709 92 9 10.91/6.94 0.012/0.041 87 13 11.21/5.61 Naringenin 0.014/0.041 70 10 4.06/9.19 Quercetin 0.011/0.036 80 8 10.08/11.21 0.002/0.008 85 8 2.72/5.7 Genistein 0.006/0.021 113 8 0.44/4.44

Kuhnle et Buffer, celery, red LC‐MS/MS SPE (200 µL, 40% Buffer 1 89‐107 al. 2007 cabbage & orange MeOH) biochanin A 1/3 [1] (100 mg F‐D) dadizein 6/13 formononetin 1/1

genistein 4/3

glycitein 4/12 matairesinol 3/3

secoisolariciresinol 4/4

coumestrol 5/9 Milder et Bread (1g, F‐D) LC‐APCI‐MS/MS L‐L (1 mL, 30% Matairesinol 2.4 55‐102 al. 2004 Tea (1mL) (50 µL) MeOH) Secoisolariciresinol 2.3 82‐77 21/33 [2] Vila‐Donat Lentil & other pulses LC‐ESI‐MS/MS SPE (4 mL, MeOH) Daidzein 0.33/1 88‐114 4‐12/0.4‐1

et al. 2015 (1g) (5 µL) Genistein 0.33/1 91‐106 [3] Bioachanin A 0.03/0.01 69‐105 Shim et al. Black, red bean, soy LC‐PDA (3 µL) DMSO (12 mL, 70% Daidzein 0.5/2 mg/100g 94‐104 6‐12/5‐8

2015 [4] bean & soybean MeOH) Glycitein 1/3 mg/100g 91‐94 4‐9/3‐4 paste (1g) Genistein 0.6/2 mg/100g 92‐106 3‐8/3‐5

Angeloni Coffee (2 mL) LC‐ESI‐MS/MS Enzymatic hydrolysis Secoisolariciresinol 2‐5 70‐98 6‐12/2‐7 et al. 2018 (2 µL) (2.5 mL) Matairesinol 3‐10 53‐93 [5] Nørskov et Bread (20‐25 mg) LC‐ESI‐MS/MS SPE (1.6 mL, 25% Secoisolariciresinol 0.246/0.76 85‐99 1‐14 /0.2‐5 al. 2016 (5 µL) ACN) Enterodiol [6] Matairesinol 0.17/0.366 79‐109 Enterolactone ACN; acetonitrile, APCI; atmospheric pressure chemical ionization, DAD; diode‐array detector, DMSO; dimethyl sulfoxide, ESI; electrospray ionization, F‐ D; freeze‐dried, LC; liquid chromatography, L‐L; liquid‐liquid extraction, MeOH; methanol, MS(/MS); mass spectrometry (tandem), PDA; photo diode array detector, SPE; solid‐phase extraction. This table is not exhaustive and relevant information has been included in order to compare with the validated HPLC‐DAD‐ESI‐MS method with previously developed methods.

