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, acetonitril/water (50:50, v/v); solvent B, acetonitril/water (80:20, v/v) with the following gradient program: 15 % solvent B for 15 min, followed by a linear increase of solvent B up to 66.6 % within 25 min, by a linear increase of solvent B up to 100 % within 13.4 min and an isocratic run at 100 % solvent B for 26.6 min. The flow rate is 0.3 ml/min.
For analysis of the aldehydes, the absorbances at 366 nm indicating the dinitrophenylhydrazon chromophore is recorded. All substances are identified by their characteristic UV spectra. For quantification, calibration curves (five point measurements) for all dinitrophenylhydrazon derivatives of the substances were established and 1.2 nmol di-n-butylacetaldehyde is used as internal standard to determine the recovery of the aldehydes.
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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