Electronic Supplementary Material (ESI) for Food & Function. This journal is © The Royal Society of Chemistry 2015 Electronic Supplementary Information Supplemental Table 1. Fecal and urine content of ellagic acid and urolithin A, B analyzed by HPLC. Baseline Week 4 Fecal Fecal Fecal Urine Fecal Fecal Fecal Urine EA UA UB UA EA UA UB UA µg/g µg/g µg/g µg/mg µg/g µg/g µg/g µg/mg stool stool stool creatinine stool stool stool creatinine Non- 0-6.4 0 0 0-2.9 0-174.2 0 0 0-0.6 Producers Producers 0-3.7 0-71 0-36 0.1-28 0-19 14-317 0-85 2.8-36 Supplemental Table 2. Fecal content of punicalagin A/B and punicalin analyzed by HPLC. Baseline Week 4 Fecal Fecal Fecal Fecal Pun A/B Punicalin Pun A/B Punicalin µg/g µg/g µg/g µg/g stool stool stool stool Non- 0 0 0-49 0-7.4 Producers Producers 0 0 0-27 0-5.7 LC-MS/MS analysis of pomegranate extract Five milligrams of pomegranate extract was dissolved in 5mL DMSO and further diluted 10 times in DMSO. The LC-MS/MS system consisted of a LCQ Advantage Finnigan instrument with ion-trap mass spectrometer equipped with an electrospray ionization (ESI) interface, and a Surveyor HPLC unit consisting of an autosampler/injector, quaternary pump, column heater, and diode array detector operated by Xcalibur software (Thermo Finnigan Corp). A Zorbax column (SB-C18 5µm 2.1x 150 mm column, Agilent) was used for the separation of analytes using a gradient elution condition by increasing the percentage of acetonitrile (with 1% acetic acid) in water (with 1% acetic acid) from 2% to 25% in 25 minutes at a flow rate of 0.2 mL/min. The MS conditions for the detection of ellagic acid and punicalagin A/B were as follows: ESI in negative mode; scan range 200 to 1200 amu; scan rate 1scan/second; cone voltage 17 eV. The identity of ellagic acid and punicalagin A/B was determined by matching the peak retention time and molecular ions [M-H]- obtained by ESI/MS and tandem mass spectrometry (MS-MS) with the expected theoretical molecular weights and the data from literature (Nunez-Sanchez MA et al 2014). The deprotonated [M-H]- ions were m/z 301 and 1083, and the fragment ions were m/z 257, 229 and 781, 601 for ellagic acid and punicalagin A/B, respectively. Nunez-Sanchez, M. A., Garcia-Villalba, R., Monedero-Saiz, T., Garcia-Talavera, N. V., Gomez- Sanchez, M. B., Sanchez-Alvarez, C., Garcia-Albert, A. M., Rodriguez-Gil, F. J., Ruiz-Marin, M. et al. (2014) Targeted metabolic profiling of pomegranate polyphenols and urolithins in plasma, urine and colon tissues from colorectal cancer patients. Mol. Nutr. Food Res. 58: 1199- 1211. Figure 1. LC-MS/MS analysis of standards (A) and pomegranate supplement (B). A. Standards A-1 A-2 A-3 A-4 Punicalagin AB and EA standards: 1) UV @ 360 nm 2) Full Scan 200-1200 @ M-H M/Z=1083 & M/Z=301 3)MS/MS of 1083 4) MS/MS 0f 301 pun STD_05212015A'' #418-450 RT: 9.19-9.61 AV: 11 NL: 2.79E8 T: - c ESI Full ms [ 200.00-1200.00] 1083.1 100 95 90 85 80 1084.1 75 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 30 541.3 25 1085.1 20 15 1094.2 10 1095.2 542.1 5 1102.6 575.1 1192.6 232.5 301.1 594.1 653.0 728.3 844.8 349.7 430.7 442.1 482.7 786.8 922.7 966.6 1018.6 