Supplement Table 2. Comparison of relevant methods for the quantification of phytoestrogens in serum, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analysed, limits of detection / limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter / intra‐ precision (%) Author, Matrix System Extraction Phytoestrogens LOD/LOQ RE% Accuracy Inter/Intra‐ year assay Palma‐ serum, human LC‐DAD‐ESI‐ SPE (200 µL, Resveratrol 0.018/0.06 92 5 9.43/6.67 Duran et al. (1 mL) MS (50 µL) MeOH/mobile Biochanin A 0.002/0.008 77 11 8.5/3.87 2020 phase, 40/60, v/v Secoisolariciresinol 0.008/0.027 99 4 3.73/2.05 Luteolin 0.028/0.096 82 6 10.55/5.92 Coumestrol 0.001/0.003 30 13 14.46/2.33 Formononetin 0.003/0.011 73 12 11.03/3.2 Daidzein 0.042/0.141 102 0.3 11.79/11.31 Enterodiol 0.039/0.133 98 9 6.09/3.73 Equol 0.068/0.227 104 1 4.11/0.88 Kaempferol 0.003/0.012 76 10 14.25/3.9 Glycitein 0.075/0.251 90 12 14.84/2.72 Matairesinol 0.059/0.198 93 1 4.14/1.27 Naringenin 0.005/0.019 84 2 12.21/4.75 Quercetin 0.061/0.206 83 15 14.87/3.18 Enterolactone 0.001/0.003 80 0.5 6.08/5.61 Genistein 0.001/0.006 101 2 14.05/3.74 Grace et al. Serum, LC‐MS/MS (10 SPE (100 µL, 40% Daidzein <0.010 96‐101 3.6/3.7 2003 [7] human (200 µL) MeOH) Enterodiol <0.010 96‐97 3.7/3.2 µL) Equol 0.100 96 3.5/5.6 Glycitein <0.010 94‐96 4.3/5.7 Enterolactone <0.010 98‐101 3.3/2.8 Genistein <0.010 96‐99 3/2.9 Jian et al. Serum, rat LC‐MS/MS (50 L‐L (120 µL, 2% ACN) Biochanin A 0.1 62‐107 2016 [8] (100 µL) µL) Coumestrol 0.7 28‐52 Daidzein 1.4 108‐123 Enterodiol 1.3 75‐94 Equol 1.5 84‐89 Glycitein 3.5 60 Matairesinol 0.6 84‐89 Naringenin 1.5 63‐79 Enterolactone 2.2 73‐91 Genistein 1.3 105‐123 L‐L (80% MeOH) Biochanin A 2 65.6 5 4.5/4.7

Prasain et Serum, LC‐ESI‐MS/MS Coumestrol 77.1 6.1 7.7/8.2 al. 2010 [9] human (200 (10 µL) Formononetin 85.2 2.5 5.6/5.6 µL) Daidzein 82.1 1.1 6.6/6.3 Enterodiol 86.7 7 7.1/7.3 Equol 83.2 4.3 3.7/3.8 Glycitein 86.2 10.3 8.6/9.0 Enterolactone 81.4 5.1 5.6/5.8 Genistein 57.5 4.5 4.9/4.7 Valentin‐ Serum, LC‐APCI‐ SPE (100 µL, 10mM Daidzein 0.5 100.8 <13 9.8 Blasini et al. human, fetal MS/MS (25 µL) ammonium Enterodiol 1.1 90.9 5.1 2000 [10] bovine serum acetate/ACN/MeOH, Equol 2.4 96 13.7 (1 mL) 70/15/15, v/v/v) Matairesinol 0.5 88.2 7.9 Enterolactone 0.1 89.5 4.5 Genistein 0.6 93 19.3 Wyns et al. Serum, LC‐APCI‐MS SPE (100 µL, MeOH) Secoisolariciresinol 132.6 95.8 14.8 15.7/2.8 2010 [11] human (800 (25 & 50 µL) Coumestrol 6.2 19.7 14.4 13.1/9.3 µL) Daidzein 1.9 95.2 4 12.8/2.9 Enterodiol 3.1 95.6 15 11.2/3.2 Equol 13.8 95.5 1.7 11/3.3 Matairesinol 17.8 98 12.9 13.4/2.9 Enterolactone 2.4 95 17.3 10.5/3.2 Genistein 2.2 90.6 3.1 13.7/3.0 Soukup et Plasma, LC‐ESI‐MS/MS SPE (200 µL, 30% Daidzein 0.076/0.25 91 1 5.2/2.6 al. 2014 human (500 (20 µL) MeOH) Equol 9.6/319 88 7 5.1/4.3 [12] µL) Genistein 0.03/0.1 87 3 6.1/3.4 Nørskov et Plasma, LC‐ESI‐MS/MS SPE (600 µL, 25% Secoisolariciresinol /0.067 86.3 2.1 3 al. 2015 human & pig ACN) Enterodiol /0.081 93 4.9 4.2 [13] (500 µL) Matairesinol /0.068 96.5 2.9 2.8 Enterolactone /0.16 101.9 5.9 7.6 Cao et al. Plasma, LC‐DAD (30 SPE (100 µL, Luteolin 0.62 ‐0.70 97.26 5.77 2010 [14] human (1 mL) µL) MeOH/0.1% Kaempferol 97.31 6.01 Phosphoric acid, Quercetin 104.25 6.84 3/2, v/v) Preindl et Serum, LC‐ESI‐MS/MS Protein precipitation Resveratrol 4.8/16 81 20/36 al. 2019 human (200 (200 µL, 10% ACN) Coumestrol 0.4/1.3 87 5/7

