The straight phase HPLC (SP-HPLC) analysis is carried out using an Agilent 1100 HPLC system coupled to a diode array detector equipped with an Zorbax Rx-SIL column (150 x 2.1 mm, 5 µm particle size; Agilent). A solvent system of n-hexane:2-propanol:acetic acid (100:1:0.1, v/v/v) and a flow rate of 0.2 ml/min are used.
For detection of the hydroperoxy fatty acids and hydroxy fatty acids, the absorbance at 234 nm indicating the conjugated diene system is recorded. For detection of keto fatty acids, the absorbance at 270 nm is recorded. All substances are identified by their characteristic UV spectra. For quantification, calibration curves (five point measurements) for 13-HOD, 13-HOT, 11-HHT, 13-KOD and 13-KOT were established. (6Z,9Z,11E,13S)-13-hydroxy-6,9,11-octadecatrienoic acid is used as internal standard to determine the recovery of the hydroperoxy fatty acids, hydroxy fatty acids and keto fatty acids.
standards are available from Sigma (Germany) Larodan (Sweden), Cayman Chemical (USA), kindly provided by Mats Hamberg (Stockholm, Sweden) and Otto Miersch (Halle/Salle, Germany) or enzymatically synthesized, respectively
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