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Methods


RP-HPLC






Description:

The reversed phase HPLC (RP-HPLC) analysis is carried out using an Agilent 1100 HPLC system coupled to a diode array detector equipped with an ET250/2 Nucleosil 120-5 C18 column (250 x 2.1 mm, 5 µm particle size; Macherey & Nagel). A binary gradient system is used: solvent A, methanol/water/acetic acid (80:20:0.1, v/v/v); solvent B, methanol/acetic acid (100:0.1, v/v/v) with the following gradient program: 100 % solvent A for 10 min, followed by a linear increase of solvent B up to 100 % within 15 min and by an isocratic run at 100 % solvent B for 15 min. The flow rate is 0.18 ml/min.
Oxylipins are purified by collecting the respective fractions. The following authentic standards are used to determine the retention times of the different oxylipin classes: JA-Me, dinor-oPDA-Me, oPDA-Me, 11-HHT-Me, 13-HOT-Me, 13-HOD-Me, dinor-CnA-Me, CA-Me, and CnA-Me. JA-Me is detected by monitoring the absorbance at 202 nm. For analysis of dinor-oPDA-Me and oPDA-Me, the absorbance at 224 nm is recorded. For detection of hydroperoxy fatty acid methyl esters and hydroxy fatty acid methyl esters, the absorbance of the conjugated diene system at 234 nm is recorded. For analysis of divinyl ether methyl esters, the absorbance at 250 nm and 268 nm indicating either one or two conjugated diene systems in conjugation to an ether bond of the fatty acids is recorded.

standards:
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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