[15] µL) Formononetin 0.015/0.05 92 2/5

Daidzein 0.1/0.4 90 4/6

Enterodiol 0.05/0.15 91 3/10 Equol 0.2/0.7 89 2/5

Glycitein 0.15/0.6 94 3/10

Matairesinol 0.6/2.0 88 5/10

Enterolactone 0.2/0.7 88 5/13

Genistein 0.15/0.6 88 14/12

Milk, breast Salting out (250 µL, Resveratrol 7.5/25 11 63/80 (250 µL) 10% ACN) Coumestrol 0.3/1 91 10/3

Formononetin 0.02/0.06 89 8/3 Daidzein 0.05/0.15 93 9/4

Enterodiol 0.06/0.20 56 32/34

Equol 0.1/0.4 78 10/9

Glycitein 0.01/0.03 92 8/6

Matairesinol 0.75/2.5 93 6/5

Enterolactone 0.1/0.4 91 5/6

Genistein 0.09/0.3 84 7/12 ACN; acetonitrile, APCI; atmospheric pressure chemical ionization, DAD; diode‐array detector, ESI; electrospray ionization, LC; liquid chromatography, L‐L; liquid‐liquid extraction, MeOH; methanol, MS(/MS); mass spectrometry (tandem), SPE; solid‐phase extraction. This table is not exhaustive and relevant information has been included in order to compare with the validated HPLC‐DAD‐ESI‐MS method with previously developed methods.

Supplement Table 3. Comparison of relevant methods for the quantification of phytoestrogens in urine, including matrix (g), system (injection volume), extraction (reconstitution) used, phytoestrogens analysed, limits of detection / limits of quantification (LOD/LOQ, ng/mL), recoveries (RE%), accuracy (%), and inter / intra‐ precision (%) Author, year Matrix System Extraction Phytoestrogens LOD/LOQ RE% Accuracy Inter/Intra‐assay Palma‐Duran Urine, human LC‐DAD‐ESI‐ SPE (200 µL, Resveratrol 0.018/0.06 50 10 15.71/5.82 et al. 2020 (2 mL) MS (50 µL) MeOH/Mobile Biochanin A 0.002/0.008 97 2 8.01/2.19 phase) Secoisolariciresinol 0.0080/027 59 5 4.63/4.7 Luteolin 0.028/0.096 80 5 12.99/3.65 Coumestrol 0.001/0.003 101 2 3.74/1.97 Formononetin 0.003/0.011 92 2 10.24/1.89 Daidzein 0.042/0.141 76 6 3.31/6.61 Enterodiol 0.039/0.133 103 6 1.32/5.22 Equol 0.068/0.227 90 7 3.28/4.07 Kaempferol 0.003/0.012 76 3 11.49/3.98 Glycitein 0.075/0.251 111 4 2.43/4.25 Matairesinol 0.059/0.198 98 3 11.59/3.33 Naringenin 0.005/0.019 83 5 5.18/4.41 Quercetin 0.061/0.206 84 5 8.1/2.38 Enterolactone 0.001/0.003 110 4 3.47/3.54 Genistein 0.001/0.006 87 4 6.25/2.88 Grace et al. Urine, human LC‐ESI‐MS/MS SPE (100 µl, 40% Secoisolariciresinol 89 6.1‐7.9/2‐3.3 2007 [16] (200 µL) (10 µL) MeOH) Daidzein 93 5.5‐6.6/2.4‐2.5 Enterodiol 89 4.2‐5.8/2.3‐2.5 Equol 91 4.7‐6.6/1.7‐3.7 Glycitein 92 4.4‐6.7/1.3‐2.6 Naringenin 95 5.9‐7.3/3.9‐4.7 Enterolactone 95 4.3‐6.2/2.2‐2.6 Genistein 93 6.1‐6.6/2.4‐3 Rybak et al. Urine, human LC‐ESI(APCI)‐ SPE (100 µL, Daidzein 0.3 (0.3) 86‐114 6.1‐6.5 (5.3‐23) 2008 [17] (1 mL) MS/MS (25 MeOH/ACN/10mM Enterodiol 0.06 (0.2) (95‐105) 7.7‐8.9 (21‐28) µL) ammonium Equol 0.3 (2.7) 5.9‐6.4 (6.6‐30) acetate, 15:15:70) Enterolactone 0.4 (0.3) 7‐11 (6.4‐13) Genistein 0.06 (0.3) 4.4‐7 (5.3‐13) Valentin‐ Urine, human LC‐APCI‐ SPE(100 µL, Coumestrol 1.5 91.7 <13 13.8 Blasini et al. (2 mL) MS/MS (25 MeOH/ACN/10mM Daidzein 9.3 101.5 7.2 2000 [10] µL) Enterodiol 0.5 97.6 9.7

