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

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

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

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

Pisacane et al NP-HPLC OF TOCOLS

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

Avenanthramides (ca 30 different compounds in oats)

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

Avenanthramides UV-spectra

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

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

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

Molecular weights of avenanthramides anthranilic acid5-OH5-OH, 4-MeOH4-OH4-OH, 5-OH p-coumaric p ferulic f sinapic s caffeic c p-coumaric p diene ferulic f diene sinapic s diene caffeic c diene

Effect of germination

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Antioxidant activity of different avenanthramides

Avenanthramides in crown rust susceptible and resistant cultivars

Radical scavenging activity of different avenanthramides 1 min 5 min

Radical scavenging activity of different avenanthramides 1 min 5 min

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