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Spectrum of M/Z=1083 pun STD_05212015A'' #1070-1092 RT: 18.93-19.18 AV: 7 NL: 2.27E8 T: - c ESI Full ms [ 200.00-1200.00] 301.2 100 95 90 85 80 75 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 30 25 20 15 302.2 10 624.8 299.1 612.1 5 303.2 625.8 700.6 722.6 948.7 384.8 240.9 330.7 413.2 472.8 543.1 577.1 814.9 835.3 902.5 963.6 1055.1 1118.0 1147.5 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Spectrum of M/Z=301 pun STD_05212015A'' #256-274 RT: 6.83-7.04 AV: 6 NL: 4.84E7 F: - c ESI Full ms2 [email protected] [ 295.00-1100.00] 601.1 781.1 100 95 90 85 80 75 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 721.0 30 25 575.1 20 549.2 15 763.0 10 449.1 744.8 5 393.3 404.9 459.3 719.0 301.2 337.0 511.1 677.5 613.0 807.1 851.0 0 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 m/z Spectrum of 1083 MS/MS pun STD_05212015A'' #1100-1112 RT: 19.32-19.50 AV: 5 NL: 4.62E5 F: - c ESI Full ms2 [email protected] [ 120.00-305.00] 257.2 100 95 90 85 80 75 70 65 229.2 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 30 25 20 15 226.6 258.1 266.0 251.3 218.9 284.0 10 174.8 181.8 161.3 300.9 5 217.7 173.0 239.1 273.2 201.0 0 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 m/z Spectrum of 301 MS/MS B. Pomegranate extract B-1 B-2 B-3 B-4 1) UV @ 360 nm 2) Full Scan 200-1200 @ M-H M/Z=1083 & M/Z=301 3)MS/MS of 1083 4) MS/MS 0f 301 POMX_0521205A'' #434-451 RT: 9.45-9.66 AV: 6 NL: 8.98E7 T: - c ESI Full ms [ 200.00-1200.00] 1083.0 100 95 90 85 80 75 70 65 1083.9 60 e c n 55 a d n u b A 50 541.2 e v i t a l e 45 R 40 35 1084.9 30 25 20 542.4 1094.6 15 1106.0 633.0 10 717.6 935.1 1198.2 692.2 884.7 1135.7 563.0 781.7 1064.3 623.7 749.6 832.0 280.9 298.9 634.1 5 328.8 372.2 498.9 791.7 925.2 238.0 483.2 679.4 952.9 1024.0 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Spectrum of M/Z=1083 POMX_0521205A'' #1083-1111 RT: 19.01-19.39 AV: 10 NL: 2.45E8 T: - c ESI Full ms [ 200.00-1200.00] 301.2 100 95 90 85 80 75 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 30 25 20 302.2 15 10 611.3 624.7 5 299.0 303.1 382.7 610.5 224.6 304.0 426.0 466.9 626.8 719.5 980.8 274.7 570.0 769.0 788.9 832.6 911.5 1042.6 1114.2 1153.9 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Spectrum of M/Z=301 POMX_0521205A'' #423-449 RT: 9.25-9.60 AV: 9 NL: 1.77E7 F: - c ESI Full ms2 [email protected] [ 295.00-1100.00] 601.1 100 95 781.1 90 85 80 75 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 40 35 721.1 30 25 575.2 20 15 763.0 10 549.1 501.2 5 449.0 719.0 300.9 393.1 406.8 529.1 744.7 337.2 467.1 617.0 676.9 806.9 866.8 1021.2 1047.3 1065.0 0 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 m/z Spectrum of 1083 MS/MS POMX_0521205A'' #1108-1135 RT: 19.36-19.71 AV: 9 NL: 4.38E5 F: - c ESI Full ms2 [email protected] [ 120.00-305.00] 257.0 100 95 90 85 80 75 229.2 70 65 60 e c n 55 a d n u b A 50 e v i t a l e 45 R 258.3 40 35 30 185.0 25 239.9 20 245.2 15 269.1 10 184.5 204.8 214.0 201.0 271.2 222.3 256.2 189.7 301.9 5 157.1 238.9 258.9 299.7 141.0 146.9 171.0 212.3 255.0 265.1 0 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 m/z Spectrum of 301 MS/MS Figure 2.