Supporting Information. Rec. Nat. Prod. 7:4 (2013)

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1 Supporting Information Rec. Nat. Prod. 7:4 (213) Phenolic Compounds of Pinus brutia Ten.: Chemical Investigation and Quantitative Analysis Using an Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry with Electrospray Ionization Source İbrahim Kıvrak *1,2, Şeyda Kıvrak 1, Mansur Harmandar 2 and Yunus Çetintaş 2 1 Muğla Sıtkı Koçman University, Research Laboratory Center, MGL Food Analysis Laboratory, 48 Kötekli, Muğla, Turkey 2 Department of Chemistry, Faculty of Science, Muğla Sıtkı Koçman University, 48 Kötekli, Muğla, Turkey Table of Contents Page S1: Calibration Curve and UPLC-MS/MS Chromatogram of Gallic acid 3 S2: Calibration Curve and UPLC-MS/MS Chromatogram of Gentisic acid 3 S3: Calibration Curve and UPLC-MS/MS Chromatogram of Protocatechuic acid 3 S4: Calibration Curve and UPLC-MS/MS Chromatogram of 4-hydroxy benzoic acid 4 S5: Calibration Curve and UPLC-MS/MS Chromatogram of Catechin hydrate 4 S6: Calibration Curve and UPLC-MS/MS Chromatogram of Vanillic acid 4 S7: Calibration Curve and UPLC-MS/MS Chromatogram of Caffeic acid 5 S8: Calibration Curve and UPLC-MS/MS Chromatogram of Vanillin 5 S9: Calibration Curve and UPLC-MS/MS Chromatogram of p-coumaric acid 5 S1: Calibration Curve and UPLC-MS/MS Chromatogram of Ferulic acid 6 S11: Calibration Curve and UPLC-MS/MS Chromatogram of Catechin gallate 6 S12: Calibration Curve and UPLC-MS/MS Chromatogram of Myricetin 6 S13: Calibration Curve and UPLC-MS/MS Chromatogram of Resveratrol 7 S14: Calibration Curve and UPLC-MS/MS Chromatogram of trans-cinnamic acid 7 1

2 S15: Calibration Curve and UPLC-MS/MS Chromatogram of Luteolin 7 S16: Calibration Curve and UPLC-MS/MS Chromatogram of Naringenin 8 S17: Calibration Curve and UPLC-MS/MS Chromatogram of Kaempferol 8 S18. Analytical Parameters of the Method 9 2

3 S1: Calibration Curve and UPLC-MS/MS Chromatogram of Gallic acid Compound name: Gentisic acid sodium salt Correlation coefficient: r = , r^2 = Calibration curve: * x Gentisic acid sodium salt F1:MRM of 3 channels,es > e F1:MRM of 3 channels,es > 81 Gentisic acid sodium salt;1.86; e+3 ; F1:MRM of 3 channels,es > Gentisic acid sodium salt e S2: Calibration Curve and UPLC-MS/MS Chromatogram of Gentisic acid Compound name: Protocatechuic acid Correlation coefficient: r = , r^2 = Calibration curve: * x Calvatia_gigantea_121129_EtAc Smooth(Mn,2x3) Calvatia_gigantea_121129_EtAc Protocatechuic acid F11:MRM of 4 channels,es > e+7 35 Calvatia_gigantea_121129_EtAc Smooth(Mn,2x3) Calvatia_gigantea_121129_EtAc Protocatechuic acid F11:MRM of 4 channels,es > e Calvatia_gigantea_121129_EtAc Smooth(Mn,2x3) F11:MRM of 4 channels,es- 2 Calvatia_gigantea_121129_EtAc Protocatechuic acid > e Calvatia_gigantea_121129_EtAc Smooth(Mn,2x3) Calvatia_gigantea_121129_EtAc F11:MRM of 4 channels,es > 81.1 Protocatechuic acid 3.68e S3: Calibration Curve and UPLC-MS/MS Chromatogram of Protocatechuic acid 3

4 Compound name: 4-Hydroxy benzoic acid Correlation coefficient: r = , r^2 = Calibration curve: * x Hydroxy benzoic acid F6:MRM of 2 channels,es > e R e sp onse Hydroxy benzoic acid F6:MRM of 2 channels,es > e S4: Calibration Curve and UPLC-MS/MS Chromatogram of 4-hydroxy benzoic acid Compound name: Catechin hydrate Correlation coefficient: r = , r^2 = Calibration curve: * x Catechin hydrate F26:MRM of 3 channels,es > e F26:MRM of 3 channels,es- 225 Catechin hydrate > e R e s p o n s e F26:MRM of 3 channels,es Catechin hydrate > e S5: Calibration Curve and UPLC-MS/MS Chromatogram of Catechin hydrate Compound name: Vanillic acid Correlation coefficient: r = , r^2 = Calibration curve: * x Vanillic acid F14:MRM of 3 channels,es > e F14:MRM of 3 channels,es > Vanillic acid;3.63; ; e+4 14 Response F14:MRM of 3 channels,es > Vanillic acid;3.63; e+4 6 ; S6: Calibration Curve and UPLC-MS/MS Chromatogram of Vanillic acid 4

