The chiral phase HPLC (CP-HPLC) analysis is carried out to determine the enantiomer composition of the hydroperoxy fatty acids as well as hydroxy fatty acids. The analysis is carried out using an Agilent 1100 HPLC system coupled to a diode array detector equipped with a Chiral OD-H column (150 x 2.1 mm, 5 µm particle size; Baker). A solvent system of n-hexane:2-propanol:acetic acid (100:5:0.1, v/v/v) and a flow rate of 0.1 ml/min is used.
For detection of hydroperoxy fatty acids and hydroxy fatty acids, the absorbance at 234 nm indicating the conjugated diene system is recorded. All substances are identified by their characteristic UV spectra.
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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