2,4-Dinitrophenylhydrazine (2,4-DNPH) was purchased from Sigma-Aldrich (St. Louis, USA), hydrochloric acid (HCl, 37%) was purchased from Merck (Darmstadt, Germany), all solvents (e.g. methanol, ACN, acetone) used were of HPLC grade and were purchased from Merck (Darmstadt, Germany), acetic acid was purchased from Roth (Karlsruhe, Germany), triolein was purchased from FLUKA (Buchs, Switzerland), β-carotene and α-tocopherol were from Sigma-Aldrich (St. Louis, USA).
Analysis of secondary oxidation products in oil
Oxidation of triolein with Rancimat
The triolein oil samples were subjected to oxidation in a Rancimat (679, Metrohm, Herisau, Switzerland). Eleven grammes of sample was used for the oxidation experiments. The temperature was set to 120 °C and the air flow to 20 dm3/h. Triolein was treated for up to 10 h. The oxidized samples were cooled immediately after the oxidation and stored under nitrogen below −18 °C.
Derivatization with 2,4-dinitrophenylhydrazine (DNPH)
To 1 cm3 of the oxidized oil samples 4 cm3 of acetonitrile was added and mixed with 3 cm3 of the reagent 2,4-DNPH (3.48 mg/cm3). The reaction mixture was kept in the dark for 1 h. After completion of the reaction, 2 cm3 ethyl acetate was added for extraction and 1 g KCl for better phase separation. This mixture was thoroughly shaken for 30 s and centrifuged for phase separation. The organic layer was analysed without further treatment by HPLC.
Liquid chromatography–mass spectrometry condition for aldehydes identification
The analyses of the DNPH derivatives of the carbonyls formed during oxidation were done by HPLC (Agilent 1100, Waldbronn, Germany) using a reversed phase column (Kinetex, EVO C18, 150 × 3 mm, 5 µm, Phenomenex, Aschaffenburg, Switzerland). For elution a gradient was used starting with methanol (45%), water (30%), and acetonitrile (25%) changing to methanol (6%), water (4%), and acetonitrile (90%) linearly within 15 min. The absorption of the eluent was measured at 400 nm for the presence of the DNPH derivatives.
For mass selective detection, a QTRAP 2000 (AB Sciex, Framingham, MA, USA) was used. Ionization was done using the APCI mode with a gas drying temperature of 250 °C, capillary voltage of 4000 V, and a fragmentor potential of 150 V.
Analyses of α-tocopherol and β-carotene
For the analyses of α-tocopherol and β-carotene 25 mg of the oil (triolein, triolein with β-carotene and triolein with α-tocopherol) was extracted with 1 cm3 of methanol in 2-cm3 reactions vials (Eppendorf, Wien, Austria). The samples were shaken for 2 min vigorously and centrifuged. Under these conditions, both α-tocopherol and β-carotene were extracted quantitatively. The methanolic extract was used directly for HPLC analysis on a reversed phase column (Kinetex, EVO C18, 150 × 3 mm, 5 µm, Phenomenex, Aschaffenburg, Switzerland) using a flow of 0.6 cm3/min. α-Tocopherol was separated isocratically using 5% water in methanol detecting it at 292 nm. β-Carotene was chromatographed with 100% acetonitrile with detection at 450 nm.