5 Compound name: Caffeic acid Correlation coefficient: r = , r^2 = Calibration curve: * x Caffeic acid F17:MRM of 4 channels,es > e+5 2 F17:MRM of 4 channels,es > Caffeic acid;3.67; ; e F17:MRM of 4 channels,es- 25 Caffeic acid;3.68;893.5 ; > e F17:MRM of 4 channels,es- 1 1 Caffeic acid;3.68; ; > e S7: Calibration Curve and UPLC-MS/MS Chromatogram of Caffeic acid Compound name: Vanillin Correlation coefficient: r = , r^2 = Calibration curve: * x Vanillin F9:MRM of 3 channels,es > e F9:MRM of 3 channels,es > Vanillin;4.51; e+5 ; R espo n se F9:MRM of 3 channels,es > 18.1 Vanillin;4.51; e+5 1 ; S8: Calibration Curve and UPLC-MS/MS Chromatogram of Vanillin Compound name: p-coumaric acid Correlation coefficient: r = , r^2 = Calibration curve: * x p-coumaric acid F12:MRM of 3 channels,es > e F12:MRM of 3 channels,es > p-coumaric acid e R esp on se F12:MRM of 3 channels,es- 4 p-coumaric acid > e S9: Calibration Curve and UPLC-MS/MS Chromatogram of p-coumaric acid 5

6 Compound name: Ferrulic acid Correlation coefficient: r =.99924, r^2 = Calibration curve: * x Ferrulic acid F18:MRM of 3 channels,es > e Ferrulic acid;5.37; ;1.83 F18:MRM of 3 channels,es > e Ferrulic acid;5.37; ;2.41 F18:MRM of 3 channels,es > e S1: Calibration Curve and UPLC-MS/MS Chromatogram of Ferulic acid Compound name: Catechin gallate Correlation coefficient: r = , r^2 = Calibration curve: * x Catechin gallate F31:MRM of 2 channels,es- 441 > e Catechin gallate F31:MRM of 2 channels,es- 441 > e S11: Calibration Curve and UPLC-MS/MS Chromatogram of Catechin gallate Compound name: Myricetin Correlation coefficient: r = , r^2 = Calibration curve: * x R e s p o n se Myricetin Myricetin;6.89; ;.94 Myricetin;6.89; ; F29:MRM of 4 channels,es > e F29:MRM of 4 channels,es > e F29:MRM of 4 channels,es > e Myricetin;6.89;73.99 ;3.62 F29:MRM of 4 channels,es > e S12: Calibration Curve and UPLC-MS/MS Chromatogram of Myricetin 6

7 Compound name: Resveratrol Correlation coefficient: r = , r^2 = Calibration curve: * x Resveratrol F2:MRM of 3 channels,es > e+6 14 F2:MRM of 3 channels,es > Resveratrol 9.21e F2:MRM of 3 channels,es > Resveratrol e S13: Calibration Curve and UPLC-MS/MS Chromatogram of Resveratrol Compound name: trans-cinnamic acid Correlation coefficient: r = , r^2 = Calibration curve: * x trans-cinnamic acid F8:MRM of 2 channels,es > e Response S14: Calibration Curve and UPLC-MS/MS Chromatogram of trans-cinnamic acid Compound name: Luteolin Correlation coefficient: r =.99592, r^2 = Calibration curve: * x Luteolin F25:MRM of 4 channels,es > e+5 12 F25:MRM of 4 channels,es- 11 Luteolin;8.32; > e+5 ; F25:MRM of 4 channels,es- 7 6 Luteolin;8.32; ; > e F25:MRM of 4 channels,es > Luteolin;8.33;479.8 ; e S15: Calibration Curve and UPLC-MS/MS Chromatogram of Luteolin 7

8 Compound name: Naringenin Correlation coefficient: r = , r^2 = Calibration curve: * x Naringenin F23:MRM of 4 channels,es > e+5 18 F23:MRM of 4 channels,es > Naringenin;9.14; e+6 16 ; F23:MRM of 4 channels,es- 1 Naringenin;9.14; ; > e F23:MRM of 4 channels,es- 4 Naringenin;9.14; > e+5 ; S16: Calibration Curve and UPLC-MS/MS Chromatogram of Naringenin Compound name: Kaempferol Correlation coefficient: r =.99652, r^2 = Calibration curve: * x Kaempferol F24:MRM of 4 channels,es > e F24:MRM of 4 channels,es > Kaempferol;9.51; ; e F24:MRM of 4 channels,es- 8 Kaempferol;9.51; ; > e F24:MRM of 4 channels,es > Kaempferol;9.51; ; e S17: Calibration Curve and UPLC-MS/MS Chromatogram of Kaempferol 8

9 S18. Analytical Parameters of the Method LOD LOQ Compound (mg 1 ml -1 ) (mg 1 ml -1 CV a CV b Recovery Stability Accuracy ) () b CV c () d Calibration equations r 2 4-hydroxy benzoic acid y= x trans-cinnamic acid y= x Resveratrol y= x Catechin gallate y= x Homogentisic acid y=4.5441x Gentisic acid y= x Vanillin y=129.68x Vanillic acid y=137.65x Catechin hydrate y=87.461x Chlorogenic acid y= x p-coumaric acid y=58.683x Ferulic acid y= x Hesperetin y= x Chrysin y=575.5x Protocatechuic acid y=137.52x trans-2-hydroxy cinnamic acid y= x Gallic acid y= x

10 LOD LOQ Compound (mg 1 ml -1 ) (mg 1 ml -1 CV a CV b Recovery Stability Accuracy ) () b CV c () d Calibration equations r 2 Myricetin y= x Naringenin y=764.46x Caffeic acid y= x Pyrogallol y= x Luteolin y=66.354x Kaempferol y= x a Intra-day repeatability. b Inter-day reproducibility and recovery from once a day of three consecutive days. c Stability were measured by standard solutions over three consecutive days. d Accuracy represents the ratio of the measured concentration to the theoretical concentration from medium concentration value of the calibration range. 1