ammonium Equol 1.1 93.6 9.7 acetate, 15:15:70) Matairesinol 0.2 98.3 12.6 Enterolactone 3.3 103.7 7 Genistein 4.2 92.8 6.8 Wyns et al. Urine, human LC‐APCI‐MS L‐L (100 µL, MeOH) Secoisolariciresinol 65.1 67.5 10.4 14.8/7.4 2010 [11] (2 mL) (25 µL) Coumestrol 0.4 89.7 0.9 13.37.3 Daidzein 1 119 1.6 17.8/6.9 Enterodiol 2.7 99.8 6.9 10.7/8.1 Equol 5.8 91.1 2.7 14.3/5.6 Matairesinol 7.5 87.8 4.7 12.3/7.1 Enterolactone 2.5 115.5 11 14/9.4 Genistein 0.8 99.6 1.8 11.7/7.4 Soukup et al. Urine, human LC‐ESI‐MS/MS SPE (200 µL, 30% Daidzein 1.8/2.67 93 1 2.3/2.2 2014 [12] (500 µL) (20 µL) MeOH) Equol 75.2/251 91 6 3.8/3.4 Genistein 0.42/1.41 89 1 2.9/1.8 Parker et al. Urine, human LC‐APPI‐ NA, (390 µL) Daidzein 0.4/1 109 3 3.3/3/3 2012 [18] (200 µL) MS/MS (5‐20 Enterodiol 0.04/0.1 102 11 2.8/4.1 µL) Equol 0.06/0.2 103 3 2.7/3.5 Enterolactone 0.1/0.3 108 3 4.0/4.1 Genistein 0.2/0.6 112 7 3.9/5.2 Nørskov et Urine, human LC‐ESI‐MS/MS SPE (1.2 mL) Secoisolariciresinol /0.067 68 8 0.3 al. 2015 [13] & pigs (250 Enterodiol /0.081 97 4 0.9 µL) Matairesinol /0.068 80 10 1.2 Enterolactone /0.16 94 11.5 0.4 Min et al. Urine, human LC‐ESI‐MS/MS L‐L (80% MeOH) Daidzein 0.08‐0.5/0.38‐8.99 76.1‐115.7 1.18‐4.8/0.6‐1.8 2020 [19] (5 µL) Equol Glycitein Enterolactone Genistein Chen et al. Urine, human LC‐MS L‐L Daidzein 0.1 2020 [20] Equol 30 Glycitein 0.0007 Genistein 0.07 Preindl et al. Urine, human LC‐ESI‐MS/MS Protein Resveratrol 0.15/0.5 107 6/4 2019 [15] (200 µL) precipitation (200 Coumestrol 0.15/0.5 99 2/6 µL, 10% ACN) Formononetin 0.05/0.15 102 3/7

Daidzein 0.15/0.5 103 4/6 Enterodiol 0.15/0.5 101 3/5 Equol 0.2/0.5 102 4/6 Glycitein 0.2/0.5 105 1/6 Matairesinol 1.0/3.0 103 6/9 Enterolactone 0.15/0.5 104 2/3 Genistein 0.2/0.5 94 10/10 ACN; acetonitrile, APCI; atmospheric pressure chemical ionization, APPI; atmospheric pressure photoionization, DAD; diode‐array detector, ESI; electrospray ionization, LC; liquid chromatography, L‐L; liquid‐liquid extraction, MeOH; methanol, MS(/MS); mass spectrometry (tandem), NA, not apply, SPE; solid‐phase extraction. This table is not exhaustive and relevant information has been included in order to compare with the validated HPLC‐DAD‐ESI‐MS method with previously developed methods.

