1. AVEQ Analysis of Tocopherols/Tocotrienols and Avenanthramides in Oats WP 8 Rita Redaelli and Lena Dimberg

2. Tocopherols/ Tocotrienols Tocopherols Tocotrienols IsomersR'R'' αCH 3 β H γH δHH

3. Pisacane et al., 2004 Solvent: Methanol Flour/solvent ratio: 1 g/14 ml Time: 2*1 min Solubilized: Methanol (or hexane/1,4-dioxane, 95/5) SLU-lab Solvent - Methanol Flour/solvent ratio:(1 g/21 ml) Time: 3*20 min Solubilized - Hexane Peterson et al., 2007 Solvent: Methanol (or saponified KOH in ethanol + antioxidant) Flour/solvent ratio: 1 g/28 ml Time: 2* 20 min Solubilized: Hexane (or hexane/2-propanol, 99.5/0.5) Extraction of Tocopherols/Tocotrienols Nielsen and Hansen, 2008 Solvent: Hexane Flour/solvent ratio: 1 g/30 ml Time: 1* 10 min Solubilized: Hexane

4. SLU-lab NP- column: 250 mm Isocratic solvent: Hexane/1,4-dioxane (96:4 v/v) Flow: 1.5 ml/min Time: 21 min Fluorescence detector: ex 294 nm; em 326 nm Reference: 8 isomers Nielsen and Hansen, 2008 NP- column: 125 mm Isocratic solvent: Hexane/ethyl acetate/acetic acid (94.6/3.6/1.8) Flow: 1 ml/min Time: 16 min Fluorescence detector: ex 290 nm; em 330 nm References: 8 isomers Pisacane et al., 2004 NP- column: 250 mm Isocratic solvent: Hexane/1,4-dioxane (95:5 v/v) Flow: 1,5 ml/min Time: 11 min Fluorescence detector: ex 294 nm; em 326 nm Reference: 8 isomers Peterson et al., 2007 NP- column: 250 mm Isocratic solvent: Hexane/2-propanol (99.5/0.5 v/v) Flow: 1 ml/min Time:? Fluorescence detector: ex 295 nm; em 330 nm References: α-tocopherol HPLC analysis of Tocopherols/ Tocotrienols

5. Pisacane et al. 2004 NP-HPLC OF TOCOLS

6. Avenanthramides R= H, OH or OCH 3 n= 1 or 2

7. Avenanthramides (ca 30 different compounds in oats)

8. HPLC-chromatogram (UV 340 nm) oat extract and reference compounds

9. Avenanthramides UV-spectra

10. SLU-lab Solvent - 80% Ethanol (pH 2) Flour/solvent ratio:(1 g/21 ml) Temperature: 50  C Time: 3*20 min Solubilized - Methanol Extraction of Avenanthramides Wise et al., 2009 Solvent: 80% Ethanol Flour/solvent ratio: 1 g/30 ml Temperature: 50  C Time: 3*20 min Solubilized: Methanol Matsukawa et al., 2000 Solvent - Methanol Flour/solvent ratio:(1 g/30 ml) Temperature: ambient? Time: 3*? min Solubilized -Methanol Collins Solvent - 80% Ethanol (acidified) Flour/solvent ratio:(1 g/25 ml) Temperature: 100  C Time: 3*20 min Purification: Octyl-sepharose, Sephadex-LH20 Solubilized – 50% Ethanol

11. SLU-lab RP C-18 column: 125 mm Solvent: A. = 0.01 M formic acid (pH 2.9), 5% acetonitrile B. acetonitrile Gradient: 5 to 38% B in A in 35 min Flow: 1 ml/min Time: 30 min Diod array: 340 nm Reference: 17 compounds Wise et al., 2009 RP C-18 : 50 mm Solvent: A. 0.1% formic acid, 5% acetonitrile B. acetonitrile, 0.1% formaric acid Gradient: 13-30% B in A for 20 min Flow: 1 ml/min Time: 20 min Diode array detector: 280 and 330 nm References: 3 compounds Collins et al., 2004 RP C-18 column: ? mm Solvent: A. 5% acetic acid B. methanol Gradient: 40 to 100% B in A for 88 min Flow: 0.8 ml/min Time: 50 min Diod array: 330 nm Reference: ? compounds Matsukawa et al., 2000 RP C-18 column: 150 mm Solvent: A. 0.5% TFA, B. acetonitrile Gradient: 15 to 30% B in A for 60 min Flow: ? ml/min Time: ? min Diod array: 340 nm Reference ? compounds HPLC analysis of Avenanthramides

12. Analysis of Avenanthramides LC-MS b-g Selective ion monitoring (SIM) detection [M+H] + for 2c, 2p, 2f, 3f, 2p d and 2f d, respectively a UV detection, 340 nm

13. Molecular weights of avenanthramides anthranilic acid5-OH5-OH, 4-MeOH4-OH4-OH, 5-OH 12345 p-coumaric p283299329299315 ferulic f313329359329345 sinapic s343359389359375 caffeic c299315345315331 p-coumaric p diene 309325355325341 ferulic f diene 339355385355371 sinapic s diene 369385415385401 caffeic c diene 325341371341357

14. Effect of germination

15. Thank you

16. Antioxidant activity of different avenanthramides

17. Avenanthramides in crown rust susceptible and resistant cultivars

18. Radical scavenging activity of different avenanthramides 1 min 5 min

19. Radical scavenging activity of different avenanthramides 1 min 5 min

20. HPLC-chromatogram (UV-detection 340 nm) - oat extract and reference compounds-