Supplement Table 4. Matrix Effect (%) at low and high quality control level in four different blank serums. Phytoestrogen 16 ng/mL 100 ng/mL Resveratrol 106.0 109.3 Biochanin A 60.1 61.2 Secoisolariciresinol 114.3 109.0 Luteolin 78.9 73.6 Coumestrol 61.1 61.0 Formononetin 64.0 60.5 Daidzein 100.9 82.2 Enterodiol 97.3 103.1 Equol 107.9 92.3 Kaempferol 97.4 81.0 Glycitein 96.8 89.7 Matairesinol 111.5 92.2 Naringenin 90.3 68.1 Quercetin 103.9 104.4 Enterolactone 73.4 64.5 Genistein 101.9 75.0 The matrix effect (ME) was analyzed using the strategy described by Matuszewski et al. [21], wherein the results for the serum spiked after extraction (C) were compared to the standards (A) (Equation 1:𝑀𝐸% 𝐶⁄ 𝐴 100). The ME was determined by analyzing the sample in four different blank serum batches. An ME value at < 100% indicated ion suppression, and values > 100% indicated ion enhancement.

Supplement Table 5. Comparison of phytoestrogens content (µg/ 100 g) found in this study with relevant previously studies and public databases Food Tomatoe Mandarin Beans Mea soy‐ Beef Pu * Pu ‡ Ora Tan Pinto* * W Blac Oth Pint bla Turke t base Lettuce, raw stea re re nge geri hit k er o* ck* y subs d k* e e n e‡ ham titut burge es rs Reference P‐D [1] [22] [23] P‐D et al. [23] [22] P‐D et [1] [22] [23] P‐D et [1] [22] [23] P‐D [24] [25] et al. 2020 al. al. et al. 2020 2020 2020 2020

Phytoestrog 10 ‐ ens 28,7 58 Resveratrol nd nd nd nd nd nd

Biochanin A nd 2 nd nd 0.06 37 0.04 nd nd nd

Secoisolaric 0.64 148 0.18 nd nd 0.06 2.51 151 0.08 14.24 66 0. 0.08 nd iresionol 12

Luteolin nd 0.02 0.03 0.15 0. 0. 0. 0.1 nd nd 6 01 04 Coumestrol nd <1 0.18 0. 0.02 15 0.59 nd nd nd 05 Formonone nd nd 0.41 0. 0.04 2 nd nd nd 5.26 tin 36 Daidzein 0.04 <1 nd 0. nd 25 3.5 7 0. 0.8 395.2 259 7 3 4 Enterodiol 0.21 nd nd 4.87 nd nd

Equol nd nd nd 11.6 nd nd

Kaempferol 0.01 0.05 0.15 0.07 1. 0. 0. 0.43 nd 406.4 0. 2.68 1.22 0.9 0.1 0.64 95 01 08 1 76 8 1 Glycitein nd <1 nd nd 0.53 nd nd 1 172.3 9 Matairesino nd 6 <0.0 1.32 1. <0.0 0.02 1 0.02 nd 2 <0 nd l 1 91 1 .0 1 Naringenin 0.06 140. 32 27.1 83.9 10.0 81.97 13.17 6 2. 2 39 Quercetin 0.02 1.63 1.42 0.33 16 0. 4. 13.7 0.99 1 0. 0.98 0.68 0.1 7.0 nd .5 7 12 5 29 7

Enterolacto nd nd 0. 0.03 nd 0.09 ne 02 Genistein 0.03 <1 0.24 0. nd 9 1.96 10 0. 0.6 0.2 0.0 356.2 272 89 2 7 4 * cooked or ‡ adjusted for processing [26], nd; not detected or below the limit of quantification. This table is not exhaustive and relevant information has been included in order to compare with the validated HPLC‐DAD‐ESI‐MS method with previously published studies and databases.

References

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